WO2009109146A1 - HNF4α诱导分化治疗人体恶性实体瘤 - Google Patents

HNF4α诱导分化治疗人体恶性实体瘤 Download PDF

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WO2009109146A1
WO2009109146A1 PCT/CN2009/071590 CN2009071590W WO2009109146A1 WO 2009109146 A1 WO2009109146 A1 WO 2009109146A1 CN 2009071590 W CN2009071590 W CN 2009071590W WO 2009109146 A1 WO2009109146 A1 WO 2009109146A1
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hnf4
differentiation
cells
protein
tumor
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PCT/CN2009/071590
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English (en)
French (fr)
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谢渭芬
尹川
林勇
陈岳祥
曾欣
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中国人民解放军第二军医大学
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Priority to US12/921,369 priority Critical patent/US20110077206A1/en
Publication of WO2009109146A1 publication Critical patent/WO2009109146A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • HNF4a induces differentiation to treat human malignant solid tumors
  • the invention relates to the fields of molecular biology, cell biology and medicine. Specifically, the present invention relates to a method and use for the treatment of solid tumors by using Hepatocyte Nuclear Factor-4 ⁇ (HNF4 ⁇ ) to induce differentiation of human malignant solid tumor cells.
  • HNF4 ⁇ Hepatocyte Nuclear Factor-4 ⁇
  • Tumor-induced differentiation therapy is an important breakthrough in the clinical treatment of cancer in the past 20 years. Inducing differentiation therapy promotes differentiation of tumor cells to the mature stage by inducing differentiation, restores normal cell phenotype and function, and inhibits proliferation of malignant cells. Inducing differentiation therapy has broken the traditional understanding of irreversible tumor development, and has strongly promoted the development of the entire cancer research field.
  • the present inventors have studied the regulation of all-trans retinoic acid, somatostatin, tumor necrosis factor and arsenic trioxide on the differentiation of hepatoma cell lines in vitro, and have not screened drugs or proteins having a clear differentiation therapeutic effect, and in vivo studies. It has also been found that these substances are not effective in inducing differentiation of solid tumors.
  • the object of the present invention is to provide a specific gene-HNF4 ⁇ gene and its encoded product HNF4 ci protein which are closely related to the induction of differentiation of malignant solid tumor cells, and the use of HNF4 ⁇ gene/protein in the induction of differentiation of solid tumors.
  • Another object of the present invention is to provide a method for treating a tumor by inducing a malignant solid tumor by HNF4 CI gene/protein differentiation.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises (a) a HNF4a protein, a HNF4 ⁇ coding sequence or an expression vector comprising the coding sequence, and (b) a pharmaceutically acceptable carrier or excipient.
  • the expression vector comprises a viral vector and a non-viral vector.
  • the non-viral vector is a liposome.
  • the solid tumor is selected from the group consisting of: liver cancer, gastric cancer, intestinal cancer, pancreatic cancer, lung cancer, prostate cancer or gonad tumor.
  • the pharmaceutical composition is also useful for inhibiting the formation of solid tumors in vivo.
  • the hepatocyte nuclear factor 4 ⁇ is human hepatocyte nuclear factor 4 ⁇ .
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition further comprises a chemotherapeutic agent.
  • a method of inducing or promoting differentiation of a solid tumor in a mammal comprising the steps of: administering to a mammalian subject in need of treatment a hepatocyte nuclear factor 4 alpha protein, a coding sequence thereof, or An expression vector for the coding sequence.
  • the mammal is a human.
  • Figure 1 RT-PCR detection of HNF4 a gene and expression of hepatocyte-related functional genes in human liver tumor cell lines.
  • Figure 2. HNF4 a cDNA fragment obtained by RT-PCR.
  • Figure 3 In vitro ligation of the shuttle plasmid pAdTrack-CMV-HNF4 a Bgl II and EcoRV digestion.
  • Figure 4 Recombinant adenovirus plasmid pAdHNF4 ⁇ was identified by Pac I digestion.
  • Figure 8 Quantitative analysis of HNF4 a protein expression 3 days after AdHNF4 a infection of liver tumor cells.
  • Figure 9 Quantitative analysis of HNF4 a gene and hepatocyte-related functional gene mRNA expression in human liver tumor cell lines.
  • Figure 10. Ammonia metabolism test after 3 days of AdHNF4 a infection of liver tumor cells.
  • FIG. 11 and Figure 12. CD133 expression assay after infection of liver tumor cells with AdHNF4a.
  • Figure 13 and Figure 14. Effect of exogenous introduction of HNF4 ci on colony formation of human liver tumor cells.
  • FIG. 1 ICG uptake staining assay after AdHNF4 alpha infection of liver tumor cells.
  • HNF4 ci is a key gene/protein which can effectively induce or promote the differentiation of malignant solid tumors into normal cells in vivo.
  • HNF4 ci not only affects tumor cell apoptosis, but also induces or promotes the differentiation of solid tumor cells (cell morphology, hepatocyte-related functional gene expression is up-regulated, and tumor prevention, intervention and treatment in vivo) effect;). Therefore, HNF4 CI has potential application prospects as the first proven gene/protein that can effectively induce or promote the differentiation of malignant solid tumors into normal cells in vivo.
  • the inventors have completed the present invention on this basis.
  • the term "gene/protein” refers to a gene and/or protein.
  • HNF4 ci protein As used herein, the terms “HNF4 ci protein”, “HNF4 ci polypeptide”, “polypeptide of the invention”, and “protein of the invention” are used interchangeably and refer to hepatocyte nuclear factor-4 alpha protein. They may include HNF4 alpha with or without the initial methionine (Met). In a narrow sense, the term refers to human HNF4 ⁇ ; broadly speaking, the term includes not only human HNF4 ⁇ but also HNF4 ci of other mammals, especially primate HNF4 a, such as HNF4 of baboons or monkeys. a. The term also encompasses active fragments, active derivatives and analogs of the HNF4a protein.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, either a chemically synthesized product, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells).
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol. Polypeptides of the invention may also or may not include an initial methionine residue.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of a natural HNF4 ci protein of the invention (e.g., human HNF4 ci ).
  • polypeptide fragments, derivatives or analogs may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may be It may also not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylidene) a polypeptide formed by fusion of a diol) or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or with an antigen IgG) The fusion protein formed by the fragment).
  • the term "human HNF4 a polypeptide” refers to a polypeptide having a wild-type HNF4 a sequence.
  • the term also encompasses variant forms of wild-type sequences that have the same function as the human HNF4a protein to induce solid tumor differentiation. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually 20 or less, preferably 10) at the C-terminus and/or N-terminus More preferably, within 5 amino acids.
  • the function of the protein is generally not altered.
  • the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the human HNF4 CI protein.
  • the invention also includes analogs of human HNF4a proteins or polypeptides.
  • the difference between these analogs and the natural human HNF4a polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids).
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the "human HNF4a protein conservative variant polypeptide” means up to 10, preferably up to 8, more preferably up to 5, optimally up to 3 amino acids compared to the wild type amino acid sequence.
  • the polypeptide is replaced by an amino acid of similar or similar nature.
  • These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • HNF4 ci gene or “gene of the invention” are used interchangeably and refer to a polynucleotide sequence encoding a HNF4 ci protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the wild type coding region sequence or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence that encodes a protein having a wild-type amino acid sequence but differs from the wild-type coding region sequence.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include a polynucleotide encoding additional and/or non-coding sequences.
  • the present invention also relates to variants of the above polynucleotides, as long as these polynucleotide variants encode the above-described
  • HNF4a polypeptide or active fragment thereof, active analog and active derivative.
  • the full length sequence of human HNF4 CI nucleotide or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed based on the nucleotide sequence of human HNF4 ci, particularly the open reading frame sequence, and used as a cDNA library prepared by a commercially available cDNA library or a conventional method known to those skilled in the art.
  • the template is amplified and related to the sequence.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the invention also relates to vectors (especially viral vectors) comprising a HNF4 CI coding sequence, and host cells genetically engineered using the vector of the invention or the HNF4 CI coding sequence, and methods of producing the polypeptides of the invention by recombinant techniques.
  • vectors especially viral vectors
  • host cells genetically engineered using the vector of the invention or the HNF4 CI coding sequence
  • the HNF4 ci polynucleotide sequence can be used to express or produce a recombinant HNF4 a polypeptide by conventional recombinant DNA techniques. Generally there are the following steps:
  • the human HNF4 ci polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to, T7-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988).
  • any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein
  • GFP tetracycline
  • ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; fungal cells such as yeast; animal cells such as CH0, COS, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as Escherichia coli, it can be carried out by the CaCl 2 method or the electroporation method.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express a HNF4 a polypeptide.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant HNF4a polypeptide can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography
  • the recombinant HNF4 a polypeptide can directly induce or promote the differentiation of malignant solid tumors.
  • a polynucleotide encoding a HNF4a protein or a vector carrying the HNF4a coding sequence can also be used to induce or promote solid tumor differentiation.
