WO2024125641A1 - Series of polypeptides/proteins and uses thereof - Google Patents

Series of polypeptides/proteins and uses thereof Download PDF

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Publication number
WO2024125641A1
WO2024125641A1 PCT/CN2023/139218 CN2023139218W WO2024125641A1 WO 2024125641 A1 WO2024125641 A1 WO 2024125641A1 CN 2023139218 W CN2023139218 W CN 2023139218W WO 2024125641 A1 WO2024125641 A1 WO 2024125641A1
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seq
polypeptide
group
skin
amino acid
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PCT/CN2023/139218
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French (fr)
Chinese (zh)
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陈敏
赵俊
刘维达
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陈敏
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention belongs to the technical field of biomedicine and skin care products, and specifically relates to a series of amino acid sequences, polypeptides, proteins and compositions comprising the amino acid sequences, nucleic acid sequences encoding the amino acid sequences, polypeptides, proteins and compositions, and applications of products comprising the amino acid sequences, polypeptides, proteins, compositions or nucleic acid sequences in the field of skin disease prevention and treatment.
  • Skin is the most external organ of the human body and can reflect the health and aging of the body.
  • the occurrence of skin aging is believed to be the result of the combined action of internal and external factors. Skin aging is often manifested as thinning of the epidermis, shrinkage and reduction of dermis, thinning of collagen fibers, and even disordered arrangement and breakage.
  • the decline in cell proliferation ability is an important feature of aging. Aged cells often show a trend of declining proliferation ability due to decreased protein synthesis ability and dysregulated expression of immune system-related genes.
  • the sebum membrane is a transparent film covering the surface of the skin. It is mainly formed by the emulsification of sebum secreted by sebaceous glands, lipids produced by the disintegration of stratum corneum cells and sweat secreted by sweat glands. It is weakly acidic. Its main components are squalene, linoleic acid, linolenic acid and lipid components, which have the function of locking moisture and having certain anti-inflammatory effects.
  • the skin barrier is composed of sebum membrane, stratum corneum keratin, lipids, "sandwich” structure, brick wall structure, dermal mucopolysaccharides, mucopolysaccharides, etc., which resist the entry of harmful external factors, irritants and sunlight, and have moisturizing and regulating anti-inflammatory effects. Damage to the skin barrier will cause dry skin, skin aging, pigmentation, dermatitis, etc. Tissue repair includes wound healing, repair of skin barrier function, etc.
  • the restoration of skin barrier function is also beneficial for preventing and treating local bacterial infections of the skin, assisting in the treatment of acute and chronic skin inflammation, and preventing the recurrence of skin inflammation, including seborrheic dermatitis, glucocorticoid-dependent dermatitis, eczema, atopic dermatitis and psoriasis.
  • Alopecia areata is a non-scarring hair loss. Epidemiological surveys in my country show that the prevalence of androgenic alopecia in men is 21.3% and in women is 6.0%. Alopecia areata usually presents as sudden hair loss patches. In severe cases, it may involve the entire scalp, which is called alopecia totalis (AT). When it involves all the hair on the body, including axillary hair and pubic hair, it is called alopecia universalis (AU), which can easily have a serious impact on the patient's appearance and psychology. Abnormal or unstable autoimmune function and neuropsychiatric factors are considered to be important related factors. The earlier the treatment of alopecia areata is initiated, the higher the chance of cure.
  • Minoxidil can promote skin vasodilation, improve local blood circulation, and promote hair growth. It is a common topical drug for the treatment of alopecia areata. Glucocorticoids are commonly used for severe alopecia areata, mainly including prednisolone, compound betamethasone, etc., which can be taken orally, applied topically, or injected intradermally. For patients who are not suitable for glucocorticoid drugs, immunosuppressants can be used for treatment. Common drugs include cyclosporine and methotrexate. Glucocorticoids and immunosuppressants have many side effects.
  • AGA Androgenic alopecia
  • SA seborrheic alopecia
  • male pattern baldness or hereditary alopecia is an androgen-dependent hereditary hair loss.
  • Male pattern baldness often presents a horseshoe-shaped appearance. The skin at the site of hair loss is shiny, the pores are reduced or a few fine and soft hairs remain. The speed, range and severity of hair loss are affected by genetics and individuals. Women often experience diffuse hair loss on the top of the head. The etiology and pathogenesis of androgenic alopecia are still unclear. It is generally believed that androgens and their receptors play a key role in the occurrence of this disease.
  • androgens in the body Under normal physiological conditions, androgens in the body have a certain stimulating and promoting effect on the growth and development of hair, but can induce hair loss in certain specific parts; testosterone is the main androgen in the body, which is converted into dihydrotestosterone by 5 ⁇ -reductase, which can cause the transformation of terminal hair to vellus hair, ultimately leading to hair loss. Androgenetic alopecia is a difficult-to-treat type of hair loss disease, and the animal model of the disease is usually used as a representative model of hair loss diseases.
  • Minoxidil is a non-specific drug for the treatment of hair loss and is a first-line topical drug approved by the FDA for the treatment of hair loss.
  • Finasteride is a type II 5 ⁇ -reductase selective inhibitor.
  • the FDA approves oral finasteride for the treatment of androgenic alopecia, which can continuously improve hair growth.
  • finasteride has adverse reactions such as causing sexual dysfunction, transient sperm reduction, and abnormal male breast development. It has been found to have teratogenic effects in animal experiments, so it is not suitable for children and women of childbearing age. Cimetidine needs to be taken for 5 months or longer, and the side effects are male breast development, impotence, and decreased libido.
  • Oral contraceptives The main ones are sogogestrel, levonorgestrel (levonorgestrel), norgestrel, norethindrone, norgestimate (norgestrel oxime), diester norgestrel and norgestrel acetate, etc., which are often used to treat AGA in women. The hair will improve after 6 to 12 months of treatment.
  • Hair loss caused by anti-tumor drugs is the most common type of growth aging alopecia. While anti-tumor drugs eliminate rapidly dividing cancer cells, they also attack rapidly dividing cells around hair follicles, causing hair loss. The above hair loss treatments are difficult and prone to recurrence. Therefore, it is necessary to find more safe and effective products for treating hair loss.
  • Acne is commonly known as pimples, and is a chronic inflammatory disease that is prone to occur in the hair follicle sebaceous glands, with an incidence rate of about 9.4%, which is more common in adolescence.
  • Clinical manifestations mainly include acne, papules, pustules, nodules, cysts, scars, etc., and the healing time is long, which has a serious impact on the patient's appearance and psychology.
  • Acne is related to multiple pathogenesis, and abnormal keratinization of the hair follicle mouth is an important basis for the onset of this disease, and inflammation and infection are the pathogenic factors of acne.
  • the sebaceous glands of acne patients are larger, and the secretion of sebaceous glands increases.
  • the level of linoleic acid in sebum is relatively reduced, which affects the synthesis of fat, resulting in a lack of fatty acids in the follicular epithelium, thereby inducing excessive keratinization of the follicles, so that epithelial cells cannot fall off normally, the hair follicle sebaceous gland mouth is excessively reduced, and sebum cannot be discharged smoothly, forming acne, and secondary infection forms papules, pustules, nodules, cysts, scars, etc.
  • the human body contains a variety of collagen, accounting for 1/3 of the total protein in the human body, which is responsible for the normal functioning of the human body.
  • the skin is the largest organ in the human body, and 80% of the dermis is collagen, mainly type I and type III collagen. With age, the total amount of collagen tends to decrease. Modern medicine has proven that the essence of aging is the continuous and accelerated loss of soft and elastic type III collagen, which cannot be regenerated after adulthood. Therefore, decreased skin elasticity, sagging, and reduced repair effect are one of the reasons for human skin aging.
  • Type III collagen has the functions of promoting cell proliferation, increasing cell activity, helping the body repair aging and damaged skin, and playing an anti-aging and wound repair role.
  • Natural collagen molecules form a special superhelical structure, consisting of three polypeptide chains that are not formed by intrachain hydrogen bonds but are only supported by interchain hydrogen bonds. This helical structure is a left-handed helix with three amino acid residues as the basic repeating body.
  • Kotch and Raines synthesized a polypeptide with the characteristic sequence of (Gly-XY) n collagen by chemical method, which can successfully form a triple helical structure through self-assembly.
  • Recombinant humanized collagen synthesized by genetic engineering technology has the advantages of low cost and high efficiency, and can be industrialized and promoted through bacterial fermentation technology, attracting more and more researchers' attention.
  • Recombinant humanized collagen is a re-optimized gene sequence based on the characteristics and main functional domains of human collagen, and the expressed target protein has similar structural characteristics to human collagen.
  • recombinant humanized collagen Compared with animal collagen extracted by traditional extraction methods, recombinant humanized collagen has the advantages of high yield, batch stability, strong water resistance, good biocompatibility, and no immune virus risk, making it an ideal biomedical material.
  • the present invention discloses an amino acid sequence with multiple functions, and utilizes DNA recombination technology combined with biosynthesis technology to produce polypeptides or recombinant collagen, which well guarantees the biological activity of polypeptides/proteins and enhances multiple functions. It not only ensures the safety of medical applications, but also has more stable performance in biocompatibility, bioabsorbability, and cell adhesion.
  • extension sequence includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, which is at least 70% homologous to any of the aforementioned extension sequences and has the same or similar functions, extension sequences, fragments or mutants,
  • the mutant has the same amino acid sequence as the amino acid sequence (for example, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22).
  • the mutant is an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% homologous to the amino acid sequence.
  • the present invention provides a product.
  • the product includes any one of the amino acid sequences, for example, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22.
  • the product is a polypeptide or a protein.
  • polypeptide is A+ polypeptide
  • protein is A+ polypeptide recombinant human type III collagen.
  • polypeptide comprises an amino acid sequence (e.g., SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22), it may contain additional amino acids at its N and/or C-terminus beyond the reference sequence, for example, the polypeptide may contain additional amino acids at its N-terminus. Likewise, where the polypeptide comprises a fragment, mutant or derivative of the amino acid sequence according to the reference sequence, it may comprise additional amino acids at its N- and/or C-terminus.
  • amino acid sequence e.
  • polypeptide may comprise or consist of a mutant or a fragment of the mutant according to the amino acid sequence of the reference sequence, and such a mutant may not exist naturally.
  • polypeptide or protein also comprises immunoglobulin.
  • the present invention provides a nucleic acid sequence.
  • nucleic acid sequence encodes the amino acid sequence of any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, or encodes the polypeptide or protein.
  • the present invention provides a composition.
  • composition includes an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, the polypeptide, the protein, the nucleic acid sequence, a derivative of the amino acid sequence/polypeptide/protein/nucleic acid sequence or a pharmaceutically acceptable salt thereof.
  • the derivatives are obtained by replacing hydrogen atoms, hydroxyl groups, carboxyl groups, and imino groups in the polypeptide with known substituents.
  • Available salts are not particularly limited, and examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic, neutral, or acidic amino acids.
  • the polypeptide or protein preparation method of the present invention can adopt the commonly used methods in the prior art, such as the well-known chemical synthesis method or biological synthesis method or Synthesis by genetic engineering, etc.
  • the method for synthesizing the polypeptide or protein of the present invention belongs to a biosynthesis method based on genetic engineering technology, including a prokaryotic or eukaryotic host cell under conditions suitable for expressing the nucleic acid encoding the polypeptide, and recovering the polypeptide or protein from the host cell, wherein the host cell is prokaryotic or eukaryotic.
  • composition is composed of the amino acid sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SE Q ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, the polypeptide, the protein, the nucleic acid sequence, the amino acid sequence/polypeptide/protein/nucleic acid sequence mutant, the amino acid sequence/polypeptide/protein/nucleic acid sequence fusion, the amino acid sequence/polypeptide/protein/nucleic acid sequence derivative or its pharmaceutically acceptable salt is the active ingredient or main active ingredient, supplemented by a
  • the pharmaceutically acceptable carrier is selected from at least one of water, physiological saline, a physiologically compatible buffer, a physiologically compatible solution and a physiologically compatible suspension.
  • mutants of the polypeptide include insertions, deletions and substitutions, which are either conservative or non-conservative.
  • Conservative substitutions refer to the substitution of an amino acid within the same general class (e.g., acidic amino acids, basic amino acids, non-polar amino acids, polar amino acids, or aromatic amino acids) with another amino acid within the same class. The meanings of conservative and non-conservative amino acid substitutions are well known in the art.
  • polypeptide or protein or composition can be prepared as a solution, tincture, spirit, liniment, spray, emulsion, paste, gel, patch, aqueous solution, tablet, granule, oral solution, capsule, pill, enema, film, injection or other pharmaceutical dosage forms.
  • polypeptide or protein or composition can be used alone or in combination with one or more products.
  • the present invention provides a method for preparing the polypeptide or protein.
  • the method comprises: culturing a host cell, and then recovering the polypeptide or protein from the host cell, wherein the host cell is prokaryotic or eukaryotic.
  • the present invention proposes the use of the amino acid sequence, polypeptide, protein, nucleic acid sequence and composition in the preparation of skin products.
  • the products include but are not limited to medicines, medical devices, health products or skin care products.
  • the uses of the product include but are not limited to anti-aging, anti-inflammatory, tissue repair, hair growth, scar removal and acne removal products.
  • polypeptide or protein or composition of the present invention can be prepared into any pharmaceutically acceptable dosage form, for example, a preparation suitable for external use on the skin, oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intrafatty, intraarticular, intraperitoneal or intrathecal administration.
  • the polypeptide or protein or composition of the present invention is in the form of a solution, tincture, spirit, liniment, spray, emulsion, paste, gel, patch, aqueous solution, tablet, granule, oral solution, capsule, pill, enema, film or injection.
  • the present invention provides a method for treating or preventing skin diseases.
  • the method comprises administering a therapeutically effective amount of a product comprising the amino acid sequence, polypeptide, protein or composition.
  • the skin diseases include but are not limited to skin aging, skin inflammation, skin damage, hair loss, scars and acne.
  • the product is administered to humans.
  • the A+ polypeptide referred to in the present invention refers to a polypeptide comprising but not limited to an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, or a fragment or mutant thereof.
  • the A+ polypeptide recombinant human type III collagen referred to in the present invention refers to but is not limited to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ Recombinant human type III collagen having an amino acid sequence shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, a fragment thereof or a mutant thereof.
  • amino acid sequence or its mutant described in the present invention can significantly repair the skin barrier, and has significant effects in anti-aging, anti-inflammation, tissue repair, hair growth, scar removal and acne removal, and is safe with few side effects.
  • the series A+ polypeptides including this amino acid sequence have high biological activity and good anti-aging, anti-inflammatory, tissue repair, hair growth, scar removal and acne removal effects. They are superior to most of the currently known polypeptide/protein products, are safe, stable, biocompatible, and have low production costs. They are particularly suitable for the field of functional skin care product raw materials and are suitable for the production of a variety of functional skin care products that can be used for a long time.
  • A+ polypeptide is integrated with the human collagen synthesis system.
  • the A+ recombinant type III collagen synthesized by genetic engineering technology can significantly enhance the efficacy of the A+ polypeptide series, including but not limited to anti-aging, anti-inflammation, tissue repair, hair growth, scar removal and acne removal.
  • Controllable quality The purification method of recombinant human type III collagen is to heat-treat at 65-75°C, and the fermentation broth obtained by fermentation of recombinant yeast with the ability to express recombinant human type III collagen effectively prevents the degradation of recombinant human type III collagen in the fermentation broth into small molecular peptides, and reduces the production of endotoxins in the fermentation broth, greatly improving the purity of recombinant human type III collagen.
  • Genetic engineering technology can express human-like collagen fragments of specific molecular weight, with good reproducibility between batches, while collagen extracted from animals is usually a mixed product of different types of collagen, with large differences between batches, which is not conducive to quality control; chemical synthesis cannot guarantee the biological activity of polypeptides/proteins.
  • Figure 1 SDS-PAGE detection of the expression of A+ recombinant collagen engineering bacteria. Lane 1: before induction; Lanes 2-4: expression of 3 different recombinant collagen engineering bacteria (diluted 2 times).
  • Figure 2 SDS-PAGE detection of the expression form of A+ polypeptide 3 recombinant collagen, lane 1: whole expression bacteria; lane 2: ultrasonic supernatant; lane 3: ultrasonic precipitate.
  • Figure 3 SDS-PAGE detection of A+ polypeptide 3 recombinant collagen purification results, lane 1: purification flow-through; lane 2: after purification (10-fold dilution); lane 3: after purification (20-fold dilution).
  • Figure 5 72h culture sample microscopy observation shows that the number of 3T3 cells in the A+ polypeptide 3 test group is more than that in the negative control group when compared with that at 0h (100 ⁇ ).
  • Figure 6 72h culture sample under microscopy, the negative control group 3T3 cells were mostly dead compared with 0h cells (100 ⁇ ).
  • Figure 7 Cell migration at 0h and 24h in the blank control group (NC), fibronectin group (FN) and experimental groups (40 ⁇ ):
  • the cell migration rate of the FN group was 100%, and the cell migration rates of the A+ polypeptide 3 recombinant collagen group, A+ polypeptide 1 group and A+ polypeptide 3 group were significantly higher than those of the blank control group, and the cell migration rate of the A+ polypeptide 3 recombinant collagen group was significantly higher than that of the type III collagen group.
  • Figure 8 shows the hair growth of mice in each group on the 45th day after modeling.
  • the hair growth of A+ polypeptide 3 recombinant collagen group, A+ polypeptide 1 and A+ polypeptide 3 groups was faster than that of the model group, and there was no significant difference compared with the negative control; the hair growth of A+ polypeptide 3 recombinant collagen group was significantly faster than that of the collagen group.
  • the immunohistochemistry results in Figure 9 showed that the expression of VEGF protein in the skin hair follicles of the model group was reduced.
  • the expression of VEGF protein in the A+ polypeptide 3 recombinant collagen group, A+ polypeptide 1 group, A+ polypeptide 2 group, A+ polypeptide 3 group, A+ polypeptide 4 group, A+ polypeptide 5 group, A+ polypeptide 6 group and A+ polypeptide 7 group was significantly increased, and there was no significant difference compared with the negative control; the expression of VEGF protein in the A+ polypeptide 3 recombinant collagen group was significantly higher than that in the collagen group.
  • Figure 10 An acne patient's erythema, papules and comedones improved after 8 weeks of treatment with 0.5% A+ polypeptide 3 solution.
  • Figure 11 A patient with moderate acne had significant improvement in papules, pustules and comedones after 8 weeks of treatment with 0.5% A+ polypeptide 9 recombinant collagen solution.
  • Figure 12 A patient with moderate acne showed significant improvement in papules, pustules and comedones after 4 weeks of treatment with 0.5% A+ polypeptide 18 recombinant collagen solution.
  • test materials used in the present invention were purchased from conventional biochemical reagent stores.
  • E.coli DH5 ⁇ competent cells BL21 (DE3.0) competent cells, BALB/c 3T3 cells; plasmids were synthesized by General Biotechnology; fermentation medium was purchased from OXOID.
  • -80°C ultra-low temperature refrigerator clean bench, constant temperature incubator, constant temperature shaker, high pressure sterilizer, nucleic acid electrophoresis instrument, protein electrophoresis instrument, metal bath, PCR instrument, water bath, gel imager, desktop centrifuge, refrigerated centrifuge, high pressure homogenizer, GE purifier, ice maker, desktop centrifuge, carbon dioxide incubator, etc.
  • a functional region fragment of type III human collagen (COL3A1, NCBI, GenBank: AGL34959.1) with a total of 30 amino acids was selected, namely, amino acids 483 to 512 of type III alpha-1 subtype of human collagen, SEQ ID NO.23: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.
  • the amino acid sequence of the recombinant type III humanized collagen fusion protein includes (483G-512G) ⁇ n.
  • n represents the amino acids 483 to 512 of human collagen type III alpha-1 subtype directly and continuously repeated n times, and n is an integer greater than or equal to 1.
  • Synthesized recombinant type III humanized collagen fusion protein, n 13, specific sequence: SEQ ID NO.24:
  • the fusion protein plasmids were transformed into E.coli DH5 ⁇ competent cells, and positive transformants were selected for culture after resistance screening. The plasmids were then extracted for sequencing. The correct sequencing proved that the expression plasmid was successfully obtained.
  • the engineered bacteria showed red bacilli, with a size of about 0.3 ⁇ m ⁇ 1.0 ⁇ m.
  • the engineered bacteria can grow normally on LB plates containing corresponding resistances, and after culturing at 37°C for 24h, they form round, smooth, protruding, milky white and shiny colonies, which are typical Escherichia coli colony morphology.
  • Ferment 2L induce expression, collect the cells, homogenize under high pressure, collect the supernatant by centrifugation, filter, and purify by affinity chromatography to obtain the target A+ recombinant human type III collagen fusion protein with a purity greater than 95% (see Figure 3).
  • the A+ polypeptide recombinant human type III collagen fusion protein engineering bacteria were successfully constructed, and the A+ polypeptide recombinant human type III collagen fusion protein was successfully induced to express. After expression form detection, the target protein was expressed in the supernatant with a purity greater than 95%.
  • A+ polypeptide series and DZ polypeptide series synthesized by Shanghai Biotech, with a purity greater than 95%.
  • the amino acid sequences of the A+ polypeptide series are as shown in the above invention content, and the representative hydrogen spectrum analysis results of the A+ polypeptide are shown in Figure 4.
  • DZ polypeptide 1 EYPYKHSGYYHR (SEQ ID NO.25)
  • DZ polypeptide 2 EYTYEGAGYYHRP (SEQ ID NO.26)
  • DZ polypeptide 3 EYTYNGAGYYHR (SEQ ID NO.27)
  • DZ polypeptide 4 EYTYKGSGYYHR (SEQ ID NO.28)
  • DZ polypeptide 5 EYPWKGSGYYHRP (SEQ ID NO.29)
  • DZ polypeptide 6 EYTFKHSGYYHRP (SEQ ID NO.30 )
  • DZ polypeptide 7 EYPYDYSGYYHRP (SEQ ID NO.31)
  • DZ polypeptide 8 EFPFKWSGYYHR (SEQ ID NO.32)
  • DZ polypeptide 9 EYAFAGAGYYHRP (SEQ ID NO.33)
  • DZ polypeptide 10 EFPWPHAGYYHRP (SEQ ID NO.34)
  • Example 1 Synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); A-alcohol: also known as retinol (produced by Nanjing Zhongyi Xuyuan Biotechnology Co., Ltd.); Fibronectin: produced by Wuhan Aimejie Technology Co., Ltd.
  • the fibroblast proliferation experiment is a relatively mature and effective means of evaluating the anti-wrinkle and firming efficacy of the skin in vitro.
  • the experiment designed in the present invention detects the effect of the A+ polypeptide on promoting the proliferation of cells by promoting the proliferation of 3T3 cells.
  • Retinol retinol
  • Retinol is a well-recognized ingredient with good anti-aging effect in the list of ingredients that can be added to skin care products approved by the State Food and Drug Administration, and is used as a positive control in this experiment.
  • the proliferation activity of skin fibroblasts was determined by MTT colorimetric method.
  • Fibroblast BALB/C 3T3 cell line was cultured in DMEM medium, and cell activity was detected for 72 hours. Only a few cells survived in the negative control wells, while the number of cells in the A+ polypeptide group increased, as shown in Figures 5 and 6.
  • the results of the detection of the OD values of the series A+ polypeptides, series DZ polypeptides, human type III collagen, A+ recombinant collagen and experimental groups are shown in Table 1.
  • the results of cell proliferation activity are shown in Table 2 below.
  • the series of A+ polypeptides, DZ polypeptides and A+ polypeptide recombinant collagen can inhibit fibroblast aging, promote fibroblast proliferation, and have anti-aging effects.
  • the series of A+ polypeptides inhibit fibroblast aging and promote fibroblast proliferation significantly better than the series of DZ polypeptides; the combination of A+ polypeptide and type III collagen recombinant can significantly improve the anti-aging effect; fibronectin has almost no effect on promoting fibroblast proliferation.
  • the experimental results show that the series of A+ polypeptides and A+ polypeptide recombinant human type III collagen disclosed in the present invention have a strong effect on promoting fibroblast proliferation.
  • the combination of A+ polypeptide and human type III collagen can significantly enhance the proliferation of fibroblasts and has anti-aging, anti-wrinkle and firming effects.
  • fibronectin produced by Wuhan Aimejie Technology Co., Ltd.
  • a alcohol also known as retinol (produced by Nanjing Zhongyi Xuyuan Biotechnology Co., Ltd.)
  • series A+ polypeptides and series DZ polypeptides synthesized by Shanghai Shenggong, with a purity greater than 95%.
  • the amino acid sequence of the series A+ polypeptides is as shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4; the amino acid sequence of the series DZ polypeptides is as shown in Example 2, and is used as the experimental control group in the following experiments; A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1).
  • Cell line Balb/c 3T3 cells were purchased from ATCC, USA.
  • Average scratch width scratch gap area / length
  • the in vitro cell migration rate was calculated (see Table 3). It can be seen that the migration rates of Balb/c 3T3 cells in the experimental groups of series A+ polypeptide, series DZ polypeptide, human type III collagen, A+ recombinant collagen 1 and A+ recombinant collagen 3 were all higher than those in the negative control group, with statistical differences (all P ⁇ 0.01). There was no statistical difference in the migration rate of Balb/c 3T3 cells between A+ and the negative control group.
  • the series A+ polypeptide was higher than the series DZ polypeptide group, with statistical differences (P ⁇ 0.05).
  • the A+ polypeptide recombinant collagen group was higher than the type III collagen group, with statistical differences (P ⁇ 0.01).
  • the fibroblast migration or migration experiment is a relatively mature and effective means of evaluating tissue repair and reconstruction functions in vitro.
