WO2012113236A1 - 肝细胞核因子1α治疗慢性肝病的用途及方法 - Google Patents
肝细胞核因子1α治疗慢性肝病的用途及方法 Download PDFInfo
- Publication number
- WO2012113236A1 WO2012113236A1 PCT/CN2011/081172 CN2011081172W WO2012113236A1 WO 2012113236 A1 WO2012113236 A1 WO 2012113236A1 CN 2011081172 W CN2011081172 W CN 2011081172W WO 2012113236 A1 WO2012113236 A1 WO 2012113236A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hnfla
- chronic liver
- liver disease
- expression
- liver
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
Definitions
- the present invention relates to the fields of molecular biology, cell biology, genetic engineering, and clinical medicine such as disease treatment technology.
- the invention relates to the use and method of treating chronic liver disease using hepatocyte nuclear factor la (HNFla).
- HNFla hepatocyte nuclear factor la
- Hepatic fibrosis is a necessary stage for the development of various chronic liver diseases to cirrhosis. It is a repairing response of the liver to chronic injury. It is characterized by excessive deposition of extracellular matrix (ECM) in the liver. . It is currently believed that liver fibrosis is a dynamic process and is a reversible lesion. Therefore, blocking, inhibiting or reversing liver fibrosis is an important means of treating chronic liver disease.
- liver fibrosis The central part of liver fibrosis is the activation of hepatic stellate cells (HSC) and transformation into myofibroblasts (MFs), inhibition of HSC activation, proliferation and migration, and induction of apoptosis.
- HSC hepatic stellate cells
- MFs myofibroblasts
- EMT Epithelial-to-mesenchymal transition
- Hepatocyte nuclear factor 1 belongs to the POU-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. Although previous studies have confirmed that HNFla plays an important role in maintaining liver function and liver development, the relationship between this transcription factor and liver fibrosis is still unclear. Whether it can block or liver reversal fibrosis has not been confirmed. The therapeutic effect on liver fibrosis is also unclear. The up-regulation of HNFla expression has not been studied at home and abroad as a means of treating liver fibrosis.
- a second object of the present invention is to provide a use of the gene sequence of human HNFla for the preparation of a medicament for treating chronic liver disease.
- a third object of the present invention is to provide a use of a protein encoded by the human HNFla gene sequence for the treatment of chronic liver disease.
- a fourth object of the present invention is to provide a use of a protein encoded by the human HNFla gene sequence for the preparation of a medicament for the treatment of chronic liver disease.
- a fifth object of the present invention is to provide a pharmaceutical composition for treating chronic liver disease.
- a sixth object of the present invention is to provide a method of treating chronic liver disease.
- a seventh object of the present invention is to provide a gene therapy method for chronic liver disease.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- a pharmaceutical composition for treating chronic liver disease comprising the gene sequence of human HNFla and/or a protein encoded thereby, and/or a pharmaceutically acceptable carrier or excipient, and/or other known Treatment of chronic liver disease drugs.
- the pharmaceutical composition is delivered by oral, intramuscular, intravenous, subcutaneous, topical, transdermal routes.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- a method of treating chronic liver disease comprising administering an effective amount of the human HNFla gene sequence and/or its encoded protein and/or the above pharmaceutical composition to a patient.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- a gene therapy method for chronic liver disease comprising introducing a HNFla gene into liver parenchyma cells and interstitial cells for expression.
- the method of introducing the HNFla gene into liver parenchymal cells and mesenchymal cells includes mediated by plasmid transfection, adenovirus or adeno-associated virus.
- the chronic liver disease includes liver fibrosis and cirrhosis.
- the invention has the advantages of: using genetic engineering technology to regulate the expression of the important transcription factor HNFla gene in HSC in vitro, and confirming its inhibitory effect on HSC activation and proliferation and EMT process, and further studying its possible molecular biological mechanism; Viral vector injection clears the up-regulation of HNFla expression to block and reverse the process of liver fibrosis. This is a new exploration in the field of research on chronic liver disease using transcription factors, providing a new method for the treatment of chronic liver disease.
- FIG. 1 Immunohistochemistry and real-time PCR were used to detect the expression of HNFla protein and mRNA in human cirrhotic tissues and relative normal liver tissues.
- FIG. 1 Schematic diagram of HNF1 a treatment of DMN liver injury rat model.
- FIG. 3 Schematic diagram of HNF1 a treatment of rat model of BDL liver injury.
- FIG. 4 Recombinant adenovirus expressing HNFla AdHNFla was injected into the different types of hepatic fibrosis rats by tail vein injection to inhibit liver fibrosis.
- HNFla inhibits the expression of a-SMA, TGF- ⁇ in the liver of rats with BDL hepatic fibrosis, and reverses the EMT process.
- Figure 7 HNFla inhibits the expression of a-SMA, TGF- ⁇ in liver of DMN liver fibrosis rats, and reverses the EMT process.
- Figure 8 HNFla inhibits the expression of TIMP-1, IL-6 and JAK-STAT pathway proteins in the liver of rats with BDL and DMN liver fibrosis.
- Figure 9 HNFla up-regulates the expression of SHP1 in the liver of BDL and DMN liver fibrosis rats, promotes SHP1 membrane translocation, increases SHP1 activity, and down-regulates a-SMA expression.
- FIG. 10 Activation of HSC by AdHNFla-infected rats up-regulated the expression of SHP1 and inhibited JAK-STAT pathway, regulated HSC activation and mRNA expression of collagen synthesis-related genes.
- Figure 11 HNFla down-regulates JAK-STAT pathway-associated phosphorylation protein expression and represses JAK-STAT pathway.
- Figure 12 Down-regulation of SHP1 expression by siRNA, HNFla inhibits the expression of a-SMA, type I and type III collagen mRNA.
- FIG. 13 HNFla promotes miR-194 expression, and miR-194 inhibits downstream target gene SET and STAT5 expression.
- Figure 14 miR-194 regulates SET and STAT5 protein expression, and HNFla down-regulates SET after inhibition of miR-194 The role is repressed.
- the present invention relates to the isolation and functional studies of the human transcription factor HNFla, and also includes a method for introducing HNFla gene and protein into HSC and a means for increasing HNFla expression in HSC.
- HNFla is an important transcriptional protein that regulates liver differentiation and function. It binds to cis-acting elements in the form of homologous or heterodimers with HNF1P, and interacts with some transcriptional activators to alter the vicinity of promoters or enhancers. The structure of the chromosome, thereby achieving regulation of gene expression at the transcriptional level.
- HNFla may play an important role in the development of liver fibrosis and may become an important target for the treatment of chronic liver disease and liver fibrosis.
- AdHNFla was introduced into different types of liver fibrosis rats by tail vein injection to study the inhibitory effect of HNFla expression on liver fibrosis.
- Immunohistochemistry and Real-time PCR were used to detect the differential expression of HNFla protein and mRNA in normal liver tissues and liver cirrhosis tissues.
- the ⁇ -actin was used as an internal reference for amplification reaction, and the PCR product was subjected to 1.5% agarose gel electrophoresis and scanned by a gel imager to detect reverse transcription.
- the reaction system is as follows:
- the reaction conditions were as follows: pre-denaturation at 94 ° C for 30 s, followed by 94 ° C l0 s, 60 ° C for 30 s, and a total of 40 cycles were carried out to determine the dissolution curve.
- the fluorescence background signal and threshold are generally the default values of the instrument, which are automatically generated after each run. The number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold is defined as the Ct value; each pair of primers
- HNF1 a expression inhibits liver fibrosis in rats.
- DMN-induced liver fibrosis model Male SD rats were randomly divided into 4 groups, 10 in each group. Ordinary feed feeding, free drinking water, day and night alternate lighting. Group 1 was given intraperitoneal injection of saline as a negative control group; Groups 2 to 4 were a model of liver fibrosis, and 1% DMN solution was intraperitoneally injected at a dose of 10 ⁇ /kg, and three times a week, a total of 4 injections. w, Preparation of DMN-induced rat liver fibrosis model. BDL liver fibrosis model: 40 male SD rats were divided into 4 groups, 10 in each group. Ordinary feed feeding, free drinking water, day and night alternate lighting.
