【作者】 肖亮；

【导师】 杨镇；

【作者基本信息】 华中科技大学 ， 老年医学， 2009， 博士

【摘要】 目的：1、构建稳定、可靠、符合肝硬化门脉高压疾病自然演变过程的大鼠动物模型，研究该模型肝脏病变和门静脉高压性血管病变的进展情况。2、研究胰岛素样生长因子1(IGF-1)在肝硬化门脉高压大鼠中的表达改变，探讨IGF-1在肝硬化门脉高压性血管病变中的作用。3、研究IGF-1基因启动子的多态性，以及启动子的多态性对IGF-1基因表达的影响和在肝硬化门脉高压性血管病变机制中的作用。方法：1、应用CCL4(四氯化碳)腹腔注射法制作肝硬化门脉高压性血管病变模型，以HE染色、Masson三色染色、透射电镜、CT扫描观察大鼠肝脏及其门静脉的病变，并测量门静脉压力以了解该模型血液动力学变化情况。2、应用RT-RealTime-PCR和免疫组织化学检测病变大鼠肝脏组织IGF-1的mRNA和蛋白水平，应用ELISA方法测定病变大鼠血清IGF蛋白的含量水平，并与正常大鼠组相比较。3、应用序列特异性引物PCR和非对称性PCR扩增肝硬化组大鼠IGF-1基因启动子，应用基因芯片技术检验IGF-1基因启动子多态性。结果：1、腹腔注射CCL4后大鼠肝脏组织经HE染色后可见肝小叶结构紊乱，肝小叶重建，纤维组织增生，肝内假小叶广泛形成，同时伴有明显的汇管区纤维组织增生等肝硬化病理表现；门静脉血管内皮细胞呈结节样增生，平滑肌细胞肥大，成纤维细胞和胶原纤维增多；经CT扫描发现肝硬化组和对照组大鼠肝脏体积分别为9741±2715mm~3和12898±3081 mm~3，两组差异有统计学意义；经测压后发现肝硬化组和正常组大鼠门静脉压力分别为7．6±2．6mmHg，12．4±3．6 mmHg，两组间差异有显著性意义。2、经免疫组化检测两组肝脏组织IGF-1蛋白表达分别为0．40±0．13和0．68±0．19，差异有统计学意义；经RT-Real Time-PCR检测两组肝脏组织IGF-1 mRNA分别为3．95±2．26和6．12±3．14，差异有统计学意义；经ELISA方法测定两组大鼠血清IGF蛋白分别为687±137 ng／ml和919±183 ng／ml，差异有统计学意义；经相关线性分析，肝细胞IGF-1浓度、血清IGF-1浓度以及肝细胞IGF-1 mRNA浓度对门静脉压力值存在负相关。3、经序列特异性引物PCR和非对称性PCR扩增肝硬化组大鼠I6F-1基因启动子后，基因芯片技术检验IGF-1基因启动子发现，在肝硬化组大鼠，-10位点碱基多态性导致肝脏IGF-1蛋白含量分别为0．44±0．13(AI)和0．34±0．10(AI)，差异有统计学意义；-35位点碱基多态性导致门静脉压力分别为13．0±3．5(mmHg)和9．6±2．6(mmHg)，两组的差异有统计学意义。结论：1、CCL4性肝硬化门脉高压血管病变模型稳定、类似人类自然病程；门静脉系统血流动力学改变明显，是研究门脉高压症的理想模型。2、在肝硬化门脉高压大鼠模型中，随着肝脏体积的不断缩小、门静脉压力的不断升高，肝脏组织及血清IGF-1蛋白和基因转录水平不断下降。IGF-1是肝硬化门脉高压的保护性因子。3、IGF-1基因启动子区域-10，-35位点碱基的变异可以导致大鼠IGF-1基因启动子活性的改变，从而影响到IGF-1基因的表达改变引起的门脉高压血管病变的变化。IGF-1基因启动子多态性导致不同的个体在肝硬化门脉高压病程中的反应多态性。更多还原