  • Fibronectin has a strong effect of promoting cell migration and tissue repair, and is used as a positive control in this experiment.
  • A+ polypeptide is conducive to cell recognition and migration, thereby playing a role in tissue repair and reconstruction.
  • a blank area is artificially created on the fused monolayer cells, called a "scratch".
  • the cells at the edge of the scratch will gradually enter the blank area to heal the "scratch". Images are captured at the beginning and regularly during the cell migration process, and the cell migration rate is determined by comparing the images.
  • the experimental results show that the series A+ polypeptides and A+ polypeptide recombinant human type III collagen have the ability to promote fibroblast migration, and the combination of A+ polypeptide and human type III collagen can significantly improve tissue repair function.
  • Series A+ polypeptides and series DZ polypeptides synthesized by Shanghai Biotech, with a purity greater than 95%.
  • the amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4.
  • the amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
  • A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1).
  • Fibronectin (FN): produced by Wuhan Aimijie Technology Co., Ltd.
  • Ovalbumin (OVA): Prepared at 20 g/L in PBS and stored at -20°C.
  • Calcipotriol ointment (Dalixi ointment): a product of LEO Pharma Ltd. of Denmark.
  • IL-4 ELISA kit purchased from Raybiotech, USA.
  • PCSK9 ELISA kit Human Proprotein Convertase 9/PCSK9 Quantikine ELISA Kit from American R&D company.
  • the base components include methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), octadecyl alcohol (5%), glycerol (20%), alkyl aryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl paraben (0.1%), distilled water (about 31-55%), and form mixed emulsions of different concentrations with an appropriate amount of polypeptide solution.
  • the cream base used in this example refers to the base component of the polypeptide cream without the active ingredient.
  • A+ polypeptide No. 10 cream A+ polypeptide group 8 (skin applied 1% A+ polypeptide No. 11 cream), A+ polypeptide group 9 (skin applied 1% A+ polypeptide No. 12 cream), A+ polypeptide group 10 (skin applied 1% A+ polypeptide No. 13 cream), A+ polypeptide group 11 (skin applied 1% A+ polypeptide No. 15 cream), A+ polypeptide group 12 (skin applied 1% A+ polypeptide No. 16 cream), A+ polypeptide group 13 (skin applied 1% A+ polypeptide No. 17 cream), A+ polypeptide group 14 (skin applied 1% A+ polypeptide No.
  • A+ polypeptide group 15 skin applied 1% A+ polypeptide No. 20 cream
  • A+ polypeptide group 16 skin applied 1% A+ polypeptide No. 21 cream
  • series DZ polypeptide groups 0.25% DZ polypeptide series creams were applied to the skin
  • Modeling 14.3ul of 75% ethanol was applied to both ears of mice in the negative control group. At the same time, 14.3ul of 1nmoI/L calcipotriol ointment was applied to both ears of mice in other groups on a regular basis every day. After air-drying, 25ul of 20g/L OVA was applied once a day for 12 consecutive days to establish the model.
  • the cream base was applied on the ear skin of mice in the blank control group and the model group, the ear skin of mice in the positive drug group was applied with eloson, and the ear skin of mice in the type III collagen group, fibronectin group, each polypeptide group and A+ polypeptide recombinant type III collagen group was applied with the corresponding cream once a day for 10 consecutive days. Pictures were taken every day for scoring.
  • mice were randomly tested with a skin tester, and the transepidermal water loss (TEWL) values were recorded and the average value was taken.
  • TEWL transepidermal water loss
  • the thickness of the mouse auricle was measured and recorded with an ear thickness meter before modeling and on day 14. After the measurement was completed on day 14, the mice were killed by dislocation of the neck, and blood was collected to separate serum and plasma.
  • the ELISA plate was coated with rabbit anti-mouse interleukin (IL)-4 antibody, incubated at 4°C overnight, stained and the reaction was terminated to detect serum IL-4 levels.
  • concentration of PCSK9 in plasma was determined according to the ELISA kit instructions.
  • Serum IL-4 concentration The serum IL-4 levels in the peripheral blood of mice in each group are shown in Table 6. There was no statistically significant difference between the model group and the collagen group and fibronectin group (P>0.05). Except for the collagen group and fibronectin group, the levels of serum IL-4 in other groups were significantly lower than the model group (all P ⁇ 0.01), the levels of serum IL-4 in each A+ polypeptide group were significantly lower than the series of DZ polypeptide groups (all P ⁇ 0.05), and the levels of serum IL-4 in the A+ polypeptide recombinant collagen group were significantly lower than the collagen group (all P ⁇ 0.01).
  • mice Ear thickness and TEWL values of mice in each group after modeling
  • Series A+ polypeptides and series DZ polypeptides synthesized by Shanghai Bioengineering, with a purity greater than 95%.
  • the amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4.
  • the amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
  • A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
  • Preparation method add appropriate amount of distilled water to each group of sample powders and mix well.
  • SPF Wistar rats were selected and randomly divided into negative control group (apply distilled water), model control group (apply distilled water), fibronectin group (apply 1% fibronectin solution on the skin), positive control group (apply 5% minoxidil solution on the skin), A+ polypeptide recombinant collagen group 1 (apply 1% A+ polypeptide 1 recombinant type III collagen solution on the skin), A+ polypeptide recombinant collagen group 3 (apply 1% A+ polypeptide 3 recombinant type III collagen solution on the skin), collagen group (apply 1% type III collagen solution on the skin), A+ polypeptide group 1 (apply 1% A+ polypeptide No.
  • the invention relates to a group of 1+ polypeptide groups: (a) applying 1% A+ polypeptide solution No. 8 to the skin, (b) applying 1% A+ polypeptide solution No. 9 to the skin, (c) applying 1% A+ polypeptide solution No. 10 to the skin, (d) applying 1% A+ polypeptide solution No. 11 to the skin, (d) applying 1% A+ polypeptide solution No.
  • each rat selected an area of 4cmx5cm on the back and removed the hair as the observation area. Except for the negative control group, the rats in other groups were subcutaneously injected with testosterone propionate injection [5ml/(kg ⁇ d) once a day for 60 consecutive days to establish the AGA model. After 4 weeks of continuous subcutaneous injection of testosterone propionate, the rats gradually lost their hair. The remaining hair became thin and brittle, proving that the androgenic alopecia model was successfully established.
  • the skin of the observation area on the back of the corresponding treatment group rats was smeared with the solution of each treatment group, 1mL/(rat ⁇ time), once a day, for 60 consecutive days.
  • the negative control group and the model control group were smeared with distilled water 1mL/(rat ⁇ time), once a day, for 60 consecutive days.
  • the grading standards are as follows: “1" indicates normal structure of skin dermis tissue cells and subcutaneous hair follicles and sebaceous glands: “ ⁇ ” indicates no obvious hyperplasia of skin dermis tissue, obvious cystic changes of hair follicles and sebaceous glands, and no inflammation of subcutaneous tissue: “+” indicates segmental hyperplasia of skin dermis tissue, which is not obvious, cystic changes of a small number of hair follicles, and mild hyperplasia and hypertrophy of sebaceous glands.
  • the hair growth length of the series DZ polypeptide group, series A+ polypeptide and A+ polypeptide recombinant collagen experimental group on the 15th, 30th, 45th and 60th days of administration were all longer than that of the model control group, with statistical differences (all P ⁇ 0.01); there were no statistical differences between the series DZ polypeptide group, series A+ polypeptide group and A+ recombinant collagen group and the negative control group and the positive control group; the hair length of the series A+ polypeptide group was longer than that of the series DZ polypeptide group, with statistical differences (P ⁇ 0.05); the hair length of the A+ recombinant collagen group was longer than that of the human type III collagen group, with statistical differences (P ⁇ 0.05); there were no statistical differences between the fibronectin group and the type III collagen group and the model control group.
  • the series of A+ polypeptides and A+ recombinant type III collagen have obvious hair growth effects on the hair of rats with androgenic alopecia (AGA), the hair growth effects of the series A+ polypeptides are significantly stronger than those of the series DZ polypeptides, and the combination of the A+ polypeptides and type III collagen can significantly improve the hair growth effect; type III collagen and fibronectin have no obvious hair growth effect.
  • Example 6 Effects of A+ polypeptide or A+ recombinant type III collagen on a mouse hair loss model
  • Series A+ polypeptides and series DZ polypeptides synthesized by Shanghai Bioengineering, with a purity greater than 95%.
  • the amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4.
  • the amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
  • A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
  • Preparation method add appropriate amount of distilled water to each group of sample powders and mix well.
  • SPF grade C57BL/6 mice were selected and randomly divided into negative control group (applying distilled water), model control group (applying distilled water), fibronectin group (applying 1% fibronectin solution on the skin), positive control group (applying 2% minoxidil solution on the skin), A+ polypeptide recombinant collagen group 1 (applying 1% A+ polypeptide 1 recombinant type III collagen solution on the skin), A+ polypeptide recombinant collagen group 3 (applying 1% A+ polypeptide 3 recombinant type III collagen solution on the skin), collagen group (applying 1% type III collagen solution on the skin), A+ polypeptide group 1 (applying 1% A+ polypeptide No.
  • A+ polypeptide group 2 (applying 1% A+ polypeptide No. 3 solution on the skin), A+ polypeptide group 3 (applying 1% A+ polypeptide No. 5 solution on the skin), A+ polypeptide group 4 (applying 1% A+ polypeptide No. 7 solution on the skin), A+ polypeptide group 5 (applying 1% A+ polypeptide No. 8 solution on the skin) , A+ polypeptide group 6 (1% A+ polypeptide No. 9 solution was applied to the skin), A+ polypeptide group 7 (1% A+ polypeptide No. 10 solution was applied to the skin), A+ polypeptide group 8 (1% A+ polypeptide No.
  • A+ polypeptide group 9 (1% A+ polypeptide No. 12 solution was applied to the skin)
  • A+ polypeptide group 10 (1% A+ polypeptide No. 14 solution was applied to the skin)
  • A+ polypeptide group 11 (1% A+ polypeptide No. 16 solution was applied to the skin)
  • A+ polypeptide group 12 (1% A+ polypeptide No. 18 solution was applied to the skin)
  • A+ polypeptide group 13 (1% A+ polypeptide No. 19 solution was applied to the skin)
  • A+ polypeptide group 14 (1% A+ polypeptide No. 20 solution was applied to the skin),
  • A+ polypeptide group 15 (1% A+ polypeptide No.
  • A+ polypeptide group 16 1% A+ polypeptide No. 22 solution was applied to the skin
  • a series of DZ polypeptide groups 1% DZ polypeptide series solutions were applied to the skin respectively.
  • Each group had 10 rats, half of which were male and half were female.
  • the grading standards are as follows: “1" indicates normal structure of skin dermis tissue cells and subcutaneous hair follicles and sebaceous glands: “ ⁇ ” indicates no obvious hyperplasia of skin dermis tissue, obvious cystic changes of hair follicles and sebaceous glands, and no inflammation of the subcutaneous tissue; “+” indicates segmental hyperplasia of skin dermis tissue, which is not obvious, cystic changes of a small number of hair follicles, and mild hyperplasia and hypertrophy of sebaceous glands.
  • mice in the series A+ polypeptide and A+ recombinant type III collagen groups on days 15, 30, 45, and 60 of drug administration was longer than that of the model control group, and the differences were statistically significant (P ⁇ 0.01). Compared with the series DZ polypeptide treatment group, the differences were statistically significant (P ⁇ 0.05). See Table 7. The hair growth of mice in each group on day 45 of modeling is shown in Figure 8.
  • mice had segmental thickening to varying degrees, and there was mild lymphocytic hyperplasia under the skin of mice; some rats had obvious cystic changes in subcutaneous hair follicles, and the hair follicles were of different sizes. There was detached keratin in the enlarged hair follicle cavity. There was mild fibrosis around the hair follicles, and the cells around the hair follicles disappeared or the cell layers were significantly reduced. There seemed to be calcifications in the cavity that were stained blue. The number of sebaceous glands increased, and some glands were hypertrophic. The nuclei of hypertrophic glandular cells were significantly reduced, and the number of normal hair follicles decreased.
  • the lesions of the skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands of mice in the polypeptide group and minoxidil solution group were alleviated to varying degrees compared with those in the model group.
  • the lesions of the skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands of rats in the polypeptide group and A+ recombinant collagen group were significantly alleviated (P ⁇ 0.05).
  • Example 7 Effects of A+ polypeptide or A+ recombinant type III collagen on rabbit ear acne model
  • Series A+ polypeptides and series DZ polypeptides synthesized by Shanghai Bioengineering, with a purity greater than 95%.
  • the amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4.
  • the amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
  • A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1), A+ polypeptide 2 recombinant human type III collagen (referred to as A+ recombinant collagen 2) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
  • Positive treatment drug 0.1% adapalene gel (trade name: Differin, produced by Galderma Pharmaceuticals, France)
  • the excipient matrix components include methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), octadecyl alcohol (5%), glycerol (20%), alkyl aryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl paraben (0.1%), distilled water (about 31-55%), respectively, and mixed with appropriate amounts of the above fibronectin or series of polypeptides or proteins to form a 0.5% mixed emulsion.
  • the cream matrix used in the embodiment of this patent refers to the matrix component of the cream without the active ingredient.
  • the subjects were divided into negative control group (applying cream base), model control group (applying cream base), fibronectin group (applying 0.5% fibronectin cream on the skin), positive control group (applying Dafuwen on the skin), A+ recombinant collagen group (applying 0.5% A+ polypeptide recombinant type III collagen cream on the skin), collagen group (applying 0.5% type III collagen cream on the skin), A+ polypeptide group 1 (applying 0.5% A+ polypeptide No. 1 cream on the skin), A+ polypeptide group 2 (applying 0.5% A+ polypeptide No. 3 cream on the skin), A+ polypeptide group 3 (applying 0.5% A+ polypeptide No.
  • A+ polypeptide group 9 skin applied with 0.5% A+ polypeptide cream No. 9
  • A+ polypeptide group 7 skin applied with 0.5% A+ polypeptide cream No. 10
  • A+ polypeptide group 8 skin applied with 1% A+ polypeptide cream No. 11
  • A+ polypeptide group 9 skin applied with 1% A+ polypeptide cream No.
  • A+ polypeptide group 10 (skin applied with 1% A+ polypeptide cream No. 13), A+ polypeptide group 11 (skin applied with 1% A+ polypeptide cream No. 15), A+ polypeptide group 12 (skin applied with 1% A+ polypeptide cream No. 17), A+ polypeptide group 13 (skin applied with 1% A+ polypeptide cream No. 18), A+ polypeptide group 14 (skin applied with 1% A+ polypeptide cream No. 20), A+ polypeptide group 15 (skin applied with 1% A+ polypeptide cream No. 21) and A+ polypeptide group 16 (skin applied with 1% A+ polypeptide cream No.
  • the area of about 2cm ⁇ 2cm at the opening of the ear tube on the inner side of the rabbit's ear was evenly applied with a sterile cotton swab once a day, 0.5mL each time, and the previous application site was wiped with warm water.
  • the application was continued for 14 days to establish an acne micro-acne model.
  • the changes in the local skin were observed with the naked eye, including the thickness, hardness, roughness of the ear, and the presence or absence of black horn plugs at the hair follicle opening.
  • the samples were sacrificed 18 hours after the last application, fixed with 10% formaldehyde, embedded in paraffin, and sliced. After HE staining, pathological histological observation and analysis were performed.
  • the acne model histological grading standard is as follows: the histological grade is 3.
  • Grade 0 " ⁇ " means that there are only loose keratinized cells in the infundibulum, and no acne is generated;
  • Grade 1 means that the skin on the surface of the rabbit ear is red, or a small amount of dense keratinized material is seen in the infundibulum of the hair follicle, and the infundibulum is not dilated "+”;
  • Grade 2 means that medium-dense keratinized material is seen in the infundibulum of the hair follicle, and extends to the sebaceous gland, accompanied by hyperplasia of the sebaceous gland duct, and the infundibulum is dilated "2+”;
  • Grade 3 means that there are extensive keratinized materials in the hair follicles, and the tight keratin plugs in the hair follicles cause severe dilation of the hair follicles, obvious
  • the histopathological changes were observed under a microscope, and the thickness of the epidermis at 5 different locations on a slice was measured using the Biomia 99 image analysis system, and the average value was calculated.
  • the areas of the two hair follicles with the most complete structure and the diameters of the four sebaceous glands in the same position in the four slices were detected, and their respective average values were calculated.
  • the data of the left and right external auditory canals of each group of rabbits were subtracted to obtain the difference in epidermal thickness, hair follicle area and sebaceous gland diameter between the left and right ears of each rabbit.
  • SPSS 22 software was used for statistical analysis. Paired t-test was used for left-right comparison and t-test was used for comparison between groups. P ⁇ 0.05 was considered statistically significant.
  • the right ears of rabbits in the positive treatment group had mild redness, a little desquamation, a small amount of keratin plugs and acne, the acne was reduced and flattened, the pores were reduced, and the skin was slightly dry.
  • the right ears of the remaining treatment groups showed varying degrees of soft skin, reduced acne, The pores are significantly smaller, there is no desquamation, and it is basically close to a normal rabbit ear.
  • the left ear of the model group showed a thinner epidermis, with visible hair follicles and a clear junction between the dermis and the epidermis.
  • the right ear of the model group showed thickening of the epidermis, hyperkeratosis, thickening of the granular layer and the spinous layer, enlarged hair follicles, keratin plugs blocking the hair follicle openings and extending to the sebaceous glands, and the funnel of the hair follicles was filled with keratinized substances and enlarged into a pot shape; the capillaries in the upper dermis were dilated, and there were scattered inflammatory cell infiltrations around the hair follicles, and a small amount of neutrophils infiltrated; the number of sebaceous glands increased, and the volume of sebaceous glands increased.
  • the epidermal thickness, hair follicle area and sebaceous gland diameter of the right ear of the rabbits in the model group were compared with those of their left ears (negative control), and the differences were statistically significant (P ⁇ 0.01), indicating that the rabbit ear acne model was successfully replicated; there were no statistically significant differences between the right ears of the type III collagen group and the fibronectin group and the right ears of the model group (all P > 0.05); the epidermal thickness, hair follicle area and sebaceous gland diameter of the right ears of the rabbits in the other treatment groups were compared with those of the right ears of the rabbits in the model group, and the differences were statistically significant (all P ⁇ 0.01); the A+ polypeptide group was lower than the DZ polypeptide group (all P ⁇ 0.05); the A+ recombinant collagen group was lower than the series A+ polypeptide group (all P ⁇ 0.05). (See Table 10).
  • Rabbit ears are an animal model commonly used to measure the intensity of acne-causing substances.
  • the size of the hair follicle sebaceous glands of rabbits varies greatly, just like humans. As animals age, their ability to form acne also increases. Therefore, adult male rabbits are selected to replicate the acne model so that the male hormones in their bodies have a certain stimulating effect on the skin.
  • Adapalene can remove keratin plugs by regulating the abnormal keratinization process of the hair follicle sebaceous gland epithelium, thereby preventing and eliminating acne skin lesions. Therefore, the present invention selects adapalene as a positive control.
  • the series of polypeptides and A+ recombinant type III collagen can significantly inhibit the symptoms of the coal tar-induced rabbit ear acne model, reduce pore blockage and blackhead acne formation, and have a therapeutic effect on acne;
  • the series A+ polypeptide group is significantly better than the series DZ polypeptide group;
  • the collagen group and the fibronectin group have no obvious effect on acne;
  • the combination of A+ polypeptide and type III collagen can significantly improve the therapeutic effect on acne.
  • Example 8 Clinical observation on the treatment of facial rash in acne patients with peptides and recombinant collagen
  • amino acid sequence of A+ polypeptide 3 EYPYKHSGYYHRAV
  • amino acid sequence of A+ polypeptide 18 EYPYDYSGYYHRPAV
  • amino acid sequence of DZ polypeptide 1 EYPYKHSGYYHR were synthesized by Shanghai Biotech; polypeptide 3 recombinant human type III collagen and polypeptide 18 recombinant human type III collagen (the synthesis method is the same as that in Example 1).
  • Preparation of A+ polypeptide 3, DZ polypeptide 1, polypeptide 3 recombinant human type III collagen and polypeptide 18 recombinant human type III collagen solutions mix with appropriate amount of purified water to prepare 0.5% A+ polypeptide 3 or 0.5% DZ polypeptide 1 or 0.5% polypeptide 3 recombinant human type III collagen or polypeptide 18 recombinant human type III collagen solution.
  • Group A Placebo group (6 people), half male and half female, applied purified water externally on facial rash twice a day.
  • Group B Group A + polypeptide 3 (8 people), half male and half female, applied 0.5% polypeptide 3 solution to facial rashes, twice a day.
  • Group C DZ polypeptide 1 group (6 people), half male and half female, applied 0.5% DZ polypeptide 1 solution to the facial rash, twice a day.
  • Group D polypeptide 3 recombinant collagen group (8 people), half male and half female, applied 0.5% polypeptide 3 recombinant human type III collagen solution to the facial rash, twice a day.
  • Group E polypeptide 18 recombinant collagen group (8 people), half male and half female, applied 0.5% polypeptide 18 recombinant human type III collagen solution to the facial rash, twice a day.
  • the trial period was 8 weeks, with follow-up every 2 weeks to record facial rash and adverse reactions.
  • SPSS 21.0 software was used to process the data.
  • the t-test was used for quantitative data (x ⁇ s), and the ⁇ 2 test was used for counting data (%).
  • P ⁇ 0.05 was considered to be statistically significant.
  • the effective rates of A+ polypeptide 3 group, A+ polypeptide 3 recombinant collagen group and A+ polypeptide 18 recombinant collagen group were 87.5%, 100% and 100% respectively, which were significantly different from the placebo group (P ⁇ 0.01); the effective rates of A+ polypeptide 3 group, A+ polypeptide 3 recombinant collagen group and A+ polypeptide 18 recombinant collagen group were also significantly different from the DZ polypeptide 1 group (P ⁇ 0.05); there was no statistically significant difference between the DZ polypeptide 1 group and the placebo group (P > 0.05). See Table 11 for details.
  • the TEWL of the mandible in the A+ polypeptide 3 group, the A+ polypeptide 3 recombinant collagen group and the A+ polypeptide 18 recombinant collagen group was significantly different from that in the placebo group (P ⁇ 0.01); after 4 and 8 weeks of treatment, the A+ polypeptide 3 group and the recombinant collagen group were significantly different from those in the DZ polypeptide 1 group (P ⁇ 0.05); see Table 12 for details.
  • Both A+ polypeptide and A+ recombinant type III collagen can significantly improve facial rashes in acne patients, reduce pore blockage and blackhead formation, and significantly reduce scars (acne marks); the A+ polypeptide group is significantly better than the DZ polypeptide group; the combination of A+ polypeptide and type III collagen can significantly enhance the therapeutic effect on acne.
  • Series A+ polypeptides and series DZ polypeptides synthesized by Shanghai Bioengineering, with a purity greater than 95%.
  • the amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4.
  • the amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
  • A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1), A+ polypeptide 2 recombinant human type III collagen (referred to as A+ recombinant collagen 2) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
  • the excipient matrix components include methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), octadecyl alcohol (5%), glycerol (20%), alkyl aryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl paraben (0.1%), distilled water (about 31-55%), respectively, and mixed with appropriate amounts of the above fibronectin or series of polypeptides or proteins to form a 0.5% mixed emulsion.
  • the cream matrix used in the embodiment of this patent refers to the matrix component of the cream without the active ingredient.
  • SPF male rats weighing (210 ⁇ 28) g, were obtained from the Animal Center of Nanjing Medical University. According to the weight number, they were divided into negative control group (applying cream base), model control group (applying cream base), fibronectin group (applying 0.5% fibronectin cream on the skin), A+ recombinant collagen group (applying 0.5% A+ polypeptide recombinant type III collagen cream on the skin), collagen group (applying 0.5% type III collagen cream on the skin), A+ polypeptide group 1 (applying 0.5% A+ polypeptide No. 1 cream on the skin), A+ polypeptide group 2 (applying 0.5% A+ polypeptide No.
  • A+ polypeptide group 3 (applying 0.5% A+ polypeptide No. 5 cream on the skin), A+ polypeptide group 4 (applying 0.5% A+ polypeptide No. 6 cream on the skin), A+ polypeptide group 5 (applying 0.5% A+ polypeptide No. 8 cream on the skin), A+ polypeptide group 6 (applying 0.5% A+ polypeptide No. 9 cream on the skin).
  • Cream A+ polypeptide group 7 (0.5% A+ polypeptide No. 10 cream applied on the skin), A+ polypeptide group 8 (1% A+ polypeptide No. 11 cream applied on the skin), A+ polypeptide group 9 (1% A+ polypeptide No.
  • A+ polypeptide group 10 cream applied on the skin
  • A+ polypeptide group 11 1% A+ polypeptide No. 15 cream applied on the skin
  • A+ polypeptide group 12 1% A+ polypeptide No. 17 cream applied on the skin
  • A+ polypeptide group 13 1% A+ polypeptide No. 18 cream applied on the skin
  • A+ polypeptide group 14 1% A+ polypeptide No. 20 cream applied on the skin
  • A+ polypeptide group 15 (1% A+ polypeptide No. 21 cream applied on the skin
  • A+ polypeptide group 16 1% A+ polypeptide No. 21 cream applied on the skin
  • 0.5% DZ polypeptide cream was applied on the skin.
  • mice There were 10 mice in each group, half male and half female, and the topical application was once a day.
  • Each group of rats was anesthetized by intraperitoneal injection of 2% sodium pentobarbital (120 mg/kg) and fixed on the operating table. Then a 4 ⁇ 5 cm piece of intact skin was selected on the left side of the back, and 8% sodium sulfide was used to depilate the hair.
  • a circular wound with a diameter of 2.4 cm and deep to the muscle fascia was cut at the depilated area with tissue scissors to destroy part of the muscle surface fascia.
  • the animals were kept in separate cages to prevent the rats from biting and licking.
  • the wound surface was routinely disinfected with 2% iodine tincture every day, and the healing of the rats' wounds was observed.
  • the wounds were routinely disinfected every day, and the wounds of rats were observed on the 1st, 3rd, 5th, 7th, 12th, and 20th days. Since the 6th day, the wounds of the A+ polypeptide and A+ recombinant collagen series treatment groups began to recover, and the speed was significantly faster than that of the model group, and the wound area gradually became smaller. On the 15th day, the wounds of each treatment group had basically recovered, while the model group still had a wound of about 0.6 cm2 . By the 20th day, the wounds of each group had recovered, and the model control group left obvious scars, while the series A+ polypeptide and recombinant collagen treatment groups only left varying amounts of pigmentation. A+ recombinant collagen is better than A+ polypeptide. The A+ polypeptide group is significantly better than the DZ polypeptide group.
  • Both A+ polypeptide and A+ recombinant collagen can significantly promote skin wound healing and reduce scar formation.
  • the A+ polypeptide group is significantly better than the DZ polypeptide control group;
  • A+ polypeptide and type III collagen recombinant can significantly improve the therapeutic effect on scars.