  • the method of introducing a polynucleotide into a tissue or a cell comprises: injecting the polynucleotide directly into the tissue in vivo; or introducing the polynucleotide into the cell by a vector (such as a virus, a phage or a plasmid, etc.) in vitro. , then transplant the cells into the body and so on.
  • a vector such as a virus, a phage or a plasmid, etc.
  • Recombinant gene therapy vectors such as viral vectors
  • Viral-derived expression vectors such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, parvoviruses and the like can be used to transfer the HNF4 ci gene into cells.
  • a method for constructing a recombinant viral vector carrying the HNF4a gene can be found in the literature (Sambro 0 k, et al.).
  • the recombinant human HNF4 a gene can be packaged into liposomes and then transferred into cells.
  • the HNF4 ci protein, HNF4 ci polynucleotide and vector of the present invention when administered (administered) to a mammalian subject (e.g., a human), induce or promote differentiation of a malignant solid tumor.
  • these substances can be formulated into non-toxic, inert And a pharmaceutically acceptable carrier medium (including an aqueous carrier medium) to form a pharmaceutical composition.
  • a pharmaceutically acceptable carrier medium including an aqueous carrier medium
  • the pH of the aqueous carrier medium will generally be from about 5 to about 8, preferably from about 6 to about 8, although the pH may vary depending on the nature of the substance being formulated and the condition being treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for inducing differentiation (treatment) of solid tumors, and representative examples include, but are not limited to, liver cancer, gastric cancer, intestinal cancer, lung cancer, pancreatic cancer, renal cancer, prostate cancer, and gonads. Tumors, etc.
  • HNF4a gene/protein or pharmaceutical composition of the present invention is used, other therapeutic agents such as cisplatin, TNF and the like may be simultaneously or additionally used.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount (e.g., 0.0001 to 90% by weight) of the HNF4 ci protein, HNF4 ci polynucleotide or vector of the present invention, and (b) pharmaceutically acceptable Carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • Other therapeutic agents such as chemotherapeutic agents, may also be included in the pharmaceutical compositions of the
  • a safe and effective amount of a HNF4 ci protein, HNF4 ci polynucleotide or vector is administered to a mammal, wherein the safe and effective amount is usually at least about 1 microgram per kilogram of body weight, and in most cases does not exceed About 10 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the invention also provides a method of inducing and/or promoting differentiation of a malignant solid tumor, the method comprising administering to a mammalian subject, such as a human, a HNF4 ci protein, HNF4 ci polynucleotide or vector of the invention, thereby The subject induces and/or promotes solid tumor differentiation in vivo.
  • a mammalian subject such as a human, a HNF4 ci protein, HNF4 ci polynucleotide or vector of the invention.
  • the present invention also provides a method for gene therapy of tumor cells (especially malignant solid tumors), which comprises introducing a HNF4 a gene into a tumor cell for expression, wherein the method for introducing the HNF4 a gene into a tumor cell comprises using a plasmid Transfection, adenovirus or adeno-associated virus mediated.
  • tumor cells especially malignant solid tumors
  • the method for introducing the HNF4 a gene into a tumor cell comprises using a plasmid Transfection, adenovirus or adeno-associated virus mediated.
  • RT-PCR Take 4 ⁇ g RNA, 2 ⁇ 1 random primer, DEPC water to 33 ⁇ 1 at 70 ° C for 5 min, 0 ° C for 5 min, then add 10 ⁇ 15 X Buffer 3 ⁇ 1 dNTP, 2 ⁇ 1 RNA reverse transcriptase and P 2 ⁇ 1 RNase inhibitor were mixed and placed at 37 ° C for 1.5 h to obtain a reverse transcription product. The reverse transcription product was diluted, and ⁇ ⁇ ⁇ was used as a template for PCR amplification.
  • the detected gene primer sequences, reaction conditions, and reaction systems are shown in Tables 2 and 3.
  • HNF4a sense strand 5'- -TTGAAAATGTGCAGGTGTTGAC-3 ' 1 429bp 55 °C 29 antisense strand 5'- -CAGAGATGGGAGAGGTGATCTG-3 ' 2
  • Hepatocyte nuclear factor 4 ⁇ (hepatocyte nuclear factor 4 ⁇ HNF4 a ) glucose-6-phosphatase
  • glucose-6-phosphatase G-6-P
  • albumin albumin
  • glutamine synthetase GS
  • cytochrome P450 family la2 cytochrome P450 la2 CYPla2
  • PEPCK phosphoenol Phospholipid carboxykinase
  • TTR thyroid hormone-binding protein
  • AFP alpha fetoprotein
  • CI l Kapol ipoprotein cI II APOCI II
  • HNF4 a 1425bp cDNA fragment Design and synthesize primers based on human HNF4 a cDNA sequence.
  • Just primer (addition of Bgl II restriction site at the 5' end): 5 ' - CCG AGA TCT AGA ATG CGA CTC TCC AAA ACC -3 ' (SEQ ID NO: 21).
  • Antisense primer (addition of EcoR V cleavage site at the 5' end): 5 ' - CGC GAT ATC GGC TTG CTA GAT AAC TTC CTG CT - 3 ' (SEQ ID NO: 22).
  • the HNF4 a cDNA fragment was amplified by PCR, and the product was subjected to 1% agarose gel electrophoresis, and the fragment size was identified, and the tapping recovery was carried out into an Eppendorf tube, and the weight of the gel was weighed.
  • Add 200 ml/100 mg of NT solution to the Eppendorf tube melt at 50 °C for 5-10 min, centrifuge the liquid, centrifuge at 13 000 rpm for 1 min, add 600 ⁇ l ⁇ 3 buffer, centrifuge at 13 000 rpm for 2 min.
  • the DNA fragment was eluted by 30 ⁇ l of double distilled water and centrifuged for 1 min at 1 000 13 rpm. Carefully remove the eluate into a clean Eppendorf tube.
  • the spectrophotometer measures 0D 26 . Value, 1. 5% agarose gel electrophoresis to identify fragment size (Figure 2).
  • Reaction conditions 94 ° C 30 s 60 ° C 30 s 72 ° C 90 s 35 cycles.
  • Construction of the adenoviral plasmid pAdHNF4 a expressing HNF4 a EcoR V, Bgl ll digestion of the shuttle plasmid pAdTrack-CMV (purchased from Howard-Hughes Medical Institute, USA) and HNF4 a cDNA for 4 h and purification, taking 0 1 ⁇ g of plasmid pAdTrack_CMV, 0.4 ⁇ g of HNF4 a cDNA, 10 XT 4 buffer 2 ⁇ l, T 4 DNA ligase 1 ⁇ l (2U) and dd 0, total volume 20 ⁇ l, 16 °C ligated overnight.
  • the ligation product was added to a conventional competent E. coli DH5 a transformation, plated with LB medium plate containing kanamycin, and thermostated at 37 ° C overnight to select a single colony clone, and the colony clone of the HNF4a cDNA fragment was amplified by Qiagen.
  • the plasmid pAdTrack_CMV-HNF4 ⁇ was obtained and identified (Fig. 3). Pme I endonuclease-directed linearization of pAdTrack- CMV-HNF4 ⁇ , respectively, 0.4 ⁇ g linear pAdTrack-CMV- HNF4 ⁇ and 0.
  • Packaging, amplifying adenovirus AdHNF4 a Resuscitation of conventional 293 cells, inoculated in a 10-cm tissue culture dish at 4.8 ⁇ 10 6 / dish, and added to DMEM at 37 ° C, 5% CO 2 , and cultured for 24 h. The density is increased to 60% to 80%. Pac I was digested to linearize pAdHNF4 ⁇ , mixed with serum-free DMEM solution 250 ⁇ l, and mixed into sputum; take Lipofectamin 20 ⁇ 1, add serum-free DMEM solution 250 ⁇ 1 and mix.
  • the sputum, sputum and B solution were thoroughly mixed, and placed at room temperature for 30 min, then added to the 293 cells to be transfected, and the medium was replaced after 4 h. After 7 days, 293 cells and supernatant were collected, repeatedly frozen and thawed in liquid nitrogen and 37 ° C water bath for 4 times, centrifuged at 5,000 rpm for 5 min, virus supernatant was collected, and the virus supernatant was re-infected with 293 cells for amplification.
  • Example 3 Efu/ml of AdHNF4 ⁇ , stored at -80 ° C for later use.
  • the reaction conditions were the same as before, and PCR reaction was carried out under the same reaction conditions with ⁇ -actin.
  • the reaction system is as follows.
  • Dd3 ⁇ 40 11.3 ⁇ 1 Reaction conditions are 95 ° C for 30 s, 55 V 30 s, 72 V 90 s, 27 cycles.
  • the RT-PCR products were identified by 1.5% agarose gel electrophoresis, scanned, and then subjected to optical density scanning and sequencing analysis using Multy-Analasyst image analysis software.
  • AdHNF4 a infects pG2 and Hep3B, respectively, and the whole cell protein is collected from the cell lysate. After standardization of the protein, 10 ⁇ g of each protein is separated by 10% SDS-PAGE to form a polyvinylidene fluoride membrane (PVDF membrane) ddH20. Rinse, equilibrate the electrophoresis gel, PVDF membrane, and filter paper in Transfer Buffer, and place in an electrotransfer tank at 18 V for 40 min.
