WO2012068936A1 - 一种肝细胞核因子1α在制备治疗恶性实体瘤疾病药物中的应用 - Google Patents

一种肝细胞核因子1α在制备治疗恶性实体瘤疾病药物中的应用 Download PDF

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WO2012068936A1
WO2012068936A1 PCT/CN2011/081163 CN2011081163W WO2012068936A1 WO 2012068936 A1 WO2012068936 A1 WO 2012068936A1 CN 2011081163 W CN2011081163 W CN 2011081163W WO 2012068936 A1 WO2012068936 A1 WO 2012068936A1
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nuclear factor
solid tumor
cancer
hnfla
hepatocyte nuclear
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French (fr)
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谢渭芬
曾欣
林勇
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中国人民解放军第二军医大学
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Priority to US13/824,691 priority Critical patent/US20140010786A1/en
Publication of WO2012068936A1 publication Critical patent/WO2012068936A1/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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—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

  • hepatocyte nuclear factor 1 ⁇ Application of hepatocyte nuclear factor 1 ⁇ in preparing medicine for treating malignant solid tumor diseases
  • the present invention relates to the use of hepatocyte nuclear factor 1 ⁇ , and more particularly to the use of a hepatocyte nuclear factor 1 ⁇ for the preparation of a medicament for the treatment of malignant solid tumor diseases.
  • malignant solid tumors are one of the current clinical difficulties. Especially for malignant solid tumors that cannot be removed by surgery, there is still no effective treatment in clinical practice. Selective targeting of key proteins, molecules and genes closely related to the development of tumor cells is one of the core issues in the treatment of malignant solid tumors.
  • Hepatocyte nuclear factor 1 belongs to the P0U-homologous domain family and 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, of which HNFla is HNF1. An important subtype. Studies on HNFla knockout mice have revealed that HNFla is an essential transcription factor in liver development and is closely related to the establishment and maintenance of the final normal differentiation and development of embryonic liver. HNFla knockout mice may have severe liver and kidney damage. Death within a few days after birth.
  • HNFla binds to cis-acting elements either homologously or in the form of a heterodimer with HNFip, interacts with some transcriptional activators to alter the chromosomal structure near the promoter or enhancer, thereby enabling differentiation and function at the transcriptional level. Regulation of gene expression. Despite studies in foreign countries, the expression level of HNFla is closely related to the differentiation of hepatocellular carcinoma. After down-regulating the expression of HNFla, the expression of some hepatocyte functional genes in tumor cells is decreased. But can HNFla improve tumor cell biology? Learning characteristics, reducing the tumorigenicity of liver cancer and reversing its poorly differentiated state have not been confirmed; the regulation of other malignant solid tumors is not clear; nor has the up-regulation of HNFla expression as a treatment for malignant solid tumors.
  • Chinese Patent Application No. 200810034200.3 discloses a method for inducing differentiation of HNF4a in the treatment of malignant solid tumors of human body, and relates to the method and use of the treatment of malignant solid tumors by using hepatocyte nuclear factor 4a to induce differentiation of human malignant solid tumor cells, and studies have shown that by regulating malignancy The expression of HNF4a gene in solid tumor cells can effectively induce differentiation of tumor cells, thus providing a new means of tumor differentiation therapy.
  • the application of a hepatocyte nuclear factor la gene and/or protein and its expression vector in the preparation of a medicament for treating malignant solid tumor diseases has not been reported yet.
  • the object of the present invention is to provide a use of a hepatocyte nuclear factor 1 ⁇ gene and/or protein for the preparation of a medicament for treating malignant solid tumor diseases, in view of the deficiencies in the prior art.
  • Another object of the present invention is to provide a recombinant expression vector containing hepatocyte nuclear factor lot for the preparation of a medicament for treating malignant solid tumor diseases.
  • a fourth object of the present invention is to provide a use of a recombinant expression vector containing hepatocyte nuclear factor 1 ⁇ for the preparation of an inducing differentiation agent or composition for inducing differentiation of malignant solid tumor cells.
  • a fifth object of the present invention is to provide a method of inducing or promoting differentiation of solid tumors in a mammal.
  • the technical solution adopted by the present invention is: The use of a hepatocyte nuclear factor 1 ⁇ gene and/or protein in the preparation of a medicament for treating malignant solid tumor diseases.
  • the technical solution adopted by the present invention is: The use of a hepatocyte nuclear factor lex and/or protein for the preparation of an inducing differentiation agent or composition for inducing differentiation of malignant solid tumor cells.
  • 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 composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises (a) a HNFla protein, an HNFla coding sequence or an expression vector comprising the coding sequence, and (b) a pharmaceutically acceptable carrier or excipient.
  • the expression vector includes a viral vector and a non-viral vector.
  • the hepatocyte nuclear factor 1 ⁇ is human hepatocyte nuclear factor 1 ⁇ .
  • the technical solution adopted by the present invention is: The use of a recombinant expression vector containing hepatocyte nuclear factor 1 ⁇ for the preparation of a medicament for treating malignant solid tumor diseases.
  • the technical solution adopted by the present invention is: The use of a recombinant expression vector containing hepatocyte nuclear factor 1 ⁇ for the preparation of an inducing differentiation agent or composition for inducing differentiation of malignant solid tumor cells.
  • 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 expression vector includes a viral vector and a non-viral vector.
  • the technical solution adopted by the present invention is: A method for inducing or promoting differentiation of solid tumors in a mammal, the method comprising the steps of: administering a hepatocyte nuclear factor 1 ⁇ to a mammalian subject in need of treatment A protein, a coding sequence thereof or an expression vector comprising the coding sequence.
  • the invention provides a new use of hepatocyte nuclear factor le, and proposes a new treatment for the treatment of malignant solid tumors.
  • Method. utilizes genetic engineering technology to regulate the expression of HNFl gene in solid tumor cells, and proves that it improves the biological characteristics of tumor cells and blocks the growth of tumor cells; and the therapeutic effect of up-regulation of HNFla expression on the animal model of malignant solid tumors by injection of HNFla adenovirus vector in vivo This is a new exploration in the field of treatment of malignant solid tumors.
  • FIG. 1 Real-time RT-PCR detection of HNF1 a gene expression in human liver tumor cell lines.
  • HNFla expression in adjacent tissues is higher than that in HCC tissues; II: HNFla expression in liver cancer and adjacent tissues is equivalent; HNFla expression in paracancerous tissues is lower than that in HCC tissues ).
  • FIG. 4 Immunohistochemical detection of HNFla gene expression was gradually down-regulated during DEN-induced liver cancer model in rats.
  • Figure 5. HNFla cDNA fragment obtained by RT-PCR.
  • Figure 6 In vitro ligation obtained shuttle plasmid pAdTrack-CMV-HNFla, Bgl ll, I restriction enzyme digestion.
  • Figure 7. Identification of recombinant adenovirus plasmid pAdHNFl by Pac I digestion
  • AdHNFla is infected with Hep3B (A, B), Huh7 (C, D), MHCC-H (E, F), respectively;
  • MHCC-L (G, H) cells were expressed after 3 days of GFP.
  • FIG. 1 Quantitative analysis of HNFla protein expression after 3 days of AdHNFla infection in liver tumor cells.
  • FIG. 14 Apoptosis changes after 3 days of AdHNFla infection in liver tumor cells.
  • FIG. 1 Cell cycle changes after 3 days of AdHNFla infection in liver tumor cells.
  • Figure 17 Effect of exogenous introduction of HNFl on cell proliferation of different human liver tumor cell lines.
  • Figure 18 Effect of exogenous introduction of H Fl a on cell clone formation in different human liver tumor cell lines.
  • FIG. 20 AdHNFla infection in liver tumor cells Huh7 in vivo inoculation tumor formation experiments.
  • FIG. 21 AdHNFla intratumoral injection gene therapy for subcutaneous tumor formation.
  • FIG. 22 HNFla gene treatment of hepatic tumor cells in situ by injection into an experimental liver tumor model. detailed description
  • Liver tumor cell lines Huh-7, Hep3B, MHCC-H, MHCC-L, PLC, YY, 7721 were inoculated into 6-well plates at 5> ⁇ 10 5 / dish, and freshly cultured with 10% fetal bovine serum.
  • the RNA was extracted on the next day, and the OD 26Q value was measured by a spectrophotometer to prepare a working concentration (1 ⁇ ⁇ / ⁇ 1 and 0.1 ⁇ ⁇ / ⁇ 1), and RNA integrity was detected by 1% agarose gel electrophoresis.
