WO2022135523A1 - Drug for treating and preventing fibroproliferative diseases - Google Patents

Drug for treating and preventing fibroproliferative diseases Download PDF

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WO2022135523A1
WO2022135523A1 PCT/CN2021/140837 CN2021140837W WO2022135523A1 WO 2022135523 A1 WO2022135523 A1 WO 2022135523A1 CN 2021140837 W CN2021140837 W CN 2021140837W WO 2022135523 A1 WO2022135523 A1 WO 2022135523A1
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bmp4
seq
bmp4 protein
protein
vector
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PCT/CN2021/140837
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French (fr)
Chinese (zh)
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卢文菊
管瑞娟
王健
蔡宙
徐静仪
李媛媛
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广州医科大学附属第一医院（广州呼吸中心）
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Publication of WO2022135523A1 publication Critical patent/WO2022135523A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to the medical use of bone morphogenetic protein 4 (Bone Morphogenetic Protein 4, BMP4), in particular to the application of BMP4 in the treatment of fibroproliferative diseases.
  • BMP4 bone morphogenetic protein 4
  • Fibrosis is the end-stage change of a large class of diseases characterized by the proliferation of fibroblasts and the accumulation of a large amount of extracellular matrix accompanied by inflammatory damage and tissue structure destruction. abnormal.
  • Pulmonary Fibrosis is an interstitial lung disease with unknown etiology, rapid progression, poor prognosis, and high fatality rate. Its typical pathological features are diffuse pulmonary interstitial collagen deposition, scarring, progressive and irreversible alveolar structure destruction and diffuse fibrosis. Most patients die of respiratory failure due to ventilation dysfunction. Common causes of the disease include infections, dust, drugs, smoking, and autoimmune diseases.
  • IPF Idiopathic Pulmonary Fibrosis
  • pulmonary fibroblast/myofibroblast hyperproliferation and collagen deposition that disrupt lung structure and function. Fibroblasts and myofibroblasts activate and aggregate in fibroblast foci, producing excess extracellular matrix.
  • the lung function of patients with IPF shows a progressive decline and eventually death due to respiratory failure.
  • the average survival time after diagnosis is only 3-5 years, and the 5-year mortality rate exceeds 40%.
  • the present invention provides the use of a BMP4 protein in the manufacture of a medicament for treating or preventing a fibroproliferative disease in a subject.
  • the BMP4 protein is free BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
  • Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a BMP4 protein of the present invention, wherein the BMP4 protein is free BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis. In some embodiments, the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
  • Another aspect of the present invention provides a recombinant vector for expressing BMP4 protein, which comprises a vector comprising or carrying a polynucleotide encoding BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant vector of the present invention, wherein the recombinant vector comprises a vector, wherein the The vector comprises or carries a polynucleotide encoding the BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Another aspect of the present invention provides the use of the recombinant vector of the present invention in the preparation of a medicament for the treatment or prevention of fibroproliferative diseases in a subject, wherein the recombinant vector comprises a vector comprising or carrying a multinucleus encoding BMP4 protein Glycosides.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Another aspect of the present invention provides a pharmaceutical composition for treating or preventing a fibroproliferative disease in a subject, wherein the pharmaceutical composition comprises a recombinant carrier and a pharmaceutically acceptable excipient, wherein the recombinant carrier comprises a carrier,
  • the vector comprises or carries a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises recombinant A carrier and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof.
  • the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Another aspect of the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing fibroproliferative diseases in a subject, wherein the pharmaceutical composition comprises a recombinant carrier and a pharmaceutically acceptable excipient , wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • administration of the BMP4 protein, recombinant vector or pharmaceutical composition of the invention to a subject results in increased expression of the BMP4 protein in the subject's cells. In some embodiments, administration of the BMP4 protein, recombinant vector or pharmaceutical composition of the invention to a subject results in increased expression of the BMP4 gene in the subject's cells. In some embodiments, the BMP4 protein, recombinant vector or pharmaceutical composition of the present invention inhibits the proliferation of fibroblasts and promotes apoptosis of fibroblasts after being administered to a subject. In some embodiments, the BMP4 protein, recombinant vector or pharmaceutical composition of the invention inhibits the transformation of fibroblasts to myofibroblasts after administration to a subject.
  • Figure 1 The expression of BMP4 was significantly reduced in the lung tissue of IPF patients compared to the normal control lung tissue.
  • A protein band map
  • B band grayscale analysis statistical results
  • C mRNA expression statistical results
  • D immunofluorescence image of human lung tissue sections
  • FIG. 2 Bleomycin (Bleomycin, BLM) induces decreased expression of BMP4 protein in mouse lung tissue.
  • Figure 3 Effects of BMP4 knockout on bleomycin-induced mouse survival and body weight.
  • A Survival rate curve of mice treated with bleomycin;
  • B Changes in body weight of mice.
  • Figure 5 Effects of BMP4 knockout on bleomycin-induced lung function in mice.
  • A Dynamic lung compliance;
  • Figure 6 Effects of BMP4 knockout on bleomycin-induced mouse HE and Masson staining results.
  • n 10.
  • Figure 8 The effect of BMP4 gene knockout on fibronectin (Fibronectin), alpha-smooth muscle actin (alpha-smooth muscle actin, alpha-SMA), type I collagen (Collagen 1, Col 1), Effects of changes in protein expression of type III collagen (Collagen 3, Col 3).
  • Figure 9 Effects of BMP4 overexpression on the preventive and therapeutic effects of bleomycin-induced pulmonary fibrosis in mice.
  • A The effect of BMP4 overexpression on lung function in mice with bleomycin-induced pulmonary fibrosis;
  • B The effect of BMP4 overexpression on lung pathology in mice with bleomycin-induced pulmonary fibrosis.
  • Figure 10A Effects of BMP4 knockout on TGF- ⁇ 1-induced transformation and ECM synthesis in mouse primary fibroblasts.
  • Figures 10B-10D Effects of exogenous addition of BMP4 recombinant protein on TGF-beta1-induced transformation of mouse primary fibroblasts.
  • FIGS 11A-11C Effects of BMP4 knockout on TGF-beta1-induced proliferation of mouse primary fibroblasts.
  • A Protein band map;
  • FIGS 11D-10F Effects of exogenous addition of BMP4 recombinant protein on TGF-beta1-induced transformation of mouse primary fibroblasts.
  • Figure 12 Effect of BMP4 knockout on TGF- ⁇ 1-induced apoptosis in mouse primary fibroblasts.
  • B: Statistics of apoptosis rate, n 5.
  • treatment refers to therapeutic as well as prophylactic measures which prevent or slow the development of undesired physiological changes or disorders in a subject, such as the development of pulmonary fibrosis or the progression of cancer.
  • Treatment includes, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization (ie, no exacerbation) of disease state, delay or slowing of disease progression, reduction or alleviation of disease state, and partial or All healed regardless of whether the above effects were detectable.
  • Treatment can also mean prolonging survival as compared to no treatment.
  • Objects in need of treatment include those already suffering from the disease or disorder, as well as those likely to suffer from the disease or disorder, or for which the disease or disorder is to be prevented.
  • Subject or “patient”, “individual” refers to any subject for which diagnosis, prognosis or treatment is desired, particularly mammalian subjects. Mammals include humans, domestic animals, farm animals, zoo animals, sports animals or pets such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, cows, and the like. The subject referred to herein is preferably a human.
  • the term "patient in need of treatment” or “subject in need of treatment” includes a subject, such as a mammalian subject, who would benefit from administration of a polypeptide of the present invention, for example, for detection, diagnostic and/or therapeutic use, or a composition thereof .
  • the term "therapeutically effective amount” or “effective amount” refers to a medicament or pharmaceutical composition of the present invention which is administered to a cell, tissue or subject when administered alone or in combination with an additional therapeutic agent.
  • a therapeutically effective dose further refers to an amount of the compound sufficient to cause alleviation of symptoms, such as to treat, cure, prevent or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention or alleviation of the condition .
  • the therapeutically effective amount refers to that ingredient alone.
  • a therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether it is administered in combination, consecutively, or simultaneously.
  • a therapeutically effective amount will generally reduce symptoms by at least 10%; usually by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.
  • “about” means that the index value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, “about” or “substantially comprising” can mean a range of up to 20%. Also, with respect to a biological system or process, the term can mean up to an order of magnitude or up to 5 times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about” or “substantially comprising” should be assumed to be within an acceptable error range for the specific value.
  • genes can be modified such that they differ in nucleotide sequence from the modified polynucleotides from which they are derived.
  • a polynucleotide or nucleotide sequence derived from a given DNA sequence may be similar, eg, have a certain percentage identity to the starting sequence, eg, it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% the same.
  • nucleotide or amino acid substitutions, deletions or insertions can be made to make conservative substitutions or changes in "non-essential" regions.
  • a polypeptide or amino acid sequence derived from a specified protein except for one or more individual amino acid substitutions, insertions, or deletions (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 The remainder may be identical to the starting sequence except for one or more single amino acid substitutions, insertions or deletions).
  • the polypeptide or amino acid sequence derived from a given protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 individual amino acid substitutions, insertions or deletions relative to the starting sequence.
  • Bone morphogenetic proteins belong to the transforming growth factor (TGF) family and are a large class of multifunctional growth factors that can affect embryogenesis and tissue formation in organs such as kidneys and lungs, and regulate cell proliferation, transformation, and apoptosis. apoptosis and secretion of ECM components.
  • TGF transforming growth factor
  • BMP4 refers to bone morphogenetic protein 4, a member of the BMP, one of the key factors in lung development.
  • the amino acid sequences of BMP4 proteins can be obtained from NCBI with accession numbers NP_001334841.1, NP_001334845.1, NP_001334843.1, NP_001193.2, NP_570911.2, NP_570912.2, NP_001334846.1, NP_001334842.1, and NP_0013.
  • the amino acid sequence of the BMP4 protein is shown in any one of SEQ ID NO:1 to SEQ ID NO:3.
  • the nucleotide sequence encoding the BMP4 protein product is set forth in any of SEQ ID NO:4 to SEQ ID NO:9.
  • the NCBI sequence accession numbers of the messenger RNA (mRNA) of BMP4 are NM_001347912.1 (SEQ ID NO:19), NM_001347916.1 (SEQ ID NO:20), NM_001347914.2 (SEQ ID NO:21), NM_001202.6 ( SEQ ID NO:22), NM_130850.5 (SEQ ID NO:23), NM_130851.4 (SEQ ID NO:24), NM_001347917.1 (SEQ ID NO:25), NM_001347913.2 (SEQ ID NO:26) , and NM_001347915.2 (SEQ ID NO:27).
  • BMP4 plays an important role in maintaining pulmonary vascular homeostasis, and inhibition of BMP4 can inhibit the pathological changes of hypoxic pulmonary hypertension. Studies have shown that BMP4 inhibits the proliferation of human lung fibroblasts and induces the proliferation and dedifferentiation of lung epithelial cells.
  • polynucleotide and “nucleic acid” are used interchangeably herein to refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. These terms include single-, double- or triple-stranded DNA, genomic DNA, cDNA, genomic RNA, mRNA, DNA-RNA hybrids, or polymers; the polymers contain purine and pyrimidine bases, or other natural, chemical, Biochemically modified, non-natural or derived nucleotide bases.
  • the backbone of a polynucleotide may contain sugar and phosphate groups (commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • the backbone of the polynucleotide may comprise a polymer of synthetic subunits (eg, phosphoramidate), and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate- Phosphodiester oligomers.
  • synthetic subunits eg, phosphoramidate
  • P-NH2 oligodeoxynucleoside phosphoramidate
  • Phosphodiester oligomers e.g, P-NH2
  • the term “encoding” refers to any process by which information in a polymeric macromolecule or sequence string is used to direct the production of a second molecule or sequence string that differs from the first molecule or sequence string. As used herein, the term is widely used and can have various applications. In one aspect, the term “encoding” describes the process of semi-conservative DNA replication in which one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In another aspect, the term “encoding” refers to any process by which information in one molecule is used to direct the production of a second molecule having different chemical properties than the first molecule.
  • a DNA molecule can encode an RNA molecule (eg, by participating in a DNA-dependent RNA polymerase transcription process).
  • RNA molecules can encode polypeptides, as during translation. When used to describe the process of translation, the term “encoding” also extends to triplet codons encoding amino acids.
  • an RNA molecule can encode a DNA molecule, eg, by participating in the reverse transcription process of an RNA-dependent DNA polymerase.
  • a DNA molecule may encode a polypeptide, where it is understood that "encoding" as used in this context encompasses both transcription and translation processes.
  • variant refers to a nucleus having at least 60%, 70%, 80%, 90%, 95%, 98% sequence identity to a sequence with conservative substitutions or changes in "non-essential" regions nucleotide or amino acid sequence.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence shares less than 40% identity with one of the sequences of the invention, but preferably less than 25% identity.
  • a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%) of another sequence or 99%) "sequence identity", meaning that when aligned, the percentage of bases (or amino acids) that are identical when the two sequences are compared. Such alignment and percent homology or sequence identity can be determined using software programs known in the art.
  • Viral vectors can bring genetic material into cells by using the molecular mechanism of viruses to transmit their genomes into other cells for infection.
  • Viral vectors may also be referred to as vectors, vector virions, or vector particles.
  • Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • Retroviral vectors can be or can be derived from any suitable retrovirus.
  • a large number of different retroviruses have been identified. Examples include, but are not limited to: Murine Leukemia Virus (MLV), Human T-Cell Leukemia Virus (HTLV), Mouse Mammary Tumor Virus (MMTV), Rous Sarcoma Virus (RSV), Fujinami Sarcoma Virus (FuSV), Moloney Murine leukemia virus (Mo MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloma virus-29 (MC29) and avian polycythemia virus (AEV).
  • MMV Murine Leukemia Virus
  • HTLV Human T-Cell Leukemia Virus
  • MMTV Mouse Mammary Tumor Virus
  • RSV Rous Sarcoma Virus
  • FuSV Fujin
  • Adenoviruses are double-stranded, linear DNA viruses that do not replicate through an RNA intermediate.
  • Adenoviruses are double-stranded DNA non-enveloped viruses capable of transducing a wide range of human and non-human derived cell types in vivo, ex vivo and in vitro. These cells include airway airway epithelial cells, hepatocytes, muscle cells, cardiomyocytes, synoviocytes, primary mammary epithelial cells, and post-mitotic terminally differentiated cells (eg, neurons).
  • Adenoviruses have been used as vectors for gene therapy and heterologous gene expression.
  • the large (36kb) genome can accommodate up to 8kb of exogenous insert DNA and can replicate efficiently in complementary cell lines to yield very high titers of up to 1012 transduction units per milliliter.
  • Adenoviruses are therefore one of the best systems for studying gene expression in primary non-replicating cells.
  • the expression of viral genes or foreign genes from the adenoviral genome does not require replicating cells.
  • Adenoviral vectors enter cells through receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into the host chromosome. Instead, they exist as episomes (independent of the host genome), as a linear genome in the host cell nucleus.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Dependovirus of the family Parvoviridae, and is the simplest single-stranded DNA-deficient virus discovered so far.
  • Recombinant AAV vectors have been successfully used for in vitro, ex vivo and in vivo transduction of marker genes and genes involved in human disease.
  • Certain AAV vectors have been developed that can efficiently bind large payloads (up to 8-9 kb).
  • AAV serotypes Multiple adeno-associated virus (AAV) serotypes have now been identified, including 12 human serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12) and more than 100 serotypes from non-human primates.
  • the present invention contemplates that any of these serotypes can be used.
  • Herpes simplex virus is an enveloped double-stranded DNA virus that naturally infects neurons. It can accommodate large segments of foreign DNA and has been adopted as a vector for gene delivery to neurons. The use of HSV during therapy requires attenuating the strains so that they cannot establish a lytic cycle.
  • the polynucleotide is preferably inserted into the essential gene. This is because if the viral vector encounters the wild-type virus, the heterologous gene can be transferred to the wild-type virus by recombination.
  • the recombinant virus is constructed in a way that prevents its replication, this can be achieved by inserting oligonucleotides into the viral genes necessary for replication.
  • the viral vector of the present invention may be a vaccinia virus vector such as MVA or NYVAC.
  • vaccinia vectors include, for example, the fowlpox (avipox) vector known as ALVAC or canarypox, and strains derived therefrom that can infect and express recombinant proteins in human cells but cannot replicate . It will be appreciated that portions of the viral genome may remain intact following insertion of the recombinant gene. This means that the notion that a viral vector can retain the ability to infect cells and subsequently express additional genes that support its replication and possibly promote lysis and death of infected cells.
  • Lentiviruses are part of a larger group of retroviruses. Can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV).
  • the non-primate lentivirus population includes the prototype "lentivirus" visna/maedi virus (VMV), as well as the related goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus ( FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV related goat arthritis-encephalitis virus
  • EIAV equine infectious anemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • Lentiviral vector generally refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1).
  • the lentiviral vector contains the genetic information required for packaging, transfection, and stable integration, and is the main component of the lentiviral vector system. component.
  • Lentiviral vectors carrying foreign genes are packaged into infectious virus particles with the help of lentiviral packaging plasmids and cell lines. By infecting cells or living tissues, the exogenous genes can be expressed in cells or living tissues. .
  • Non-viral vectors use the physicochemical properties of non-viral vector materials to mediate gene transfer. Any suitable non-viral vector can be used to introduce the BMP4 gene into cells of a subject. Examples of non-viral vectors include, but are not limited to, plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes.
  • Plasmids are small circular DNA molecules. As the most commonly used and simplest vector in genetic engineering, plasmids must include three parts: genetic marker gene, replication region, and target gene. Plasmids are found in all bacterial groups, and they are DNA molecules that replicate independently of bacteria extrachromosomally. Examples of plasmid vectors include, but are not limited to, E. coli plasmid vectors, Bacillus subtilis plasmid vectors, yeast plasmid vectors, Agrobacterium plasmid vectors, and cyanobacterial plasmid vectors.
  • Liposome is a liposome (hollow) made from lecithin and ceramide, etc. It has a bilayer structure and is an artificial membrane.
  • the composition of liposomes is usually a combination of phospholipids (especially high phase transition temperature phospholipids), usually in combination with steroids (especially cholesterol). Other phospholipids or other lipids can also be used.
  • the physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • the transduction efficiency of liposomes can be increased by using dioleoylphosphatidylethanolamine during transduction. High potency liposomes are commercially available. Examples of liposomes include, but are not limited to, neutral liposomes, negatively charged liposomes, and positively charged liposomes.
  • An episomal carrier is a substance that allows and facilitates the penetration and transfer of water-soluble substances into the cell through the lipid layer of the cell membrane. It is a protein in the membrane, a component that regulates the active uptake of ions.
  • Episomal vectors can include one or more pluripotency genes operably linked to at least one regulatory sequence for expressing the factor.
  • Episomal vectors of the present invention may also include components that allow the vector to replicate in cells.
  • the Epstein Barr oriP/nuclear antigen-1 (EBNA-1) combination can support self-replication of vectors in mammalian cells, especially primate cells.
  • the EBNA1 trans-element and OriP cis-element from the EBV genome enable simple plasmid replication and maintenance as episomes in proliferating human cells.
  • Cationic polymers for gene transfer include, but are not limited to, polylysine, polyethyleneimine, and dendrimers.
  • chitosan-DNA system can not be degraded by electrostatic interaction with DNA, and can enter cells completely.
  • chitosan As a gene carrier, chitosan has the characteristics of low cytotoxicity, good biocompatibility, low gene immunity and high transfection efficiency.
  • Chitosan-DNA complexes are mainly divided into DNA complexes of chitosan and its derivatives, chitosan-DNA nano-microspheres and chitosan self-polymer-DNA according to the preparation method.
  • Inorganic nanoparticles play a role in the treatment of diseases mainly by transporting drugs or biomolecules into organisms through cell membranes.
  • Examples of inorganic nanoparticles for gene delivery include, but are not limited to, silicon, iron oxides, carbon nanotubes, calcium phosphates, metal nanoparticles, quantum dots, and the like.
  • the recombinant vectors disclosed herein are recombinant vectors expressing a BMP4 protein comprising a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the vector is a viral vector or a non-viral vector.
  • the polynucleotide encoding the BMP4 protein is administered via a vector, wherein the vector comprises a viral vector and a non-viral vector.
  • the vector comprises a viral vector and a non-viral vector.
  • One aspect of the present invention provides a recombinant vector for treating or preventing a fibroproliferative disease in a subject, comprising a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof.
  • the vector is a viral vector.
  • the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof.
  • the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof.
  • the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof.
