脑出血后凝血酶诱导自噬细胞的种类研究

The Study of Cell Types in Thrombin Induced Autophagy after Intracerebral Hemorrhage

目的探讨脑出血后凝血酶诱导的自噬细胞类型。方法建立凝血酶脑损伤模型,大鼠脑内基底节注射凝血酶,收集脑损伤标本,免疫荧光双染检测beclin1^+与神经胶质纤维酸性蛋白(GFAP^+)以及神经元特异性烯醇化酶(NSE^+)的重合率;电镜下直接观察含有自噬泡的细胞。分别培养星形细胞和神经细胞,经凝血酶处理后,用Western blot法测量微管相关蛋白轻链3 (LC3)Ⅰ型向Ⅱ型转化,单丹磺酰戊二胺(MDC)染色观察细胞内自噬空泡的变化。结果大鼠基底节注射凝血酶后,免疫荧光双染显示beclin1+细胞主要为星形细胞;电镜显示,自噬空泡均位于星形细胞内,未能在神经细胞中发现。离体实验表明,凝血酶可以明显上调星形细胞中LC3Ⅱ/LC3Ⅰ比值,增加MDC染色标记的自噬空泡数量,神经细胞未见类似改变。结论脑出血后凝血酶诱导的细胞自噬主要发生于星形细胞,而不是神经细胞。

Aim To study the cell types in thrombin induced autophagy. Methods Rats received infusion of rat thrombin into the right caudate nucleus and were killed 3 days later. Brains were used for immunofluorescence double staining and electron microscopy analyses. Primary cultured astrocytes from rat pups and neurons from fetal rats were exposed to thrombin or vehicle. The cells were collected for Western blot and monodansylcadaverin( MDC) labeling counting. Results Intra-caudate injection of thrombin specifically elevated the astrocytic expression of beclin 1,an autophagic marker,and promoted the formation of autophagic vacuoles within astrocytes rather than neurons in the ipsilateral basal ganglia. In vitro,thrombin enhanced the LC3Ⅱ/LC3 Ⅰ ratio and increased the number of MDC-labeled autophagic vacuoles in cultured astrocytes,not in neurons. Conclusion The results indicated that thrombin induced autophagy preferentially activated in the astrocyte after intracerebral hemorrhage,which provided novel insights into the pathophysiological mechanisms and therapeutic targets for hemorrhage stroke and brain trauma.