  • Realtime RT-PCR Take 4 g RNA, 2 ⁇ 1 Randomprimer, add DEPC water to 33 ⁇ 1 at 70 °C for 5 min, and at 0 °C for 5 min, then add ⁇ 5xBuffer, 3 ⁇ 1 ⁇ 2 lRNA reverse transcriptase and 2 ⁇ 1 RNA. After the enzyme inhibitor was mixed and placed at 37 ° C for 1.5 h, the reverse transcription product was obtained, as shown in Table 1. The reverse transcription product was diluted, and ⁇ was used as a template for Realtime PCR amplification.
  • the gene primer sequence is shown in Table 2.
  • the reaction system was as follows.
  • the reaction conditions were: 94 ° C pre-denaturation for 30 s, then 94 ° C l0 s, 60 ° C 30 s, The detection of the dissolution curve was carried out after 40 cycles. The results showed that the expression of HNFla m NA in each liver tumor cell line was significantly down-regulated. see picture 1.
  • Example 2 Realtime RT-PCR and immunohistochemistry for detection of HNFla gene and protein expression in human hepatocellular carcinoma tissues and adjacent tissues
  • RNA from human liver cancer and paracancerous tissues, and determines its OD 26Q value by spectrophotometer to prepare working concentration ( ⁇ / ⁇ and 0. ⁇ ⁇ / ⁇ ), 1% agarose Gel electrophoresis was used to detect RNA integrity. 4 ⁇ ⁇ RNA was used for reverse transcription and Realtime PCR amplification (reverse transcription reaction, Realtime PCR reaction conditions and primer sequences as before). The results showed that in 11 pairs of human hepatocarcinoma tissues/cancer tissues, the expression of H Fla in liver cancer tissues of 7 patients (63.64%) was lower than that of adjacent tissues. See Figure 2. 2.
  • Immunohistochemistry 4 mm serial section of human liver cancer and paracancerous tissue wax block, baked in oven at 60 °C for 30 min, dewaxed to water, 3 % H 2 0 2 at room temperature for 10 min to eliminate endogenous Peroxidase, citrate buffer microwave for antigen retrieval, add 1: 10 normal rabbit serum for 30 min at room temperature, add HNFla antibody (1:200) at 4 °C overnight; next day PBS (0.01 M, pH 7.4) Wash 3 times, each time 5 min; add secondary antibody to incubate for 30 min at room temperature; wash with PBS, add SABC (1:100) for 20 min at room temperature, DAB color, routine resin sealing, observe under light microscope.
  • DE was prepared by intraperitoneal injection of 70 mg/kg. Rats were sacrificed before modeling and 10 w, 18 w and 22 w after modeling.
  • Rat liver tissue was taken and fixed in 10% neutral formalin overnight. The tissue was trimmed into 1.0x 1.0x0.5 cm block, soaked in running water for 12 h, ethanol gradient (50%-75%-80%- 95%-anhydrous ethanol) is dehydrated, and xylene is transparent and then waxed to form a tissue wax block. The wax blocks were serially sectioned and subjected to immunohistochemical staining. The results showed that the expression of HNFla in rat liver tissue gradually decreased with the prolongation of modeling time, and the expression of HNFla was the weakest in liver cancer tissues. See Figure 4.
  • HNFla 1896bp cDNA fragment Design and synthesize primers based on the human HNFla cDNA sequence. Add a Bgi II restriction site at the 5' end: Sense 5'- GGAAGATCTCGAGCCATGGTTTCTAAACTGAG -3 ' ( SEQ ID N0.5 ); antisense adds a Kpn l restriction site at the 5' end: 5'- CGGGGTACCTTACTGGGAGGAAGAGGCCAT - 3' (SEQ ID 0.6 is shown in Figure 5, HNFla cDNA fragment was obtained by PCR amplification, product 1% agarose gel electrophoresis, fragment size was identified, and the gel was recovered and placed in an Eppendorf tube, and the weight of the gel was weighed.
  • the NT solution was added to the Eppendorf tube. 200 ml/100 mg gel, 50 °C 5-10 min to melt the gel, centrifuge the liquid through the column, centrifuge at 13,000 rpm for 1 min, add 600 ⁇ l ⁇ 3 buffer, centrifuge at 13,000 rpm for 2 min. 30 ⁇ l double steamed column wash The DNA fragment was removed, allowed to stand for 1 min, centrifuged at 13,000 rpm for 1 min, and the eluate was carefully removed into a clean Eppendorf tube. The OD 26Q value was measured by a spectrophotometer, and the fragment size was identified by 1.5% agarose gel electrophoresis.
  • the full length sequence of the HNFla cDNA is the nucleotide sequence set forth in SEQ ID N0.45. table 3
  • Reaction conditions 98 ° C for 10 s, 68 ° C for 8 min, 35 cycles.
  • HNFla-expressing adenoviral plasmid pAdHNFla Kpn I, restriction of shuttle plasmid pAdTrack-CMV and HNFla cDNA for 4 h and purification, and take 0.1 plasmid pAdTrack-CMV, 0.4 g HNFla cDNA, 10xT 4 buffer 2 ⁇ 1, T 4 DNA ligase ⁇ ⁇ (2U) and ddH 2 0, total volume 20 ⁇ 1, connected overnight at 16 °C.
  • the ligation product was added to competent bacteria DH501 for 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 amplified HNFla cDNA fragment was ligated with Qiagen-tip 100 reagent.
  • the plasmid pAdTrack-CMV-HNFl was obtained and identified. See Figure 6.
  • Pme I endonuclease-directed linearization of pAdTrack-CMV-HNFla respectively, 0.4 ⁇ ⁇ linear pAdTrack-CMV-HNFla and O.
  • Example 5 Realtime RT-PCR, Western blot and cellular immunofluorescence assay for expression and localization of HNFla after AdHNFla infection in human liver tumor cell lines 1.
  • Hep3B, Huh7, MHCC-H, and MHCC-L were inoculated into 35mm culture dishes at 5> 10 5 / dish, and the virus was infected with MOI 100, 500, 300, 300 respectively. After 24 hours, 10% fetal cattle were replaced. Fresh MEM or DMEM in serum was observed, and GFP expression was observed after 3 days.
  • AdHNFla is infected with Hep3B, Huh7, MHCC-H and MHCC-L, respectively.
  • Cell lysate collects whole-cell protein. After protein standard quantitation, each protein is separated by 10 ⁇ 8 on 10% SDS-PAGE to form polyvinylidene fluoride.
  • HNFla monoclonal antibody (1:200) 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. Min, after washing twice with PBST, fluorescence was detected by Odyssey infrared laser imaging system and grayscale scanning was performed. The results showed that the expression of HNFla protein was up-regulated after AdHNFla infection of human liver tumor cells. See Figure 10.
  • the cells were washed twice with pre-cooled PBS, fixed with 4 PFA (w/v) /0.1% Triton-X-100/PBS lml at 4 ° C for 30 min ; 0.05 % PBST washed 3 times; 5% horse serum at room temperature in a wet box Close the 2h; aspirate the blocking solution in the Petri dish, draw a box around the coverslip with a crayon, and draw a line along the middle of the slide to make the left and right halves one big and one small; half plus 5% horse Serum, the other half plus primary antibody dilution (HNFla antibody 1:200 was prepared with blocking solution); incubate at 4 ° C overnight in the humid chamber; next day
  • Example 6 Effect of exogenous introduction of HNFla on hepatocyte-associated functional genes in human liver tumor cells
  • Realtime RT-PCR was used to detect the expression of hepatocyte-related functional genes: Hep3B, Huh7, MHCC-H, and MHCC-L were inoculated into 35mm culture dishes at 5 ⁇ 10 5 / dish, and the virus was infected with MOI 100, 500, 300, 300 respectively. The cells were replaced with fresh MEM or DMEM containing 10% fetal bovine serum after 24 hours, 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. Realtime PCR amplification, reaction conditions and reaction system are the same as before, the primer sequences are shown in Table 4. The results showed that the expression of some hepatocyte-related functional genes in the AdHNFla-infected group was significantly up-regulated compared with the control group, including glucose-6-phosphatase.
