SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义  

The Expression and Significance of SIRT3 in Mouse Cortical Neurons after Oxygen-glucose Deprivation/Reperfusion

作　　者：王元欣 张磊[1] 岳康异 黑悦 鱼洋 武秀权 蒋晓帆[1] WANG Yuan-xin1,2, ZHANG Lei1, YUE Kang-yi1, HEI Yue1, YU Yang1, WU Xiu-quan1, JIANG Xiao-fan1(1 Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi＇an, Shaanxi, 710032, China; 2 Department ofNeurosurgery, Fufeng CountyPeople＇s Hospital, Baoji, Shaanxi, 722200, Chin)

机构地区：[1]第四军医大学西京医院神经外科,陕西西安710032 [2]扶风县人民医院神经外科,陕西宝鸡722200

出　　处：《现代生物医学进展》2018年第5期817-821,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(8167050366)

摘　　要：目的:通过建立体外脑缺血模型,探讨沉默信息因子3(SIRT3)在小鼠皮层神经元氧糖剥夺再灌注(OGD/R)损伤后的表达和意义。方法:C57BL/6J小鼠皮层神经元原代培养7天后,以氧糖剥夺不同时长(2 h、4 h、6 h、8 h)再灌注24 h作为观察时间点,利用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)检测细胞活力;小鼠乳酸脱氢酶(LDH)试剂盒检测LDH释放;蛋白印迹法(Western blot WB)观察微管相关蛋白1轻链3(LC3-Ⅱ)、活化凋亡蛋白3(Cleaved caspase-3)、以及SIRT3的表达变化;免疫荧光下进一步观察LC3-II、SIRT3表达。结果:与正常组比,随着氧糖剥夺时间的延长,LDH释放量呈台阶式升高(P<0.01),而神经元活性进展性下降(P<0.01);蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调,并于OGD 4h达峰值,SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状,而Cleaved caspase-3整体上调;相应的,细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达,与此同时SIRT3荧光强度亦增高。结论:神经元缺血时间越长损伤越重;LC3-Ⅱ和SIRT3表达呈现相似性;SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。Objective： To verify the expression and significance of silent information regulator 3 （SIRT3） in cerebral ischaemia using the oxygen glucose deprivation/reperfusion （OGD/R） model in vitro. Methods： Primary cultured cortical neurons from C57BL/6J exposed to oxygen-glucose deprivation for 2 h, 4 h, 6 h, 8h, and followed by 24 h of reperfusion. Additionally, the cell viability was detected by Cell Counting Kit-8 （CCK-8） assay, the cytotoxicity was measured by LDH kit assay, the expression of LC3-II, cleaved caspase-3 and SIRT3 was determined by western blot and the levels of LC3-II and SIRT3 were further asssayed by immunofluorescence. Results： The results showed that there was a time-dependent increase of LDH release and decrease of cell viability （P〈0.01）. The results of Western blot demonstrated that LC3-II and SIRT3 increased and peaked at OGD 4h while the expression of Cleaved caspase-3 was elevated all the time. The results of immunofluorescence indicated that LC3-II and SIRT3 were significantly expressed in somas and neurites compared with the control. Conclusion： We observed that the expression of LC3 and SIRT3 changes in a similar pattern through a OGD/R model. Hence, SIRT3 might protects neurons against ischemic injury via regulating autophagy.

关 键 词：SIRT3 LC3 Cleavedcaspase-3 自噬 凋亡 

分 类 号：R-33[医药卫生]