高糖环境对视网膜血管内皮细胞自噬的影响及机制  

Effect and related mechanism of high-glucose level on the autophagy of retinal vascular endothelial cells

作　　者：姚国敏[1] 李蓉[1] 姚杨[2] 杜军辉[3] 王小娣[1] 邓颖[1] Yao Guomin;Li Rong;Yao Yang;Du Junhui;Wang Xiaodi;Deng Ying(Department of Ophthalmology, the First Affiliated Hospital of Xi'an Medical University, Xi'an, Shaanxi 710077,China;Department of Central laboratory, the First Affiliated Hospital of Xi'an Medical University, Xi'an, Shaanxi 710077,China;Department of Ophthalmology, Xi'an Ninth Hospital, Affiliated to Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710054,China)

机构地区：[1]西安医学院第一附属医院眼科,西安710077 [2]西安医学院第一附属医院中心实验室,西安710077 [3]西安交通大学附属西安市第九医院眼科,西安710054

出　　处：《临床眼科杂志》2019年第4期361-366,共6页Journal of Clinical Ophthalmology

基　　金：国家自然科学基金(81500726);陕西省卫生健康科研基金(No.2018D074);西安市科技局医学研究项目(No.201805097YX5SF31(4))

摘　　要：目的研究高糖培养环境对恒河猴脉络膜视网膜血管内皮细胞(RF/6A)自噬的影响及机制。方法将体外培养的RF/6A细胞随机分为对照组、高糖组和3-甲基腺嘌呤(3-MA)+高糖组,对照组细胞在无糖培养基中培养,高糖组细胞在培养基中加入25mMD-glucose进行培养,3-MA+高糖组用5mg/ml3-MA预处理1.5h,然后置于含25mMD-glucose的培养基中继续培养。培养48h后,采用Westernblot法检测各组细胞的自噬相关蛋白微管相关蛋白1轻链3β(LC3B)、Atg5及关键信号通路蛋白PI3K、AKT、mTOR的表达,采用可表达mCherry-GFP-LC3B融合蛋白的腺病毒感染细胞,激光共聚焦显微镜下观察细胞内自噬的变化。结果①对照组、高糖组、3-MA+高糖组细胞中的LC3B-II/LC3B-I值、Atg5值分别为(0.19±0.02、0.58±0.06和0.43±0.04)和(0.35±0.05、0.96±0.06和0.83±0.06),组间总体比较差异均有统计学意义(LC3B-II/LC3B-I:F=55.58,P=0.000;Atg5:F=100.35,P=0.000)。高糖组细胞中LC3B-II/LC3B-I值和Atg5值均明显高于对照组和3-MA+高糖组,差异均有统计学意义(均P<0.05),3-MA+高糖组的LC3B-II/LC3B-I值和Atg5值均高于对照组,差异均有统计学意义(均P<0.05)。②对照组、高糖组、3-MA+高糖组细胞中的PI3K值、AKT值和mTOR值分别为(0.93±0.06、0.94±0.07和0.89±0.07)、(0.95±0.05、0.94±0.04和0.90±0.02)和(0.81±0.04、0.82±0.04和0.80±0.02),组间总体比较差异均无统计学意义(均P>0.05);三组磷酸化蛋白p-PI3K值、p-AKT值和p-mTOR值分别为(0.74±0.02、0.40±0.02和0.70±0.02)、(0.87±0.02、0.47±0.02和0.77±0.06)和(0.76±0.03、0.38±0.02和0.70±0.02),组间总体比较差异均有统计学意义(p-PI3K:F=249.92,P=0.000;p-AKT:F=100.94,P=0.000;p-mTOR:F=218.21,P=0.000)。高糖组细胞中p-PI3K、p-AKT和p-mTOR值均明显低于对照组和3-MA+高糖组,差异均有统计学意义(均P<0.05),3-MA+高糖组的p-PI3K、p-AKT和p-mTOR值均低于对照组,差异均有统计学意义(均P<0.05)。③对照组细胞浆中出�Objective To investigate the effect and related mechanism of high-glucose level on the autophagy of choroid-retinal endothelial cells(RF/6 A) in rhesus.Methods In vitro RF/6 A cells were randomly divided to the control group, high-glucose group and 3-methyladenine(3-MA)+high-glucose group. Cells in the control group were cultured in the glucose-free medium, cells in the high glucose group were cultured in the medium with 25 mM D-glucose, and cells in the 3-MA+high-glucose group were pre-treated with 5 mg/ml 3-MA for 1.5 hours and then cultured in the medium with 25 mM D-glucose. After 48 hours, western blot was performed to detect the expression of autophagy-associated proteins in different groups, including microtubule associated protein 1 light chain 3 B(LC3 B), Atg5 and key factors in autophagy signaling pathway(PI3 K, AKT and mTOR). RF/6 A cells were then transfected with Ad-mCherry-GFP-LC3 B and the changes of autophagy were observed under a laser confocal microscope. Results The value of LC3 B-II/LC3 B-I in the control group, high-glucose group and 3-MA+high-glucose group was 0.19±0.02, 0.58±0.06 and 0.43±0.04, respectively. The value of Atg5 in the above three groups was 0.35±0.05, 0.96±0.06 and 0.83±0.06, respectively. Significant differences exited among the groups(LC3 B-II/LC3 B-I:F=55.58, P=0.000;Atg5:F=100.35, P=0.000). The values of LC3 B-II/LC3 B-I and Atg5 were significantly higher in the high-glucose group than the control group and the 3-MA+high-glucose group(all with P<0.05). These two values were also higher in the 3-MA+high-glucose group as compared with the control group(all with P<0.05). The value of PI3 K, AKT and mTOR in the control group, high-glucose group and 3-MA+high-glucose group was(0.93±0.06, 0.94±0.07 and 0.89±0.07),(0.95±0.05, 0.94±0.04 and 0.90±0.02) and(0.81±0.04, 0.82±0.04 and 0.80±0.02), respectively. There were no significant differences among the groups(all with P>0.05). The value of p-PI3 K, p-AKT and p-mTOR in the control group, high-glucose group and 3-MA+

关 键 词：自噬 高糖 RF/6A PI3K/AKT/MTOR 

分 类 号：R774.1[医药卫生—眼科] R587.2[医药卫生—临床医学]