【作者】 郑磊；

【导师】 罗蒙；

【作者基本信息】 上海交通大学 ， 外科学（普外）， 2020， 博士

【摘要】 第一部分:门静脉高压症与动脉重构目的:探索肝硬化和非肝硬化门静脉高压症模型中是否存在血管重构表型。方法:分别采用CCl₄吸入诱导肝硬化和PPVL手术诱导非肝硬化门静脉高压症大鼠模型,判定其成模情况后检测肠系膜血管重构表型。结果:两种模型平均动脉压均降低,门静脉压力均升高,肝脏组织学显示肝硬化门静脉高压症大鼠肝脏明显硬化改变,非肝硬化组大鼠肝脏无明显差别。在肝硬化门静脉高压症中肠系膜动脉变薄,非肝硬化门静脉高压症中无明显改变。结论:两种模型造模是成功的。肝硬化门静脉高压症模型存在显著动脉重构表型,非肝硬化门静脉高压症模型中不存在此表型。第二部分:门静脉高压症动脉重构的结构表现目的:阐明门静脉高压症动脉重构的血管成分异常。方法:通过电镜、WB、免疫组化和荧光检测肠系膜动脉主要结构相关蛋白的表达。结果:门静脉高压症大鼠肠系膜动脉中血管内皮凹凸不平,内皮细胞发生脱落,p-e NOS表达增多,钙调蛋白、弹性蛋白和α-SMA表达减少。结论:肠系膜动脉重构的主要改变以内皮细胞脱落、基底膜间隙增宽、收缩蛋白（钙调蛋白、弹性蛋白、α-SMA）表达减少,扩张蛋白p-e NOS表达增多。第三部分:VEGFR-3与门静脉高压症动脉重构目的:研究VEGFR-3与肠系膜动脉重构的关系。方法:运用PCR和免疫荧光检测门静脉高压症的肠系膜动脉相关基因和蛋白表达。结果:门静脉高压症的大鼠肠系膜动脉上VEGF-D和VEGFR-3表达增多,同时Ki-67增殖信号和cleaved caspase-3凋亡信号表达增多。结论:在门静脉高压症的肠系膜动脉重构中,VEGFR-3信号表达增加,其配体VEGF-D表达同时增加。血管增殖和凋亡信号均有所增加,但血管平滑肌细胞凋亡明显。第四部分:抑制VEGFR-3与门静脉高压症动脉重构目的:证明抑制VEGFR-3信号能够改善门静脉高压症中肠系膜动脉重构。方法:运用VEGFR-3抑制剂MAZ-51治疗门静脉高压症大鼠后检测重构表型。结果:两组大鼠门静脉压力、平均动脉压和VEGFR-3表达无明显差异,MAZ-51改善门静脉高压症大鼠肠系膜动脉重构,血管厚度和面积增加,肠系膜动脉平滑肌细胞凋亡减少。结论:运用VEGFR-3抑制剂MAZ-51能够通过减少肠系膜动脉平滑肌细胞凋亡,缓解门静脉高压症的肠系膜动脉重构。第五部分:VEGFR-3信号通路与门静脉高压症动脉重构目的:初探VEGFR-3信号在肠系膜动脉上内皮细胞层的信号通路。方法:原代分离肠系膜动脉内皮细胞,运用不同时间和浓度VEGF-D刺激内皮细胞,以及MAZ-51联合VEGF-D刺激内皮细胞,检测相关蛋白表达。结果:随着VEGF-D刺激时间和浓度的增加,VEGFR-3下游信号磷酸化e NOS、AKT和ERK的表达逐渐增加,在10min和50ng/ml磷酸化程度最为显著。MAZ-51组较VEGF-D组,下游蛋白AKT、ERK和e NOS磷酸化程度明显减弱。结论:运用VEGFR-3抑制剂MAZ-51能够减少肠系膜动脉内皮细胞的VEGFR-3信号下游磷酸化AKT、ERK、e NOS的表达。更多还原

