摘要
目的研究细菌脂多糖(LPS)对大鼠肺微血管内皮细胞(rat pulmonary microvascular en- dothelial cell,RPMVEC)Src抑制的蛋白激酶C底物(Src-suppressed C kinase substrate,SSeCKS)表达和细胞内定位的影响,探讨SSeCKS参与细胞骨架结构改变的可能机制。方法用植块培养法体外培养大鼠肺微血管内皮细胞,用抗大鼠CD31抗体进行细胞鉴定。LPS刺激体外培养的RPMVEC,用定量PCR、免疫印迹方法检测LPS刺激RPMVEC不同时间SSeCKS mRNA和蛋白的表达情况;用0.05μmol/L蛋白激酶C(PKC)抑制剂(Calphostin c)预处理RPMVEC 30 min后再用LPS刺激6 h,免疫荧光细胞化学法观察Calphostin C埘LPS诱导SSeCKS与纤维状肌动蛋白(filamentous-actin,F-actin)细胞内定位和结构改变的影响。结果定量PCR结果显示LPS刺激RPMVEC 1 h后SSeCKS表达水平达到最高, Western blot结果与定量PCR结果相一致,同时,LPS以时间依赖的方式诱导SSeCKS磷酸水平增加。免疫荧光结果显示LPS刺激后,F-actin发生重构,细胞内形成应力纤维,SSeCKS向核周、细胞膜纤维、板状伪足末端聚集;Calphostin C部分抑制LPS对内皮细胞F-actin和SSeCKS细胞内定位改变的影响。结论LPS能够诱导内皮细胞SSeCKS表达增加和细胞内定位改变,PKC参与LPS诱导内皮细胞F-ac- tin的重构和SSeCKS重新分布;提示SSeCKS可能与LPS诱导内皮细胞F-actin的重构有关。
Objective To study the effects of LPS on the expression and subcellular locahzation of SSeCKS at RPMVEC, so as to explore the role of SSeCKS in cytoskeleton remodeling. Methods Primary culture was conducted with explant/outgrowth method, the RPMVEC was identified by cell morphology and immunofluorescence. RPMVEC was stimulated by LPS, the expression of SSeCKS was detected by real-time PCR and Western blot. Immunofluorescent staining method with confocal laser-scanning fluorescence microscopy was used to observe the effects of Calphostin C on cytoskeleton remodeled and SSeCKS redistributed induced by LPS. Results LPS induced the expressions of SSeCKS in a time dependent manner. LPS induced phosphorylation of SSeCKS in endothehal cell in a time dependent manner. 0.05 μmol/L PKC inhibitor （Calphostin C） partly blocked the effects of LPS on cytoskeleton and subcellular localization of SSeCKS in endothehal cells. Conclusion LPS induces the change of expression and localization SSeCKS in RPMVEC. PKC plays a role in the reorganization of SSeCKS and F-actin in RPMVEC induced by LPS. The mechanism of cytoskeleton remodeled induced by LPS in RPMVEC is partially related to the SSeCKS.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2007年第10期893-897,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助(30300099
30770488)
江苏省自然科学基金资助(BK2003035)
江苏省高校自然科学基金资助(03KJB180109)