  • the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
  • the viral vector is an adeno-associated viral vector.
  • the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein.
  • the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises as SEQ ID NO: 1 to Any one of the sequences shown in SEQ ID NO: 3 or a variant thereof.
  • the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID Any one of the sequences shown in NO:4 to SEQ ID NO:9 or a variant thereof.
  • the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID Any one of the sequences shown in NO: 10 to SEQ ID NO: 27 or a variant thereof.
  • the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID The sequence shown in NO:28 or SEQ ID NO:29 or a variant thereof.
  • the vector is a non-viral vector
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, capsids Glycan polymers, inorganic nanoparticles and exosomes.
  • the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof.
  • the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof.
  • the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof.
  • the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
  • the non-viral vector is an exosome.
  • the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein.
  • the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof.
  • the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof.
  • the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof.
  • the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
  • the non-viral vector is a plasmid.
  • the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein.
  • the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3 any of the sequences or variants thereof.
  • the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO:4 to SEQ ID NO: Any one of the sequences shown in 9 or a variant thereof.
  • the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 10 to SEQ ID NO: Any one of the sequences shown in 27 or a variant thereof.
  • the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO:28 or SEQ ID NO: 29 or a variant thereof.
  • the non-viral vector is a liposome.
  • the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein.
  • the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof.
  • the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof.
  • the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof.
  • the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
  • “Pharmaceutical composition” refers to a pharmaceutical formulation for use in humans.
  • the pharmaceutical composition comprises the medicament of the present invention together with suitable formulations of carriers, stabilizers and/or excipients.
  • One aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant vector and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the pharmaceutical composition can be used to treat or prevent fibroproliferative diseases in a subject.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising free BMP4 protein and a pharmaceutically acceptable excipient.
  • the amino acid sequence of the free BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
  • the recombinant carrier is mixed with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers.
  • compositions include materials that, when combined with an active ingredient of a composition, allow the ingredient to remain biologically active and not cause a damaging reaction with the subject's immune system. These may include stabilizers, preservatives, salts or sugar complexes or crystals, and the like. “Pharmaceutically acceptable” refers to molecules and ingredients that do not produce allergic or similar undesired reactions when administered to humans. It is known in the art how to prepare aqueous compositions containing as an active ingredient. Typically, these compositions are prepared as injectables or sprays, such as liquid solutions or suspensions; solid forms suitable for solution or suspension prior to injection or spraying can also be prepared.
  • Fibrosis can occur in a variety of organs, and the main pathological changes are the increase of fibrous connective tissue and the decrease of parenchymal cells in the organ tissue. Continuous progress can lead to structural damage and functional decline of organs, and even failure, which seriously threatens human health and life.
  • fibroproliferative diseases include, but are not limited to: fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow and skin.
  • Pulmonary fibroproliferative diseases include, but are not limited to: inorganic dust occupational diseases (such as silicosis, asbestosis, coal lung, etc.), organic dust and hypersensitivity pneumonitis (such as farmers' lung), primary lung diseases (such as idiopathic interstitial disease) pneumonia, bronchiolitis obliterans with organizing pneumonia, etc.), idiopathic pulmonary fibrosis, progressive system sclerosis, lymphocytic interstitial pneumonia, familial pulmonary fibrosis, etc.
  • inorganic dust occupational diseases such as silicosis, asbestosis, coal lung, etc.
  • organic dust and hypersensitivity pneumonitis such as farmers' lung
  • primary lung diseases such as idiopathic interstitial disease) pneumonia, bronchiolitis obliterans with organizing pneumonia, etc.
  • idiopathic pulmonary fibrosis progressive system sclerosis
  • lymphocytic interstitial pneumonia familial pulmonary fibrosis, etc.
  • Fibroproliferative diseases of the cardiovascular system include, but are not limited to, replacement and interstitial fibrosis following myocardial infarction.
  • Fibroproliferative diseases of the liver include, but are not limited to, viral cirrhosis (eg, viral hepatitis B, C and D), dystrophic cirrhosis, and the like.
  • viral cirrhosis eg, viral hepatitis B, C and D
  • dystrophic cirrhosis e.g., dystrophic cirrhosis, and the like.
  • Renal fibroproliferative diseases include, but are not limited to, chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, systemic lupus erythematosus nephropathy, and the like.
  • Ocular fibroproliferative diseases include, but are not limited to, ocular trauma and post-surgical ocular fibroproliferative diseases.
  • Fibroproliferative diseases of the nervous system include, but are not limited to, fibroproliferative diseases after spinal cord trauma and the like.
  • Myelofibroproliferative diseases include, but are not limited to, idiopathic and drug-induced myelofibrosis, among others.
  • Dermatofibroproliferative disorders include, but are not limited to, oral mucosal fibrosis and the like.
  • a method of treating or preventing a fibroproliferative disease in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a BMP4 protein of the present invention, wherein the BMP4 protein is free BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • the present invention provides the use of the BMP4 protein of the present invention in the preparation of a medicament for treating or preventing fibroproliferative diseases in a subject, wherein the BMP4 protein is a free BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a BMP4 protein of the present invention, wherein the BMP4 protein is expressed A recombinant vector for the BMP4 protein, the recombinant vector comprising a vector comprising or carrying a polynucleotide encoding the BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof.
  • the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • the present invention provides the application of the BMP4 protein of the present invention in the preparation of a medicine for treating or preventing fibroproliferative diseases in an object, wherein the BMP4 protein is a recombinant vector expressing the BMP4 protein, and the recombinant vector comprises a vector , the vector comprises or carries a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises A recombinant vector and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof.
  • the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for the treatment or prevention of fibroproliferative diseases in a subject, wherein the pharmaceutical composition comprises a recombinant carrier and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein.
  • the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  • the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof.
  • the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector.
  • the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes .
  • the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
  • the viral vector is an adeno-associated virus.
  • the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin.
  • the fibroproliferative disease is pulmonary fibroproliferative disease.
  • the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  • Suitable routes of administration include parenteral (eg, intramuscular, intravenous or subcutaneous) and oral administration.
  • the BMP4 protein, recombinant vector or pharmaceutical composition of the methods of the present invention can be administered in a variety of conventional ways, such as by endotracheal intubation, oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, gastrointestinal Extracorporeal, intraarterial or intravenous injection.
  • the BMP4 protein, recombinant vector or pharmaceutical composition of the invention is administered via endotracheal intubation.
  • the BMP4 protein, recombinant vector or pharmaceutical composition of the invention is administered by intravenous injection.
  • Appropriate doses are determined by the clinician, eg, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment. Typically, the starting dose is slightly lower than the optimal dose and is increased by small amounts thereafter until the desired or optimal effect is achieved with respect to any adverse side effects.
  • Important diagnostic measurements include measuring, for example, inflammatory symptoms or levels of inflammatory cytokines produced.
  • the BMP4 protein, recombinant vector, or pharmaceutical composition of the present invention can be administered by continuous administration or by administration at intervals (eg, one day, one week, or 1-7 times per week). Dosages can be provided by endotracheal intubation, intravenously, subcutaneously, intraperitoneally, transdermally, topically, orally, nasally, rectally, intramuscularly, intracerebrally or intraspinal.
  • a preferred dosage regimen is one that includes the maximum dose or frequency of administration that avoids significant undesirable side effects.
  • the methods of treating or preventing fibroproliferative diseases of the present invention prevent the progression of fibrosis or the onset of diseases caused by fibrosis.
  • the present invention provides a method of arresting the progression of fibrosis and/or the onset of diseases caused by fibrosis, the method comprising administering to a subject in need thereof an effective amount of a BMP4 protein of the present invention , recombinant vector or pharmaceutical composition.
  • these methods of treating or preventing fibroproliferative diseases prevent the onset, progression and/or recurrence of symptoms associated with fibrosis.
  • the present invention provides a method of treating or preventing symptoms associated with fibrosis, the method comprising administering to a subject in need thereof an effective amount of a BMP4 protein, recombinant vector or pharmaceutical composition of the present invention.
  • SPF wild-type C57BL/6 mice were purchased from the Animal Experiment Center of Guangzhou University of Traditional Chinese Medicine, and BMP4 +/- heterozygous knockout mice with C57BL/6 background were purchased from the Jackson Laboratory in the United States. All animals were reared and bred in the SPF animal room of the Experimental Animal Center of Guangzhou Medical University. The mice were randomly divided into groups when they were 6-8 weeks old for experiments. Ethics committee review and approval.
  • GraphPad Prism 5.0 software was used for statistical analysis and software mapping, and the experimental results were expressed as mean ⁇ standard error (Mean ⁇ SEM). The t test was used for the comparison of the means between the two groups, and the single factor was used for the comparison of the means of multiple groups. Analysis of variance (one way-ANOVA) test, P ⁇ 0.05 was considered statistically significant.
  • Example 1 Significantly reduced expression of BMP4 in lung tissue of IPF patients and bleomycin-induced pulmonary fibrosis mice
  • Extraction of total protein from lung tissue Take the cryopreserved lung tissue out of the -80°C refrigerator, wash the lung tissue with normal saline, and dry the water. Weigh the lung tissue, add about 1 ml of protein lysis solution per 100 mg of lung tissue, put it into a homogenization tube, crush the lung tissue with a high-speed homogenizer, and lyse the lysis solution into a thick soup sample. Centrifuge in a high-speed refrigerated centrifuge at 12,000g at 4°C for 25 minutes, suck the upper liquid into a new EP tube for subsequent protein concentration measurement, and discard the precipitate.
  • Bleomycin induces the reduction of BMP4 protein expression in lung tissue of mice with pulmonary fibrosis
  • Intratracheal administration of bleomycin induced pulmonary fibrosis in wild-type mice at different stages of the development of pulmonary fibrosis, namely at 0, 7, 14, and 14 days after bleomycin was administered.
  • Lung tissue samples were collected on day 21; Western blotting was used to analyze the expression of BMP4 protein in mouse lung tissue at different stages of pulmonary fibrosis. It was found that compared with the control group mice, the BMP4 level in the lung tissue of the model group mice was significantly reduced, and with the increase of the action time of bleomycin, the BMP4 protein expression in the lung tissue of the pulmonary fibrosis mice was further reduced (Figure 2 ).
  • Example 2 The effect of BMP4 gene knockout on bleomycin-induced pulmonary fibrosis in mice
  • NEO primers are used to amplify BMP4 mutant gene
  • WT primers are used to amplify BMP4 wild gene.
  • F upstream primer
  • R downstream primer
  • the PCR amplification reaction is performed after the reaction system is fully mixed.
  • mice were divided into four groups: wild-type mice control group, BMP4 +/- mice control group, wild-type mice treated with bleomycin, and BMP4 +/- mice treated with bleomycin.
  • Bleomycin was instilled by intratracheal intubation via laryngotracheal intubation.
  • the dose of bleomycin was 2.0 mg/kg, and the control group was given the corresponding dose of normal saline.
  • the mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g), and the anesthesia should not be too deep.
  • the mice were fixed on the surgical table, perpendicular to the table top.
  • mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g), and the anesthetized mice were placed in a CT scanning box, and the chest CT scan imaging was performed on the mice with LCT-200 Micro CT.
  • mice On the 21st day after bleomycin modeling, the lung function of mice was detected by small animal pulmonary function analyzer.
  • the small animal spirometer was calibrated, and the test was started after the machine parameters reached the standard.
  • the mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g), the neck of the mice was wiped with alcohol, the skin of the neck was incised, the muscle tissue was immediately separated, the trachea was exposed, and the trachea was cut and connected to the tracheal intubation. After entering the body tracing box, connect the small animal pulmonary function instrument and perform mechanical ventilation. Pulmonary function was measured after the changes of the respiration and various parameters of the mice became stable, and various pulmonary ventilation function indexes were recorded.
  • the isolated mouse lung tissue was fixed with paraformaldehyde, then dehydrated, embedded, sectioned, and cut into slices with a thickness of about 3 ⁇ m.
  • mice lung tissue was fixed with formaldehyde, it was routinely dehydrated and embedded in sections. After the sections were deparaffinized, they were placed in a fixative solution overnight at room temperature and rinsed with running water. It was stained with celestine blue solution and hematoxylin semen for 2 minutes each, differentiated with 1% hydrochloric acid ethanol, and rinsed with running water for 10 minutes. It was treated in magenta solution and 1% aluminophosphate solution for 10 minutes, then dyed in 2% aniline blue solution for 5 minutes, and treated in 0.2% glacial acetic acid solution for 2 minutes. 95% alcohol dehydration, gradient alcohol dehydration, xylene transparent, neutral gum seal.
  • the activity status of the mice in each group was observed.
  • the mice in the control group (Saline) had sensitive responses, stable breathing, and normal coat color and gloss; while the mice in the model group (BLM) had poor activity status, dark coat color, dullness, and shortness of breath.
  • mice were treated with bleomycin (2.0 mg/kg), and the body weight and death of the mice during the 21-day modeling process were recorded.
  • the survival rate of the model group (BLM) mice was decreased.
  • the survival rate of BMP4 +/- mice decreased significantly (Fig. 3A).
  • the body weight of the model group (BLM) mice gradually decreased within 10 days after modeling, and became stable and gradually recovered after 10 days.
  • the weight loss of BMP4 +/- mice was more significant compared with wild-type mice (Fig. 3B).
  • mice On the 14th day after the bleomycin model was established in mice, in vivo chest CT scans were performed to evaluate the degree of pulmonary fibrosis in mice. As shown in FIG. 4 , compared with the control group, the model group (BLM) mice had significant lung consolidation and fibrosis. In the model group (BLM) mice, compared with wild-type mice, BMP4 +/- mice had more significant lung consolidation and fibrosis.
  • hydroxyproline is unique to collagen fibers, which is used as an indicator of pulmonary fibrosis.
  • the content of hydroxyproline in the lung tissue of mice was detected (Fig. 7) to judge the degree of fibrosis.
  • Example 3 The effect of BMP4 gene knockout on bleomycin-induced transformation of mouse fibroblasts to myofibroblasts and synthesis of ECM components
  • ⁇ -SMA is a marker for the transformation of fibroblasts to myofibroblasts, and Fibronectin and Col 1 and Col 3, the main components of collagen, are the main components of ECM.
  • bleomycin modeling we detected the expression of ⁇ -SMA, Fibronectin, Col 1, and Col 3 in the lung tissue of mice in each group.
  • Example 4 The effect of BMP4 overexpression on the preventive and therapeutic effects of bleomycin-induced pulmonary fibrosis in mice
  • AAV9-BMP4 adeno-associated virus overexpressing BMP4 (SEQ ID NO:34) two weeks before intratracheal injection of bleomycin and after Nine days later, adeno-associated virus AAV-BMP4 was administered via tracheal intubation. After 23 days of bleomycin-induced modeling, the lung function of the mice in each group was measured, and the tissues were obtained by HE staining and Masson staining to observe the effect of BMP4 overexpression on bleomycin-induced pulmonary fibrosis in mice. preventive and therapeutic effects.
  • mice were divided into four groups: control group (control), group treated with bleomycin and AAV9 empty vector (BLM+AAV-Empty), and group treated with AAV9-BMP4 after bleomycin treatment (BLM+AAV9-BMP4) , AAV9-BMP4-treated BLM-treated group (AAV9-BMP4+BLM).
  • Example 5 BMP4 gene knockout can promote TGF- ⁇ 1-induced transformation and ECM synthesis of mouse primary lung fibroblasts
  • mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g). After anesthesia, the mice were soaked in alcohol for 3-5 minutes. The mice were placed in the ultra-clean table, the abdominal aorta was cut open for bloodletting, and the chest cavity was opened to take out Lung tissue, discard the lung tissue near the large airway, wash the lung tissue in a large dish containing PBS for 2-3 times, transfer it into a new large dish, quickly cut the lung tissue into about 1mm3 pieces, wash the tissue with PBS Block until liquid is clear, discard PBS. Add trypsin to the tissue block for 15min digestion, suck the digested tissue block and trypsin into a filter, filter, and rinse with DMEM without FBS.
  • Avertin 0.2ml/10g
  • 2ml DMEM containing 10% FBS transfer the tissue blocks into two culture flasks, aspirate and discard the liquid, and place the tissue blocks evenly on the side wall of the culture flask to make the tissue blocks adhere to the bottle wall, at least 0.5 from the bottom edge of the flask. cm away, add 4ml of DMEM containing 10% FBS to the bottle, put the culture bottle vertically into the cell incubator, and gently put the bottle down after 3 hours so that the medium does not cover the tissue block and ensure that the tissue block still adheres to the bottle on the wall. After 3 days, the medium was changed, the floating tissue blocks were aspirated and discarded, and the adherent tissue blocks were continued to be cultured, and passaged after 7-10 days.
  • BMP4 knockout The primary lung fibroblasts (BMP4 +/- and BMP4 +/+ ) extracted and cultured from the lung tissues of BMP4 +/- mice and wild-type mice were treated with TGF- ⁇ 1 for 48 hours to establish a pulmonary fibrosis cell model.
  • BMP4 gene knockout affects the outcome of pulmonary fibrosis.
  • the expressions of fibroblast transformation markers Fibronectin, ⁇ -SMA, Col 3 and Col 1 in mouse primary fibroblasts were detected.
  • Example 6 Exogenous addition of BMP4 recombinant protein can inhibit the transformation and ECM production of mouse primary lung fibroblasts induced by TGF- ⁇ 1
  • mouse primary fibroblasts were stimulated with TGF- ⁇ 1 to establish a fibrotic cell model, and on this basis, BMP4 was treated for 48 h , to detect the protein expression changes of fibroblast transformation markers Fibronectin, ⁇ -SMA, Col 1 and Col 3 in cells.
  • Example 7 BMP4 knockout can promote TGF- ⁇ 1-induced mouse lung primary fibroblast proliferation
  • Fibroblast activation and transformation are the main features of pulmonary fibrosis. Activation of fibroblasts includes hyperproliferation and resistance to apoptosis.
  • Example 8 Exogenous addition of BMP4 recombinant protein can inhibit the proliferation of mouse lung primary fibroblasts induced by TGF- ⁇ 1
  • Example 9 BMP4 knockout inhibits TGF- ⁇ 1-mediated apoptosis resistance of fibroblasts and promotes fibroblast apoptosis
  • AnnexinV FITC Apoptosis Detection Kit The specific steps are as follows: wash the adherent cells once with PBS, digest the cells with trypsin, add DMEM containing 10% FBS to stop the digestion, and pipet the cell suspension into 15ml EP tube, 800rpm, centrifugation for 5min. Wash the cells twice with pre-cooled PBS, centrifuge at 800 rpm for 5 min, discard the PBS, resuspend the cells with 1 ⁇ Bingding Buffer solution, count the cells in parallel, and dilute the cell suspension to make the cell concentration 1 ⁇ 10 6 /ml.
  • the AnnexinV FITC apoptosis detection kit was used to detect the apoptosis of mouse primary fibroblasts treated with TGF- ⁇ 1, and the apoptosis rate was calculated.
  • BMP4 can inhibit the apoptosis resistance of fibroblasts mediated by TGF- ⁇ 1 and promote the apoptosis of fibroblasts.

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Abstract

Disclosed in the present invention is an application of a BMP4 protein in preparation of a drug for treating or preventing a fibroproliferative disease of an object, wherein the BMP4 protein is a free BMP4 protein or a recombinant vector expressing the BMP4 protein. The recombinant vector expressing the BMP4 protein comprises a vector, and the vector comprises or carries a polynucleotide encoding the BMP4 protein. The BMP4 can inhibit fibroblast proliferation, promote apoptosis of fibroblasts, and inhibit the transformation of fibroblasts into muscle fibroblasts. These results indicate that fibroproliferative diseases can be treated or prevented by targeting BMP4.

Description

用于治疗或预防纤维增生性疾病的药物Medications used to treat or prevent fibroproliferative disorders 技术领域technical field
本发明涉及骨形态发生蛋白4(Bone Morphogenetic Protein 4,BMP4)的医药用途，具体涉及BMP4在纤维增生性疾病的治疗中的应用。The present invention relates to the medical use of bone morphogenetic protein 4 (Bone Morphogenetic Protein 4, BMP4), in particular to the application of BMP4 in the treatment of fibroproliferative diseases.
背景技术Background technique
纤维化(fibrosis)是以成纤维细胞增殖及大量细胞外基质聚集并伴炎症损伤、组织结构破坏为特征的一大类疾病的终末期改变，也就是正常的组织被损坏后经过异常修复导致结构异常。Fibrosis is the end-stage change of a large class of diseases characterized by the proliferation of fibroblasts and the accumulation of a large amount of extracellular matrix accompanied by inflammatory damage and tissue structure destruction. abnormal.
