凝血酶导致的星形细胞死亡中自噬激活以及作用研究  

The Effect of Thrombin Induced Autophagy in Astrocytes

作　　者：胡枢坤[1] 吴刚[1] 常川[1] 郑康[1] 

机构地区：[1]复旦大学附属华山医院神经外科,200040

出　　处：《中国临床神经科学》2014年第6期608-613,共6页Chinese Journal of Clinical Neurosciences

基　　金：国家自然科学基金课题(编号:81272374)

摘　　要：目的探讨凝血酶导致的星形细胞死亡中是否存在细胞自噬,以及自噬在此过程中的作用。方法孕21 d SD大鼠原代培养星形细胞分为:生理盐水组,凝血酶组(凝血酶处理),自噬抑制剂3-甲基腺嘌呤(3-MA)组(3-MA干预),凝血酶+3-MA组(凝血酶+3-MA共处理)。应用Western blot法检测微管相关蛋白轻链3(LC3)转化,免疫组化学染色检测beclin 1表达水平,单丹磺酰尸胺(MDC)染色观察细胞自噬空泡;乳酸脱氢酶(LDH)和活/死细胞计数评估细胞死亡。结果星形细胞经凝血酶处理24 h后,1LC3Ⅰ向LC3Ⅱ转化和beclin 1表达明显上升(P<0.05)。2MDC染色示自噬空泡数显著增加;3-MA可降低LC3Ⅰ向LC3Ⅱ的转化,下调beclin 1表达(P<0.01)和减少MDC染色自噬空泡(P<0.05)。3细胞死亡检测,星形细胞经凝血酶处理后LDH水平和活/死细胞计数与生理盐水组比较,死亡细胞比例上升(均P<0.05);3-MA干预后,LDH水平和活/死细胞计数中的死亡细胞比例较凝血酶组进一步上升(均P<0.05)。结论凝血酶导致的星形细胞死亡过程中存在细胞自噬,抑制细胞自噬加重细胞死亡。Aim To explore the effect of autophagy in astrocyte death induced by thrombin in brain and the relation of thrombin and autophagy in astrocytes. Methods Primary cultured cortex astrocytes from rat pups were exposed to saline, 3-MA （an autophagy inhibitor）, rat thrombin or rat thrombin with3-MA, the cells were collected for Western blot, immunocytochemistry, MDC labeling,LDH assay and live/death cell counting. Results With thrombin added astrocytes were founded an enhancement in the conversion of LC3 I to LC3Ⅱ and the immunoreactivity of beclin 1. MDC labeling in cultured astrocytes showed a peaked accumulation of autophagy bubbles at 24 hours. The conversion of LC3 I to LC3Ⅱ, the immunoreactivity of beclin 1 and MDC accumulation decreased with 3-MA added. 3-MA increased LDH levels and the ratio of dead cells in live/death cell counting in astrocytes. Conclusion Thrombin induced autophagy in astrocytes. 3-MA could suppress the thrombin-induced activation of autophagy and promoted the cell death. Autophagy may protect thrombin related death in astrocytes.

关 键 词：凝血酶 自噬 星形细胞 细胞死亡 

分 类 号：R7[医药卫生—临床医学]