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Abstract

The present invention discloses a series of amino acid sequences, polypeptides, proteins, and compositions comprising said amino acid sequences, nucleic acid sequences encoding the amino acid sequences, polypeptides, proteins, and compositions, and uses of the compositions or pharmaceutically acceptable salts thereof in the preparation of skin products. An amino acid sequence of the present invention is made up of alanine-valine-glutamate-tyrosine-proline-tyrosine-lysine-histidine-serine-glycine-tyrosine-tyrosine-histidine-arginine. A product containing said amino acid sequence of the present invention has remarkable therapeutic effect with respect to anti-aging, inflammation reduction, tissue repair, hair growth, acne removal, and scar removal, and is safe, reliable, and has mild side effects.

Description

一系列多肽/蛋白质及其应用A series of peptides/proteins and their applications 技术领域Technical Field
本发明属于生物医药和护肤品技术领域，具体涉及一系列氨基酸序列，包含所述氨基酸序列的多肽，蛋白和组合物，编码所述氨基酸序列、多肽、蛋白和组合物的核酸序列，以及包含所述氨基酸序列，多肽，蛋白，组合物或核酸序列的产品在皮肤病预防和治疗领域的应用。The present invention belongs to the technical field of biomedicine and skin care products, and specifically relates to a series of amino acid sequences, polypeptides, proteins and compositions comprising the amino acid sequences, nucleic acid sequences encoding the amino acid sequences, polypeptides, proteins and compositions, and applications of products comprising the amino acid sequences, polypeptides, proteins, compositions or nucleic acid sequences in the field of skin disease prevention and treatment.
技术背景technical background
皮肤是人体最外在的器官，可反映机体的健康和衰老状况。皮肤衰老的发生被认为是内在和外在因素共同作用的结果。皮肤衰老往往表现为表皮变薄，真皮皱缩，厚度减少，胶原纤维变细甚至排列紊乱、断裂。在衰老过程中，细胞增殖能力下降是衰老的重要特征，衰老的细胞由于蛋白质合成能力下降、免疫***相关基因表达失调等，往往表现为增殖能力下降的趋势。Skin is the most external organ of the human body and can reflect the health and aging of the body. The occurrence of skin aging is believed to be the result of the combined action of internal and external factors. Skin aging is often manifested as thinning of the epidermis, shrinkage and reduction of dermis, thinning of collagen fibers, and even disordered arrangement and breakage. During the aging process, the decline in cell proliferation ability is an important feature of aging. Aged cells often show a trend of declining proliferation ability due to decreased protein synthesis ability and dysregulated expression of immune system-related genes.
皮脂膜为覆盖于皮肤表面的一层透明薄膜，主要由皮脂腺分泌的皮脂、角质层细胞崩解产生的脂质与汗腺分泌的汗液乳化形成，呈弱酸性，其主要成分角鲨烯、亚油酸、亚麻酸及脂质成分，具有锁住水分及一定的抗炎作用。皮肤屏障由皮脂膜、角质层角蛋白、脂质、"三明治"结构、砖墙结构、真皮粘多糖类、粘多糖类等共同构成，抵御外界有害、刺激物、日光进入，同时具有保湿及调节抗炎作用。皮肤屏障受损将引起皮肤干燥，皮肤老化、色素沉着、皮炎等。组织修复包括伤口愈合、皮肤屏障功能的修复等。皮肤屏障功能的恢复也有利于防治皮肤局部细菌感染，辅助治疗急慢性皮肤炎症，防止皮肤炎症的反复发作，包括脂溢性皮炎、糖激素依赖性皮炎、湿疹、特异性皮炎和银屑病等。The sebum membrane is a transparent film covering the surface of the skin. It is mainly formed by the emulsification of sebum secreted by sebaceous glands, lipids produced by the disintegration of stratum corneum cells and sweat secreted by sweat glands. It is weakly acidic. Its main components are squalene, linoleic acid, linolenic acid and lipid components, which have the function of locking moisture and having certain anti-inflammatory effects. The skin barrier is composed of sebum membrane, stratum corneum keratin, lipids, "sandwich" structure, brick wall structure, dermal mucopolysaccharides, mucopolysaccharides, etc., which resist the entry of harmful external factors, irritants and sunlight, and have moisturizing and regulating anti-inflammatory effects. Damage to the skin barrier will cause dry skin, skin aging, pigmentation, dermatitis, etc. Tissue repair includes wound healing, repair of skin barrier function, etc. The restoration of skin barrier function is also beneficial for preventing and treating local bacterial infections of the skin, assisting in the treatment of acute and chronic skin inflammation, and preventing the recurrence of skin inflammation, including seborrheic dermatitis, glucocorticoid-dependent dermatitis, eczema, atopic dermatitis and psoriasis.
斑秃(alopecia areata，AA)是一种非瘢痕性脱发，我国流行病学调查显示男性雄激素性脱发患病率为21.3％，女性为6.0％。斑秃通常情况下表现为突发的脱发斑，严重时可累及整个头皮，此时称为全秃(alopecia totalis，AT)，当累及包括腋毛、***在内的全身所有毛发时，称为普秃(alopecia universalis，AU)，容易给患者的容貌和心理带来严重的影响。自身免疫功能异常或不稳定、神经精神因素被认为是重要相关因素。斑秃越早介入治疗，治愈几率越高。米诺地尔可促进皮肤血管扩张、改善局部血液循环、促进毛发生长，是一种治疗斑秃常见的外用药物。严重斑秃常用糖皮质激素，主要包括***龙、复方倍他米松等等，可以口服、外用或皮内注射。对于不适用糖皮质激素类药物的患者，可采用免疫抑制剂治疗，常见的药物有环孢素、甲氨蝶呤。糖皮质激素和免疫抑制剂副作用多。Alopecia areata (AA) is a non-scarring hair loss. Epidemiological surveys in my country show that the prevalence of androgenic alopecia in men is 21.3% and in women is 6.0%. Alopecia areata usually presents as sudden hair loss patches. In severe cases, it may involve the entire scalp, which is called alopecia totalis (AT). When it involves all the hair on the body, including axillary hair and pubic hair, it is called alopecia universalis (AU), which can easily have a serious impact on the patient's appearance and psychology. Abnormal or unstable autoimmune function and neuropsychiatric factors are considered to be important related factors. The earlier the treatment of alopecia areata is initiated, the higher the chance of cure. Minoxidil can promote skin vasodilation, improve local blood circulation, and promote hair growth. It is a common topical drug for the treatment of alopecia areata. Glucocorticoids are commonly used for severe alopecia areata, mainly including prednisolone, compound betamethasone, etc., which can be taken orally, applied topically, or injected intradermally. For patients who are not suitable for glucocorticoid drugs, immunosuppressants can be used for treatment. Common drugs include cyclosporine and methotrexate. Glucocorticoids and immunosuppressants have many side effects.
雄性激素源性脱发(androgenetic alopecia，AGA)，又名雄激素性脱发，脂溢性脱发(Seborrheic alopecia，SA)，男性脱发或遗传性脱发，是一种雄激素依赖性的遗传性毛发脱落，男性脱发多呈马蹄形外观。脱发处皮肤光亮，毛孔缩小或残留少许细软毳毛。脱发的速度、范围和严重程度，受遗传和个体影响。女性多为发生于头顶的弥漫性脱发。雄激素性脱发的病因及发病机制尚不明确，一般认为雄激素及其受体在本病的发生中起关键作用。正常生理状态下，雄激素在体内对毛发的生长发育起一定的刺激促进作用，但在某些特定部位能诱导毛发脱失；睾酮是体内主要的雄激素，它经5α-还原酶转化为二氢睾酮，后者可引起终毛向毳毛转变，最终导致脱发。雄激素源性秃发是脱发性疾病难治的类型，该病的动物模型通常作为脱发性疾病的代表模型。米诺地尔是一种非特异性治疗脱发的药物，是FDA批准用于治疗脱发的一线外用药物，但使用过程中可能引起面部和四肢多毛症，停用后治疗效果逐渐消失。非那雄胺是一种Ⅱ型5α-还原酶选择性抑制剂，FDA批准口服非那雄胺用于治疗雄激素性脱发，可持续改善头发的生长情况，但非那雄胺存在引起性功能异常，***一过性减少和男性***发育异常等不良反应，在动物实验中发现有致畸作用，故不宜用于小儿和育龄妇女。西咪替丁需连服5个月或更久，副作用为男性***发育、阳痿、***降低等。口服避孕药：主要有的索高诺酮、左炔诺孕酮(左旋甲基炔诺孕酮)、炔诺孕酮、炔诺酮、诺孕酯(肟炔诺酯)、双酯炔诺醇和醋炔诺酮等，常用来治疗女性AGA，治疗6～12个月后头发会有所改善。Androgenic alopecia (AGA), also known as androgenic alopecia, seborrheic alopecia (SA), male pattern baldness or hereditary alopecia, is an androgen-dependent hereditary hair loss. Male pattern baldness often presents a horseshoe-shaped appearance. The skin at the site of hair loss is shiny, the pores are reduced or a few fine and soft hairs remain. The speed, range and severity of hair loss are affected by genetics and individuals. Women often experience diffuse hair loss on the top of the head. The etiology and pathogenesis of androgenic alopecia are still unclear. It is generally believed that androgens and their receptors play a key role in the occurrence of this disease. Under normal physiological conditions, androgens in the body have a certain stimulating and promoting effect on the growth and development of hair, but can induce hair loss in certain specific parts; testosterone is the main androgen in the body, which is converted into dihydrotestosterone by 5α-reductase, which can cause the transformation of terminal hair to vellus hair, ultimately leading to hair loss. Androgenetic alopecia is a difficult-to-treat type of hair loss disease, and the animal model of the disease is usually used as a representative model of hair loss diseases. Minoxidil is a non-specific drug for the treatment of hair loss and is a first-line topical drug approved by the FDA for the treatment of hair loss. However, it may cause facial and limb hirsutism during use, and the therapeutic effect gradually disappears after discontinuation. Finasteride is a type II 5α-reductase selective inhibitor. The FDA approves oral finasteride for the treatment of androgenic alopecia, which can continuously improve hair growth. However, finasteride has adverse reactions such as causing sexual dysfunction, transient sperm reduction, and abnormal male breast development. It has been found to have teratogenic effects in animal experiments, so it is not suitable for children and women of childbearing age. Cimetidine needs to be taken for 5 months or longer, and the side effects are male breast development, impotence, and decreased libido. Oral contraceptives: The main ones are sogogestrel, levonorgestrel (levonorgestrel), norgestrel, norethindrone, norgestimate (norgestrel oxime), diester norgestrel and norgestrel acetate, etc., which are often used to treat AGA in women. The hair will improve after 6 to 12 months of treatment.
抗肿瘤药物引起的脱发是最常见的生长期秃发，抗肿瘤药物在消除迅速***的癌细胞的同时，也攻击毛囊四周迅速***的细胞引起脱发。以上脱发治疗较困难，容易反复。因此，需要寻找更多安全、有效的治疗脱发的产品。Hair loss caused by anti-tumor drugs is the most common type of growth aging alopecia. While anti-tumor drugs eliminate rapidly dividing cancer cells, they also attack rapidly dividing cells around hair follicles, causing hair loss. The above hair loss treatments are difficult and prone to recurrence. Therefore, it is necessary to find more safe and effective products for treating hair loss.
痤疮(acne)俗称青春豆，是好发于毛囊皮脂腺的慢性炎症性疾病，发病率约9.4％，于***多见。临床表现主要有粉刺、丘疹、脓疱、结节、囊肿、疤痕等，愈合时间长，给患者的容貌和心理带来严重的影响。痤疮与多个发病机制相关，毛囊口角化异常是本病发病的重要基础，炎症和感染是痤疮的发病因素。痤疮患者的皮脂腺较大，皮脂腺分泌增加，皮脂中亚油酸水平相对减少，影响脂肪的合成，导致滤泡上皮缺乏脂肪酸，从而诱导滤泡过度角化，使上皮细胞不能正常脱落，毛囊皮脂腺口过度变小，皮脂不能顺畅排出，形成粉刺，继发感染形成丘疹、脓疱、结节、囊肿、疤痕等。 Acne (acne) is commonly known as pimples, and is a chronic inflammatory disease that is prone to occur in the hair follicle sebaceous glands, with an incidence rate of about 9.4%, which is more common in adolescence. Clinical manifestations mainly include acne, papules, pustules, nodules, cysts, scars, etc., and the healing time is long, which has a serious impact on the patient's appearance and psychology. Acne is related to multiple pathogenesis, and abnormal keratinization of the hair follicle mouth is an important basis for the onset of this disease, and inflammation and infection are the pathogenic factors of acne. The sebaceous glands of acne patients are larger, and the secretion of sebaceous glands increases. The level of linoleic acid in sebum is relatively reduced, which affects the synthesis of fat, resulting in a lack of fatty acids in the follicular epithelium, thereby inducing excessive keratinization of the follicles, so that epithelial cells cannot fall off normally, the hair follicle sebaceous gland mouth is excessively reduced, and sebum cannot be discharged smoothly, forming acne, and secondary infection forms papules, pustules, nodules, cysts, scars, etc.
目前人们已经发现和分离出一百多种存在于人体的肽和蛋白，对维护细胞、组织及器官的正常生理功能有着重要作用，被广泛的应用于医学材料及药物方面、美容化妆品、保健品以及各种实用工业中。但是对上诉问题效果好、安全的产品很少，已有的产品远不能满足临床需求，因此需要寻找更多疗效好、副作用少、价格便宜的新产品来控制疾病进展，减少复发和并发症。At present, people have discovered and separated more than 100 peptides and proteins existing in the human body, which play an important role in maintaining the normal physiological functions of cells, tissues and organs, and are widely used in medical materials and drugs, beauty cosmetics, health products and various practical industries. However, there are few products with good effects and safety for the above problems, and the existing products are far from meeting clinical needs. Therefore, it is necessary to find more new products with good efficacy, few side effects and low prices to control the progression of the disease and reduce recurrence and complications.
人体内含有多种胶原蛋白，占人体蛋白质总量的1/3，负责人体机能的正常运转。皮肤是人体最大的器官，其中真皮层里80％都是胶原蛋白，主要是I型和Ⅲ型胶原蛋白。随着年龄增加胶原总量呈下降趋势，现代医学证明，衰老本质是柔软富有弹性的Ⅲ型胶原蛋白持续加速流失，且成年后无法自体再生。因此皮肤弹性下降、松弛、修复效果降低、是人皮肤衰老的原因之一。Ⅲ型胶原蛋白具有促进细胞增生、增加细胞活性、帮助机体修复老化受损皮肤，起到抗衰老、修复创口的作用。The human body contains a variety of collagen, accounting for 1/3 of the total protein in the human body, which is responsible for the normal functioning of the human body. The skin is the largest organ in the human body, and 80% of the dermis is collagen, mainly type I and type III collagen. With age, the total amount of collagen tends to decrease. Modern medicine has proven that the essence of aging is the continuous and accelerated loss of soft and elastic type III collagen, which cannot be regenerated after adulthood. Therefore, decreased skin elasticity, sagging, and reduced repair effect are one of the reasons for human skin aging. Type III collagen has the functions of promoting cell proliferation, increasing cell activity, helping the body repair aging and damaged skin, and playing an anti-aging and wound repair role.
从结构上来说，人体天然的胶原蛋白的结构非常的复杂，所以才导致人源胶原蛋白极难通过常规手段表达和大量制备。天然胶原分子形成了一种特殊的超螺旋结构，由三条无链内氢键形成而仅受链间氢键支撑的多肽链组成。这种螺旋性的结构是以3个氨基酸残基为基本重复体的左手螺旋。Kotch和Raines于2006年通过化学法合成了具有(Gly-X-Y)n胶原蛋白特征序列的多肽，该多肽可通过自组装成功形成三螺旋结构。Structurally, the structure of natural collagen in the human body is very complex, which is why it is extremely difficult to express and mass-produce human collagen by conventional means. Natural collagen molecules form a special superhelical structure, consisting of three polypeptide chains that are not formed by intrachain hydrogen bonds but are only supported by interchain hydrogen bonds. This helical structure is a left-handed helix with three amino acid residues as the basic repeating body. In 2006, Kotch and Raines synthesized a polypeptide with the characteristic sequence of (Gly-XY) n collagen by chemical method, which can successfully form a triple helical structure through self-assembly.
利用基因工程技术合成的重组人源化胶原蛋白具有成本低、效率高等优点，且能通过菌种发酵技术获得产业化推广，受到越来越多研究者的关注。重组人源化胶原蛋白是基于人胶原蛋白的特征和主要功能域重新优化设计基因序列，表达的目标蛋白具有与人胶原蛋白相似的结构特征。与传统提取法提取的动物胶原蛋白相比，重组人源化胶原蛋白具有产量高、批次稳定、水性强、生物相容性好、无免疫病毒风险等优点，是一种理想的生物医用材料。Recombinant humanized collagen synthesized by genetic engineering technology has the advantages of low cost and high efficiency, and can be industrialized and promoted through bacterial fermentation technology, attracting more and more researchers' attention. Recombinant humanized collagen is a re-optimized gene sequence based on the characteristics and main functional domains of human collagen, and the expressed target protein has similar structural characteristics to human collagen. Compared with animal collagen extracted by traditional extraction methods, recombinant humanized collagen has the advantages of high yield, batch stability, strong water resistance, good biocompatibility, and no immune virus risk, making it an ideal biomedical material.
发明内容Summary of the invention
本发明披露了具有多种功效的氨基酸序列，利用DNA重组技术结合生物合成技术生产的多肽或重组胶原蛋白，很好保证了多肽/蛋白的生物活性，同时增强了多方面的功效。不仅保障医学应用安全，而且在生物相容性、生物吸收性、细胞黏附性上具有更稳定的表现。The present invention discloses an amino acid sequence with multiple functions, and utilizes DNA recombination technology combined with biosynthesis technology to produce polypeptides or recombinant collagen, which well guarantees the biological activity of polypeptides/proteins and enhances multiple functions. It not only ensures the safety of medical applications, but also has more stable performance in biocompatibility, bioabsorbability, and cell adhesion.
为实现所述目的，本申请采用了以下技术方案：To achieve the above objectives, this application adopts the following technical solutions:
进一步地，所述延伸序列包括SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22与前述任意一个延伸序列至少70％同源的且功能相同或相似的延伸序列、片段或突变体，

Further, the extension sequence includes SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, which is at least 70% homologous to any of the aforementioned extension sequences and has the same or similar functions, extension sequences, fragments or mutants,

