mtDNA氧化损伤在人视网膜血管内皮细胞高糖代谢记忆过程中的作用  

Mitochondrial DNA oxidative damage induced by high glucose in“metabolic memory”of human retinal vascular endothelial cells

作　　者：杨金波 谢琳 YANG Jinbo;XIE Lin(Jiangxi Medical College,Nanchang University,the Department of Ophthalmology,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi Province,China)

机构地区：[1]南昌大学医学部,南昌大学第二附属医院眼科,江西省南昌市330006

出　　处：《眼科新进展》2020年第8期701-705,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号81160123,81460088);江西省青年科学基金(编号20152ACB21025);江西省科技计划项目(编号20141BBG70043)。

摘　　要：目的探讨mtDNA氧化损伤在人视网膜血管内皮细胞(hRECs)高糖代谢记忆过程中的作用。方法选择30 mmol·L^-1葡萄糖作为作用浓度,含浓度为5.5 mmol·L^-1葡萄糖的DMEM培养基作为正常培养基。将hRECs分为4组空白对照组对hRECs不做特殊处理,用正常培养基培养;高糖诱导3 h组用高糖作用hRECs 3 h,高糖诱导12 h组用高糖作用12 h,高糖诱导24 h组用高糖作用24 h,之后均转至正常培养基培养2 d。采用流式细胞仪检测hRECs细胞凋亡情况及hRECs内活性氧自由基(ROS)含量,Western blot检测hRECs细胞色素C蛋白表达,实时荧光定量PCR检测线粒体呼吸链复合物COX1 mRNA的表达,ELISA法检测hRECs线粒体内mtDNA氧化损伤标志物8-OHdG含量。结果流式细胞仪检测结果显示,对照组细胞凋亡率为6.94%。高糖诱导3 h组、12 h组及24 h组细胞凋亡率分别为10.30%、12.08%、13.55%;高糖诱导24 h组细胞凋亡率高于高糖诱导12 h组,差异有统计学意义(P<0.05),高糖诱导12 h组细胞凋亡率高于高糖诱导3 h组,差异有统计学意义(P<0.05);各高糖诱导组细胞凋亡率均高于对照组,差异均有统计学意义(均为P<0.05)。Western blot结果显示,各高糖诱导组细胞线粒体氧化损伤相关蛋白细胞色素C相对表达量均高于对照组,差异均有统计学意义(均为P<0.05);高糖诱导3 h组细胞色素C相对表达量低于12 h组和24 h组,差异均有统计学意义(均为P<0.05)。高糖诱导3 h组COX1 mRNA相对表达量高于12 h组和24 h组,差异均有统计学意义(均为P<0.05);各高糖诱导组COX1 mRNA相对表达量均低于对照组,差异均有统计学意义(均为P<0.05)。各高糖诱导组细胞ROS含量高于对照组,差异均有统计学意义(均为P<0.05);高糖诱导3 h组ROS含量低于12 h组,差异有统计学意义(P<0.05);高糖诱导12 h组ROS含量低于24 h组,差异有统计学意义(P<0.05)。各高糖诱导组线粒体氧化损伤标志物8-OHdG含量高于对照组,差异均有统计�Objective To investigate the effect of mitochondrial DNA(mtDNA)oxidative damage on hyperglycaemic memory in human retinal microvascular endothelial cells(hRECs).Methods We chose 30 mmol·L^-1 as the treatment concentration,and the DMEM medium with a concentration of 5.5 mmol·L^-1 glucose as a normal medium.The hRECs were divided into 4 groups the blank control group did not do special treatment for hRECs and cultivated with normal medium;the 3-hour group used high glucose for 3 hours,the 12-hour group used high glucose for 12 hours,and the 24-hour group used high glucose for 24 hours,and all of them were transferred to normal medium for 2 days.Flow cytometry was used to detect the apoptosis of hRECs and the content of reactive oxygen species(ROS)in hRECs.Western blot was used to detect the expression of cytochrome C protein.Real-time fluorescence quantitative PCR was used to detect the expression of COX1mRNA in the mitochondrial respiratory chain complex,and the ELISA method was used to detect the content of 8-OHdG in hRECs mitochondria.Results The results of flow cytometry showed that the apoptosis rate of the control group was 6.94%.The apoptosis rates of 3-hour group,12-hour group and 24-hour group were 10.30%,12.08%and 13.5%,respectively;the apoptosis rate in 24-hour group was higher than that of the 12-hour group,and the difference statistically significant(P<0.05),the 12-hour group was higher than the 3-hour group,and the difference was statistically significant(P<0.05).The apoptosis rate of each high glucose-induced group was higher than that of the control group,and the differences were statistically significant(all P<0.05).Western blot results showed that the relative expression of cytochrome C in each high glucose-induced group was higher than that in the control group,and the differences were statistically significant(all P<0.05);the 3-hour group was lower than the 12-hour group and the 24-hour group,and the differences were statistically significant(both P<0.05).The relative expression of COX1 mRNA i

关 键 词：糖尿病视网膜病变 高糖代谢记忆 MTDNA 活性氧自由基 氧化应激 

分 类 号：R774.1[医药卫生—眼科]