长链非编码RNA MEG3在高糖诱发大鼠神经细胞损伤中的作用:与线粒体途径凋亡的关系  

Role of long non-coding RNA MEG3 in hyperglycose-induced neurocyte damage in rats:relationship with mitochondrion-dependent apoptosis

作　　者：玛丽江·木拉提 王志华[2] 陈诚[1] 黄炎 夏萍萍[1] 张帆[1] 王锷[1] 郭曲练[1] 叶治[1] Marjan Murat;Wang Zhihua;Chen Cheng;Huang Yan;Xia Pingping;Zhang Fan;Wang E;Guo Qulian;Ye Zhi(Department of Anesthesiology,Xiangya Hospital of Central South University,Changsha 410005,China;Department of Anesthesiology,Hainan General Hospital,Haikou 570311,China)

机构地区：[1]中南大学湘雅医院麻醉科,长沙410005 [2]海南省人民医院麻醉科,海口570311

出　　处：《中华麻醉学杂志》2019年第10期1176-1180,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81771422);湖南省自然科学基金(2019JJ50931)。

摘　　要：目的评价长链非编码RNA母系表达基因3(MEG3)在高糖诱发大鼠神经细胞损伤中的作用及其与线粒体途径凋亡的关系。方法正常培养PC12细胞,采用随机数字表法分为5组(n=18):正常浓度葡萄糖对照组(C组):用含25 mmol/L葡萄糖的培养基培养;正常浓度葡萄糖+MEG3组(C+MEG3组):MEG3慢病毒载体(LV-MEG3)转染PC12细胞后,用含25 mmol/L葡萄糖的培养基培养;高浓度葡萄糖组(HG组):用含250 mmol/L葡萄糖的培养基孵育;高浓度葡萄糖+MEG3组(HG+MEG3组):经LV-MEG3转染后用含250 mmol/L葡萄糖的培养基孵育;高浓度葡萄糖+阴性慢病毒载体(LV-NC)(HG+NC组):经LV-NC转染后用含250 mmol/L葡萄糖的培养基孵育。培养或孵育1 d后,采用CCK-8法检测细胞活力,流式法检测细胞凋亡率和ROS水平,采用DCFH-DA法检测LDH漏出量;Western blot法测定细胞色素c(Cyt c)、caspase-3、caspase-9、Bcl-2、Bax和Apaf-1的表达水平,计算Blc-2/Bax比值;荧光法检测线粒体膜通透性转换孔(mPTP)的开放程度。结果与C组比较,HG组、HG+MEG3组和HG+NC组细胞活力降低,LDH漏出量、ROS水平和细胞凋亡率升高,mPTP开放增加,caspase-3、caspase-9、Cyt c、Bax、Bcl-2、Apaf-1表达上调,HG+MEG3组Bcl-2/Bax比值升高,HG组和HG+NC组Bcl-2/Bax比值降低(P<0.05);与HG组和HG+NC组比较,HG+MEG3组细胞活力升高,LDH漏出量、ROS水平和细胞凋亡率降低,mPTP开放减少,caspase-3、caspase-9、Cyt c、Bax、Apaf-1表达下调,Bcl-2表达上调,Blc-2/Bax比值升高(P<0.01)。结论MEG3可能通过抑制线粒体途径凋亡,参与高糖诱发神经细胞损伤时的内源性保护机制。Objective To evaluate the role of long non-coding RNA maternally expressed gene 3(MEG3)in hyperglycose-induced neurocyte damage and the relationship with mitochondrion-dependent apoptosis in rats.Methods Normally cultured PC12 cells were divided into 5 groups(n=18 each)using a random number table method:normal concentration of glucose control group(C group),normal concentration of glucose plus MEG3 group(C+MEG3 group),high-concentration glucose group(HG group),high-concentration glucose plus MEG3 group(HG+MEG3 group),and high-concentration glucose plus negative lentiviral vector(LV-NC)group(HG+NC group).PC12 cells were cultured in DMEM medium with 25 mmol/L glucose in group C.PC12 cells were cultured in DMEM medium with 25 mmol/L glucose after being transfected with MEG3 lentiviral vector(LV-MEG3)in C+MEG3 group.PC12 cells were cultured in DMEM medium with 250 mmol/L glucose in HG group.PC12 cells were incubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-MEG3 in HG+MEG3 group.PC12 cells were incubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-NC in HG+NC group.After the cells were cultured or incubated for 1 day,the cell viability was measured by CCK8 assay,the apoptosis rate and level of reactive oxygen species(ROS)were determined by flow cytometry,and the amount of lactic dehydrogenase(LDH)released was measured by DCFH-DA,the expression of Cyt c,caspase-3,caspase-9,Bcl-2,Bax and Apaf-1 was determined by Western blot,and the opening of mitochondrial permeability transition pore(mPTP)was determined by fluorescent method.Blc-2/Bax ratio was calculated.Results Compared with group C,the cell viability was significantly decreased,the amount of LDH released,ROS level and apoptosis rate were increased,the opening of mPTP was increased,and the expression of caspase-3,caspase-9,Cyt c,Bax,Bcl-2 and Apaf-1 was up-regulated in HG,HG+MEG3 and HG+NC groups,and Bcl-2/Bax ratio was increased in HG+MEG3 group and decreased in HG and HG+NC groups(P<0.05).Compare

关 键 词：RNA 长链非编码 糖尿病 神经元 细胞凋亡 

分 类 号：R587.1[医药卫生—内分泌]