进一步地，所述突变体具有与所述氨基酸序列(例如，SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22)。或其片段具有至少70％同源，例如该突变体为与所述氨基酸序列至少70％,71％,72％,73％,74％,75％,76％,77％,78％,79％,80％,81％,82％,83％,84％,85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,或至少99％同源的氨基酸序列。Further, the mutant has the same amino acid sequence as the amino acid sequence (for example, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22). or a fragment thereof having at least 70% homology, for example the mutant is an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% homologous to the amino acid sequence.
第二方面，本发明提出一种产品。In a second aspect, the present invention provides a product.
进一步地，所述产品包括所述任意一个氨基酸序列，例如，SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22。Further, the product includes any one of the amino acid sequences, for example, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22.
进一步地，所述产品为多肽或蛋白质。Furthermore, the product is a polypeptide or a protein.
进一步地，所述多肽为A+多肽，所述蛋白为A+多肽重组人源Ⅲ型胶原蛋白。Furthermore, the polypeptide is A+ polypeptide, and the protein is A+ polypeptide recombinant human type III collagen.
进一步地，在多肽包含氨基酸序列(例如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22)的情况中，其可包含超出参照序列的在其N和/或C端的额外氨基酸，例如多肽可以包含在其N端的额外氨基酸。同样地，在多肽包含依照参照序列的氨基酸序列的片段、突变体或衍生物的情况中，其可包含在其N和/或C端的额外氨基酸。Further, where the polypeptide comprises an amino acid sequence (e.g., SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22), it may contain additional amino acids at its N and/or C-terminus beyond the reference sequence, for example, the polypeptide may contain additional amino acids at its N-terminus. Likewise, where the polypeptide comprises a fragment, mutant or derivative of the amino acid sequence according to the reference sequence, it may comprise additional amino acids at its N- and/or C-terminus.
进一步地，所述多肽可以包含依照参照序列的氨基酸序列的突变体或所述突变体的片段或由所述突变体或所述突变体的片段组成，而此类突变体可以是非天然存在的。Furthermore, the polypeptide may comprise or consist of a mutant or a fragment of the mutant according to the amino acid sequence of the reference sequence, and such a mutant may not exist naturally.
进一步地，所述多肽或蛋白质还包含免疫球蛋白。Furthermore, the polypeptide or protein also comprises immunoglobulin.
第三方面，本发明提出一种核酸序列。In a third aspect, the present invention provides a nucleic acid sequence.
进一步地，所述核酸序列编码SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22中任意一项所述的氨基酸序列，或编码所述多肽或蛋白质。Further, the nucleic acid sequence encodes the amino acid sequence of any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, or encodes the polypeptide or protein.
第四方面，本发明提出一种组合物。In a fourth aspect, the present invention provides a composition.
进一步地，所述组合物包括如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22所示的氨基酸序列，所述多肽，所述蛋白质，所述核酸序列，所述氨基酸序列/多肽/蛋白质/核酸序列的衍生物或其可药用盐。Further, the composition includes an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, the polypeptide, the protein, the nucleic acid sequence, a derivative of the amino acid sequence/polypeptide/protein/nucleic acid sequence or a pharmaceutically acceptable salt thereof.
进一步地，所述衍生物为对于多肽中的氢原子、羟基、羧基、亚氨基用能够取代的公知的取代基取代而成。作为可用盐，没有特别限定，可列举出例如与无机碱的盐、与有机碱的盐、与无机酸的盐、与有机酸的盐、与碱性、中性或酸性氨基酸的盐等。Furthermore, the derivatives are obtained by replacing hydrogen atoms, hydroxyl groups, carboxyl groups, and imino groups in the polypeptide with known substituents. Available salts are not particularly limited, and examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic, neutral, or acidic amino acids.
本发明所述的多肽或蛋白质制备方法可以采用现有技术常用的方法，如公知的化学合成法或生物合成法或 遗传工程学的合成法等。优选地，本发明所述多肽或蛋白质的合成方法属于基于基因工程技术的生物合成法，包括在适于表达编码所述多肽的核酸的条件下的原核或真核的宿主细胞，由宿主细胞回收所述多肽或蛋白质，其中所述宿主细胞是原核的或真核的。The polypeptide or protein preparation method of the present invention can adopt the commonly used methods in the prior art, such as the well-known chemical synthesis method or biological synthesis method or Synthesis by genetic engineering, etc. Preferably, the method for synthesizing the polypeptide or protein of the present invention belongs to a biosynthesis method based on genetic engineering technology, including a prokaryotic or eukaryotic host cell under conditions suitable for expressing the nucleic acid encoding the polypeptide, and recovering the polypeptide or protein from the host cell, wherein the host cell is prokaryotic or eukaryotic.
进一步地，所述组合物以所述氨基酸序列SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22，所述多肽，所述蛋白质，所述核酸序列，所述氨基酸序列/多肽/蛋白质/核酸序列突变体、所述氨基酸序列/多肽/蛋白质/核酸序列融合物、所述氨基酸序列/多肽/蛋白质/核酸序列衍生物或其可药用盐为活性成分或主要活性成分，辅以药学上可接受的载体。Furthermore, the composition is composed of the amino acid sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SE Q ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, the polypeptide, the protein, the nucleic acid sequence, the amino acid sequence/polypeptide/protein/nucleic acid sequence mutant, the amino acid sequence/polypeptide/protein/nucleic acid sequence fusion, the amino acid sequence/polypeptide/protein/nucleic acid sequence derivative or its pharmaceutically acceptable salt is the active ingredient or main active ingredient, supplemented by a pharmaceutically acceptable carrier.
进一步地，所述药学上可接受的载体选自水、生理盐水、生理上可匹配的缓冲液、生理上可匹配的溶液和生理上可匹配的悬浮液中的至少一种。Furthermore, the pharmaceutically acceptable carrier is selected from at least one of water, physiological saline, a physiologically compatible buffer, a physiologically compatible solution and a physiologically compatible suspension.
进一步地，所述多肽的“突变体”包括***、缺失和取代，其或是保守的或是非保守的。保守取代指将相同通用类别(例如酸性氨基酸、碱性氨基酸、非极性氨基酸、极性氨基酸或芳香族氨基酸)内的氨基酸用相同类别内的另一种氨基酸的取代。保守氨基酸取代和非保守氨基酸取代的意义是本领域中公知的。Further, "mutants" of the polypeptide include insertions, deletions and substitutions, which are either conservative or non-conservative. Conservative substitutions refer to the substitution of an amino acid within the same general class (e.g., acidic amino acids, basic amino acids, non-polar amino acids, polar amino acids, or aromatic amino acids) with another amino acid within the same class. The meanings of conservative and non-conservative amino acid substitutions are well known in the art.
进一步地，所述的多肽或蛋白质或组合物可制备为溶液、酊剂、醑剂、搽剂、喷雾剂、乳剂、膏剂、凝胶剂、贴剂、水剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂、注射剂或其他药用剂型。Furthermore, the polypeptide or protein or composition can be prepared as a solution, tincture, spirit, liniment, spray, emulsion, paste, gel, patch, aqueous solution, tablet, granule, oral solution, capsule, pill, enema, film, injection or other pharmaceutical dosage forms.
进一步地，所述的多肽或蛋白质或组合物可单独使用，也可与一种或多种产品组合使用。Furthermore, the polypeptide or protein or composition can be used alone or in combination with one or more products.
第五方面，本发明提出一种制备所述多肽或蛋白质的方法。In a fifth aspect, the present invention provides a method for preparing the polypeptide or protein.
进一步地，所述方法包括：培养宿主细胞，后从所述宿主细胞回收所述多肽或蛋白质，其中所述宿主细胞是原核的或真核的。Furthermore, the method comprises: culturing a host cell, and then recovering the polypeptide or protein from the host cell, wherein the host cell is prokaryotic or eukaryotic.
第六方面，本发明提出所述氨基酸序列，多肽，蛋白质，核酸序列及组合物在制备皮肤类产品中的应用。In a sixth aspect, the present invention proposes the use of the amino acid sequence, polypeptide, protein, nucleic acid sequence and composition in the preparation of skin products.
进一步地，所述产品包括但不限于药品、医疗器械、保健品或护肤品。Furthermore, the products include but are not limited to medicines, medical devices, health products or skin care products.
进一步地，所述产品的用途包括但不限于抗衰、抗炎、组织修复、生发、祛疤和祛痘产品。Furthermore, the uses of the product include but are not limited to anti-aging, anti-inflammatory, tissue repair, hair growth, scar removal and acne removal products.
本发明所述的多肽或蛋白质或组合物可制备为药学上允许的任何剂型，例如为适于皮肤外用、口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、***、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。The polypeptide or protein or composition of the present invention can be prepared into any pharmaceutically acceptable dosage form, for example, a preparation suitable for external use on the skin, oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intrafatty, intraarticular, intraperitoneal or intrathecal administration.
在一种优选的实施方式中，本发明所述多肽或蛋白质或组合物的剂型为溶液、酊剂、醑剂、搽剂、喷雾剂、乳剂、膏剂、凝胶剂、贴剂、水剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。In a preferred embodiment, the polypeptide or protein or composition of the present invention is in the form of a solution, tincture, spirit, liniment, spray, emulsion, paste, gel, patch, aqueous solution, tablet, granule, oral solution, capsule, pill, enema, film or injection.
第七方面，本发明提出一种用于治疗或预防皮肤病的方法。In a seventh aspect, the present invention provides a method for treating or preventing skin diseases.
进一步地，所述方法包括施用治疗有效量的包含所述氨基酸序列，多肽，蛋白或组合物的产品。Furthermore, the method comprises administering a therapeutically effective amount of a product comprising the amino acid sequence, polypeptide, protein or composition.
进一步地，所述皮肤病包括但不限于皮肤衰老、皮肤炎症、皮肤损伤、脱发、疤痕和痤疮。Furthermore, the skin diseases include but are not limited to skin aging, skin inflammation, skin damage, hair loss, scars and acne.
进一步地，所述产品的施用对象是人。Furthermore, the product is administered to humans.
本发明所指的A+多肽，是指包含但不限于如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22所示的氨基酸序列，其片段或其突变体的多肽。The A+ polypeptide referred to in the present invention refers to a polypeptide comprising but not limited to an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, or a fragment or mutant thereof.
本发明所指的A+多肽重组人源Ⅲ型胶原蛋白，是指包含但不限于如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ  ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22所示的氨基酸序列，其片段或其突变体的重组人源Ⅲ型胶原蛋白。The A+ polypeptide recombinant human type III collagen referred to in the present invention refers to but is not limited to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ Recombinant human type III collagen having an amino acid sequence shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, a fragment thereof or a mutant thereof.
与现有技术相对比，本申请具有以下优点：Compared with the prior art, this application has the following advantages:
(1)本发明所述的氨基酸序列或其突变体，能显著修复皮肤屏障，在抗衰、抗炎、组织修复、生发、祛疤和祛痘等方面效果显著，且安全、副作用小。(1) The amino acid sequence or its mutant described in the present invention can significantly repair the skin barrier, and has significant effects in anti-aging, anti-inflammation, tissue repair, hair growth, scar removal and acne removal, and is safe with few side effects.
(2)包括该氨基酸序列的系列A+多肽生物活性高，同时具有较好的抗衰、抗炎、组织修复、生发、祛疤和祛痘等多种功效，优于目前所知的绝大多数多肽/蛋白产品，安全、稳定、生物相容性好、生产成本不高，尤其适用于功效性护肤品原料领域，适合生产出多种功效性护肤品，可以长期使用。(2) The series A+ polypeptides including this amino acid sequence have high biological activity and good anti-aging, anti-inflammatory, tissue repair, hair growth, scar removal and acne removal effects. They are superior to most of the currently known polypeptide/protein products, are safe, stable, biocompatible, and have low production costs. They are particularly suitable for the field of functional skin care product raw materials and are suitable for the production of a variety of functional skin care products that can be used for a long time.
(3)重组蛋白显著提高功效：A+多肽与人胶原蛋白合成体系相融，采用基因工程技术，通过生物法合成的A+重组Ⅲ型胶原蛋白能够明显增强系列A+多肽的功效，包括但不限于抗衰、抗炎、组织修复、生发、祛疤和祛痘等，A+重组人Ⅲ型胶原蛋白增加了较好的抗炎和生发功能(迄今未见其他重组人胶原蛋白报道有此功效)，达到了1+1＞2的效果，而且安全性好；其中SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21的的抗炎、祛疤和生发作用明显优于ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10。(3) Recombinant protein significantly improves efficacy: A+ polypeptide is integrated with the human collagen synthesis system. The A+ recombinant type III collagen synthesized by genetic engineering technology can significantly enhance the efficacy of the A+ polypeptide series, including but not limited to anti-aging, anti-inflammation, tissue repair, hair growth, scar removal and acne removal. A+ recombinant human type III collagen has added better anti-inflammatory and hair growth functions (no other recombinant human collagen has been reported to have such effects so far), achieving the effect of 1+1＞2, and has good safety; among them, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, S The anti-inflammatory, scar removal and hair growth effects of EQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 are significantly better than those of ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.
(4)质量可控：重组人源Ⅲ型胶原蛋白的纯化方法通过在温度为65～75℃条件下热处理，由具有表达出重组人源Ⅲ型胶原蛋白能力的重组酵母菌发酵得到的发酵液,有效地防止了发酵液中的重组人源Ⅲ型胶原蛋白降解成小分子肽,并降低发酵液中内毒素的产生,大大提高了重组人源Ⅲ型胶原蛋白纯度。基因工程技术可表达特定分子量的类人胶原片段，批次之间重复性好，而从动物提取的胶原蛋白通常为不同种类胶原蛋白的混合产物，批次之间差异大，不利于质量控制；化学合成不能保证多肽/蛋白的生物活性。(4) Controllable quality: The purification method of recombinant human type III collagen is to heat-treat at 65-75°C, and the fermentation broth obtained by fermentation of recombinant yeast with the ability to express recombinant human type III collagen effectively prevents the degradation of recombinant human type III collagen in the fermentation broth into small molecular peptides, and reduces the production of endotoxins in the fermentation broth, greatly improving the purity of recombinant human type III collagen. Genetic engineering technology can express human-like collagen fragments of specific molecular weight, with good reproducibility between batches, while collagen extracted from animals is usually a mixed product of different types of collagen, with large differences between batches, which is not conducive to quality control; chemical synthesis cannot guarantee the biological activity of polypeptides/proteins.
(5)较低的排异反应：类人胶原蛋白以人体胶原蛋白基因为基础构建，其免疫排异反应比动物来源的胶原蛋白小。(5) Lower rejection reaction: Human-like collagen is constructed based on human collagen genes, and its immune rejection reaction is smaller than that of collagen from animal sources.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1SDS-PAGE检测A+重组胶原蛋白工程菌的表达情况。泳道1：诱导前；泳道2-4：3种不同重组胶原蛋白工程菌表达情况(稀释2倍)。Figure 1 SDS-PAGE detection of the expression of A+ recombinant collagen engineering bacteria. Lane 1: before induction; Lanes 2-4: expression of 3 different recombinant collagen engineering bacteria (diluted 2 times).
图2SDS-PAGE检测A+多肽3重组胶原蛋白表达形式，泳道1：表达全菌；泳道2：超声破碎上清液；泳道3：超声破碎沉淀。Figure 2 SDS-PAGE detection of the expression form of A+ polypeptide 3 recombinant collagen, lane 1: whole expression bacteria; lane 2: ultrasonic supernatant; lane 3: ultrasonic precipitate.
图3SDS-PAGE检测A+多肽3重组胶原蛋白纯化结果，泳道1：纯化流穿液；泳道2：纯化后(10倍稀释)；泳道3：纯化后(20倍稀释)。Figure 3 SDS-PAGE detection of A+ polypeptide 3 recombinant collagen purification results, lane 1: purification flow-through; lane 2: after purification (10-fold dilution); lane 3: after purification (20-fold dilution).
图4具有代表性的A+多肽的氢谱分析结果。Figure 4 shows representative hydrogen spectrum analysis results of A+ peptides.
图5培养72h样品孔镜下观察A+多肽3试验组较阴性对照组3T3细胞较0h细胞增多(100×)。Figure 5: 72h culture sample microscopy observation shows that the number of 3T3 cells in the A+ polypeptide 3 test group is more than that in the negative control group when compared with that at 0h (100×).
图6培养72h样品孔镜下观察阴性对照组3T3细胞较0h细胞大部分死亡(100×)。Figure 6: 72h culture sample under microscopy, the negative control group 3T3 cells were mostly dead compared with 0h cells (100×).
图7空白对照组(NC)、纤连蛋白组(FN)和各实验组0h和24h细胞迁移情况(40×)：显示FN组细胞迁移率为100％，A+多肽3重组胶原蛋白组、A+多肽1组和A+多肽3组细胞迁移率均较空白对照组明显增高，A+多肽3重组胶原蛋白组细胞迁移率明显高于Ⅲ型胶原蛋白组。Figure 7 Cell migration at 0h and 24h in the blank control group (NC), fibronectin group (FN) and experimental groups (40×): The cell migration rate of the FN group was 100%, and the cell migration rates of the A+ polypeptide 3 recombinant collagen group, A+ polypeptide 1 group and A+ polypeptide 3 group were significantly higher than those of the blank control group, and the cell migration rate of the A+ polypeptide 3 recombinant collagen group was significantly higher than that of the type III collagen group.
图8建模第45天各组小鼠的毛发生长情况，A+多肽3重组胶原蛋白组、A+多肽1和A+多肽3组比较模型组毛发生长更快，与阴性对照比较无显著差异；A+多肽3重组胶原蛋白组毛发生长明显快于胶原蛋白组。Figure 8 shows the hair growth of mice in each group on the 45th day after modeling. The hair growth of A+ polypeptide 3 recombinant collagen group, A+ polypeptide 1 and A+ polypeptide 3 groups was faster than that of the model group, and there was no significant difference compared with the negative control; the hair growth of A+ polypeptide 3 recombinant collagen group was significantly faster than that of the collagen group.
图9免疫组化结果显示模型组皮肤毛囊中VEGF蛋白表达减少，比较模型组，A+多肽3重组胶原蛋白组、A+多肽1组、A+多肽2组、A+多肽3组、A+多肽4组、A+多肽5组、A+多肽6组和A+多肽7组VEGF蛋白表达明显增加，与阴性对照比较无显著差异；A+多肽3重组胶原蛋白组VEGF蛋白表达明显高于胶原蛋白组。The immunohistochemistry results in Figure 9 showed that the expression of VEGF protein in the skin hair follicles of the model group was reduced. Compared with the model group, the expression of VEGF protein in the A+ polypeptide 3 recombinant collagen group, A+ polypeptide 1 group, A+ polypeptide 2 group, A+ polypeptide 3 group, A+ polypeptide 4 group, A+ polypeptide 5 group, A+ polypeptide 6 group and A+ polypeptide 7 group was significantly increased, and there was no significant difference compared with the negative control; the expression of VEGF protein in the A+ polypeptide 3 recombinant collagen group was significantly higher than that in the collagen group.
图10某痤疮患者接受0.5％A+多肽3溶液治疗8周后红斑、丘疹、粉刺好转。Figure 10: An acne patient's erythema, papules and comedones improved after 8 weeks of treatment with 0.5% A+ polypeptide 3 solution.
图11某中度痤疮患者接受0.5％A+多肽9重组胶原蛋白溶液治疗8周后丘疹、脓疱、粉刺明显好转。Figure 11 A patient with moderate acne had significant improvement in papules, pustules and comedones after 8 weeks of treatment with 0.5% A+ polypeptide 9 recombinant collagen solution.
图12某中度痤疮患者接受0.5％A+多肽18重组胶原蛋白溶液治疗4周后丘疹、脓疱、粉刺明显好转。 Figure 12 A patient with moderate acne showed significant improvement in papules, pustules and comedones after 4 weeks of treatment with 0.5% A+ polypeptide 18 recombinant collagen solution.
具体实施方式Detailed ways
为了进一步了解本申请，下面结合最佳实施例对本申请作进一步的详细说明。In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.
以下的实施例便于更好地理解本发明，但并不限定本发明。The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.
如无特别说明，本发明实施例中的实验方法，均为常规方法。Unless otherwise specified, the experimental methods in the embodiments of the present invention are all conventional methods.
如无特别说明，本发明所用的试验材料，均为自常规生化试剂商店购买得到的。Unless otherwise specified, the test materials used in the present invention were purchased from conventional biochemical reagent stores.
如无特别说明，本发明所用制剂配方中的百分比均为重量百分比。Unless otherwise specified, the percentages in the formulations used in the present invention are all percentages by weight.
实施例1制备A+多肽重组人源Ⅲ型胶原蛋白的方法Example 1 Method for preparing A+ polypeptide recombinant human type III collagen
1.材料1. Materials
E.coli DH5α感受态细胞、BL21(DE3.0)感受态细胞、BALB/c 3T3细胞；质粒由通用生物公司合成；发酵培养基购自OXOID公司。E.coli DH5α competent cells, BL21 (DE3.0) competent cells, BALB/c 3T3 cells; plasmids were synthesized by General Biotechnology; fermentation medium was purchased from OXOID.
2.设备与仪器2. Equipment and Instruments
-80℃超低温冰箱、超净工作台、恒温培养箱、恒温摇床、高压灭菌锅、核酸电泳仪、蛋白质电泳仪、金属浴、PCR仪、水浴锅、凝胶成像仪、台式离心机、冷冻离心机、高压均质仪、GE纯化仪、制冰机、台式离心机、二氧化碳培养箱等。-80℃ ultra-low temperature refrigerator, clean bench, constant temperature incubator, constant temperature shaker, high pressure sterilizer, nucleic acid electrophoresis instrument, protein electrophoresis instrument, metal bath, PCR instrument, water bath, gel imager, desktop centrifuge, refrigerated centrifuge, high pressure homogenizer, GE purifier, ice maker, desktop centrifuge, carbon dioxide incubator, etc.
3方法3 Methods
3.1目的基因的获取3.1 Acquisition of target genes
合成目的基因并获得表达质粒和工程菌：根据Col3A1多肽序列，在羧基端分别添加A+多肽1的氨基酸序列：AVEYPYKHSGYYHR，或A+多肽2的氨基酸序列：AVEFPFKWSGYYHR，或A+多肽3的氨基酸序列：EYPYKHSGYYHRAV。以大肠杆菌为原核表达***，构建工程菌。目的基因送公司进行优化合成并连接相应表达质粒。同样的方法合成并连接A+多肽4～22氨基酸序列的相应表达质粒。Synthesize the target gene and obtain the expression plasmid and engineered bacteria: According to the Col3A1 polypeptide sequence, add the amino acid sequence of A+ polypeptide 1: AVEYPYKHSGYYHR, or the amino acid sequence of A+ polypeptide 2: AVEFPFKWSGYYHR, or the amino acid sequence of A+ polypeptide 3: EYPYKHSGYYHRAV at the carboxyl end. Use Escherichia coli as the prokaryotic expression system to construct the engineered bacteria. Send the target gene to the company for optimization synthesis and connection to the corresponding expression plasmid. Use the same method to synthesize and connect the corresponding expression plasmid of the amino acid sequence of A+ polypeptide 4 to 22.
3.2Ⅲ型人源化胶原融合蛋白的合成3.2 Synthesis of humanized collagen type III fusion protein
选取Ⅲ型人源胶原蛋白(COL3A1,NCBI,GenBank:AGL34959.1)功能区片段共30个氨基酸，即人源胶原蛋白Ⅲ型alpha-1亚型483位到512位氨基酸，SEQ ID NO.23：GERGAPGFRGPAGPNGIPGEKGPAGERGAP。重组Ⅲ型人源化胶原融合蛋白的氨基酸序列包括(483G-512G)×n。A functional region fragment of type III human collagen (COL3A1, NCBI, GenBank: AGL34959.1) with a total of 30 amino acids was selected, namely, amino acids 483 to 512 of type III alpha-1 subtype of human collagen, SEQ ID NO.23: GERGAPGFRGPAGPNGIPGEKGPAGERGAP. The amino acid sequence of the recombinant type III humanized collagen fusion protein includes (483G-512G)×n.
其中，n表示为人源胶原蛋白Ⅲ型alpha-1亚型483位到512位氨基酸直接连续重复n次，n为大于等于1的整数。Wherein, n represents the amino acids 483 to 512 of human collagen type III alpha-1 subtype directly and continuously repeated n times, and n is an integer greater than or equal to 1.
合成的重组Ⅲ型人源化胶原融合蛋白，n＝13，具体序列为：SEQ ID NO.24：
Synthesized recombinant type III humanized collagen fusion protein, n=13, specific sequence: SEQ ID NO.24:
3.3表达工程菌构建3.3 Construction of expression engineering bacteria
将融合蛋白质粒分别转化到E.coli DH5α感受态细胞中，经抗性筛选后挑取阳性转化子进行培养。再提取质粒进行测序。测序无误证明成功获得表达质粒。 The fusion protein plasmids were transformed into E.coli DH5α competent cells, and positive transformants were selected for culture after resistance screening. The plasmids were then extracted for sequencing. The correct sequencing proved that the expression plasmid was successfully obtained.
3.4工程菌形态、生长特性鉴定3.4 Identification of engineered bacteria morphology and growth characteristics
对工程菌进行革兰氏染色镜检，结果呈红色杆菌形态，大小约为0.3μm×1.0μm左右。工程菌能在含有相应抗性的LB平板上正常生长，37℃培养24h，形成圆形、边缘整齐、突起、乳白色有光泽的光滑菌落，呈典型大肠埃希菌落形态。Gram staining of the engineered bacteria showed red bacilli, with a size of about 0.3μm×1.0μm. The engineered bacteria can grow normally on LB plates containing corresponding resistances, and after culturing at 37℃ for 24h, they form round, smooth, protruding, milky white and shiny colonies, which are typical Escherichia coli colony morphology.
3.5表达量及表达形式的鉴定3.5 Identification of expression level and expression form
菌种用LB培养基复壮，经37℃扩大培养后，以终浓度为1Mm IPTG、30℃培养诱导5h。收集菌体后进行超声破碎、离心，分别留取破碎后的菌体、离心后的上清和沉淀做SDS-PAGE电泳分析，并用软件分析目的蛋白表达水平。The strain was rejuvenated with LB medium, expanded and cultured at 37°C, and induced with a final concentration of 1 Mm IPTG at 30°C for 5 h. The bacterial cells were collected and ultrasonically disrupted and centrifuged, and the disrupted bacterial cells, supernatant after centrifugation and precipitate were retained for SDS-PAGE electrophoresis analysis, and the expression level of the target protein was analyzed using software.
3.6蛋白纯化3.6 Protein purification
离心收集培养液上清，用0.22μm滤膜过滤用以上样。使用GE Healthcare公司Chelating Sepharose TM Fast Flow镍离子螯合亲和层析填料自行装柱，用3个柱体积的纯化水清洗Ni2+螯合亲和层析柱，再用PBS平衡2-3个柱体积。在线检测电导率值及280nm波长吸收值，待两者都稳定后开始上样，设置样品经过泵过层析柱的流速5-6ml/min。再用PBS过层析柱，洗去未与层析柱结合的杂蛋白，直到OD280nm稳定。再以含500Mm咪唑PBS缓冲液过层析柱，梯度洗脱并收集洗脱峰对应的蛋白。Collect the culture supernatant by centrifugation and filter with a 0.22μm filter membrane for the above sample. Use GE Healthcare's Chelating Sepharose TM Fast Flow nickel ion chelate affinity chromatography filler to load the column by yourself, wash the Ni2+ chelate affinity chromatography column with 3 column volumes of purified water, and then balance it with PBS for 2-3 column volumes. Detect the conductivity value and the 280nm wavelength absorbance value online, and start loading the sample after both are stable. Set the flow rate of the sample through the chromatography column to 5-6ml/min. Then use PBS to pass through the chromatography column to wash away the impurities that are not bound to the chromatography column until OD280nm is stable. Then pass the chromatography column with PBS buffer containing 500Mm imidazole, gradient elute and collect the protein corresponding to the elution peak.
4.结果4. Results
4.1 A+多肽重组人源Ⅲ型胶原融合蛋白工程菌表达鉴定4.1 Identification of the expression of A+ polypeptide recombinant human type III collagen fusion protein in engineered bacteria
挑取多个单菌落，培养、转接，诱导表达。经SDS-PAGE电泳检测相应优势表达条带，说明工程菌成功表达(见图1)。超声破碎后，分别取破碎全菌、上清和沉淀，检测结果显示90％为上清表达(见图2)。Pick multiple single colonies, culture, transfer, and induce expression. Detect the corresponding dominant expression bands by SDS-PAGE electrophoresis, indicating that the engineered bacteria are successfully expressed (see Figure 1). After ultrasonic disruption, take the whole broken bacteria, supernatant and precipitate respectively, and the test results show that 90% is expressed in the supernatant (see Figure 2).
4.2表达与纯化4.2 Expression and purification
发酵2L，诱导表达。收集菌体，经高压均质，离心收集上清液。过滤，经亲和层析纯化可得到纯度大于95％的目的A+重组人源Ⅲ型胶原融合蛋白(见图3)。Ferment 2L, induce expression, collect the cells, homogenize under high pressure, collect the supernatant by centrifugation, filter, and purify by affinity chromatography to obtain the target A+ recombinant human type III collagen fusion protein with a purity greater than 95% (see Figure 3).
成功构建A+多肽重组人源Ⅲ型胶原融合蛋白工程菌，成功诱导表达A+多肽重组人源Ⅲ型胶原融合蛋白。经表达形式检测，目的蛋白为上清表达，纯度大于95％。The A+ polypeptide recombinant human type III collagen fusion protein engineering bacteria were successfully constructed, and the A+ polypeptide recombinant human type III collagen fusion protein was successfully induced to express. After expression form detection, the target protein was expressed in the supernatant with a purity greater than 95%.
同样的方法合成纯化重组Ⅲ型人源化胶原蛋白1和2～22。The same method was used to synthesize and purify recombinant humanized type III collagen 1 and 2-22.
实施例2系列A+多肽对皮肤成纤维细胞增殖的影响Example 2 Effects of Series A+ Peptides on the Proliferation of Skin Fibroblasts
1.材料1. Materials
BALB/C 3T3细胞株；BALB/C 3T3 cell line;
A+多肽系列和DZ多肽系列：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示。系列DZ多肽氨基酸序列如下：DZ多肽1：EYPYKHSGYYHR(SEQ ID NO.25)，DZ多肽2：EYTYEGAGYYHRP(SEQ ID NO.26)，DZ多肽3：EYTYNGAGYYHR(SEQ ID NO.27)，DZ多肽4：EYTYKGSGYYHR(SEQ ID NO.28)，DZ多肽5：EYPWKGSGYYHRP(SEQ ID NO.29)，DZ多肽6：EYTFKHSGYYHRP(SEQ ID NO.30)，DZ多肽7：EYPYDYSGYYHRP(SEQ ID NO.31)，DZ多肽8：EFPFKWSGYYHR(SEQ ID NO.32)，DZ多肽9：EYAFAGAGYYHRP(SEQ ID NO.33)，DZ多肽10：EFPWPHAGYYHRP(SEQ ID NO.34)在以下实验中作为实验对照组；A+多肽1重组人源Ⅲ型胶原融合蛋白(简称A+重组胶原蛋白1)和A+多肽3重组人源Ⅲ型胶原融合蛋白(简称A+重组胶原蛋白3)：A+ polypeptide series and DZ polypeptide series: synthesized by Shanghai Biotech, with a purity greater than 95%. The amino acid sequences of the A+ polypeptide series are as shown in the above invention content, and the representative hydrogen spectrum analysis results of the A+ polypeptide are shown in Figure 4. The amino acid sequences of the DZ polypeptide series are as follows: DZ polypeptide 1: EYPYKHSGYYHR (SEQ ID NO.25), DZ polypeptide 2: EYTYEGAGYYHRP (SEQ ID NO.26), DZ polypeptide 3: EYTYNGAGYYHR (SEQ ID NO.27), DZ polypeptide 4: EYTYKGSGYYHR (SEQ ID NO.28), DZ polypeptide 5: EYPWKGSGYYHRP (SEQ ID NO.29), DZ polypeptide 6: EYTFKHSGYYHRP (SEQ ID NO.30 ), DZ polypeptide 7: EYPYDYSGYYHRP (SEQ ID NO.31), DZ polypeptide 8: EFPFKWSGYYHR (SEQ ID NO.32), DZ polypeptide 9: EYAFAGAGYYHRP (SEQ ID NO.33), DZ polypeptide 10: EFPWPHAGYYHRP (SEQ ID NO.34) were used as experimental control groups in the following experiments; A+ polypeptide 1 recombinant human type III collagen fusion protein (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen fusion protein (referred to as A+ recombinant collagen 3):
由实施例1所示方法合成；人源Ⅲ型胶原蛋白(同实施例1所示)；A醇：又名视黄醇(南京中益旭元生物科技公司生产)；纤连蛋白：武汉艾美捷科技有限公司生产。Synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); A-alcohol: also known as retinol (produced by Nanjing Zhongyi Xuyuan Biotechnology Co., Ltd.); Fibronectin: produced by Wuhan Aimejie Technology Co., Ltd.
2.方法2. Methods
在体外功效模型中，成纤维细胞增殖实验属于较成熟且有效的体外评测皮肤抗皱紧致功效的手段。本发明中设计的试验通过促进3T3细胞的增殖试验，来检测包含所属A+多肽对细胞的增值促进效果。A醇(视黄醇)是目前国家药监局批准的护肤品可添加成份目录中公认抗衰效果好的成分，在本实验中作为阳性对照。In the in vitro efficacy model, the fibroblast proliferation experiment is a relatively mature and effective means of evaluating the anti-wrinkle and firming efficacy of the skin in vitro. The experiment designed in the present invention detects the effect of the A+ polypeptide on promoting the proliferation of cells by promoting the proliferation of 3T3 cells. Retinol (retinol) is a well-recognized ingredient with good anti-aging effect in the list of ingredients that can be added to skin care products approved by the State Food and Drug Administration, and is used as a positive control in this experiment.
MTT比色法测定促皮肤成纤维细胞增殖活性。The proliferation activity of skin fibroblasts was determined by MTT colorimetric method.
3.结果 3. Results
在DMEM培养基条件下培养成纤维细胞BALB/C 3T3细胞株，72h检测细胞活性。阴性对照孔细胞仅有极少数细胞存活，而A+多肽组的细胞数量增多，见图5和6。检测系列A+多肽、系列DZ多肽、人源Ⅲ型胶原蛋白和A+重组胶原蛋白和实验组OD值结果如表1所示。细胞增殖活性结果如下表2所示。可见系列A+多肽、系列DZ多肽、人源Ⅲ型胶原蛋白和A+重组胶原蛋白实验组的OD值和细胞增殖活性均高于阴性对照组，有统计学差异(P均＜0.01)；系列A+多肽实验组的OD值和细胞增殖活性均优于系列DZ多肽组，有统计学差异(P均＜0.05)。A+重组胶原蛋白组的OD值和细胞增殖活性均明显优于人源Ⅲ型胶原蛋白组，有统计学差异(P＜0.01)。纤连蛋白组与阴性对照组比较无统计学差异。Fibroblast BALB/C 3T3 cell line was cultured in DMEM medium, and cell activity was detected for 72 hours. Only a few cells survived in the negative control wells, while the number of cells in the A+ polypeptide group increased, as shown in Figures 5 and 6. The results of the detection of the OD values of the series A+ polypeptides, series DZ polypeptides, human type III collagen, A+ recombinant collagen and experimental groups are shown in Table 1. The results of cell proliferation activity are shown in Table 2 below. It can be seen that the OD values and cell proliferation activities of the series A+ polypeptides, series DZ polypeptides, human type III collagen and A+ recombinant collagen experimental groups were higher than those of the negative control group, with statistical differences (all P < 0.01); the OD values and cell proliferation activities of the series A+ polypeptide experimental group were better than those of the series DZ polypeptide group, with statistical differences (all P < 0.05). The OD value and cell proliferation activity of the A+ recombinant collagen group were significantly better than those of the human type III collagen group, with statistical differences (P < 0.01). There was no statistical difference between the fibronectin group and the negative control group.
表1各组OD值检测结果
Table 1 OD value test results of each group
表2各组细胞增殖活性效价测定结果