  • G-6-P glucose-6-phosphatase, G-6-P
  • ADH1 alcohol dehydrogenase 1
  • BR biliverdin reductase
  • APOCIII apolipoprotein CIII
  • TTR Transthyretin
  • PPCK C-reactive protein
  • CRP C-reactive protein
  • NTCP cytochrome oxidase P450 7A1
  • CYP7A1 cytochrome P4507A1, CYP7A1
  • G-6-P was up-regulated by 4.67 ⁇ 1.18 times (P ⁇ 0.05) and 2.03 ⁇ 0.51 times (P ⁇ 0.05); ADH1 was up-regulated by 1.91 ⁇ 0.24 times.
  • Antisense strand 5 -AAATCCCAGAACTCAGAGAAC-3 ' SEQ ID NO 8.
  • Antisense strand 5 - TTCAGCTGCACAGCCCAGAA-3 (SEQ ID NO. 10)
  • Antisense strand 5 -CTCGATGAACTTCGGGATGA-3 ' ( SEQ ID NO. 12)
  • Antisense strand 5 -GAAAAGTCGTTGATGTTGGA-3 ' ( SEQ ID N0.14)
  • CYP1A2 sense strand 5 -CTGGCCTCTGCCATCTTCTG-3' (SEQ ID NO.15)
  • Antisense strand 5 -TTAGCCTCCTTGCTCACATGC-3' (SEQ ID NO.16)
  • Antisense strand 5 -CCCTCTTCAGCAAAGCAGAC-3' (SEQ ID N0.22)
  • CYP2E sense strand 5 -CGTCATAGCCGACATCCT-3 ' SEQ ID NO 23
  • Antisense strand 5 -CTCCATTTCCACGAGCAG-3 ' ( SEQ ID N0.24)
  • Antisense strand 5 -TCACTTCATACAGCACGATC-3 ' ( SEQ ID N0.28)
  • CYP7A2 sense strand 5 -GCTTGCGACACTTCACG-3 ' ( SEQ ID N0.29)
  • Antisense strand 5 -TCACTTCATACAGCACGATC-3 ' ( SEQ ID NO.30)
  • Flow cytometry to determine the apoptotic rate of human liver tumor cells Hep3B, Huh7, MHCC-H, MHCC-L were inoculated into 35mm culture dishes at 5 ⁇ 10 5 / dish, and the virus was MOI 100, 500, 300, respectively. The cells were infected with 300, fresh MEM/DMEM containing 10% fetal bovine serum was replaced after 24 h, and cells were collected on the 3rd day. 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. The results showed that: After up-regulating the expression of HNFla in liver tumor cells, except for MHCC-L, the apoptosis rate of each tumor cell line did not increase significantly. See Figure 14.
  • Flow cytometry to determine cell cycle changes in human liver tumor cells Hep3B, Huh7, MHCC-H, and MHCC-L were inoculated into 35mm culture dishes at 5 ⁇ 10 5 / dish, and the viruses were MOI 100, 500, 300, respectively. The cells were infected with 300, and fresh MEM/DMEM containing 10% fetal bovine serum was replaced after 24 hours. The cells were collected on the 3rd day, and the apoptosis rate was measured by EPICS XL flow cytometry (Coulter) and statistical analysis was performed. Each group was set up with 2 sets and repeated 3 times.
  • Realtime RT-PCR and western blot were used to detect the expression of cell cycle-associated protein mRNA and protein: AdGFP and AdHNFla were infected with Hep3B and Huh7 for 72 h, respectively. Total RNA was extracted with Trizol kit, and whole cell protein was collected from cell lysate.
  • Realtime RT-PCR (primer sequence is shown in Table 5) and western blot analysis showed that after HNFla up-regulation, cell cycle-associated proteins cyclinA2 and cyclinB1 were up-regulated, cell division cycle 2 (CDC2) was down-regulated, and P21 was up-regulated ( ⁇ 0.05); There was no significant difference in cyclinD and cyclinE (P>0.05).
  • Antisense strand 5 '- CTCTGGTGGGTTGAGGAGAG -3 ' SEQ ID NO 34
  • cyclinB 1 sense strand 5 '- CGGGAAGTCACTGGAAACAT -3 '
  • antisense strand 5 '- AAACATGGCAGTGACACCAA -3 ' SEQ ID N0.36
  • Antisense strand 5'- GCCATTAGCGCATCACAGT -3' SEQ ID NO. 40
  • cyclinD sense strand 5'- GACCTTCGTTGCCCTCTGT -3 ' ( SEQ ID N0.41 )
  • Antisense strand 5 '- TGAGGCGGTAGTAGGACAGG -3 ' SEQ ID N0.42
  • cyclinE sense strand 5 '- GAAATGGCCAAAATCGACAG -3 ' SEQ ID N0.43
  • antisense strand 5'- GAGTTTGGGTAAACCCGGTC -3' SEQ ID N0.44
  • Dual luciferase reporter detection system detects P21 reporter gene expression: 2x10 6 Hep3B cells were inoculated into the market, and 24 hours later, the adherent growth was observed to be about 60% to 70% full, and serum-free and antibiotic-free culture was performed. Liquid culture; Take 10 P21 reporter gene expression plasmid WWP, lg internal reference plasmid SV40 and OPTI-MEM 250 ⁇ 1 mixed, and prepare sputum; Lipofectamine 2000 20 ⁇ 1, add OPTI-MEM 250 ⁇ 1 to mix, prepare sputum; sputum and The sputum was mixed well, and placed in Hep3B cells after being placed at room temperature for 30 min.
  • the serum-containing MEM solution was replaced. After 4 h, the MEM solution was aspirated, and the cells were washed twice with PBS, 1 > ⁇ trypsin digestion for 10 min, added The serum was neutralized with serum MEM, and the cells were counted. The cells were plated to a 24-well plate with IX 10 5 /plate; after adhering to the cells, the AdGFP and AdHNFla were infected with MOI 100, respectively, and each group was set up with 3 wells.
  • the human liver tumor cell line, gastric cancer cell line and colon cancer cell line were inoculated into the 96-well plate at 5 ⁇ 10 3 /well, respectively, and infected with the virus AdHNFla after 24 h. Thereafter, the absorbance at 450 nm was detected by CCK8 reagent every day to judge the activity. The number of cells. The results showed that HNFla expression significantly inhibited the proliferation of solid tumor cells. At the same time, it was found that with the increase of virus infection titer, the up-regulation of HNFla inhibited the proliferation of some solid tumor cells in a time- and dose-dependent manner. See Figure 17.
  • 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.
  • AdHNFla virus
  • 8 10 3 cells were inoculated into 10 cm culture dishes every 3 days.
  • the medium was changed, cultured for 3-4 w, until obvious clones were visible, 4% PFA was fixed, crystal violet staining, and counting clones.
  • AdHNFla infection The clones formed by human solid tumor cell lines were all decreased compared with the control group, and the up-regulation of HNFla could significantly reduce the colony forming ability of solid tumor cell lines. See Figure 18.
  • Hep3B 5 ⁇ 10 6 and Huh7 2 ⁇ 10 6 after infection with AdHNFla 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.
  • the results showed that: in the subcutaneous Hep3B tumor formation experiment, the control side (AdGFP side) had tumor growth from the 12th day, and all 7 mice had tumor growth on the 37th day; while the AdHNFla group was treated until 7 weeks. There was no tumor growth on the side of nude mice. See Figure 19.
  • the control side (AdGFP side) showed tumors from the 10th day, and all the 8 mice had tumor growth under the skin on the 28th day, and died at 6 w; On the 42nd day, only one nude mouse had tumor growth. See Figure 20.
  • Hep3B was resuspended in 200 ul of serum-free MEM and inoculated subcutaneously into the neck of nude mice to construct an experimental liver tumor nude mouse model. After the tumors on both sides of the naked eye were identified, the nude mice of the same size were selected, and the recombinant replication-deficient adenoviruses AdGFP and AdHNFla were injected into the left and right tumors at a dose of 2 ⁇ 10 9 pfu, 3 times a week. Persist for 2 weeks; the long diameter and width of the subcutaneous tumor were measured with vernier calipers before and after treatment and during treatment, and the tumor volume was calculated to determine the tumor growth.
  • the nude mice were sacrificed at 6 weeks, and the size and weight of subcutaneous tumors were measured. Paraffin sections were prepared from tumor tissues and stained with HE. The expression of H Fl o PCNA and Ki67 in tumor tissues was detected by immunohistochemistry. The immunohistochemical staining methods and conditions were the same as before. . The results showed that the average tumor size of the HNFla gene treatment side of the 7 nude mice was significantly lower than that of the control side at different time points. See Figure 21.