- Group 1 is the sham operation group, and groups 2 to 4 are the BDL group.
- BDL rats were anesthetized with a 10% chloral hydrate solution at a dose of 35 mg/kg body weight, and the limbs were fixed on the mouse plate.
- a skin incision of about 1.5 ⁇ 2 cm was cut from the midline of the abdomen, and the lower abdominal muscles were cut along the abdominal muscle white line until the peritoneum, and the liver was exposed.
- Use a cotton swab to remove the liver from the top of the rat, and fully expose the hepatic hilum. It can be seen that the white tube accompanying the portal vein is the common bile duct, and the bile duct is bluntly separated.
- the three "0" silk thread is used to pass through the common bile duct.
- the cholestasis causes the ligature line to fall off.
- the upper end of the common bile duct is ligated with two lines. The upper two are as close as possible to the left and right hepatic ducts and merged into the common bile duct.
- the lower end of the common bile duct is ligated one at a distance from the upper two lines. Cut the bile duct.
- the abdominal cavity was cleaned, and the line 4 was closed layer by layer, that is, the preparation of the common bile duct ligation model was completed.
- the common bile duct was separated only by open surgery, and the abdomen was directly closed without ligature.
- the full length sequence of human HNFla cDNA is shown in SEQ ID N0.33.
- synthetic primers (synthesized by Shanghai Shenggong Bioengineering Co., Ltd.), Bgl ll restriction site was added at the 5' end, Kpn l restriction site was added at the 3' end, and PCR primers were added.
- the sequence is:
- Upstream arch I 5'- GGAAGATCTCGAGCCATGGTTTCTAAACTGAG -3 ' ( SEQ ID N0.31 ) ; Downstream 5'-CGGGGTACCTTACTGGGAGGAAGAGGCCAT-3' (SEQ ID N0.32).
- the HNFla cDNA fragment was obtained by PCR amplification under the conditions of 98 ° C for 10 s, 68 ° C for 8 min, and 35 cycles.
- the reaction system is shown in Table 2.
- the PCR product was electrophoresed on a 1% agarose gel, and the fragment size was identified, and the tapping recovery was placed in an Eppendorf tube, and the weight of the gel was weighed.
- the DNA fragment was eluted by 30 ⁇ l of double distilled water, allowed to stand for 1 min, centrifuged at 13,000 rpm for 1 min, and carefully pipet the eluate 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.
- Kpn I and Bgl II were digested by shuttle plasmid pAdTrack-CMV (purchased from Stratagene) and purified by P HNFla cDNA for 4 h, and the OD 26Q value was determined by spectrophotometer, and the fragment size was identified by 1% agarose gel electrophoresis.
- HNFla cDNA 10> ⁇ T 4 buffer 2 ⁇ l, T 4 DNA ligase ⁇ (2U) and dd3 ⁇ 40, total volume 20 ⁇ l, and ligated overnight at 16 °C.
- the ligation product was added to the competent bacterial Escherichia coli DH5a transformation, plated with LB medium plates containing kanamycin, and thermostated overnight at 37 ° C to select single colony clones. A small number of colonies were picked and dissolved in ddH 2 0.
- the HNFla cDNA fragment was amplified by PCR using the bacterial solution as a template.
- the colony clone from which the HNFla cDNA fragment was amplified was extracted with Qiagen-tip 100 kit to obtain plasmid pAdTrack-CMV-HNFla, and the plasmid concentration was calculated and formulated into a working concentration (0.5 ⁇ / ⁇ 1).
- pAdTrack-CMV-HNF 1 ⁇ 2 ⁇ 1, Kpn I and Bgl II endonuclease were digested for 4 h, and the fragment size was identified by 1% agarose gel electrophoresis.
- the plasmid was sent to Shanghai Shenggong Bioengineering Co., Ltd. for sequencing.
- pAdTrack-CMV-HNFla plasmid 1 pAdTrack-CMV-HNFla plasmid was obtained, and Pme I endonuclease was digested for 4 h. Dephosphorylation was carried out by adding bovine intestinal alkaline phosphatase CIP for 1 h, 1% agarose gel electrophoresis, and tapping recovery linearized pAdTrack-CMV-HNFla. Formulated to work concentration (0.1 ⁇ / ⁇ 1). A total of 20 ⁇ l of competent BJ5183 bacteria were co-transformed with 0.4 linear pAdTrack-CMV-HNFla and O. lg supercoiled pAdEasy-1 plasmids at 2,000 V, 200 Ohms, and 25 ⁇ electroporation.
- the plasmid pAdHNFla was selected and transformed into the competent bacteria DH5a, and the plasmid was ligated to a working concentration (1 ⁇ / ⁇ 1), and the plasmid was sequenced and identified.
- Resuscitation 293 cells inoculated into a 10 cm tissue culture dish at 4.8 ⁇ 10 6 / dish, and added DMEM (containing 10% fetal bovine serum, 100 U/ml penicillin, 100 g/ml streptomycin) 37 ° C, 5% C0 2 After culture, the cell density grew to 60% to 80% after 24 hours.
- pAdHNFla was linearized with restriction endonuclease Pacl and the plasmid was recovered by ethanol precipitation.
- the lOg linearized pAdHNFla was mixed with serum-free DMEM solution 250 ⁇ 1 to prepare a sputum.
- Take Lipofectamin 2 ( ⁇ l, add serum-free DMEM solution 250 ⁇ 1, mix well, prepare sputum, sputum and sputum fully mixed, leave it at room temperature for 30min, add to 293 cells to be transfected, and change culture after 4h After 4 days, GFP expression was observed by fluorescence microscope. After 7 days, 293 cells and supernatant were collected, and repeatedly frozen and thawed in liquid nitrogen and 37 ° C water bath for 4 times, centrifuged at 5,000 rpm for 5 min, and the virus supernatant was collected.
- DMN group 40 male Sprague-Dawley rats were randomly divided into 4 groups, 10 in each group. Ordinary feed feeding, free drinking water, day and night alternate lighting. Group 1 was given intraperitoneal injection of saline as a negative control group; Groups 2 to 4 were a model of liver fibrosis, and 1% DMN solution was intraperitoneally injected at a dose of 10 ⁇ /kg, and three times a week for a total of 4 weeks. , Preparation of rat liver fibrosis model induced by DMN.
- the second group was set as the model control group; the third group was the blank virus AdGFP control group; the fourth group was set as the AdHNFla introduction group, and the first group, the second group, the third group and the fourth group were injected with DMN 2 w after Intravenous injection of PBS, PBS, 5> ⁇ 10 9 pfU AdGFP and 5x10 9 pfu AdHNFla, 2 weeks later, the rats were sacrificed, and the same part of the liver tissue was fixed with neutral formaldehyde, which was prepared as paraffin. Sectioning; residual liver tissue cryopreservation (see Figure 2).
- BDL group 48 male Sprague-Dawley rats were randomly divided into 4 groups, 12 in each group.
- the first group was the sham operation group, and the 2 ⁇ 4 groups were the BDL model group, which were sequentially set as the PBS control group, the blank virus AdGFP control group, and the AdHNFla introduction group.
- Group 1, Group 2, Group 3 and Group 4 were injected intravenously PBS, PBS, 5x l0 9 pfh AdGFP and 5x l0 9 pfh AdHNFla; W after modeling, the rats were sacrificed 3
- the organization processes the same DMN model (see Figure 3).
- AdHNFla treatment The amount of ECM in the liver of the group was significantly lower than that of the control group.
- DN model ECM in the AdHNFla treatment group was 57% in the AdGFP group
- BDL model The ECM in the AdHNFla treatment group was 51% in the AdGFP group (see Figure 4).
- DN model hydroxyproline content in AdHNFla treatment group (163 ⁇ 42.57 g/g) was significantly lower than AdGFP group (259.33 ⁇ 57.95 g/g); BDL model: AdHNFla treatment group hydroxyproline (227.60 ⁇ 39.94 g/ g) was significantly less than the AdGFP group (304.22 ⁇ 38.36 g/g) ( ⁇ 0.05, see Figure 4).