肺纤维化(Pulmonary Fibrosis,PF)是一种病因不明、进展迅速、预后极差、致死率极高的间质性肺疾病，缺乏有效治疗方法。其典型病理特征为弥漫性肺间质胶原沉积、瘢痕形成，进行性、不可逆的肺泡结构破坏及弥漫性纤维化，多数病人因换气功能障碍而死于呼吸衰竭。该病的常见病因包括感染、粉尘、药物、吸烟和自身免疫性疾病等。Pulmonary Fibrosis (PF) is an interstitial lung disease with unknown etiology, rapid progression, poor prognosis, and high fatality rate. Its typical pathological features are diffuse pulmonary interstitial collagen deposition, scarring, progressive and irreversible alveolar structure destruction and diffuse fibrosis. Most patients die of respiratory failure due to ventilation dysfunction. Common causes of the disease include infections, dust, drugs, smoking, and autoimmune diseases.
特发性肺纤维化(Idiopathic Pulmonary Fibrosis,IPF)，是一种病因不明的肺纤维化，也是肺纤维化中最常见和最严重的一种，有时呈急性恶化。IPF的特征是肺成纤维细胞/肌成纤维细胞过度增殖和胶原沉积，破坏肺部结构和功能。成纤维细胞和肌成纤维细胞在成纤维细胞灶中激活并聚集，产生过量的细胞外基质。IPF患者肺功能呈进行性下降，最终因呼吸衰竭死亡，确诊后的平均存活期仅为3-5年，5年病死率超过40％。Idiopathic Pulmonary Fibrosis (IPF), a pulmonary fibrosis of unknown etiology, is the most common and severe form of pulmonary fibrosis, sometimes with acute exacerbation. IPF is characterized by pulmonary fibroblast/myofibroblast hyperproliferation and collagen deposition that disrupt lung structure and function. Fibroblasts and myofibroblasts activate and aggregate in fibroblast foci, producing excess extracellular matrix. The lung function of patients with IPF shows a progressive decline and eventually death due to respiratory failure. The average survival time after diagnosis is only 3-5 years, and the 5-year mortality rate exceeds 40%.
尽管对纤维化(包括肺纤维化)进行了大量的研究，但其病因和发病机制尚未完全清楚，至今仍缺乏有效的治疗手段。针对IPF的上市药物只有两个，分别是吡非尼酮和尼达尼布，但其都不能有效提高患者生存期，且治疗费用高。目前，肺移植是唯一能够有效延长生存期的治疗方法，但也受到诸多应用条件的限制。Despite extensive research on fibrosis (including pulmonary fibrosis), its etiology and pathogenesis are not fully understood, and effective treatments are still lacking. There are only two marketed drugs for IPF, namely pirfenidone and nintedanib, but neither of them can effectively improve the survival time of patients, and the treatment cost is high. At present, lung transplantation is the only treatment method that can effectively prolong survival, but it is also limited by many application conditions.
因此，有必要提供更多能够有效治疗或预防纤维化的方法和手段。Therefore, it is necessary to provide more methods and means that can effectively treat or prevent fibrosis.
发明内容SUMMARY OF THE INVENTION
本发明的一方面提供BMP4蛋白在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用。在一些实施方式中，所述BMP4蛋白是游离的BMP4蛋白。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。在一些实施方式中，所述游离的BMP4蛋白是从天然来源纯化得到的，或者是通过基因工程方法得到的 重组BMP4蛋白。One aspect of the present invention provides the use of a BMP4 protein in the manufacture of a medicament for treating or preventing a fibroproliferative disease in a subject. In some embodiments, the BMP4 protein is free BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis. In some embodiments, the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
本发明的另一方面提供了一种治疗或预防对象的纤维增生性疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的BMP4蛋白，其中所述BMP4蛋白是游离的BMP4蛋白。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。在一些实施方式中，所述游离的BMP4蛋白是从天然来源纯化得到的，或者是通过基因工程方法得到的重组BMP4蛋白。Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a BMP4 protein of the present invention, wherein the BMP4 protein is free BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis. In some embodiments, the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
本发明的另一方面提供了一种表达BMP4蛋白的重组载体，其包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。Another aspect of the present invention provides a recombinant vector for expressing BMP4 protein, which comprises a vector comprising or carrying a polynucleotide encoding BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus.
本发明的另一方面提供了一种治疗或预防对象的纤维增生性疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的重组载体，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列 或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant vector of the present invention, wherein the recombinant vector comprises a vector, wherein the The vector comprises or carries a polynucleotide encoding the BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
本发明的另一方面提供了本发明的重组载体在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，该载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides the use of the recombinant vector of the present invention in the preparation of a medicament for the treatment or prevention of fibroproliferative diseases in a subject, wherein the recombinant vector comprises a vector comprising or carrying a multinucleus encoding BMP4 protein Glycosides. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
本发明的另一方面提供了一种在对象中治疗或预防纤维增生性疾病的药物组合物，其中该药物组合物包含重组载体和药学上可接受的赋形剂，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含 如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides a pharmaceutical composition for treating or preventing a fibroproliferative disease in a subject, wherein the pharmaceutical composition comprises a recombinant carrier and a pharmaceutically acceptable excipient, wherein the recombinant carrier comprises a carrier, The vector comprises or carries a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
本发明的另一方面提供了一种治疗或预防对象的纤维增生性疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的药物组合物，其中该药物组合物包含重组载体和药学上可接受的赋形剂，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises recombinant A carrier and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
本发明的另一方面提供了本发明的药物组合物在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用，其中该药物组合物包含重组载体和药学上可接受的赋形剂，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中， BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing fibroproliferative diseases in a subject, wherein the pharmaceutical composition comprises a recombinant carrier and a pharmaceutically acceptable excipient , wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
在一些实施方式中，本发明的BMP4蛋白、重组载体或药物组合物被施用于对象后引起BMP4蛋白在所述对象的细胞中表达增加。在一些实施方式中，本发明的BMP4蛋白、重组载体或药物组合物被施用于对象后引起BMP4基因在所述对象的细胞中表达升高。在一些实施方式中，本发明的BMP4蛋白、重组载体或药物组合物被施用于对象后抑制成纤维细胞增殖，促进了成纤维细胞的凋亡。在一些实施方式中，本发明的BMP4蛋白、重组载体或药物组合物被施用于对象后抑制成纤维细胞向肌纤维母细胞转化。In some embodiments, administration of the BMP4 protein, recombinant vector or pharmaceutical composition of the invention to a subject results in increased expression of the BMP4 protein in the subject's cells. In some embodiments, administration of the BMP4 protein, recombinant vector or pharmaceutical composition of the invention to a subject results in increased expression of the BMP4 gene in the subject's cells. In some embodiments, the BMP4 protein, recombinant vector or pharmaceutical composition of the present invention inhibits the proliferation of fibroblasts and promotes apoptosis of fibroblasts after being administered to a subject. In some embodiments, the BMP4 protein, recombinant vector or pharmaceutical composition of the invention inhibits the transformation of fibroblasts to myofibroblasts after administration to a subject.
附图说明Description of drawings
图1：相对正常对照肺组织而言，IPF患者肺组织中BMP4的表达显著降低。A：蛋白条带图；B：条带灰度分析统计结果；C：mRNA表达情况统计结果；D：人肺组织切片免疫荧光图；Control：正常对照组，n＝19；IPF：肺纤维化组，n＝16。 *P＜0.05， **P＜0.01。 Figure 1: The expression of BMP4 was significantly reduced in the lung tissue of IPF patients compared to the normal control lung tissue. A: protein band map; B: band grayscale analysis statistical results; C: mRNA expression statistical results; D: immunofluorescence image of human lung tissue sections; Control: normal control group, n=19; IPF: pulmonary fibrosis Group, n=16. * P<0.05, ** P<0.01.
图2：博莱霉素(Bleomycin，BLM)诱导小鼠肺组织中BMP4蛋白表达降低。A：条带灰度分析统计结果；B：蛋白条带图。 ***P<0.001，与0day比较； ###P<0.001，与0day比较，n＝6。 Figure 2: Bleomycin (Bleomycin, BLM) induces decreased expression of BMP4 protein in mouse lung tissue. A: Statistical results of band grayscale analysis; B: protein band map. *** P<0.001, compared with 0day; ### P<0.001, compared with 0day, n=6.
图3：BMP4基因敲除对博莱霉素诱导的小鼠生存率和体重的影响。A：给予博来霉素处理后小鼠生存率曲线；B：小鼠体重变化情况。BMP4 +/++Saline：野生型小鼠对照组，n＝12；BMP4 +/-+Saline：BMP4 +/-小鼠对照组，n＝10；BMP4 +/++BLM：野生型小鼠予以博来霉素处理组，n＝25；BMP4 +/-+BLM：BMP4 +/-小鼠予以博来霉素处理组，n＝37。 Figure 3: Effects of BMP4 knockout on bleomycin-induced mouse survival and body weight. A: Survival rate curve of mice treated with bleomycin; B: Changes in body weight of mice. BMP4 +/+ +Saline: wild-type mice control, n=12; BMP4 +/- +Saline: BMP4 +/- mice control, n=10; BMP4 +/+ +BLM: wild-type mice Bleomycin-treated group, n=25; BMP4 +/ -+BLM: BMP4 +/- mice received bleomycin-treated group, n=37.
图4：BMP4基因敲除对博来霉素诱导的小鼠Micro-CT肺影像结果的影响。n＝10。Figure 4: The effect of BMP4 gene knockout on bleomycin-induced mouse Micro-CT lung imaging results. n=10.
图5：BMP4基因敲除对博来霉素诱导的小鼠肺功能的影响。A：动态肺顺应性；B：气道阻力。 *P<0.05， ***P<0.001，与BMP4 +/++Saline比较，n＝6； #P<0.05， ##P<0.01，BMP4 +/++BLM与BMP4 +/-+BLM比较，n＝6。 Figure 5: Effects of BMP4 knockout on bleomycin-induced lung function in mice. A: Dynamic lung compliance; B: Airway resistance. * P<0.05, *** P<0.001 vs BMP4 +/- Saline, n=6; # P<0.05, ## P<0.01, BMP4 +/- BLM vs BMP4 +/- + BLM , n=6.
图6：BMP4基因敲除对博来霉素诱导的小鼠HE和Masson染色结果的影响。A：HE染色结果，B：Masson染色结果。n＝10。Figure 6: Effects of BMP4 knockout on bleomycin-induced mouse HE and Masson staining results. A: HE staining results, B: Masson staining results. n=10.
图7：BMP4基因敲除对博来霉素诱导的小鼠肺组织中羟脯胺酸含量的影响。 *P<0.05，与BMP4 +/++Saline比较，n＝10； ###P<0.001，与BMP4 +/-+BLM比较，n＝10。 Figure 7: Effects of BMP4 knockout on hydroxyproline content in bleomycin-induced mouse lung tissue. * P<0.05 vs. BMP4 +/- +Saline, n=10; ### P<0.001, vs. BMP4 +/ -+BLM, n=10.
图8：BMP4基因敲除对小鼠肺组织中纤连蛋白(Fibronectin)、α-平滑肌肌动蛋白(Alpha-smooth muscle actin，α-SMA)、I型胶原蛋白(Collagen 1，Col 1)、III型胶原蛋白(Collagen 3，Col 3)的蛋白表达变化的影响。Figure 8: The effect of BMP4 gene knockout on fibronectin (Fibronectin), alpha-smooth muscle actin (alpha-smooth muscle actin, alpha-SMA), type I collagen (Collagen 1, Col 1), Effects of changes in protein expression of type III collagen (Collagen 3, Col 3).
图9：BMP4过表达对博来霉素诱导的肺纤维化小鼠的预防和治疗作用的影响。(A)BMP4过表达对博来霉素诱导肺纤维化小鼠肺功能的影响；(B)BMP4过表达对博来霉素诱导肺纤维化小鼠肺病理的影响。Figure 9: Effects of BMP4 overexpression on the preventive and therapeutic effects of bleomycin-induced pulmonary fibrosis in mice. (A) The effect of BMP4 overexpression on lung function in mice with bleomycin-induced pulmonary fibrosis; (B) The effect of BMP4 overexpression on lung pathology in mice with bleomycin-induced pulmonary fibrosis.
图10A：BMP4基因敲除对TGF-β1诱导的小鼠原代成纤维细胞转化和合成ECM的影响。Figure 10A: Effects of BMP4 knockout on TGF-β1-induced transformation and ECM synthesis in mouse primary fibroblasts.
图10B-10D：外源性加入BMP4重组蛋白对TGF-β1诱导的小鼠原代成纤维细胞转化的影响。Figures 10B-10D: Effects of exogenous addition of BMP4 recombinant protein on TGF-beta1-induced transformation of mouse primary fibroblasts.
图11A-11C：BMP4基因敲除对TGF-β1诱导的小鼠原代成纤维细胞增殖的影响。A：蛋白条带图；B、C：条带灰度分析统计结果，n＝5。Figures 11A-11C: Effects of BMP4 knockout on TGF-beta1-induced proliferation of mouse primary fibroblasts. A: Protein band map; B, C: Statistical results of gray-scale analysis of bands, n=5.
图11D-10F：外源性加入BMP4重组蛋白对TGF-β1诱导的小鼠原代成纤维细胞转化的影响。E、F：条带灰度分析统计结果，n＝5。Figures 11D-10F: Effects of exogenous addition of BMP4 recombinant protein on TGF-beta1-induced transformation of mouse primary fibroblasts. E, F: Statistical results of band grayscale analysis, n=5.
图12：BMP4基因敲除对TGF-β1诱导的小鼠原代成纤维细胞凋亡的影响。A：细胞凋亡流式图；B：细胞凋亡率统计结果，n＝5。Figure 12: Effect of BMP4 knockout on TGF-β1-induced apoptosis in mouse primary fibroblasts. A: Flow chart of apoptosis; B: Statistics of apoptosis rate, n=5.
具体实施方式Detailed ways
定义definition
在本发明中，术语“治疗”是指疗法上的以及预防性的措施，其阻止或减缓对象发生不期望的生理学改变或病症，例如肺部纤维化的发生或癌症进展。有利或期望的临床效果包括，但不限于，症状的缓解、疾病程度的降低、疾病状态的稳定化(即不恶化)、疾病进展的延迟或减缓、疾病状态的减轻或缓和以及疾病的部分或全部治愈，而不论上述效果是否可检测到。“治疗”也可指与不治疗相比生存期延长。需要治疗的对象包括已患有该疾病或病症的对象，以及有可能患有该疾病或病症的对象，或要预防该疾病或病症的对象。In the present invention, the term "treatment" refers to therapeutic as well as prophylactic measures which prevent or slow the development of undesired physiological changes or disorders in a subject, such as the development of pulmonary fibrosis or the progression of cancer. Favorable or desired clinical effects include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization (ie, no exacerbation) of disease state, delay or slowing of disease progression, reduction or alleviation of disease state, and partial or All healed regardless of whether the above effects were detectable. "Treatment" can also mean prolonging survival as compared to no treatment. Objects in need of treatment include those already suffering from the disease or disorder, as well as those likely to suffer from the disease or disorder, or for which the disease or disorder is to be prevented.
“对象”或“患者”、“个体”是指任何期望进行诊断、预后或治疗的对象，特别是哺乳动物对象。哺乳动物包括人、家畜、农畜、动物园动物、竞技动物或宠物，例如狗、猫、猪、兔、大鼠、小鼠、马、牛、奶牛等。本文所称的对象优选是人。"Subject" or "patient", "individual" refers to any subject for which diagnosis, prognosis or treatment is desired, particularly mammalian subjects. Mammals include humans, domestic animals, farm animals, zoo animals, sports animals or pets such as dogs, cats, pigs, rabbits, rats, mice, horses, cows, cows, and the like. The subject referred to herein is preferably a human.
本文所用的术语“有治疗需要的患者”或“有治疗需要的对象”包括因施用本发明用于例如检测、诊断和/或治疗用途的多肽或其组合物而受益的对象，如哺乳动物对象。As used herein, the term "patient in need of treatment" or "subject in need of treatment" includes a subject, such as a mammalian subject, who would benefit from administration of a polypeptide of the present invention, for example, for detection, diagnostic and/or therapeutic use, or a composition thereof .
如本文中所使用的，术语“治疗有效量”或“有效量”是指当将本发明的药物或药物组合物单独给予或与另外的治疗剂联合给予细胞、组织或受治疗者时，其有效防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指所述化合物足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。当施用给个体单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；优选至少约30％；更优选至少40％和最优选至少50％。As used herein, the term "therapeutically effective amount" or "effective amount" refers to a medicament or pharmaceutical composition of the present invention which is administered to a cell, tissue or subject when administered alone or in combination with an additional therapeutic agent. An amount effective to prevent or slow the disease or disorder to be treated. A therapeutically effective dose further refers to an amount of the compound sufficient to cause alleviation of symptoms, such as to treat, cure, prevent or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention or alleviation of the condition . When an active ingredient is administered to a subject alone, the therapeutically effective amount refers to that ingredient alone. When a combination is administered, a therapeutically effective amount refers to the combined amount of the active ingredients that produces the therapeutic effect, regardless of whether it is administered in combination, consecutively, or simultaneously. A therapeutically effective amount will generally reduce symptoms by at least 10%; usually by at least 20%; preferably by at least about 30%; more preferably by at least 40% and most preferably by at least 50%.
在本发明中，“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，对于生物学***或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。In the present invention, "about" means that the index value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (ie, the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. Also, with respect to a biological system or process, the term can mean up to an order of magnitude or up to 5 times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for the specific value.
本领域普通技术人员还应该理解，可以修饰基因的基因组，使得它们在核苷酸序列上与它们所衍生出的修饰的多核苷酸不同。例如，从指定的DNA序列衍生的多核苷酸或核苷酸序列可以是相似的，例如与起始序列具有一定的百分比同一性，例如它可以与起始序列60％、70％、75％、80％、85％、90％、95％、98％或99％相同。It will also be understood by those of ordinary skill in the art that the genomes of genes can be modified such that they differ in nucleotide sequence from the modified polynucleotides from which they are derived. For example, a polynucleotide or nucleotide sequence derived from a given DNA sequence may be similar, eg, have a certain percentage identity to the starting sequence, eg, it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% the same.
此外，可以进行核苷酸或氨基酸取代、缺失或***，以在“非必需”区域进行保守取代或改变。例如，衍生自指定蛋白质的多肽或氨基酸序列，除了一个或多个单独的氨基酸取代、***或缺失(例如1、2、3、4、5、6、7、8、9、10、15、20或更多个单个氨基酸取代、***或缺失)之外，其余部分可以与起始序列相同。在某些实施方式中，衍生自指定蛋白的多肽或氨基酸序列相对于起始序列具有1至5个、1至10个、1至15个或1至20个单独的氨基酸取代、***或缺失。In addition, nucleotide or amino acid substitutions, deletions or insertions can be made to make conservative substitutions or changes in "non-essential" regions. For example, a polypeptide or amino acid sequence derived from a specified protein, except for one or more individual amino acid substitutions, insertions, or deletions (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 The remainder may be identical to the starting sequence except for one or more single amino acid substitutions, insertions or deletions). In certain embodiments, the polypeptide or amino acid sequence derived from a given protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 individual amino acid substitutions, insertions or deletions relative to the starting sequence.
BMP4BMP4
骨形态发生蛋白(Bone morphogenetic proteins,BMPs)属于转化生长因子(TGF)家族，是 一大类多功能生长因子，能影响肾、肺等器官胚胎发生和组织生成，并调节细胞增殖、转化、凋亡和ECM成分的分泌等。在肺发育和再生过程中，TGF-β1和BMP信号活性之间的平衡至关重要。Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) family and are a large class of multifunctional growth factors that can affect embryogenesis and tissue formation in organs such as kidneys and lungs, and regulate cell proliferation, transformation, and apoptosis. apoptosis and secretion of ECM components. The balance between TGF-β1 and BMP signaling activities is critical during lung development and regeneration.