【Abstract】 PartⅠ.SMA remodeling in PHTAim:Explore whether SMA remodeling exist in cirrhotic and non-cirrhotic PHT.Methods:Establish CCl₄ inhalation induced cirrhotic and PPVL induced non-cirrhotic PHT rat model,and check condition of model then check SMA remodeling phenotype.Results:MAP were decreased while PP were elevated in two kinds of PHT modeling.Liver gross observation,HE,Masson and Sirius Red staining showed liver cirrhosis changes in cirrhotic PHT,but there were no difference between non-cirrhotic PHT and control rats.SMA thinning occurred in cirrhotic PHT rats but not in non-cirrhotic PHT rats.Conclusion:Both models were successful.A significant arterial remodeling phenotype existed in cirrhotic PHT model,which did not exist in the non-cirrhotic PHT model.PartⅡ.Vascular structural changes in PHT arterial remodelingAim:To elucidate the changes of main components in cirrhotic PHT arterial remodeling.Methods:The expression levels of main structural-related proteins in SMA were detected by electron microscopy,WB,immunohistochemistry and fluorescence.Results:The inner surface of SMA in cirrhotic PHT rats was uneven and EC was shed,while p-e NOS expression increased,and calmodulin,elastin andα-SMA expression decreased.Conclusion:The main changes in SMA remodeling were the endothelial layer shedding,increased basement membrane space,and decreased contractile proteins such as calmodulin,elastin,andα-SMA,while the expression of p-e NOS associated with vasodilation increased.Part Ⅲ.VEGFR-3 expression and PHT arterial remodelingAim:To study the role of VEGFR-3 in vascular remodeling of SMA.Methods:The expression of SMA-related genes and proteins in cirrhotic PHT model was detected by PCR and immunofluorescence.Results:The expression of VEGFR-3 on SMA increased in rats with cirrhotic PHT,while the proliferation signaling represented by Ki-67 and the apoptosis signal represented by cleaved caspase-3 increased.Conclusion:In the SMA arterial remodeling of PHT in liver cirrhosis,the expression of VEGFR-3 signal increased,and the expression of its ligand VEGF-D also increased.Vascular proliferation and apoptotic signaling were increased,and the smooth muscle cell layer is dominated by apoptosis.Part Ⅳ.Effect of VEGFR-3 inhibition in PHT arterial remodeling Aim:To prove whether inhibition of VEGFR-3 signaling pathway can improve SMA thinning in PHT.Methods:The SMA phenotype was measured after treatment with the VEGFR-3 inhibitor MAZ-51 in cirrhotic PHT rats.Results:There was no significant difference in PP,MAP and VEGFR-3 expression in two groups.MAZ-51 improved SMA remodeling,increased wall thickness and wall area,and reduced SMA smooth muscle layer apoptosis in cirrhotic PHT rats.Conclusion:The treatment of the VEGFR-3 inhibitor MAZ-51 could alleviate SMA arterial remodeling and reduce apoptosis of SMA smooth muscle layer in cirrhotic PHT.Part Ⅴ.VEGFR-3 signaling pathway in PHT arterial remodeling Aim:To explore the VEGFR-3 signaling pathway in the SMA EC layer.Methods:The primary isolation of mesenteric arterial EC was used,to stimulate EC with different time and concentration of VEGF-D,and MAZ-51 combined with VEGF-D stimulated EC.Results:With the increase of VEGF-D stimulation time and concentration,the expression of VEGFR-3downstream such as phosphorylated e NOS,AKT and ERK increased gradually,reaching the highest peak at 10 min and 50 ng/ml.Compared with the VEGF-D group,the MAZ-51 group significantly reduced the phosphorylation of downstream proteins AKT,ERK and e NOS.Conclusion:The treatment of the VEGFR-3 inhibitor MAZ-51 can reduce the expression of phosphorylated AKT,ERK,and e NOS,which were downstream of VEGFR-3 signaling in the mesenteric arterial EC.更多还原