Table 2 Results of cell proliferation activity titer determination in each group

所述一系列A+多肽、DZ多肽和A+多肽重组胶原蛋白均能抑制成纤维细胞衰老，促进成纤维细胞增殖，具有抗衰的作用，系列A+多肽抑制成纤维细胞衰老，促进成纤维细胞增殖明显好于系列DZ多肽；A+多肽与Ⅲ型胶原蛋白重组结合能明显提高抗衰作用；纤连蛋白几乎没有促进成纤维细胞增殖的作用。The series of A+ polypeptides, DZ polypeptides and A+ polypeptide recombinant collagen can inhibit fibroblast aging, promote fibroblast proliferation, and have anti-aging effects. The series of A+ polypeptides inhibit fibroblast aging and promote fibroblast proliferation significantly better than the series of DZ polypeptides; the combination of A+ polypeptide and type III collagen recombinant can significantly improve the anti-aging effect; fibronectin has almost no effect on promoting fibroblast proliferation.
实验结果表明，本发明公开的一系列A+多肽和A+多肽重组人源Ⅲ型胶原蛋白均具有较强促进成纤维细胞增殖作用，A+多肽和人源Ⅲ型胶原蛋白结合能明显提高对成纤维细胞的增殖作用，具有抗衰抗皱紧致作用。The experimental results show that the series of A+ polypeptides and A+ polypeptide recombinant human type III collagen disclosed in the present invention have a strong effect on promoting fibroblast proliferation. The combination of A+ polypeptide and human type III collagen can significantly enhance the proliferation of fibroblasts and has anti-aging, anti-wrinkle and firming effects.
实施例3 A+多肽对体外细胞划痕修复的作用Example 3 Effect of A+ polypeptide on in vitro cell scratch repair
1.材料1. Materials
供试品：纤连蛋白(FN)：武汉艾美捷科技有限公司生产；A醇：又名视黄醇(南京中益旭元生物科技公司生产)；系列A+多肽和系列DZ多肽：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示；系列DZ多肽氨基酸序列如实施例2所示，在以下实验中作为实验对照组；A+多肽1重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白1)和A+多肽3重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白3)：由实施例1所示方法合成；人源Ⅲ型胶原蛋白(同实施例1所示)。Test products: fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.; A alcohol: also known as retinol (produced by Nanjing Zhongyi Xuyuan Biotechnology Co., Ltd.); series A+ polypeptides and series DZ polypeptides: synthesized by Shanghai Shenggong, with a purity greater than 95%. The amino acid sequence of the series A+ polypeptides is as shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4; the amino acid sequence of the series DZ polypeptides is as shown in Example 2, and is used as the experimental control group in the following experiments; A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1).
细胞株：Balb/c 3T3细胞，购自美国ATCC。 Cell line: Balb/c 3T3 cells were purchased from ATCC, USA.
其他试剂：细胞营养液、无血清培养基、PBS、0.25％胰蛋白酶Other reagents: cell nutrient solution, serum-free culture medium, PBS, 0.25% trypsin
其他物品：96孔细胞培养板、TIP头和微量移液器，试验前进行灭菌或过滤除菌处理。Other items: 96-well cell culture plates, TIP tips and micropipettes should be sterilized or filtered before the experiment.
2.方法2. Methods
(1)用Marker笔在6孔板背后，用直尺对齐，均匀得划横线，大约每隔0.5～1cm一道，横穿过孔。(1) Use a marker pen to align with a ruler on the back of the 6-well plate and draw horizontal lines evenly across the holes, approximately every 0.5 to 1 cm.
(2)向6孔板孔中加入浓度为5×105个/ml的细胞悬液2ml。(2) Add 2 ml of cell suspension with a concentration of 5×105 cells/ml to each well of a 6-well plate.
(3)第二天观察到6孔板内细胞均长满单层，用枪头比着直尺，垂直于背后的横线划痕，每孔内划2道。(3) On the second day, observe that all cells in the 6-well plate have grown into a monolayer. Use the tip of the gun to measure the ruler and make two scratches perpendicular to the horizontal line on the back of the plate.
(4)用PBS洗细胞3次，洗掉划下的悬浮细胞。(4) Wash the cells three times with PBS to remove the suspended cells.
(5)根据分组往孔中加入1.8ml无血清培养液，之后加入200μl的样品，细胞对照孔加入等量的PBS溶液。(5) Add 1.8 ml of serum-free culture medium to each well according to the grouping, then add 200 μl of sample, and add an equal amount of PBS solution to the cell control well.
(6)放入37度5％CO2培养箱，培养。0时拍照，记录每孔内拍照位置。后续观察时对固定位置进行观察、拍照。(6) Place the cells in a 37°C 5% CO2 incubator and culture. Take photos at 0:00 and record the photo location in each well. During subsequent observations, observe and take photos of the fixed locations.
3.结果3. Results
细胞对照孔与各个样品孔0h与24h观察结果如图7所示。The observation results of the cell control well and each sample well at 0 h and 24 h are shown in FIG7 .
数据处理：面积检测方法(划痕距离测量为等效测量)Data processing: Area detection method (scratch distance measurement is equivalent measurement)
划痕宽度平均值＝划痕空隙面积/长度Average scratch width = scratch gap area / length
细胞迁移率＝(0h划痕宽度-培养后划痕宽度)/0h划痕宽度×100％Cell migration rate = (0h scratch width - scratch width after culture) / 0h scratch width × 100%
由此计算出体外促细胞迁移率(见表3)，可见系列A+多肽、系列DZ多肽、人源Ⅲ型胶原蛋白、A+重组胶原蛋白1和A+重组胶原蛋白3实验组Balb/c 3T3细胞迁移率均高于阴性对照组，比较有统计学差异(P均＜0.01)。A醇Balb/c 3T3细胞迁移率与阴性对照组比较没有统计学差异。系列A+多肽高于系列DZ多肽组，有统计学差异(P＜0.05)。A+多肽重组胶原蛋白组高于Ⅲ型胶原蛋白组，有统计学差异(P＜0.01)。The in vitro cell migration rate was calculated (see Table 3). It can be seen that the migration rates of Balb/c 3T3 cells in the experimental groups of series A+ polypeptide, series DZ polypeptide, human type III collagen, A+ recombinant collagen 1 and A+ recombinant collagen 3 were all higher than those in the negative control group, with statistical differences (all P < 0.01). There was no statistical difference in the migration rate of Balb/c 3T3 cells between A+ and the negative control group. The series A+ polypeptide was higher than the series DZ polypeptide group, with statistical differences (P < 0.05). The A+ polypeptide recombinant collagen group was higher than the type III collagen group, with statistical differences (P < 0.01).
表3各组细胞迁移率

Table 3 Cell migration rate in each group

在体外功效模型中，成纤维细胞迁移或移行实验属于较成熟有效的体外评测组织修复和重建功能的手段。纤连蛋白具有较强的促细胞迁移和组织修复功效，在本实验中作为阳性对照。基于体外细胞划痕修复实验模型，检测A+多肽是否有利于细胞的识别、移行，从而起到组织修复和重建的功效。当细胞长到融合成单层状态时，在融合的单层细胞上人为制造一个空白区域，称为“划痕”。划痕边缘的细胞会逐渐进入空白区域使“划痕”愈合。在细胞迁移过程中的开始时和定期捕获图像，通过比较图像以确定细胞迁移速率。In the in vitro efficacy model, the fibroblast migration or migration experiment is a relatively mature and effective means of evaluating tissue repair and reconstruction functions in vitro. Fibronectin has a strong effect of promoting cell migration and tissue repair, and is used as a positive control in this experiment. Based on the in vitro cell scratch repair experimental model, it is detected whether A+ polypeptide is conducive to cell recognition and migration, thereby playing a role in tissue repair and reconstruction. When the cells grow to a fused monolayer state, a blank area is artificially created on the fused monolayer cells, called a "scratch". The cells at the edge of the scratch will gradually enter the blank area to heal the "scratch". Images are captured at the beginning and regularly during the cell migration process, and the cell migration rate is determined by comparing the images.
实验结果表明系列A+多肽和A+多肽重组人源Ⅲ型胶原蛋白均具有促进成纤维细胞迁移能力，A+多肽和人源Ⅲ型胶原蛋白结合能明显提高组织修复功能。The experimental results show that the series A+ polypeptides and A+ polypeptide recombinant human type III collagen have the ability to promote fibroblast migration, and the combination of A+ polypeptide and human type III collagen can significantly improve tissue repair function.
实施例4 A+系列多肽或蛋白对小鼠耳部炎症模型的影响Example 4 Effects of A+ series peptides or proteins on mouse ear inflammation model
1.材料1. Materials
系列A+多肽和系列DZ多肽：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示。系列DZ多肽氨基酸序列如实施例2所示，在以下实验中作为实验对照组。Series A+ polypeptides and series DZ polypeptides: synthesized by Shanghai Biotech, with a purity greater than 95%. The amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4. The amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
A+多肽1重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白1)和A+多肽3重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白3)：由实施例1所示方法合成；人源Ⅲ型胶原蛋白(同实施例1所示)。A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1).
纤连蛋白(FN)：武汉艾美捷科技有限公司生产。Fibronectin (FN): produced by Wuhan Aimijie Technology Co., Ltd.
卵清蛋白(OVA)：PBS配成20g/L，保存于-20℃。Ovalbumin (OVA): Prepared at 20 g/L in PBS and stored at -20°C.
卡泊三醇搽剂(达力士搽剂)：丹麦利奥制药有限公司产品。Calcipotriol ointment (Dalixi ointment): a product of LEO Pharma Ltd. of Denmark.
阳性药：糠酸莫米松乳膏(艾洛松)，上海先灵葆雅制药有限公司产品。Positive drug: Mometasone furoate cream (Elosone), a product of Shanghai Schering-Plough Pharmaceutical Co., Ltd.
IL-4ELISA试剂盒：购自美国Raybiotech公司。IL-4 ELISA kit: purchased from Raybiotech, USA.
PCSK9ELISA试剂盒：美国R&D公司的Human Proprotein Convertase 9/PCSK9Quantikine ELISA Kit试剂盒。PCSK9 ELISA kit: Human Proprotein Convertase 9/PCSK9 Quantikine ELISA Kit from American R&D company.
各组乳膏和基质制备方法：基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，与适量多肽溶液形成不同浓度的混合乳剂。本实施例所用的乳膏基质是指多肽乳膏除去活性成分的基质成分。Preparation method of each group of creams and bases: the base components include methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), octadecyl alcohol (5%), glycerol (20%), alkyl aryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl paraben (0.1%), distilled water (about 31-55%), and form mixed emulsions of different concentrations with an appropriate amount of polypeptide solution. The cream base used in this example refers to the base component of the polypeptide cream without the active ingredient.
动物：SPF级健康纯系小鼠(C57BL/6)；8周龄。Animals: SPF-grade healthy pure strain mice (C57BL/6); 8 weeks old.
2.方法2. Methods
购SPF级雌性8周龄C57BL/6小鼠，随机分为阴性对照组、模型组、阳性对照组、A+重组胶原蛋白组1 (皮肤涂抹0.25％A+多肽1重组Ⅲ型胶原蛋白乳膏)、A+重组胶原蛋白组3(皮肤涂抹0.25％A+多肽3重组Ⅲ型胶原蛋白乳膏)、胶原蛋白组(皮肤涂抹0.25％Ⅲ型胶原蛋白乳膏)、纤连蛋白组(皮肤涂抹0.25％纤连蛋白乳膏)、多肽组1(皮肤涂抹0.25％A+多肽1号乳膏)、多肽组2(皮肤涂抹0.25％A+多肽3号乳膏)、多肽组3(皮肤涂抹0.25％A+多肽5号乳膏)、多肽组4(皮肤涂抹0.25％A+多肽6号乳膏)、多肽组5(皮肤涂抹0.25％A+多肽7号乳膏)、多肽组6(皮肤涂抹0.25％A+多肽9号乳膏)、多肽组7(皮肤涂抹0.25％A+多肽10号乳膏)、A+多肽组8(皮肤涂抹1％A+多肽11号乳膏)、A+多肽组9(皮肤涂抹1％A+多肽12号乳膏)、A+多肽组10(皮肤涂抹1％A+多肽13号乳膏)、A+多肽组11(皮肤涂抹1％A+多肽15号乳膏)、A+多肽组12(皮肤涂抹1％A+多肽16号乳膏)、A+多肽组13(皮肤涂抹1％A+多肽17号乳膏)、A+多肽组14(皮肤涂抹1％A+多肽18号乳膏)、A+多肽组15(皮肤涂抹1％A+多肽20号乳膏)和A+多肽组16(皮肤涂抹1％A+多肽21号乳膏)，系列DZ多肽组(皮肤分别涂抹0.25％DZ多肽系列乳膏)，每组各6只。SPF female C57BL/6 mice aged 8 weeks were purchased and randomly divided into negative control group, model group, positive control group, A+ recombinant collagen group 1 (apply 0.25% A+ polypeptide 1 recombinant type III collagen cream on the skin), A+ recombinant collagen group 3 (apply 0.25% A+ polypeptide 3 recombinant type III collagen cream on the skin), collagen group (apply 0.25% type III collagen cream on the skin), fibronectin group (apply 0.25% fibronectin cream on the skin), polypeptide group 1 (apply 0.25% A+ polypeptide 1 cream on the skin), polypeptide group 2 (apply 0.25% A+ polypeptide 3 cream on the skin), polypeptide group 3 (apply 0.25% A+ polypeptide 5 cream on the skin), polypeptide group 4 (apply 0.25% A+ polypeptide 6 cream on the skin), polypeptide group 5 (apply 0.25% A+ polypeptide 7 cream on the skin), polypeptide group 6 (apply 0.25% A+ polypeptide 9 cream on the skin), polypeptide group 7 (apply 0.25% A+ polypeptide 1 cream on the skin). % A+ polypeptide No. 10 cream), A+ polypeptide group 8 (skin applied 1% A+ polypeptide No. 11 cream), A+ polypeptide group 9 (skin applied 1% A+ polypeptide No. 12 cream), A+ polypeptide group 10 (skin applied 1% A+ polypeptide No. 13 cream), A+ polypeptide group 11 (skin applied 1% A+ polypeptide No. 15 cream), A+ polypeptide group 12 (skin applied 1% A+ polypeptide No. 16 cream), A+ polypeptide group 13 (skin applied 1% A+ polypeptide No. 17 cream), A+ polypeptide group 14 (skin applied 1% A+ polypeptide No. 18 cream), A+ polypeptide group 15 (skin applied 1% A+ polypeptide No. 20 cream) and A+ polypeptide group 16 (skin applied 1% A+ polypeptide No. 21 cream), series DZ polypeptide groups (0.25% DZ polypeptide series creams were applied to the skin), 6 mice in each group.
造模：阴性对照组小鼠两侧耳部涂抹75％乙醇14.3ul，同时其余各组每日定时先在两侧耳部涂抹1nmoI/L卡泊三醇搽剂14.3ul，风干后涂抹20g/L的OVA 25ul，每日1次，连续涂抹12d造模。Modeling: 14.3ul of 75% ethanol was applied to both ears of mice in the negative control group. At the same time, 14.3ul of 1nmoI/L calcipotriol ointment was applied to both ears of mice in other groups on a regular basis every day. After air-drying, 25ul of 20g/L OVA was applied once a day for 12 consecutive days to establish the model.
造模开始后4天起，在空白对照组和模型组小鼠耳部皮肤上涂抹乳膏基质，阳性药组小鼠耳部皮肤上涂抹艾洛松，Ⅲ型胶原蛋白组、纤连蛋白组、各多肽组和A+多肽重组Ⅲ型胶原蛋白组小鼠耳部皮肤上分别涂抹上诉相应乳膏，每天1次，连续10天，每天拍照，进行评分。Starting from 4 days after the start of modeling, the cream base was applied on the ear skin of mice in the blank control group and the model group, the ear skin of mice in the positive drug group was applied with eloson, and the ear skin of mice in the type III collagen group, fibronectin group, each polypeptide group and A+ polypeptide recombinant type III collagen group was applied with the corresponding cream once a day for 10 consecutive days. Pictures were taken every day for scoring.
分别在造模前和第14天用皮肤测试仪随机检测小鼠背部受试区3处部位，记录经皮水分丢失量(TEWL)值，取平均值。Before modeling and on the 14th day, three test areas on the back of the mice were randomly tested with a skin tester, and the transepidermal water loss (TEWL) values were recorded and the average value was taken.
分别在造模前和第14天用耳厚度测量仪测量记录小鼠耳廓厚度。于第14天测量完毕后脱颈处死小鼠，取血分离血清和血浆。The thickness of the mouse auricle was measured and recorded with an ear thickness meter before modeling and on day 14. After the measurement was completed on day 14, the mice were killed by dislocation of the neck, and blood was collected to separate serum and plasma.
用兔抗小鼠白介素(IL)-4抗体包被酶标板，4℃过夜，染色并终止反应，检测血清IL-4水平。按照ELISA试剂盒说明书操作测定血浆中PCSK9浓度。The ELISA plate was coated with rabbit anti-mouse interleukin (IL)-4 antibody, incubated at 4°C overnight, stained and the reaction was terminated to detect serum IL-4 levels. The concentration of PCSK9 in plasma was determined according to the ELISA kit instructions.
3.结果3. Results
(1)小鼠耳厚度比较：造模前，各组间耳厚度差异无统计学意义(P＞0.05)。造模后，各组小鼠耳厚度见表6。胶原蛋白组和纤连蛋白组与模型组比较差异无统计学意义(P＞0.05)，其它各组明显低于模型组(P均＜0.01)，各A+多肽组明显低于各DZ多肽组(P均＜0.05)，A+多肽重组胶原蛋白组明显低于胶原蛋白组(P＜0.01)。(1) Comparison of ear thickness of mice: Before modeling, there was no significant difference in ear thickness among the groups (P>0.05). After modeling, the ear thickness of mice in each group is shown in Table 6. There was no significant difference between the collagen group and the fibronectin group and the model group (P>0.05), and the thickness of the other groups was significantly lower than the model group (all P<0.01), the thickness of each A+ polypeptide group was significantly lower than that of each DZ polypeptide group (all P<0.05), and the thickness of the A+ polypeptide recombinant collagen group was significantly lower than that of the collagen group (P<0.01).
(2)各组小鼠皮肤经皮水分丢失量(TEWL)比较：造模前，各组间TEWL值差异无统计学意义(P＞0.05)。造模14天后，各组小鼠TEWL值见表6。胶原蛋白组和纤连蛋白组与模型组比较差异无统计学意义(P＞0.05)，其它各组明显低于模型组(P均＜0.01)，各A+多肽组明显低于各DZ多肽组(P均＜0.05)，A+多肽重组胶原蛋白组明显低于胶原蛋白组(P＜0.01)。(2) Comparison of transepidermal water loss (TEWL) of mice in each group: Before modeling, there was no significant difference in TEWL values among the groups (P>0.05). 14 days after modeling, the TEWL values of mice in each group are shown in Table 6. There was no significant difference between the collagen group and the fibronectin group and the model group (P>0.05), and the TEWL values of the other groups were significantly lower than the model group (all P<0.01), the A+ polypeptide groups were significantly lower than the DZ polypeptide groups (all P<0.05), and the A+ polypeptide recombinant collagen group was significantly lower than the collagen group (P<0.01).
(3)血清IL-4浓度：各组小鼠外周血中血清IL-4水平见表6。模型组与胶原蛋白组和纤连蛋白组比较差异无统计学意义(P＞0.05)，除胶原蛋白组和纤连蛋白组外，其它各组明显低于模型组(P均＜0.01)，各A+多肽组明显低于系列DZ多肽组(P均＜0.05)，A+多肽重组胶原蛋白组明显低于胶原蛋白组(P均＜0.01)。(3) Serum IL-4 concentration: The serum IL-4 levels in the peripheral blood of mice in each group are shown in Table 6. There was no statistically significant difference between the model group and the collagen group and fibronectin group (P>0.05). Except for the collagen group and fibronectin group, the levels of serum IL-4 in other groups were significantly lower than the model group (all P<0.01), the levels of serum IL-4 in each A+ polypeptide group were significantly lower than the series of DZ polypeptide groups (all P<0.05), and the levels of serum IL-4 in the A+ polypeptide recombinant collagen group were significantly lower than the collagen group (all P<0.01).
(4)血浆中PCSK9的表达：除各DZ多肽组外，其余各组小鼠外周血血浆中PCSK9的表达比较差异无统计学意义(P均＞0.05)。(4) Expression of PCSK9 in plasma: Except for the DZ polypeptide groups, there was no statistically significant difference in the expression of PCSK9 in the peripheral blood plasma of the mice in the other groups (all P＞0.05).
表4造模后各组小鼠耳厚度和TEWL值