  • PVDF membrane polyvinylidene fluoride membrane
  • HNF4 ⁇ polyclonal antibody (1:500) was incubated overnight at 4 °C, and after PBST washing the next day, it was incubated with scorpion anti-sheep fluorescent secondary antibody (1:2000) at room temperature. After 30 min washing with PBST, the fluorescence was detected by Odyssey infrared laser imaging system and grayscale scanning was performed.
  • RT-PCR was used to detect the expression of hepatocyte-related functional genes: the conventional human liver tumor cell lines HepG2 and Hep3B were inoculated into a six-well plate in 5 ⁇ 107 dishes, and the virus AdHNF4a was infected with M0I 40, 100, respectively, 24 h later. Fresh DMEM containing 10% fetal bovine serum was replaced, and GFP expression was observed after 3 days. Total RNA was extracted with Trizol kit, reverse transcription reaction for 2 h, and 1 ⁇ diluted reverse transcript was used as template for PCR amplification. The detected gene primer sequence and reaction conditions were the same as in Example 1, and the reaction system was as follows. Ingredient volume ( ⁇ )
  • AdHNF4a group AdHNF4a group
  • AdGFP group AdGFP group
  • Flow cytometry to determine the apoptotic rate of human liver tumor cells The conventional liver cancer cell lines pG2 and Hep3B were inoculated into a six-well plate in 5 ⁇ 107 dishes, and the virus AdHNF4 a was infected with M0I 40, 100, respectively. After h, fresh DMEM containing 10% fetal bovine serum was replaced, cells were collected on day 3, and apoptosis rate was determined by EPICS XL flow cytometry (Coulter) and statistical analysis was performed. Each group was set up with 2 sets and repeated 3 times.
  • Human liver tumor cell line, gastric cancer cell line and colon cancer cell line were inoculated into 96-well plate with 5 ⁇ 107 wells respectively, and infected with virus AdHNF4 a after 24 h. Thereafter, the absorbance at 450 nm was detected by CCK8 reagent every day to judge the active cells. quantity.
  • HNF4 a expression significantly inhibited the proliferation of solid tumor cells.
  • the proliferation of tumor cells in the AdHNF4 a infection group began to decrease, and the number of cells decreased significantly on the fifth day.
  • the inhibition rate was as high as 50. %-68%.
  • the up-regulation of HNF4 ci inhibited the proliferation of some solid tumor cells in a time- and dose-dependent manner.
  • Human liver tumor cells HepG2 and H ⁇ 3B were inoculated into 6-well plates at 5 ⁇ 10 5 /d, and the virus AdHNF4 a was infected with MOI 40, 100, respectively. After 24 h, fresh DMEM containing 10% fetal bovine serum was replaced. On the third day, cells were collected, CD133/l-PE (Mi ltenyi Biotec, Auburn, CA) as a primary antibody, and the ratio of flow cytometry CD 133+ cells.
  • CD133 + is a specific marker of cancer stem cells, and a significant decrease in the proportion of CD133+ cells indicates that AdHNF4a promotes the differentiation of tumor stem cells, and the proportion is significantly decreased.
  • all-trans retinoic acid or arsenic trioxide was not significantly induced to differentiate into tumor stem cells (data not shown).
  • Human liver tumor cell line, gastric cancer cell line and colon cancer cell line were inoculated into a 35 mm culture dish at 2 ⁇ 10 5 , respectively. After infection with virus AdHNF4 a for 24 h, 8 ⁇ 10 3 cells were inoculated into 10 cm culture dishes, each. The cells were changed for 3 days, cultured for 3-4 weeks, until obvious clones were visible, 4% PFA was fixed, crystal violet staining, and counting clones.
  • ⁇ 3 ⁇ and ⁇ G2 were inoculated into 6-well plates at 2 ⁇ 10 5 , respectively, and cells were fixed at 4 h PFA 72 h, 96 h after infection, and then stained with a newly configured senescence-associated ⁇ -gal solution, 37 ° C, 4 ⁇ 6h, washed with PBS, photographed under an optical microscope.
  • Solution formulation 1 mg/ml 5-bromo-4-chloro-3-indolyl ⁇ -D-galactos ide (X ⁇ Gal), 40 mM c itric aci d/ sodium phosphate (pH 6. 0), 5 mM K 4 Fe (CN) 6 (potassium ferrocyanide), 5 mM K 3 Fe (CN) 6 (potassium ferricyanide), 150 mM NaCl, and 2 mM MgCl 2 .
  • Hpress 3B 5 X 10 6 and H-print G2 1 X 10 7 after infection with AdHNF4 a for 24 h were inoculated into the armpit of nude mice to observe the tumor formation in vivo, and the size of the new tumor was measured with a vernier caliper.
  • HNF4 a gene induced differentiation therapy experimental liver tumor model
  • Hep3B was resuspended in 200 ul of serum-free MEM, injected into the nude mouse through the spleen, and 5 ⁇ 10 9 pfu of AdHNF4 a was introduced into the animal by tail vein injection at 0 d and 2 d after cell injection. After 8 weeks, the nude mice were sacrificed, the liver was removed, frozen sections were taken for HE staining and pathological analysis.
  • HNF4 a gene induced differentiation therapy experimental liver tumor model
  • the results of immunohistochemistry showed that the tumor cells in the treatment group had obvious changes in their atypia (relative rules, small nuclei, nuclear malformation and nuclear fissure increase); the expression of apoptosis-related proteins such as Bcl_2 and Bax did not change significantly. This result indicates that the HNF4 ⁇ gene/protein can effectively induce or promote the differentiation of solid tumors into normal cells in vivo.
  • Hepatocyte nuclear factor 4 is a transcription factor of the nuclear hormone receptor family. It is an important transcriptional protein that regulates hepatocyte differentiation and maintains the biological functions of hepatocytes. It is highly expressed in differentiated and mature hepatocytes. , wherein HNF4 a is an important subtype of HNF4.
  • the wild type human HNF4 a sequence has accession number (GenelD: 419198).
  • HNF4 a knockout mice have revealed that a large number of functional gene expression is down-regulated in hepatocytes at different developmental stages. These genes not only affect hepatocyte differentiation phenotype, but also affect hepatocyte involvement in fat metabolism, albumin synthesis, and drugs. Important gene expression such as detoxification. HNF4 ci binds to a cis-acting element in the form of a dimer.
  • the zinc finger DNA binding domain recognizes the DNA sequence, and It can regulate its own activity by acetylation, phosphorylation and binding to SMADS 3 or 4, and interact with some transcriptional activators such as SRC-1, GRIP-1 and CBP/p300 to change the chromosome structure near the promoter or enhancer. Thereby regulation of differentiation and functional gene expression is achieved at the transcriptional level.
  • HNF4 a regulates the expression of many cell differentiation genes and functional genes, such as up-regulation of HNF4 a expression in embryonic stem cells, which can significantly enhance some important functional genes such as apolipoprotein, aldolase B, phenylalanine hydroxylase, TFN and visual Expression of flavanol-binding protein and the like.
  • the present invention provides a novel method for tumor differentiation and differentiation treatment by injecting HNF4 a adenoviral vector into the in vivo to clarify the effect of up-regulation of HNF4 a expression on the induction and differentiation of a human malignant solid tumor animal model.

