自噬抑制剂3-MA对高糖诱导的人视网膜色素上皮细胞增生的抑制作用  

Inhibitory effects of autophagy inhibitor 3-MA on high glucose-induced proliferation of human retinal pigment epithelium cells

作　　者：侯文文[1] 石焕琦[1] 张真[1] 张晓梅[1] 

机构地区：[1]哈尔滨医科大学附属第一医院眼科医院,150001

出　　处：《中华实验眼科杂志》2017年第1期5-9,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：2013年黑龙江省人社厅省级领军人才梯队后备带头人资助（创新研发类）资金项目

摘　　要：背景糖尿病视网膜病变（DR）是糖尿病常见的并发症之一，视网膜色素上皮（RPE）细胞作为视网膜重要的组成细胞在DR的发生和发展中至关重要。目的探讨自噬抑制剂3-MA对高糖培养的人RPE细胞（hRPECs）增生的影响。方法将体外培养的hRPECs分为对照组、高糖组和3-MA＋高糖组，对照组细胞用含5mmol／L葡萄糖的DMEM／F12培养基进行培养，高糖组采用含30mmol／L葡萄糖的DMEM／F12培养基进行培养，干预组细胞先在DMEM／F12培养基中添加10mmol／L的3-MA处理1h后，再添加30mmol／L葡萄糖溶液进行培养。将培养的细胞以1×10^5／孔的密度接种于24孔细胞培养板中，待细胞80％融合后用含体积分数0．5％胎牛血清的培养基继续孵育24h，使细胞周期同步化。倒置光学显微镜和透射电子显微镜下观察各组jRPECs的形态及超微结构；采用CCK-8法检测各组hRPECs的增生率；采用Western blot法检测各组hRPECs中自噬相关基因微管相关蛋白轻链3B（LC3B）蛋白的表达。结果对照组细胞生长状态良好，细胞排列整齐；高糖组细胞体积稍增大，细胞数量明显多于对照组，而3-MA＋高糖组细胞形态不规则，排列紊乱。透射电子显微镜下可见对照组细胞核呈圆形或卵圆形，亚细胞器形态均正常，未发现自噬小体；高糖组细胞中可见自噬溶酶体，而3-MA＋高糖组可见自噬小体。对照组、高糖组、3-MA＋高糖组细胞的增生率分别为（100．0±2．0）％、（116．9±5．2）％和（103．7±4．7）％，总体比较差异有统计学意义（F=13．526，P=0．006），高糖组的细胞增生率明显高于对照组和3-MA＋高糖组，差异均有统计学意义（均P〈0．05），3-MA＋高糖组与对照组间细胞增生率的差异无统计学意义（P〉0．05）。与对照组比较，高糖组细胞中LC3B—Ⅰ蛋白表达减弱，LC3B-Ⅱ蛋白表达增强，而3-MA＋高糖组细胞中LC3B—Ⅰ�Background Diabetic retinopathy （DR） is one of the common complications of diabetes. Retinal pigment epithelium （RPE） cells, as important constituent cells of the retina, play important roles in the development and progression of DR. Objective This study was to investigate the suppressive effects of autophagy inhibitor 3-MA on the proliferation of human retinal pigment epithelium cells （hRPECs） following high glucose culture. Methods HRPECs were divided into control group,high-glucose group and 3-MA＋high glucose group. The cells were cultured by the DMEM/F12 with 5 mmol/L glucose in the control group,and by the DMEM/F12 with 5 mmol/L glucose in the high glucose group and by the DMEM/F12 with 10 mmol/L 3-MA （for 1 hour firstly） and 30 mmol/L glucose in the 3-MA＋high glucose group. The cells were inoculated into 24-well plate with the content of 1×105/well,and then the cells were consecutively cultured for 24 hours with DMEM/F12 containing 0. 5% fetal bovine serum after achieved attached 80% confluence. The morphology and ultrastructure of the cells were examined by optics microscope and transmission electronic microscope. The proliferative rate of the ceils was assayed by CCK -8 kit. The expression of autophagy-related gene microtubule associated protein light chain 3B （LC3B） in the ceils was detected by Western blot and LC3B-Ⅱ/LC3B- Ⅰ value was quantitatively evaluated among the groups. Results The cells grew well with uniform size and regulatory arrangement in the control group. The cells in the high glucose group enlarged and the number of cells evidently increased. In the 3-MA＋high glucose group,the ceils decreased with a disorder arrangement. Under the transmission electron microscope,the cells were normal with the roundor oval-like nucleus and normal organelles in the control group,and autolysosome could be seen in the cells in the high glucose group. In the 3-MA ＋high glucose group, some autophagic bodies were found. The proliferative rate of the cells was （ 100. 0±2.0 �

关 键 词：自噬/药物作用 葡萄糖/药理 视网膜色素上皮细胞/药物作用 超微结构 生物标志物 人 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]