【作者】 缪玉兰；

【导师】 夏照帆； 付晋凤； 王广庆； 贾一韬；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 博士

【摘要】 目的:利用RNA干扰(RNAi)方法探讨凝血酶敏感蛋白-1(TSP-1)对脓毒症肝损伤的影响及其作用机制。方法:雄性Balb/c小鼠通过盲肠结扎穿刺成功制作脓毒症模型,观察腹腔及肝脏大体解剖,检测血清转氨酶含量、肝脏TSP-1mRNA表达水平及肝脏TSP-1蛋白表达水平,探讨脓毒症时肝脏TSP-1的变化规律;利用RNAi技术靶向降解TSP-1 mRNA,引起转录后基因沉默,从而下调肝脏TSP-1表达水平,检测血清转氨酶含量、血清TNF-α含量,观察肝细胞凋亡情况,检测肝脏p38 MAPK蛋白表达变化,探讨TSP-1在脓毒症肝损伤中的作用及其机制。结果:与对照组相比,制作脓毒症模型后6小时肝脏TSP-1 mRNA及TSP-1蛋白表达明显增高,同时伴有肝脏炎症加重、血清ALT和AST含量明显升高,之后在制模24小时观察期内TSP-1维持于较高水平;利用流体尾静脉注射法予以siRNA-TSP1有效片段进行RNA干扰,制作脓毒症模型后血清ALT和AST含量、血清TNF-α含量均明显降低,肝细胞凋亡明显减少,同时肝脏磷酸化p38 MAPK蛋白表达减少。结论:脓毒症时肝脏TSP-1表达增加,作为炎症介质可能参与介导脓毒症肝损伤发生,导致血清转氨酶含量升高、血清TNF-α含量升高、肝细胞凋亡增加,同时磷酸化p38 MAPK蛋白活化,TSP-1可能通过p38 MAPK信号转导通路的激活介导其肝损伤作用。更多还原

【Abstract】 Objective:To explore the impact and mechanism of the thrombospondin-1 in septic liver injury by application of RNA interference.Methods:The model for sepsis was developed by cecal ligation and puncture(CLP) in male Balb/c mice.Then the general anatomical feature of abdominal cavity and liver was observed.Serum aminotransferase,expression level of liver TSP-1 mRNA and TSP-1 protein were detected.The pattern of liver TSP-1 in sepsis was investigated.By application of RNA interference targeted degradation of TSP-1 mRNA to induce post transcriptional gene silencing(PTGS),the downregulation of liver TSP-1 mRNA and protein was achieved.Serum aminotransferase and serum TNF-αcontents were detected.Apoptosis of liver cell was observed.The change of liver p38 mitogen activated protein kinase was detected.The role and mechanism of TSP-1 in septic liver injury were investigated.Results:In comparison with control group,the expression of liver TSP-1 mRNA and TSP-1 protein was significantly increased accompanied with more severe liver inflammation as well as dramatically increasing serum ALT and AST contents 6 hours after the CLP model for sepsis.TSP-1 maintained a relatively high level during 24 hours observation period.Through hydrodynamic tail vein injection effective fragments of siRNA specific for TSP-1 were deliveried to make RNA inference,and then the CLP model for sepsis was developed.Serum ALT,AST and TNF-αcontents were significantly decreased,apoptosis of liver cell reduced markedly,and the expression of liver phospho-p38 MAPK was decreased.Conclusion:In sepsis the expression of liver TSP-1 is increased which may induce septic liver injury as a mediator of inflammation.This results in increased serum aminotransferase,serum TNF-αcontents and increased apoptosis of liver cell,with the activation of phospho-p38 MAPK.TSP-1 may induce liver injury by the activation of p38MAPK signal transduction pathway.更多还原