- Rat liver tissue total RNA 0.5 ⁇ 0.8 g liver tissue, adding TRizol reagent (1 ml / 100 mg tissue), the tissue was crushed to homogenate, placed at room temperature for 5 min; Add chloroform 0.2 ml/ml TRizol, shake vigorously for 15 s, and let stand for 3 min at room temperature; centrifuge at 12,000 rpm for 15 min at 4 °C; take the upper aqueous phase, add isopropanol 0.5 ml/ml TRizol, and mix by inversion.
- liver fibrosis marker proteins such as a-SMA and TGF- ⁇ decreased significantly with the improvement of fibrosis after AdHNFla induction; the expression of E-cadherin was significantly increased, and the interstitial phenotype protein The expression of vimentin was reduced and the EMT process was reversed; the expression of Jak2, IL-6 and TIMP-1 was significantly decreased in liver tissue, and the JAK-STAT pathway was significantly inhibited (see Figures 6, 7, and 8).
- Tissue paraffin section baked at 60 °C, 30 min; dewaxed to water; remove endogenous peroxidase: 3 % H 2 0 2 , room temperature 10 min; PBS (0.01 M, pH 7.4) Wash 3 Times, 3 min each time; antigen retrieval: citrate buffer microwave repair; PBS (0.01 M, pH 7.4) 3 washes, 3 min each time; blocking: 5 % horse serum (diluted in PBS), placed in a wet box, Blocked at 30 ° C for 1 h; add primary antibody at 4 ° C overnight (rabbit anti-rat SHP1 and mouse anti-rat a-SMA primary antibody concentration is 1: 100); PBS (0.01 M, pH 7.4) wash 3 Times, 5 min each time; add fluorescent secondary antibody: 37 °C, 30 min; wash PBS (0.01 M, pH 7.4) 3 times for 5 min each time; nuclear DNA coloration [J DAPI (4'6'- Diamidino-2-phenylindole) Dilute with blocking solution 1 : 1000; take
- the supernatant was discarded and centrifuged twice with D-Hanks for 1700 rpm/min for 7 min .
- the supernatant was discarded and the precipitate was mixed with a 1:2 volume of 18% Nycodenz. Density gradient centrifugation, 3400 rpm/min, 17 min; The cells at the interface were washed twice with DMEM at 1700 rpm/min for 7 min.
- the pellet was suspended in DMEM containing 10% calf serum and the survival rate was determined by trypan blue staining; the cells were treated with lxl0 5 /cm 2 was inoculated in a 35 mm 2 culture dish, placed in a 5% C0 2 , 37 ° C incubator, and the cells were changed after 24 h adherence, and then changed every 2 days; cell identification: observation under an inverted microscope The morphology of the isolated HSC and the morphological changes after culture; the autofluorescence of HSC was observed under the excitation of ultraviolet light at 328 nm fluorescence microscope; the expression of desmin was confirmed by immunofluorescence.
- Ad HNFla significantly affected the expression of HSC-related genes in HSC a-SMA, type I collagen, type III collagen, TIMP-1 and IL-6 were down-regulated by 93% ( ⁇ 0.01), 81% ( ⁇ 0.01), 68% ( ⁇ 0.01), 29% ( ⁇ 0.05) and 70% ( ⁇ 0.01); ⁇ -9 and MMP-13 were up-regulated by 1.7 times and 10.8 times.
- AdHNFla was infected with HSC for 48h, and after 20% fetal bovine serum stimuli for 0, 1, 2, 4h, the whole cell protein was collected from the cell lysate. After the protein was quantified, each protein was separated by 10 ⁇ ⁇ 10% SDS-PAGE.
- Polyvinylidene fluoride film (PVDF film) ddH 2 0 rinse, the electrophoresis gel, PVDF film, filter paper were placed in the Transferring Buffer and equilibrated, placed in an electric transfer tank, 300 mA, 70 min.
- SHP1 expression was down-regulated by siRNA-SHP1, and the effect of down-regulation of SHP1 on HNFla inhibition of hepatic fibrosis was detected by real-time PCR.
- the PCR primers used are shown in Table 5.
- AdHNFla+siRNA-SHP1 group compared with AdHNFla+siRNA-NC group: a-SMA, I
- the expressions of collagen and type III collagen mRNA were up-regulated by 58% (P ⁇ 0.05), 33% (P ⁇ 0.05) and 122% CP ⁇ 0.05, respectively.