Table 4 Ear thickness and TEWL values of mice in each group after modeling

A+系列多肽和A+重组胶原蛋白均能通过抑制炎症因子IL-4来减轻炎症，同时减少皮肤经皮水分丢失量(TEWL)；Ⅲ型胶原蛋白和纤连蛋白几乎无抗炎功能，A+多肽与人源Ⅲ型胶原蛋白重组能明显增强抗炎作用。系列A+多肽和A+重组胶原蛋白对血浆中PCSK9的含量均无影响。A+ series peptides and A+ recombinant collagen can reduce inflammation by inhibiting the inflammatory factor IL-4, while reducing the transepidermal water loss (TEWL) of the skin; type III collagen and fibronectin have almost no anti-inflammatory function, and A+ peptides and human type III collagen recombinant can significantly enhance the anti-inflammatory effect. Series A+ peptides and A+ recombinant collagen have no effect on the content of PCSK9 in plasma.
实施例5 A+系列多肽或蛋白对雄性激素性脱发(AGA)大鼠模型的影响Example 5 Effects of A+ series polypeptides or proteins on the androgenic alopecia (AGA) rat model
1.材料1. Materials
系列A+多肽和系列DZ多肽：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示。系列DZ多肽氨基酸序列如实施例2所示，在以下实验中作为实验对照组。Series A+ polypeptides and series DZ polypeptides: synthesized by Shanghai Bioengineering, with a purity greater than 95%. The amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4. The amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
A+多肽1重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白1)和A+多肽3重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白3)：由实施例1所示方法合成；人源Ⅲ型胶原蛋白(同实施例1所示)；纤连蛋白(FN)：武汉艾美捷科技有限公司生产。A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
1％A+多肽或1％DZ多肽溶液、1％人源Ⅲ型胶原蛋白、1％A+重组人源Ⅲ型胶原蛋白和1％纤连蛋白溶液、配制方法：各组样品粉末分别加适量蒸馏水混匀配成。1% A+ polypeptide or 1% DZ polypeptide solution, 1% human type III collagen, 1% A+ recombinant human type III collagen and 1% fibronectin solution. Preparation method: add appropriate amount of distilled water to each group of sample powders and mix well.
5％米诺地尔液(曼迪)：由浙江万晟药业生产。5% Minoxidil Solution (Mandy): Produced by Zhejiang Wansheng Pharmaceutical.
2.动物分组与造模2. Animal grouping and modeling
选取SPF级Wistar大鼠，采用随机排列表法分为阴性对照组(涂抹蒸馏水)、模型对照组(涂抹蒸馏水)、纤连蛋白组(皮肤涂抹1％纤连蛋白溶液)、阳性对照组(皮肤涂抹5％米诺地尔液)、A+多肽重组胶原蛋白组1(皮肤涂抹1％A+多肽1重组Ⅲ型胶原蛋白溶液)、A+多肽重组胶原蛋白组3(皮肤涂抹1％A+多肽3重组Ⅲ型胶原蛋白溶液)、胶原蛋白组(皮肤涂抹1％Ⅲ型胶原蛋白溶液)，A+多肽组1(皮肤涂抹1％A+多肽1号溶液)、A+多肽组2(皮肤涂抹1％A+多肽3号溶液)、A+多肽组3(皮肤涂抹1％A+多肽5号溶液)、A+多肽组4(皮肤涂抹1％A+多肽7 号溶液)、A+多肽组5(皮肤涂抹1％A+多肽8号溶液)、A+多肽组6(皮肤涂抹1％A+多肽9号溶液)、A+多肽组7(皮肤涂抹1％A+多肽10号溶液)、A+多肽组8(皮肤涂抹1％A+多肽11号溶液)、A+多肽组9(皮肤涂抹1％A+多肽12号溶液)、A+多肽组10(皮肤涂抹1％A+多肽13号溶液)、A+多肽组11(皮肤涂抹1％A+多肽15号溶液)、A+多肽组12(皮肤涂抹1％A+多肽17号溶液)、A+多肽组13(皮肤涂抹1％A+多肽18号溶液)、A+多肽组14(皮肤涂抹1％A+多肽20号溶液)、A+多肽组15(皮肤涂抹1％A+多肽21号溶液)和A+多肽组16(皮肤涂抹1％A+多肽22号溶液)，以及系列DZ多肽组(皮肤分别涂抹1％DZ多肽系列溶液)。每组10只，雌雄各半。SPF Wistar rats were selected and randomly divided into negative control group (apply distilled water), model control group (apply distilled water), fibronectin group (apply 1% fibronectin solution on the skin), positive control group (apply 5% minoxidil solution on the skin), A+ polypeptide recombinant collagen group 1 (apply 1% A+ polypeptide 1 recombinant type III collagen solution on the skin), A+ polypeptide recombinant collagen group 3 (apply 1% A+ polypeptide 3 recombinant type III collagen solution on the skin), collagen group (apply 1% type III collagen solution on the skin), A+ polypeptide group 1 (apply 1% A+ polypeptide No. 1 solution on the skin), A+ polypeptide group 2 (apply 1% A+ polypeptide No. 3 solution on the skin), A+ polypeptide group 3 (apply 1% A+ polypeptide No. 5 solution on the skin), A+ polypeptide group 4 (apply 1% A+ polypeptide No. 7 solution on the skin). The invention relates to a group of 1+ polypeptide groups: (a) applying 1% A+ polypeptide solution No. 8 to the skin, (b) applying 1% A+ polypeptide solution No. 9 to the skin, (c) applying 1% A+ polypeptide solution No. 10 to the skin, (d) applying 1% A+ polypeptide solution No. 11 to the skin, (d) applying 1% A+ polypeptide solution No. 12 to the skin, (d) applying 1% A+ polypeptide solution No. 13 to the skin, (d) applying 1% A+ polypeptide solution No. 15 to the skin, (d) applying 1% A+ polypeptide solution No. 17 to the skin, (d) applying 1% A+ polypeptide solution No. 18 to the skin, (d) applying 1% A+ polypeptide solution No. 20 to the skin, (d) applying 1% A+ polypeptide solution No. 21 to the skin, (d) applying 1% A+ polypeptide solution No. 22 to the skin, and a series of DZ polypeptide groups (1% DZ polypeptide series solutions are applied to the skin respectively). There were 10 mice in each group, half male and half female.
实验前每只大鼠选取背部4cmx5cm面积的区域将毛脱去作为观察区。除阴性对照组外，其他各组大鼠颈后皮下注射丙酸睾酮注射液[5ml/(kg·d)，每天1次，连续60d，建立AGA模型。连续皮下注射丙酸***4周后大鼠逐渐出现毛发脱落.残存毛发变得纤细、质脆，证明雄性激素性脱发模型成功建立。造模同时于对应治疗组大鼠背部观察区皮肤分别涂抹各治疗组溶液，涂抹1mL/(只·次)，每日1次，连续60d。阴性对照组和模型对照组涂抹蒸馏水1mL/(只·次)，每日1次，连续60d。Before the experiment, each rat selected an area of 4cmx5cm on the back and removed the hair as the observation area. Except for the negative control group, the rats in other groups were subcutaneously injected with testosterone propionate injection [5ml/(kg·d) once a day for 60 consecutive days to establish the AGA model. After 4 weeks of continuous subcutaneous injection of testosterone propionate, the rats gradually lost their hair. The remaining hair became thin and brittle, proving that the androgenic alopecia model was successfully established. At the same time as the modeling, the skin of the observation area on the back of the corresponding treatment group rats was smeared with the solution of each treatment group, 1mL/(rat·time), once a day, for 60 consecutive days. The negative control group and the model control group were smeared with distilled water 1mL/(rat·time), once a day, for 60 consecutive days.
3.观察指标及测试方法3. Observation indicators and test methods
给药每15天于每只大鼠背部观察区拔取10根毛发，用游标卡尺测量毛发长度。给药60d后，取实验观察区皮肤，进行常规组织脱水、石蜡包埋、HE染色，光镜镜检，观察大鼠皮肤毛囊和皮脂腺组织病理学改变。对各组病变进行半定量分析。分级标准如下：皮肤真皮组织细胞和皮下毛囊、皮脂腺结构正常记为“一”：皮肤真皮未见有增生，毛囊、皮脂腺病变局限，皮下未见有炎症记为“±”：皮肤真皮组织未见有明显增生，毛囊明显囊性变.皮脂腺未见有明显增生，皮下未见有炎症记为“+”：皮肤真皮组织有节段性增生，不明显，少部分毛囊有囊性变，皮脂腺有轻度增生肥大.皮下未见有明显炎症记为“++”：皮肤真皮组织细胞有不同程度节段性增生，部分毛囊囊性变，表现毛囊大小不均匀，周边部无细胞。皮脂腺有增生，增生腺体内细胞核较少，个别大鼠皮下有轻度炎性增生记为“+++”。Every 15 days after administration, 10 hairs were plucked from the observation area on the back of each rat, and the hair length was measured with a vernier caliper. After 60 days of administration, the skin of the experimental observation area was taken, and routine tissue dehydration, paraffin embedding, HE staining, and light microscopy were performed to observe the pathological changes of the rat skin hair follicles and sebaceous glands. Semi-quantitative analysis of the lesions in each group was performed. The grading standards are as follows: "1" indicates normal structure of skin dermis tissue cells and subcutaneous hair follicles and sebaceous glands: "±" indicates no obvious hyperplasia of skin dermis tissue, obvious cystic changes of hair follicles and sebaceous glands, and no inflammation of subcutaneous tissue: "+" indicates segmental hyperplasia of skin dermis tissue, which is not obvious, cystic changes of a small number of hair follicles, and mild hyperplasia and hypertrophy of sebaceous glands. No obvious inflammation of subcutaneous tissue: "++" indicates segmental hyperplasia of skin dermis tissue cells to varying degrees, cystic changes of some hair follicles, uneven size of hair follicles, and no cells in the periphery. Sebaceous glands have hyperplasia, fewer nuclei in the hyperplastic glands, and mild inflammatory hyperplasia of subcutaneous tissue of some rats: "+++" indicates mild inflammatory hyperplasia of subcutaneous tissue of some rats.
4.结果4. Results
如表5所示，系列DZ多肽组、系列A+多肽和A+多肽重组胶原蛋白实验组在给药第15、30、45、60天毛发生长长度均高于模型对照组，比较有统计学差异(P均＜0.01)；系列DZ多肽组、系列A+多肽组和A+重组胶原蛋白组与阴性对照组和阳性对照组比较均无统计学差异；系列A+多肽组毛发长度长于系列DZ多肽组，比较有统计学差异(P＜0.05)；A+重组胶原蛋白组毛发长度长于人源Ⅲ型胶原蛋白组，比较有统计学差异(P＜0.05)；纤连蛋白组和Ⅲ型胶原蛋白组与模型对照组比较无统计学差异。As shown in Table 5, the hair growth length of the series DZ polypeptide group, series A+ polypeptide and A+ polypeptide recombinant collagen experimental group on the 15th, 30th, 45th and 60th days of administration were all longer than that of the model control group, with statistical differences (all P < 0.01); there were no statistical differences between the series DZ polypeptide group, series A+ polypeptide group and A+ recombinant collagen group and the negative control group and the positive control group; the hair length of the series A+ polypeptide group was longer than that of the series DZ polypeptide group, with statistical differences (P < 0.05); the hair length of the A+ recombinant collagen group was longer than that of the human type III collagen group, with statistical differences (P < 0.05); there were no statistical differences between the fibronectin group and the type III collagen group and the model control group.
表5建模后不同时间各组大鼠毛发生长长度


*与模型组比较差异均有统计学意义(P均＜0.01)Table 5 Hair growth length of rats in each group at different time after modeling


*Compared with the model group, the differences were statistically significant (P < 0.01)
所述一系列A+多肽和A+重组Ⅲ型胶原蛋白对雄性激素性脱发(AGA)大鼠的毛发具有明显的生发作用，系列A+多肽的生发作用明显强于系列DZ多肽，A+多肽与Ⅲ型胶原蛋白结合能明显提高生发作用；Ⅲ型胶原蛋白和纤连蛋白无明显生发作用。The series of A+ polypeptides and A+ recombinant type III collagen have obvious hair growth effects on the hair of rats with androgenic alopecia (AGA), the hair growth effects of the series A+ polypeptides are significantly stronger than those of the series DZ polypeptides, and the combination of the A+ polypeptides and type III collagen can significantly improve the hair growth effect; type III collagen and fibronectin have no obvious hair growth effect.
实施例6 A+多肽或A+重组Ⅲ型胶原蛋白对小鼠脱发模型的影响Example 6 Effects of A+ polypeptide or A+ recombinant type III collagen on a mouse hair loss model
1.实验方法1. Experimental Methods
系列A+多肽和系列DZ多肽：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示。系列DZ多肽氨基酸序列如实施例2所示，在以下实验中作为实验对照组。Series A+ polypeptides and series DZ polypeptides: synthesized by Shanghai Bioengineering, with a purity greater than 95%. The amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4. The amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
A+多肽1重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白1)和A+多肽3重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白3)：由实施例1所示方法合成；人源Ⅲ型胶原蛋白(同实施例1所示)；纤连蛋白(FN)：武汉艾美捷科技有限公司生产。A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
1％A+多肽或1％DZ多肽溶液、1％人源Ⅲ型胶原蛋白、1％A+重组人源Ⅲ型胶原蛋白和1％纤连蛋白溶液、配制方法：各组样品粉末分别加适量蒸馏水混匀配成。1% A+ polypeptide or 1% DZ polypeptide solution, 1% human type III collagen, 1% A+ recombinant human type III collagen and 1% fibronectin solution. Preparation method: add appropriate amount of distilled water to each group of sample powders and mix well.
2％米诺地尔液：由医科院皮肤病研究所生产。2% minoxidil solution: produced by the Institute of Dermatology, Academy of Medical Sciences.
2.动物分组与造模2. Animal grouping and modeling
选取SPF级C57BL/6小鼠，采用随机排列表法分为阴性对照组(涂抹蒸馏水)、模型对照组(涂抹蒸馏水)、纤连蛋白组(皮肤涂抹1％纤连蛋白溶液)、阳性对照组(皮肤涂抹2％米诺地尔液)、A+多肽重组胶原蛋白组1(皮肤涂抹1％A+多肽1重组Ⅲ型胶原蛋白溶液)、A+多肽重组胶原蛋白组3(皮肤涂抹1％A+多肽3重组Ⅲ型胶原蛋白溶液)、胶原蛋白组(皮肤涂抹1％Ⅲ型胶原蛋白溶液)，A+多肽组1(皮肤涂抹1％A+多肽1号溶液)、A+多肽组2(皮肤涂抹1％A+多肽3号溶液)、A+多肽组3(皮肤涂抹1％A+多肽5号溶液)、A+多肽组4(皮肤涂抹1％A+多肽7号溶液)、A+多肽组5(皮肤涂抹1％A+多肽8号溶液)、A+多肽组6(皮肤涂抹1％A+多肽9号溶液)、A+多肽组7(皮肤涂抹1％A+多肽10号溶液)、A+多肽组8(皮肤涂抹1％A+多肽11号溶液)、A+多肽组9(皮肤涂抹1％A+多肽12号溶液)、A+多肽组10(皮肤涂抹1％A+多肽14号溶液)、A+多肽组11(皮肤涂抹1％A+多肽16号溶液)、A+多肽组12(皮肤涂抹1％A+多肽18号溶液)、A+多肽组13(皮肤涂抹1％A+多肽19号溶液)、A+多肽组14(皮肤涂抹1％A+多肽20号溶液)、A+多肽组15(皮肤涂抹1％A+多肽21号溶液)和A+多肽组16(皮肤涂抹1％A+多肽22号溶液)，以及系列DZ多肽组(皮肤分别涂抹1％DZ多肽系列溶液)。每组10只，雌雄各半。SPF grade C57BL/6 mice were selected and randomly divided into negative control group (applying distilled water), model control group (applying distilled water), fibronectin group (applying 1% fibronectin solution on the skin), positive control group (applying 2% minoxidil solution on the skin), A+ polypeptide recombinant collagen group 1 (applying 1% A+ polypeptide 1 recombinant type III collagen solution on the skin), A+ polypeptide recombinant collagen group 3 (applying 1% A+ polypeptide 3 recombinant type III collagen solution on the skin), collagen group (applying 1% type III collagen solution on the skin), A+ polypeptide group 1 (applying 1% A+ polypeptide No. 1 solution on the skin), A+ polypeptide group 2 (applying 1% A+ polypeptide No. 3 solution on the skin), A+ polypeptide group 3 (applying 1% A+ polypeptide No. 5 solution on the skin), A+ polypeptide group 4 (applying 1% A+ polypeptide No. 7 solution on the skin), A+ polypeptide group 5 (applying 1% A+ polypeptide No. 8 solution on the skin) , A+ polypeptide group 6 (1% A+ polypeptide No. 9 solution was applied to the skin), A+ polypeptide group 7 (1% A+ polypeptide No. 10 solution was applied to the skin), A+ polypeptide group 8 (1% A+ polypeptide No. 11 solution was applied to the skin), A+ polypeptide group 9 (1% A+ polypeptide No. 12 solution was applied to the skin), A+ polypeptide group 10 (1% A+ polypeptide No. 14 solution was applied to the skin), A+ polypeptide group 11 (1% A+ polypeptide No. 16 solution was applied to the skin), A+ polypeptide group 12 (1% A+ polypeptide No. 18 solution was applied to the skin), A+ polypeptide group 13 (1% A+ polypeptide No. 19 solution was applied to the skin), A+ polypeptide group 14 (1% A+ polypeptide No. 20 solution was applied to the skin), A+ polypeptide group 15 (1% A+ polypeptide No. 21 solution was applied to the skin) and A+ polypeptide group 16 (1% A+ polypeptide No. 22 solution was applied to the skin), and a series of DZ polypeptide groups (1% DZ polypeptide series solutions were applied to the skin respectively). Each group had 10 rats, half of which were male and half were female.
3.观察指标及测试方法3. Observation indicators and test methods
给药每15天于每只小鼠背部观察区拔取10根毛发，用游标卡尺测量毛发长度。给药60d后，取实验观察区皮肤进行常规组织脱水、石蜡包埋、HE染色，光镜镜检，观察小鼠皮肤毛囊和皮脂腺组织病理学改变。对各组病变进行半定量分析。分级标准如下：皮肤真皮组织细胞和皮下毛囊、皮脂腺结构正常记为“一”：皮肤真皮未见有增生，毛囊、皮脂腺病变局限，皮下未见有炎症记为“±”：皮肤真皮组织未见有明显增生，毛囊明显囊性变.皮脂腺未见有明显增生，皮下未见有炎症记为“+”：皮肤真皮组织有节段性增生，不明显，少部分毛囊有囊性变，皮脂腺有轻度增生肥大.皮下未见有明显炎症记为“++”：皮肤真皮组织细胞有不同程度节段性增生，部分毛囊囊性变，表现毛囊大小不均匀，周边部无细胞。皮脂腺有增生，增生腺体内细胞核较少，个别皮下有轻度炎性增生记为 “+++”。Every 15 days after administration, 10 hairs were plucked from the observation area on the back of each mouse, and the hair length was measured with a vernier caliper. After 60 days of administration, the skin of the experimental observation area was taken for routine tissue dehydration, paraffin embedding, HE staining, and light microscopy to observe the pathological changes of the mouse skin hair follicles and sebaceous glands. Semi-quantitative analysis of the lesions in each group was performed. The grading standards are as follows: "1" indicates normal structure of skin dermis tissue cells and subcutaneous hair follicles and sebaceous glands: "±" indicates no obvious hyperplasia of skin dermis tissue, obvious cystic changes of hair follicles and sebaceous glands, and no inflammation of the subcutaneous tissue; "+" indicates segmental hyperplasia of skin dermis tissue, which is not obvious, cystic changes of a small number of hair follicles, and mild hyperplasia and hypertrophy of sebaceous glands. No obvious inflammation of the subcutaneous tissue is recorded as "++" indicates segmental hyperplasia of skin dermis tissue cells to varying degrees, cystic changes of some hair follicles, uneven size of hair follicles, and absence of cells in the periphery. Sebaceous glands hyperplasia, with fewer cell nuclei in the hyperplastic glands, and mild inflammatory hyperplasia of some subcutaneous tissues is recorded as "+++".
4.实验结果4. Experimental results
4.1各组对小鼠毛发生长的影响4.1 Effects of each group on hair growth in mice
系列A+多肽和A+重组Ⅲ型胶原蛋白组小鼠在给药第15、30、45、60天的毛发长度均长于模型对照组，差异均有统计学意义(P<0.01)，与系列DZ多肽治疗组比较，差异均有统计学意义(P<0.05)。见表7。建模第45天各组鼠的毛发生长情况见图8。
The hair length of mice in the series A+ polypeptide and A+ recombinant type III collagen groups on days 15, 30, 45, and 60 of drug administration was longer than that of the model control group, and the differences were statistically significant (P<0.01). Compared with the series DZ polypeptide treatment group, the differences were statistically significant (P<0.05). See Table 7. The hair growth of mice in each group on day 45 of modeling is shown in Figure 8.
表7建模后不同时间各组鼠毛发生长长度*与模型组比较差异均有统计学意义(P均＜0.01)Table 7 Hair growth length of mice in each group at different time after modeling *The differences were statistically significant compared with the model group (all P < 0.01)
4.2对小鼠观察区皮肤组织真皮浅层毛囊形态的影响4.2 Effects on the morphology of superficial dermal hair follicles in the observation area of mice
模型组小鼠部分皮肤真皮组织细胞有不同程度节段性增厚，小鼠皮下有轻度淋巴细胞增生；部分大鼠皮下毛囊有明显囊性变，毛囊大小不等，增大毛囊腔内有脱落角化物.周边有轻度纤维化，毛囊周边细胞消失或细胞层次明显减少，腔内似有钙化物染成蓝色，皮脂腺数目增多，部分腺体有肥大，肥大腺体细胞核明显减少，正常毛囊数减少。多肽组和米诺地尔液组小鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变与模型组比较有不同程度减轻。与模型对照组比较，多肽组和A+重组胶原蛋白组大鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变明显减轻(P均 <0.01)，与阴性对照和阳性对照组比较无显著差异(P＞0.05)；系列A+多肽组与系列DZ多肽组比较明显减轻(P均＜0.05)；A+重组胶原蛋白组大鼠皮肤损伤毛囊数与系列A+多肽组比较差异有统计学意义(P均＜0.05)。纤连蛋白组和Ⅲ型胶原蛋白组与模型对照组比较无显著差异(P＞0.05)。见表8。免疫组化结果显示模型组皮肤毛囊中VEGF蛋白表达减少；比较模型组，A+重组胶原蛋白组和各多肽组VEGF蛋白表达均明显增加(P均＜0.01)，与阴性对照和阳性对照组比较无显著差异(P＞0.05)；A+重组胶原蛋白组VEGF蛋白表达高于系列A+多肽组(P＜0.05)；系列A+多肽组VEGF蛋白表达高于系列DZ多肽组，比较有统计学差异(P＜0.05)；Ⅲ型胶原蛋白组、纤连蛋白组与模型对照组比较无显著差异(P＞0.05)。见图9。In the model group, some skin dermal tissue cells of mice had segmental thickening to varying degrees, and there was mild lymphocytic hyperplasia under the skin of mice; some rats had obvious cystic changes in subcutaneous hair follicles, and the hair follicles were of different sizes. There was detached keratin in the enlarged hair follicle cavity. There was mild fibrosis around the hair follicles, and the cells around the hair follicles disappeared or the cell layers were significantly reduced. There seemed to be calcifications in the cavity that were stained blue. The number of sebaceous glands increased, and some glands were hypertrophic. The nuclei of hypertrophic glandular cells were significantly reduced, and the number of normal hair follicles decreased. The lesions of the skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands of mice in the polypeptide group and minoxidil solution group were alleviated to varying degrees compared with those in the model group. Compared with the model control group, the lesions of the skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands of rats in the polypeptide group and A+ recombinant collagen group were significantly alleviated (P < 0.05). <0.01), and there was no significant difference compared with the negative control and positive control groups (P>0.05); the series A+ polypeptide group was significantly relieved compared with the series DZ polypeptide group (all P<0.05); the number of damaged hair follicles in the skin of rats in the A+ recombinant collagen group was statistically significant compared with the series A+ polypeptide group (all P<0.05). There was no significant difference between the fibronectin group and the type III collagen group and the model control group (P>0.05). See Table 8. The results of immunohistochemistry showed that the expression of VEGF protein in the hair follicles of the model group decreased; compared with the model group, the expression of VEGF protein in the A+ recombinant collagen group and each polypeptide group increased significantly (all P < 0.01), and there was no significant difference compared with the negative control and positive control groups (P >0.05); the expression of VEGF protein in the A+ recombinant collagen group was higher than that in the series A+ polypeptide group (P <0.05); the expression of VEGF protein in the series A+ polypeptide group was higher than that in the series DZ polypeptide group, and there was a statistical difference (P <0.05); there was no significant difference between the type III collagen group and the fibronectin group and the model control group (P > 0.05). See Figure 9.
表8各组对小鼠皮肤毛囊和皮脂腺的影响(只)