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Description

HNF4a诱导分化治疗人体恶性实体瘤 技术领域
本发明涉及分子生物学、 细胞生物学以及医学领域。 具体地， 本发明涉及利用肝细 胞核因子 4 a (Hepatocyte Nuclear Factor-4 α , HNF4 α )诱导人体恶性实体瘤细胞发生 分化， 从而应用于实体肿瘤治疗的方法和用途。 背景技术
肿瘤诱导分化治疗（differentiation therapy)是近 20年来肿瘤临床治疗方面的重要 突破。诱导分化治疗是通过诱导分化方法促进肿瘤细胞向成熟阶段分化， 恢复正常的细胞 表型和功能并抑制恶性肿瘤细胞的增殖。 诱导分化治疗打破了肿瘤发展不可逆的传统认 识， 有力的推动了整个癌症研究领域的发展。
我国学者曾率先运用全反式维甲酸诱导分化治疗急性早幼粒细胞性白血病，取得了较 好的疗效。然而， 尽管白血病的分化治疗取得了很大进展， 但恶性实体瘤的分化治疗仍然 是肿瘤治疗研究领域的难题。 迄今为止， 对于诸如肝癌、 胃癌、 肠癌、 肾癌以及胰腺癌等 人体恶性实体肿瘤的诱导分化治疗仍未见明确报道。迄今实验表明，全反式维甲酸对恶性 实体瘤的分化无明显作用。
选择合适的药物或相关物质进行特异性靶向调控是肿瘤诱导分化治疗的难点。有研究 利用药物或蛋白体外调控实体肿瘤的分化状态， 但效果有限。 体内实验大多提示： 上述方 法在诱导肿瘤细胞凋亡、促进肿瘤组织坏死方面有一定的作用，但是很难实现显著调控或 逆转实体瘤低分化程度。
在众多候选物质中筛选出有效的诱导肿瘤分化的试剂是非常困难的， 也是收效甚微 的。 因此， 选择出与肿瘤细胞诱导分化密切相关的关键蛋白、分子和基因进行特异性靶向 调控是肿瘤诱导分化治疗的核心问题之一。
近年来， 随着人类基因组计划研究的不断深入， 为人们利用基因技术手段调控甚至改 变细胞重要基因的表达以改变其表型、 分化状态和生物学功能创造了条件。然而， 虽然已 经证实有一些物质或基因在体外实验中可改善肿瘤细胞的某些生物性特性（如增殖及克隆 形成能力降低、 部分正常细胞功能基因表达上调）， 一些物质甚至证实了可降低癌细胞在 动物体内的成瘤作用，但是往往发现这些物质对正常细胞也有影响（副作用）， 并不能特异 性地在体内诱导实体肿瘤发生分化。本发明人曾研究了全反式维甲酸、 生长抑素、肿瘤坏 死因子以及三氧化二砷等物质体外对肝癌细胞株分化的调控作用，均未筛选出有明确分化 治疗作用的药物或蛋白， 同时体内研究也发现这些物质不能有效地诱导实体肿瘤发生分 化。
因此，本领域迫切需要开发与恶性实体瘤细胞诱导分化密切相关的特定蛋白或基因， 能够作为特异性调控的靶标， 从而有效地诱导分化实体瘤。 发明内容 本发明的目的在于提供一种与恶性实体瘤细胞诱导分化密切相关的特定的基因 -HNF4 α 基因及其编码产物 HNF4 ci 蛋白，以及 HNF4 α基因 /蛋白在诱导分化实体瘤中的用 途。
本发明的另一个目的在于提供一种通过 HNF4 CI基因 /蛋白分化诱导恶性实体瘤从而 ***的方法。 在本发明的第一方面， 提供了一种肝细胞核因子 4 a 0tepatOCyte Nuclear Factor-4 α , HNF4 α )基因和 /或蛋白的用途， 它们被用于制备诱导恶性实体瘤细胞分化的 诱导分化试剂或组合物。
在另一优选例中， 所述的组合物是药物组合物。
在另一优选例中， 所述的药物组合物含有（a) HNF4 a 蛋白、 HNF4 α 编码序列或含所 述编码序列的表达载体以及 (b)药学上可接受的载体或赋形剂。
在另一优选例中， 所述的表达载体包括病毒载体和非病毒载体。 较佳地， 所述的非 病毒载体为脂质体。
在另一优选例中， 所述的实体瘤选自： 肝癌、 胃癌、 肠癌、 胰腺癌、 肺癌、 *** 癌或生殖腺肿瘤。
在另一优选例中， 所述的药物组合物还用于体内抑制实体瘤的形成。
在另一优选例中， 所述的肝细胞核因子 4 α 是人的肝细胞核因子 4 α。
在另一优选例中， 所述的药物组合物的剂型为注射剂。
在另一优选例中， 所述的药物组合物还含有化疗剂。
在本发明的第二方面， 提供了一种诱导或促进哺乳动物中实体瘤分化的方法， 它包 括步骤： 给需要治疗的哺乳动物对象施用肝细胞核因子 4 α 蛋白、其编码序列或含所述编 码序列的表达载体。
在另一优选例中， 所述的哺乳动物是人。 附图说明
图 1. RT-PCR检测人肝肿瘤细胞株 HNF4 a 基因和肝细胞相关功能基因的表达。 图 2. RT-PCR 获得 HNF4 a cDNA 片段。
图 3. 体外连接获得穿梭质粒 pAdTrack-CMV- HNF4 a Bgl II和 EcoRV酶切鉴定。 图 4. Pac I酶切鉴定重组腺病毒质粒 pAdHNF4 α 。
图 5. 酶切鉴定重组腺病毒质粒 pAdHNF4 α 。
图 6. AdHNF4 α 分别感染 Η印 G2 (A、 B)禾 P H印 3B (C、 D)细胞 3 天后 GFP表达。
图 7. AdHNF4 α 感染肝肿瘤细胞 3 天后 Western blot检测 HNF4 a 蛋白表达。
图 8. AdHNF4 a 感染肝肿瘤细胞 3 天后 HNF4 a 蛋白表达定量分析。
图 9. 检测人肝肿瘤细胞株 HNF4 a 基因和肝细胞相关功能基因 mRNA表达定量分析。 图 10. AdHNF4 a 感染肝肿瘤细胞 3 天后氨代谢检测。
图 11和图 12. AdHNF4 a 感染肝肿瘤细胞后 CD133表达测定。 图 13和图 14. 外源导入 HNF4 ci 对人肝肿瘤细胞克隆形成的影响。
图 15. AdHNF4 α 感染肝肿瘤细胞后 ICG吸收染色测定。
图 16. AdHNF4 α 感染肝肿瘤细胞后体内接种成瘤实验。
图 17. HNF4 a 基因诱导分化治疗实验性肝肿瘤模型。 具体实施方式
本发明人经过广泛而深入的研究，首次发现了一种可有效地在体内诱导或促进恶性实 体瘤向正常细胞分化的关键基因 /蛋白即 HNF4 ci 基因 /蛋白。 实验表明， HNF4 ci 不仅对肿 瘤细胞凋亡有影响， 更可对实体瘤细胞的分化产生诱导或促进作用 (细胞形态变化、 肝细 胞相关功能基因表达明显上调及体内对肿瘤的预防、干预和治疗作用；)。 因此 HNF4 CI 作为 首次被证实的可有效地在体内诱导或促进恶性实体瘤向正常细胞分化的关键基因 /蛋白， 具有潜在的应用前景。 本发明人在此基础上完成了本发明。 如本文所用， 术语 "基因 /蛋白"指基因和 /或蛋白。
如本文所用， 术语 " HNF4 ci 蛋白" 、 "HNF4 ci 多肽" 、 "本发明多肽" 、 "本发明 蛋白"可互换使用， 都指肝细胞核因子 4 α (Hepatocyte Nuclear Factor-4 α )蛋白。 它 们可包括含有或不含起始甲硫氨酸 (Met)的 HNF4 α。狭义上， 所述术语指人的 HNF4 α； 广 义上， 所述术语不仅包括人的 HNF4 α， 还包括其他哺乳动物的 HNF4 ci， 尤其是灵长类动 物的 HNF4 a， 如猿或猴的 HNF4 a。 该术语还包括 HNF4 a 蛋白的活性片段、 活性衍生物和 类似物。
本发明多肽可以是重组多肽、 天然多肽、 合成多肽， 优选重组多肽。 本发明的多肽 可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞）中产生。 根据重组生产方案所用的宿主， 本发明的多肽可以是糖基化的， 或可以是非糖基化的。本发明的多肽还可包括或不包括起 始的甲硫氨酸残基。
如本文所用， 术语 "片段" 、 "衍生物"和 "类似物"是指基本上保持本发明的天 然 HNF4 ci 蛋白（如人 HNF4 ci )相同的生物学功能或活性的多肽。 这些多肽片段、 衍生物或 类似物可以是 (i)有一个或多个保守或非保守性氨基酸残基 (优选保守性氨基酸残基)被取 代的多肽， 而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的， 或 (i i)在一 个或多个氨基酸残基中具有取代基团的多肽，或（i i i)成熟多肽与另一个化合物（比如延长 多肽半衰期的化合物， 例如聚乙二醇）融合所形成的多肽， 或（iv)附加的氨基酸序列融合 到此多肽序列而形成的多肽 (如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序 列， 或与抗原 IgG片段的形成的融合蛋白）。 根据本文的教导， 这些片段、 衍生物和类似 物属于本领域熟练技术人员公知的范围。
例如， 在本发明中， 术语 "人 HNF4 a 多肽"指具有野生型 HNF4 a 序列的多肽。 该术 语还包括具有与人 HNF4 a 蛋白相同的诱导实体瘤分化功能的、 野生型序列的变异形式。 这些变异形式包括 (但并不限于）： 一个或多个 (通常为 1-50个， 较佳地 1-30个， 更佳地 1-20个， 最佳地 1-10个)氨基酸的缺失、 ***和 /或取代， 以及在 C末端和 /或 N末端添 加一个或数个 (通常为 20个以内，较佳地为 10个以内，更佳地为 5个以内）氨基酸。例如， 在本领域中， 用性能相近或相似的氨基酸进行取代时， 通常不会改变蛋白质的功能。又比 如， 在 C末端和 /或 N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。 同样， 该术语还包括人 HNF4 CI 蛋白的活性片段和活性衍生物。
