脑出血后凝血酶诱导自噬细胞的种类研究
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摘要
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.
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.
引文
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[10]胡枢坤,吴靖宇,黄峰平.凝血酶在脑出血后脑损伤过程中促进细胞自噬性死亡的研究[J].中华实验外科杂志,2014,31:322-324
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[14]Mizushima N,Komatsu M.Autophagy:renovation of cells and tissues[J].Cell,2011,147:728-741
[15]王莉,刘媛,伍亚民.自噬在神经系统疾病中的作用研究进展[J].中国临床神经科学,2015,3:344-349
[16]Halassa MM,Haydon PG.Integrated brain circuits:astrocytic networks modulate neuronal activity and behavior[J].Annu Rev Physiol,2010,72:335-355
[2]Gong Y,Xi G,Hu H,Gu Y,et al.Increase in brain thrombin activity after experimental intracerebral hemorrhage[J].Acta Neurochir Suppl,2008,105:47-50
[3]Xi G,Keep RF,Hoff JT.Mechanisms of brain injury after intracerebral haemorrhage[J].Lancet Neurol,2006,5:53-63
[4]Xi G,Reiser G,Keep RF.The role of thrombin and thrombin receptors in ischemic,hemorrhagic and traumatic brain injury:deleterious or protective?[J].J Neurochem,2003,84:3-9
[5]Hua Y,Keep RF,Hoff JT,et al.Brain injury after intracerebral hemorrhage:the role of thrombin and iron[J].Stroke,2007,38:759-762
[6]Yang S,Song S,Hua Y,et al.Effects of thrombin on neurogenesis after intracerebral hemorrhage[J].Stroke,2008,39:2079-2084
[7]He Y,Wan S,Hua Y,et al.Autophagy after experimental intracerebral hemorrhage[J].J Cereb Blood Flow Metab,2008,28:897-905
[8]Mizushima N,Komatsu M.Autophagy:renovation of cells and tissues[J].Cell,2011,147:728-741
[9]Hu S,Xi G,Jin H,et al.Thrombin-induced autophagy:a potential role in intracerebral hemorrhage[J].Brain Res,2011,1424:60-66
[10]胡枢坤,吴靖宇,黄峰平.凝血酶在脑出血后脑损伤过程中促进细胞自噬性死亡的研究[J].中华实验外科杂志,2014,31:322-324
[11]胡枢坤,吴刚,常川,等.凝血酶导致的星形细胞死亡中自噬激活以及作用研究[J].中国临床神经科学,2014,22:608-613
[12]Striggow F,Riek M,Breder J,et al.The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations[J].Proc Natl Acad Sci U S A,2000,97:2264-2269
[13]Fisher MJ.Brain regulation of thrombosis and hemostasis:from theory to practice[J].Stroke,2013,44:3275-8325
[14]Mizushima N,Komatsu M.Autophagy:renovation of cells and tissues[J].Cell,2011,147:728-741
[15]王莉,刘媛,伍亚民.自噬在神经系统疾病中的作用研究进展[J].中国临床神经科学,2015,3:344-349
[16]Halassa MM,Haydon PG.Integrated brain circuits:astrocytic networks modulate neuronal activity and behavior[J].Annu Rev Physiol,2010,72:335-355