如本文所用，术语“BMP4”是指骨形态发生蛋白4，是BMP的一员，是肺发育过程中的关键因子之一。BMP4蛋白的氨基酸序列可以从NCBI获得，登录号为NP_001334841.1、NP_001334845.1、NP_001334843.1、NP_001193.2、NP_570911.2、NP_570912.2、NP_001334846.1、NP_001334842.1、以及NP_001334844.1。BMP4蛋白的氨基酸序列如SEQ ID NO:1至SEQ ID NO:3任一个所示。编码BMP4蛋白产物的核苷酸序列如SEQ ID NO:4至SEQ ID NO:9任一个所示。BMP4的信使RNA(mRNA)的NCBI序列登陆号为NM_001347912.1(SEQ ID NO:19)、NM_001347916.1(SEQ ID NO:20)、NM_001347914.2(SEQ ID NO:21)、NM_001202.6(SEQ ID NO:22)、NM_130850.5(SEQ ID NO:23)、NM_130851.4(SEQ ID NO:24)、NM_001347917.1(SEQ ID NO:25)、NM_001347913.2(SEQ ID NO:26)、以及NM_001347915.2(SEQ ID NO:27)。BMP4的DNA序列的NCBI序列登陆号为NC_000014.9(SEQ ID NO:28)，基因ID为652。BMP4在维持肺血管稳态中起重要作用，抑制BMP4能抑制低氧性肺动脉高压的病理改变。有研究表明，BMP4抑制人肺成纤维细胞增殖，诱导肺上皮细胞增殖和去分化。As used herein, the term "BMP4" refers to bone morphogenetic protein 4, a member of the BMP, one of the key factors in lung development. The amino acid sequences of BMP4 proteins can be obtained from NCBI with accession numbers NP_001334841.1, NP_001334845.1, NP_001334843.1, NP_001193.2, NP_570911.2, NP_570912.2, NP_001334846.1, NP_001334842.1, and NP_0013. The amino acid sequence of the BMP4 protein is shown in any one of SEQ ID NO:1 to SEQ ID NO:3. The nucleotide sequence encoding the BMP4 protein product is set forth in any of SEQ ID NO:4 to SEQ ID NO:9. The NCBI sequence accession numbers of the messenger RNA (mRNA) of BMP4 are NM_001347912.1 (SEQ ID NO:19), NM_001347916.1 (SEQ ID NO:20), NM_001347914.2 (SEQ ID NO:21), NM_001202.6 ( SEQ ID NO:22), NM_130850.5 (SEQ ID NO:23), NM_130851.4 (SEQ ID NO:24), NM_001347917.1 (SEQ ID NO:25), NM_001347913.2 (SEQ ID NO:26) , and NM_001347915.2 (SEQ ID NO:27). The NCBI sequence accession number of the DNA sequence of BMP4 is NC_000014.9 (SEQ ID NO: 28), and the gene ID is 652. BMP4 plays an important role in maintaining pulmonary vascular homeostasis, and inhibition of BMP4 can inhibit the pathological changes of hypoxic pulmonary hypertension. Studies have shown that BMP4 inhibits the proliferation of human lung fibroblasts and induces the proliferation and dedifferentiation of lung epithelial cells.
编码BMP4的多核苷酸Polynucleotide encoding BMP4
本文可互换使用的术语“多核苷酸”和“核酸”是指任何长度的核苷酸(核糖核苷酸或脱氧核糖核苷酸)的聚合形式。这些术语包括单链、双链或三链DNA，基因组DNA、cDNA、基因组RNA、mRNA、DNA-RNA杂交体、或聚合物；该聚合物包含嘌呤和嘧啶碱基，或其他天然的，化学、生物化学修饰的，非天然或衍生的核苷酸碱基。多核苷酸的骨架可包含糖和磷酸基团(通常可在RNA或DNA中发现)，或经修饰或取代的糖或磷酸基团。或者，多核苷酸的主链可以包含合成亚单位的聚合物(例如氨基磷酸酯(phosphoramidate))，并且因此可以是寡聚脱氧核苷氨基磷酸酯(P-NH2)或混合的氨基磷酸酯-磷酸二酯寡聚体。The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. These terms include single-, double- or triple-stranded DNA, genomic DNA, cDNA, genomic RNA, mRNA, DNA-RNA hybrids, or polymers; the polymers contain purine and pyrimidine bases, or other natural, chemical, Biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide may comprise a polymer of synthetic subunits (eg, phosphoramidate), and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate- Phosphodiester oligomers.
如本文所用，术语“编码”指这样的任何过程，借助该过程聚合物大分子或序列串中的信息被用来指导产生不同于第一分子或序列串的第二分子或序列串。如本文所用，所述术语被广泛使用，并且可以具有各种应用。在一个方面，术语“编码”描述了半保守DNA复制的过程，其中双链DNA分子的一条链被用作模板来通过DNA依赖性的DNA聚合酶来编码新合成的互补姐妹链。在另一个方面，术语“编码”是指这样的任何过程，借助该过程一个分子中的信息被用来指导产生与第一分子具有不同的化学性质的第二分子。例如，DNA分子可以编码RNA分子(例如，通过参与DNA依赖性的RNA聚合酶的转录过程)。 而且，RNA分子可以编码多肽，如在翻译过程中那样。当用于描述翻译过程时，术语“编码”也延伸至编码氨基酸的三联体密码子。在一些方面，RNA分子可以编码DNA分子，例如通过参与RNA依赖性的DNA聚合酶的逆转录过程。在另一个方面，DNA分子可以编码多肽，其中应理解的是，在该情况下使用的“编码”包含转录和翻译过程。As used herein, the term "encoding" refers to any process by which information in a polymeric macromolecule or sequence string is used to direct the production of a second molecule or sequence string that differs from the first molecule or sequence string. As used herein, the term is widely used and can have various applications. In one aspect, the term "encoding" describes the process of semi-conservative DNA replication in which one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In another aspect, the term "encoding" refers to any process by which information in one molecule is used to direct the production of a second molecule having different chemical properties than the first molecule. For example, a DNA molecule can encode an RNA molecule (eg, by participating in a DNA-dependent RNA polymerase transcription process). Furthermore, RNA molecules can encode polypeptides, as during translation. When used to describe the process of translation, the term "encoding" also extends to triplet codons encoding amino acids. In some aspects, an RNA molecule can encode a DNA molecule, eg, by participating in the reverse transcription process of an RNA-dependent DNA polymerase. In another aspect, a DNA molecule may encode a polypeptide, where it is understood that "encoding" as used in this context encompasses both transcription and translation processes.
如本文所用，术语“变体”是指在“非必需”区域进行保守取代或改变使其与序列具有至少60％、70％、80％、90％、95％、98％序列同一性的核苷酸或氨基酸序列。As used herein, the term "variant" refers to a nucleus having at least 60%, 70%, 80%, 90%, 95%, 98% sequence identity to a sequence with conservative substitutions or changes in "non-essential" regions nucleotide or amino acid sequence.
同一性identity
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。同源性能够通过比较每个序列中的位置来确定，该序列可以为了比较的目的而被比对。当比较的序列中的位置被相同的碱基或氨基酸占据时，那么分子在该位置是同源的。序列间同源性的程度是序列共有的匹配或同源位置的数量的函数。“无关的”或“非同源的”序列与本发明的序列之一共享小于40％的同一性，但优选地小于25％的同一性。"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity with one of the sequences of the invention, but preferably less than 25% identity.
多核苷酸或多核苷酸区域(或者多肽或多肽区域)与另一个序列具有一定百分比(例如60％、65％、70％、75％、80％、85％、90％、95％、98％或者99％)的“序列同一性”，意味着当比对时，在比较两个序列时该百分比的碱基(或氨基酸)是相同的。这种比对和百分比同源性或序列同一性可以使用本领域已知的软件程序来确定。A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%) of another sequence or 99%) "sequence identity", meaning that when aligned, the percentage of bases (or amino acids) that are identical when the two sequences are compared. Such alignment and percent homology or sequence identity can be determined using software programs known in the art.
病毒载体viral vector
病毒载体可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入其他细胞，进行感染的分子机制。病毒载体也可以称为载体、载体病毒粒子或载体粒子。病毒载体的实例包括但不限于：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。Viral vectors can bring genetic material into cells by using the molecular mechanism of viruses to transmit their genomes into other cells for infection. Viral vectors may also be referred to as vectors, vector virions, or vector particles. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
逆转录病毒载体可以使衍生自或能够衍生自任何适合的逆转录病毒。大量的不同的逆转录病毒已经被鉴定。例子包括但不限于：鼠白血病病毒(MLV)、人T-细胞白血病病毒(HTLV)、小鼠乳腺肿瘤病毒(MMTV)、劳氏肉瘤病毒(RSV)、Fujinami肉瘤病毒(FuSV)、莫洛尼鼠白血病病毒(Mo MLV)、FBR鼠骨肉瘤病毒(FBR MSV)、莫洛尼鼠肉瘤病毒(Mo-MSV)、Abelson鼠白血病病毒(A-MLV)、禽骨髓细胞瘤病毒-29(MC29)和禽红细胞增多症病毒(AEV)。Retroviral vectors can be or can be derived from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include, but are not limited to: Murine Leukemia Virus (MLV), Human T-Cell Leukemia Virus (HTLV), Mouse Mammary Tumor Virus (MMTV), Rous Sarcoma Virus (RSV), Fujinami Sarcoma Virus (FuSV), Moloney Murine leukemia virus (Mo MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloma virus-29 (MC29) and avian polycythemia virus (AEV).
腺病毒是双链的线性DNA病毒，其不通过RNA中间体复制。腺病毒是双链的DNA无包膜病毒，其能够在体内、离体和体外转导大范围的人和非人来源的细胞类型。这些细胞包括呼吸道气道上皮细胞、肝细胞、肌肉细胞、心肌细胞、滑膜细胞、原代乳腺上皮细胞和有丝***后终末分化的细胞(例如神经元)。腺病毒已被用作用于基因治疗和异源基因表达 的载体。大(36kb)基因组可以容纳高达8kb的外源***DNA，并且能够在互补细胞系中有效地复制以产生每毫升高达1012个转导单位的非常高的效价。腺病毒因此是研究原代非复制细胞中基因表达的最佳***之一。来自腺病毒基因组的病毒基因或外源基因的表达不需要复制细胞。腺病毒载体通过受体介导的内吞作用进入细胞。一旦进入细胞内，腺病毒载体很少整合到宿主染色体中。相反，它们作为附加体(独立于宿主基因组)存在，作为宿主细胞核中的线性基因组。Adenoviruses are double-stranded, linear DNA viruses that do not replicate through an RNA intermediate. Adenoviruses are double-stranded DNA non-enveloped viruses capable of transducing a wide range of human and non-human derived cell types in vivo, ex vivo and in vitro. These cells include airway airway epithelial cells, hepatocytes, muscle cells, cardiomyocytes, synoviocytes, primary mammary epithelial cells, and post-mitotic terminally differentiated cells (eg, neurons). Adenoviruses have been used as vectors for gene therapy and heterologous gene expression. The large (36kb) genome can accommodate up to 8kb of exogenous insert DNA and can replicate efficiently in complementary cell lines to yield very high titers of up to 1012 transduction units per milliliter. Adenoviruses are therefore one of the best systems for studying gene expression in primary non-replicating cells. The expression of viral genes or foreign genes from the adenoviral genome does not require replicating cells. Adenoviral vectors enter cells through receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into the host chromosome. Instead, they exist as episomes (independent of the host genome), as a linear genome in the host cell nucleus.
腺相关病毒(adeno-associated virus，AAV)，也称腺伴随病毒，属于微小病毒科依赖病毒属，是目前发现的一类结构最简单的单链DNA缺陷型病毒。重组的AAV载体已成功地被用于标记基因和涉及人类疾病的基因的体外、离体和体内的转导。已经开发了某些AAV载体，其可以有效地结合大的有效载荷(高达8-9kb)。现在已经鉴定了多种腺相关病毒(AAV)的血清型，包括12种人血清型(AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11和AAV12)和超过100种来自非人灵长动物的血清型。本发明预期可使用这些血清型中的任何一个。Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependovirus of the family Parvoviridae, and is the simplest single-stranded DNA-deficient virus discovered so far. Recombinant AAV vectors have been successfully used for in vitro, ex vivo and in vivo transduction of marker genes and genes involved in human disease. Certain AAV vectors have been developed that can efficiently bind large payloads (up to 8-9 kb). Multiple adeno-associated virus (AAV) serotypes have now been identified, including 12 human serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12) and more than 100 serotypes from non-human primates. The present invention contemplates that any of these serotypes can be used.
单纯性疱疹病毒(HSV)是包膜的双链DNA病毒，其天然地感染神经元。它可以容纳外源DNA的大区段，并且已经被采用为用于对神经元的基因递送的载体。在治疗过程中HSV的使用需要使毒株减毒，从而它们不能建立裂解周期。特别地，如果HSV载体用于人类的基因治疗，则优选地将多核苷酸***必需基因中。这是因为如果病毒载体遭遇野生型病毒，则可以通过重组将异源基因转移至野生型病毒。然而，如果以防止其复制的方式构建重组病毒，则这可以通过将寡核苷酸***对复制必需的病毒基因来实现。Herpes simplex virus (HSV) is an enveloped double-stranded DNA virus that naturally infects neurons. It can accommodate large segments of foreign DNA and has been adopted as a vector for gene delivery to neurons. The use of HSV during therapy requires attenuating the strains so that they cannot establish a lytic cycle. In particular, if the HSV vector is used for gene therapy in humans, the polynucleotide is preferably inserted into the essential gene. This is because if the viral vector encounters the wild-type virus, the heterologous gene can be transferred to the wild-type virus by recombination. However, if the recombinant virus is constructed in a way that prevents its replication, this can be achieved by inserting oligonucleotides into the viral genes necessary for replication.
本发明的病毒载体可以是牛痘病毒载体，例如MVA或NYVAC。牛痘载体的替代物包括例如称为ALVAC的鸡痘或金丝雀痘的禽痘(avipox)载体，以及由其衍生的毒株，该毒株可以在人类细胞中感染和表达重组蛋白但不能复制。应当理解的是，在重组基因的***之后病毒基因组的部分可以保持完整。这意味着病毒载体可以保留感染细胞并随后表达额外的基因的能力的概念，该额外的基因支持其复制并可能促进被感染细胞的裂解和死亡。The viral vector of the present invention may be a vaccinia virus vector such as MVA or NYVAC. Alternatives to vaccinia vectors include, for example, the fowlpox (avipox) vector known as ALVAC or canarypox, and strains derived therefrom that can infect and express recombinant proteins in human cells but cannot replicate . It will be appreciated that portions of the viral genome may remain intact following insertion of the recombinant gene. This means that the notion that a viral vector can retain the ability to infect cells and subsequently express additional genes that support its replication and possibly promote lysis and death of infected cells.
慢病毒是更大群体的逆转录病毒的一部分。可以分为灵长类动物和非灵长类动物群体。灵长类慢病毒的例子包括但不限于：人免疫缺陷病毒(HIV)、人自身免疫缺陷综合症(AIDS)的病原体以及猿猴免疫缺陷病毒(SIV)。非灵长类动物慢病毒群体包括原型“慢病毒”visna/maedi病毒(VMV)，以及相关的山羊关节炎-脑炎病毒(CAEV)、马传染性贫血病毒(EIAV)、猫免疫缺陷病毒(FIV)和牛免疫缺陷病毒(BIV)。慢病毒载体一般是指以人类免疫缺陷病毒-1(HIV-1)来源的一种病毒载体，慢病毒载体包含了包装、转染、稳定整合所需要的遗传信息，是慢病毒载体***的主要组成部分。携带有外源基因的慢病毒载 体在慢病毒包装质粒、细胞系的辅助下，经过病毒包装成为有感染力的病毒颗粒，通过感染细胞或活体组织，实现外源基因在细胞或活体组织中表达。Lentiviruses are part of a larger group of retroviruses. Can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus population includes the prototype "lentivirus" visna/maedi virus (VMV), as well as the related goat arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus ( FIV) and bovine immunodeficiency virus (BIV). Lentiviral vector generally refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1). The lentiviral vector contains the genetic information required for packaging, transfection, and stable integration, and is the main component of the lentiviral vector system. component. Lentiviral vectors carrying foreign genes are packaged into infectious virus particles with the help of lentiviral packaging plasmids and cell lines. By infecting cells or living tissues, the exogenous genes can be expressed in cells or living tissues. .
非病毒载体non-viral vector
非病毒载体是利用非病毒的载体材料的物化性质来介导基因的转移。任何合适的非病毒载体可被用于将BMP4基因引入对象的细胞中。非病毒载体的例子包括但不限于，质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。Non-viral vectors use the physicochemical properties of non-viral vector materials to mediate gene transfer. Any suitable non-viral vector can be used to introduce the BMP4 gene into cells of a subject. Examples of non-viral vectors include, but are not limited to, plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes.
质粒是小型环状DNA分子，在基因工程中作为最常用，最简单的载体，必须包括三部分：遗传标记基因，复制区，目的基因。质粒在所有的细菌类群中都可发现，它们是独立于细菌染色体外自我复制的DNA分子。质粒载体的例子包括但不限于：大肠杆菌质粒载体、枯草杆菌质粒载体、酵母质粒载体、农杆菌质粒载体和蓝细菌质粒载体。Plasmids are small circular DNA molecules. As the most commonly used and simplest vector in genetic engineering, plasmids must include three parts: genetic marker gene, replication region, and target gene. Plasmids are found in all bacterial groups, and they are DNA molecules that replicate independently of bacteria extrachromosomally. Examples of plasmid vectors include, but are not limited to, E. coli plasmid vectors, Bacillus subtilis plasmid vectors, yeast plasmid vectors, Agrobacterium plasmid vectors, and cyanobacterial plasmid vectors.
脂质体由卵磷脂和神经酰胺等制得的脂质体(空心)，具有的双分子层结构，是一种人工膜。脂质体的组成通常是磷脂(特别是高相转变温度的磷脂)的组合，通常与类固醇(尤其是胆固醇)组合。也可以使用其他磷脂或其他脂质。脂质体的物理特性取决于pH、离子强度和二价阳离子的存在。通过在转导期间通过使用二油酰磷脂酰乙醇胺可以增加脂质体的转导效率。高效脂质体可商购获得。脂质体的例子包括但不限于：中性脂质体、负电荷脂质体和正电荷脂质体。Liposome is a liposome (hollow) made from lecithin and ceramide, etc. It has a bilayer structure and is an artificial membrane. The composition of liposomes is usually a combination of phospholipids (especially high phase transition temperature phospholipids), usually in combination with steroids (especially cholesterol). Other phospholipids or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations. The transduction efficiency of liposomes can be increased by using dioleoylphosphatidylethanolamine during transduction. High potency liposomes are commercially available. Examples of liposomes include, but are not limited to, neutral liposomes, negatively charged liposomes, and positively charged liposomes.
附加型载体是一种允许和帮助水溶性物质渗入并穿过细胞膜的脂质层而转移到细胞中去的物质。它是膜上的一种蛋白质，调节着离子主动吸收的一种成分。附加型载体可以包括一种或多种多能性基因，其可操作地连接至少一个用于表达因子的调控序列。本发明的附加型载体还可包括允许该载体在细胞中复制的组分。例如，Epstein Barr oriP/核抗原-1(EBNA-1)组合可以支持哺乳动物细胞(特别是灵长类动物细胞)中载体的自我复制。来自于EBV基因组的EBNA1反式元件和OriP顺式元件使得简单质粒能够在增殖的人类细胞中作为附加体进行复制和维持。An episomal carrier is a substance that allows and facilitates the penetration and transfer of water-soluble substances into the cell through the lipid layer of the cell membrane. It is a protein in the membrane, a component that regulates the active uptake of ions. Episomal vectors can include one or more pluripotency genes operably linked to at least one regulatory sequence for expressing the factor. Episomal vectors of the present invention may also include components that allow the vector to replicate in cells. For example, the Epstein Barr oriP/nuclear antigen-1 (EBNA-1) combination can support self-replication of vectors in mammalian cells, especially primate cells. The EBNA1 trans-element and OriP cis-element from the EBV genome enable simple plasmid replication and maintenance as episomes in proliferating human cells.
用于基因转移的阳离子多聚物包括但不限于：多聚赖氨酸、聚乙烯亚胺和树突状聚合物。Cationic polymers for gene transfer include, but are not limited to, polylysine, polyethyleneimine, and dendrimers.
壳聚糖作为一种天然阳离子聚合物，通过与DNA以静电方式作用使壳聚糖-DNA体系不被降解，完全进入细胞。作为基因载体，壳聚糖具有细胞毒性低、生物相容性好、基因免疫性低和转染效率较高等特点。壳聚糖-DNA复合物按制备方法主要分为壳聚糖及其衍生物的DNA复合物、壳聚糖-DNA纳米微球和壳聚糖自聚体-DNA。As a natural cationic polymer, chitosan-DNA system can not be degraded by electrostatic interaction with DNA, and can enter cells completely. As a gene carrier, chitosan has the characteristics of low cytotoxicity, good biocompatibility, low gene immunity and high transfection efficiency. Chitosan-DNA complexes are mainly divided into DNA complexes of chitosan and its derivatives, chitosan-DNA nano-microspheres and chitosan self-polymer-DNA according to the preparation method.