  • Hep3B was resuspended in 200 ul of serum-free MEM and injected into NOD/SCID mice by in situ injection. After 14 days, laparotomy was performed to observe the growth of white spotted tumors at the site of liver injection.
  • AdGFP and B AdHNFla were injected into the tail vein at a dose of 2 ⁇ 10 9 ⁇ /rat, twice a week for 3 weeks. The mice were sacrificed, the body weight and liver weight of the mice were measured, and the local tumor growth was observed. , Paraffin sections were prepared and subjected to HE staining and pathological analysis. The results showed that there were tumor growth in the liver of 6 mice in the AdGFP group, 4 of them had ascites and 3 had bloody ascites.

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Description

一种肝细胞核因子 1α在制备治疗恶性实体瘤疾病药物中的应用 技术领域
本发明涉及一种肝细胞核因子 1α的应用， 具体地说， 是关于一种肝细胞核因子 1α 在制备治疗恶性实体瘤疾病药物中的应用。
背景技术
恶性实体瘤的治疗是目前临床的难点之一， 尤其对于手术无法切除的恶性实体瘤， 临床尚缺乏有效的治疗手段。 选择与肿瘤细胞发生发展密切相关的关键蛋白、 分子和基 因进行特异性靶向调控是恶性实体瘤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，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， 使肿瘤细胞发生调亡、 生长阻滞从而发生抗肿瘤作用将成为可 能。
肝细胞核因子 1 (hepatocyte nuclear factor, HNF1 )属于 P0U-同源结构域家族， 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 HNFla是 HNF1的重要亚型。 对 HNFla基因敲除小鼠研究发现： HNFla是肝脏发 生发育中必需的转录因子，与建立和维持胚胎肝脏的最终正常分化发育密切相关， HNFla 基因敲除小鼠可出现严重肝肾功能损害， 多于出生后数天内死亡。 HNFla 以同源或与 HNFip形成异源二聚体的形式与顺式作用元件结合， 与一些转录激活蛋白相互作用来改 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。 尽管国外有研究报道， HNFla的表达水平与肝细胞癌的分化密切相关， 下调肝癌细 胞株 HNFla的表达后， 肿瘤细胞的某些肝细胞功能基因表达降低； 但 HNFla能否起到 改善肿瘤细胞生物学特性， 降低肝癌成瘤性， 逆转其低分化状态的作用仍未得到证实； 对其它恶性实体肿瘤的调控作用亦不明确；更未将上调 HNFla表达作为恶性实体瘤治疗 手段加以研究。
中国专利申请号 200810034200.3公开了一种 HNF4a诱导分化治疗人体恶性实体瘤， 涉及利用肝细胞核因子 4a诱导人体恶性实体瘤细胞发生分化，从而应用于恶性实体瘤治 疗的方法和用途， 研究表明通过调控恶性实体肿瘤细胞 HNF4a基因表达， 可有效地对肿 瘤细胞的产生诱导分化作用， 从而提供了一种肿瘤诱导分化治疗的新手段。 但是关于一 种肝细胞核因子 la基因和 /或蛋白及其表达载体在制备治疗恶性实体瘤疾病药物中的应 用目前还未见报道。
发明内容 本发明的目的是针对现有技术中的不足， 提供一种肝细胞核因子 1α基因和 /或蛋白 在制备治疗恶性实体瘤疾病药物中的应用。
本发明的再一的目的是， 提供一种肝细胞核因子 lex和 /或蛋白在制备诱导恶性实体 瘤细胞分化的诱导分化试剂或组合物中的应用。
本发明的另一的目的是，提供一种含肝细胞核因子 lot的重组表达载体在制备治疗恶 性实体瘤疾病药物中的应用。
本发明的第四个目的是，提供一种含肝细胞核因子 1α的重组表达载体在制备诱导恶 性实体瘤细胞分化的诱导分化试剂或组合物中的应用。
本发明的第五个目的是， 提供一种诱导或促进哺乳动物中实体瘤分化的方法。 为实现上述目的， 本发明采取的技术方案是： 一种肝细胞核因子 1α基因和 /或蛋白 在制备治疗恶性实体瘤疾病药物中的应用。
为实现上述第二个目的， 本发明采取的技术方案是： 一种肝细胞核因子 lex和 /或蛋 白在制备诱导恶性实体瘤细胞分化的诱导分化试剂或组合物中的应用。
所述的实体瘤选自： 肝癌、 胃癌、 肠癌、 胰腺癌、 肺癌、 前列腺癌或生殖腺肿瘤。 所述的组合物是药物组合物。
所述的药物组合物含有 (a)HNFla 蛋白、 HNFla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。
所述的表达载体包括病毒载体和非病毒载体。
所述的药物组合物在制备体内抑制实体瘤的形成药物中的应用。
所述的肝细胞核因子 1α是人的肝细胞核因子 1α。
为实现上述第三个目的， 本发明采取的技术方案是： 一种含肝细胞核因子 1α的重组 表达载体在制备治疗恶性实体瘤疾病药物中的应用。
为实现上述第四个目的， 本发明采取的技术方案是： 一种含肝细胞核因子 1α的重组 表达载体在制备诱导恶性实体瘤细胞分化的诱导分化试剂或组合物中的应用。
所述的实体瘤选自： 肝癌、 胃癌、 肠癌、 胰腺癌、 肺癌、 前列腺癌或生殖腺肿瘤。 所述的表达载体包括病毒载体和非病毒载体。
为实现上述第五个目的， 本发明采取的技术方案是： 一种诱导或促进哺乳动物中实 体瘤分化的方法， 所述的方法包括以下步骤： 给需要治疗的哺乳动物对象施用肝细胞核 因子 1α蛋白、 其编码序列或含所述编码序列的表达载体。
本发明优点在于：
本发明提供了肝细胞核因子 le 的新用途，为恶性实体瘤的治疗提出了一种新的治疗 方法。本发明利用基因工程技术调控实体肿瘤细胞 HNFl a基因表达， 证实其改善肿瘤细 胞生物学特性， 阻滞肿瘤细胞生长； 通过体内 HNFla腺病毒载体注射明确 HNFla表达 上调对恶性实体瘤动物模型的治疗作用， 这是在恶性实体瘤治疗研究领域的全新探索。 附图说明
图 1. Real-time RT-PCR检测人肝肿瘤细胞株 HNFl a基因表达。
图 2. Real-time RT-PCR检测人肝癌及癌旁组织 HNFla基因表达。
图 3. 免疫组化检测人肝癌及癌旁组织 HNFla蛋白表达（ I： 癌旁组织 HNFla表达 高于肝癌组织； II： 肝癌及癌旁组织 HNFla表达相当； ΙΠ癌旁组织 HNFla表达低于肝 癌组织）。
图 4. 免疫组化检测大鼠 DEN诱导肝癌模型过程中 HNFla基因表达逐步下调。 图 5. RT-PCR获得 HNFla cDNA片段。
图 6. 体外连接获得穿梭质粒 pAdTrack-CMV- HNFla， Bgl ll , I酶切鉴定。 图 7. Pac I酶切鉴定重组腺病毒质粒 pAdHNFl o
图 8. 酶切鉴定重组腺病毒质粒 pAdHNFlo
图 9. AdHNFla分别感染 Hep3B ( A、 B)、 Huh7 ( C、 D)、 MHCC-H (E、 F ) ; 及
MHCC-L ( G、 H) 细胞 3 d后 GFP表达。
图 10. AdHNFla感染肝肿瘤细胞 3 d后 western blot检测 HNFla蛋白表达。
图 11. AdHNFla感染肝肿瘤细胞 3 d后 HNFla 蛋白表达定量分析。
图 12. AdHNFla感染肝肿瘤细胞 3 d后免疫荧光检测 HNFla蛋白表达定位。
图 13. AdHNFl a感染人肝肿瘤细胞株后 HNFl a基因和肝细胞相关功能基因 mRNA 表达定量分析
图 14. AdHNFla感染肝肿瘤细胞株 3 d后细胞凋亡变化。
图 15. AdHNFla感染肝肿瘤细胞株 3 d后细胞周期变化。
图 16. Real-time RT-PCR及 western blot检测 AdHNFla感染肝肿瘤细胞株 3 d对细胞 周期相关蛋白的影响。
图 17. 外源导入 HNFl a对不同人肝肿瘤细胞株细胞增殖能力的影响。
图 18. 外源导入 H Fl a对不同人肝肿瘤细胞株细胞克隆形成的影响。
图 19. AdHNFla感染肝肿瘤细胞 Hep3B后体内接种成瘤实验。
图 20. AdHNFla感染肝肿瘤细胞 Huh7后体内接种成瘤实验。
图 21. AdHNFla瘤内注射基因治疗皮下成瘤模型。
图 22. HNFla基因治疗肝肿瘤细胞肝脏原位注射致实验性肝肿瘤模型。 具体实施方式
以下结合附图和实施例对本发明作进一步说明， 但本发明的范围并不仅限于下列实 验所公开的范围。
