精细化检测原代皮质神经元细胞氧糖剥夺再复氧后自噬流的变化  

The changes of autophagic flux in primary cortical neurons after oxygen and glucose deprivation reoxygenation were detected

作　　者：张磊[1,2] 戴朝 韩延峰[3] 李阳 胡玉奇[1] 陈富雷 赵冬 Zhang Lei;Dai Chao;Han Yanfeng;Li Yang;Hu Yuqi;Chen Fulei;Zhao Dong(Dept of Neurosurgery, The First Affiliated Hospital of Shihezi University, Shihezi 832000;Dept of Neurosurgery, Xinjiang Production and Construction Corps Tenth Division Beitun Hospital, Beitun 836000;Xinjiang Production and Construction Corps Tenth Division Disease Prevention and Control Center,Beitun 836000)

机构地区：[1]石河子大学第一附属医院神经外科,石河子832000 [2]新疆生产建设兵团第十师北屯医院外科,北屯836000 [3]新疆生产建设兵团第十师疾病预防控制中心,北屯836000

出　　处：《安徽医科大学学报》2022年第2期182-186,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81960222);石河子大学国际科技合作促进计划(编号:GJHZ201704);兵团财政科技计划项目(编号:2020CB011)。

摘　　要：目的用几种高度精细灵敏的方法检测氧糖剥夺再复氧(OGD/R)后神经元自噬流不同阶段的变化。方法原代皮质神经元细胞经过OGD/R后将实验分为OGD/R组及OGD/R+bafilomycin A1(BafA1)组,用RFP-GFP串联荧光标记LC3基因转染检测自噬小体和溶酶体的融合情况,透射电子显微镜(TEM)观察自噬的超微结构,SQSTM1/p62结合LC3蛋白翻转实验检测p62与LC3蛋白的定量,p62免疫染色观测其分布与含量。结果荧光显微镜下OGD/R组自噬溶酶体与自噬小体比值明显增高;TEM可观测到不同阶段的自噬结构变化;可溶性p62的比值结合LC3Ⅱ/Ⅰ的比值共同反映了自噬流的活化;p62荧光染色后在BafA1组中分布居多。结论每种方法各有其优点,不同方法和指标能够精准地反映神经元OGD/R后自噬流在不同阶段的具体变化,掌握并应用好这些方法能有效从自噬角度探索中枢神经系统疾病。Objective To detect the changes of autophagic flux at different stages after oxygen-glucose deprivation-reoxygenation(OGD/R)with several highly sensitive methods.Methods Primary cortical neurons after oxygen deprivation of sugar after reoxygenation(OGD/R)were divided into the experimental OGD/R group and OGD/R+bafilomycinA1(BafA1)group,using an RFP-GFP series fluorescent tags LC3 gene transfection detection cytolysosome and fusion of lysosomes,transmission electron microscope(TEM)observation the ultrastructure of autophagy,p62/SQSTM1 combining LC3 protein to flip the experimental testing p62 and LC3 protein quantitative,p62 immune staining observing the distribution and content.Results Under fluorescence microscope,the ratio of autophagy lysosome to autophagosome increased significantly in OGD/R group,and the changes of autophagy structure in different stages could be observed in TEM.The ratio of soluble p62 and LC3Ⅱ/Ⅰreflected the activation of autophagic flux,and p62 was mainly distributed in BafA1 group after fluorescence staining.Conclusion Each method has its own advantages,and different methods and indicators can accurately reflect the specific changes of autophagic flux in different stages after neuronal OGD/R.Mastering and applying these methods can effectively explore central nervous system diseases from the perspective of autophagy.

关 键 词：皮质神经元细胞 自噬 自噬流 OGD/R 检测 

分 类 号：R331[医药卫生—人体生理学]