【作者】 陈思文；

【导师】 谢渭芬； 陈岳祥；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 研究背景及目的肝纤维化是肝脏对不同病因所致的慢性损伤共有的过修复反应,其病理特征是肝脏细胞外基质（extracellular matrix,ECM）过度沉积及分布异常。肝星状细胞（hepaticstellate cell,HSC）是纤维化肝脏中产生ECM的最主要细胞,HSC的活化是肝纤维化发生的核心环节,设法抑制HSC激活、增殖与迁移是肝纤维化治疗的重要对策。血小板衍生生长因子（platelet-derived growth factor,PDGF）是最强的促进HSC增殖的细胞因子,由A、B、C、D四条高度同源的多肽链组成,其中A、B亚基互相组合可以表现为AA、BB、AB三种分子形式。PDGF受体（platelet-derived growthfactor receptor,PDGFR）由两种亚单位α及β构成,静止的HSC表面只有α亚单位,HSC活化后才表达β亚单位,而且以β亚单位为主,与PDGF-BB具有较强的亲和力。PDGF-BB以及PDGFR-β在肝纤维化过程中发挥重要作用,设法抑制PDGF及其受体表达,阻断PDGF细胞内信号转导是抗肝纤维化的治疗策略之一。RNA干扰（RNA interference,RNAi）是一种介导转录后基因沉默的强大工具。它是通过小干扰RNA（small interfering RNA,siRNA）降解生物体或细胞内同源mRNA,导致序列特异性同源基因沉默,使细胞表现出特定基因缺失表型的过程。本研究应用RNAi技术沉默PDGFR-β表达,阻断PDGF的作用,观察在体外对HSC生物学特性及大鼠实验性肝纤维化的影响;并构建胶质纤维酸性蛋白（glial fibrillaryacidic protein,GFAP）基因启动子介导的PDGFR-βsiRNA表达载体,观察其靶向治疗大鼠实验性肝纤维化的疗效。实验方法1、筛选高效沉默PDGFR-β基因表达的siRNA设计3对针对大鼠PDGFR-βmRNA开放阅读框的siRNA分子,用脂质体转染法将siRNA转染HSC-T6细胞,RT-PCR法和Western Blot法检测HSC-T6中PDGFR-βmRNA和蛋白的表达,筛选高效沉默PDGFR-β基因表达的siRNA。2、siRNA干扰PDGFR-β表达对HSC-T6生物学特性的影响PDGFR-βsiRNA转染HSC-T6后进行PDGF-BB刺激,48小时后收集细胞,用RT-PCR法检测细胞中与纤维化相关的基因表达变化;用BrdU掺入试验检测细胞增殖能力的变化;用Annexin IV/PI染色法检测凋亡细胞数目的变化;用Western Blot检测ERK的磷酸化水平,RT-PCR检测c-fos的表达水平来观察PDGFR-βsiRNA对HSC细胞内MAPK信号转导通路的影响。3、构建表达PDGFR-βsiRNA的质粒载体siRNA分子在体内具有半衰期短、不稳定等缺点,为了观察PDGFR-βsiRNA在体内对动物实验性肝纤维化的影响,我们将PDGFR-βsiRNA对应的短发夹RNA（shorthairpin RNA,shRNA）插入商品化质粒pCMR30的miR30结构中,构建表达PDGFR-βshRNA的质粒载体（pCMV-shRNA-LacZ）,以CMV基因启动子启动下游PDGFR-βshRNA的表达,β-半乳糖苷酶（β-galactosidase,β-gal）基因作为报告基因。另外构建阴性对照质粒（pCMV-NC-LacZ）,以CMV基因启动子启动下游对PDGFR-β基因无干扰作用的小RNA的表达。4、pCMV-shRNA-LacZ对动物实验性肝纤维化的影响构建胆总管结扎（bile duct ligation,BDL）和二甲基亚硝胺（dimethylnitrosamine,DMN）大鼠肝纤维化模型,将pCMV-shRNA-LacZ和pCMV-NC-LacZ质粒在造模前1天通过尾静脉高压注射至大鼠体内。为了保证PDGFR-βshRNA在大鼠体内的稳定表达,以后每5天重新注射一次。造模结束后取血检测血清肝功能,取肝组织用免疫组化法和Western Blot方法检测α平滑肌激动蛋白（α-smooth muscle actin,α-SMA）和PDGFR-β蛋白表达水平,用HE染色和Masson三色染色法观察肝脏的病理变化。5、构建含胶质纤维酸性蛋白基因启动子的PDGFR-βshRNA质粒表达载体质粒pGfa2-clac包含人的GFAP基因启动子。用PCR克隆的方法将pGfa2-clac中的GFAP基因启动子克隆出来,插入pCMV-shRNA-LacZ的酶切位点之间,取代CMV基因启动子,命名为pGfa-shRNA-LacZ。同理构建阴性对照质粒,命名为pGfa-NC-LacZ。6、体内验证pGfa-shRNA-LacZ的HSC靶向性由于在正常肝脏中HSC的数量较少,为了增加肝脏中HSC的数目以便于我们观察,我们用四氯化碳（carbon tetrachloride,CCl₄）腹腔内注射制备大鼠急性肝损伤模型,用BDL方法制备大鼠慢性肝损伤模型。将pGfa-shRNA-LacZ和pCMV-shRNA-LacZ分别通过尾静脉高压注射入模型大鼠体内,24小时后处死小鼠,取肝组织用免疫荧光染色和激光共聚焦显微镜技术检测表达β-gal的细胞在两种模型大鼠肝组织中的分布,并观察肝脏中β-gal蛋白和GFAP蛋白、β-gal蛋白和α-SMA蛋白的细胞共定位情况。7、pGfa-shRNA-LacZ对大鼠实验性肝纤维化的影响构建BDL大鼠肝纤维化模型,将构建好的pGfa-shRNA-LacZ及其相应的阴性对照质粒pGfa-NC-LacZ在造模前1天通过尾静脉高压注射至各组大鼠体内。造模结束后取血检测血清肝功能,取肝组织用免疫组化法检测α-SMA和PDGFR-β蛋白表达水平,用HE染色和Masson三色染色法观察肝脏的病理变化,测定组织羟脯氨酸含量来定量评价PDGFR-βshRNA对大鼠肝纤维化的影响。实验结果1、针对PDGFR-β基因957位点的siRNA能高效沉默靶基因的表达我们合成了针对PDGFR-β基因不同位点的小干扰RNA及其阴性对照RNA,分别命名为PDGFR-βsiRNA₂₅₉₇、PDGFR-βsiRNA₂₆₈₇、PDGFR-βsiRNA₉₅₇和PDGFR-βsiRNA_con。