注：*为与模型对照组比较有显著差异(P＜0.01)Table 8 Effects of each group on mouse skin hair follicles and sebaceous glands (n=1)

Note: * indicates significant difference compared with the model control group (P < 0.01)
实施例7 A+多肽或A+重组Ⅲ型胶原蛋白对兔耳痤疮模型的影响 Example 7 Effects of A+ polypeptide or A+ recombinant type III collagen on rabbit ear acne model
1.材料1. Materials
系列A+多肽和系列DZ多肽：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示。系列DZ多肽氨基酸序列如实施例2所示，在以下实验中作为实验对照组。Series A+ polypeptides and series DZ polypeptides: synthesized by Shanghai Bioengineering, with a purity greater than 95%. The amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4. The amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
A+多肽1重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白1)、A+多肽2重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白2)和A+多肽3重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白3)：由实施例1所示方法合成；人Ⅲ型胶原蛋白(同实施例1所示)；纤连蛋白(FN)：武汉艾美捷科技有限公司生产。A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1), A+ polypeptide 2 recombinant human type III collagen (referred to as A+ recombinant collagen 2) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
阳性治疗药：0.1％阿达帕林凝胶(商品名：达芙文，法国高德美制药公司生产)Positive treatment drug: 0.1% adapalene gel (trade name: Differin, produced by Galderma Pharmaceuticals, France)
实验动物：普通级新西兰家兔，1.9～2.4kg，雄性，来源于上海斯莱克实验动物有限责任公司。Experimental animals: Ordinary New Zealand rabbits, 1.9-2.4 kg, male, sourced from Shanghai Slake Experimental Animal Co., Ltd.
乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量以上纤连蛋白或系列多肽或蛋白质混匀形成0.5％混合乳剂。本专利实施例所用的乳膏基质是指乳膏除去活性成分的基质成分。Preparation method of cream: The excipient matrix components include methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), octadecyl alcohol (5%), glycerol (20%), alkyl aryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl paraben (0.1%), distilled water (about 31-55%), respectively, and mixed with appropriate amounts of the above fibronectin or series of polypeptides or proteins to form a 0.5% mixed emulsion. The cream matrix used in the embodiment of this patent refers to the matrix component of the cream without the active ingredient.
2.动物分组与造模2. Animal grouping and modeling
按体重编号，采用随机排列表法分为阴性对照组(涂抹乳膏基质)、模型对照组(涂抹乳膏基质)、纤连蛋白组(皮肤涂抹0.5％纤连蛋白乳膏)、阳性对照组(皮肤涂抹达芙文)、A+重组胶原蛋白组(皮肤涂抹0.5％A+多肽重组Ⅲ型胶原蛋白乳膏)、胶原蛋白组(皮肤涂抹0.5％Ⅲ型胶原蛋白乳膏)、A+多肽组1(皮肤涂抹0.5％A+多肽1号乳膏)、A+多肽组2(皮肤涂抹0.5％A+多肽3号乳膏)、A+多肽组3(皮肤涂抹0.5％A+多肽5号乳膏)、A+多肽组4(皮肤涂抹0.5％A+多肽6号乳膏)、A+多肽组5(皮肤涂抹0.5％A+多肽8号乳膏)、A+多肽组6(皮肤涂抹0.5％A+多肽9号乳膏)、A+多肽组7(皮肤涂抹0.5％A+多肽10号乳膏)、A+多肽组8(皮肤涂抹1％A+多肽11号乳膏)、A+多肽组9(皮肤涂抹1％A+多肽12号乳膏)、A+多肽组10(皮肤涂抹1％A+多肽13号乳膏)、A+多肽组11(皮肤涂抹1％A+多肽15号乳膏)、A+多肽组12(皮肤涂抹1％A+多肽17号乳膏)、A+多肽组13(皮肤涂抹1％A+多肽18号乳膏)、A+多肽组14(皮肤涂抹1％A+多肽20号乳膏)、A+多肽组15(皮肤涂抹1％A+多肽21号乳膏)和A+多肽组16(皮肤涂抹1％A+多肽22号乳膏)，系列DZ多肽组(皮肤分别涂抹各0.5％DZ多肽乳膏)。每组10只，雌雄各半，外用每天1次。取兔右耳内侧脱毛处理作为观察区，所有家兔左耳作为自身阴性对照，涂抹95％酒精，模型组和实验组右耳内侧均涂2％煤焦油(Alfa Aesar中国公司，用95％酒精配制成2％的煤焦油乳膏)，用无菌棉签均匀涂于家兔耳内侧面耳导管开口处约2cm×2cm范围，每天1次，每次0.5mL，并且用温水擦拭前次涂药部位，连续涂14d，建立痤疮微粉刺模型。肉眼观察局部皮肤的变化，包括耳厚薄、硬度、粗糙程度和毛囊口有无黑色角栓等。末次涂18h后处死取材，10％甲醛固定，石蜡包埋切片，HE染色后，进行病理组织学观察分析。According to the body weight, the subjects were divided into negative control group (applying cream base), model control group (applying cream base), fibronectin group (applying 0.5% fibronectin cream on the skin), positive control group (applying Dafuwen on the skin), A+ recombinant collagen group (applying 0.5% A+ polypeptide recombinant type III collagen cream on the skin), collagen group (applying 0.5% type III collagen cream on the skin), A+ polypeptide group 1 (applying 0.5% A+ polypeptide No. 1 cream on the skin), A+ polypeptide group 2 (applying 0.5% A+ polypeptide No. 3 cream on the skin), A+ polypeptide group 3 (applying 0.5% A+ polypeptide No. 5 cream on the skin), A+ polypeptide group 4 (applying 0.5% A+ polypeptide No. 6 cream on the skin), A+ polypeptide group 5 (applying 0.5% A+ polypeptide No. 8 cream on the skin), A+ polypeptide group 6 (applying 0.5% A+ polypeptide No. 7 cream on the skin). A+ polypeptide group 9 (skin applied with 0.5% A+ polypeptide cream No. 9), A+ polypeptide group 7 (skin applied with 0.5% A+ polypeptide cream No. 10), A+ polypeptide group 8 (skin applied with 1% A+ polypeptide cream No. 11), A+ polypeptide group 9 (skin applied with 1% A+ polypeptide cream No. 12), A+ polypeptide group 10 (skin applied with 1% A+ polypeptide cream No. 13), A+ polypeptide group 11 (skin applied with 1% A+ polypeptide cream No. 15), A+ polypeptide group 12 (skin applied with 1% A+ polypeptide cream No. 17), A+ polypeptide group 13 (skin applied with 1% A+ polypeptide cream No. 18), A+ polypeptide group 14 (skin applied with 1% A+ polypeptide cream No. 20), A+ polypeptide group 15 (skin applied with 1% A+ polypeptide cream No. 21) and A+ polypeptide group 16 (skin applied with 1% A+ polypeptide cream No. 22), series DZ polypeptide group (skin applied with 0.5% DZ polypeptide cream respectively). Ten mice in each group, half male and half female, external application once a day. The inner side of the right ear of the rabbit was depilated as the observation area, and the left ear of all rabbits was used as the negative control. 95% alcohol was applied. 2% coal tar (Alfa Aesar China Company, 2% coal tar cream prepared with 95% alcohol) was applied to the inner side of the right ear of the model group and the experimental group. The area of about 2cm×2cm at the opening of the ear tube on the inner side of the rabbit's ear was evenly applied with a sterile cotton swab once a day, 0.5mL each time, and the previous application site was wiped with warm water. The application was continued for 14 days to establish an acne micro-acne model. The changes in the local skin were observed with the naked eye, including the thickness, hardness, roughness of the ear, and the presence or absence of black horn plugs at the hair follicle opening. The samples were sacrificed 18 hours after the last application, fixed with 10% formaldehyde, embedded in paraffin, and sliced. After HE staining, pathological histological observation and analysis were performed.
3.观察指标3. Observation indicators
痤疮模型组织学判定分级标准：按组织学级别为3级。0级“一”为漏斗部仅有松散的角化的细胞，无粉刺生成；1级为兔耳表面皮肤发红，或毛囊漏斗部见少量致密角化物质，漏斗部不扩张“+”；2级为毛囊漏斗部见中等致密角化物质，并向皮脂腺延伸，伴随皮脂腺导管的增生，漏斗扩张“2+”；3级为毛囊内有广泛的角化物质，毛囊中紧密的角质栓塞引起毛囊重度扩张，皮脂腺导管上皮明显增生，皮肤凸起、瘢痕，部分皮脂腺发生退行变“3+”。The acne model histological grading standard is as follows: the histological grade is 3. Grade 0 "一" means that there are only loose keratinized cells in the infundibulum, and no acne is generated; Grade 1 means that the skin on the surface of the rabbit ear is red, or a small amount of dense keratinized material is seen in the infundibulum of the hair follicle, and the infundibulum is not dilated "+"; Grade 2 means that medium-dense keratinized material is seen in the infundibulum of the hair follicle, and extends to the sebaceous gland, accompanied by hyperplasia of the sebaceous gland duct, and the infundibulum is dilated "2+"; Grade 3 means that there are extensive keratinized materials in the hair follicles, and the tight keratin plugs in the hair follicles cause severe dilation of the hair follicles, obvious hyperplasia of the sebaceous gland duct epithelium, skin bulges, scars, and some sebaceous glands undergo degeneration "3+".
在显微镜下观察其组织病理改变情况，并用Biomias99图像分析***测量一张切片上5处不同表皮的厚度，计算平均值；检测4张切片中位置相同而结构最完整的2个毛囊的面积和4个皮脂腺的直径，计算各自的平均值，然后将各组兔左、右外耳道数据相减，即得各兔的左、右耳表皮厚度差、毛囊面积差和皮脂腺直径差。The histopathological changes were observed under a microscope, and the thickness of the epidermis at 5 different locations on a slice was measured using the Biomia 99 image analysis system, and the average value was calculated. The areas of the two hair follicles with the most complete structure and the diameters of the four sebaceous glands in the same position in the four slices were detected, and their respective average values were calculated. Then the data of the left and right external auditory canals of each group of rabbits were subtracted to obtain the difference in epidermal thickness, hair follicle area and sebaceous gland diameter between the left and right ears of each rabbit.
4统计学处理4 Statistical analysis
用SPSS22软件进行统计分析。自身左右对照采用配对t检验，各组间比较用t检验，P<0.05为差异有统计学意义。SPSS 22 software was used for statistical analysis. Paired t-test was used for left-right comparison and t-test was used for comparison between groups. P<0.05 was considered statistically significant.
5.结果5. Results
肉眼观察：涂煤焦油14d后，所有组兔左耳皮肤柔软，其外耳道毛囊口排列整齐，未见粉刺、丘疹及脓疱等。模型对照组兔右耳涂煤焦油后耳厚度增加、***，表面粗燥，毛囊口有黑色角栓，形成黑头粉刺，毛囊口***呈丘疹状，触之较硬，部分融合成片。阳性治疗组兔右耳与左耳相比，皮肤轻度发红，有少许脱屑，少量毛囊角栓和粉刺，粉刺减少变平，毛孔缩小，皮肤稍干燥。其余各治疗组右耳表现为不同程度的皮肤柔软，粉刺减少， 毛孔明显缩小，无脱屑，基本接近正常兔耳。Macroscopic observation: 14 days after the application of coal tar, the skin of the left ears of rabbits in all groups was soft, and the openings of the hair follicles in the external auditory canals were arranged neatly, without acne, papules, or pustules. After the right ears of rabbits in the model control group were applied with coal tar, the ear thickness increased and became hard, the surface was rough, there were black keratin plugs at the openings of the hair follicles, forming blackhead comedones, the openings of the hair follicles were raised and papular, hard to the touch, and some were fused into pieces. Compared with the left ears, the right ears of rabbits in the positive treatment group had mild redness, a little desquamation, a small amount of keratin plugs and acne, the acne was reduced and flattened, the pores were reduced, and the skin was slightly dry. The right ears of the remaining treatment groups showed varying degrees of soft skin, reduced acne, The pores are significantly smaller, there is no desquamation, and it is basically close to a normal rabbit ear.
组织切片观察：模型组左耳显示表皮较薄，可见毛囊，真皮与表皮交界清楚。模型组右耳造模后见表皮增厚，角化过度，颗粒层和棘层增厚，毛囊扩大，角栓堵塞毛囊口，并向皮脂腺延伸，毛囊漏斗部充满角化物质并扩大呈壶状；真皮上层毛细血管扩张，毛囊周围散在炎症细胞浸润，少量中性粒细胞浸润；皮脂腺数量增多，皮脂腺体积增大。Tissue section observation: The left ear of the model group showed a thinner epidermis, with visible hair follicles and a clear junction between the dermis and the epidermis. After modeling, the right ear of the model group showed thickening of the epidermis, hyperkeratosis, thickening of the granular layer and the spinous layer, enlarged hair follicles, keratin plugs blocking the hair follicle openings and extending to the sebaceous glands, and the funnel of the hair follicles was filled with keratinized substances and enlarged into a pot shape; the capillaries in the upper dermis were dilated, and there were scattered inflammatory cell infiltrations around the hair follicles, and a small amount of neutrophils infiltrated; the number of sebaceous glands increased, and the volume of sebaceous glands increased.
各组镜下实验性粉刺组织学分级(见表9)：模型组兔右耳与其左耳(阴性对照)进行比较，差异有统计学意义(P＜0.01)，提示兔耳痤疮模型复制成功；Ⅲ型胶原蛋白组和纤连蛋白组右耳与模型组右耳比较，差异均无统计学意义(P均＞0.05)；其余各治疗组兔右耳与模型组兔右耳进行比较，差异均有统计学意义(P均＜0.01)；A+多肽组明显低于DZ多肽组(P均＜0.05)；A+重组胶原蛋白组明显低于系列A+多肽组(P均＜0.05)。Microscopic histological grading of experimental acne in each group (see Table 9): The right ear of the rabbits in the model group was compared with their left ears (negative control), and the difference was statistically significant (P < 0.01), indicating that the rabbit ear acne model was successfully replicated; there was no statistically significant difference between the right ears of the type III collagen group and the fibronectin group and the right ear of the model group (all P > 0.05); the right ears of the rabbits in the other treatment groups were compared with the right ears of the rabbits in the model group, and the differences were statistically significant (all P < 0.01); the A+ polypeptide group was significantly lower than the DZ polypeptide group (all P < 0.05); the A+ recombinant collagen group was significantly lower than the series A+ polypeptide group (all P < 0.05).
模型组兔右耳表皮厚度、毛囊画积和皮脂腺直径与其左耳(阴性对照)进行比较，差异有统计学意义(P＜0.01)，提示兔耳痤疮模型复制成功；Ⅲ型胶原蛋白组和纤连蛋白组右耳与模型组右耳比较，差异均无统计学意义(P均＞0.05)；其余各治疗组兔右耳表皮厚度、毛囊画积和皮脂腺直径与模型组兔右耳进行比较，差异均有统计学意义(P均＜0.01)；A+多肽组低于DZ多肽组(P均＜0.05)；A+重组胶原蛋白组低于系列A+多肽组(P均＜0.05)。(见表10)。The epidermal thickness, hair follicle area and sebaceous gland diameter of the right ear of the rabbits in the model group were compared with those of their left ears (negative control), and the differences were statistically significant (P < 0.01), indicating that the rabbit ear acne model was successfully replicated; there were no statistically significant differences between the right ears of the type III collagen group and the fibronectin group and the right ears of the model group (all P > 0.05); the epidermal thickness, hair follicle area and sebaceous gland diameter of the right ears of the rabbits in the other treatment groups were compared with those of the right ears of the rabbits in the model group, and the differences were statistically significant (all P < 0.01); the A+ polypeptide group was lower than the DZ polypeptide group (all P < 0.05); the A+ recombinant collagen group was lower than the series A+ polypeptide group (all P < 0.05). (See Table 10).
表9各组痤疮粉刺的组织学分级

Table 9 Histological grading of acne in each group

表10各组耳表皮厚度、毛囊面积和皮脂腺直径


(*为与模型组右耳比较P<0.01)Table 10 Ear epidermal thickness, hair follicle area and sebaceous gland diameter in each group


(*Compared with the right ear of the model group, P<0.01)
兔耳是一种常用来衡量引起粉刺物质作用强度的动物模型，兔和人一样毛囊皮脂腺大小变化很大。动物随着年龄的增加，形成粉刺的能力也增加，因此选择成年雄性家兔来复制痤疮模型，使其体内的雄性激素对皮肤具有一定的刺激作用。阿达帕林可通过调节毛囊皮脂腺上皮角化异常过程来去除角质栓，从而起到防止及消除粉刺皮损的作用，故本发明选择阿达帕林作为阳性对照品。Rabbit ears are an animal model commonly used to measure the intensity of acne-causing substances. The size of the hair follicle sebaceous glands of rabbits varies greatly, just like humans. As animals age, their ability to form acne also increases. Therefore, adult male rabbits are selected to replicate the acne model so that the male hormones in their bodies have a certain stimulating effect on the skin. Adapalene can remove keratin plugs by regulating the abnormal keratinization process of the hair follicle sebaceous gland epithelium, thereby preventing and eliminating acne skin lesions. Therefore, the present invention selects adapalene as a positive control.
所述一系列多肽和A+重组Ⅲ型胶原蛋白均能明显抑制煤焦油诱导的兔耳痤疮模型症状，减少毛孔堵塞和黑头粉刺形成，对痤疮具有治疗作用；系列A+多肽组明显优于系列DZ多肽组；胶原蛋白组和纤连蛋白组对痤疮无明显作用；A+多肽与Ⅲ型胶原蛋白结合能明显提高对痤疮的治疗效果。The series of polypeptides and A+ recombinant type III collagen can significantly inhibit the symptoms of the coal tar-induced rabbit ear acne model, reduce pore blockage and blackhead acne formation, and have a therapeutic effect on acne; the series A+ polypeptide group is significantly better than the series DZ polypeptide group; the collagen group and the fibronectin group have no obvious effect on acne; the combination of A+ polypeptide and type III collagen can significantly improve the therapeutic effect on acne.
实施例8：多肽和重组胶原蛋白治疗痤疮患者面部皮疹的临床观察Example 8: Clinical observation on the treatment of facial rash in acne patients with peptides and recombinant collagen
1.材料1. Materials
A+多肽3的氨基酸序列：EYPYKHSGYYHRAV，A+多肽18的氨基酸序列：EYPYDYSGYYHRPAV和DZ多肽1的氨基酸序列：EYPYKHSGYYHR由上海生工合成；多肽3重组人源Ⅲ型胶原蛋白和多肽18重组人源Ⅲ型胶原蛋白(合成方法同实施例1)。The amino acid sequence of A+ polypeptide 3: EYPYKHSGYYHRAV, the amino acid sequence of A+ polypeptide 18: EYPYDYSGYYHRPAV and the amino acid sequence of DZ polypeptide 1: EYPYKHSGYYHR were synthesized by Shanghai Biotech; polypeptide 3 recombinant human type III collagen and polypeptide 18 recombinant human type III collagen (the synthesis method is the same as that in Example 1).
A+多肽3、DZ多肽1、多肽3重组人源Ⅲ型胶原蛋白和多肽18重组人源Ⅲ型胶原蛋白溶液配制：分别与适量纯净水混合配成0.5％A+多肽3或0.5％DZ多肽1或0.5％多肽3重组人源Ⅲ型胶原蛋白或多肽18重组人源Ⅲ型胶原蛋白溶液。Preparation of A+ polypeptide 3, DZ polypeptide 1, polypeptide 3 recombinant human type III collagen and polypeptide 18 recombinant human type III collagen solutions: mix with appropriate amount of purified water to prepare 0.5% A+ polypeptide 3 or 0.5% DZ polypeptide 1 or 0.5% polypeptide 3 recombinant human type III collagen or polypeptide 18 recombinant human type III collagen solution.
2.方法2. Methods
2.1分组：2.1 Grouping:
本次试验共招募28名面部痤疮患者，所有患者被随机分为四组，四组分别为A total of 28 patients with facial acne were recruited in this trial and all patients were randomly divided into four groups:
A组：安慰剂组(6人)，男女各半，面部皮疹外用纯净水，每天2次外用。Group A: Placebo group (6 people), half male and half female, applied purified water externally on facial rash twice a day.
B组：A+多肽3组(8人)，男女各半，面部皮疹外用0.5％多肽3溶液，每天2次外用。Group B: Group A + polypeptide 3 (8 people), half male and half female, applied 0.5% polypeptide 3 solution to facial rashes, twice a day.
C组：DZ多肽1组(6人)，男女各半，面部皮疹外用0.5％DZ多肽1溶液，每天2次外用。Group C: DZ polypeptide 1 group (6 people), half male and half female, applied 0.5% DZ polypeptide 1 solution to the facial rash, twice a day.
D组：多肽3重组胶原蛋白组(8人)，男女各半，面部皮疹外用0.5％多肽3重组人源Ⅲ型胶原蛋白溶液，每天2次外用。Group D: polypeptide 3 recombinant collagen group (8 people), half male and half female, applied 0.5% polypeptide 3 recombinant human type III collagen solution to the facial rash, twice a day.
E组：多肽18重组胶原蛋白组(8人)，男女各半，面部皮疹外用0.5％多肽18重组人源Ⅲ型胶原蛋白溶液，每天2次外用。Group E: polypeptide 18 recombinant collagen group (8 people), half male and half female, applied 0.5% polypeptide 18 recombinant human type III collagen solution to the facial rash, twice a day.
2.2患者纳入和排除标准2.2 Patient inclusion and exclusion criteria
纳入标准：Inclusion criteria:
(1)所有患者均为寻常性痤疮，发病部位为面部，据Pillsbury分级标准，患者为轻中度。(2)就诊前2周内未接受***和外用药治疗者。(3)患者知情，签署了知情同意书。(1) All patients had acne vulgaris, with the affected area being the face. According to the Pillsbury grading system, the patients were classified as mild to moderate. (2) They had not received systemic or topical medication within 2 weeks before the visit. (3) The patients were informed and signed the informed consent form.
排除标准：Exclusion criteria:
(1)对多肽或蛋白过敏者。(2)排除患有面外伤的患者。(3)排除患有免疫***、器官功能障碍的患者。(4)6个月内口服或外用过维甲酸类药物患者。(5)排除其他类型痤疮患者。(6)未同时参加其他试验。 (1) Patients who are allergic to peptides or proteins. (2) Patients with facial trauma are excluded. (3) Patients with immune system or organ dysfunction are excluded. (4) Patients who have taken oral or topical retinoids within 6 months. (5) Patients with other types of acne are excluded. (6) Not participating in other trials at the same time.
试验期为8周，每2周随访一次，记录面部皮疹情况和不良反应。The trial period was 8 weeks, with follow-up every 2 weeks to record facial rash and adverse reactions.
2.3观察指标2.3 Observation indicators
(1)评价两各组患者临床疗效，统计患者皮损总数，减少60％以上为显效；皮损总数减少超过30％为有效；皮损总数减少不足30％，为无效。(1) The clinical efficacy of the two groups of patients was evaluated by counting the total number of skin lesions. A reduction of more than 60% was considered significant efficacy; a reduction of more than 30% was considered effective; and a reduction of less than 30% was considered ineffective.
(2)检测患者TEWL(经表皮失水率)，使用1.5.3皮肤生理检测仪进行检测，将探头放置在颧骨最高点，读取数值，计算出平均值。(2) Detect the patient's TEWL (transepidermal water loss) using a 1.5.3 skin physiological detector. Place the probe at the highest point of the zygomatic bone, read the value, and calculate the average value.
2.4统计学方法2.4 Statistical methods
采用SPSS 21.0软件处理数据，使用t检验计量资料(x±s)，使用χ2检验计数资料(％)，P＜0.05视为差异有统计学意义。SPSS 21.0 software was used to process the data. The t-test was used for quantitative data (x±s), and the χ2 test was used for counting data (%). P < 0.05 was considered to be statistically significant.
3.结果3. Results
3.1各组临床疗效对比3.1 Comparison of clinical efficacy among groups
A+多肽3组、A+多肽3重组胶原蛋白组和A+多肽18重组胶原蛋白组治疗有效率分别为87.5％、100％和100％，与安慰剂组对比差异显著(P＜0.01)；A+多肽3组、A+多肽3重组胶原蛋白组和A+多肽18重组胶原蛋白组治疗有效率与DZ多肽1组比较差异也显著(P＜0.05)；DZ多肽1组与安慰剂组比较差异无统计学意义(P＞0.05)。详见表11。The effective rates of A+ polypeptide 3 group, A+ polypeptide 3 recombinant collagen group and A+ polypeptide 18 recombinant collagen group were 87.5%, 100% and 100% respectively, which were significantly different from the placebo group (P < 0.01); the effective rates of A+ polypeptide 3 group, A+ polypeptide 3 recombinant collagen group and A+ polypeptide 18 recombinant collagen group were also significantly different from the DZ polypeptide 1 group (P < 0.05); there was no statistically significant difference between the DZ polypeptide 1 group and the placebo group (P > 0.05). See Table 11 for details.
表11各组痤疮治疗8周后疗效比较