- SHP1 expression is one of the main mechanisms by which HNFla inhibits liver fibrosis (see Figure 12).
- HNFla up-regulated the expression of miR-194, which in turn down-regulated the expression of miR-194 target gene SET and STAT5, and increased the catalytic activity of SHP1.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
肝细胞核因子 1α治疗慢性肝病的用途及方法
技术领域
本发明涉及分子生物学、细胞生物学、基因工程以及临床医学如疾病治疗技术领域。 具体地说， 本发明涉及利用肝细胞核因子 la (HNFla) 治疗慢性肝病的用途及方法。 背景技术
慢性肝病、 肝硬化是临床常见的慢性疾病， 治疗难度大， 临床预后差， 占用了巨大 的医疗资源， 是危害国民健康的重大疾病。 肝纤维化 （hepatic fibrosis) 是各种慢性肝病 发展至肝硬化的必经阶段， 是肝脏对慢性损伤的一种修复反应， 以细胞外基质 (extracellular matrix, ECM)在肝内过多沉积为特征。 目前认为，肝纤维化为一动态过程， 属可逆性病变。 因此， 阻断、 抑制或逆转肝纤维化是治疗慢性肝病的一个重要手段。 肝 纤维化发生的中心环节是肝星状细胞（hepatic stellate cell, HSC)激活并向肌成纤维样细 胞 （myofibroblasts, MFs) 转化， 抑制 HSC激活、 增殖与迁移、 诱导凋亡是肝纤维化治 疗的重要策略。 上皮细胞间质转型 （epithelial-to-mesenchymal transition, EMT) 主要是指 上皮细胞在细胞形态、 细胞结构、 细胞功能以及细胞粘附、 迁移能力等方面获得间质细 胞特性的过程。一系列研究表明，肝细胞、 HSC、胆管上皮细胞也通过 EMT转化为 MFs， 是肝纤维化进展过程中的重要环节。 这些研究是肝纤维化机制与治疗领域新的突破。
肝细胞核因子 1 (hepatocyte nuclear factor, HNF1 ) 属于 POU-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。尽管既往研究证实 HNFla在维持肝脏功能和肝脏发育过程中发挥重要作用， 但是该 转录因子与肝纤维化之间的关系尚不明确， 其能否起到阻断或肝逆转纤维化仍未得到证 实， 对肝纤维化的治疗作用也不明确。 国内外亦未将上调 HNFla表达作为抗肝纤维化治 疗手段加以研究。
发明内容
本发明的目的在于提供一种人 HNFla的基因序列在治疗慢性肝病中的用途。
本发明的第二个目的是提供一种人 HNFla 的基因序列在制备治疗慢性肝病药物中 的用途。
本发明的第三个目的是提供一种人 HNFla基因序列编码的蛋白在治疗慢性肝病中 的用途。
本发明的第四个目的是提供一种人 HNFla基因序列编码的蛋白在制备治疗慢性肝 病药物中的用途。
本发明的第五个目的是提供一种用于治疗慢性肝病的药物组合物。
本发明的第六个目的是提供一种治疗慢性肝病的方法。
本发明的第七个目的是提供一种慢性肝病的基因治疗方法。
为实现上述目的， 本发明采取的技术方案是：
人 HNFla的基因序列在治疗慢性肝病中的用途。
所述的慢性肝病包括肝纤维化和肝硬化。
为实现上述第二个目的， 本发明采取的技术方案是：
人 HNFla的基因序列在制备治疗慢性肝病药物中的用途。
所述的慢性肝病包括肝纤维化和肝硬化。
为实现上述第三个目的， 本发明采取的技术方案是：
人 HNFla基因序列编码的蛋白在治疗慢性肝病中的用途。
所述的慢性肝病包括肝纤维化和肝硬化。
为实现上述第四个目的， 本发明采取的技术方案是：
人 HNFla基因序列编码的蛋白在制备治疗慢性肝病药物中的用途。
所述的慢性肝病包括肝纤维化和肝硬化。
为实现上述第五个目的， 本发明采取的技术方案是：
一种用于治疗慢性肝病的药物组合物，所述的药物组合物包括人 HNFla的基因序列 和 /或其编码的蛋白， 和 /或药用载体或赋形剂， 和 /或其他已知的治疗慢性肝病药物。
所述的药物组合物通过口服， 肌内， 静脉内， 皮下， 局部， 经皮途径来传送。
所述的慢性肝病包括肝纤维化和肝硬化。
为实现上述第六个目的， 本发明采取的技术方案是：
一种治疗慢性肝病的方法， 包括将有效量的人 HNFla 的基因序列和 /或其编码的蛋 白和 /或上述药物组合物给药于患者。
所述的慢性肝病包括肝纤维化和肝硬化。
为实现上述第七个目的， 本发明采取的技术方案是：
一种慢性肝病的基因治疗方法， 包括将 HNFla基因导入肝脏实质细胞和间质细胞， 使之表达。
所述的将 HNFla基因导入肝脏实质细胞和间质细胞的方法包括用质粒转染、腺病毒 或腺相关病毒介导。
所述的慢性肝病包括肝纤维化和肝硬化。
本发明的优点在于： 利用基因工程技术体外调控 HSC中重要转录因子 HNFla基因 表达， 证实其对 HSC活化增殖以及 EMT进程的抑制作用， 同时进一步研究了其可能的 分子生物学机制； 通过体内 HNFla腺病毒载体注射明确 HNFla表达上调对肝纤维化进 程的阻断和逆转作用， 这是利用转录因子治疗慢性肝病研究领域的全新探索， 为慢性肝 病的治疗提供了一种新方法。
附图说明
附图 1 免疫组化法及 real-time PCR检测人肝硬化组织及相对正常肝组织中 HNFla 蛋白及 mRNA的表达。
附图 2 HNF1 a治疗 DMN肝损伤大鼠模型示意图。
附图 3 HNF1 a治疗 BDL肝损伤大鼠模型示意图。
附图 4 表达 HNFla的重组腺病毒 AdHNFla经尾静脉注射导入不同类型肝纤维化大 鼠体内对肝纤维化进程的抑制作用。
附图 5体内研究 HNFla抑制 IL-6、 TIMP-lmRNA表达。
附图 6 HNFla抑制 BDL肝纤维化大鼠肝脏 a-SMA、 TGF-βΙ等表达， 逆转 EMT进 程。
附图 7 HNFla抑制 DMN肝纤维化大鼠肝脏 a-SMA、 TGF-βΙ等表达， 逆转 EMT进 程。
附图 8 HNFla抑制 BDL及 DMN肝纤维化大鼠肝脏 TIMP-1、 IL-6和 JAK-STAT通 路蛋白表达。
附图 9 HNFla上调 BDL及 DMN肝纤维化大鼠肝脏 SHP1表达，促进 SHP1膜转位、 提高 SHP1活性， 同时下调 a-SMA表达。
附图 10 AdHNFla感染大鼠活化 HSC后，上调 SHP1表达， 并抑制 JAK-STAT通路、 调控 HSC活化及胶原合成相关基因 mRNA表达。
附图 11 HNFla下调 JAK-STAT通路相关磷酸化蛋白表达， 阻遏 JAK-STAT通路。 附图 12利用 siRNA下调 SHP1表达， HNFla抑制 a-SMA、 I型及 III型胶原 mRNA 表达的作用被阻遏。
附图 13 HNFla促进 miR-194表达， miR-194抑制下游靶基因 SET及 STAT5表达。 附图 14 miR-194调控 SET及 STAT5蛋白表达， 抑制 miR-194后， HNFla下调 SET
的作用被阻遏。
具体实施方式
本发明涉及人转录因子 HNFla的分离和功能研究， 还包括 HNFla基因和蛋白导入 HSC的方法和提高 HSC内 HNFla表达的手段。 HNFla是调控肝脏分化和功能的重要转 录蛋白， 其以同源或与 HNF1P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基 因表达的调控。 我们在 mRNA及蛋白水平检测了人肝硬化组织及相对正常肝组织中 HNFla的表达水平， 证实了肝纤维化发展过程中， HNFla表达下调； 更为重要的是， 上 调 HNFla表达可抑制 HSC活化及 HSC胶原合成相关基因的表达； 并在实验性肝纤维化 大鼠体内发挥抑制肝纤维化进程的作用。 因此， 我们认为， HNFla可能在肝纤维化发展 进程中发挥重要作用， 可能成为慢性肝病、 肝纤维化治疗的重要靶点。
以下为本发明的基本思路：
1. 对比测定人肝硬化组织和相对正常肝组织中 HNFla表达情况。
2. AdHNFla经尾静脉注射导入不同类型肝纤维化大鼠体内， 研究上调 HNFla表达 对肝纤维化进程的抑制作用。
3. 检测上调 HNFla对原代大鼠 HSC活化及胶原合成相关基因表达的影响。
4. 体外通过 Real-time PCR等方法筛选 HNFla调控的肝纤维化相关信号转导通路中 的重要基因， 并用 westen-blot检测其蛋白表达水平。
5. 体内进一步验证及研究 HNFla调控的靶基因及肝纤维化相关信号通路。
6. 体外重组获取高纯度 HNFla蛋白， 利用脂质体等非病毒载体介导， 研发新型生 物药物， 开展 HNFla治疗慢性肝病的临床研究。