RT-PCR结果显示三对siRNA均能不同程度抑制PDGFR-βmRNA的表达,其中以PDGFR-βsiRNA₉₅₇的基因沉默效率最高,达到（72±4.20）%,且转染HSC-T6细胞72小时的基因沉默效率明显高于转染24（40±3.14）%和48小时（55±4.67）%,Western Blot检测显示PDGFR-βsiRNA₉₅₇转染HSC-T6细胞72小时后PDGFR-β蛋白水平降低约（60±7.59）%。以后的实验均用PDGFR-βsiRNA₉₅₇进行。2、PDGFR-βsiRNA能抑制HSC-T6细胞的活化和胶原合成RT-PCR和Western Blot结果表明PDGF-BB能提高HSC-T6细胞的活化水平,使α-SMA的基因表达水平升高,而PDGFR-βsiRNA能抑制PDGF-BB的这种效应,使α-SMA的基因表达水平降低约（50±2.20）%;此外,RT-PCR结果还提示PDGF-BB能明显促进HSC-T6 PDGF-B链、结缔组织生长因子（connective tissue growth factor,CTGF）、金属蛋白酶组织抑制因子-1（Tissue Inhibitor of Metalloproteinase-1,TIMP-1）和I型胶原基因表达,而PDGFR-βsiRNA能使PDGF-B、CTGF、TIMP-1和I型胶原基因表达分别下降约（43±3.40）%、（50±5.10）%、（45±1.80）%和（40±2.40）%。但无论是PDGF-BB还是PDGFR-βsiRNA对转化生长因子（Transforming GrowthFactor-β₁,TGF-β₁）和基质金属蛋白酶-2（Matrix Metalloproteinases-2,MMP-2）的基因表达均无明显影响。3、PDGFR-βsiRNA抑制HSC-T6细胞的增殖BrdU掺入试验表明PDGF-BB明显促进HSC细胞增殖,而PDGFR-βsiRNA能明显抑制PDGF-BB的促增殖效应（流式细胞法显示抑制40%的细胞增殖,细胞免疫荧光法显示抑制35%的细胞增殖）。Western Blot和RT-PCR检测显示PDGF-BB能激活细胞内MAPK信号转导通路,使ERK蛋白磷酸化和c-fos的基因表达明显增加,而PDGF-BB的这种效应被PDGFR-βsiRNA明显抑制,其中c-fos的基因表达减少约35%,ERK蛋白磷酸化减少约50%。Annexin/PI流式细胞检测表明无论PDGF-BB还是PDGFR-βsiRNA对HSC凋亡均无明显影响。4、pCMV-shRNA-LacZ能改善肝纤维化大鼠肝功能,减轻其肝纤维化程度血清肝功能检测显示BDL模型大鼠的血清丙氨酸氨基转移酶（alanineaminotransferase,ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase,AST）、总胆红素（total bilirubin,TB）和间接胆红素（indirect bilirubin,IDB）水平明显高于假手术组和正常组,而pCMV-shRNA-LacZ处理后转氨酶和胆红素水平均明显降低。DMN模型大鼠的ALT、AST和TB水平明显高于正常组大鼠,而pCMV-shRNA-LacZ处理后转氨酶水平和胆红素均明显下降。HE染色和Masson染色显示pCMV-shRNA-LacZ能明显减轻BDL和DMN引起的大鼠肝损伤和肝纤维化程度。Western Blot检测和免疫组化染色显示BDL模型组和DMN模型组肝脏的PDGFR-β蛋白和α-SMA蛋白水平明显升高,而pCMV-shRNA-LacZ处理后两种蛋白的表达水平均明显降低。5、GFAP基因启动子能驱动下游基因在HSC特异表达免疫荧光染色显示在CCl₄介导的急性肝损伤和BDL介导的慢性肝损伤模型中,无论是pCMV-shRNA-LacZ处理组还是pGfa-shRNA-LacZ处理组,大鼠肝脏中β-gal蛋白均主要表达在中央静脉周围及汇管区的细胞中。免疫荧光双重染色显示:pCMV-shRNA-LacZ处理组大鼠的β-gal蛋白不仅表达在GFAP蛋白表达阳性的细胞中,还表达在GFAP蛋白表达阴性的细胞中;而pGfa-shRNA-LacZ处理组大鼠肝脏中β-gal蛋白仅表达在GFAP蛋白表达阳性的细胞中。β-gal是我们构建的质粒的报告基因,GFAP是HSC特异表达的标志蛋白,提示GFAP基因启动子能介导下游基因在HSC特异表达。6、pGfa-shRNA-LacZ改善BDL大鼠的肝功能,减轻肝纤维化程度血清生化检测显示BDL模型组大鼠转氨酶（ALT和AST）和胆红素（TB和IDB）水平明显升高,而pGfa-shRNA-LacZ处理后升高的转氨酶和胆红素水平明显降低。α-SMA和PDGFR-β免疫组化染色以及Masson染色显示单纯BDL大鼠的PDGFR-β蛋白和α-SMA蛋白表达水平明显强于正常大鼠,并出现明显的胶原沉积和胆管增生,而pGfa-shRNA-LacZ处理后PDGFR-β蛋白和α-SMA蛋白表达水平均有明显降低,肝纤维化和胆管增生情况均有改善。BDL大鼠肝组织羟脯氨酸含量较正常大鼠增加约2倍,而pGfa-shRNA-LacZ可使BDL大鼠的肝组织羟脯氨酸含量下降约45%。结论1、用RNAi技术抑制HSC中PDGFR-β基因表达,阻断PDGFR-β与PDGF-BB的结合,可阻断细胞内MAPK信号转导通路进而抑制细胞的增殖,并可显著抑制HSC的活化和胶原分泌;2、用RNAi技术抑制PDGFR-β基因表达,可明显改善BDL和DMN诱导的肝纤维化模型大鼠的肝功能,减轻肝纤维化程度;3、GFAP基因启动子可介导下游基因特异性地在HSC中表达;4、GFAP基因启动子能介导针对PDGFR-β基因的小干扰RNA特异性地表达在HSC中,并能有效改善BDL大鼠肝功能,减轻肝纤维化程度。更多还原