本发明还包括人 HNF4 a 蛋白或多肽的类似物。 这些类似物与天然人 HNF4 a 多肽的 差别可以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者兼而有 之。 类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨基酸）的类似物， 以及具有非 天然存在的或合成的氨基酸 (如 β、 Υ -氨基酸）的类似物。
修饰 (通常不改变一级结构）形式包括：体内或体外的多肽的化学衍生形式如乙酰化或 羧基化。修饰还包括糖基化， 如那些在多肽的合成和加工中或进一步加工步骤中进行糖基 化修饰而产生的多肽。 这种修饰可以通过将多肽暴露于进行糖基化的酶 (如哺乳动物的糖 基化酶或去糖基化酶）而完成。修饰形式还包括具有磷酸化氨基酸残基 (如磷酸酪氨酸，磷 酸丝氨酸， 磷酸苏氨酸）的序列。 还包括被修饰从而提高了其抗蛋白水解性能或优化了溶 解性能的多肽。
在本发明中， "人 HNF4 a 蛋白保守性变异多肽 "指与野生型氨基酸序列相比， 有至 多 10个， 较佳地至多 8个， 更佳地至多 5个， 最佳地至多 3个氨基酸被性质相似或相近 的氨基酸所替换而形成多肽。 这些保守性变异多肽最好根据表 1进行氨基酸替换而产生。
表 1
Figure imgf000005_0001
如本文所用， 术语 "HNF4 ci 基因"或 "本发明基因"可互换使用， 都指编码 HNF4 ci 蛋白的多核苷酸序列。本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、 基因组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编 码链。编码成熟多肽的编码区序列可以与野生型编码区序列相同或者是简并的变异体。如 本文所用， "简并的变异体"在本发明中是指编码具有野生型氨基酸序列的蛋白质， 但与 野生型编码区序列有差别的核酸序列。
术语 "编码多肽的多核苷酸"可以是包括编码此多肽的多核苷酸， 也可以是还包括 附加编码和 /或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，只要这些多核苷酸变异体编码与本发明上述的
HNF4 a 多肽或其的活性片段、 活性类似物和活性衍生物。
在本发明中， 人 HNF4 CI 核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或 人工合成的方法获得。 对于 PCR扩增法， 可根据人 HNF4 ci 的核苷酸序列， 尤其是开放阅 读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人员已知的常规方法所制备的 cDNA库作为模板， 扩增而得有关序列。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明也涉及包含 HNF4 CI 编码序列的载体 (尤其是病毒载体）， 以及用本发明的载体 或 HNF4 CI 编码序列经基因工程产生的宿主细胞， 以及经重组技术产生本发明所述多肽的 方法。
通过常规的重组 DNA技术， 可利用 HNF4 ci 多核苷酸序列可用来表达或生产重组的 HNF4 a 多肽。 一般来说有以下步骤：
(1) .用编码人 HNF4 a 多肽的多核苷酸 (或变异体），或用含有该多核苷酸的重组表达 载体转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养的宿主细胞；
(3) .从培养基或细胞中分离、 纯化蛋白质。
本发明中， 人 HNF4 ci 多核苷酸序列可***到重组表达载体中。 术语"重组表达载体" 指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病 毒、逆转录病毒或其他载体。在本发明中适用的载体包括但不限于： 在细菌中表达的基于 T7的表达载体（Rosenberg, et al. Gene, 1987， 56: 125)；在哺乳动物细胞中表达的 pMSXND 表达载体（Lee and Nathans, J Bio Chem. 263： 3521, 1988)。 总之， 只要能在宿主体内 复制和稳定， 任何质粒和载体都可以用。 表达载体的一个重要特征是通常含有复制起点、 启动子、 标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含人 HNF4 ci 编码 DNA序列和合适的转录 / 翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体内重组技 术等 (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989)。 所述的 DNA序列可有效连接到表达载体中的适当启动子 上， 以指导 mRNA合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启动子； λ 噬菌体 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启动子、 早期和 晚期 SV40启动子、反转录病毒的 LTRs和其他一些已知的可控制基因在原核或真核细胞或 其病毒中表达的启动子。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿 主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白
(GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于转化适当 的宿主细胞， 以使其能够表达蛋白质。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等 真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 真菌细胞如酵母； 动 物细胞如 CH0、 COS, 293细胞等。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为原核 生物如大肠杆菌时， 可用 CaCl2法或电穿孔方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。
获得的转化子可以用常规方法培养， 表达 HNF4 a 多肽。 根据所用的宿主细胞， 培 养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿 主细胞生长到适当的细胞密度后，用合适的方法 (如温度转换或化学诱导)诱导选择的启动 子， 将细胞再培养一段时间。
重组的 HNF4 a 多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法 是本领域技术人员所熟知的。这些方法的例子包括但并不限于： 常规的复性处理、用蛋白 沉淀剂处理 (盐析方法）、 离心、 渗透破菌、 超处理、 超离心、 分子筛层析 (凝胶过滤）、 吸 附层析、离子交换层析、高效液相层析 (HPLC)和其它各种液相层析技术及这些方法的结合。
重组的 HNF4 a 多肽可直接做为诱导分化试剂诱导或促进恶性实体瘤发生分化。此外， 编码 HNF4 a蛋白的多核苷酸或携带 HNF4 a编码序列的载体也可用于诱导或促进实体瘤分 化。
将多核苷酸导入组织或细胞内的方法包括： 将多聚核苷酸直接注入到体内组织中； 或在体外通过载体 (如病毒、噬菌体或质粒等)先将多聚核苷酸导入细胞中， 再将细胞移植 到体内等。
重组的基因治疗载体（如病毒载体)可设计成表达野生型的 HNF4 a 蛋白， 以增加实体 瘤中 HNF4 ci 蛋白的数量和活性。 来源于病毒的表达载体如逆转录病毒、 腺病毒、 腺病毒 相关病毒、 单纯疱疹病毒、 细小病毒等可用于将 HNF4 ci 基因转移至细胞内。 构建携带 HNF4 a 基因的重组病毒载体的方法可见于已有文献（Sambro0k，et al. )。 另外重组人 HNF4 a 基因可包装到脂质体中， 然后再转入细胞内。
本发明 HNF4 ci 蛋白、 HNF4 ci 多核苷酸及载体， 当施用（给药）于哺乳动物对象（如 人）时， 可诱导或促进恶性实体瘤发生分化。 通常， 可将这些物质配制于无毒的、 惰性的 和药学上可接受的载体介质（包括水性载体介质）， 形成药物组合物。 水性载体介质的 pH 通常约为 5-8， 较佳地 pH约为 6-8， 尽管 pH值可随被配制物质的性质以及待治疗的病症 而有所变化。 配制好的药物组合物可以通过常规途径进行给药， 其中包括 (但并不限于）： 瘤内、 肌内、 腹膜内、 静脉内 、 皮下、 皮内、 或局部给药。
本发明的药物组合物可直接用于诱导实体瘤的分化 (治疗），代表性的例子包括 (但并 不限于）： 肝癌、 胃癌、 肠癌、 肺癌、 胰腺癌、 肾癌、 ***癌及生殖腺肿瘤等。 在使用 本发明 HNF4 a 基因 /蛋白或药物组合物时，还可同时或辅助使用其他治疗剂，如顺铂、 TNF 等。
