蛋白激酶G介导烧伤休克后内皮细胞骨架的变化

Effect of cGMP-dependent protein kinase on endothelial cytoskeleton changes

目的 :探讨蛋白激酶G(PKG)在烧伤休克发病机制中的作用。方法 :用 10 %人烧伤血清刺激培养的内皮细胞后 ,通过细胞裂解和离心获得细胞裂解液 ,用放射性同位素法测定PKG的活性。同时采用特异性荧光染色法检测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 ,再检测烧伤血清介导的细胞内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 :人烧伤血清和PKG的激动剂 8-Br -cGMP都可以时间依赖性地激活内皮细胞的PKG ,并且诱导细胞内F -actin呈极性分布。KT5 82 3预处理则明显抑制了这种变化。结论 :烧伤血清可以介导血管内皮细胞PKG的激活和F -actin的应力性变化 ;烧伤休克时的血管通透性升高与PKG的活性变化密切正相关 。

AIM: To study the effect of cGMP-dependent protein kinase (PKG) on the pathogenesis of burn shock. METHODS: Confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with 10% burn serum 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 were used to pretreat the endothelial cells before the administration of burn serum or PKG activator 8-Br-cGMP. RESULTS: Exposures to burn serum and 8-Br-cGMP led to a rapid time-dependent increase in endothelial PKG activity and the polar distribution of intracellular filamentous actin, and preincubation with KT5823 abolished those effects. CONCLUSIONS: The results suggest that burn serum induces PKG activation and the stress variety of filamentous actin in the vascular endothelial cells, which probably contributes to the endothelial hyperpermeability after burn shock. [