【Abstract】 Background and ObjectiveHepatic fibrosis,characterized by the overproduction and accumulation of hepatic extracellular matrix（ECM）,was the common and important pathological changes in the chronic liver diseases,through which the chronic hepatitis developed into cirrhosis.It was acknowledged that hepatic cirrhosis couldn’t be reversed,while hepatic fibrosis could. Hepatic stellate cell（HSC） was the main resource of hepatic ECM and the cellular basis for hepatic fibrosis.The activation of HSC was recognized as the key event of the pathogenesis of hepatic fibrosis.Inhibiting the activation,proliferation and migration of HSCs is one of the most important strategies to prevent the hepatic fibrogenesis.In fibrotic liver,PDGF is the most potent mitogen for HSCs.PDGF binds to structurally related tyrosine kinase receptors that is a homodimer or heterodimer ofαorβsubunits.PDGFR-αis constitutively expressed in quiescent HSCs,whereas PDGFR-βis acquired as the activation of HSCs,the major cell type expressing PDGFR-βin liver. PDGF and PDGFR-βplay important roles in hepatic fibrogenesis,therefore,targeting to PDGF,PDGF receptor and molecules related to PDGF intracellular signaling transduction pathway is attractive for therapeutic intervention in hepatic fibrosis.RNAi（RNA interference） is one of the gene-silencing technologies,which is considered as an antisense mechanism of action that utilizes double strand RNase to promote hydrolysis of target RNA.In this study,we inhibited the PDGFR-βexpression by small interference RNA（siRNA） to block the binding of PDGF,and investigated its effect on biological characteristics of HSCs in vitro.Furthermore,we constructed a PDGFR-βshRNA（short hairpin RNA） expression vector and investigated its antifibrogenic effect on experimental hepatic fibrosis.To avoid the non-specific interference of PDGFR-βgene expression in other tissues and cells,we used cell-specific glial fibrillary acidic protein （GFAP） promoter to drive the expression of PDGFR-βshRNA in HSCs of liver,and investigated its antifibrogenic effect in vivo.Methods1.Screening for the PDGFR-βsiRNA with high gene-silencing efficacy The targeted region of siRNA was the coding sequence of PDGFR-βcDNA.We selected 3 pairs 21-nucleotide RNAs and 1 pairs non-specific nucleotide RNA.Transient transfection of siRNA by lipofection method was carried out in the rat HSC-T6 cell line, whose phenotype was activated HSC.Reverse transcription-polymerase chain reaction （RT-PCR） and Western Blot were used to detect the expressions of PDGFR-β.2.Investigate the effect of PDGFR-βsiRNA on biological characteristics of HSCCells were transfected with PDGFR-βsiRNA and were stimulated by PDGF-BB,and then were collected.RT-PCR was used to detect the expression of fibrogenesis- related genes.BrdU incorporation was performed to determine cell proliferation.Annexin V/PI staining method was utilized to measure cell