以下结合附图和具体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
免疫组化法及 Real-time PCR法检测正常人肝脏组织及肝硬化组织中 HNFla蛋白及 mRNA的差异表达。
1 免疫组织化学方法检测肝组织中 HNFla蛋白变化。
人正常肝组织及肝硬化组织蜡块 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 lO 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 :20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。
2 Real-time PCR检测肝组织中 HNFla mRNA变化。
Trizol法抽提人正常肝组织及肝硬化组织 RNA， 以分光光度计测定其 OD26Q值， 配 成工作浓度 （ ^g/μΐ及 0.1μ§/μ1)， 1%琼脂糖凝胶电泳检测 RNA完整性。 取 2μ§ RNA进 行逆转录及 Real-time PCR扩增。 制备 cDNA:
RT反应体系及步骤如下：
Random primer 1 μΐ
RNA
力口 DEPC水至总体积 16.5 μ1， PCR仪上 70°C i min后快速置于冰上冷却 5 min, 加 入：
Inhibitor 1 μΐ
5 χ buffer 5 μΐ
dNTPmix 1.5 μΐ
M-MLV (逆转录酶） 1 μΐ
混匀， 37 °C， 2 h， 一20 °C保存备用。
逆转录成 cDNA后， 以 beta-actin为内参照弓 |物扩增反应， PCR产物经 1.5 %琼脂糖 凝胶电泳， 凝胶成像仪扫描成像， 以检测逆转录情况。
Real-time PCR
反应体系如下:
ddH20 8.2 μΐ
SYBR 10 μΐ
primer F 0.4 μΐ
primer R 0.4 μΐ
cDNA 1 μΐ
Total 20 μΐ
反应条件为： 94°C预变性 30s， 之后 94°C l0s， 60°C 30s, 共进行 40个循环后进行溶 解曲线的检测。 荧光本底信号和阈值一般采用仪器默认数值， 每次运行结束以后自动生 成， 每个反应管内的荧光信号达到设定的阈值时所经历的循环数定义为 Ct值； 每对引物
(基因） 在每个模板中做 3个重复管， 得到的 α值取平均值； 每个目的基因的 ct平均
值减去对应模板的内参基因 （ACTB ) 的 α平均值， 得到 ACt。 实验组的 ACt减去对照 组的 ACt， 得到 ΔΔα值， 对照组和实验组中的待测基因的倍数关系用 2_ΔΔα表示。 PCR 引物如表 1所示。
表 1
结果表明： 肝硬化组织与相对正常肝组织相比， HNFl a蛋白及 mRNA表达下调， 说 明 HNFl a表达下调与肝纤维化发展相关 （见附图 1 )。
实施例 2
上调 HNFl a表达对大鼠肝纤维化进程的抑制作用。
1 肝纤维化模型制备：
DMN损伤肝纤维化模型： 将雄性 SD大鼠随机分为 4组， 各组 10只。 普通饲料喂 养， 自由饮水， 昼夜交替照明。 第 1组予生理盐水腹腔注射作为阴性对照组； 第 2~4组 为肝纤维化模型组，予 1 %DMN溶液按照 10 μΒ/kg的剂量腹腔注射，每周连续注射 3次， 共注射 4 w, 制备 DMN诱导的大鼠肝纤维化模型。 BDL肝纤维化模型： 将 40只雄性 SD 大鼠分成 4组， 每组 10只。 普通饲料喂养， 自由饮水， 昼夜交替照明。 第 1组为假手术 组， 第 2~4组为 BDL组。 BDL大鼠予 10 %水合氯醛溶液按照 35 mg/kg体重的剂量腹腔 注射麻醉后，将四肢固定于鼠板上。腹部碘伏消毒后，从腹部正中剑突下剪开约 1.5~2 cm 的皮肤切口， 沿腹肌白线剪开下面腹肌直至腹膜， 暴露肝脏。 用棉签将肝叶向大鼠的上 方拨开， 充分暴露肝门， 可见与门静脉伴行的白色管道即为胆总管， 钝性分离胆总管， 用三根 "0"丝线穿过胆总管， 为防治胆汁淤积导致结扎线脱落， 胆总管上端结扎两根线， 上面两根尽量接近左右肝管汇合成胆总管处结扎， 胆总管下端结扎一根， 与上面两根线 留取一段距离， 在此中间剪断胆管。清理腹腔， 4号线逐层关腹， 即完成胆总管结扎模型 的制备。 假手术组仅开腹分离胆总管而不结扎切断就直接关腹。
2 构建重组腺病毒 AdHNFla和 AdGFP:
人 HNFla cDNA的全长序列如 SEQ ID N0.33所示。根据人 HNFla cDNA序列设计、 合成引物 （上海生工生物工程有限公司合成）， 在 5'端加入 Bgl ll限制性酶切位点， 在 3 ' 端加入 Kpn l限制性酶切位点， PCR引物序列为：
上游弓 I物 5'- GGAAGATCTCGAGCCATGGTTTCTAAACTGAG -3 ' ( SEQ ID N0.31 ) ;
下游弓 I物 5'- CGGGGTACCTTACTGGGAGGAAGAGGCCAT - 3' ( SEQ ID N0.32)。 PCR扩增获取 HNFla cDNA片段， 反应条件为 98 °C 10s, 68 °C 8min, 35个循环。 反应体系如表 2所示。
表 2
成分 体积 ( μθ
上游引物 2μ1
下游引物 2μ1
正常肝细胞 cDNA 2μ1
PrimerSTAR酶 Ιμΐ
5χ缓冲液 20μ1
dNTPs 8μ1
ddH20 65 μΐ
PCR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 l 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。 Kpn I、 Bgl II酶切穿梭质粒 pAdTrack-CMV (购于 Stratagene公司） 禾 P HNFla cDNA 4 h后并纯 化， 分光光度计测定 OD26Q值， 1%琼脂糖凝胶电泳鉴定片段大小。 取 0.1μ§ 质粒 pAdTrack-CMV 0.4 g HNFla cDNA、 10><T4缓冲液 2μ1、 T4DNA连接酶 Ιμΐ ( 2U) 以及 dd¾0, 总体积 20μ1， 16°C连接过夜。 将连接产物加入感受态细菌大肠杆菌 DH5a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。 挑取少量菌落， 溶于 ddH20中， 以菌液为模板， PCR扩增 HNFla cDNA片段。 将扩增出 HNFla cDNA 片段的菌落克隆用 Qiagen-tip 100试剂盒抽提， 获取质粒 pAdTrack-CMV- HNFla, 计算 质粒浓度， 并配成工作浓度 （0.5μ§/μ1)。 取 p AdTrack-CMV-HNF 1 α 2μ1, Kpn I、 Bgl II 内切酶酶切 4 h， 1%琼脂糖凝胶电泳鉴定片段大小。 质粒送上海生工生物工程有限公司 测序。 取 1 pAdTrack-CMV-HNFla质粒， Pme I内切酶酶切 4 h， 加入牛小肠碱性磷 酸酶 CIP去磷酸化 l h ， 1%琼脂糖凝胶电泳， 割胶回收线性化 pAdTrack-CMV-HNFla， 配成工作浓度 （0.1μ§/μ1)。 分别取 0.4 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。 500 μΐ LB稀释后， 37°C静置 45 min, 卡那霉素 LB培养基平板筛选， 37°C过夜生长。 选择 10-20个孤立小克隆， 分别用 5 ml含 5(^g/ml卡那霉素 LB溶解， 37°C、 300 rpm摇床摇
过夜， 小剂量抽提质粒， 分别用 Pac l和 Bxt l酶切， 1%琼脂糖凝胶电泳显示片段大小。 选择病毒质粒 pAdHNFla转化至感受态细菌 DH5a中抽质粒， 配成工作浓度 （ 1μ§/μ1)， 质粒测序鉴定。 复苏 293细胞， 以 4.8χ 106/皿接种于 10cm的组织培养皿， 加入 DMEM (含 10%胎牛血清、 100U/ml青霉素、 lOO g/ml链霉素） 37°C、 5%C02培养， 24h后细 胞密度生长至 60%~80%。 pAdHNFla用限制性内切酶 Pacl酶切线性化， 并用乙醇沉淀 DNA方法回收质粒；取 lO g线性化的 pAdHNFla与不含血清的 DMEM培液 250μ1混匀， 配成 Α液。 取 Lipofectamin 2(^l， 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 30min后加入待转染的 293细胞中， 4h后更换培液， 4d 后荧光显微镜观察 GFP表达。 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， 5,000rpm离心 5min， 收集病毒上清。 以 2ml /皿病毒上清再次感染 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， 置 于 -80°C保存备用。 同法获得空载病毒 AdGFP (无外源基因片段插入， 仅表达 GFP)。