无机纳米粒子主要通过穿过细胞膜将药物或生物分子转运到生物体中而起到治疗疾病的作用。应用于基因转运的无机纳米粒子的例子包括但不限于硅、铁氧化物、碳纳米管、 磷酸钙、金属纳米粒子、量子点等。Inorganic nanoparticles play a role in the treatment of diseases mainly by transporting drugs or biomolecules into organisms through cell membranes. Examples of inorganic nanoparticles for gene delivery include, but are not limited to, silicon, iron oxides, carbon nanotubes, calcium phosphates, metal nanoparticles, quantum dots, and the like.
重组载体recombinant vector
本文所公开的重组载体是表达BMP4蛋白的重组载体，其包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述载体是病毒载体或非病毒载体。The recombinant vectors disclosed herein are recombinant vectors expressing a BMP4 protein comprising a vector comprising or carrying a polynucleotide encoding a BMP4 protein. In some embodiments, the vector is a viral vector or a non-viral vector.
表达BMP4蛋白的重组载体Recombinant vector expressing BMP4 protein
在本文公开的任何重组载体、药物组合物和方法中，编码BMP4蛋白的多核苷酸通过载体来施用，其中载体包含病毒载体和非病毒载体。将编码BMP4蛋白的核酸序列包装进载体的方法是本领域技术人员已知的。In any of the recombinant vectors, pharmaceutical compositions and methods disclosed herein, the polynucleotide encoding the BMP4 protein is administered via a vector, wherein the vector comprises a viral vector and a non-viral vector. Methods for packaging nucleic acid sequences encoding BMP4 proteins into vectors are known to those skilled in the art.
本发明的一个方面提供一种在对象中治疗或预防纤维增生性疾病的重组载体，其包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。One aspect of the present invention provides a recombinant vector for treating or preventing a fibroproliferative disease in a subject, comprising a vector comprising or carrying a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof.
在一些实施方式中，所述载体是病毒载体。在一些实施方式中，所述病毒载体选自逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus.
在一些实施方式中，所述重组载体包含病毒载体，其中所述病毒载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述重组载体包含病毒载体，其中所述病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含病毒载体，其中所述病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含病毒载体，其中所述病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含病毒载体，其中所述病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。In some embodiments, the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof. In some embodiments, the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof. In some embodiments, the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof. In some embodiments, the recombinant vector comprises a viral vector, wherein the viral vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
在一些实施方式中，所述病毒载体是腺相关病毒载体。在一些实施方式中，所述重组载体包含腺相关病毒载体，其中所述腺相关病毒载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述重组载体包含腺相关病毒载体，其中所述腺相关病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含腺相关病毒载体，其中所述腺相关病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含腺相关病毒载体，其中所述腺相关病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含腺相关病毒载体，其中所述腺相关病毒载体包含或携带编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。In some embodiments, the viral vector is an adeno-associated viral vector. In some embodiments, the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises as SEQ ID NO: 1 to Any one of the sequences shown in SEQ ID NO: 3 or a variant thereof. In some embodiments, the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID Any one of the sequences shown in NO:4 to SEQ ID NO:9 or a variant thereof. In some embodiments, the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID Any one of the sequences shown in NO: 10 to SEQ ID NO: 27 or a variant thereof. In some embodiments, the recombinant vector comprises an adeno-associated virus vector, wherein the adeno-associated virus vector comprises or carries a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID The sequence shown in NO:28 or SEQ ID NO:29 or a variant thereof.
在一些实施方式中，所述载体是非病毒载体，在一些实施方式中，所述非病毒载体选自质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。In some embodiments, the vector is a non-viral vector, in some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, capsids Glycan polymers, inorganic nanoparticles and exosomes.
在一些实施方式中，所述重组载体包含非病毒载体，其中所述非病毒载体包含编码BMP4蛋白的多核苷酸。在一些实施方式中，所述重组载体包含非病毒载体，其中所述非病毒载体包含编码BMP4蛋白的多核苷酸，其中所述BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含非病毒载体，其中所述非病毒载体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含非病毒载体，其中所述非病毒载体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含非病毒载体，其中所述非病毒载体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。In some embodiments, the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof. In some embodiments, the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof. In some embodiments, the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof. In some embodiments, the recombinant vector comprises a non-viral vector, wherein the non-viral vector comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
在一些实施方式中，所述非病毒载体是外泌体。在一些实施方式中，所述重组载体包含外泌体，其中所述外泌体包含编码BMP4蛋白的多核苷酸。在一些实施方式中，所述重组载体包含外泌体，其中所述外泌体包含编码BMP4蛋白的多核苷酸，其中所述BMP4蛋白 的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含外泌体，其中所述外泌体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含外泌体，其中所述外泌体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含外泌体，其中所述外泌体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。In some embodiments, the non-viral vector is an exosome. In some embodiments, the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof. In some embodiments, the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof. In some embodiments, the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof. In some embodiments, the recombinant vector comprises exosomes, wherein the exosomes comprise a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
在一些实施方式中，所述非病毒载体是质粒。在一些实施方式中，所述重组载体包含质粒，其中所述质粒包含编码BMP4蛋白的多核苷酸。在一些实施方式中，所述重组载体包含质粒，其中所述质粒包含编码BMP4蛋白的多核苷酸，其中所述BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含质粒，其中所述质粒包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含质粒，其中所述质粒包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含质粒，其中所述质粒包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。In some embodiments, the non-viral vector is a plasmid. In some embodiments, the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3 any of the sequences or variants thereof. In some embodiments, the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO:4 to SEQ ID NO: Any one of the sequences shown in 9 or a variant thereof. In some embodiments, the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 10 to SEQ ID NO: Any one of the sequences shown in 27 or a variant thereof. In some embodiments, the recombinant vector comprises a plasmid, wherein the plasmid comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO:28 or SEQ ID NO: 29 or a variant thereof.
在一些实施方式中，所述非病毒载体是脂质体。在一些实施方式中，所述重组载体包含脂质体，其中所述脂质体包含编码BMP4蛋白的多核苷酸。在一些实施方式中，所述重组载体包含脂质体，其中所述脂质体包含编码BMP4蛋白的多核苷酸，其中所述BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含脂质体，其中所述脂质体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含脂质体，其中所述脂质体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，所述重组载体包含脂质体，其中所述脂质体包含编码BMP4蛋白的多核苷酸，其中所述编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其 变体。In some embodiments, the non-viral vector is a liposome. In some embodiments, the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein. In some embodiments, the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the amino acid sequence of the BMP4 protein comprises, for example, SEQ ID NO: 1 to SEQ ID NO: 3 Any one of the sequences shown or a variant thereof. In some embodiments, the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises, for example, SEQ ID NO: 4 to Any one of the sequences set forth in SEQ ID NO: 9 or a variant thereof. In some embodiments, the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as in SEQ ID NO: 10 to Any one of the sequences set forth in SEQ ID NO: 27 or a variant thereof. In some embodiments, the recombinant vector comprises a liposome, wherein the liposome comprises a polynucleotide encoding a BMP4 protein, wherein the sequence of the polynucleotide encoding a BMP4 protein comprises as SEQ ID NO: 28 or The sequence shown in SEQ ID NO: 29 or a variant thereof.
药物组合物pharmaceutical composition
“药物组合物”是指用于人的药物制剂。该药物组合物包含本发明的药物以及载体、稳定剂和/或赋形剂的合适制剂。"Pharmaceutical composition" refers to a pharmaceutical formulation for use in humans. The pharmaceutical composition comprises the medicament of the present invention together with suitable formulations of carriers, stabilizers and/or excipients.
本发明的一个方面提供包含重组载体以及药学上可接受的赋形剂的药物组合物，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。该药物组合物可用于治疗或预防对象的纤维增生性疾病。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。One aspect of the present invention provides a pharmaceutical composition comprising a recombinant vector and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein. The pharmaceutical composition can be used to treat or prevent fibroproliferative diseases in a subject. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus.
本发明的另一个方面提供包含游离的BMP4蛋白以及药学上可接受的赋形剂的药物组合物。在一些实施方式中，所述游离的BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述游离的BMP4蛋白是从天然来源纯化得到的，或者是通过基因工程方法得到的重组BMP4蛋白。Another aspect of the present invention provides a pharmaceutical composition comprising free BMP4 protein and a pharmaceutically acceptable excipient. In some embodiments, the amino acid sequence of the free BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering.
为了制备药物组合物或无菌组合物，让重组载体与可药用载体或赋形剂混合。可通过与生理学上可接受的载体、赋形剂或稳定剂混合，来制备呈例如冻干粉、浆液、水溶液或混悬剂形式的治疗及诊断药物的制剂。To prepare a pharmaceutical or sterile composition, the recombinant carrier is mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers.
药学上可接受的赋形剂是本领域熟知的。如本文所用，“药学上可接受的赋形剂”包括当与组合物的活性成分组合时允许该成分保持生物活性并且不会引起与对象的免疫***的破坏性反应的材料。这些可以包括稳定剂、防腐剂、盐或糖配合物或晶体等。“药学上可接受的”是指当施用至人体时不会产生过敏反应或类似的不期望的反应的分子和成分。本领域已知如何制备包含作为活性组分的水性组合物。通常，这些组合物被制备成注射剂或喷雾剂，例如液态溶液或悬浮液；也可以制备成适于在注射或喷雾之前配制成溶液或悬浮液的固体形 式。Pharmaceutically acceptable excipients are well known in the art. As used herein, "pharmaceutically acceptable excipients" include materials that, when combined with an active ingredient of a composition, allow the ingredient to remain biologically active and not cause a damaging reaction with the subject's immune system. These may include stabilizers, preservatives, salts or sugar complexes or crystals, and the like. "Pharmaceutically acceptable" refers to molecules and ingredients that do not produce allergic or similar undesired reactions when administered to humans. It is known in the art how to prepare aqueous compositions containing as an active ingredient. Typically, these compositions are prepared as injectables or sprays, such as liquid solutions or suspensions; solid forms suitable for solution or suspension prior to injection or spraying can also be prepared.
纤维增生性疾病fibroproliferative disease
纤维增生可发生于多种器官，主要病理改变为器官组织内纤维***增多，实质细胞减少，持续进展可致器官结构破坏和功能减退，乃至衰竭，严重威胁人类健康和生命。Fibrosis can occur in a variety of organs, and the main pathological changes are the increase of fibrous connective tissue and the decrease of parenchymal cells in the organ tissue. Continuous progress can lead to structural damage and functional decline of organs, and even failure, which seriously threatens human health and life.
在本发明中，纤维增生性疾病包括但不限于：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。In the present invention, fibroproliferative diseases include, but are not limited to: fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow and skin.
肺纤维增生性疾病包括但不限于：无机粉尘职业病(例如矽肺、石棉肺、煤肺等)，有机粉尘和过敏性肺炎(例如农民肺)，肺原发性疾病(例如特发性间质性肺炎、闭塞性细支气管炎伴机化性肺炎等)，特发性肺纤维化，进行性***硬化症，淋巴细胞间质性肺炎，家族性肺纤维化等。Pulmonary fibroproliferative diseases include, but are not limited to: inorganic dust occupational diseases (such as silicosis, asbestosis, coal lung, etc.), organic dust and hypersensitivity pneumonitis (such as farmers' lung), primary lung diseases (such as idiopathic interstitial disease) pneumonia, bronchiolitis obliterans with organizing pneumonia, etc.), idiopathic pulmonary fibrosis, progressive system sclerosis, lymphocytic interstitial pneumonia, familial pulmonary fibrosis, etc.
心血管***纤维增生性疾病包括但不限于心肌梗死后的替代性和间质性纤维化。Fibroproliferative diseases of the cardiovascular system include, but are not limited to, replacement and interstitial fibrosis following myocardial infarction.
肝纤维增生性疾病包括但不限于：病毒性肝硬化(例如乙、丙和丁型肝炎病毒性肝炎)，营养不良性肝硬化等。Fibroproliferative diseases of the liver include, but are not limited to, viral cirrhosis (eg, viral hepatitis B, C and D), dystrophic cirrhosis, and the like.
肾纤维增生性疾病包括但不限于：慢性肾小球肾炎、慢性肾盂肾炎、阻塞性肾病、***性红斑狼疮性肾病等。Renal fibroproliferative diseases include, but are not limited to, chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, systemic lupus erythematosus nephropathy, and the like.
眼睛纤维增生性疾病包括但不限于眼睛外伤和手术后眼睛纤维增生性疾病等。Ocular fibroproliferative diseases include, but are not limited to, ocular trauma and post-surgical ocular fibroproliferative diseases.
神经***纤维增生性疾病包括但不限于脊髓外伤后纤维增生性疾病等。Fibroproliferative diseases of the nervous system include, but are not limited to, fibroproliferative diseases after spinal cord trauma and the like.
骨髓纤维增生性疾病包括但不限于特发性和药物引起的骨髓纤维化等。Myelofibroproliferative diseases include, but are not limited to, idiopathic and drug-induced myelofibrosis, among others.
皮肤纤维增生性病症包括但不限于口腔粘膜纤维化等。Dermatofibroproliferative disorders include, but are not limited to, oral mucosal fibrosis and the like.
方法和治疗methods and treatments
本发明的一方面，提供了一种治疗或预防对象的纤维增生性疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的BMP4蛋白，其中所述BMP4蛋白是游离的BMP4蛋白。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，所述游离的BMP4蛋白是从天然来源纯化得到的，或者是通过基因工程方法得到的重组BMP4蛋白。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。In one aspect of the present invention, there is provided a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a BMP4 protein of the present invention, wherein the BMP4 protein is free BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
进一步地，本发明提供了本发明的BMP4蛋白在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用，其中所述BMP4蛋白是游离的BMP4蛋白。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列 或其变体。在一些实施方式中，所述游离的BMP4蛋白是从天然来源纯化得到的，或者是通过基因工程方法得到的重组BMP4蛋白。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Further, the present invention provides the use of the BMP4 protein of the present invention in the preparation of a medicament for treating or preventing fibroproliferative diseases in a subject, wherein the BMP4 protein is a free BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the free BMP4 protein is purified from a natural source, or a recombinant BMP4 protein obtained by genetic engineering. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
本发明的另一方面，提供了一种治疗或预防对象的纤维增生性疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的BMP4蛋白，其中所述BMP4蛋白是表达BMP4蛋白的重组载体，所述重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a BMP4 protein of the present invention, wherein the BMP4 protein is expressed A recombinant vector for the BMP4 protein, the recombinant vector comprising a vector comprising or carrying a polynucleotide encoding the BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
进一步地，本发明提供了本发明的BMP4蛋白在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用，其中所述BMP4蛋白是表达BMP4蛋白的重组载体，所述重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO: 29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Further, the present invention provides the application of the BMP4 protein of the present invention in the preparation of a medicine for treating or preventing fibroproliferative diseases in an object, wherein the BMP4 protein is a recombinant vector expressing the BMP4 protein, and the recombinant vector comprises a vector , the vector comprises or carries a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
本发明的另一方面，提供了一种治疗或预防对象的纤维增生性疾病的方法，所述方法包括向有需要的对象施用治疗有效量的本发明的药物组合物，其中该药物组合物包含重组载体和药学上可接受的赋形剂，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Another aspect of the present invention provides a method of treating or preventing a fibroproliferative disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention, wherein the pharmaceutical composition comprises A recombinant vector and a pharmaceutically acceptable excipient, wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
进一步地，本发明提供了本发明的药物组合物在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用，其中该药物组合物包含重组载体和药学上可接受的赋形剂，其中该重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。在一些实施方式中，所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。在一些实施方式中，BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至 SEQ ID NO:9所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。在一些实施方式中，编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。在一些实施方式中，所述载体是病毒载体或非病毒载体。在一些实施方式中，所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。在一些实施方式中，所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。在一些实施方式中，所述病毒载体是腺相关病毒。在一些实施方式中，所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。在一些实施方式中，所述纤维增生性疾病是肺纤维增生性疾病。在一些实施方式中，所述肺纤维增生性疾病是特发性肺纤维化。Further, the present invention provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for the treatment or prevention of fibroproliferative diseases in a subject, wherein the pharmaceutical composition comprises a recombinant carrier and a pharmaceutically acceptable excipient, Wherein the recombinant vector comprises a vector comprising or carrying a polynucleotide encoding a BMP4 protein. In some embodiments, the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein. In some embodiments, the amino acid sequence of the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO:4 to SEQ ID NO:9, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences set forth in SEQ ID NO: 10 to SEQ ID NO: 27, or a variant thereof. In some embodiments, the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence set forth in SEQ ID NO:28 or SEQ ID NO:29 or a variant thereof. In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles, and exosomes . In some embodiments, the viral vector is selected from the group consisting of: retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus, and lentivirus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow, and skin. In some embodiments, the fibroproliferative disease is pulmonary fibroproliferative disease. In some embodiments, the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
合适的给药途径包括胃肠外给药(例如肌内、静脉内或皮下给药)及口服给药。可按多种常规方式给予本发明方法的BMP4蛋白、重组载体或药物组合物，这些方法例如有经气管插管给予、经口摄取、吸入、局部施用或经皮肤、皮下、腹膜内、胃肠外、动脉内或静脉内注射。此外，人们可以在靶向药物递送***中给予本发明的BMP4蛋白、重组载体或药物组合物。在一个实施方式中，经气管插管给予本发明的BMP4蛋白、重组载体或药物组合物。在一个实施方式中，经静脉内注射给予本发明的BMP4蛋白、重组载体或药物组合物。Suitable routes of administration include parenteral (eg, intramuscular, intravenous or subcutaneous) and oral administration. The BMP4 protein, recombinant vector or pharmaceutical composition of the methods of the present invention can be administered in a variety of conventional ways, such as by endotracheal intubation, oral ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal, gastrointestinal Extracorporeal, intraarterial or intravenous injection. Furthermore, one can administer the BMP4 protein, recombinant vector or pharmaceutical composition of the invention in a targeted drug delivery system. In one embodiment, the BMP4 protein, recombinant vector or pharmaceutical composition of the invention is administered via endotracheal intubation. In one embodiment, the BMP4 protein, recombinant vector or pharmaceutical composition of the invention is administered by intravenous injection.
由临床医生例如用本领域已知或怀疑影响治疗或预期影响治疗的参数或因子来测定合适的剂量。通常，开始剂量比最佳剂量稍低，此后少量增加直到达到相对于任何不良副作用所要的或最佳的作用效果。重要的诊断测量包括测量例如炎性症状或所产生的炎性细胞因子的水平。Appropriate doses are determined by the clinician, eg, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment. Typically, the starting dose is slightly lower than the optimal dose and is increased by small amounts thereafter until the desired or optimal effect is achieved with respect to any adverse side effects. Important diagnostic measurements include measuring, for example, inflammatory symptoms or levels of inflammatory cytokines produced.
可通过连续给药或通过以一定间隔(例如一天、一周或每周1-7次)给药来施用本发明的BMP4蛋白、重组载体或药物组合物。可通过气管插管、静脉内、皮下、腹膜内、经皮肤、局部、经口、经鼻、经直肠、肌内、大脑内或脊柱内来提供剂量。优选剂量方案是包括避免显著的不合乎需要的副作用的最大剂量或给药频率的方案。The BMP4 protein, recombinant vector, or pharmaceutical composition of the present invention can be administered by continuous administration or by administration at intervals (eg, one day, one week, or 1-7 times per week). Dosages can be provided by endotracheal intubation, intravenously, subcutaneously, intraperitoneally, transdermally, topically, orally, nasally, rectally, intramuscularly, intracerebrally or intraspinal. A preferred dosage regimen is one that includes the maximum dose or frequency of administration that avoids significant undesirable side effects.
在一些实施方式中，本发明的治疗或预防纤维增生性疾病的方法阻止了纤维化的进展或由纤维化导致的疾病的发作。因此，在一些实施方式中，本发明提供一种阻止纤维化的进展和/或由纤维化导致的疾病的发作的方法，该方法包括向有此需要的对象施用有效量的本发明的BMP4蛋白、重组载体或药物组合物。在一些实施方式中，这些治疗或预防纤维增生性疾病的方法阻止了与纤维化有关的症状的发作、进展和/或复发。因此，在一些实施方式中，本发明提供了治疗或预防与纤维化有关的症状的方法，该方法包括向有此需要的对象 施用有效量的本发明的BMP4蛋白、重组载体或药物组合物。In some embodiments, the methods of treating or preventing fibroproliferative diseases of the present invention prevent the progression of fibrosis or the onset of diseases caused by fibrosis. Accordingly, in some embodiments, the present invention provides a method of arresting the progression of fibrosis and/or the onset of diseases caused by fibrosis, the method comprising administering to a subject in need thereof an effective amount of a BMP4 protein of the present invention , recombinant vector or pharmaceutical composition. In some embodiments, these methods of treating or preventing fibroproliferative diseases prevent the onset, progression and/or recurrence of symptoms associated with fibrosis. Accordingly, in some embodiments, the present invention provides a method of treating or preventing symptoms associated with fibrosis, the method comprising administering to a subject in need thereof an effective amount of a BMP4 protein, recombinant vector or pharmaceutical composition of the present invention.
