蛋白激酶对内毒素休克后内皮细胞骨架的作用  

Effect of protein kinase on endothelial cytoskeleton induced by septic shock

作　　者：吴杰裕[1] 肖能坎[1] 黄巧冰[2] 黄绪亮[2] 石胜军[1] 肖添有[1] 刘洪[1] 

机构地区：[1]第一军医大学珠江医院烧伤科,广州510282 [2]第一军医大学全军休克微循环重点实验室

出　　处：《中华外科杂志》2003年第3期193-196,共4页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目 ( 39870 80 8);广东省自然科学基金资助项目 ( 980 2 2 0 )

摘　　要：目的　探讨内毒素休克发病机制中蛋白激酶G(PKG)的作用。　方法　用脂多糖 (LPS)刺激培养的内皮细胞 ,通过细胞裂解和离心获得细胞裂解液 ,用放射性同位素法标记法检测PKG的活性。同时采用特异性荧光染色法检测LPS刺激后细胞内肌动蛋白微丝 (F actin)的结构和分布变化。用PKG特异性抑制剂KT5 82 3预处理细胞后 ,再检测LPS介导的细胞内PKG活性和F actin的变化。以空白组为阴性对照 ,以PKG激动剂 8 Br cGMP刺激细胞作为阳性对照组。　结果　LPS分别刺激 5、10、30和 6 0min后细胞内PKG活力呈时间依赖性的增高 (与空白组相比P <0 .0 1) ,细胞内的F actin出现极性分布 ;而KT5 82 3预刺激 2 0min后再用LPS刺激没有出现上述变化。PKG的激动剂 8 Br cGMP刺激细胞后的变化与LPS的刺激相似。　结论　LPS可以介导血管内皮细胞PKG的激活和F actin的应力性变化 ;内毒素休克后内皮细胞通透性增高与cGMP PKG通路的激活有关。Objective To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of septic shock. Methods Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with LPS or PKG activator 8-Br-cGMP. PKG activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2B by PKG, and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study, the PKG specific inhibitor KT5823 was used to pretreat the endothelial cells before the administration of LPS or PKG activator 8-Br-cGMP. Results Exposure to LPS for 5, 10, 30 and 60 minutes led to a rapid time-dependent increase in endothelial PKG activity ( P<0.01 compared to the blank ) and the polar distribution of intracellular filamentous actin and preincubation with KT5823 abolished these effects. 8-Br-cGMP was similar to LPS. Conclusions The results suggested that LPS can mediate PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably induce the endothelial hyperpermeability after septic shock.

关 键 词：内毒素休克 内皮细胞 发病机制 蛋白激酶G 脂多糖 信号传导 细胞骨架 

分 类 号：R605.971[医药卫生—急诊医学]