实施例 1 Realtime RT-PCR检测人肝肿瘤细胞株 HNFla基因表达
1. 肝肿瘤细胞株 Huh-7、 Hep3B、 MHCC-H、 MHCC-L、 PLC、 YY、 7721均以 5>< 105/ 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 OD26Q值， 配成工作浓度 （1μ§/μ1及 0.1μ§/μ1)， 1%琼脂糖凝胶电泳检测 RNA 完整性。
2. Realtime RT-PCR: 取 4 g RNA、 2μ1 Randomprimer, DEPC水加至 33μ1于 70°C置 5 min、 0°C置 5 min后， 加入 ΙΟμΙ 5xBuffer、 3μ1 άΝΤΡ 2 lRNA逆转录酶和 2μ1 RNA酶 抑制剂混匀后， 37°C置 1.5 h， 即可得到逆转录产物， 见表 1。 将逆转录产物稀释后取 Ιμΐ 为模版进行 Realtime PCR扩增，基因引物序列见表 2， 反应体系如下， 反应条件为： 94°C 预变性 30 s， 之后 94°C l0 s， 60°C 30 s, 共进行 40个循环后进行溶解曲线的检测。 结果 表明： 各肝肿瘤细胞株的 HNFla m NA表达均明显下调。 见图 1。
表 1
Figure imgf000006_0001
表 2 人引物序列
Figure imgf000006_0002
实施例 2 Realtime RT-PCR及免疫组化检测人肝癌组织及癌旁组织 HNFla基因及 蛋白表达
1. Realtime RT-PCR: Trizol法抽提人肝癌及癌旁组织 RNA， 以分光光度计测定其 OD26Q值， 配成工作浓度（ §/^及0. ^§/^)， 1%琼脂糖凝胶电泳检测 RNA完整性。取 4μδ RNA进行逆转录及 Realtime PCR扩增（逆转录反应、 Realtime PCR反应条件及引物 序列同前）。结果表明： 11对人肝癌组织 /癌旁组织中， 7例（63.64%)肝癌组织中 H Fla 表达较癌旁组织降低。 见图 2。 2. 免疫组化：人肝癌及癌旁组织蜡块 4 mm连续切片， 60°C烤箱中烘烤 30 min固定， 脱蜡至水后， 3 %H202室温放置 10 min清除内源性过氧化物酶， 柠檬酸缓冲液微波进行 抗原修复， 加 1 : 10正常兔血清室温封闭 30 min, 滴加 HNFla抗体 （1 :200) 4 °C过夜； 次日 PBS (0.01 M, pH 7.4) 洗涤 3次， 每次 5 min; 加二抗室温孵育 30 min; PBS洗 涤后， 加 SABC ( 1 : 100)室温孵育 20 min， DAB显色， 常规树脂封片， 光镜下观察。 根 据阳性染色范围， 将免疫组化切片用图像分析仪行半定量分析， 每张切片扫 4个视野， 用图像分析系统测量阳性染色面积并自动计算其与总面积的百分比。 结果显示： 17对人 肝癌组织 /癌旁组织 HNFla表达情况， 结果显示： 52.94% (9/17) 患者肝癌组织 HNFla 表达较癌旁组织降低。 见图 3。
实施例 3 免疫组化法检测 DE 致大鼠原发性肝癌模型肝组织 HNFla基因及蛋白 表达
1. DE 以 70 mg/kg腹腔注射制备大鼠原发性肝癌模型， 造模过程中， 于造模前、 造模第 10 w、 18 w、 22 w分别处死大鼠。
2. 取大鼠肝组织，于 10%中性福尔马林固定过夜，组织修剪成 1.0x 1.0x0.5 cm块状， 流水浸泡 12 h， 乙醇梯度（50%-75%-80%-95%-无水乙醇）脱水， 二甲苯透明后浸蜡制成 组织蜡块。 蜡块连续切片后进行免疫组化染色。 结果显示随着造模时间延长， 大鼠肝组 织中 HNFla表达逐渐减弱， 肝癌组织中 HNFla表达最弱。 见图 4。
实施例 4 构建表达 HNFla的重组复制缺陷型腺病毒 AdHNFla
1. 获取 HNFla 1896bp cDNA片段： 根据人 HNFla cDNA序列设计、 合成引物。 在 5' 端 加 入 Bgi II 限 制 性 酶 切 位 点 ： Sense 5'- GGAAGATCTCGAGCCATGGTTTCTAAACTGAG -3 ' ( SEQ ID N0.5 ); antisense在 5' 端加入 Kpn l限制性酶切位点： 5'- CGGGGTACCTTACTGGGAGGAAGAGGCCAT - 3' ( SEQ ID 0.6 见图 5， PCR扩增获取 HNFla 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 600μ1 ΝΤ3缓冲液， 13,000 rpm离心 2 min。 30μ1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 OD26Q值， 1.5%琼脂糖凝胶电泳鉴定片段大小。 需要说明的是， 所述的 HNFla cDNA的 全长序列为 SEQ ID N0.45所述的核苷酸序列。 表 3
Figure imgf000008_0001
反应条件： 98°C 10s， 68 °C 8min, 35个循环。
2. 构建表达 HNFla 的腺病毒质粒 pAdHNFla : Kpn I、 酶切穿梭质粒 pAdTrack-CMV和 HNFla cDNA 4 h后并纯化，取 0.1 质粒 pAdTrack-CMV、 0.4 g HNFla cDNA、 10xT4缓冲液 2μ1、 T4DNA连接酶 Ι μΐ (2U) 以及 ddH20， 总体积 20μ1， 16°C连 接过夜。 将连接产物加入感受态细菌 DH501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 HNFla cDNA片段的菌落克隆用 Qiagen-tip 100试剂盒中抽， 获取质粒 pAdTrack-CMV- HNFl a并鉴定。 见图 6。 Pme I内切酶酶切 线性化 pAdTrack-CMV-HNFla， 分别取 0.4μδ线性 pAdTrack-CMV- HNFla和 O. l g超螺 旋 pAdEasy-1质粒 2,000 V、 200 Ohms、 25μΡϋ电穿孔共转化 20μ1感受态 BJ5183细菌， 卡那霉素 LB培养基平板筛选， 选择病毒质粒 pAdHNFl a测序鉴定。
3. 包装、 扩增腺病毒 AdHNFla: 复苏 293细胞， 以 4.8χ 106/皿接种于 10cm 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 pAdHNFla, 与不含血清的 DMEM培液 250μ1混匀， 配成 Α液； 取 Lipofectamin
20μ1， 加入不含血清的 DMEM培液 250μ1混匀， 配成 Β液， Α液与 Β液充分混匀， 室温 放置 30 mm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 l x l01Q efb/ml的 AdHNFla，置于 -80Ό 保存备用。 见图 7和图 8。
实施例 5 Realtime RT-PCR, western blot及细胞免疫荧光法检测 AdHNFla感染 人肝肿瘤细胞株后 HNFla表达及定位 1. Hep3B、 Huh7、 MHCC-H、 MHCC-L均以 5> 105/皿接种于 35mm培养皿， 将病毒 分别以 MOI 100、 500、 300、 300感染细胞， 24h后更换含 10%胎牛血清的新鲜 MEM或 DMEM培液， 3d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 HNFla realtime PCR扩增，同时以 β-actin在相同反应条 件进行 realtime PCR反应作为内参照， 反应条件及反应体系同前。 结果表明： AdHNFla 感染人肝肿瘤细胞株后 HNFlt mRNA在肿瘤细胞中表达明显上调。 见图 9。
2. AdHNFla分别感染 Hep3B、 Huh7、 MHCC-H、 MHCC-L, 细胞裂解液收取全细胞 蛋白， 蛋白标准定量后， 各取 10μ8于 10% SDS -PAGE电泳分离蛋白， 将聚偏二氟乙烯 膜 （PVDF膜） dd¾0冲洗， 将电泳胶、 PVDF膜、 滤纸放于 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， HNFla 单抗（1 :200) 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红外激光成像系统检测荧光并进行灰度扫描。结 果显示， AdHNFla感染人肝肿瘤细胞株后 HNFla蛋白表达均明显上调。 见图 10。
3. 细胞免疫荧光： 将玻片以 75%乙醇浸泡后， 酒精灯烧灼， 冷却后置于 35mm培养 皿内。 Hep3B、 Huh7、 MHCC-H、 MHCC-L分别以 5χ 105/皿接种于 35mm培养皿， 将病 毒分别以 MOI 100、 500、 300、 300感染细胞， 3d后观察 GFP表达。 将细胞以预冷 PBS 洗涤 2次，加入 4 PFA (w/v) /0.1 % Triton-X-100/PBS lml 4°C固定 30min; 0.05 % PBST 洗涤 3次； 5 %马血清室温湿盒内封闭 2h; 吸去培养皿中的封闭液，用蜡笔沿盖玻片的四 周描一个方框，并沿玻片的中间画一条线，使左右两半面积一大一小；一半加 5 %马血清， 另一半加一抗稀释液（HNFla抗体 1 :200用封闭液配制）； 置湿盒内 4°C孵育过夜； 次日