实施例Example
实验动物laboratory animal
SPF级野生型C57BL/6小鼠购自广州中医药大学动物实验中心，C57BL/6背景的BMP4 +/-杂合子基因敲除小鼠购自于美国Jackson Laboratory。所有动物均在广州医科大学实验动物中心SPF级动物房进行饲养和繁殖，在小鼠6-8周龄时将小鼠随机分组进行实验，所有动物的使用和处理等均经过广州医科大学实验动物伦理委员会的审查批准。 SPF wild-type C57BL/6 mice were purchased from the Animal Experiment Center of Guangzhou University of Traditional Chinese Medicine, and BMP4 +/- heterozygous knockout mice with C57BL/6 background were purchased from the Jackson Laboratory in the United States. All animals were reared and bred in the SPF animal room of the Experimental Animal Center of Guangzhou Medical University. The mice were randomly divided into groups when they were 6-8 weeks old for experiments. Ethics committee review and approval.
统计学处理Statistical processing
使用GraphPad Prism 5.0软件进行统计学分析和软件作图，实验结果以均数±标准误差表示(Mean±SEM)，两组样本之间均数的比较应用t检验，多组均数比较应用单因素方差分析(one way-ANOVA)检验，P<0.05为有统计学意义。GraphPad Prism 5.0 software was used for statistical analysis and software mapping, and the experimental results were expressed as mean ± standard error (Mean ± SEM). The t test was used for the comparison of the means between the two groups, and the single factor was used for the comparison of the means of multiple groups. Analysis of variance (one way-ANOVA) test, P<0.05 was considered statistically significant.
实施例1在IPF患者和博莱霉素诱导的肺纤维化小鼠肺组织中BMP4的表达显著降低Example 1 Significantly reduced expression of BMP4 in lung tissue of IPF patients and bleomycin-induced pulmonary fibrosis mice
免疫印迹检测肺组织中相关目的蛋白的表达量Western blotting to detect the expression of related target proteins in lung tissue
(1)肺组织总蛋白提取：将冻存的肺组织从-80℃冰箱拿出，用生理盐水清洗肺组织，并吸干水。称量肺组织重量，大约每100mg肺组织加入1ml蛋白裂解液，装入匀浆管中，用高速匀浆机破碎肺组织，裂解至裂解液成浓汤样。于高速冷冻离心机中离心，4℃12000g，离心25分钟，吸取上层液体至一新的EP管中，用于后续测蛋白浓度，弃去沉淀。(1) Extraction of total protein from lung tissue: Take the cryopreserved lung tissue out of the -80°C refrigerator, wash the lung tissue with normal saline, and dry the water. Weigh the lung tissue, add about 1 ml of protein lysis solution per 100 mg of lung tissue, put it into a homogenization tube, crush the lung tissue with a high-speed homogenizer, and lyse the lysis solution into a thick soup sample. Centrifuge in a high-speed refrigerated centrifuge at 12,000g at 4°C for 25 minutes, suck the upper liquid into a new EP tube for subsequent protein concentration measurement, and discard the precipitate.
(2)测蛋白浓度：按碧云天BCA蛋白浓度测定试剂盒说明书操作。加入总体积为25μl的梯度稀释的标准品。将1.25μl的样品用水稀释20倍，使总体积为25μl并加入96孔板。试剂A、试剂B按50：1比例混匀，将混合液200μl加入已加标准品和样品的96孔板中，37℃避光孵育30min。全波长酶标仪于562nm波长下测定每个孔的OD值，制作标准曲线，并计算出每孔样品蛋白浓度。(2) Determination of protein concentration: operate according to the instructions of Biyuntian BCA protein concentration determination kit. Add serially diluted standards to a total volume of 25 μl. 1.25 μl of the sample was diluted 20-fold with water to make a total volume of 25 μl and added to a 96-well plate. Mix reagent A and reagent B at a ratio of 50:1, add 200 μl of the mixture to a 96-well plate to which standards and samples have been added, and incubate at 37°C for 30 minutes in the dark. The OD value of each well was measured by a full-wavelength microplate reader at a wavelength of 562 nm, a standard curve was made, and the protein concentration of each well was calculated.
(3)制备上样蛋白：以上样20ng为总量，根据所测蛋白浓度，算出每个样品上样体积，用蛋白裂解液稀释样品，使每个样品最终上样体积一致。按照比例加入5*loading buffer。100℃沸水煮样品5min使蛋白变性，煮完将样品置于冰上冷却后于-20℃保存。(3) Preparation of loading protein: 20 ng of the sample was taken as the total amount, and the loading volume of each sample was calculated according to the measured protein concentration, and the samples were diluted with protein lysis buffer so that the final loading volume of each sample was the same. Add 5*loading buffer in proportion. The samples were boiled in boiling water at 100°C for 5 min to denature the protein. After boiling, the samples were cooled on ice and stored at -20°C.
(4)蛋白免疫印迹：提前配制好10％分离胶和5％浓缩胶，向电泳槽内加入电泳液，垂直向上拔出加样梳，将样品加入上样孔。先以70v电压恒压电泳，至样品经浓缩胶压成一条直线，marker开始分离，加压至120v，恒压电泳至溴酚蓝跑至底部约0.5cm时结束。用甲醇浸泡PVDF膜，取出放入装有电转液的盘子中，弃去电泳液，从电泳槽中取出凝胶，按照“海绵-滤纸-凝胶-PVDF膜-滤纸-海绵”的顺序制作电转“三明治”。将三明治夹放入电转 槽中，将预冷的电转液倒入电转仪中，放入冰袋使电转液保持低温。以300mA，120min条件电转并用冰覆盖电转槽。电转完将PVDF膜取出，将目的蛋白相应条带加入对应的一抗置于摇床上慢摇，4℃孵育过夜，第二天用TBST洗膜3次，每次10min，加入对应二抗室温孵育1到2个小时，TBST洗膜3次，每次10分钟。用全自动化学发光显影***显影，用Image J图像分析软件对目的条带灰度值进行分析并用Graph-Pad软件进行统计。(4) Western blotting: Prepare 10% separating gel and 5% stacking gel in advance, add electrophoresis solution to the electrophoresis tank, pull out the sample loading comb vertically upward, and add the sample to the sample hole. First, electrophoresis at a constant voltage of 70v, until the sample is pressed into a straight line by the stacking gel, the marker begins to separate, pressurized to 120v, and the constant voltage electrophoresis ends when the bromophenol blue runs to about 0.5cm from the bottom. Soak the PVDF membrane with methanol, take it out and put it into a plate with electroporation solution, discard the electrophoresis solution, take out the gel from the electrophoresis tank, and make electroporation in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge" "sandwich". Put the sandwich clip into the electroporation tank, pour the pre-cooled electroporation solution into the electroporator, and put it in an ice pack to keep the electroporation solution at a low temperature. Electroporation was performed at 300 mA for 120 min and the electroporation tank was covered with ice. After electroporation, take out the PVDF membrane, add the corresponding band of the target protein to the corresponding primary antibody and place it on a shaker, incubate overnight at 4°C, wash the membrane 3 times with TBST the next day, each time for 10 min, add the corresponding secondary antibody and incubate at room temperature For 1 to 2 hours, wash the membrane 3 times with TBST for 10 minutes each. It was developed with a fully automatic chemiluminescence developing system, and the gray value of the target band was analyzed with Image J image analysis software and counted with Graph-Pad software.
结果result
IPF患者肺组织中BMP4的表达显著降低The expression of BMP4 was significantly decreased in the lung tissue of IPF patients
收集癌旁正常对照肺组织和IPF患者肺组织，观察IPF患者和正常对照肺组织中BMP4的表达变化(n＝20)。如图1A和图1B所示，IPF患者肺组织中BMP4蛋白水平显著降低。荧光定量PCR(Real-time PCR)的结果显示，IPF患者肺组织中BMP4 mRNA水平显著降低(图1C)。免疫荧光染色的结果显示，IPF患者肺组织中BMP4的荧光强度显著降低(图1D)。综上，根据图1所示的结果可知，相对正常对照肺组织而言，肺纤维化患者肺组织中BMP4的表达显著降低。The adjacent normal control lung tissues and the lung tissues of IPF patients were collected, and the expression changes of BMP4 in the lung tissues of IPF patients and normal controls were observed (n=20). As shown in Figure 1A and Figure 1B, BMP4 protein levels were significantly reduced in the lung tissue of IPF patients. Fluorescence quantitative PCR (Real-time PCR) results showed that BMP4 mRNA levels were significantly reduced in the lung tissue of IPF patients (Fig. 1C). The results of immunofluorescence staining showed that the fluorescence intensity of BMP4 in the lung tissue of IPF patients was significantly reduced (Fig. 1D). In conclusion, according to the results shown in Figure 1, it can be seen that the expression of BMP4 in the lung tissue of patients with pulmonary fibrosis is significantly reduced compared with the normal control lung tissue.
博来霉素在肺纤维化小鼠肺组织中诱导BMP4蛋白表达的降低Bleomycin induces the reduction of BMP4 protein expression in lung tissue of mice with pulmonary fibrosis
博莱霉素气管内给药(2.5mg/kg)诱导野生型小鼠发生肺纤维化，分别在肺纤维化发展的不同阶段，即给予博来霉素造模后第0、7、14、21天收集肺组织标本；应用蛋白免疫印迹技术，分析在肺纤维化不同阶段，小鼠肺组织中BMP4蛋白表达情况。发现，与对照组小鼠相比，模型组小鼠肺组织中BMP4水平显著降低，且随着博来霉素的作用时间增加，肺纤维化小鼠肺组织中BMP4蛋白表达进一步降低(图2)。Intratracheal administration of bleomycin (2.5 mg/kg) induced pulmonary fibrosis in wild-type mice at different stages of the development of pulmonary fibrosis, namely at 0, 7, 14, and 14 days after bleomycin was administered. Lung tissue samples were collected on day 21; Western blotting was used to analyze the expression of BMP4 protein in mouse lung tissue at different stages of pulmonary fibrosis. It was found that compared with the control group mice, the BMP4 level in the lung tissue of the model group mice was significantly reduced, and with the increase of the action time of bleomycin, the BMP4 protein expression in the lung tissue of the pulmonary fibrosis mice was further reduced (Figure 2 ).
实施例2 BMP4基因敲除对博莱霉素诱导小鼠肺纤维化的影响Example 2 The effect of BMP4 gene knockout on bleomycin-induced pulmonary fibrosis in mice
BMP4基因敲除小鼠(BMP4 +/-)的鉴定 Identification of BMP4 knockout mice (BMP4 +/- )
(1)剪取小鼠尾巴并裂解：给小鼠编号，用洁净的剪刀剪取小鼠尾巴，并置于1.5ml EP管内，每个EP管中加入200μl鼠尾裂解液，55℃水浴过夜(约10h左右)，再85℃水浴1h使蛋白酶K灭活(标本可置于4℃保存一周)。(1) Cut the mouse tail and lyse: Number the mice, cut the mouse tail with clean scissors, and place it in a 1.5ml EP tube, add 200μl of mouse tail lysis solution to each EP tube, and water bath at 55°C overnight (about 10h), and then water bath at 85°C for 1h to inactivate proteinase K (specimens can be stored at 4°C for a week).
(2)引物序列如下表所示：(2) The primer sequences are shown in the following table:
表1 BMP4基因敲除小鼠(BMP4 +/-)基因型鉴定引物序列 Table 1 BMP4 knockout mouse (BMP4 +/- ) genotype identification primer sequences
Figure PCTCN2021140837-appb-000001
Figure PCTCN2021140837-appb-000001
Figure PCTCN2021140837-appb-000002
Figure PCTCN2021140837-appb-000002
注：NEO引物用于扩增BMP4突变基因；WT引物用于扩增BMP4野生基因。Note: NEO primers are used to amplify BMP4 mutant gene; WT primers are used to amplify BMP4 wild gene.
(3)按照下表配制PCR反应体系(总体积25μl)：(3) Prepare a PCR reaction system (total volume 25 μl) according to the following table:
表2 BMP4 +/-小鼠基因鉴定反应体系 Table 2 BMP4 +/- mouse gene identification reaction system
主要成分main ingredient 加样体积Injection volume
2×PCR Master Mix2×PCR Master Mix 12.5μl12.5μl
DNA模板DNA template 2μl2μl
ddH 2O ddH 2 O 8.5μl8.5μl
FF 1μl1μl
RR 1μl1μl
注：F：上游引物；R：下游引物。Note: F: upstream primer; R: downstream primer.
(4)PCR扩增的反应条件(4) Reaction conditions for PCR amplification
表3 BMP4 +/-小鼠基因型鉴定反应条件 Table 3 BMP4 +/- mouse genotyping reaction conditions
Figure PCTCN2021140837-appb-000003
Figure PCTCN2021140837-appb-000003
注：将反应体系充分混匀后再行PCR扩增反应。Note: The PCR amplification reaction is performed after the reaction system is fully mixed.
(5)琼脂糖凝胶电泳(5) Agarose gel electrophoresis
称取适量琼脂糖粉末至锥形瓶中，按比加入1xTAE轻轻摇匀，用微波炉加热至沸腾后，轻轻摇匀，使琼脂糖完全溶解。待其稍微冷却后加入EB替代物(每100ml琼脂糖凝胶液加入5μl EB替代物)，混匀后倒入加样槽中，并垂直***加样梳，待琼脂糖凝胶彻底凝固后，拔出加样梳，取出琼脂糖凝胶并放入琼脂糖电泳槽中，加入1xTAE至没过琼脂糖凝胶。按序加入DL1000marker和样品，按120V、30min的条件进行电泳。电泳结束后，将琼脂糖凝胶置于凝胶成像***中拍照成像，保存并分析结果。Weigh an appropriate amount of agarose powder into a conical flask, add 1xTAE in proportion, and shake gently. After heating to boiling in a microwave oven, shake gently to completely dissolve the agarose. After it cools down slightly, add EB substitute (add 5 μl EB substitute for every 100ml of agarose gel), mix well, pour it into the sample addition tank, and insert the sample addition comb vertically. After the agarose gel is completely solidified, Pull out the loading comb, take out the agarose gel and put it into the agarose electrophoresis tank, add 1xTAE to cover the agarose gel. DL1000marker and samples were added in sequence, and electrophoresis was carried out under the conditions of 120V and 30min. After electrophoresis, place the agarose gel in a gel imaging system to take pictures, save and analyze the results.
小鼠肺纤维化模型建立Establishment of a mouse model of pulmonary fibrosis
将小鼠分为四组：野生型小鼠对照组，BMP4 +/-小鼠对照组，野生型小鼠予以博来霉素处理组，BMP4 +/-小鼠予以博来霉素处理组。采用经喉气管插管的方式气管内滴注博来霉素，博来霉素给药剂量为2.0mg/kg，对照组给予相应剂量的生理盐水。用1.2％阿佛丁(0.2ml/10g)腹腔注射麻醉小鼠，麻醉不宜过深。将小鼠固定于手术实验台上，垂直于桌面。将小鼠喉镜***部***小鼠口腔，将整个喉镜向上提起，暴露声门，于声门张开时顺势将22g静脉 留置针***气管内，并用注射器头向里推注约0.1ml的气体，观察小鼠是否仍能正常均匀呼吸。若小鼠出现短暂的呼吸停止，表明气管插管成功。将稀释好的博来霉素注入气管内，并轻捏小鼠尾巴，刺激小鼠呼吸，进一步将留置针内的液体吸入肺部，轻晃小鼠身体，促进博来霉素在肺内均匀分布。给药后，观察小鼠的一般情况并记录小鼠体重以及死亡情况。 The mice were divided into four groups: wild-type mice control group, BMP4 +/- mice control group, wild-type mice treated with bleomycin, and BMP4 +/- mice treated with bleomycin. Bleomycin was instilled by intratracheal intubation via laryngotracheal intubation. The dose of bleomycin was 2.0 mg/kg, and the control group was given the corresponding dose of normal saline. The mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g), and the anesthesia should not be too deep. The mice were fixed on the surgical table, perpendicular to the table top. Insert the insertion part of the mouse laryngoscope into the mouth of the mouse, lift the whole laryngoscope up to expose the glottis, insert a 22g intravenous indwelling needle into the trachea when the glottis is opened, and inject about 0.1ml of gas with the syringe tip. Observe whether the mice can still breathe normally and evenly. If the mouse has a brief respiratory arrest, it indicates that the tracheal intubation was successful. Inject the diluted bleomycin into the trachea, and gently pinch the mouse's tail to stimulate the mouse to breathe, further inhale the liquid in the indwelling needle into the lungs, and gently shake the body of the mouse to promote the uniform distribution of bleomycin in the lungs. distributed. After administration, the general condition of the mice was observed and the body weight and death of the mice were recorded.
小鼠活体CT扫描In vivo CT scan of mice
腹腔注射1.2％阿佛丁(0.2ml/10g)麻醉小鼠，将麻醉后的小鼠置于CT扫描箱中，用LCT-200 Micro CT对小鼠进行胸部CT扫描成像。The mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g), and the anesthetized mice were placed in a CT scanning box, and the chest CT scan imaging was performed on the mice with LCT-200 Micro CT.
小鼠肺功能检测Pulmonary function test in mice
于博来霉素造模后第21天，用小动物肺功能仪检测小鼠肺功能情况。校正小动物肺功能仪，使机器参数达标后开始检测。腹腔注射1.2％阿佛丁(0.2ml/10g)麻醉小鼠，用酒精擦拭小鼠颈部，切开颈部皮肤，顿性分离肌肉组织，暴露气管，剪开气管并接气管插管，放入体描箱后，连接小动物肺功能仪并进行机械通气。待小鼠呼吸及各参数变化趋于稳定后测定肺功能，记录各项肺通气功能指标。On the 21st day after bleomycin modeling, the lung function of mice was detected by small animal pulmonary function analyzer. The small animal spirometer was calibrated, and the test was started after the machine parameters reached the standard. The mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g), the neck of the mice was wiped with alcohol, the skin of the neck was incised, the muscle tissue was immediately separated, the trachea was exposed, and the trachea was cut and connected to the tracheal intubation. After entering the body tracing box, connect the small animal pulmonary function instrument and perform mechanical ventilation. Pulmonary function was measured after the changes of the respiration and various parameters of the mice became stable, and various pulmonary ventilation function indexes were recorded.
小鼠肺组织HE染色HE staining of mouse lung tissue
将分离的小鼠肺组织用多聚甲醛固定，再进行脱水、包埋、切片，切成约3μm厚的切片。将切片置于烤片机上烤2h，使石蜡充分熔化；脱蜡：二甲苯10min*2次，100％酒精5min*2次，95％酒精5min*2次，流水冲洗5min；苏木素染核10min，流水冲洗3min；2％盐酸酒精转化15s，流水冲洗2min；伊红染色1min，流水冲洗4min；脱水透明：95％酒精5min*2次，100％酒精5min*2次，二甲苯2次；待玻片晾干，用中性树脂封片；扫片机全视野扫描后观察肺部病变情况。The isolated mouse lung tissue was fixed with paraformaldehyde, then dehydrated, embedded, sectioned, and cut into slices with a thickness of about 3 μm. Bake the slices on a microtome for 2 hours to fully melt the paraffin; dewaxing: xylene 10min*2 times, 100% alcohol 5min*2 times, 95% alcohol 5min*2 times, rinsed with running water for 5min; nuclear staining with hematoxylin for 10min, Rinse with running water for 3min; 2% hydrochloric acid alcohol conversion for 15s, rinse with running water for 2min; eosin staining for 1min, rinse with running water for 4min; dehydration and transparency: 95% alcohol 5min*2 times, 100% alcohol 5min*2 times, xylene 2 times; wait for glass The slides were air-dried and sealed with neutral resin; the lung lesions were observed after full-field scanning with a scanning machine.
