摘要
目的探讨体外模拟缺血再灌注(ischemia/reperfusion,IS/RE)后大鼠脑皮质神经元内质网应激凋亡的机制及抑制细胞色素c(Cytc)表达对内质网应激的影响。方法体外培养SD乳鼠脑皮质神经元,用免疫组织化学、免疫荧光染色鉴定神经元纯度;用流式细胞术Annexin V、PI双标检测调亡率及活性Caspase-3,-7,-9表达水平;用Western-blot免疫印迹方法检测Caspase-12、GRP78、Bcl-2、Cytc蛋白表达水平。结果SD乳鼠皮质神经元可纯化体外培养;空转染组神经元模拟缺血6h再灌注24h及48h(IS6h/RE24h、48h)细胞凋亡率分别是17.95%、22.62%;Cytc基因静默后凋亡率分别降至10.26%、9.37%,空转染组与转染组比较有统计学意义(P<0.05);空转染组神经元模拟缺血再灌注后GRP78、Cytc、活性Caspase-3、caspase-9、caspase-12表达均增加,Bcl-2表达减少;siRNACytc基因静默后神经元Bcl-2表达增加,GRP78、活性caspase-12、caspase-3、caspase-9表达明显降低,2组比较有统计学意义(P<0.05);空转染组和转染组均未检测到caspase-7表达。结论体外模拟缺血再灌注后脑皮质神经元发生凋亡,线粒体、内质网应激参与了神经元凋亡;抑制细胞色素c释放对内质网应激凋亡有抑制作用。
Objective To study the apoptotic induction effect of analogous ischemia/reperfusion in vitro on rat cortical neurons and the effect of analogous ischemia/reperfusion initiated ER stress by silencing of cyto chrome c（Cyt c）. Methods Primary cortical neurons were cultured in vitro,and NSE-positive cells were detected by immunohistological shemistry and immunofluorescence staining. Protein levels of GRP78, Bcl-2, Cyt c and active Caspasc-12 were assessed by immnnoblotting. The percentage of apoptotic （annexin V positive and propidium iodide negative） cells and protein expression of active Caspase-3, 7,-9 were determined by flow cytometric analysis. Results As expected,GRP78 and Caspase12 protein levels were elevated in cortical neurons in response to ischemia/reperfusion （ischemia 6 h, reperfusion 24 h or 48 h） but not to control. The rate of apoptosis induced after reperfusion 24 h and 48 h was 17.95% and 22. 62%,and was depressed to 10. 26% and 9. 37% after silencing of Cyt c. Activated Caspase-3,-9, 12 was detected at 24 h and 48 h after reperfusion and was reduced by silencing of Cyt c,and silencing of Cyt c increased the expression of Bcl-2 caused by ischemia/ reperfusion. Conclusions ER stress is involved in neuronal apoptosis induced by analogous ischemia/reperfusion in vitro. Silencing of cyt c suppresses ER stress-induced apoptosis of neurons.
出处
《卒中与神经疾病》
2008年第5期302-306,共5页
Stroke and Nervous Diseases