3 HNFla体内导入肝纤维化动物模型
DMN组： 将 40只雄性 SD大鼠随机分为 4组， 各组 10只。 普通饲料喂养， 自由饮 水， 昼夜交替照明。 第 1组予生理盐水腹腔注射作为阴性对照组； 第 2~4组为肝纤维化 模型组，予 1 %DMN溶液按照 10 μΒ/kg的剂量腹腔注射，每周连续注射 3次，共注射 4w， 制备 DMN诱导的大鼠肝纤维化模型。 其中第 2组设为模型对照组； 第 3组为空白病毒 AdGFP对照组； 第 4组设为 AdHNFla导入组， 第 1组、 第 2组、 第 3组和第 4组于注 射 DMN 2 w后经尾静脉分别注入 PBS、 PBS、 5>< 109pfU AdGFP和 5x l09pfu AdHNFla, 2 w后处死， 留取大鼠血清病本， 同时取相同部位肝组织中性甲醛固定， 备做石蜡切片； 余肝组织液氮冻存 （见附图 2)。 BDL组： 将雄性 48只 SD大鼠随机分成 4组， 每组 12 只。 分别为： 第 1组为假手术组， 2~4组为 BDL模型组， 依次设为 PBS对照组、 空白病 毒 AdGFP对照组和 AdHNFla导入组。 BDL术后 2 d， 第 1组、 第 2组、 第 3组和第 4 组分别经尾静脉注入 PBS、 PBS、 5x l09pfh AdGFP和 5x l09pfh AdHNFla; 造模 3 w后处 死大鼠， 组织处理同 DMN模型 （见附图 3 )。
4 肝组织石蜡切片分别进行苏木精 -伊红 （hematoxylin-eosin, HE) 染色、 苦味酸-酸 性品红（Van Gieson, VG)染色及 Masson's trichrome染色测定肝脏 ECM沉积情况， 结果 表明： AdHNFla治疗组大鼠肝脏 ECM量较对照组明显减少。 （DMN模型： AdHNFla 治疗组 ECM为 AdGFP组的 57%; BDL模型： AdHNFla治疗组 ECM为 AdGFP组的 51% (见附图 4)。
5 碱水解法测定各组肝组织羟脯氨酸含量
随机选取各组肝组织各 6例， 碱水解法测肝组织中羟脯氨酸含量 （实验方法具体见 南京凯基生物公司羟脯氨酸测定试剂盒说明书， CAT number: KGT030-2), 结果表明： AdHNFla 治疗组大鼠肝脏羟脯氨酸含量较对照组明显减少 （附图 4)。 (DMN模型： AdHNFla治疗组羟脯氨酸含量 （163±42.57 g/g) 较 AdGFP组 （259.33±57.95 g/g) 明 显减少； BDL 模型： AdHNFla 治疗组羟脯氨酸 （227.60±39.94 g/g ) 较 AdGFP 组 ( 304.22±38.36 g/g) 明显减少 ( < 0.05 , 见附图 4)。
实施例 3
Real-time PCR和免疫组织化学法证实： HNFla在体内明显抑制 HSC活化和细胞外 基质沉积， 并阻断肝纤维化组织 EMT过程。
1 提取 DMN及 BDL肝损伤模型各组大鼠肝组织总 RNA: 0.5〜0.8 g肝组织， 加入 TRizol试剂 （1 ml/100 mg组织） ， 将组织碾碎至匀浆状， 室温放置 5 min; 加入氯仿 0.2 ml/ml TRizol, 剧烈振荡 15 s后， 室温下静置 3 min; 4 °C， 12000 rpm离心 15 min; 取上 层水相， 加入异丙醇 0.5 ml/ml TRizol, 颠倒混匀， 室温静置 10 min; 4 °C， 12000 rpm离 心 10 min; 弃上清，加入无水乙醇 1 ml/ml TRizol, 涡流混匀； 4。C， 7500 rpm离心 5 min, 弃去乙醇， 室温下自然晾干； 加 50μ1 DEPC水溶解 RNA; 待 RNA充分溶解后， 各取 Ιμΐ 进行 1.5 %的琼脂糖凝胶电泳， 并用紫外分光光度仪测定 260 nm、 280 nm波长处的光密 度值， 换算浓度。 逆转录后 Real-time PCR检测肝纤维化相关基因 mRNA表达变化。 结 果显示， AdHNFla导入组， HNFla及 SHP1表达明显上调； a-SMA、 collagen I、 IL-6 及 TIMP-1表达下调。 BDL模型组结果与 DMN模型组一致（见附图 5、 6、 7)。所用 PCR 引物如表 3所示。
表 3
2 免疫组化检测 DMN和 BDL肝纤维化大鼠模型肝脏组织肝纤维化指标及相关通路 逆转情况。
免疫组化结果表明， AdHNFla导入后肝纤维化标志蛋白如 a-SMA、 TGF-βΙ表达随 纤维化程度的改善而显著减少； 上皮表型标志物 E-cadherin表达明显增多， 间质表型蛋 白 vimentin表达减少， EMT进程得到逆转； 肝组织中 Jak2、 IL-6及 TIMP-1表达明显减 少， JAK-STAT通路明显抑制 （见附图 6、 7、 8)。
3 免疫组织荧光双标检测 DMN和 BDL肝纤维化大鼠模型肝脏组织 SHP1与 a-SMA 表达情况及位置关系。
组织石蜡切片， 60 °C烘烤固片， 30 min; 脱蜡至水； 清除内源性过氧化物酶： 3 % H202， 室温 10 min; PBS (0.01 M， pH 7.4) 洗 3次， 每次 3 min; 抗原修复： 柠檬酸缓 冲液微波修复； PBS (0.01 M， pH 7.4) 洗 3次， 每次 3 min; 封闭： 5 %马血清 （PBS稀 释），放入湿盒， 30°C封闭 1 h;，加一抗， 4 °C过夜（兔抗大鼠 SHP1及小鼠抗大鼠 a-SMA 一抗浓度为 1 : 100); PBS (0.01 M， pH 7.4) 洗 3次， 每次 5 min; 加荧光二抗： 37 °C， 30 min ; PBS ( 0.01 M， pH 7.4 ) 洗 3 次， 每次 5 min ; 核 DNA 显色齐 [J DAPI (4'6'-diamidino-2-phenylindole) 用封闭液 Mounting Solution 1 : 1000稀释； 取干净的载玻 片一块， 作上标记， 滴上约 30 μΐ mounting solution (含 DAPI); 用镊子取出盖玻片， 边 缘搭在吸水纸上吸去液体， 细胞面朝下， 盖在载玻片上； 盖玻片的边缘四周刷上无色指 甲油， 待干； 荧光显微镜或共聚焦显微镜观察。
免疫荧光结果表明， AdHNFla导入后肝组织中 SHP1蛋白表达明显增加， 汇管区及 窦周隙 a-SMA表达明显减少， SHP1膜表达明显增多， 因其底物主要分布在细胞膜附近， SHP1与底物结合后向细胞膜转移， 提示其活性增强 （见附图 9)。
实施例 4
Real-time PCR检测 AdHNFla感染大鼠 HSC后肝纤维化及相关通路基因变化。 1 分离制备 SD大鼠原代 HSC: 以戊巴比妥钠 30 mg/kg腹腔注射麻醉大鼠， 1%肝素 钠 1 ml/kg使大鼠全身肝素化；固定，消毒，开腹，暴露门静脉，插管；以 37°C无钙 D-HanK's 灌流液预灌流； 迅速剪断下腔静脉以备灌流液顺畅流出， 灌流速度 40 50 ml/min, 持续 约 10 min; 取出肝脏， 剔除血管、 筋膜， 剪碎， 倒入 50 ml离心管， 加含 0.05%IV胶原酶 及 0.1%链酶蛋白酶消化液 40~50 ml， 37°C水浴， 振荡消化 30 min; 依次用 100和 200目 筛过滤，滤液离心， 1700 rpm/min, 7 min;弃上清，用 D-Hanks清洗离心 2次， 1700 rpm/min, 7min;弃上清，沉淀以 1 :2体积的 18%的 Nycodenz混匀，行密度梯度离心， 3400 rpm/min,
17 min; 取界面处细胞， 用 DMEM清洗 2次， 1700 rpm/min， 7min; 取沉淀细胞悬浮于 含 10%小牛血清的 DMEM中，用台盼蓝染色判存活率;将细胞以 lxl05/cm2接种于 35 mm2 培养皿， 置 5%C02、 37°C培养箱培养， 24 h细胞贴壁后换液， 以后每隔 2天换液 1次； 细胞鉴定:倒置显微镜下观察所分离的 HSC形态和培养后的形态变化;在荧光显微镜 328 nm波长的紫外光激发下，观察 HSC的自发荧光；免疫荧光测定 desmin表达进一步证实。
2 原代培养大鼠 HSC培养 72h后， 用含 10%FCS的 DMEM换液后， 以 MOI 300加 入 AdGFP及 AdHNFla感染 HSC， 培养 48h后抽提总 RNA， 制备 cDNA， real-time PCR 检测各组肝纤维化及相关通路基因变化。 所用 PCR引物如表 4所示。
表 4