小鼠肺组织Masson染色Masson staining of mouse lung tissue
小鼠肺组织用甲醛固定后，常规脱水包埋切片，切片脱蜡后，置于固定液中于常温下过夜，流水冲洗。与天青石蓝液、苏木***中各染2分钟，用1％的盐酸乙醇分化，流水冲洗10分钟。于品红溶液、1％磷铝酸盐溶液各处理10分钟，再于2％的苯胺蓝溶液中染5分钟，于0.2％冰醋酸溶液中处理2分钟。95％酒精脱水，梯度酒精脱水，二甲苯透明，中性树胶封片。After the mouse lung tissue was fixed with formaldehyde, it was routinely dehydrated and embedded in sections. After the sections were deparaffinized, they were placed in a fixative solution overnight at room temperature and rinsed with running water. It was stained with celestine blue solution and hematoxylin semen for 2 minutes each, differentiated with 1% hydrochloric acid ethanol, and rinsed with running water for 10 minutes. It was treated in magenta solution and 1% aluminophosphate solution for 10 minutes, then dyed in 2% aniline blue solution for 5 minutes, and treated in 0.2% glacial acetic acid solution for 2 minutes. 95% alcohol dehydration, gradient alcohol dehydration, xylene transparent, neutral gum seal.
肺组织中羟脯氨酸(hydroxyproline，HYP)含量测定Determination of hydroxyproline (HYP) content in lung tissue
(1)肺组织的水解：称取约20mg左右的肺组织至15ml的EP管中，加入500μl碱性水解液，混匀后放置于95℃水浴锅中水解20min，间中多次摇晃混匀。(1) Hydrolysis of lung tissue: Weigh about 20 mg of lung tissue into a 15 ml EP tube, add 500 μl of alkaline hydrolyzate, mix well, place it in a 95°C water bath for hydrolysis for 20 minutes, and shake it for several times. .
(2)调节PH至6.0～6.8左右：水解后用流水冲洗EP管使其冷却，加入指示剂后充分振荡摇匀，准确加入调PH甲液500μl后充分振荡摇匀，液体呈红色；将调PH的乙液加入各EP管，边加边充分振荡摇匀，直至EP管中的液体由红色变黄绿色；往EP管中加双蒸 水至5ml后充分振荡摇匀；取1.5-1.6ml经稀释的水解液于2ml的EP管中，称量10-15mg活性炭于水解液中，充分振荡摇匀后，3500rpm/min离心10min，用加样枪吸取离心后的上清液500μl以待检测。(2) Adjust the pH to about 6.0-6.8: after hydrolysis, rinse the EP tube with running water to cool it, add the indicator and shake it well, add 500 μl of the pH-adjusting solution, and shake it well, the liquid turns red; Add the PH solution B into each EP tube, shake and shake well while adding, until the liquid in the EP tube turns from red to yellow-green; add double-distilled water to the EP tube to 5ml and shake well; take 1.5-1.6ml The diluted hydrolyzate was placed in a 2ml EP tube, and 10-15mg of activated carbon was weighed into the hydrolyzate, fully shaken and shaken, centrifuged at 3500rpm/min for 10min, and 500μl of the centrifuged supernatant was sucked with a sample gun for testing. .
(3)检测反应体系：(3) Detection reaction system:
表4反应体系Table 4 Reaction system
   空白管blank tube 标准管standard tube 检测管Detection tube
双蒸水(μl)Double distilled water (μl) 500500 ———— ————
5ug/ml标准应用液(μl)5ug/ml standard application solution (μl) ———— 500500 ————
检测液(μl)Detection solution (μl) ———— ———— 500500
试剂一(μl)Reagent one (μl) 250250 250250 250250
试剂二(μl)Reagent II (μl) 250250 250250 250250
试剂三(μl)Reagent three (μl) 250250 250250 250250
注：加完试剂一后充分振荡摇匀后，静置10min，再加入试剂二，充分振荡摇匀后，静置5min，再加入试剂三，充分振荡摇匀冷却后，置于60℃的水浴锅中反应15min，再3500rpm/min离心10min。Note: After adding Reagent 1, shake well, let stand for 10 minutes, then add Reagent 2, shake well, let stand for 5 minutes, add Reagent 3, shake well and cool, put in a water bath at 60°C React in the pot for 15 min, and then centrifuge at 3500 rpm/min for 10 min.
(4)样品OD值测量：用加样枪吸取200μl的样品于96孔板中，置于酶标仪中以550nm的波长测量各样品的OD值。(4) Measurement of the OD value of the sample: 200 μl of the sample was drawn into a 96-well plate with a sample gun, and placed in a microplate reader to measure the OD value of each sample at a wavelength of 550 nm.
结果result
一般状态观察General status observation
我们将小鼠随机分为四组，BMP4 +/++Saline：野生型小鼠对照组，n＝12；BMP4 +/-+Saline：BMP4 +/-小鼠对照组，n＝10；BMP4 +/++BLM：野生型小鼠予以博来霉素处理组，n＝25；BMP4 +/-+BLM：BMP4 +/-小鼠予以博来霉素处理组，n＝37。博来霉素造模后，对各组小鼠的活动状态进行观察。对照组(Saline)小鼠的反应灵敏，呼吸平稳，毛色及光泽正常；而模型组(BLM)小鼠的活动状态差，毛色发暗，无光泽，呼吸急促。 We randomly divided the mice into four groups, BMP4 +/+ +Saline: wild-type mice control, n=12; BMP4 +/ -+Saline: BMP4 +/- mice control, n=10; BMP4 + /+ +BLM: wild-type mice treated with bleomycin, n=25; BMP4 +/ -+BLM: BMP4 +/- mice treated with bleomycin, n=37. After the bleomycin model was established, the activity status of the mice in each group was observed. The mice in the control group (Saline) had sensitive responses, stable breathing, and normal coat color and gloss; while the mice in the model group (BLM) had poor activity status, dark coat color, dullness, and shortness of breath.
小鼠生存率和体重Mouse survival and body weight
小鼠给予博来霉素(2.0mg/kg)处理，记录造模21天过程中小鼠的体重及死亡情况。与对照组(Saline)小鼠相比，模型组(BLM)小鼠的生存率下降。模型组(BLM)小鼠中，与野生型小鼠相比，BMP4 +/-组小鼠生存率下降更显著(图3A)。博来霉素造模后，与对照组(Saline)相比，模型组(BLM)小鼠的体重于造模后的10天内逐渐下降，10天后趋于平稳以及逐渐复升。模型组(BLM)小鼠中，与野生型小鼠相比，BMP4 +/-组小鼠体重下降更显著(图3B)。 The mice were treated with bleomycin (2.0 mg/kg), and the body weight and death of the mice during the 21-day modeling process were recorded. Compared with the control group (Saline) mice, the survival rate of the model group (BLM) mice was decreased. In the model group (BLM) mice, compared with wild-type mice, the survival rate of BMP4 +/- mice decreased significantly (Fig. 3A). After the bleomycin modeling, compared with the control group (Saline), the body weight of the model group (BLM) mice gradually decreased within 10 days after modeling, and became stable and gradually recovered after 10 days. In the model group (BLM) mice, the weight loss of BMP4 +/- mice was more significant compared with wild-type mice (Fig. 3B).
小鼠肺部CT结果Lung CT results of mice
小鼠博来霉素造模后第14天，进行了小鼠活体胸部CT扫描，评估小鼠肺纤维化程度。如图4所示，与对照组相比，模型组(BLM)小鼠的肺实变及纤维化显著。模型组(BLM)小鼠 中，与野生型小鼠相比，BMP4 +/-小鼠肺实变及纤维化更显著。 On the 14th day after the bleomycin model was established in mice, in vivo chest CT scans were performed to evaluate the degree of pulmonary fibrosis in mice. As shown in FIG. 4 , compared with the control group, the model group (BLM) mice had significant lung consolidation and fibrosis. In the model group (BLM) mice, compared with wild-type mice, BMP4 +/- mice had more significant lung consolidation and fibrosis.
小鼠肺功能结果Mouse lung function results
造模后第21天，对小鼠进行肺功能检测，如图5所示：与对照组(Saline)相比，模型组(BLM)小鼠的肺功能下降，表现为肺顺应性下降(n＝6，P＜0.05)和气道阻力升高(n＝6，P＜0.001)。模型组(BLM)小鼠中，与野生型小鼠相比，BMP4 +/-组肺功能下降更显著。 On the 21st day after modeling, the mice were tested for lung function, as shown in Figure 5: Compared with the control group (Saline), the lung function of the mice in the model group (BLM) decreased, manifested as decreased lung compliance (n = 6, P < 0.05) and increased airway resistance (n = 6, P < 0.001). In the model group (BLM) mice, the lung function decreased more significantly in the BMP4 +/- group compared with wild-type mice.
小鼠肺组织HE和Masson染色结果HE and Masson staining results of mouse lung tissue
小鼠博来霉素造模后第21天，小鼠肺组织HE染色结果如图6A所示，Masson染色结果如图6B所示，予以生理盐水处理组的小鼠肺泡结构完整，气道及肺泡周围无炎症细胞浸润，未见纤维组织增生和胶原沉积；模型组(BLM)小鼠肺泡结构破坏，肺泡壁及肺泡间隔增厚，可见气道及肺泡周围有炎症细胞浸润，肺泡间隔有纤维组织增生，BMP4 +/-+BLM组肺组织破坏最显著。 On the 21st day after the mouse bleomycin was established, the results of HE staining of the mouse lung tissue are shown in Figure 6A, and the results of Masson staining are shown in Figure 6B. The alveolar structure of the mice in the saline-treated group was complete, and the airways and There was no inflammatory cell infiltration around the alveoli, and no fibrous tissue proliferation and collagen deposition; the alveolar structure of the model group (BLM) mice was destroyed, the alveolar wall and alveolar septum were thickened, and inflammatory cell infiltration around the airway and alveoli was seen, and there were fibers in the alveolar septum. Tissue hyperplasia, with the most significant destruction of lung tissue in the BMP4 +/ -+BLM group.
肺组织中羟脯氨酸(hydroxyproline，HYP)的含量The content of hydroxyproline (HYP) in lung tissue
肺纤维化时，肺组织内胶原纤维增加，而羟脯胺酸为胶原纤维所特有，以此作为肺纤维化的指标。博来霉素造模后第21天，检测了小鼠肺组织中羟脯胺酸含量(图7)，以此判断纤维化程度。与对照组相比，模型组(BLM)小鼠的肺组织中的羟脯胺酸含量增多(n＝10，P<0.05)，BMP4 +/-+BLM含量最高，其差别具有统计学意义(n＝10，P<0.001)。 In pulmonary fibrosis, collagen fibers in lung tissue increase, and hydroxyproline is unique to collagen fibers, which is used as an indicator of pulmonary fibrosis. On the 21st day after bleomycin modeling, the content of hydroxyproline in the lung tissue of mice was detected (Fig. 7) to judge the degree of fibrosis. Compared with the control group, the content of hydroxyproline in the lung tissue of the model group (BLM) mice increased (n=10, P<0.05), and the content of BMP4 +/- +BLM was the highest, and the difference was statistically significant ( n=10, P<0.001).
实施例3 BMP4基因敲除对博来霉素诱导小鼠成纤维细胞向肌纤维母细胞转化和合成ECM成分的影响Example 3 The effect of BMP4 gene knockout on bleomycin-induced transformation of mouse fibroblasts to myofibroblasts and synthesis of ECM components
α-SMA是成纤维细胞向肌纤维母细胞转化的标记物，Fibronectin、和胶原主要成分Col 1、Col 3是ECM的主要成分。博来霉素造模后，我们检测了各组小鼠肺组织α-SMA、Fibronectin、Col 1、Col 3蛋白表达情况。α-SMA is a marker for the transformation of fibroblasts to myofibroblasts, and Fibronectin and Col 1 and Col 3, the main components of collagen, are the main components of ECM. After bleomycin modeling, we detected the expression of α-SMA, Fibronectin, Col 1, and Col 3 in the lung tissue of mice in each group.
结果result
如图8所示，与对照组相比，予以博来霉素处理21天后小鼠肺组织中的Fibronectin、α-SMA、Col 1、Col 3蛋白含量显著增加，说明博来霉素诱导了小鼠成纤维细胞向肌纤维母细胞转化和合成大量的ECM。模型组(BLM)小鼠中，与野生型小鼠相比，BMP4 +/-小鼠肺组织中的Fibronectin、α-SMA、Col 1、Col 3表达更高，说明BMP4基因敲除加重了博来霉素诱导的小鼠成纤维细胞向肌纤维母细胞转化以及ECM的沉积。 As shown in Figure 8, compared with the control group, the protein contents of Fibronectin, α-SMA, Col 1 and Col 3 in the lung tissue of mice treated with bleomycin for 21 days were significantly increased, indicating that bleomycin induced a small amount of protein in the lung tissue. Murine fibroblasts transform into myofibroblasts and synthesize large amounts of ECM. In the model group (BLM) mice, compared with wild-type mice, the expression of Fibronectin, α-SMA, Col 1, and Col 3 in the lung tissue of BMP4 +/- mice was higher, indicating that BMP4 gene knockout aggravated the disease. Leomycin-induced transformation of mouse fibroblasts to myofibroblasts and deposition of ECM.
实施例4 BMP4过表达对博来霉素诱导肺纤维化小鼠的预防和治疗作用的影响Example 4 The effect of BMP4 overexpression on the preventive and therapeutic effects of bleomycin-induced pulmonary fibrosis in mice
为了进一步确定BMP4在肺纤维化中的保护作用，我们包装了过表达BMP4(SEQ ID NO:34)的腺相关病毒(AAV9-BMP4)，分别在气管内注射博莱霉素前两周以及造模9天后，经气管插管给予腺相关病毒AAV-BMP4。在给予博莱霉素造模23天后对各组小鼠进行肺功能 测定，并对取材后的组织进行HE染色和Masson染色，观察BMP4过表达对博莱霉素诱导的小鼠肺纤维化的预防和治疗作用。将小鼠分为四组：对照组(control)，博来霉素和AAV9空载体处理组(BLM+AAV-Empty)，博来霉素处理后AAV9-BMP4处理组(BLM+AAV9-BMP4)，AAV9-BMP4处理后BLM处理组(AAV9-BMP4+BLM)。To further determine the protective role of BMP4 in pulmonary fibrosis, we packaged an adeno-associated virus (AAV9-BMP4) overexpressing BMP4 (SEQ ID NO:34) two weeks before intratracheal injection of bleomycin and after Nine days later, adeno-associated virus AAV-BMP4 was administered via tracheal intubation. After 23 days of bleomycin-induced modeling, the lung function of the mice in each group was measured, and the tissues were obtained by HE staining and Masson staining to observe the effect of BMP4 overexpression on bleomycin-induced pulmonary fibrosis in mice. preventive and therapeutic effects. Mice were divided into four groups: control group (control), group treated with bleomycin and AAV9 empty vector (BLM+AAV-Empty), and group treated with AAV9-BMP4 after bleomycin treatment (BLM+AAV9-BMP4) , AAV9-BMP4-treated BLM-treated group (AAV9-BMP4+BLM).
结果result
如图9A所示，与博莱霉素模型组(BLM+AAV-Empty)相比，BMP4过表达预防组(AAV9-BMP4+BLM)和治疗组(BLM+AAV9-BMP4)小鼠的肺功能都明显改善，表现为肺动态顺应性增加和肺阻力降低；图9B所示，与博莱霉素模型组(BLM+AAV-Empty)相比，BMP4过表达预防组(AAV9-BMP4+BLM)和治疗组(BLM+AAV9-BMP4)小鼠的肺纤维化病理程度也显著降低，进一步说明了BMP4在肺纤维化中能够起到抗纤维化的作用。As shown in Figure 9A, compared with the bleomycin model group (BLM+AAV-Empty), the lung function of BMP4 overexpression prevention group (AAV9-BMP4+BLM) and treatment group (BLM+AAV9-BMP4) mice Both were significantly improved, manifested as increased pulmonary dynamic compliance and decreased pulmonary resistance; as shown in Figure 9B, compared with the bleomycin model group (BLM+AAV-Empty), the BMP4 overexpression prevention group (AAV9-BMP4+BLM) The pathological degree of pulmonary fibrosis was also significantly reduced in mice in the and treatment groups (BLM+AAV9-BMP4), further indicating that BMP4 can play an anti-fibrotic role in pulmonary fibrosis.
实施例5 BMP4基因敲除能够促进TGF-β1诱导的小鼠原代肺成纤维细胞转化和合成ECMExample 5 BMP4 gene knockout can promote TGF-β1-induced transformation and ECM synthesis of mouse primary lung fibroblasts
小鼠肺原代成纤维细胞的提取和培养Extraction and culture of mouse lung primary fibroblasts
将小鼠用1.2％阿佛丁(0.2ml/10g)腹腔注射麻醉，麻醉后用酒精浸泡小鼠3-5分钟，将小鼠放入超净台中，剪开腹主动脉放血，打开胸腔取出肺组织，弃掉靠近大气道的肺组织，将肺组织放入含PBS的大皿中清洗2-3遍，移入新的大皿中，将肺组织快速剪成约1mm 3碎块，用PBS清洗组织块至液体澄清，弃去PBS。往组织块中加入胰酶消化15min，将消化后的组织块和胰酶吸入滤网中过滤，用无FBS的DMEM冲洗。将肺组织移入培养瓶中，不带有残留的液体。加入含有10％FBS的DMEM 4ml，放入37℃细胞培养箱中过夜。第二天将培养瓶中的组织块吹打均匀并吸至50ml EP管中，将液体吸弃，留组织块于管中，用DMEM清洗组织块3遍，吸弃液体，往50ml EP管中加入2ml含10％FBS的DMEM，将组织块移入2个培养瓶中，吸弃液体，往培养瓶一侧瓶壁上均匀摆放组织块，使组织块黏附于瓶壁，距离瓶底下缘至少0.5cm远，往瓶中加入4ml含10％FBS的DMEM，将培养瓶垂直放入细胞培养箱中，3h后轻轻将瓶放倒，使培养基没过组织块并确保组织块仍然黏附于瓶壁上。3天后更换培养基，吸弃漂浮的组织块，贴壁组织块继续培养，7-10天后传代。 The mice were anesthetized by intraperitoneal injection of 1.2% Avertin (0.2ml/10g). After anesthesia, the mice were soaked in alcohol for 3-5 minutes. The mice were placed in the ultra-clean table, the abdominal aorta was cut open for bloodletting, and the chest cavity was opened to take out Lung tissue, discard the lung tissue near the large airway, wash the lung tissue in a large dish containing PBS for 2-3 times, transfer it into a new large dish, quickly cut the lung tissue into about 1mm3 pieces, wash the tissue with PBS Block until liquid is clear, discard PBS. Add trypsin to the tissue block for 15min digestion, suck the digested tissue block and trypsin into a filter, filter, and rinse with DMEM without FBS. Transfer the lung tissue into a culture flask without residual liquid. Add 4 ml of DMEM containing 10% FBS and place in a 37°C cell incubator overnight. The next day, the tissue pieces in the culture flask were pipetted evenly and sucked into a 50ml EP tube, the liquid was aspirated and discarded, the tissue pieces were kept in the tube, the tissue pieces were washed three times with DMEM, the liquid was aspirated, and the liquid was added to the 50ml EP tube. 2ml DMEM containing 10% FBS, transfer the tissue blocks into two culture flasks, aspirate and discard the liquid, and place the tissue blocks evenly on the side wall of the culture flask to make the tissue blocks adhere to the bottle wall, at least 0.5 from the bottom edge of the flask. cm away, add 4ml of DMEM containing 10% FBS to the bottle, put the culture bottle vertically into the cell incubator, and gently put the bottle down after 3 hours so that the medium does not cover the tissue block and ensure that the tissue block still adheres to the bottle on the wall. After 3 days, the medium was changed, the floating tissue blocks were aspirated and discarded, and the adherent tissue blocks were continued to be cultured, and passaged after 7-10 days.
细胞总蛋白的提取Extraction of total cell protein
将细胞孔板或细胞培养皿中培养基吸弃，加入PBS清洗一遍，弃去PBS，加入配好的蛋白裂解液，于冰上裂解30min。用细胞刮刮取细胞，将蛋白裂解液及细胞碎片吸入1.5ml EP管中，置于事先预冷的离心机中，4℃，12000rpm，离心30min。将上清吸至新的1.5ml EP管中，弃去沉淀，-80℃保存用于后续实验。Aspirate and discard the medium in the cell well plate or cell culture dish, add PBS to wash once, discard the PBS, add the prepared protein lysis solution, and lyse on ice for 30 min. The cells were scraped with a cell scraper, and the protein lysate and cell debris were sucked into a 1.5ml EP tube, placed in a pre-cooled centrifuge, 4°C, 12000rpm, and centrifuged for 30min. Aspirate the supernatant into a new 1.5ml EP tube, discard the pellet, and store at -80°C for subsequent experiments.
