miR-29a调控水通道蛋白4抑制过氧化氢诱导的星形胶质细胞凋亡  

miR-29a inhibits hydrogen peroxide-induced astrocyte apoptosis by regulating aquaporin-4

作　　者：朱琳[1] 郭世武 刘锦波[2] 邹红军 Zhu Lin;Guo Shiwu;Liu Jinbo;Zou Hongjun(Department of Cardiology,The First People's Hospital of Changzhou,Changzhou 213003,Jiangsu Province,China;Department of Spine Surgery,The First People's Hospital of Changzhou,Changzhou 213003,Jiangsu Province,China)

机构地区：[1]常州市第一人民医院心内科,江苏省常州市213003 [2]常州市第一人民医院脊柱外科,江苏省常州市213003

出　　处：《中国组织工程研究》2023年第19期2993-2998,共6页Chinese Journal of Tissue Engineering Research

基　　金：江苏省卫生健康委医学科研项目(Z2019014),项目负责人:邹红军;常州市应用基础研究计划(CJ20220108),项目负责人:邹红军;常州市2020年青苗工程培养对象(CZQM2020046),项目负责人:邹红军;常州市卫生计生委青年人才科技项目(QN201704),项目负责人:邹红军。

摘　　要：背景:一些研究已经证实了miR-29a、水通道蛋白4在星形胶质细胞损伤过程中的作用,但其是否存在相互作用以及分子机制尚未见报道。目的:研究miR-29a调控水通道蛋白4表达对过氧化氢诱导星形胶质细胞损伤的影响。方法:①以小鼠原代培养的星形胶质细胞为研究对象,用不同浓度的过氧化氢诱导小鼠星形胶质细胞损伤反应,细胞分为对照组、过氧化氢组、过氧化氢+空载体组、过氧化氢+miR-29a过表达组。采用MTT法检测小鼠星形胶质细胞存活率;Western blot法检测小鼠星形胶质细胞凋亡相关蛋白(Bcl-2、Bax、cleaved-Caspase-3)、水通道蛋白4和胶质纤维酸性蛋白的蛋白表达水平;实时定量PCR检测小鼠星形胶质细胞中miR-29a的表达水平;流式细胞仪检测小鼠星形胶质细胞凋亡水平。②将12只雌性BALB/c小鼠随机分为空载体组、过表达miR-29a组,每组6只。制备小鼠T10脊髓撞击损伤动物模型,建模后分别注射以下重组慢病毒(空载体、miR-29a过表达载体)。术后第7天,采用免疫组织荧光染色检测小鼠脊髓损伤部位水通道蛋白4和胶质纤维酸性蛋白表达水平。结果与结论:①过氧化氢诱导后星形胶质细胞活性下降,促凋亡相关蛋白Bax、cleaved-caspase-3水平升高,抑凋亡相关蛋白Bcl-2水平降低,且具有浓度依赖效应。在过氧化氢诱导损伤的小鼠星形胶质细胞中,miR-29a表达降低而水通道蛋白4和胶质纤维酸性蛋白的蛋白表达显著增加;过表达miR-29a能抑制水通道蛋白4的蛋白表达,抑制细胞凋亡。信息学软件预测水通道蛋白4可能是miR-29a的下游靶基因。②在小鼠脊髓撞击损伤模型中,与注射空载体病毒组比较,损伤部位过表达miR-29a能抑制水通道蛋白4和胶质纤维酸性蛋白的蛋白表达。③以上研究结果表明,miR-29a调控水通道蛋白4能够抑制过氧化氢诱导的星形胶质细胞凋亡。BACKGROUND:The role of miR-29a and aquaporin-4 in astrocyte injury has been demonstrated.However,the underlying mechanisms remain largely unknown.OBJECTIVE:To investigate the effects of the regulation of aquaporin-4 by miR-29a on hydrogen peroxide-induced astrocyte injury.METHODS:(1)Primary cultured mouse astrocytes were used to study the damage response of mouse astrocytes induced by different concentrations of hydrogen peroxide,and the cells were divided into control,hydrogen peroxide,hydrogen peroxide+empty vector,and hydrogen peroxide+miR-29a overexpression groups.The survival rate of mouse astrocytes was detected by MTT assay.The protein expression of apoptosis-related proteins(Bcl-2,Bax,and cleaved-Caspase-3),aquaporin-4 and glial fibrillary acidic protein was detected by western blotting.The expression of miR-29a in mouse astrocytes was detected by quantitative reverse transcription-polymerase chain reaction.Mouse astrocyte apoptosis was detected by flow cytometry.(2)Twelve female BALB/c mice were randomly divided into empty vector and miR-29a overexpression groups with six mice in each group.A T_(10) spinal cord contusion injury mouse model was established,and the injection of recombinant lentiviral vectors(empty and miR-29a overexpression vectors)was administered after modeling.The expression of aquaporin-4 and glial fibrillary acidic protein at the spinal cord injury site in mice was detected by immunohistochemical staining at 7 days after surgery.RESULTS AND CONCLUSION:(1)The activity of astrocytes and the expression of anti-apoptosis protein Bcl-2 were decreased,while the expression of proapoptosis proteins Bax and cleaved-caspase-3 was increased in a concentration-dependent manner after hydrogen peroxide-induced injury.In mouse astrocytes with hydrogen peroxide-induced injury,miR-29a expression was decreased,while protein expression of aquaporin-4 and glial fibrillary acidic protein was significantly increased;overexpression of miR-29a inhibited protein expression of aquaporin-4 and apoptosis.Aquapor

关 键 词：MIRNAS 水通道蛋白4 星形胶质细胞 凋亡 慢病毒 过氧化氢 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R826.64