*与安慰剂组比较有显著差异(P＜0.01)Table 11 Comparison of efficacy of acne treatment in each group after 8 weeks

*Significantly different from the placebo group (P＜0.01)
3.2各组患者TEWL对比3.2 Comparison of TEWL among groups of patients
A+多肽3组、A+多肽3重组胶原蛋白组和A+多肽18重组胶原蛋白组治疗4周和8周后下颌部TEWL与安慰剂组对比差异显著(P＜0.01)；A+多肽3组和重组胶原蛋白组治疗4周和8周后与DZ多肽1组比较差异显著(P＜0.05)；详见表12。After 4 and 8 weeks of treatment, the TEWL of the mandible in the A+ polypeptide 3 group, the A+ polypeptide 3 recombinant collagen group and the A+ polypeptide 18 recombinant collagen group was significantly different from that in the placebo group (P < 0.01); after 4 and 8 weeks of treatment, the A+ polypeptide 3 group and the recombinant collagen group were significantly different from those in the DZ polypeptide 1 group (P < 0.05); see Table 12 for details.
表12各组患者TEWL对比

*与安慰剂组比较有显著差异(P＜0.05)Table 12 Comparison of TEWL in each group of patients

*Significantly different from the placebo group (P＜0.05)
3.3 A+多肽组和重组胶原蛋白组患者治疗8周前后对比 3.3 Comparison of patients in the A+ polypeptide group and the recombinant collagen group before and after 8 weeks of treatment
某患者，女，22岁，轻度寻常性痤疮，接受0.5％A+多肽3溶液治疗，治疗8周前后对比如图10所示：A patient, female, 22 years old, with mild acne vulgaris, was treated with 0.5% A+ peptide 3 solution. The comparison before and after 8 weeks of treatment is shown in Figure 10:
某患者，女，23岁，中度寻常性痤疮，接受0.5％A+多肽3重组胶原蛋白溶液治疗，治疗8周前后对比如图11所示。A patient, female, 23 years old, with moderate acne vulgaris, was treated with 0.5% A+ polypeptide 3 recombinant collagen solution. The comparison before and after 8 weeks of treatment is shown in Figure 11.
某患者，女，20岁，中度寻常性痤疮，接受0.5％A+多肽18重组胶原蛋白溶液治疗，治疗4周前后对比如图12所示。A patient, female, 20 years old, with moderate acne vulgaris, was treated with 0.5% A+ polypeptide 18 recombinant collagen solution. The comparison before and after 4 weeks of treatment is shown in Figure 12.
4结论4 Conclusion
A+多肽和A+重组Ⅲ型胶原蛋白均能明显改善痤疮患者面部皮疹，减少毛孔堵塞和黑头粉刺形成，明显减少疤痕(痘印)；A+多肽组明显优于DZ多肽组；A+多肽与Ⅲ型胶原蛋白结合能明显提高对痤疮的治疗作用。Both A+ polypeptide and A+ recombinant type III collagen can significantly improve facial rashes in acne patients, reduce pore blockage and blackhead formation, and significantly reduce scars (acne marks); the A+ polypeptide group is significantly better than the DZ polypeptide group; the combination of A+ polypeptide and type III collagen can significantly enhance the therapeutic effect on acne.
实施例9 A+多肽和重组胶原蛋白对大鼠疤痕模型的影响Example 9 Effects of A+ polypeptide and recombinant collagen on the rat scar model
1.实验方法1. Experimental Methods
1.1材料1.1 Materials
系列A+多肽和系列DZ多肽：由上海生工合成，纯度大于95％。系列A+多肽氨基酸序列如上述发明内容中所示，具有代表性的A+多肽氢谱分析结果如图4所示。系列DZ多肽氨基酸序列如实施例2所示，在以下实验中作为实验对照组。Series A+ polypeptides and series DZ polypeptides: synthesized by Shanghai Bioengineering, with a purity greater than 95%. The amino acid sequence of the series A+ polypeptides is shown in the above invention content, and the representative A+ polypeptide hydrogen spectrum analysis results are shown in Figure 4. The amino acid sequence of the series DZ polypeptides is shown in Example 2, and is used as the experimental control group in the following experiments.
A+多肽1重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白1)、A+多肽2重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白2)和A+多肽3重组人源Ⅲ型胶原蛋白(简称A+重组胶原蛋白3)：由实施例1所示方法合成；人Ⅲ型胶原蛋白(同实施例1所示)；纤连蛋白(FN)：武汉艾美捷科技有限公司生产。A+ polypeptide 1 recombinant human type III collagen (referred to as A+ recombinant collagen 1), A+ polypeptide 2 recombinant human type III collagen (referred to as A+ recombinant collagen 2) and A+ polypeptide 3 recombinant human type III collagen (referred to as A+ recombinant collagen 3): synthesized by the method shown in Example 1; human type III collagen (same as shown in Example 1); fibronectin (FN): produced by Wuhan Aimejie Technology Co., Ltd.
乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量以上纤连蛋白或系列多肽或蛋白质混匀形成0.5％混合乳剂。本专利实施例所用的乳膏基质是指乳膏除去活性成分的基质成分。Preparation method of cream: The excipient matrix components include methyl silicone oil (15%), stearic acid (6%), white vaseline (5%), liquid paraffin (5%), octadecyl alcohol (5%), glycerol (20%), alkyl aryl polyethanol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethyl paraben (0.1%), distilled water (about 31-55%), respectively, and mixed with appropriate amounts of the above fibronectin or series of polypeptides or proteins to form a 0.5% mixed emulsion. The cream matrix used in the embodiment of this patent refers to the matrix component of the cream without the active ingredient.
1.2实验动物分组与造模1.2 Experimental animal grouping and modeling
SPF级雄性大鼠，体重(210±28)g，来源于南医大动物中心。按体重编号，采用随机排列表法分为阴性对照组(涂抹乳膏基质)、模型对照组(涂抹乳膏基质)、纤连蛋白组(皮肤涂抹0.5％纤连蛋白乳膏)、A+重组胶原蛋白组(皮肤涂抹0.5％A+多肽重组Ⅲ型胶原蛋白乳膏)、胶原蛋白组(皮肤涂抹0.5％Ⅲ型胶原蛋白乳膏)、A+多肽组1(皮肤涂抹0.5％A+多肽1号乳膏)、A+多肽组2(皮肤涂抹0.5％A+多肽3号乳膏)、A+多肽组3(皮肤涂抹0.5％A+多肽5号乳膏)、A+多肽组4(皮肤涂抹0.5％A+多肽6号乳膏)、A+多肽组5(皮肤涂抹0.5％A+多肽8号乳膏)、A+多肽组6(皮肤涂抹0.5％A+多肽9号乳膏)、A+多肽组7(皮肤涂抹0.5％A+多肽10号乳膏)、A+多肽组8(皮肤涂抹1％A+多肽11号乳膏)、A+多肽组9(皮肤涂抹1％A+多肽12号乳膏)、A+多肽组10(皮肤涂抹1％A+多肽13号乳膏)、A+多肽组11(皮肤涂抹1％A+多肽15号乳膏)、A+多肽组12(皮肤涂抹1％A+多肽17号乳膏)、A+多肽组13(皮肤涂抹1％A+多肽18号乳膏)、A+多肽组14(皮肤涂抹1％A+多肽20号乳膏)、A+多肽组15(皮肤涂抹1％A+多肽21号乳膏)、A+多肽组16(皮肤涂抹1％A+多肽21号乳膏)，皮肤分别涂抹各0.5％DZ多肽乳膏。每组10只，雌雄各半，外用每天1次。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，然后在其背部中左侧选择一块4×5cm的完整皮肤，8％硫化钠脱毛，用组织剪在脱毛处各剪成一直径为2.4cm圆形深达肌筋膜的伤口，破坏部分肌肉表面筋膜。动物分笼饲养防止大鼠撕咬、舔蹭。创面每日涂2％碘酊常规消毒，观察大鼠创面愈合情况。SPF male rats, weighing (210±28) g, were obtained from the Animal Center of Nanjing Medical University. According to the weight number, they were divided into negative control group (applying cream base), model control group (applying cream base), fibronectin group (applying 0.5% fibronectin cream on the skin), A+ recombinant collagen group (applying 0.5% A+ polypeptide recombinant type III collagen cream on the skin), collagen group (applying 0.5% type III collagen cream on the skin), A+ polypeptide group 1 (applying 0.5% A+ polypeptide No. 1 cream on the skin), A+ polypeptide group 2 (applying 0.5% A+ polypeptide No. 3 cream on the skin), A+ polypeptide group 3 (applying 0.5% A+ polypeptide No. 5 cream on the skin), A+ polypeptide group 4 (applying 0.5% A+ polypeptide No. 6 cream on the skin), A+ polypeptide group 5 (applying 0.5% A+ polypeptide No. 8 cream on the skin), A+ polypeptide group 6 (applying 0.5% A+ polypeptide No. 9 cream on the skin). Cream), A+ polypeptide group 7 (0.5% A+ polypeptide No. 10 cream applied on the skin), A+ polypeptide group 8 (1% A+ polypeptide No. 11 cream applied on the skin), A+ polypeptide group 9 (1% A+ polypeptide No. 12 cream applied on the skin), A+ polypeptide group 10 (1% A+ polypeptide No. 13 cream applied on the skin), A+ polypeptide group 11 (1% A+ polypeptide No. 15 cream applied on the skin), A+ polypeptide group 12 (1% A+ polypeptide No. 17 cream applied on the skin), A+ polypeptide group 13 (1% A+ polypeptide No. 18 cream applied on the skin), A+ polypeptide group 14 (1% A+ polypeptide No. 20 cream applied on the skin), A+ polypeptide group 15 (1% A+ polypeptide No. 21 cream applied on the skin), A+ polypeptide group 16 (1% A+ polypeptide No. 21 cream applied on the skin), 0.5% DZ polypeptide cream was applied on the skin. There were 10 mice in each group, half male and half female, and the topical application was once a day. Each group of rats was anesthetized by intraperitoneal injection of 2% sodium pentobarbital (120 mg/kg) and fixed on the operating table. Then a 4×5 cm piece of intact skin was selected on the left side of the back, and 8% sodium sulfide was used to depilate the hair. A circular wound with a diameter of 2.4 cm and deep to the muscle fascia was cut at the depilated area with tissue scissors to destroy part of the muscle surface fascia. The animals were kept in separate cages to prevent the rats from biting and licking. The wound surface was routinely disinfected with 2% iodine tincture every day, and the healing of the rats' wounds was observed.
2.实验结果2. Experimental results
2.1大鼠创面观测结果2.1 Observation results of rat wound surface
创面每天常规消毒，第1d、3d、5d、7d、12d、20d观察大鼠创面。自第6天左右各A+多肽和A+重组胶原蛋白系列治疗组伤口开始恢复，速度明显比模型组快，创面面积逐渐变小。第15天，各治疗组创面已经基本恢复，而模型组仍有约0.6cm2大小的创面。到第20天时，各组创面均已经恢复，模型对照组留下明显疤痕，而系列A+多肽和重组胶原蛋白治疗组只留下数量不等的色素沉着。A+重组胶原蛋白优于A+多肽。A+多肽组明显优于DZ多肽组。The wounds were routinely disinfected every day, and the wounds of rats were observed on the 1st, 3rd, 5th, 7th, 12th, and 20th days. Since the 6th day, the wounds of the A+ polypeptide and A+ recombinant collagen series treatment groups began to recover, and the speed was significantly faster than that of the model group, and the wound area gradually became smaller. On the 15th day, the wounds of each treatment group had basically recovered, while the model group still had a wound of about 0.6 cm2 . By the 20th day, the wounds of each group had recovered, and the model control group left obvious scars, while the series A+ polypeptide and recombinant collagen treatment groups only left varying amounts of pigmentation. A+ recombinant collagen is better than A+ polypeptide. The A+ polypeptide group is significantly better than the DZ polypeptide group.
3.实验结论3. Experimental conclusion
A+多肽和A+重组胶原蛋白均能明显促进皮肤创面愈合，减少疤痕(瘢痕)形成。A+多肽组明显优于DZ多肽对照组；A+多肽与Ⅲ型胶原蛋白重组能明显提高对疤痕的治疗作用。 Both A+ polypeptide and A+ recombinant collagen can significantly promote skin wound healing and reduce scar formation. The A+ polypeptide group is significantly better than the DZ polypeptide control group; A+ polypeptide and type III collagen recombinant can significantly improve the therapeutic effect on scars.

Claims (15)

  1. 一种氨基酸序列，其特征在于，所述氨基酸序列为含有第一序列的氨基酸序列、片段或突变体：An amino acid sequence, characterized in that the amino acid sequence is an amino acid sequence, a fragment or a mutant containing a first sequence:
    所述第一序列包括SEQ ID NO.1或与SEQ ID NO.1至少70％同源的且功能相同或相似的延伸序列，SEQ ID NO.1为AVEYPYKHSGYYHR；The first sequence includes SEQ ID NO.1 or an extended sequence that is at least 70% homologous to SEQ ID NO.1 and has the same or similar function, and SEQ ID NO.1 is AVEYPYKHSGYYHR;
    所述的同源为在原序列基础上增加、减少或改变至少一个氨基酸。The homology is to increase, decrease or change at least one amino acid based on the original sequence.
  2. 根据权利要求1所示的氨基酸序列，其特征在于，所述延伸序列包括SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21或者SEQ ID NO.22或者与前述任意一个延伸序列至少70％同源的且功能相同或相似的序列、片段或突变体，The amino acid sequence according to claim 1, characterized in that the extended sequence comprises SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22, or a sequence, fragment or mutant that is at least 70% homologous to any of the aforementioned extended sequences and has the same or similar functions,
    SEQ ID NO.2：AVEFPFKWSGYYHR；SEQ ID NO.2：AVEFPFKWSGYYHR;
    SEQ ID NO.3：EYPYKHSGYYHRAV；SEQ ID NO.3：EYPYKHSGYYHRAV;
    SEQ ID NO.4：AVEYTYNGAGYYHR；SEQ ID NO.4：AVEYTYNGAGYYHR;
    SEQ ID NO.5：EYTYKGSGYYHRAV；SEQ ID NO.5：EYTYKGSGYYHRAV;
    SEQ ID NO.6：AVEYTYIGAGYYHR；SEQ ID NO.6：AVEYTYIGAGYYHR;
    SEQ ID NO.7：AVEYPWKGSGYYHRP；SEQ ID NO.7：AVEYPWKGSGYYHRP;
    SEQ ID NO.8：AVEYTFKHSGYYHRP；SEQ ID NO.8：AVEYTFKHSGYYHRP;
    SEQ ID NO.9：AVEYPYDYSGYYHRP；SEQ ID NO.9：AVEYPYDYSGYYHRP;
    SEQ ID NO.10：AVEYTYEGAGYYHRP；SEQ ID NO.10：AVEYTYEGAGYYHRP;
    SEQ ID NO.11：AVEYAFAGAGYYHRP；SEQ ID NO.11：AVEYAFAGAGYYHRP;
    SEQ ID NO.12：AVEFPWPHAGYYHRP；SEQ ID NO.12：AVEFPWPHAGYYHRP;
    SEQ ID NO.13：EYTYEGAGYYHRPAV；SEQ ID NO.13：EYTYEGAGYYHRPAV;
    SEQ ID NO.14：EYTYIGAGYYHRPAV；SEQ ID NO.14：EYTYIGAGYYHRPAV;
    SEQ ID NO.15：EYTYNGAGYYHRPAV；SEQ ID NO.15：EYTYNGAGYYHRPAV;
    SEQ ID NO.16：EYPWKGSGYYHRPAV；SEQ ID NO.16：EYPWKGSGYYHRPAV;
    SEQ ID NO.17：EYTFKHSGYYHRPAV；SEQ ID NO.17：EYTFKHSGYYHRPAV;
    SEQ ID NO.18：EYPYDYSGYYHRPAV；SEQ ID NO.18：EYPYDYSGYYHRPAV;
    SEQ ID NO.19：EFPFKWSGYYHRPAV；SEQ ID NO.19：EFPFKWSGYYHRPAV;
    SEQ ID NO.20：EYAFAGAGYYHRPAV；SEQ ID NO.20：EYAFAGAGYYHRPAV;
    SEQ ID NO.21：EFPWPHAGYYHRPAV；SEQ ID NO.21：EFPWPHAGYYHRPAV;
    SEQ ID NO.22：AVEYTYKGSGYYHRP。SEQ ID NO.22：AVEYTYKGSGYYHRP.
  3. 一种包括权利要求1或2所述氨基酸序列的产品，其特征在于，所述产品为多肽或蛋白质，优选地，所述蛋白质为重组人源Ⅲ型胶原蛋白。A product comprising the amino acid sequence of claim 1 or 2, characterized in that the product is a polypeptide or a protein, preferably, the protein is recombinant human type III collagen.
  4. 根据权利要求3所述的产品，其特征在于，其为能够在抗衰、抗炎、组织修复、生发和祛痘、祛疤中应用的产品。The product according to claim 3 is characterized in that it is a product that can be used in anti-aging, anti-inflammation, tissue repair, hair growth, acne removal and scar removal.
  5. 根据权利要求3所述的产品，其特征在于，所述多肽或蛋白质还包含免疫球蛋白。The product according to claim 3, characterized in that the polypeptide or protein further comprises immunoglobulin.
  6. 一种编码如权利要求1或2所述氨基酸序列或权利要求3～5中任意一项所述产品的核酸序列。A nucleic acid sequence encoding the amino acid sequence of claim 1 or 2 or the product of any one of claims 3 to 5.
  7. 一种组合物，其特征在于，该组合物包括如权利要求1或2所述氨基酸序列、权利要求3～5中任意一项所述产品、权利要求6所述核酸序列或者前述任意一个的衍生物或其可药用盐。A composition, characterized in that it comprises the amino acid sequence according to claim 1 or 2, the product according to any one of claims 3 to 5, the nucleic acid sequence according to claim 6, or a derivative of any of the foregoing or a pharmaceutically acceptable salt thereof.
  8. 根据权利要求7所述的组合物，其特征在于，所述产品或组合物可制备为溶液、酊剂、醑剂、搽剂、喷雾剂、乳剂、膏剂、凝胶剂、贴剂、水剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂、注射剂或其他药用剂型。The composition according to claim 7, characterized in that the product or composition can be prepared as a solution, tincture, spirit, liniment, spray, emulsion, paste, gel, patch, aqueous solution, tablet, granule, oral liquid, capsule, pill, enema, film, injection or other pharmaceutical dosage form.
  9. 根据权利要求7所述的组合物，其特征在于，所述产品和组合物可单独使用，也可与一种或多种产品组合 使用。The composition according to claim 7, characterized in that the product and the composition can be used alone or in combination with one or more products use.
  10. 一种制备如权利要求3～5中任意一项所述产品的方法，其特征在于，所述方法包括：培养宿主细胞，后从所述宿主细胞回收所述多肽或蛋白质，其中所述宿主细胞是原核的或真核的。A method for preparing a product as claimed in any one of claims 3 to 5, characterized in that the method comprises: culturing host cells and then recovering the polypeptide or protein from the host cells, wherein the host cells are prokaryotic or eukaryotic.
  11. 如权利要求1或2所述氨基酸序列、权利要求3～5中任意一项所述产品或权利要求6所述的核酸序列在制备皮肤类产品中的应用，其特征在于，所述皮肤类产品包括但不限于药品、医疗器械、保健品或护肤品。Use of the amino acid sequence of claim 1 or 2, the product of any one of claims 3 to 5, or the nucleic acid sequence of claim 6 in the preparation of skin products, characterized in that the skin products include but are not limited to medicines, medical devices, health products or skin care products.
  12. 如权利要求11所述的应用，其特征在于，所述皮肤类产品包括但不限于抗衰、抗炎、组织修复、生发和祛痘产品。The use according to claim 11 is characterized in that the skin products include but are not limited to anti-aging, anti-inflammatory, tissue repair, hair growth and acne removal products.
  13. 一种用于治疗或预防皮肤病的方法，其特征在于，所述方法包括施用治疗有效量的包含依照权利要求1或2所述的氨基酸序列，或权利要求3～5中任意一项所述的产品，或权利要求6所述的组合物的产品。A method for treating or preventing a skin disease, characterized in that the method comprises administering a therapeutically effective amount of a product comprising the amino acid sequence according to claim 1 or 2, or the product according to any one of claims 3 to 5, or the composition according to claim 6.
  14. 根据权利要求13的方法，其特征在于，所述皮肤病包括但不限于皮肤衰老、皮肤炎症、皮肤损伤、脱发、斑秃、疤痕和痤疮。The method according to claim 13, characterized in that the skin diseases include but are not limited to skin aging, skin inflammation, skin damage, hair loss, alopecia, alopecia areata, scars and acne.
  15. 根据权利要求13的方法，其特征在于，所述施用对象是人。 The method according to claim 13, characterized in that the subject of administration is a human.
PCT/CN2023/139218 2022-12-16 2023-12-15 Series of polypeptides/proteins and uses thereof WO2024125641A1 (en)

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CA2328831A1 (en) * 2001-01-09 2002-07-09 Ee Been Goh Antibiotics as quorum-sensing autoinducers
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CN104740603B (en) * 2013-12-25 2017-02-15 胡卓伟 Application of polypeptide in preparation of drugs for treatment and/or prevention of rheumatoid arthritis
CN104292305B (en) * 2014-01-17 2017-06-09 河南科技大学 A kind of polypeptide, preparation method and applications
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