为研究BMP4基因敲除对小鼠原代成纤维细胞转化的影响。对BMP4 +/-小鼠和野生型 小鼠肺组织中提取培养的原代肺成纤维细胞(BMP4 +/-和BMP4 +/+)，予以TGF-β1处理48h，建立肺纤维化细胞模型，探究BMP4基因敲除是否影响肺纤维化结果。检测小鼠原代成纤维细胞中成纤维细胞转化标志物Fibronectin、α-SMA、Col 3、Col 1的表达。 To study the effect of BMP4 knockout on the transformation of mouse primary fibroblasts. The primary lung fibroblasts (BMP4 +/- and BMP4 +/+ ) extracted and cultured from the lung tissues of BMP4 +/- mice and wild-type mice were treated with TGF-β1 for 48 hours to establish a pulmonary fibrosis cell model. To explore whether BMP4 gene knockout affects the outcome of pulmonary fibrosis. The expressions of fibroblast transformation markers Fibronectin, α-SMA, Col 3 and Col 1 in mouse primary fibroblasts were detected.
结果result
如图10A所示，与空白对照组相比，予以TGF-β1处理的肺成纤维细胞中，Fibronectin、α-SMA、Col3、Col1的表达显著升高，BMP4 +/-组中表达高于BMP4 +/+组。且予以TGF-β1处理的肺成纤维细胞中BMP4表达降低，说明TGF-β1能抑制成纤维细胞中BMP4表达，BMP4基因敲除能加重TGF-β1诱导的小鼠原代肺成纤维细胞转化，加重肺纤维化。 As shown in Figure 10A, compared with the blank control group, the expressions of Fibronectin, α-SMA, Col3, and Col1 in lung fibroblasts treated with TGF-β1 were significantly increased, and the expression in BMP4 +/- group was higher than that in BMP4 ++ group. Moreover, the expression of BMP4 in lung fibroblasts treated with TGF-β1 decreased, indicating that TGF-β1 can inhibit the expression of BMP4 in fibroblasts, and BMP4 knockout can aggravate the transformation of primary lung fibroblasts induced by TGF-β1. exacerbate pulmonary fibrosis.
实施例6外源性加入BMP4重组蛋白能够抑制TGF-β1诱导的小鼠原代肺成纤维细胞转化和ECM的产生Example 6 Exogenous addition of BMP4 recombinant protein can inhibit the transformation and ECM production of mouse primary lung fibroblasts induced by TGF-β1
细胞爬片免疫荧光染色Immunofluorescence staining of cell slides
将细胞孔板中放入相应大小的圆形盖玻片，将细胞均匀铺入孔板中，置于培养箱培养3-5天并给药处理，从培养箱取出细胞孔板，用PBS轻轻清洗细胞表面1遍，弃去PBS，加入4％多聚甲醛并将孔板置于摇床上慢摇5-10min，以固定细胞。弃去固定液，加入PBS清洗3遍，弃去PBS。加入10％TRIT-100，摇床上慢摇15min以透化细胞膜，加入PBS清洗3遍，弃去PBS，加入3％BSA室温封闭1h。弃去BAS，夹出盖玻片移至湿盒，按一抗说明书稀释一抗，将稀释好的一抗滴至圆形盖玻片上，阴性对照组则加入PBS，将湿盒放至4度冰箱，一抗孵育过夜。第二天将湿盒取出，于室温复温约半小时，将玻片夹至细胞孔板中，加入PBS清洗3遍，弃去PBS。加入稀释好的二抗，室温避光孵育1h。加入PBS清洗3遍，弃去PBS。加入细胞核染色试剂DAPI，避光室温孵育5-8min。加入PBS清洗3遍，弃去PBS，用防淬灭封片剂封片。于荧光显微镜下对细胞染色情况进行观察并采集图像。Put a circular cover glass of the corresponding size in the cell well plate, spread the cells evenly into the well plate, place it in the incubator for 3-5 days and treat it with medication, take out the cell well plate from the incubator, lightly wash it with PBS. Gently wash the cell surface once, discard the PBS, add 4% paraformaldehyde, and place the plate on a shaker for 5-10 min to fix the cells. The fixative was discarded, PBS was added to wash three times, and the PBS was discarded. Add 10% TRIT-100, shake slowly on a shaker for 15 min to permeabilize the cell membrane, add PBS to wash 3 times, discard PBS, and add 3% BSA to block for 1 h at room temperature. Discard the BAS, remove the coverslip and move it to the wet box, dilute the primary antibody according to the instructions of the primary antibody, drop the diluted primary antibody on the round coverslip, add PBS to the negative control group, and place the wet box to 4 degrees Refrigerator, the primary antibody was incubated overnight. The next day, the wet box was taken out, rewarmed at room temperature for about half an hour, the slide was clamped into the cell well plate, PBS was added to wash three times, and the PBS was discarded. The diluted secondary antibody was added and incubated at room temperature for 1 h in the dark. Add PBS to wash 3 times, discard PBS. Add the nucleus staining reagent DAPI, and incubate at room temperature for 5-8 min in the dark. Add PBS to wash three times, discard PBS, and mount the slides with anti-quenching mounting medium. Cell staining was observed under a fluorescence microscope and images were collected.
为研究外源性BMP4对小鼠原代成纤维细胞转化和合成ECM能力的影响，对小鼠原代成纤维细胞予以TGF-β1刺激，建立纤维化细胞模型，在此基础上予以BMP4处理48h，检测细胞中成纤维细胞转化标志物Fibronectin、α-SMA、Col 1、Col 3蛋白表达变化。In order to study the effect of exogenous BMP4 on the transformation and ECM synthesis of mouse primary fibroblasts, mouse primary fibroblasts were stimulated with TGF-β1 to establish a fibrotic cell model, and on this basis, BMP4 was treated for 48 h , to detect the protein expression changes of fibroblast transformation markers Fibronectin, α-SMA, Col 1 and Col 3 in cells.
结果result
如图10B-10D所示，予以TGF-β1处理后，细胞中Fibronectin、α-SMA、Col 1、Col 3的蛋白表达显著升高，予以BMP4处理可以抑制TGF-β1所致Fibronectin、α-SMA、Col 1、Col 3的表达升高，其差别具有统计学意义。单独予以BMP4处理不影响Fibronectin、α-SMA、Col 1、Col 3蛋白表达。免疫荧光结果显示，予以TGF-β1处理后，细胞中α-SMA、Vimentin蛋白表达显著升高，予以BMP4处理可以抑制TGF-β1所致α-SMA、Vimentin蛋白表达升高。说明外源性BMP4能抑制TGF-β1诱导的小鼠原代成纤维细胞转化。As shown in Figures 10B-10D, after treatment with TGF-β1, the protein expressions of Fibronectin, α-SMA, Col 1, and Col 3 in cells were significantly increased, and BMP4 treatment could inhibit TGF-β1-induced Fibronectin, α-SMA The expression of Col 1 and Col 3 increased, and the difference was statistically significant. BMP4 treatment alone did not affect the protein expressions of Fibronectin, α-SMA, Col 1 and Col 3. Immunofluorescence results showed that the expression of α-SMA and Vimentin proteins in cells was significantly increased after TGF-β1 treatment, and BMP4 treatment could inhibit the increase of α-SMA and Vimentin protein expressions caused by TGF-β1. It indicated that exogenous BMP4 could inhibit the transformation of mouse primary fibroblasts induced by TGF-β1.
实施例7 BMP4基因敲除能够促进TGF-β1诱导的小鼠肺原代成纤维细胞增殖成纤维细胞的激活和转化是肺纤维化的主要特征。成纤维细胞的激活包括过度增殖和凋亡抵抗。我们研究BMP4对TGF-β1诱导的成纤维细胞增殖的影响。Example 7 BMP4 knockout can promote TGF-β1-induced mouse lung primary fibroblast proliferation Fibroblast activation and transformation are the main features of pulmonary fibrosis. Activation of fibroblasts includes hyperproliferation and resistance to apoptosis. We investigated the effect of BMP4 on TGF-β1-induced fibroblast proliferation.
结果result
如图11A-11C所示，小鼠原代成纤维细胞(BMP4 +/-和BMP4 +/+)予以TGF-β1处理后，小鼠原代成纤维细胞中PCNA、Survivin的表达显著升高，BMP4 +/-原代成纤维细胞中PCNA、Survivin的表达高于BMP4 +/+原代成纤维细胞，其差别具有统计学意义。说明BMP4基因敲除促进TGF-β1诱导的小鼠原代成纤维细胞的增殖。 As shown in Figures 11A-11C, after the mouse primary fibroblasts (BMP4 +/- and BMP4 +/+ ) were treated with TGF-β1, the expressions of PCNA and Survivin in mouse primary fibroblasts were significantly increased, The expression of PCNA and Survivin in BMP4 +/- primary fibroblasts was higher than that in BMP4 +/+ primary fibroblasts, and the difference was statistically significant. This indicated that BMP4 gene knockout promoted the proliferation of mouse primary fibroblasts induced by TGF-β1.
实施例8外源性加入BMP4重组蛋白能够抑制TGF-β1诱导的小鼠肺原代成纤维细胞增殖Example 8 Exogenous addition of BMP4 recombinant protein can inhibit the proliferation of mouse lung primary fibroblasts induced by TGF-β1
如图11D-11F所示，予以外源性BMP4处理能抑制TGF-β1所致PCNA、Survivin的表达升高，其差别具有统计学意义。As shown in Figures 11D-11F, treatment with exogenous BMP4 could inhibit the increase in the expressions of PCNA and Survivin caused by TGF-β1, and the difference was statistically significant.
实施例9 BMP4基因敲除抑制了TGF-β1介导的成纤维细胞的凋亡抵抗，促进成纤维细胞凋亡Example 9 BMP4 knockout inhibits TGF-β1-mediated apoptosis resistance of fibroblasts and promotes fibroblast apoptosis
为研究BMP4对肺成纤维细胞凋亡的影响，探究BMP4是否通过抑制TGF-β1，促进成纤维细胞的凋亡，发挥抗纤维化作用。我们对小鼠肺原代成纤维细胞(BMP4 +/-和BMP4 +/+)予以TGF-β1处理48h后，检测细胞凋亡情况。 In order to study the effect of BMP4 on the apoptosis of lung fibroblasts, to explore whether BMP4 can promote the apoptosis of fibroblasts by inhibiting TGF-β1 and play an anti-fibrotic effect. We treated mouse lung primary fibroblasts (BMP4 +/- and BMP4 +/+ ) with TGF-β1 for 48 hours, and then detected the apoptosis.
AnnexinV FITC细胞凋亡检测Apoptosis detection of AnnexinV FITC cells
按照AnnexinV FITC细胞凋亡检测试剂盒说明书操作，具体步骤如下：先用PBS清洗贴壁细胞1遍，用胰酶消化细胞，加含10％FBS的DMEM终止消化，吹打后将细胞悬液移入15ml EP管中，800rpm，离心5min。用预冷的PBS清洗细胞2遍，800rpm，离心5min，弃去PBS，用1×Bingding Buffer溶液重悬细胞，并行细胞计数，稀释细胞悬液，使得细胞浓度为1×10 6/ml。将100μl的细胞悬液移至流式管中，加入5μl的FITC Annexin和5μl的PI，轻轻混匀，室温下避光孵育10～15min。每管加入400μl的1×Bingding Buffer，上流式细胞仪检测。结果判断：在双变量流式细胞仪的散点图上，左下象限显示活细胞，为(FITC -/PI -)；其它象限为早期、晚期凋亡细胞和坏死细胞。 Follow the instructions of AnnexinV FITC Apoptosis Detection Kit. The specific steps are as follows: wash the adherent cells once with PBS, digest the cells with trypsin, add DMEM containing 10% FBS to stop the digestion, and pipet the cell suspension into 15ml EP tube, 800rpm, centrifugation for 5min. Wash the cells twice with pre-cooled PBS, centrifuge at 800 rpm for 5 min, discard the PBS, resuspend the cells with 1× Bingding Buffer solution, count the cells in parallel, and dilute the cell suspension to make the cell concentration 1×10 6 /ml. Transfer 100 μl of the cell suspension to a flow tube, add 5 μl of FITC Annexin and 5 μl of PI, mix gently, and incubate at room temperature in the dark for 10-15 min. Add 400 μl of 1× Bingding Buffer to each tube, and detect by flow cytometer. Judgment of results: On the scatter diagram of the bivariate flow cytometer, the lower left quadrant shows live cells, which is (FITC - /PI - ); the other quadrants are early and late apoptotic cells and necrotic cells.
结果result
使用AnnexinV FITC细胞凋亡检测试剂盒检测小鼠原代成纤维细胞予以TGF-β1处理后细胞凋亡情况，并计算凋亡率，结果显示，BMP4 +/-+TGF-β1组凋亡率最低，结果有统计学差异(n＝5，P<0.01)。说明TGF-β1抑制了小鼠原代成纤维细胞的凋亡，促进了细胞凋亡抵抗。BMP4能抑制TGF-β1介导的成纤维细胞的凋亡抵抗，促进成纤维细胞凋亡。 The AnnexinV FITC apoptosis detection kit was used to detect the apoptosis of mouse primary fibroblasts treated with TGF-β1, and the apoptosis rate was calculated. The results showed that the BMP4 +/- +TGF-β1 group had the lowest apoptosis rate. , the results were statistically different (n=5, P<0.01). It indicated that TGF-β1 inhibited the apoptosis of mouse primary fibroblasts and promoted apoptosis resistance. BMP4 can inhibit the apoptosis resistance of fibroblasts mediated by TGF-β1 and promote the apoptosis of fibroblasts.
应该理解的是，尽管已经通过优选实施方式和任选的特征具体公开了本发明，但是本领域技术人员可以对本文所公开的本发明进行修改、改进和变化，这些修改、改进和变化被认为在本发明的范围内。在此提供的材料、方法和实施例是优选的实施方式的代表，是示例性的，并且不旨在作为对本技术的范围的限制。It is to be understood that although the present invention has been specifically disclosed in terms of preferred embodiments and optional features, modifications, improvements and variations of the invention disclosed herein may be made by those skilled in the art, such modifications, improvements and variations being considered within the scope of the present invention. The materials, methods, and examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present technology.

Claims (19)

  1. BMP4蛋白在制备用于治疗或预防对象的纤维增生性疾病的药物中的应用。Use of BMP4 protein in the preparation of a medicament for treating or preventing fibroproliferative diseases in a subject.
  2. 根据权利要求1所述的应用，其中所述BMP4蛋白是游离的BMP4蛋白或表达BMP4蛋白的重组载体。The use according to claim 1, wherein the BMP4 protein is free BMP4 protein or a recombinant vector expressing BMP4 protein.
  3. 根据权利要求2所述的应用，其中表达BMP4蛋白的重组载体包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。The use according to claim 2, wherein the recombinant vector expressing the BMP4 protein comprises a vector comprising or carrying a polynucleotide encoding the BMP4 protein.
  4. 根据权利要求1至3任一项所述的应用，其中所述BMP4蛋白的氨基酸序列包含如SEQ ID NO:1至SEQ ID NO:3所示序列中的任何一个序列或其变体。The use according to any one of claims 1 to 3, wherein the amino acid sequence of the BMP4 protein comprises any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3 or a variant thereof.
  5. 根据权利要求3所述的应用，其中所述多核苷酸是编码BMP4蛋白的DNA、RNA、或DNA-RNA杂交体。The use of claim 3, wherein the polynucleotide is a DNA, RNA, or DNA-RNA hybrid encoding a BMP4 protein.
  6. 根据权利要求1或5所述的应用，其中编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:4至SEQ ID NO:9所示序列中的任何一个序列或其变体。The use according to claim 1 or 5, wherein the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences shown in SEQ ID NO: 4 to SEQ ID NO: 9 or a variant thereof.
  7. 根据权利要求1或5所述的应用，其中编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:10至SEQ ID NO:27所示序列中的任何一个序列或其变体。The use according to claim 1 or 5, wherein the sequence of the polynucleotide encoding the BMP4 protein comprises any one of the sequences shown in SEQ ID NO: 10 to SEQ ID NO: 27 or a variant thereof.
  8. 根据权利要求1或5所述的应用，其中编码BMP4蛋白的多核苷酸的序列包含如SEQ ID NO:28或SEQ ID NO:29所示的序列或其变体。The use according to claim 1 or 5, wherein the sequence of the polynucleotide encoding the BMP4 protein comprises the sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29 or a variant thereof.
  9. 根据权利要求3所述的应用，其中所述载体是病毒载体或非病毒载体。The use according to claim 3, wherein the vector is a viral vector or a non-viral vector.
  10. 根据权利要求9所述的应用，其中所述非病毒载体选自：质粒、脂质体、逆转录元件、转座子、附加型载体、阳离子多聚物、壳聚糖聚合物、无机纳米粒子和外泌体。The use according to claim 9, wherein the non-viral vector is selected from the group consisting of: plasmids, liposomes, retroelements, transposons, episomal vectors, cationic polymers, chitosan polymers, inorganic nanoparticles and exosomes.
  11. 根据权利要求9所述的应用，其中所述病毒载体选自：逆转录病毒、腺病毒、腺相关病毒、单纯性疱疹病毒、牛痘病毒、杆状病毒和慢病毒。The use according to claim 9, wherein the viral vector is selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, baculovirus and lentivirus.
  12. 根据权利要求11所述的应用，其中所述病毒载体是腺相关病毒。The use of claim 11, wherein the viral vector is an adeno-associated virus.
  13. 根据权利要求1所述的应用，其中所述对象是人。The use of claim 1, wherein the subject is a human.
  14. 根据权利要求1所述的应用，其中所述纤维增生性疾病选自：肺、心血管***、肝、肾、眼、神经***、骨髓和皮肤的纤维增生性疾病。The use according to claim 1, wherein the fibroproliferative disease is selected from the group consisting of fibroproliferative diseases of the lung, cardiovascular system, liver, kidney, eye, nervous system, bone marrow and skin.
  15. 根据权利要求14所述的应用，其中所述纤维增生性疾病选自：矽肺、石棉肺、煤肺、农民肺、特发性间质性肺炎、闭塞性细支气管炎伴机化性肺炎、特发性肺纤维化、进行性***硬化症、淋巴细胞间质性肺炎、家族性肺纤维化、心肌梗死后的替代性和间质性纤维化、乙、丙和丁型肝炎病毒性肝炎、营养不良性肝硬化、慢性肾小球肾炎、慢性肾盂肾炎、阻塞性肾病、***性红斑狼疮性肾病、眼睛外伤和手术后眼睛纤维增生性疾病、脊髓外伤后纤维增生性疾病、特发性和药物引起的骨髓纤维化、口腔粘膜纤维化。The use according to claim 14, wherein the fibroproliferative disease is selected from the group consisting of: silicosis, asbestosis, coal lung, farmers' lung, idiopathic interstitial pneumonia, bronchiolitis obliterans with organizing pneumonia, idiopathic interstitial pneumonia Advanced pulmonary fibrosis, progressive systemic sclerosis, lymphocytic interstitial pneumonia, familial pulmonary fibrosis, replacement and interstitial fibrosis after myocardial infarction, hepatitis B, C and D viral hepatitis, nutrition Adverse cirrhosis, chronic glomerulonephritis, chronic pyelonephritis, obstructive nephropathy, systemic lupus erythematosus nephropathy, ocular trauma and post-operative fibroproliferative disorders of the eye, post-spinal cord trauma fibroproliferative disorders, idiopathic and drug Myelofibrosis and oral mucosal fibrosis.
  16. 根据权利要求14所述的应用，其中所述纤维增生性疾病是肺纤维增生性疾病。The use according to claim 14, wherein the fibroproliferative disease is pulmonary fibroproliferative disease.
  17. 根据权利要求16所述的应用，其中所述肺纤维增生性疾病是特发性肺纤维化。The use according to claim 16, wherein the pulmonary fibroproliferative disease is idiopathic pulmonary fibrosis.
  18. 一种表达BMP4蛋白的重组载体，其包含载体，所述载体包含或携带编码BMP4蛋白的多核苷酸。A recombinant vector for expressing BMP4 protein, which comprises a vector comprising or carrying a polynucleotide encoding BMP4 protein.
  19. 一种药物组合物，其包含权利要求18所述的重组载体和药学上可接受的赋形剂。A pharmaceutical composition comprising the recombinant carrier of claim 18 and a pharmaceutically acceptable excipient.
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