apoptosis.Mitogen-activated protein kinases （MAPK） signaling transduction pathway was necessary for the HSC proliferation induced by PDGF,and extracellular-signal- related kinase（ERK） phosphorylation and c-fos expression were the two important processes of MAPK signaling transduction pathway, which were detected by Western Blot and RT-PCR in this study.3.Construct plasmid expressing PDGFR-βshRNAThe disadvantages of chemically synthesized siRNA,such as instability and easy to be degraded by serum nucleases restrict its application in vivo.To investigate the effect of PDGFR-βsiRNA on experimental hepatic fibrosis,we inserted PDGFR-βshRNA into the miR30-based shRNA expression system of pCMR30 Vector to generate pCMV-shRNA-LacZ,in which PDGFR-βshRNA was driven by CMV promoter andβ-galactosidase（β-gal） was the reporter gene.As a negative control,we generated pCMV-NC-LacZ with miR30-based PDGFR-βshRNA in pCMV-shRNA- LacZ replaced by negative control RNA.4.Investigate the effect of pCMV-shRNA-LacZ on experimental hepatic fibrosisRats model of hepatic fibrosis were induced by common bile duct ligation（BDL） and dimethylnitrosamine（DMN） treatment,pCMV-shRNA-LacZ solutions were injected once every 5 days with hydrodynamics-based transfection method.to induce stable expression of PDGFR-βshRNA.After the rats models were constructed successfully,rats were sacrificed,serum samples were collected for biochemical tests and liver tissues were taken out for histology and Western Blot analysis.5.Construct plasmid expressing PDGFR-βshRNA driven by GFAP promoterThe GFAP promoter in the plasmid named pGfa2-clac was PCR-cloned into the EcoRI and BamHI site and replaced the CMV promoter of pCMV-shRNA-LacZ to generate pGfa-shRNA-LacZ,pGfa-NC-LacZ was generated by replacement of PDGFR-βshRNA in pGfa-shRNA-LacZ with non-specific nucleotide RNA as a negative control.All the constructed plasmids were verified by PCR and sequencing.6.Locate LacZ gene expression in acute and chronic liver injury rats modelTo locate LacZ reporter gene expression driven by GFAP promoter in liver,we injected pGfa-shRNA-LacZ or pCMV-shRNA-LacZ by hydrodynamics-based transfection method into normal rats,acute liver injury rats induced by CCl4 and chronic liver injury rats induced by BDL.Twenty four hours after plasmids injection,all rats were anesthetized and perfused,livers were taken out and postfixed.Immunofluorescence and laser scanning confocal techniques were used to observe the distribution ofβ-gal protein,and the colocalization ofβ-gal andα-SMA orβ-gal and GFAP.7.Investigate the effect of pGfa-shRNA-LacZ on experimental hepatic fibrosisHepatic fibrosis was induced by BDL in rats,pGfa-shRNA-LacZ solutions