水蛭素对凝血酶所致脑微血管内皮细胞损伤的保护作用

Protective effect of hirudin against apoptosis on cultured rat brain microvascular endothelial cells

目的探讨凝血酶对体外培养的原代大鼠脑微血管内皮细胞(BMVEC)的损伤作用及凝血酶抑制剂水蛭素的保护作用。方法分离新生大鼠脑组织微血管段,原代培养BMVEC并鉴定。四唑盐(MTT)比色法检测不同浓度凝血酶及水蛭素处理后BMVEC生长抑制状况,并用TUNEL法、透射电镜、核DNA琼脂糖凝胶电泳等3种方法检测BMVEC凋亡。结果原代培养细胞经鉴定为BMVEC。150、300 U.mL-1凝血酶可明显抑制BMVEC生长并诱导BMVEC凋亡,这种作用可被水蛭素阻断。结论病理状态下异常增加的凝血酶可导致BMVEC及血脑屏障损伤。水蛭素对凝血酶所致的BMVEC损伤有明显的阻断作用。

AIM To investigate the apoptofie effect of thrombin on brain microvascular endothelial cell （BMVEC） of cultured rat, and the protective effect of hirudin, an inhibitor of thrombin. METHODS Brain microvascular fragments of neonatal rat were separated and BMVEC was cultured. After identification, BMVEC was incubated 18 h with various concentrations of thrombin, with or without hirudin. BMVEC growth inhibition and apoptosis were detected hy methyl thiazolyl tetrazolium （MTT） assay, transmission electron microscope, TUNEL assay and 1.5% agarose gel electrophoresis assay for nuclear DNA. RESULTS The cells cultured primarily were identified as BMVEC by morphological and immunological assay with anti-Ⅷ factor antibody. Significant growth inhibition and apoptosis were found in BMVEC incubated 18 h with 150 U·mL^-1 or 300 U·mL^-1 thrombin by MTT, TUNEL and 1.5% agarose gel electrophoresis assay. Hirudin was found to block these effects. CONCLUSION Increased thrombin damages BMVEC and blood brain barrier through growth-inhibition and apoptosis-induction in physiopathological condition. Hirudin can be used to protect BMVEC from these detriments significantly.