0.05 % PBST洗涤 3次后， 加二抗稀释液 （cy2标记驴抗兔抗体 1 :500， 用封闭液配制）， 放入湿盒， 室温孵育 30 min; PBST洗涤后， 滴上约 30 μΐ mounting solution (含核 DNA 显色剂 DAPI )， 指甲油封片晾干后， 共聚焦显微镜观察 HNFla表达及定位情况。 结果 显示： Ad HNFla感染后， HNFla表达明显增强， 并主要定位于核内。 见图 11和图 12。
实施例 6 外源导入 HNFla对人肝肿瘤细胞肝细胞相关功能基因的影响
Realtime RT-PCR检测肝细胞相关功能基因的表达： Hep3B、 Huh7、 MHCC-H、 MHCC-L均以 5χ 105/皿接种于 35mm培养皿， 将病毒分别以 MOI 100、 500、 300、 300 感染细胞， 24h后更换含 10%胎牛血清的新鲜 MEM或 DMEM培液， 3d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 realtime PCR扩增， 反应条件及反应体系同前， 引物序列见表 4。结果表明： AdHNFla 感染组中部分肝细胞相关功能基因表达较对照组明显上调， 主要包括葡萄糖 -6-磷酸酶
( glucose-6-phosphatase, G-6-P)、 乙醇脱氢酶 1 (alcohol dehydrogenase 1, ADH1)、 胆绿 素还原酶（biliverdin reductase, BR)、 载脂蛋白 CIII ( apolipoprotein CIII, APOCIII )、 转甲 状腺素蛋白 （ transthyretin, TTR )、 磷酸烯醇丙酮酸羧激酶 ( hosphoenolpyruvate carboxykinase, PEPCK)、 C-反应蛋白 （C-reactive protein, CRP)、 细胞色素氧化酶 P450 7A1 (cytochrome P4507A1, CYP7A1 ). 钠离子 /牛磺胆酸盐同向运转器 (Na taurocholate co-transporter, NTCP) 脂酶 A (lipase A, LIPA)等。 其中， 在 Hep3B和 Huh7中， G-6-P 分别上调 4.67±1.18倍 （尸 <0.05)、 2.03±0.51倍 （P<0.05); ADH1分别上调 1.91±0.24倍
( <0+05)、 2.91±0.94倍 (尸 <0.05)； BR分别上调 1.52±0.13倍 (尸 <0.05)、 1.12±0.33倍; APOCIII分别上调 1.90±0.18倍（尸 <0.05)、 1.97±0.17倍（尸 <0.05)； TTR分别上调 1.91士0.05 倍（尸 <0.05)、 1.32±0.25倍 (^<0.05)； PEPCK分另 lj上调 4.90士0.65倍（尸 <0.01)、 8.91士1.36 倍（P<0.05); CRP分别上调 83.65±13.06倍（P<0.01)、 42.68±18.07倍（P<0.01); CYP7A1 分别上调 31.23±3.33倍 (尸 <0+01)、 27.44±3.15倍 （尸 <0.01)； LIPA分别上调 1.42±0.22 倍 ( <0.05), 1+22±0.24倍 ( <0.05)； NTCP分别上调 2.60±0.56倍（P<0.05)、 2.65±0.32 倍 （P<0.05)。 其它肝细胞相关功能基因， 如： ALB、 GS、 CYP1A2, CYP2E、 APOA2、 INSR等均无明显上调 （P>0.05)。 见图 13。
表 4 肝细胞相关功能基因引物序列
基因 引物序列
APOCIII 正义链 5 -GGGTACTCCTTGTTGTTGC-3' ( SEQ ID N0.7)
反义链 5 -AAATCCCAGAACTCAGAGAAC-3 ' ( SEQ ID NO 8)
G-6-P 正义链 5 -GGCTCCATGACTGTGGGATC-3 ( SEQ ID NO 9)
反义链 5 - TTCAGCTGCACAGCCCAGAA-3 (SEQ ID NO.10)
ALB 正义链 5 -AGCCTAAGGCAGCTTGACTT-3 ( SEQ ID NO.11 )
反义链 5 -CTCGATGAACTTCGGGATGA-3 ' ( SEQ ID NO.12)
GS 正义链 5 -CCTGCTTGTATGCTGGAGTC-3' (SEQ ID NO.13)
反义链 5 -GAAAAGTCGTTGATGTTGGA-3 ' ( SEQ ID N0.14)
CYP1A2 正义链 5 -CTGGCCTCTGCCATCTTCTG-3' (SEQ ID NO.15)
反义链 5 -TTAGCCTCCTTGCTCACATGC-3' (SEQ ID NO.16)
PEPCK 正义链 5 -GTGTCCCTCTAGTCTATGAAGC-3 (SEQ ID NO.17) 反义链 5 -ATTGACTTGATCCTCCAGATAC-3' (SEQ IDN0.18)
TTR 正义链 5 -GCGGGACTGGTATTTGTGTCTG-3 ' (SEQ ID NO.19) 反义链 5 -TTAGTGACGACAGCCGTGGTG-3 (SEQ IDNO.20)
AFP 正义链 5 -AGCTTGGTGGTGGATGAAAC-3 ' ( SEQ IDN0.21)
反义链 5 -CCCTCTTCAGCAAAGCAGAC-3' ( SEQ ID N0.22) CYP2E 正义链 5 -CGTCATAGCCGACATCCT-3 ' ( SEQ ID NO 23 )
反义链 5 -CTCCATTTCCACGAGCAG-3 ' ( SEQ ID N0.24)
APOA2 正义链 5 -AGAAGGTCAAGAGCCCAGAG-3 ' ( SEQ ID N0.25 ) 反义链 5 -TCCAAGTTCCACGAAATAGC-3 ' ( SEQ ID N0.26 )
INSR 正义链 5 -GCTTGCGACACTTCACG-3 ' ( SEQ ID N0.27)
反义链 5 -TCACTTCATACAGCACGATC-3 ' ( SEQ ID N0.28)
CYP7A2 正义链 5 -GCTTGCGACACTTCACG-3 ' ( SEQ ID N0.29)
反义链 5 -TCACTTCATACAGCACGATC-3 ' ( SEQ ID NO.30)
LIPA 正义链 5 -GCTTGCGACACTTCACG-3 ' ( SEQ ID 0.31 )
反义链 5 -TCACTTCATACAGCACGATC-3 ' ( SEQ ID N0.32) 实施例 7 外源导入 HNFla对人肝肿瘤细胞凋亡及细胞周期的影响
1. 流式细胞仪测定人肝肿瘤细胞凋亡率： Hep3B、 Huh7、 MHCC-H、 MHCC-L均以 5χ 105/皿接种于 35mm培养皿， 将病毒分别以 MOI 100、 500、 300、 300感染细胞， 24 h 后更换含 10%胎牛血清的新鲜 MEM/DMEM培液， 第 3 d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 HNFla表达后， 除 MHCC-L夕卜， 各肿瘤细胞株凋亡率无明显增 加。 见图 14。
2. 流式细胞仪测定人肝肿瘤细胞细胞周期变化： Hep3B、 Huh7、 MHCC-H、 MHCC-L 均以 5χ 105/皿接种于 35mm培养皿， 将病毒分别以 MOI 100、 500、 300、 300感染细胞， 24 h后更换含 10%胎牛血清的新鲜 MEM/DMEM培液，第 3 d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 HNFla表达后， 各肿瘤细胞细胞株 G2/M期细胞较感染空载病毒 AdGFP 组及空白对照组明显增加， 尸<0.05。 见图 15。
3. Realtime RT-PCR及 western blot检测细胞周期相关蛋白 mRNA及蛋白的表达： AdGFP和 AdHNFla分别感染 Hep3B、 Huh7 72 h， 以 Trizol试剂盒抽提总 RNA， 细胞裂 解液收取全细胞蛋白，分别进行 Realtime RT-PCR (引物序列见表 5 )及 western blot检测， 结果表明： HNFla上调后， 细胞周期相关蛋白 cyclinA2、 cyclinBl上调， 细胞分裂周期 蛋白 2 (cell division cycle 2, CDC2)下调， P21上调 （Ρ<0.05 ); cyclinD、 cyclinE变化无 统计学意义 （P>0.05 )。 上述研究表明 HNFla对肿瘤的抑制作用可能是通过调控 P21及 CDC2, 导致肿瘤细胞发生 G2/M期阻滞。 见图 16。
细胞周期相关蛋白基因引物序列
基因 引物序列
cyclinA2 正义链 5'- TTATTGCTGGAGCTGCCTTT -3 ' ( SEQ ID 0.33 )
