【作者】 胡平方；

【导师】 谢渭芬； 林勇； 张新； 张俊平； 陈岳祥；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 [研究背景及目的]肝纤维化(hepatic fibrosis)的特征性病理改变是肝内细胞外基质(extracellular matrix, ECM)的过多沉积。减少ECM沉积、促进ECM降解是治疗肝纤维化的关键环节。由尿激酶型纤溶酶原激活剂(urokinase-type plasminogen activator, uPA)、纤溶酶(plasmin)、纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor-1, PAI-1)和基质金属蛋白酶(matrix metal loproteinases, MMPs)构成的反应体系是调节ECM降解的主要途径之一,在肝纤维化形成和发展中起重要作用。uPA位于纤维化调控的上游,可激活纤溶酶原形成纤溶酶,后者可进一步活化MMPs;纤溶酶和活化的MMPs均可降解胶原、蛋白多糖等ECM成分。PAI-1可与uPA结合,抑制uPA对纤溶酶原的激活作用,从而促进ECM沉积和肝纤维化发展。正常生理情况下,体内uPA和PAI-1表达处于平衡状态,而在肝纤维化发生过程中,uPA活性逐渐降低,PAI-1表达则不断增高,过多表达的PAI-1通过与uPA结合抑制其纤溶激活作用,进而导致uPA、纤溶酶和MMPs系统活性下调,ECM沉积明显增加,加重肝纤维化程度。本科室前期研究表明,在肝纤维化过程中上调uPA表达可以改善肝纤维化进程；此外,近期多项研究结果还显示,PAI-1基因敲除小鼠对纤维化形成具有保护作用,而PAI-1过表达小鼠可加重纤维化的发生发展。因此,我们设想通过下调肝纤维化过程中PAI-1的表达可能达到治疗肝纤维化的目的。小干扰RNA (small interfering RNA, siRNA)技术是以转录后调控为目的的新型基因调控与治疗技术。通过合成反义小分子RNA特异性抑制靶基因的转录后表达。RNA干扰(RNA interfering, RNAi)具有操作简单,可调控性和靶向性强、高效抑制靶基因表达等优点。本研究以肝纤维化过程中上调的PAI-1基因作为靶点,利用siRNA技术,通过体外、体内实验研究PAI-1 siRNA对实验性肝纤维化的治疗效果,为抗肝纤维化治疗提供新的靶点和途径。[实验方法]一、筛选PAI-1 siRNA应用RNAi设计软件,设计并合成针对PAI-1 mRNA 219、559、1061和2665位点的4对21核苷酸(nt) siRNA,分别转染大鼠肝星状细胞(hepatic stellate cell, HSC)株HSC-T6。以转染非特异siRNA (NC siRNA,与PAI-1 mRNA无同源性的21 ntsiRNA)作为阴性对照,应用实时定量RT-PCR (real-time reverse transcription polymerase chain reaction, real time RT-PCR)和Western blot方法检测四对siRNA分子对PAI-1基因及蛋白水平的阻抑效率,筛选出针对PAI-1 mRNA高效的siRNA片段。二、PAI-1 siRNA对HSC-T6生物学特性的影响通过细胞悬液转染以及构建稳定株两种方法将筛选出的PAI-1 siRNA转染至HSC-T6内,应用real time RT-PCR法检测肝纤维化基因mRNA表达；酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定HSC-T6上清Ⅰ、Ⅲ型胶原含量；四甲基偶氮唑盐(methyl thiazolyl tetrazolium, MTT)法测定细胞增殖变化；流式细胞术检测分析细胞周期等生物学特性的影响。三、针对PAI-1 siRNA的腺病毒载体——AdshPAI的构建以及鉴定合成针对219位点的PAI shRNA片段,利用pSuper载体的H1启动子,体外连接构建重组腺病毒质粒pAdshPAI,经293细胞包装后得到复制缺陷型重组腺病毒AdshPAI,同法构建对照病毒AdNC(与PAI-1 mRNA无同源性的NC shRNA)和表达绿色荧光蛋白(green fluorescent protein, GFP)病毒AdGFP。将AdshPAI和AdNC分别体外感染HSC-T6,以RT-PCR法检测AdshPAI对PAI-1的抑制效果。四、AdshPAI体内导入对实验性肝纤维化的治疗作用分别构建胆总管结扎(bile duct ligation, BDL)和二甲基亚硝胺(dimethylnitrosamine, DMN)两种大鼠肝纤维化模型。在BDL模型中,将雄性SD大鼠随机分为4组,每组10只。分设假手术组、模型对照组、病毒AdNC对照组和AdshPAI导入组。钝性分离并结扎胆总管,制备BDL肝纤维化模型。于术前48 h AdNC组和AdshPAI组分别经尾静脉导入4×109pfu／只AdNC或AdshPAI。于手术后第21 d处死大鼠。在DMN模型中,将雄性SD大鼠随机分为4组,每组8只。第1组为正常对照组；第2-4组为肝纤维化模型组,予1%DMN以10μg/kg腹腔注射,每周连续注射3次,共注射4 w。其中第2组设为模型对照组；第3组为对照病毒AdNC组,第4组为AdshPAI治疗组。于DMN注射后2 w末AdNC组和AdshPAI组分别经尾静脉导入4×109pfu／只AdNC或AdshPAI至大鼠体内。于DMN注射后第4 w末处死大鼠。分别采用组织病理方法观察各组大鼠肝纤维化程度分级改变；肝组织切片采用Masson染色和天狼猩红(sirius red)染色分析胶原沉积变化；免疫组织化学法测定PAI-1、平滑肌肌动蛋白-α(a-smooth muscle actin,α-SMA)、转化生长因子-β1 (transforming growth factor-β1, TGF-β1)、MMP-9、TIMP-1、Brdu和PCNA表达,图象分析仪半定量分析；real-time RT-PCR方法检测肝组织中PAI-1、α-SMA、TGF-β1、Ⅰ型胶原、smad3、smad7、uPA、uPAR、tPA、MMP-2、MMP-9、MMP-13和TIMP-1 mRNA水平；组织匀浆酸水解方法测定肝组织中羟脯氨酸水平；末端转移酶标记技术(TUNEL法)检测肝组织中细胞凋亡情况。五、统计学处理数据采用SPSS 11.0统计软件包进行分析,结果以X+S表示。多组间采用One-Way ANOVA分析,方差非齐性则采用非参数检验；细胞周期分析采用χ2检验。P<0.05为差异有显著性,P<0.01为差异有非常显著性。[实验结果]一、筛选PAI-1 siRNART-PCR和Western blot结果表明,219siRNA对PAI-1表达有较好的抑制效果,利用细胞悬液瞬时转染方法转染24 h和48 h后HSC PAI-1 mRNA表达较对照组明显下调(P<0.05); Western blot结果提示,转染48 h后对蛋白的抑制效率可达到85%以上(P<0.05)。559和2665位点siRNA对PAI-1 mRNA表达抑制效率分别为72%和66%,蛋白水平抑制效率分别为57%和63%(P<0.05),而1061位点则无明显基因沉默效果。