本发明还提供了一种药物组合物， 它含有（a)安全有效量（如 0. 0001-90wt%)的本发 明 HNF4 ci 蛋白、 HNF4 ci 多核苷酸或载体以及（b)药学上可接受的载体或赋形剂。 这类载 体包括 (但并不限于）： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。 药物制剂应 与给药方式相匹配。本发明药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄 糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通 过常规方法进行制备。 药物组合物如针剂、 溶液、 片剂和胶囊宜在无菌条件下制造。 本发 明药物组合物中还可含有其他治疗剂， 如化疗药物。
使用药物组合物时， 是将安全有效量的 HNF4 ci 蛋白、 HNF4 ci 多核苷酸或载体施用 于哺乳动物， 其中该安全有效量通常至少约 1微克 /千克体重， 而且在大多数情况下不超 过约 10毫克 /千克体重，较佳地该剂量是约 10微克 /千克体重-约 1毫克 /千克体重。当然， 具体剂量还应考虑给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围之内的。
本发明还提供了一种诱导和 /或促进恶性实体瘤分化的方法， 该方法包括给需要的哺 乳动物对象（如人）施用本发明的 HNF4 ci 蛋白、 HNF4 ci 多核苷酸或载体， 从而在该对象 体内诱导和 /或促进实体瘤分化。
本发明还提供了一种肿瘤细胞（尤其是恶性实体瘤）基因治疗的方法， 它包括将 HNF4 a 基因导入肿瘤细胞， 使之表达， 其中所述将 HNF4 a 基因导入肿瘤细胞的方法包括 用质粒转染、 腺病毒或腺相关病毒介导。 本发明的主要优点在于：
(a)利用多种基因工程技术方法研究并首次证实了恶性实体瘤的分化治疗。
(b)筛选并证实重要转录因子 HNF4 α 对恶性实体瘤分化的调控作用。
(c)在体内外证实恶性实体瘤分化治疗的可行性及其对临床研究的潜在意义。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条 件， 例如 Sambrook 等人， 分子克隆： 实验室手册（New York : Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1 RT-PCR检测人肝肿瘤细胞株 HNF4 α 基因以及肝细胞相关功能基因的表达
1. 将市售的常规肝肿瘤细胞株 Huh-7、 Hep3B, pG2均以 8 X lO5/皿接种于六孔板， 以含 10%胎牛血清的新鲜培养液培养， 第二天抽提细胞 RNA， 分光光度计测定 0D26。值， 配 成工作浓度（1 μ g/ μ 1及 0.1 μ g/ μ 1)， 1%琼脂糖凝胶电泳检测 RNA完整性。
2. RT-PCR: 取 4μ g RNA、 2μ 1 随机引物、 DEPC水加至 33 μ 1于 70°C置 5 min、 0 °C置 5 min后， 加入 10 μ 1 5 X Buffer 3μ 1 dNTP、 2 μ 1RNA逆转录酶禾 P 2 μ 1 RNA酶抑 制剂混匀后， 37°C置 1.5 h， 即可得到逆转录产物。 将逆转录产物稀释后取 ΐ μ ΐ为模版 进行 PCR扩增， 检测的基因引物序列、 反应条件、 和反应体系见表 2和 3。 各组 RT-PCR 产物于 1.5%琼脂糖胶电泳鉴定、 扫描图象后， 用市售的 Multy-Analasyst 图象分析软件 进行光密度扫描并测序分析。 表 2 反应体系人引物序列以及反应条件
成分 体积（μ ΐ)
正义引物 0.3μ 1
反义引物 0.3μ 1
逆转录产物 1 μ 1
Taq醇 0.2μ 1
lOXTaq缓冲液 1.5μ 1
dNTP 0.4μ 1
dd¾0 11.3μ 1 表 3 人引物序列以及反应条件
基因 引物序列 SEQ ID NO: 产物 退火温度 循环数
HNF4a 正义链 5'- -TTGAAAATGTGCAGGTGTTGAC-3 ' 1 429bp 55 °C 29 反义链 5'- -CAGAGATGGGAGAGGTGATCTG-3 ' 2
AP0CIII 正义链 5'- -GGGTACTCCTTGTTGTTGC-3 3 250bp 55 °C 31 反义链 5'- -AAATCCCAGAACTCAGAGAAC-3 4
G-6-P 正义链 5'- -GGCTCCATGACTGTGGGATC-3 ' 5 475bp 49 °C 32 反义链 5'- -TTCAGCTGCACAGCCCAGAA-3 ' 6
ALB 正义链 5'- -AGCCTAAGGCAGCTTGACTT-3 ' 7 1212bp 55 °C 29 反义链 5'- -CTCGATGAACTTCGGGATGA-3 ' 8
GS 正义链 5'- -CCTGCTTGTATGCTGGAGTC-3 ' 9 396bp 55 °C 35 反义链 5'- -GAAAAGTCGTTGATGTTGGA-3 ' 10
CYPla2 正义链 5'- -CTGGCCTCTGCCATCTTCTG-3 ' 11 464bp 49 °C 35 反义链 5'- -TTAGCCTCCTTGCTCACATGC-3 ' 12
PEPC 正义链 5'- -GTGTCCCTCTAGTCTATGAAGC-3 ' 13 485bp 49 °C 35 反义链 5'- -ATTGACTTGATCCTCCAGATAC-3 ' 14 TTR 正义链 5 '-GCGGGACTGGTATTTGTGTCTG-3 ' . 15 398bp 55 °C 27 反义链 5 '-TTAGTGACGACAGCCGTGGTG-3 ' 16
AFP 正义链 5 '-AGCTTGGTGGTGGATGAAAC-3 ' 17 248bp 55 °C 35 反义链 5 '-CCCTCTTCAGCAAAGCAGAC-3 ' 18
b-actin 正义链 5 '-CATCCTGCGTCTGGACCT-3 ' 19 499bp 55 °C 25 反义链 5 '-GTACTTGCGCTCAGGAGGAG-3 ' 20
肝细胞核因子 4 a (hepatocyte nuclear factor 4 α HNF4 a ) 葡萄糖 -6-磷酸酶
(glucose-6-phosphatase, G-6-P)； 白蛋白 (albumin, ALB)；谷胺酰氨合成酶（glutamine synthetase, GS)； 细胞色素 P450家族 la2 (cytochrome P450 la2 CYPla2)； 磷酸烯醇 丙酮酸羧激酶（phosphoenolpyr te carboxykinase, PEPCK)； 甲状腺激素结合蛋白 (transthyretin, TTR) ； 甲 胎 蛋 白 （alpha fetoprotein, AFP) ； 载月旨 蛋 白 CI l Kapol ipoprotein cI I I , APOCI I I)
结果表明：肝肿瘤细胞株 Huh-7 Hep3B以及 HepG2的 HNF4 α 表达均明显下调， HNF4 α 基因的表达与肝细胞相关的重要功能基因表达呈正相关（图 1和图 9)。 实施例 2
构建表达 HNF4 a 的重组复制缺陷型腺病毒 AdHNF4 a
1. 获取 HNF4 a 1425bp cDNA片段： 根据人 HNF4 a cDNA序列设计、 合成引物。 正义 引物（在 5 ' 端加入 Bgl II酶切位点）： 5 ' - CCG AGA TCT AGA ATG CGA CTC TCC AAA ACC -3 ' (SEQ ID NO : 21)。 反义引物（在 5 ' 端加入 EcoR V酶切位点）： 5 ' - CGC GAT ATC GGC TTG CTA GAT AAC TTC CTG CT - 3 ' (SEQ ID NO : 22)。
PCR扩增 HNF4 a cDNA片段， 产物 1%琼脂糖凝胶电泳， 鉴定片段大小，并割胶回收置 入 Eppendorf 管内，称取胶重量。 Eppendorf 管中加入 NT液 200 ml/100 mg胶， 50 °C 5-10 min至胶熔化， 将液体过柱， 13 000 rpm离心 1 min， 加入 600 μ 1 ΝΤ3缓冲液， 13 000 rpm 离心 2 min 30 μ 1 双蒸水 过柱洗脱 DNA片段， 静放 1 min 13 000 rpm离心 1 min， 小 心移取洗脱液于干净的 Eppendorf 管内。 分光光度计测定 0D26。值， 1. 5%琼脂糖凝胶电泳 鉴定片段大小（图 2)。 成分 体积（μ ΐ)
正义引物 5 μ 1
反义引物 5 μ 1
正常的人肝细胞 cDNA 2 μ 1
pfu酶 2 μ 1
lO X pfu缓冲液 10 μ 1
dNTP 10 μ 1
dd¾0 66 μ 1