were injected once every 5 days with hydrodynamics-based transfection method.to induce stable expression of PDGFR-βshRNA.After the rats models were constructed successfully,rats were sacrificed,serum samples were collected for biochemical tests and liver tissues were taken out for histology and hydroxyproline content test.Results1.PDGFR-βsiRNA could downregulate PDGFR-βexpressionAll the 3 pairs of siRNAs could suppress PDGFR-βmRNA expression,especially siRNA₉₅₇,which had the highest gene-silencing efficacy.RT-PCR results showed that siRNA₉₅₇ downregulated the PDGFR-βmRNA expression by about（40±3.14）%, （55±4.67）%,（72±4.20）%and（55±6.19）%at 24h,48h,72h and 96h,respectively.In addition,PDGFR-βprotein expression was downregulated by about（33±4.51）%, （47±5.72）%,（60±7.59）%and（82±7.13）%at 24h,48h,72h and 96h,respectively.2.PDGFR-βsiRNA could suppress the activation and proliferation of HSCThe results of RT-PCR and Western Blot revealed that theα-SMA expression in HSCs was elevated significantly after PDGF-BB stimulation,which was impeded by PDGFR-βsiRNA.Furthermore,the upregulation of PDGF-B,CTGF,TIMP-1 and collagen typeⅠmRNA induced by PDGF-BB was eliminated by siRNA₉₅₇.However,the expressions of TGF-β₁ and MMP-2 mRNA in HSCs were not affected by PDGFR-βsiRNA₉₅₇ and/or PDGF-BB. 3.PDGFR-βsiRNA inhibited HSC proliferation induced by PDGF-BB,but did not affect the apoptosis of HSCTo investigate the effect of siRNA₉₅₇ on HSC proliferation,BrdU incorporation was used and analyzed by immunofluorescence and flow cytometry.Results showed that PDGF-BB significantly promoted the proliferation of HSC,which was inhibited by PDGFR-βsiRNA₉₅₇.Additionally,Western blot analysis revealed that the phosphorylation of ERK increased significantly after PDGF-BB stimulation,which was impeded by siRNA₉₅₇,RT-PCR analysis demonstrated that siRNA₉₅₇ suppressed the upregulation of transcription factor c-fos expression induced by PDGF-BB.Annexin V/PI flow cytometry analysis showed that neither PDGF-BB nor siRNA₉₅₇ could affect the percentage of apoptotic HSC.4.pCMV-shRNA-LacZ relieved liver injury and ameliorated hepatic fibrosis in ratsIn DMN animal model,results of serum biochemical tests revealed that serum total bilirubin（TB）,alanine aminotransferase（ALT） and aspartate aminotransferase（AST） levels were elevated significantly in DMN control rats compared with the normal rats, which were reduced by pCMV-shRNA-LacZ treatment.In BDL animal model,serum TB, direct bilirubin（DB）,ALT and AST levels were elevated significantly in BDL control group compared with the sham-operated group,which was eliminated by pCMV-shRNA-LacZ treatment.HE staining and Masson’s trichrome staining of liver sections