二、PAI-1 siRNA对HSC-T6生物学特性的影响Real time RT-PCR法检测转染219 siRNA和559 siRNA组Ⅰ、Ⅲ型胶原、α-SMA、TGF-β1和TIMP-1 mRNA表达明显低于对照组(P<0.05),培养上清液中Ⅰ、Ⅲ型胶原含量较对照组显著减少(P<0.05)。MTT法动态观察HSC-T6转染219 siRNA的细胞增殖明显慢于对照组(P<0.05)。流式细胞术检测结果显示,转染219 siRNA的HSC-T6 G0/G1期时相细胞数较对照组明显增高(57.61±0.19% vs.45.49±0.93%,P<0.05),S期细胞数百分比明显低于对照组(30.29±2.17% vs.40.68±1.65%,P<0.05),表明沉默PAI-1可诱导HSC-T6周期静止,抑制其增殖。三、AdshPAI的构建和鉴定利用NotⅠ/SalⅠ酶切质粒pSuper-shPAI得到Hl+shPAI片段,与质粒pShuttle体外连接获得穿梭质粒pShuttle-shPAI,同源重组后筛选获得pAdshPAI;经293细胞包装并反复感染、冻融,获得1×1010pfu/ml滴度的AdshPAI; AdshPAI体外感染HSC-T62d后,RT-PCR检测](?)AI-1 mRNA表达抑制效率可达90%以上,证实病毒构建成功并能在细胞水平高效抑制PAI-1表达。四、AdshPAI对实验性肝纤维化的治疗作用1.AdshPAI对PAI-1表达的抑制作用：BDL和DMN大鼠肝组织real time RT-PCR结果显示,纤维化肝组织中PAI-1表达较正常肝脏分别上调6.69±0.15倍和2.62±0.08倍(P<0.01), AdshPAI治疗后PAI-1表达较对照组明显下调(P<0.01)；免疫组化结果表明AdshPAI治疗后PAI-1蛋白水平较模型对照组和AdNC组亦明显下调。2.AdshPAI对胶原沉积的影响：AdshPAI导入组肝组织羟脯氨酸含量较AdNC组明显减少(BDL:205.97±4.68μg/g vs.271.33±10.71μg/g; DMN:201.15±1.51μg/g vs. 303.20±15.11μg/g,P<0.05);胶原染色面积较对照组亦明显减少(P<0.05)。3. AdshPAI对胶原合成的影响：real time RT-PCR结果显示纤维化肝组织中Ⅰ型胶原、α-SMA、TGF-β1和smad3 mRNA水平较正常肝脏均明显上调(P<0.01), AdshPAI治疗后与病毒对照组相比均显著下调(P<0.05);α-SMA和TGF-β1免疫组化结果亦表明AdshPAI治疗后较对照组显著下调。4. AdshPAI对纤溶系统的影响：AdshPAI治疗后较AdNC组MMP9和MMP13mRNA水平明显上调,TIMP-1 mRNA水平明显下调(P<0.01),而对tPA、uPA、uPAR和MMP2 mRNA表达无明显影响；免疫组化结果显示,与AdNC组相比,AdshPAI治疗后MMP9表达明显上调,而TIMP-1表达则明显下调。5. AdshPAI对肝细胞增殖和凋亡的影响：Brdu和PCNA免疫组化结果表明,AdshPAI治疗后肝细胞增殖较AdNC组增加约5倍(P<0.01); PCNA免疫组化结果显示治疗组肝脏可见大量双核肝细胞和有丝分裂相肝细胞；TUNEL染色结果显示对照组在汇管区和胶原沉积区域周围可见大量肝细胞凋亡,AdshPAI治疗后肝细胞凋亡明显减少(P<0.05)。[结论]1.针对大鼠PAI-1 mRNA的219 siRNA,对靶基因有较好的基因沉默效率,抑制效率可达85%。2. siRNA沉默PAI-1可显著抑制HSC-T6活化、增殖,合成及分泌胶原。3.在肝纤维化发生发展过程中,PAI-1表达明显上调,AdshPAI治疗可下调PAI-1表达。4. AdshPAI治疗可促进胶原降解,减少肝纤维化大鼠肝内胶原沉积,抑制肝纤维化发展。5. AdshPAI治疗后可促进肝细胞增殖,抑制肝细胞凋亡。更多还原

【Abstract】 [Background and Objective]Hepatic fibrosis is characterized by the accumulation of excess extracellular matrix (ECM), regardless of the underlying etiology. Decreasing ECM deposition and enhancing its degradation may prove particularly valuable for the treatment of hepatic fibrosis. The plasminogen activator (PA)/plasmin system, including urokinase-type plasminogen activator (uPA), plasmin, plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinases (MMPs) and their inhibitors, exerts a major role in regulating ECM degradation. It locates upstream of the fibrolysis system and is capable of directly degrading matrix components, and indirectly inhibits the deposition of ECM by activating latent MMPs. PAI-1 is the major physiological inhibitor of both uPA and tPA, molecules that cleave plasminogen to plasmin. This inhibition decreases both fibrinolysis and matrix proteolysis, which will result in ECM deposition and hepatic fibrogenesis.Normally, there is a delicate balance between uPA and PAI-1. During liver fibrosis, the activity of uPA is gradually decreased, whereas the expression of PAI-1 is increased. Increased expression of PAI-1 weakened the activity of uPA, thus inhibiting plasmin generation and leading to excess ECM degradation. Our previous study showed that upregulation of uPA during liver fibrogenesis significantly attenuated ECM deposition in rats. Additionally, there was substantial evidence indicating that PAI-1 deficiency had a protective effect against organ fibrosis, whereas