【Abstract】 OBJECTIVE1.To construction of a stable,reliable,consistent of natural evolution of rat model withcirrhotic portal hypertension;to study liver lesions and vasculopathy induced by portalhypertension.2.To study expression change of insulin-like growth factor 1 (IGF-1) in cirrhotic portalhypertension in rats;to explore mechanism of IGF-1 in portal hypertensive vascularlesions.3.To study promoter polymorphism on IGF- 1 gene and its effect of IGF- 1 gene expression;to study the association between polymorphisms of the insulin growth factor-1 promoterand vasculopathy in rat with portal hypertension.METHODS1.To duplicate model of vasculopathy with portal hypertension by intraperitoneal injectionof CCL4;to detect liver lesion and vasculopathy by HE staining,Masson trichrome staining,transmission electron microscopy,CT scanning;to understand hemodynamicchange by portal venous pressure measurements.2 To detect mRNA and protein level of IGF- 1 in rat liver tissue by RT-Real Time-PCR andimmunohistochemistry;to detect serum IGF protein levels ELISA;and respectivelycompared the results with normal rats.3.To detect rats IGF-1 gene promoter by application of sequence-specific primer PCR anddissymmetry PCR of cirrhosis rat;to test IGF-1 gene promoter polymorphism byapplication of gene chip technology.RESULTS1.After intraperitoneal injection of CCL4,hepatic lobules structure can be seen disorders,reconstruction,fibrous tissue hyperplasia,accompanied by intrahepatic extensiveformation of false lobules by HE staining;vascular endothelial cells of portal veinhyperplasia,smooth muscle cells hypertrophy,fibroblasts and collagen fibers increase;thevolume of rat liver between liver cirrhosis group and control group is separately 9741±2715mm3 and 12898±3081 mm3 by CT scanning,the difference is statisticallysignificant;portal pressure between liver cirrhosis group and normal by manometry in ratsis 7.6±2.6mmHg and 12.9±3.7 mmHg respectively,difference between the two groupswas significant.2.IGF-1 protein expression of two groups of liver tissue by immunohistochemicaldetection is 0.40±0.13 and 0.68±0.19,and the difference has statistical significance;IGF-1 mRNA of two groups of liver is 3.95±2.26 and 6.12±3.14 By RT-Real Time-PCRDetection,and the difference has statistical significance;serum IGF protein is 687±137ng/ ml and 919±183 ng/ ml Measured by ELISA,and the difference has statisticalsignificance;there is a negative correlation between liver cells IGF-1 concentrations orserum IGF-1 concentration as well as liver cells IGF-1 mRNA concentration and portalvein pressure By the linear analysis.3.IGF-1 protein content caused different activity of gene promoter by -10 nucleotide polymorphism are 0.44±0.13 (AI) and 0.34±0.10 (AI) by sequence-specific primer PCRand asymmetric PCR and gene chip technology of liver cirrhosis in rats of liver cirrhosis,the difference has statistical significance;portal vein pressure were 13.0±3.5 (mmHg)and 9.6±2.6 (mmHg) caused by -35 nucleotide polymorphism,the difference between thetwo groups has statistical significanceCONCLUTIONS1.It is a stable model similarly to human natural course of disease established by CCL4which characterized with vasculopathy in portal hypertension;it is an ideal model forstudying portal portal hypertension which has a significant change of vein hemodynamics.2.The level of IGF-1 protein and gene transcription continues to drop according todeterioration of liver cirrhosis in rat model with cirrhotic portal hypertension.IGF-1 is aprotective factor in cirrhotic portal hypertension.3.Variation of IGF-1 gene promoter region on -10,-35 base locus can lead to activitychanges of IGF-1 gene promoter,which affect gene expression changes of IGF-1,therebyaffect the vasculopathy in portal hypertension.Polymorphism of IGF-1 gene promoterleads to individual reaction in course of cirrhotic portal hypertension.更多还原