showed that liver injury and hepatic fibrosis in both rats models were ameliorated by pCMV-shRNA-LacZ treatment.Western Blot analysis indicated that both PDGFR-βandα-SMA protein levels were elevated significantly in DMN treatment rats and BDL rats, and remarkably reduced by pCMV-shRNA-LacZ treatment.The results were confirmed by immunohistochemical examination.5.GFAP promoter targets LacZ express into HSCImmunofluorescence analysis indicated thatβ-gal-positive cells were all distributed in the pericentral and portal area of hepatic lobule in normal rats,acute liver injury rats and chronic liver injury rats for both pCMV-shRNA-LacZ and pGfa-shRNA-LacZ treatment. The results also revealed thatβ-gal proteins were detected in both hepatocytes and HSC, which were positive forα-SMA and/or GFAP staining in the liver treated with pCMV-shRNA-LacZ.While in the liver treated with pGfa-shRNA-LacZ,β-gal proteins were co-expressed with GFAP and/orα-SMA,and were located in the perisinusoidal space, suggesting that GFAP promoter targeted the reporter gene LacZ specifically expressing in HSC and CMV promoter directed LacZ expressing in not only HSCs,but also hepatocytes.6.pGfa-shRNA-LacZ relieved liver injury and ameliorated hepatic fibrosis in ratsBiochemical tests indicated that the elevated serum levels of TB,indirect bilirubin （IDB）,ALT and AST induced by BDL could be markedly reduced by pGfa- shRNA-LacZ treatment.Immunohistochemical and Masson’s trichrome staining of liver sections showed significant collagen deposition and bile duct hyperplasia in BDL rats,and increased protein expressions of PDGFR-βandα-SMA were also observed in them,while both the hepatic fibrosis and the increased expressions of PDGFR-βandα-SMA protein were significantly relieved by pGfa-shRNA-LacZ treatment.In addition,the hydroxyproline content increased more than 2-fold in BDL rats compared with the sham-operated rats,while pGfa-shRNA-LacZ treatment decreased about half of the hydroxyproline content in BDL rats.Conclusion1.siRNA against PDGFR-βcould block the binding of PDGF,inhibit the MAPK signaling transduction pathway and suppress HSC proliferation consequently.Furthermore, the activation and ECM production of HSC stimulated by PDGF-BB could also be inhibited by PDGFR-βsiRNA.2.PDGFR-βsiRNA is active as an antifibrogenic gene therapeutic method able to reduce the expression of PDGFR-βprotein and ameliorated liver injury and hepatic fibrosis induced by BDL and DMN.3.GFAP promoter was able to target genes downstream express into HSC.4.PDGFR-βshRNA driven by GFAP promoter could be targeted into HSC,and could inhibit the activation of HSC,relieve liver injury and hepatic fibrosis induced by BDL.更多还原