PAI-1 overexpression exacerbated fibrosis. Therefore, we postulated that downregulation of PAI-1 expression will exert as a potential treatment for hepatic fibrogenesis.Small interfering RNA (siRNA) is a sequence-specific, posttranscriptional gene-silencing mechanism through double-stranded RNA molecules homologous to the sequence of the target gene, which has opened new avenues in gene therapy. Compared with other antisense strategies, RNAi has been proved to be much more simple-handled and potent in terms of gene knockdown.In the present study, we used PAI-1 as the targeted gene, and evaluated the antifibrotic effect of PAI-1 siRNA on liver fibrosis in vitro and in vivo, which would present as a new target and solution for the treatment of hepatic fibrosis. [Methods]1. Screening of PAI-1 siRNAFour pairs of 21 nucleotide siRNAs directing against PAI-1 mRNA 219,559,1061 and 2665 targets were designed using RNA design software and synthesized. These siRNAs were separately transfected into activated hepatic stellate cell line (HSC-T6). Untransfected HSC-T6 and HSC-T6 transfected with nonspecific siRNA were set as blank and negative controls respectively. The inhibition efficiency of siRNA on PAI-1 expression was determined by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot.2. The effect of PAI-1 siRNA on HSC-T6The selected PAI-1 siRNA was transfected into HSC-T6 by either cell-suspension transfection method or stable cell line. Expression of fibrosis markers were evaluated by real-time RT-PCR. Collagen types I and III contents in the supernatants were measured by enzyme linked immunosorbent assay (ELISA). Cell proliferation and the cell cycle were determined by the methyl thiazolyl tetrazolium (MTT) method and flow cytometry.3. Construction of recombinant adenovirus vector AdshPAIpSUPER-shPAI, which was constructed previously was used to provide the HI promoter and shRNA sequence and cloned into the AdEasyTM system to get the recombinant adenoviral plasmid-pAdshPAI by homologous recombination. Finally, the recombinant adenovirus AdshPAI was successfully generated after being packaged in 293 cells. The control adenovirus AdNC which expresses the negative control (NC) shRNA and AdGFP which expresses green fluorescent protein (GFP), were prepared as well. The expression of PAI-1 in HSC-T6 cell line was detected by RT-PCR on the second day after infection with AdshPAI or AdNC.4. The effect of AdshPAI on experimental hepatic fibrosisTwo distinct models of experimental hepatic fibrosis were established in rats, either by bile duct ligation (BDL) or dimethylnitrosamine (DMN) administration. In BDL-induced fibrosis,40 rats were divided into four groups randomly (10 rats per group). Group 1 served as the control with sham surgery, and the rats in next three groups were hepatic fibrosis models induced by BDL for 3 w. Common bile ducts of models were dissected bluntly and ligated.48 h before BDL, rats in group 3 and 4 were infused with 4×109 plaque forming unit (PFU) AdNC or 4×109 PFU AdshPAI via the tail vein, respectively. Rats were sacrificed 3 w after BDL. In DMN-induced fibrosis, Group 1 served as a normal control, while the