结果表明： Ad HNFla可明显影响 HSC肝纤维化相关基因表达： a-SMA、 I型胶原、 III型胶原、 TIMP-1和 IL-6分别下调 93% (Ρ< 0.01)、 81% (Ρ<0.01)、 68% (Ρ<0.01)、 29%(Ρ< 0.05)和 70%(Ρ< 0.01);ΜΜΡ-9和 MMP-13分别上调 1.7倍禾 Ρ 10.8倍。 Ad HNFla 可明显影响 HSC活化增殖相关信号通路基因 mRNA表达: gpl30和 STAT3分别下调 77% ( < 0.01) 和 68% ( < 0.01)； AdHNFla可明显上调蛋白磷酸酶 SHP1的 mRNA表达
(Ρ < 0.01 )。以上结果显示 HNFla可通过上调 SHP1表达，阻遏 JAK-STAT及相关 MAPK 通路， 抑制 HSC活化及胶原合成 （见附图 10)。
实施例 5
Westen-blot检测 HNFla对 JAK-STAT相关蛋白表达的影响。
AdHNFla分别感染 HSC 48h， 20%胎牛血清剌激 0、 1、 2、 4h后， 细胞裂解液收取 全细胞蛋白， 蛋白标准定量后， 各取 10μ§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 Transferring Buffer 中平衡后， 置于电转移槽中， 300mA， 70 min。 用 5% BSA/PBST 20 ml室温封闭膜 2 h 后， p-Jak2， Jak2, STAT5 , SET等抗体 （1 :200) 4°C孵育过夜， 次日 PBST洗涤后， 与 驴抗兔荧光二抗（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 可明显下调 JAK-STAT 通路相关磷酸化蛋白表达， 阻遏 JAK-STAT通路 （见附图 11 )。
实施例 6
利用 siRNA-SHPl下调 SHP1表达， real-time PCR检测 SHP1表达下调对 HNFla抑 制肝纤维化作用的影响。
原代培养大鼠 HSC 72h后， 取 Lipofectamin ΙΟμΙ, DMEM 5ml, siRNA-SHPl 及 siRNA-NC 200pmol转染 4-6h后， 更换为含 10%FCS的 DMEM过夜， 分别加入 AdGFP 及 AdHNFla培养 48h后抽提总 RNA， 制备 cDNA， real-time PCR检测各组肝纤维化相 关基因变化。 所用 PCR引物如表 5所示。
表 5
结果表明： AdHNFla+siRNA-SHPl 组与 AdHNFla+siRNA-NC组相比： a-SMA、 I
型胶原和 III型胶原 mRNA表达分别上调 58% (P < 0.05 )、 33% (P < 0.05 ) 和 122% CP < 0.05 )， 下调 SHP1表达后， HNFla抑制肝纤维化基因表达的作用明显下降， 说 明 SHP1表达是 HNFl a抑制肝纤维化的主要机制之一 （见附图 12 ) 。
实施例 7
HNFla上调 miR- 194表达，进而下调 miR- 194靶基因 SET及 STAT5表达，提高 SHP1 的催化活性。
1 原代培养大鼠 HSC 72h后， 取 Lipofectamin ΙΟμΙ, DMEM 5ml, miR- 194 mimics 200pmol转染 4-6h， 更换为含 10%FCS的 DMEM培养 48h及 72h， 收集蛋白及总 RNA， real-time PCR及 westen blot检测 miR- 194及其靶基因的表达。 PCR引物见如表 6所示。
表 6
结果表明： 感染 AdHNFla 48h及 72h后， miR- 194表达明显上升， miR- 194 mimics 转染原代 HSC 72h后，靶基因 SET及 STAT5蛋白表达明显下降。 HNF 1 α通过上调 miR- 194 抑制 SET蛋白表达， 激活 SET-PP2A-SHP1通路， 提高 SHP1活性， 并下调 STAT5表达， 抑制 JAK-STAT通路 （见附图 13、 14)。
2 原代培养大鼠 HSC 72h后， 取 Lipofectamin 10μ1， DMEM 5ml, miR- 194 inhibitor 200pmol转染 4-6h，更换为含 10%FCS的 DMEM培养过夜，分别加入 AdGFP及 AdHNFla 培养 48h， 提取总蛋白， western-blot检测 SET蛋白变化。
结果表明， AdHNFl a可明显下调 HSC SET蛋白表达， miR- 194 inhibitor抑制 miR- 194 后， SET蛋白表达明显上升， 逆向验证 HNFla通过上调 miR-194抑制 SET蛋白表达， 提高 SHP1活性 （见附图 14)。
SEQUENCE LISTING
〈110> 中国人民解放军第二军医大学
< 120> 肝细胞核因子 1 α治疗慢性肝病的用途及方法
〈130>
< 160> 33
< 170> Patentln vers ion
<210> 1
<211> 20
<212> DNA
<213> 人工序列
〈400> 1
ccatcctcaa agagctggag 20 <210> 2
<211> 19
<212> DNA
<213> 人工序列
〈400> 2
tgttgtgctg ctgcaggta 19 <210> 3
<211> 18
<212> DNA
<213> 人工序列
〈400> 3
catcctgcgt ctggacct 18 <210> 4
<211> 20
<212> DNA
<213> 人工序列
〈■> 4
gtacttgcgc tcaggaggag 20 <210> 5
<211> 20
<212> DNA
<213> 人工序列
〈■> 5
ttatgtgtga gggtggacga 20 <210> 6
<211> 20
<212> DNA
<213> 人工序列
〈■> 6
caagacccgg ttctcaatgt 20 <210> 7
<211> 20
<212> DNA
<213> 人工序列
/s/u i/Jinosld
<212> DNA
<213> 人工序列
〈■> 15
aggccaatgg caatgtaaag 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列
〈■> 16
tattggtggg tgaaacagca 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列
〈■> 17
ttcgac ； gctg acaagaagtg 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列
〈■> 18
agggge Lgtcc tcgtggtagt 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列
〈■> 19
aggccttcag aaaagccttc 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列
〈■> 20
gagctgcttg tccaggtttc 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列
〈■> 21
tgatgtccag aacggattca 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列
〈■> 22
gtgtatgctg ccatgtggac 20
<210> 23
<211> 20
<212> DNA
<213> 人工序列
〈■> 23
cagccaaact cccagatcat 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列
〈■> 24
ggcggacaga acataggtgt 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列
〈■> 25
gtgtggactt ggagccgaga 20
<210> 26
<211> 18
<212> DNA
<213> 人工序列
〈■> 26
tggagt gctg ggtggctg 18
<210> 27
<211> 20
<212> DNA
<213> 人工序列
〈■> 27
aagagg. agga agggttggaa 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列
〈■> 28
gaggaaagga ctgcaactcg 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列
〈■> 29
ccctgctcaa gaacgagaac
<210> 30
<211> 20
<212> DNA
<213> 人工序列
81
0Z91
09ST
OOST
08ετ
0Z£l ooq.ooSSq.oo ooo¾oo .oo§ oq.SSSq.oooq.