反义链 5 '- CTCTGGTGGGTTGAGGAGAG -3 ' ( SEQ ID NO 34 ) cyclinB 1 正义链 5 '- CGGGAAGTCACTGGAAACAT -3 ' ( SEQ ID N0.35 ) 反义链 5 '- AAACATGGCAGTGACACCAA -3 ' ( SEQ ID N0.36)
CDC2 正义链 5 - AAGCCGGGATCTACCATACC -Ύ ( SEQ ID N0.37) 反义链 5 - CCTGCATAAGCACATCCTGA -3 ' ( SEQ ID N0.38 )
P21 正义链 5 - ACCGAGGCACTCAGAGGAG -3 ' ( SEQ ID N0.39)
反义链 5'- GCCATTAGCGCATCACAGT -3' ( SEQ ID NO.40) cyclinD 正义链 5'- GACCTTCGTTGCCCTCTGT -3 ' ( SEQ ID N0.41 )
反义链 5 '- TGAGGCGGTAGTAGGACAGG -3 ' ( SEQ ID N0.42 ) cyclinE 正义链 5 '- GAAATGGCCAAAATCGACAG -3 ' ( SEQ ID N0.43 ) 反义链 5'- GAGTTTGGGTAAACCCGGTC -3' ( SEQ ID N0.44)
4. 双荧光素酶报告基因检测系统检测 P21报告基因表达情况： 将 2x l06 Hep3B细胞 接种于大盘， 24 h后观察其贴壁生长至大约 60%~70%满， 换无血清无抗生素培液培养； 取 10 P21报告基因表达质粒 WWP、 l g 内参照质粒 SV40与 OPTI-MEM 250μ1混匀， 配成 Α液； Lipofectamine 2000 20μ1， 加入 OPTI-MEM 250μ1混匀， 配成 Β液； Α液与 Β 液充分混匀， 室温放置 30min后加入 Hep3B细胞中， 6h后更换含血清的 MEM培液； 4 h后吸去 MEM培液， PBS洗细胞 2次， 1 ><胰酶消化 10 min,加入含血清 MEM中和胰酶， 计数细胞， 以 I X 105/盘将细胞分盘至 24孔板； 培养至细胞贴壁后， 分别以 MOI 100感 染 AdGFP、 AdHNFla, 每组复设 3孔， 培养 72h后； 弃去培养液， PBS洗涤后， 每一孔 中加入 500 μL的 Passive Lysis Buffer (PLB ) ； 室温轻轻震荡 15 min后， 收集细胞裂解 液， 取 2( L的细胞裂解液至荧光测定管中， 加入 ΙΟΟμί萤火虫荧光素酶检测试剂， 混 匀； Luminometer荧光计量仪测定萤火虫荧光素酶的活性，每一裂解样品经 3次检测后取 其平均值，计算萤火虫荧光素酶的表达值。结果表明病毒感染 72h后，空白对照组、 AdGFP 组、 AdHNFla萤火虫荧光素酶的表达值分别为 49.47±14.41、 59.25±17.59、 85.3±29.21 , AdHNFla较 AdGFP组 P21报告基因表达明显上调。
实施例 8 外源导入 HNFlot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 103/孔接种于 96 孔板， 24 h 后感染病毒 AdHNFla, 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： HNFla表达对实体肿瘤细胞增殖有明显的抑制作用， 同时研究 发现， 随着病毒感染滴度的增高， HNFla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。 见图 17。
实施例 9 外源导入 HNFlot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 AdHNFla感染 24 h后， 各取 8 103 细胞接种于 10 cm培养皿， 每 3天换液， 培 养 3-4 w， 直至可见明显克隆， 4%PFA固定， 结晶紫染色， 计数克隆。 AdHNFla感染后 人实体肿瘤细胞株形成的克隆均较对照组减少， HNFla上调可明显降低实体肿瘤细胞株 的克隆形成能力。 见图 18。
实施例 10 上调人肝肿瘤细胞株 Hep3B和 Huh7 HNFla表达对体内成瘤的实验研 究
分别取 AdHNFla感染 24 h后的 Hep3B 5 χ 106和 Huh7 2 χ 106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： 皮下注射 Hep3B成 瘤实验中， 对照侧 （AdGFP侧） 自第 12 d起有肿瘤生长， 至第 37 d所有 7只小鼠均有 肿瘤生长； 而治疗 AdHNFla组直至 6 w时所有 7只裸鼠侧均无肿瘤生长。 见图 19。 皮 下注射 Huh7成瘤实验中， 对照侧 （AdGFP侧） 自第 10 d起即出现肿瘤， 至 28 d所有 8 只小鼠皮下均有肿瘤生长， 并于 6 w时死亡 1只； 而治疗组直至第 42 d仅 1只裸鼠有肿 瘤生长。 见图 20。
实施例 11 HNFla治疗实验性肝肿瘤模型 （1 )
5 106 Hep3B 重悬于 200ul的无血清的 MEM中， 接种于裸鼠两侧颈部皮下构建实 验性肝肿瘤裸鼠模型。 待出现两侧肉眼可辨肿瘤后， 挑选双侧肿瘤大小相当的裸鼠， 在 左右两侧瘤内分别注射重组复制缺陷型腺病毒 AdGFP和 AdHNFla, 剂量 2x l09 pfu, 每 周注射 3次， 持续 2 w; 治疗前后及治疗期间每周以游标卡尺测量皮下肿瘤长径及宽径， 计算肿瘤体积， 以判断肿瘤生长情况。 6 w时处死裸鼠， 测量皮下肿瘤大小、 重量； 并取 肿瘤组织制备石蜡切片， 行 HE染色， 免疫组织化学法检测肿瘤组织 H Fl o PCNA、 Ki67表达， 免疫组织化学染色方法及条件同前。 结果表明： 7只裸鼠 HNFla基因治疗侧 肿瘤大小平均值在不同时间节点均显著小于对照侧。 见图 21。
实施例 12 HNFla治疗实验性肝肿瘤模型 （2)
5 106 Hep3B 重悬于 200ul的无血清的 MEM中， 通过肝脏原位注射于 NOD/SCID 小鼠体内， 14d后手术开腹观察肝脏注射部位均有白色点状肿瘤生长。分别经尾静脉注射 AdGFP禾 B AdHNFla, 剂量 2χ 109 ρίίι/鼠， 每周注射 2次， 持续 3 w; 处死小鼠， 测量小 鼠体重、 肝重， 观察肝脏局部肿瘤生长情况， 并取出肝脏， 制备石蜡切片并进行 HE染 色和病理分析。 结果表明： AdGFP组 6只小鼠肝脏均有肿瘤生长， 其中 4只出现腹水， 3只为血性腹水。 AdHNFl a组 6只小鼠中， 2只小鼠肝脏有肿瘤生长， 4只未见肿瘤生长， 均未出现明显腹水。 免疫组织化学检测显示 AdHKFla 治疗组 HNFla表达明显高于 AdGFP IE, PCNA、 Ki67表达明显低于 AdGFP组。 见图 22。 SEQUENCE LISTING
<110> 中国人民解放军第二军医大学
<120> 一种肝细胞核因子 1 α在制备治疗恶性实体瘤疾病药物中的应用 <130> I
< 150> 201010559047. 3
<151> 2010- 1卜 25
<160> 45
<170> Patentln version 3. 3
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<212> DNA
<213> 人工序列
<400> 23
cgtcatagcc gacatcct 18
<210> 24
<211> 18
<212> DNA
<213> 人工序列
<400> 24
ctccatttcc acgagcag 18
<210> 25
<211> 20
<212> DNA
<213> 人工序列
<400> 25
agaaggtcaa gagcccagag 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列
<400> 26
tccaagttcc acgaaatagc 20
<210> 27
<211> 17
<212> DNA
<213> 人工序列
<400> 27
gcttgc ； gaca cttcacg 17
<210> 28
<211> 20
<212> DNA
<213> 人工序列
<400> 28
tcacttcata cagcacgatc 20
<210> 29
<211> 17
<212> DNA
<213> 人工序列
<400> 29
gcttgc ； gaca cttcacg
<210> 30 <211> 20
<212> DNA
<213> 人工序列
<400> 30
tcacttcata cagcacgatc 20
<210> 31
<211> 17
<212> DNA
<213> 人工序列
<400> 31
gcttgcgaca cttcacg 17
<210> 32
<211> 20
<212> DNA
<213> 人工序列
<400> 32
tcacttcata cagcacgatc 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列
<400> 33
ttattgctgg agctgccttt 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列
<400> 34