remaining rats were injected intraperitoneally with 1%DMN (10μg/kg body weight) for three consecutive days per week for up to 4w. At the end of the second week, rats in groups 2,3 and 4 were infused with a single dose of 4×109 PFU AdNC or 4×109 PFU AdshPAI via the tail vein, respectively. Rats were sacrificed 4 w after DMN. The effect of AdshPAI on fibrosis was evaluated by histological examination. Sirius red staining and Masson’s trichrome staining were used to determine collagen deposition. Immunohistochemical examination was carried out to detect the expression of PAI-1,α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), MMP-9, TIMP-1, Brdu and PCNA in liver tissues. The image analyzer was used to do the semiquantitative analysis. The mRNA level of PAI-1、α-SMA、TGF-β1、collagen typeⅠ、smad3、smad7、uPA、uPAR、tPA、MMP-2、MMP-9、MMP-13 and TIMP-1 were evaluated by real-time RT-PCR. The liver samples were subjected to acid hydrolysis to determine the amount of hydroxyproline. Immunofluorescence staining of apoptotic cells was performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL).5. Statistical AnalysisResults are presented as means of three independent experiments (±SD). Statitical analysis of values was performed with SPSS software (11.0 version), with a P<0.05 considered significant and P<0.01 as very significant.[Results]1. Screening of PAI-1 siRNA and construction of the stable cell lineBoth of the real time RT-PCR and Western blot results revealed that 219 siRNA exert the highest repression activity, which significantly downregulated PAI-1 mRNA expression 24 h and 48 h after transfection (P<0.05), and reduced the protein expression up to 85% (P<0.05).559 siRNA and 2665 siRNA also suppressed PAI-1 mRNA expression by 72% and 66%, and protein expression by 57%and 63%(P<0.05), respectively, whereas 1061 siRNA seemed to have no effect on PAI-1 expression.2. The effect of PAI-1 siRNA on the biological characteristic of HSC-T6Expression of collagen typesⅠandⅢ.α-SMA、TGF-β1 and TIMP-1 mRNA decreased at the 219 siRNA and 559 siRNA-transfection froup (P<0.05). And the collagen contents in the supernatant of HSC-T6 also decreased in PAI-1 gene silenced HSC-T6 (P<0.05), compared with those of the negative control. Moreover, compared with the NC group, PAI-1 siRNA significantly inhibited HSC proliferation as determined by the MTT assay (P<0.05). And Cell cycle study also indicated that cells were arrested at the G0/G1 phase (57.61±0.19%vs.45.49±0.93%, P<0.05) and cells at the S phase were significantly reduced (30.29±2.17%vs.40.68±1.65%, P<0.05) after downregulating PAI-1 in HSCs.3. Establishment of AdshPAIThe plasmid pSUPER-shPAI, which was constructed previously was used to provide the H1 promoter and shRNA sequence and cloned into the plasmid pShuttle, which is devoid of a promoter. The recombinant pAdshPAI was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. AdshPAI was amplified in 293 cells and then purified by cesium chloride gradient ultracentrifugation. Titers of viral stocks were determined by plaque assays using 293 cells, and 1×1010 pfu/ml titer of AdshPAI was obtained after amplification. After 2 days infection