反应条件： 94°C 30 s 60 °C 30 s 72 °C 90 s 35个循环。 2. 构建表达 HNF4 a 的腺病毒质粒 pAdHNF4 a ： EcoR V、 Bgl ll酶切穿梭质粒 pAdTrack-CMV (购自霍华德 -休斯医学研究所， 美国）和 HNF4 a cDNA 4 h 后并纯化， 取 0. 1 μ g质粒 pAdTrack_CMV、0.4μ gHNF4 a cDNA、 10 X T4缓冲液 2 μ 1、T4DNA连接酶 1 μ 1 (2U) 以及 dd 0， 总体积 20 μ 1， 16°C连接过夜。将连接产物加入常规的感受态大肠杆菌 DH5 a 转化， 用含卡那霉素的 LB培养基平皿铺板， 37°C恒温过夜， 选择单菌落克隆， 将扩增出 HNF4a cDNA片段的菌落克隆用 Qiagen-tip 100试剂盒中抽， 获取质粒 pAdTrack_CMV- HNF4 α 并鉴定（图 3)。 Pme I内切酶酶切线性化 pAdTrack- CMV- HNF4 α， 分别取 0.4 μ g线 性 pAdTrack-CMV- HNF4 α 禾 Ρ 0. 1 μ g超螺旋 pAdEasy-1质粒 2， 000 V、 200 0hms、 25 μ FD 电穿孔共转化 20 μ ΐ 感受态 BJ5183细菌， 卡那霉素 LB培养基平板筛选， 选择病毒质粒 pAdHNF4a 鉴定（图 4和图 5)。
3. 包装、 扩增腺病毒 AdHNF4 a： 复苏常规的 293细胞， 以 4.8X 106/皿接种于 10-cm 的组织培养皿， 加入 DMEM 37°C、 5%C02培养， 24 h后细胞密度生长至 60%〜80%。 Pac I 酶切线性化 pAdHNF4 α，与不含血清的 DMEM培液 250 μ 1混匀，配成 Α液；取 Lipofectamin 20 μ 1， 加入不含血清的 DMEM培液 250 μ 1混匀， 配成 Β液， Α液与 B液充分混匀， 室温 放置 30 min后加入待转染的 293细胞中， 4 h后更换培液。 7 d后收集 293细胞以及上清， 于液氮和 37°C水浴中反复冻融 4次， 5, 000 rpm离心 5 min， 收集病毒上清， 病毒上清再 次感染 293细胞进行扩增， 2〜3 d后收集病毒； 重复感染、 收集步骤， 将最终收集的病 毒上清分装， 测定病毒上清滴度， 最终得到滴度约为 IX 101。 efu/ml 的 AdHNF4 α， 置于 -80°C保存备用。 实施例 3
RT-PCR和 Western blot检测 AdHNF4 α 感染人肝肿瘤细胞株后 HNF4 α 的表达
1. 人肝肿瘤细胞株 pG2和 Hep3B均以 5 X 105/皿接种于六孔板， 将病毒 AdHNF4 α 分别以 ΜΟΙ 40、 100感染细胞， 24 h后更换含 10%胎牛血清的新鲜 DMEM培液， 3 d后观 察 GFP表达（图 6)。 以 Trizol试剂盒抽提总 RNA， 逆转录反应 2 h， 取 1 μ 1稀释的逆转 录产物为模板进行 HNF4 a PCR扩增， 反应条件同前， 同时以 β -actin在相同反应条件进 行 PCR反应作为内参照， 反应体系如下。
成分 体积（μ l)
正义引物 0.3 μ 1
反义引物 0.3 μ 1
逆转录产物 1 μ 1
Taq醇 0.2 μ 1
lOXTaq缓冲液 1.5 μ 1
dNTP 0.4μ 1
dd¾0 11.3 μ 1 反应条件为 95°C 30 s， 55V 30 s， 72V 90 s， 27个循环。 RT-PCR产物于 1.5%琼脂糖胶电泳鉴定、 扫描图象后， 用 Multy-Analasyst图象分析 软件进行光密度扫描并测序分析。
结果表明： AdHNF4a 感染人肝肿瘤细胞株后 HNF4 a mRNA在肿瘤细胞中表达明显上 调（图 7和 8)。
2. AdHNF4 a 分别感染 pG2和 Hep3B， 细胞裂解液收取全细胞蛋白， 蛋白标准定量 后，各取 10 μ g于 10% SDS -PAGE电泳分离蛋白，将聚偏二氟乙烯膜（PVDF膜） ddH20冲洗， 将电泳胶、 PVDF膜、 滤纸放于转移缓冲液（Transferring Buffer)中平衡后， 置于电转移 槽中， 18 V, 40 min。 用 5% BSA/PBST 20 ml室温封闭膜 2 h后， HNF4 α 多抗（1:500)4 °C孵育过夜， 次日 PBST洗涤后， 与驴抗羊荧光二抗（1:2000)室温孵育 30 min， PBST洗涤 2次后， 经 Odyssey 红外激光成像***检测荧光并进行灰度扫描。
结果显示， AdHNF4a 感染人肝肿瘤细胞株后 HNF4 α 蛋白表达分别增加约 3.4 倍 (HepG2)和 5.2倍 (Hep3B) (图 7， 8)。 实施例 4
外源导入 HNF4ci 对人肝肿瘤细胞生物学特性影响
1. RT-PCR检测肝细胞相关功能基因的表达：将常规的人肝肿瘤细胞株 HepG2和 Hep3B 均以 5X107皿接种于六孔板， 将病毒 AdHNF4a 分别以 M0I 40、 100感染细胞， 24 h后 更换含 10%胎牛血清的新鲜 DMEM培液， 3 d后观察 GFP表达；以 Trizol试剂盒抽提总 RNA， 逆转录反应 2 h， 取 1 μΐ稀释的逆转录产物为模板进行 PCR扩增， 检测的基因引物序列 以及反应条件同实施例 1， 反应体系如下。 成分 体积（μΐ)
正义引物 0.3μ 1
反义引物 0.3μ 1
逆转录产物 1 μ 1
Taq醇 0.2μ 1
lOXTaq缓冲液 1.5μ 1
dNTP 0.4μ 1
dd¾0 11.3μ 1 各组 RT-PCR产物于 1.5%琼脂糖胶电泳鉴定、 扫描图象后， 用 Multy-Analasyst图象 分析软件进行光密度扫描并测序分析。
结果表明： AdHNF4a 感染组中肝细胞相关功能基因表达较对照组明显上调， 其中 G-6-P mRNA表达上调近 50倍； ALB、TTR表达未见明显变化， AFP表达则明显下调（表 3_1)。 表 3-1 半定量分析 HNF4(x基因导入后肝细胞相关功能基因表达 细胞株
Hep3B HepG2
基 因 光密度比值 光密度比值
AdHNF4a组 AdHNF4a组
P值 P值
AdGFP组 AdGFP组
GS 2.9倍 P<0.01 12.6倍 P<0.01
PEPCK 16.7倍 P<0.01 5.0倍 P<0.01
G-6-P 52.3倍 P<0.01 2.3倍 P<0.05
CYPla 6.1倍 P<0.01 18.6倍 P<0.01
APOCIII 4.9倍 P<0.01 5.7倍 P<0.01
AFP 9.3 % P<0.01 24.7 % P<0.01
2. 流式细胞仪测定人肝肿瘤细胞凋亡率： 将常规的肝癌细胞株 pG2和 Hep3B均以 5 X 107皿接种于六孔板， 分别将病毒 AdHNF4 a 以 M0I 40、 100感染细胞， 24 h后更换含 10%胎牛血清的新鲜 DMEM培液， 第 3天收集细胞， 于 EPICS XL 流式细胞仪（Coulter)测 定细胞凋亡率并进行统计学分析。 各组复设 2盘， 重复 3次。
结果表明： 上调肝肿瘤细胞 HNF4 ci 表达后细胞的凋亡率有一定影响。
3. 流式细胞仪测定人肝肿瘤细胞细胞周期变化： 将肝癌细胞株 HepG2和 Hep3B均以 5 X 107皿接种于六孔板， 分别将病毒 AdHNF4 a 以 M0I 40、 100感染细胞， 24 h后更换含 10%胎牛血清的新鲜 DMEM培液， 第 3天收集细胞， 于 EPICS XL 流式细胞仪（Coulter)测 定细胞周期变化并进行统计学分析。
结果表明： 在病毒感染后 72h、 96h， pG2表现为 S期减少。
4.用常规试剂盒检测细胞上清的氨浓度。
结果表明， 与对照组（不加病毒或空病毒）相比， 试验组的氨代谢能力明显增强（图 10)。 实施例 5
外源导入 HNF4 a 对人实体肿瘤细胞增殖的影响
人肝肿瘤细胞株、 胃癌细胞株和结肠癌细胞株分别以 5 X 107孔接种于 96 孔板， 24 h后感染病毒 AdHNF4 a， 此后每天用 CCK8试剂检测 450 nm波长的吸光度以判断有活 性细胞的数量。
结果表明： HNF4 a 表达对实体肿瘤细胞增殖有明显的抑制作用， 最早从感染病毒后 第 3 天， AdHNF4 a 感染组肿瘤细胞的增殖开始下降， 至第 5天细胞数量明显减少， 抑制 率高达 50%-68%。 同时研究发现， 随着病毒感染滴度的增高， HNF4 ci 上调对部分实体肿瘤 细胞增殖抑制作用表现为时间和剂量的依赖性。 实施例 6
外源导入 HNF4 a 对人肝肿瘤细胞 CD133表达的影响
将人肝肿瘤细胞 HepG2和 H印 3B均以 5 X 105/皿接种于六孔板， 分别将病毒 AdHNF4 a 以 MOI 40、 100感染细胞， 24 h后更换含 10%胎牛血清的新鲜 DMEM培液， 第 3天收集细 胞， CD133/l-PE (Mi ltenyi Biotec, Auburn, CA)作为一抗孵育， 流式细胞仪 CD 133+细胞 的比例。
结果表明， AdHNF4 a 感染后肝肿瘤细胞中 CD 133+的细胞比例明显减少（图 11 和图
12) 。
CD133+是肿瘤干细胞的特异性标志物， CD133+的细胞比例明显减少表明 AdHNF4 a 促 进诱导肿瘤干细胞发生分化， 比例明显下降。 另外， 未对全反式维甲酸或三氧化二砷对肿 瘤干细胞的有明显诱导分化作用（数据未示出）。 实施例 7
