中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (19): 2993-2998.doi: 10.12307/2023.670

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

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

朱  琳1，郭世武2，刘锦波2，邹红军2   

  1. 常州市第一人民医院，1心内科，2脊柱外科，江苏省常州市   213003
  • 收稿日期:2022-07-28 接受日期:2022-09-26 出版日期:2023-07-08 发布日期:2022-11-28
  • 通讯作者: 邹红军，硕士，主治医师，常州市第一人民医院脊柱外科，江苏省常州市 213003
  • 作者简介:朱琳，女，1988年生，2015年苏州大学毕业，硕士，主治医师，主要从事分子生物学机制研究。
  • 基金资助:
    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

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

Zhu Lin1, Guo Shiwu2, Liu Jinbo2, Zou Hongjun2   

  1. 1Department of Cardiology, 2Department of Spine Surgery, The First People's Hospital of Changzhou, Changzhou 213003, Jiangsu Province, China
  • Received:2022-07-28 Accepted:2022-09-26 Online:2023-07-08 Published:2022-11-28
  • Contact: Zou Hongjun, Master, Attending physician, Department of Spine Surgery, The First People's Hospital of Changzhou, Changzhou 213003, Jiangsu Province, China
  • About author:Zhu Lin, Master, Attending physician, Department of Cardiology, The First People's Hospital of Changzhou, Changzhou 213003, Jiangsu Province, China
  • Supported by:
    A grant from the Health Commission of Jiangsu Province, No. Z2019014 (to ZHJ); the Application Basic Research Project of Changzhou City, No. CJ20220108 (to ZHJ); 2020 Qingmiao Youth Training Project of Changzhou City, No. CZQM2020046 (to ZHJ); Young Talent Science and Technology Project of Changzhou Health and Family Planning Commission, No. QN201704 (to ZHJ)

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摘要：


文题释义：

miRNA：是近年来发现的一类长度为22-24 nt 的内源、单链、非编码小RNA。成熟miRNAs 通过与mRNAs完全或不完全配对，诱导mRNAs的切割降解、翻译抑制或者其他的调节抑制，从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动。大量miRNAs已被证实在神经系统发育、神经系统内环境稳态等方面发挥作用。
细胞凋亡：是一种细胞程序性死亡过程，由细胞主动实施。在细胞凋亡过程中，细胞缩小，DNA被核酸内切酶降解成180-200 bp片段，细胞凋亡分为2个不同的阶段：启动阶段和执行阶段。启动阶段涉及到许多不同的蛋白质，由各种来自细胞外的或者细胞内的“压力”启动。启动阶段触发执行阶段，执行阶段涉及到激活拥有特殊功能的酶(半胱天冬酶和其他酶)，这些酶可以直接导致细胞死亡。

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。
https://orcid.org/0000-0003-1424-4420 (邹红军) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

Abstract: 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 T10 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 pro-apoptosis 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. Aquaporin-4 may be a downstream target gene of miR-29a as predicted using informatics software. (2) Overexpression of miR-29a at the injury site inhibited the protein expression of aquaporin 4 and glial fibrillary acidic protein in the spinal cord contusion injury mouse model compared with that of the empty vector injection group. (3) These findings suggest that the regulation of aquaporin 4 by miR-29a can inhibit hydrogen peroxide-induced astrocyte apoptosis.

Key words: miRNAs, aquaporin-4, astrocyte, apoptosis, lentivirus, hydrogen peroxide

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