by the viruses, the expression of PAI-1 in HSC-T6 was confirmed by RT-PCR. The expression of PAI-1 was inhibited by over 90%, suggesting that the adenovirus AdshPAI was successfully constructed and adequate to downregulate PAI-1 expression.4. The effect of AdshPAI on experimental hepatic fibrosis1. The effect of AdshPAI on the expression of PAI-1:Compared with the normal rats, the PAI-1 mRNA level in the fibrotic liver was increased by 6.69±0.15-fold and 2.62±0.08-fold in BDL and DMN models, respectively (P<0.01), which was significantly reduced after AdshPAI treatment (P<0.01). Moreover, compared with the control and AdNC group, the PAI-1 protein level was significantly reduced after AdshPAI treatment in terms of both quantity and intensity as determined by immunohischemistry.2. The effect of AdshPAI on collagen deposition:Quantitative analysis indicated that the hydroxyproline content in AdshPAI treated group was strongly lower than that in the AdNC group (BDL:205.97±4.68μg/g vs.271.33±10.71μg/g; DMN:201.15±1.51μg/g vs.303.20±15.11μg/g, P<0.05). Additionally, the AdshPAI group had a significant reduction in fibrosis area, as demonstrated by Masson’s trichrome staining and Sirus Red staining (P<0.05).3. The effect of AdshPAI on collagen synthesis:The expression of collagen I, a-SMA, TGF-β1 and smad3 mRNA were all increased in fibrotic livers (P<0.01), and were all significantly downregulated after AdshPAI treatment as demonstrated by real time RT-PCR (P<0.05). Furthermore, immunohistochemistry results revealed that a-SMA and TGF-β1-positive cells were significantly reduced by AdshPAI administration. 4. The effect of AdshPAI on the fibrolysis system:Compared with the AdNC group, the expression of MMP9 and MMP13 were upregulated and TIMP-1 was downregulated (P<0.01). However, PAI-1 siRNA did not affect the mRNA levels of uPA, tPA, uPAR and MMP2. Meanwhile, the immunostaining for MMP9 and TIMP-1 showed the same pattern of results.5. The effect of AdshPAI on cell proliferation and apoptosis:Compared with the AdNC group, Brdu immunostaining and PCNA indices of hepatocytes were almost 5-fold higher in the AdshPAI group (P<0.01). And there were numerous hepatocellular mitotic figures and binucleated hepatocytes after AdshPAI treatment. Moreover, TUNEL staining results revealed that the liver specimens from the control rats showed many apoptotic hepatocytes around the periportal region and fibrotic area, and AdshPAI treatment had a strong protective effect on the hepatocytes against apoptosis (P<0.05).[Conclusion]1. Chemically synthesized 219 siRNA targeting PAI-1 mRNA had excellent silencing effect on PAI-1 expression in HSC-T6.2. The inhibition of PAI-1 expression could significantly suppress the activation, proliferation of HSC-T6 and its synthesis and secretion of ECM.3. The expression of PAI-1 was remarkably upregulated during hepatic fibrogenesis, and AdshPAI could downregulate PAI-1 expression.4. AdshPAI administration facilitated matrix degradation and inhibited ECM deposition and had a protection effect on hepatic fibrosis.5. AdshPAI treatment stimulated hepatocellular proliferation and inhibited cellular apoptosis.更多还原