自噬和凋亡相关蛋白在脑缺血再灌注大鼠模型中的表达时间谱  

Expression time spectrum of autophagy-and apoptosis-related proteins in a rat model of cerebral ischemia-reperfusion

作　　者：郭涛 张永杰 赵晓明 董玲玲 何红云 邓仪昊 GUO Tao;ZHANG Yong-jie;ZHAO Xiao-ming;DONG Ling-ling;HE Hong-yun;DENG Yi-hao(Department of Anatomyy,Medical School,Kunming University of Science and Technology,Laboratory for Cerebral Stroke Pathological Mechanism Research,Kunming 650500,China)

机构地区：[1]昆明理工大学医学院解剖学教研室,脑卒中病理机制研究实验室,云南昆明650500

出　　处：《中国病理生理杂志》2021年第11期1992-2000,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81860411;No.81960418;No.82160240;No.82160241);云南省万人计划青年拔尖人才专项(No.YNWR-QNBJ-2018-034);云南省科技厅科技计划项目(No.2019FB098);云南省教育厅科研基金资助项目(No.2020J0066;No.2018JS016);大学生创新创业训练计划项目(No.2021106740077;No.2021106740080)。

摘　　要：目的:寻找脑缺血再灌注(I/R)大鼠模型半暗区中自噬与凋亡在急性期和亚急性期特征转换的时点及其神经保护机制。方法:制备大鼠大脑中动脉闭塞(MCAO)模型,脑缺血90 min后进行再灌注。分别在I/R后1~12 h、1~7 d、14 d、21 d、28 d以及假手术(sham)组,进行改良神经损伤严重程度评分(mNSS);通过Western blot检测自噬相关蛋白微管相关蛋白1轻链3(LC3)和凋亡相关蛋白cleaved caspase-3(C-caspase-3)在不同时点的表达水平,然后分析二者比值的差异变化,根据选择的特征时点用TTC染色评估脑梗死体积,免疫荧光验证LC3表达和TUNEL染色情况。结果:LC3和C-caspase-3蛋白的表达均从I/R后1~6 h逐渐增加(P<0.05),并在12 h达到最高(P<0.05)。不同的是,从12 h~2 d,LC3表达陡然下降(P<0.05),并处于相当低的表达水平,但C-caspase-3表达仅轻微降低。然而,I/R后3 d自噬再次突然升高至6 d(P<0.05),并长期维持在较高水平,但凋亡水平持续降低至28 d(P<0.05)。另外,mNSS在I/R后1 h~4 d持续增加(P<0.05)至最大后趋于稳定,脑梗死体积呈现快速增长(P<0.01)到缓慢增长的趋势(P<0.05),神经功能缺损的严重程度与脑梗死范围的增加呈相似变化。这些结果显示存在自噬和凋亡转化的特征时点--急性期(6和12 h)和亚急性期(2、7和28 d)。免疫荧光和Western blot具有类似的结果。结论:自噬和凋亡在大鼠I/R后12 h内同时被激活,而在2 d后的亚急性期可能存在从凋亡到自噬的转换。AIM:To look for the time point of the characteristic transition between autophagy and apoptosis in acute and subacute phases,and to explore its neuroprotective mechanism in the penumbra of rats with cerebral ischemia-reperfusion(I/R).METHODS:A model of middle cerebral artery occlusion(MCAO)was prepared in rats.Reperfusion was performed after 90 min of ischemia.In sham group,and at 1 to 12 h,1 to 7 d,14 d,21 d and 28 d after I/R,the neu-rological deficit was evaluated by modified neurological severity scores(mNSS).The protein levels of autophagy-related molecule microtubule-associated proteins 1 light chain 3(LC3)and apoptosis-related protein cleaved caspase-3(C-cas-pase-3)at different time points were determined by Western blot.The difference in the ratio of the autophagy-and apopto-sis-related protein levels was analyzed.According to selected characteristic time points,the volume of cerebral infarction was evaluated by TTC staining.The method of immunofluorescence was used to verify the results of LC3 expression and TUNEL staining.RESULTS:The protein levels of LC3 and C-caspase-3 were increased gradually from 1 to 6 h after I/R(P<0.05),and reached the highest level at 12 h(P<0.05).Differently,the expression of LC3 dropped abruptly from 12 h to 2 d(P<0.05),at a very low level,but the protein level of C-caspase-3 was only slightly decreased.However,at 3 d after I/R,the autophagy was suddenly increased again to 6 d(P<0.05),and maintained at a high level for a long time,but the apoptosis continued to decrease to 28 d(P<0.05).In addition,the mNSS continued to increase from 1 h to 4 d after I/R(P<0.05)to the maximum and then was stabilized.The cerebral infarct volume showed a trend of rapid increase(P<0.01)to slow increase(P<0.05).There was a certain correlation between the severity of neurological deficit and the increase in cerebral infarct volume.It was found that there were characteristic time points of autophagy and apoptosis tran-sition in acute phase(6 and 12 h)and subacute phase(2,7 and 28 d).The results of im

关 键 词：脑缺血再灌注 自噬 细胞凋亡 半暗区 动态变化 

分 类 号：R743.3[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]