COL4A1在miR-29b对N2B细胞氧糖剥夺/再灌注损伤保护作用中的调控机制研究  

Regulation and control mechanism of COL4A1 in the protective effect of mir-29b on oxygen glucose deprivation/reperfusion injury of N2B cells

作　　者：袁映楠 黄智 张帅 许敏 周石 YUAN Yingnan;HUANG Zhi;ZHANG Shuai;XU Ming;ZHOU Shi(School of Medical Imaging,Guizhou Medical University,Guiyang,Guizhou Province 550004,China)

机构地区：[1]贵州医科大学医学影像学院

出　　处：《介入放射学杂志》2019年第12期1156-1161,共6页Journal of Interventional Radiology

基　　金：贵州省普通高等学校医学影像工程研究中心建设项目(黔教合KY字2016-012);贵州医科大学医学影像工程研究中心建设项目(2016001)

摘　　要：目的通过构建N2B神经细胞氧糖剥夺(OGD)/再灌注(R)模型验证miR-29b对神经细胞的保护作用,探讨Ⅳ型胶原蛋白基因α1(COL4A1)在miR-29b对神经细胞OGD/R损伤保护作用中的调控机制。方法正常和缺氧条件培养N2B细胞,实时定量聚合酶链反应(RT-qPCR)检测miR-29b表达。细胞计数试剂盒(CCK)-8、克隆形成、Hoechst实验,流式细胞术分别检测不同处理组细胞增殖、凋亡情况。免疫印迹双荧光素酶验证miR-29b靶基因是否为COL4A1;N2B细胞转染miR-29b mimics,RT-qPCR和免疫印迹检测COL4A1表达。构建COL4A13’非翻译区(UTR)野生型和突变型载体、靶基因,设计合成COL4A1,转染N2B细胞,缺氧培养,CCK-8、克隆形成、Hoechst实验,流式细胞术检测不同处理组细胞增殖、凋亡情况。结果OGD/R细胞中miR-29b表达较正常细胞组降低。在相同缺氧/复氧条件下培养细胞中miR-29表达上调,细胞增殖能力明显增强,凋亡明显抑制。miR-29b表达上调抑制COL4A1表达,但转染COL4A1及其对照组后,COL4A1对照组细胞增殖明显高于过表达组,凋亡明显低于过表达组。结论miR-29b对N2B神经细胞OGD/R模型具有保护作用,通过抑制COL4A1表达减轻缺氧对N2B神经细胞的损伤。miR-29b可通过调节COL4A1表达减少缺血/缺氧再灌注对神经细胞的损伤。Objective To validate the protective effect of microRNA-29b on neurons by constructing oxygen and glucose deprivation/reperfusion(OGD/R) model of N2B neurons, and to explore the mechanism of COL4A1 in the protective effect of microRNA-29b on OGD/R injury of neurons. Methods N2B cells were cultured under normal and hypoxic conditions. Real-time quantitative PCR(qPCR) was used to detect the expression of microRNA-29b. CCK-8, clone formation, Hoechest testing and flow cytometry were used to detect the proliferation and apoptosis of N2B cells. Western blot(WB) double luciferase was used to verify whether the target gene of microRNA-29b was COL4A1. The microRNA-29b mimics were transfected into cells, and the expression of COL4A1 was detected by qPCR and WB. The wild and mutant vectors and target genes of COL4A1 3’untranslated region(UTR) were constructed. COL4A1 was designed and synthesized,transfected into N2B cells, cultured under hypoxia, CCK8, clone formation, Hoechest testing and flow cytometry were used to detect cell proliferation and apoptosis in different treatment groups. Results The expression of microRNA-29b in OGD/R cells was lower than that in normal cells. In the same hypoxia/reoxygenation conditions, the expression of microRNA-29b was up-regulated, the proliferation of cells was significantly enhanced, and cell apoptosis was significantly inhibited. Upregulation of the expression of microRNA-29b could inhibit the expression of COL4A1, but after transfection of both COL4A1 and its control group, the proliferation of cells in COL4A1 control group increased significantly when compared with that in overexpression group, and the apoptosis of cells in COL4A1 control group decreased significantly if compared with that in overexpression group. Conclusion The microRNA-29b has protective effect on OGD/R model of N2B neurons, it can reduce the damage of N2B neurons by inhibiting the expression of COL4A1. The microRNA-29b can reduce the damage of neurons by regulating the expression of COL4A1.(J Intervent Radiol

关 键 词：微小核糖核酸-29b 脑缺血 Ⅳ型胶原蛋口基因α1 成神经细胞瘤N2B细胞 

分 类 号：R743.3[医药卫生—神经病学与精神病学]