YAP转录激活因子在内毒素诱导血管内皮细胞凋亡中的作用

This research work is carried out on the model of mono-layer endothelium culture in vitro.In order to explore the roles of GEF-H1 and YAP in transcription of YAP/p73 induced by LPS, the intranuclear and cytosolic YAP, p73 and Bax expressions are observed and the binding activity of p73 and DNA is detected by EMSA in the model of LPS-challenged microvascular endothelium after GEF-H1 or YAP expression are inhibited by SiRNA. In the meanwhile, apoptosis of endothelium are observed by using the methods of Annexin V,Mitochondrial Membrane Potential Assay and TUNEL. The relationship between GEF-H1/ROCK and YAP is analysed by FRET and IP. Together all,the data will help us to identify GEF-H1/RhoA/YAP signalling endothelium apoptosis induced by LPS.We also try to know the possibility of alleviating LPS-induced endothelial apoptosis by down-regulated GEF-H1 and YAP expressions. Finally, the crosstalking between Fas/FADD and GEF-H1/RhoA/YAP signal pathway are also studied in the sametime. The research results are benefit to understand mechanism of endothelial apoptosis in sepsis, and to explore a potential method to prohibit LPS-related organ injury.

在LPS刺激体外培养的血管内皮细胞的基础上，利用SiRNA抑制 GEF-H1或YAP的表达，检测细胞核内外YAP、p73和Bax蛋白表达及其磷酸化水平变化，EMSA法检测p73与凋亡相关DNA的结合活性的变化，以探讨 GEF-H1和YAP在LPS介导YAP/p73转录活性变化中的作用；利用Annexin V法、线粒体膜势能检测法、TUNEL法检测细胞凋亡情况，FRET和免疫共沉淀方法观察GEF-H1、ROCK与YAP的相关性，分析调节GEF-H1和YAP表达和活性以减轻LPS造成血管内皮细胞凋亡的可能性，确认LPS通过GEF-H1/RhoA/YAP信号传递通路诱导细胞凋亡。同时研究Fas/FADD凋亡通路与GEF-H1/RhoA/YAP凋亡通路直接的内在联系。研究结果有助于深入探讨感染造成血管内皮细胞凋亡的机理，并可能为临床上干预感染性并发症提供潜在的治疗手段。

中文摘要.本课题此次主要通过探讨转录共激活因子yap蛋白在lps刺激血管内皮细胞后的活性以及yap蛋白在lps诱导血管内皮细胞凋亡和血管内皮细胞损伤后高凝中的机制，通过本课题研究发现，lps体外刺激血管内皮细胞可以诱导yap蛋白激活并由细胞浆进入细胞核，进一步通过结合并激活转录因子egr-1介导组织因子的表达诱导微循环血管内高凝，间接诱导微循环损伤。同时yap蛋白激活进入细胞核后还可以结合并激活转录因子p73蛋白，并进一步通过调控线粒体凋亡途径来诱导血管内皮细胞凋亡，本课题的科学意义在于通过体外探讨lps诱导血管内皮细胞损伤的机制，发现了转录共激活因子yap在脓毒症急性肺损伤中的重大作用，为临床防治脓毒症急性肺损伤的发生提供了新的靶点。