外源导入 HNF4 ct 对人实体肿瘤细胞克隆形成的影响
人肝肿瘤细胞株、 胃癌细胞株和结肠癌细胞株分别以 2 X 105接种于 35 mm培养皿， 病毒 AdHNF4 a 感染 24 h后， 各取 8 X 103 细胞接种于 10 cm培养皿， 每 3天换液， 培 养 3-4 周， 直至可见明显克隆， 4%PFA固定， 结晶紫染色， 计数克隆。
结果表明， AdHNF4 a 感染后人实体肿瘤细胞株形成的克隆均较对照组减少， HNF4 a 上调可明显降低实体肿瘤细胞株的克隆形成能力（图 13和 14)。 实施例 8
外源导入 HNF4 ct 对人肝肿瘤细胞衰老相关的 β -gal 染色
将 ρ3Β和 Η印 G2 分别以 2 X 105 接种于六孔板， 分别于感染后 72 h、 96 h， 4% PFA固定细胞， 然后用新配置的衰老相关的 β -gal 溶液染色， 37 °C， 4〜6h， PBS洗 涤， 光学显微镜下拍照。 溶液配方： 1 mg/ml 5-bromo-4-chloro-3-indolyl β -D-galactos ide (X~Gal)， 40 mM c itric aci d/ sodium phosphate (pH 6. 0)， 5 mM K4Fe (CN) 6 (亚铁***）， 5 mM K3Fe (CN) 6 (铁***）， 150 mM NaCl , and 2 mM MgCl2。
结果表明： HNF4 ci 基因导入后， 在 pG2 细胞中 β -gal 染色阳性的细胞较对照组 明显增多， 提示上调 HNF4 CI 对部分肝肿瘤细胞的抑制作用是通过诱导衰老来实现的（图 15)。 实施例 9
上调人肝肿瘤细胞株 HepG2和 Hep3B中 HNF4 α 表达对体内成瘤的影响
分别取 AdHNF4 a 感染 24 h后的 H印 3B 5 X 106和 H印 G2 1 X 107接种于裸鼠腋下， 观察体内成瘤情况， 同时用游标卡尺测量新生肿瘤的大小。
结果表明 Hep3B对照组早在接种后第 2 周检测出肿瘤生长， 第 3周所有裸鼠均有肿 瘤生成； HepG2对照组在接种后第 3周检测出肿瘤生长， 到第 5周 75%的裸鼠均有肿瘤生 成。 在观察的 5周中， AdHNF4 a 感染后的肝肿瘤细胞接种的裸鼠体内没有明确的肿瘤生 成（图 16和 17)。 实施例 10
HNF4 a 基因诱导分化治疗实验性肝肿瘤模型（1)
5 X 106 Hep3B 重悬于 200ul 的无血清的 MEM中， 通过脾脏注射裸鼠体内， 同时于 细胞注射后 0 d和 2d， 将 5 X 109 pfu的 AdHNF4 a 通过尾静脉注射导入动物体内。 8周 后处死裸鼠， 取出肝脏， 冰冻切片并作 HE染色和病理分析。
结果表明： 病毒通过尾静脉注射 3 天后， 取出肝脏， 冰冻切片， 荧光显微镜下观察 80% 肝细胞有 GFP表达。 8 周后， 对照组肝脏中均发现明显肿瘤生长， HNF4 a 基因治疗 组中有 2只裸鼠无肿瘤生长， 一只仅出现较小肿瘤。 进一步 HE染色分析， HNF4 a 基因治 疗组中 2只肉眼无肿瘤的裸鼠肝脏 HE染色为正常的肝脏组织结构， 对照组均可见恶性细 胞。 实施例 11
HNF4 a 基因诱导分化治疗实验性肝肿瘤模型 (2)
利用颈部皮下接种肝癌细胞株构建形成实验性肝肿瘤裸鼠模型后， 将 1 X 1010 pfu 的 AdHNF4 a 通过颈静脉注射导入动物体内。
结果显示： 病毒注射 3天后， 荧光显微镜下观察 80%以上的肿瘤细胞有 GFP表达。 治 疗 1 周后， 定期测定肿瘤大小， HNF4 ci 基因治疗组中 8只裸鼠肿瘤大小平均值在不同时 间节点均显著小于对照组； 生存时间比较， HNF4 ci 基因治疗组明显长于对照组。 免疫组 化结果显示， 治疗组肿瘤细胞异型性有明显改变 (形态相对规则， 胞核小， 核畸形及核分 裂增多少见）；与凋亡相关的蛋白如 Bcl_2、 Bax等表达未见明显变化。该结果表明， HNF4 α 基因 /蛋白可有效地在体内诱导或促进实体瘤向正常细胞分化。 实施例 12
全反式维甲酸、 生长抑素（Somatostatin)、 肿瘤坏死因子以及三氧化二砷等对肝癌细 胞株 HepG2和 ρ3Β的体外作用
人肝肿瘤细胞株 HepG2和 ρ3Β均以 5 X 105/皿接种于六孔板， 分别加入全反式维甲 酸、 生长抑素、 肿瘤坏死因子以及三氧化二砷； 以 Trizol试剂盒抽提总 RNA， 逆转录反 应 2 h， 取 1 μ ΐ稀释的逆转录产物为模板进行 PCR扩增， 检测肝细胞相关功能基因 mRNA 表达， 免疫组化测定 Cycl iru Bax, Bcl_2等增殖及凋亡相关蛋白表达。
结果表明： 各组肝细胞相关功能基因表达未见明显差别， 与肿瘤分化相关的部分基因 表达 (HNF4 a、 HNFl a、 C/EBP)表达未见明显上调， 细胞形态学无明显改变； 肿瘤坏死因 子和三氧化二砷组细胞凋亡明显增多、增殖下调。这提示这些物质对照物质对肝癌细胞没 有诱导分化作用（因为细胞形态无变化）。 讨论
肝细胞核因子 4 (h印 atocyte nuclear factor, HNF4)是一种细胞核激素受体家族的 转录因子， 是调控肝细胞分化和维护肝细胞生物学功能的重要转录蛋白，在分化成熟的肝 细胞中高表达， 其中 HNF4 a 是 HNF4 的重要亚型。 野生型的人 HNF4 a 序列的登录号为 ( GenelD: 419198)。
对 HNF4 a 基因敲除小鼠研究发现： 不同发育阶段的肝细胞中都出现大量功能基因表 达下调， 这些基因不仅影响肝细胞分化表型， 还影响肝细胞中参与脂肪代谢、 白蛋白合成 以及药物解毒等重要基因表达。 HNF4 ci 以二聚体的形式与顺式作用元件结合， 当 HNF4 ci 的 DNA及配体结合域和孕烷 X受体 a 形成二聚体时， 其利用锌指 DNA结合域识别 DNA序 列， 并可通过乙酰化、 磷酸化以及与 SMADS 3或 4 结合调节自身活性， 与一些转录激活 蛋白如 SRC-1、GRIP-1和 CBP/p300等相互作用来改变启动子或增强子附近的染色体结构， 从而在转录水平实现对分化和功能基因表达的调控。
由于绝大多数物质或基因虽然在体外实验中可改善肿瘤细胞的某些生物性特性或可 降低癌细胞在动物体内的成瘤作用，但是几乎都是通过诱导细胞凋亡实现， 并不能特异性 地在体内诱导实体肿瘤发生分化。 因此早先的研究虽认为上调肝癌细胞株 HNF4 a 的表达 可改善肿瘤细胞的某些生物性特性， 但并不相信也未证实 HNF4 a 居然具有诱导分化恶性 实体瘤， 进而逆转肿瘤低分化状态的能力； HNF4 a 对其它恶性实体肿瘤的分化调控作用 亦不明确； 更未将上调 HNF4 a 表达作为诱导分化治疗手段加以研究。
本发明的创新研究的表明： 利用基因工程技术调控实体肿瘤细胞 HNF4 a 基因表达， 可有效地对肿瘤细胞的产生诱导分化作用。 HNF4 a调控众多细胞分化基因和功能基因的表 达， 如通过上调胚胎干细胞 HNF4 a表达， 可明显增强一些重要功能基因如载脂蛋白、 醛 缩酶 B、 苯丙氨酸羟化酶、 TFN和视黄醇结合蛋白等表达。
更为重要的是， 上调 HNF4 a表达还可逆转肝癌细胞的去分化状态。 因此， 这提示， HNF4 a可能还在不同类型肿瘤的分化转录调控中发挥重要作用。 因此， 本发明通过体内 HNF4 a 腺病毒载体注射明确 HNF4 a表达上调对人体恶性实体瘤动物模型的诱导分化治疗 作用， 从而提供了一种肿瘤诱导分化治疗的新手段。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员可 以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范 围。

Claims

权 利 要 求
1 . 一种肝细胞核因子 4 α (Hepatocyte Nuc lear Factor- 4 α， HNF4 a )基因 和 /或蛋白的用途， 其特征在于， 用于制备诱导恶性实体瘤细胞分化的诱导分化 试剂或组合物。
2. 如权利要求 1所述的用途， 其特征在于， 所述的组合物是药物组合物。
3. 如权利要求 2所述的用途，其特征在于，所述的药物组合物含有（a) HNF4 α 蛋白、 匪 a 编码序列或含所述编码序列的表达载体以及（b)药学上可接受的载 体或赋形剂。
4.如权利要求 3所述的用途，其特征在于，所述的表达载体包括病毒载体和 非病毒载体。
5.如权利要求 1所述的用途， 其特征在于，所述的实体瘤选自： 肝癌、 胃癌、 肠癌、 胰腺癌、 肺癌、 ***癌或生殖腺肿瘤。
6.如权利要求 2所述的用途，其特征在于，所述的药物组合物还用于体内抑 制实体瘤的形成。
7. 如权利要求 1所述的用途， 其特征在于， 所述的肝细胞核因子 4 是人 的肝细胞核因子 4 。
8. 如权利要求 2所述的用途， 其特征在于， 所述的药物组合物的剂型为注 射剂。
9. 如权利要求 2所述的用途， 其特征在于， 所述的药物组合物还含有化疗 剂。
10.—种诱导或促进哺乳动物中实体瘤分化的方法， 其特征在于， 它包括步 骤： 给需要治疗的哺乳动物对象施用肝细胞核因子 4 蛋白、其编码序列或含所 述编码序列的表达载体。
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