ctctggtggg ttgaggagag 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列
<400> 35
cgggaagtca ctggaaacat 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列
<400> 36
aaacatggca gtgacaccaa 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列
<400> 37 aagccgggat ctaccatacc 20 <210> 38
<211> 20
<212> DNA
<213> 人工序列
<400> 38
cctgcataag cacatcctga 20 <210> 39
<211> 19
<212> DNA
<213> 人工序列
<400> 39
accgaggcac tcagaggag 19 <210> 40
<211> 19
<212> DNA
<213> 人工序列
<400> 40
gccattagcg catcacagt 19 <210> 41
<211> 19
<212> DNA
<213> 人工序列
<400> 41
gaccttcgtt gccctctgt 19 <210> 42
<211> 20
<212> DNA
<213> 人工序列
<400> 42
tgaggcggta gtaggacagg 20 <210> 43
<211> 20
<212> DNA
<213> 人工序列
<400> 43
gaaatggcca aaatcgacag 20 <210> 44
<211> 20
<212> DNA
<213> 人工序列
<400> 44
gagtttgggt aaacccggtc 20 <210> 45
<211> 1896
<212> DNA <213> 智人 (Homo sapiens )
<400> 45
atggtttcta aactgagcca gctgcagacg gagctcctgg cggccctgct cgagtcaggg 60 ctgagcaaag aggcactgat ccaggcactg ggtgagccgg ggccctacct cctggctgga 120 gaaggccccc tggacaaggg ggagtcctgc ggcggcggtc gaggggagct ggctgagctg 180 cccaatgggc tgggggagac tcggggctcc gaggacgaga cggacgacga tggggaagac 240 ttcacgccac ccatcctcaa agagctggag aacctcagcc ctgaggaggc ggcccaccag 300 aaagccgtgg tggagaccct tctgcaggag gacccgtggc gtgtggcgaa gatggtcaag 360 tcctacctgc agcagcacaa catcccacag cgggaggtgg tcgataccac tggcctcaac 420 cagtcccacc tgtcccaaca cctcaacaag ggcactccca tgaagacgca gaagcgggcc 480 gccctgtaca cctggtacgt ccgcaagcag cgagaggtgg cgcagcagtt cacccatgca 540 gggcagggag ggctgattga agagcccaca ggt gat gage taccaaccaa gaaggggcgg 600 aggaaccgtt tcaagtgggg cccagcatcc cagcagatcc tgttccaggc ctatgagagg 660 cagaagaacc ctagcaagga ggagcgagag acgctagtgg aggagtgcaa tagggcggaa 720 tgcatccaga gaggggtgtc cccatcacag gcacaggggc tgggctccaa cctcgtcacg 780 gaggtgcgtg tctacaactg gtttgccaac cggcgcaaag aagaagcctt ccggcacaag 840 ctggccatgg acacgtacag cgggcccccc ccagggccag gcccgggacc tgcgctgccc 900 gctcacagct cccctggcct gcctccacct gccctctccc ccagtaaggt ccacggtgtg 960 cgctatggac agcctgcgac cagtgagact gcagaagtac cctcaagcag cggcggtccc 1020 ttagtgacag tgtctacacc cctccaccaa gtgtccccca cgggcctgga gcccagccac 1080 agcctgctga gtacagaagc caagctggtc tcagcagctg ggggccccct cccccctgtc 1140 agcaccctga cagcactgca cagcttggag cagacatccc caggcctcaa ccagcagccc 1200 cagaacctca tcatggcctc acttcctggg gtcatgacca tcgggcctgg tgagcctgcc 1260 tccctgggtc ctacgttcac caacacaggt gcctccaccc tggtcatcgg cctggcctcc 1320 acgcaggcac agagtgtgcc ggtcatcaac agcatgggca gcagcctgac caccctgcag 1380 cccgtccagt tctcccagcc gctgcacccc tcctaccagc agccgctcat gccacctgtg 1440 cagagccatg tgacccagag ccccttcatg gccaccatgg ctcagctgca gagcccccac 1500 gccctctaca gccacaagcc cgaggtggcc cagtacaccc acacgggcct gctcccgcag 1560 actatgctca tcaccgacac caccaacctg agcgccctgg ccagcctcac gcccaccaag 1620 caggtcttca cctcagacac tgaggcctcc agtgagtccg ggcttcacac gccggcatct 1680 caggccacca ccctccacgt ccccagccag gaccctgcca gcatccagca cctgcagccg 1740 gcccaccggc tcagcgccag ccccacagtg tcctccagca gcctggtgct gtaccagagc 1800 tcagactcca gcaatggcca gagccacctg ctgccatcca accacagcgt catcgagacc 1860 ttcatctcca cccagatggc ctcttcctcc cagtaa 1896

Claims

权 利 要 求
1. 一种肝细胞核因子 1α基因和 /或蛋白在制备治疗恶性实体瘤疾病药物中的应用。
2. 一种肝细胞核因子 lot和 /或蛋白在制备诱导恶性实体瘤细胞分化的诱导分化试剂 或组合物中的应用。
3. 根据权利要求 1或 2任一所述的应用， 其特征在于， 所述的实体瘤选自： 肝癌、 胃癌、 肠癌、 胰腺癌、 肺癌、 前列腺癌或生殖腺肿瘤。
4. 根据权利要求 2所述的应用， 其特征在于， 所述的组合物是药物组合物。
5. 根据权利要求 2所述的应用， 其特征在于， 所述的药物组合物含有 (a)H Fla蛋 白、 HNFla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。
6. 根据权利要求 3所述的应用， 其特征在于， 所述的表达载体包括病毒载体和非病 毒载体。
7. 根据权利要求 2所述的应用， 其特征在于， 所述的药物组合物在制备体内抑制实 体瘤的形成药物中的应用。
8. 根据权利要求 1或 2任一所述的应用， 其特征在于， 所述的肝细胞核因子 let是 人的肝细胞核因子 1α。
9. 一种含肝细胞核因子 lot的重组表达载体在制备治疗恶性实体瘤疾病药物中的应 用。
10.一种含肝细胞核因子 la的重组表达载体在制备诱导恶性实体瘤细胞分化的诱导 分化试剂或组合物中的应用。
11.根据权利要求 9或 10任一所述的应用， 其特征在于， 所述的实体瘤选自： 肝癌、 胃癌、 肠癌、 胰腺癌、 肺癌、 前列腺癌或生殖腺肿瘤。
12.根据权利要求 9或 10任一所述的应用， 其特征在于， 所述的表达载体包括病毒 载体和非病毒载体。
13.—种诱导或促进哺乳动物中实体瘤分化的方法， 其特征在于， 所述的方法包括以 下步骤： 给需要治疗的哺乳动物对象施用肝细胞核因子 la 蛋白、 其编码序列或含所述 编码序列的表达载体。
PCT/CN2011/081163 2010-11-25 2011-10-24 一种肝细胞核因子1α在制备治疗恶性实体瘤疾病药物中的应用 WO2012068936A1 (zh)

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