09ST ΒΒ%οοΒΒΒο%
OOST
ο π o .§ .oooooo
080Τ
0S0T
096 oooq.oq.oooS
006 ooooooSSSo
0 8
08Ζ
0ZL
099
009
0 S
08^
OZ
09C
οοε
08Τ ο%ΒΒοΒΒοΒΒ
09 < < <
εε 〈o o o oo 〉
οε
Z£ <00 >
τε 〈00 〉
οε 〈00
ZLlim/llOZSiD/lDd 9εζεπ z OAV
cagg†c†tca cctcagacac tgaggcct cc ag†gag†ccg ggcttcacac gccggcatc† 1680 caggccacca ccctccacgt ccccagccag gaccct gcca gcatccagca cctgcagccg 1740 gcccaccggc tcagcgccag ccccacagtg tcct ccagca gcctggtgct gtaccagagc 1800 tcagact cca gcaatggcca gagccacctg ctgccat cca accacagcgt catcgagacc 1860 ttcatct cca cccagatggc ctcttcctcc cagtaa 1896
Claims
1. 人 HNFla的基因序列在治疗慢性肝病中的用途。
2. 人 HNFla的基因序列在制备治疗慢性肝病药物中的用途。
3. 人 HNFla基因序列编码的蛋白在治疗慢性肝病中的用途。
4. 人 HNFla基因序列编码的蛋白在制备治疗慢性肝病药物中的用途。
5. 根据权利要求 1-4任一权利要求所述的用途， 其特征在于， 所述的慢性肝病包括 肝纤维化和肝硬化。
6. 一种用于治疗慢性肝病的药物组合物， 其特征在于， 所述的药物组合物包括人 HNFla的基因序列和 /或其编码的蛋白， 和 /或药用载体或赋形剂， 和 /或其他已知的治疗 慢性肝病药物。
7. 根据权利要求 6所述的药物组合物， 其特征在于， 所述的药物组合物通过口服， 肌内， 静脉内， 皮下， 局部， 经皮途径来传送。
8. 根据权利要求 6或 7所述的药物组合物， 其特征在于， 所述的慢性肝病包括肝纤 维化和肝硬化。
9. 一种治疗慢性肝病的方法， 其特征在于， 它包括将有效量的人 HNFla的基因序 列和 /或其编码的蛋白和 /或权利要求 6-8任一所述的药物组合物给药于患者。
10. 一种慢性肝病的基因治疗方法， 其特征在于， 它包括将 HNFla基因导入肝脏实 质细胞和间质细胞， 使之表达。
11. 根据权利要求 10 的慢性肝病的基因治疗方法， 其特征在于， 所述的将 HNFla 基因导入肝脏实质细胞和间质细胞的方法包括用质粒转染、 腺病毒或腺相关病毒介导。
12. 根据权利要求 9-11任一权利要求所述的方法， 其特征在于， 所述的慢性肝病包 括肝纤维化和肝硬化。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110043321.6 | 2011-02-23 | ||
| CN201110043321.6A CN102552935B (zh) | 2011-02-23 | 2011-02-23 | 肝细胞核因子1α治疗慢性肝病的用途 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2012113236A1 true WO2012113236A1 (zh) | 2012-08-30 |
Family
ID=46400383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2011/081172 Ceased WO2012113236A1 (zh) | 2011-02-23 | 2011-10-24 | 肝细胞核因子1α治疗慢性肝病的用途及方法 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN102552935B (zh) |
| WO (1) | WO2012113236A1 (zh) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105477644A (zh) * | 2014-09-18 | 2016-04-13 | 南京大学 | Pp2a拮抗剂在制备促进肝脏再生药物中的应用 |
| CN105251022A (zh) * | 2015-09-30 | 2016-01-20 | 中国人民解放军第二军医大学 | 叉头样转录因子foxa2在制备治疗慢性肝病药物中的应用 |
| CN109481681B (zh) * | 2018-10-30 | 2020-11-03 | 中国药科大学 | Perilipin1表达干预剂在制备抗肝纤维化药物中的应用 |
| CN109432431B (zh) | 2018-12-14 | 2020-06-30 | 中国药科大学 | 一种含有sumo抑制剂的组合物及应用 |
| CN115029347B (zh) * | 2022-05-11 | 2024-02-20 | 珠海中科先进技术研究院有限公司 | 识别和调控肝肾细胞纤维化的分子监测序列、重组质粒、抑制病毒 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004016813A2 (en) * | 2002-08-16 | 2004-02-26 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Hnf1 alpha as a tumor suppressor gene, and diagnostic and therapeutic applications thereof |
| CN101890153A (zh) * | 2009-03-10 | 2010-11-24 | 中国人民解放军第二军医大学 | 肝细胞核因子4α治疗慢性肝病 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311200A (zh) * | 2000-02-29 | 2001-09-05 | 复旦大学 | 一种新的多肽——人肝细胞核因子12和编码这种多肽的多核苷酸 |
| EP1890708A4 (en) * | 2005-06-01 | 2009-07-15 | Biotec Inst For Internat | GLUCOSE INDUCIBLE INSULIN EXPRESSION AND METHODS OF TREATING DIABETES |
-
2011
- 2011-02-23 CN CN201110043321.6A patent/CN102552935B/zh active Active
- 2011-10-24 WO PCT/CN2011/081172 patent/WO2012113236A1/zh not_active Ceased
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004016813A2 (en) * | 2002-08-16 | 2004-02-26 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Hnf1 alpha as a tumor suppressor gene, and diagnostic and therapeutic applications thereof |
| CN101890153A (zh) * | 2009-03-10 | 2010-11-24 | 中国人民解放军第二军医大学 | 肝细胞核因子4α治疗慢性肝病 |
Non-Patent Citations (5)
| Title |
|---|
| DUNCAN, T.O. ET AL.: "Control of pancreas and liver gene expression by HNF transcription factors", SCIENCE, vol. 303, 27 February 2004 (2004-02-27), pages 1378 - 1381 * |
| LOU, GUIYU ET AL.: "Construction of human HNF-la expression vector and its expression", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, vol. 29, no. 13, 31 January 2007 (2007-01-31), pages 1273 - 1275 * |
| QL ANDONG ET AL.: "Expression and significance of HNF-la in human hepatocellular carcinoma tissue", SHANDONG MEDICAL JOURNAL, vol. 49, no. 3, 31 December 2009 (2009-12-31), pages 96 - 97 * |
| WANG, SU ET AL.: "Regulation of hepatitis B virus transcription and replication by liver-enriched transcriptional factors", WORLD CHINESE JOURNAL OF DIGESTOLOGY, vol. 15, no. 11, 18 April 2007 (2007-04-18), pages 1237 - 1240 * |
| XIE, WEIFEN: "New strategy for the treatment of hepatic fibrosis", THE 10TH NATIONAL CONGRESS OF GASTROENTEROLOGY OF THE CHINESE MEDICAL ASSOCIATION, 20 November 2010 (2010-11-20), pages 46 - 48 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102552935A (zh) | 2012-07-11 |
| CN102552935B (zh) | 2015-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Alshoubaki et al. | Tregs delivered post-myocardial infarction adopt an injury-specific phenotype promoting cardiac repair via macrophages in mice | |
| Wang et al. | Dysregulation of miR-342-3p in plasma exosomes derived from convalescent AMI patients and its consequences on cardiac repair | |
| WO2016197592A1 (zh) | 一种长链非编码rna hnf1a-as1在制备治疗人体恶性实体瘤药物中的应用 | |
| WO2012113236A1 (zh) | 肝细胞核因子1α治疗慢性肝病的用途及方法 | |
| CN104066452A (zh) | 治疗和预防气道损伤的含mg53组合物及方法 | |
| Dong et al. | Wnt4 is crucial for cardiac repair by regulating mesenchymal-endothelial transition via the phospho-JNK/JNK | |
| Zheng et al. | The mmu_circ_003062, hsa_circ_0075663/miR-490-3p/CACNA1H axis mediates apoptosis in renal tubular cells in association with endoplasmic reticulum stress following ischemic acute kidney injury | |
| Liang et al. | Effects of miR-145-5p on cardiomyocyte proliferation and apoptosis, GIGYF1 expression and oxidative stress response in rats with myocardial ischemia-reperfusion | |
| Sharma et al. | Noncoding vault RNA1-1 impairs intestinal epithelial renewal and barrier function by interacting with CUG-binding protein 1 | |
| CN104998249A (zh) | Sip1及其衍生物在制备预防和治疗增生性瘢痕药物中的应用 | |
| CN104721811A (zh) | 多肽在制备防治脑胶质瘤药物中的应用 | |
| CN120459275A (zh) | Dnajb6在心肌梗死预防或治疗中的医药用途 | |
| CN108187029B (zh) | 白细胞免疫球蛋白样受体亚家族b成员4在制备预防、缓解和/或治疗心肌肥厚药物的应用 | |
| WO2012068936A1 (zh) | 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 | |
| Pradeep et al. | Novel approaches to determine the functional role of cardiomyocyte specific E3 ligase, Pellino-1 following myocardial infarction | |
| CN114277119B (zh) | 一种环状RNA circ-Arsb在制备防治骨质疏松产品中的应用 | |
| CN105251020A (zh) | 泛素特异性蛋白酶4（usp4）在治疗心肌肥厚中的功能及应用 | |
| CN116327946A (zh) | Prrx1在制备糖尿病肾病防治药物中的应用 | |
| CN113769095B (zh) | 神经调节素1促进皮肤创面修复的方法及其应用 | |
| CN118356498B (zh) | Reps2的表达抑制剂在制备防治1,2-二氯乙烷引起的大脑中毒性神经炎症的药物中的应用 | |
| CN116769898B (zh) | 过表达Nudt12在制备治疗急性心肌梗死药物中的应用 | |
| CN119015428B (zh) | 一种敲低tmem91诱导肾癌细胞死亡和肿瘤免疫治疗肾癌的应用 | |
| CN119700977B (zh) | Psmg4基因在制备心脏疾病治疗药物及相关诊断产品中的应用 | |
| CN104059887A (zh) | 长链非编码RNA Ovol2-AS的应用 | |
| CN117100742B (zh) | Rhodiolin在制备治疗人甲状腺乳头状癌药物中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11859175 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 11859175 Country of ref document: EP Kind code of ref document: A1 |