自噬在高糖条件下视网膜色素上皮细胞表达血管内皮生长因子促进RF/6A细胞血管生成中的作用  

Autophagy promotes angiogenesis of RF/6A cells following upregulating the expression of vascular endothelial growth factor in retinal pigment epithelial cells under high glucose conditions

作　　者：李蓉[1] 姚杨[2] 杜军辉[3] 姚国敏[1] 王小娣[1] 张进[4] LI Rong;YAO Yang;DU Jun-Hui;YAO Guo-Min;WANG Xiao-Di;ZHANG Jin(Department of Ophthalmology,the First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,Shaanxi Province,China;Department of Central Laboratory,the First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,Shaanxi Province,China;Department of Ophthalmology,Xi’an Ninth Hospital,Affiliated to Medical College of Xi’an Jiaotong University ,Xi’an 710054,Shaanxi Province,China;Department of Ophthalmology,the First Hospital of Yulin ,Yulin 719000,Shaanxi Province,China)

机构地区：[1]西安医学院第一附属医院眼科,陕西省西安市710077 [2]西安医学院第一附属医院中心实验室,陕西省西安市710077 [3]西安交通大学附属西安市第九医院眼科,陕西省西安市710054 [4]榆林市第一医院眼科,陕西省榆林市719000

出　　处：《眼科新进展》2019年第8期714-718,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金青年基金项目(编号:81500726);陕西省卫生健康科研基金项目(编号:2018D074);西安市科技局医学研究项目[编号:201805097YX5SF31(4)]~~

摘　　要：目的研究高糖条件下诱导的人视网膜色素上皮细胞株(ARPE-19)自噬对其表达血管内皮生长因子(vascular endothelial growth factor,VEGF),及恒河猴脉络膜视网膜血管内皮细胞(RF/6A)迁移和管腔形成的影响。方法根据不同干预将ARPE-19细胞随机分为对照组、高糖组及3-甲基腺嘌呤(3-MA)+高糖组。对照组细胞在无糖DMEM培养基中常规培养24 h,高糖组细胞在DMEM培养基中加入30 mmol·L^-1葡萄糖溶液处理24 h,3-MA+高糖组细胞先用10 mmol·L^-1 3-MA处理24 h,然后更换培养基再加入30 mmol·L^-1葡萄糖溶液处理24 h。Western blot法检测细胞自噬标志性蛋白微管相关蛋白1轻链3(microtubule-related protein 1 light chain 3,LC3)II型和I型的比值(LC3-II/LC3-I)、Beclin-1及细胞中自噬相关基因3(autophagy-related gene 3,Atg3)的蛋白表达。ELISA法检测ARPE-19细胞上清液中VEGF的蛋白表达。比较三组ARPE-19细胞LC3-II/LC3-I、Beclin-1、Atg3及VEGF的蛋白表达量。然后将以上三组ARPE-19细胞上清液分别加入RF/6A细胞培养基中,将RF/6A细胞也按以上方法分组,分别采用Transwell法及Matrigel胶法检测并比较三组RF/6A细胞迁移数及细胞管腔形成数。结果对照组、高糖组和3-MA+高糖组ARPE-19细胞中LC3-II/LC3-I比值分别为0.405±0.095、0.932±0.024和0.635±0.048;Beclin-1蛋白相对表达量分别为0.205±0.035、0.590±0.120和0.425±0.082;Atg3蛋白相对表达量分别为0.277±0.035、0.539±0.071和0.389±0.019。ARPE-19细胞上清液中VEGF的蛋白表达量三组分别为(44.03±9.08)ng·L^-1、(205.70±17.90)ng·L^-1和(112.52±21.06)ng·L^-1;RF/6A细胞迁移数三组分别为(125.60±6.35)个、(153.60±19.20)个和(67.40±7.95)个;细胞管腔形成数三组分别为(12.22±0.84)个、(18.44±1.68)个和(5.44±0.51)个;整体比较差异均有统计学意义(均为P<0.01)。与对照组相比,高糖组ARPE-19细胞的LC3-II/LC3-I比值、Beclin-1及Atg3蛋白相对表达量均明显增加(均为P<0.01),VEGF表�Objective To study the effects of autophagy of human retinal pigment epithelial cell line(ARPE-19) induced by high glucose conditions on the expression of vascular endothelial growth factor(VEGF) as well as on the migration and tube formation of rhesus choroid-retinal endothelial cells(RF/6 A).Methods ARPE-19 cells were divided into the control group,high-glucose group and 3-methyladenine(3-MA)+high-glucose group.The cells in the control group were cultured in DMEM medium without glucose for 24 hours.The cells in the high-glucose group were cultured in DMEM medium adding 30 mmol·L^-1 glucose for 24 hours.The cells in the 3-MA+high-glucose group were pretreated with 10 mmol·L^-1 3-MA for 24 hours and then exposed to replaced medium adding 30 mmol·L^-1 glucose for 24 hours.The ratio of II and I form of microtubule-related protein 1 light chain 3(LC3)(LC3-II/LC3-I),Beclin-1 and autophagy-related gene 3(Atg3) protein expressions in the cells were detected by Western blot,and the expression of VEGF in the cell supernatants was examined by ELISA assay.The expressions of LC3-II/LC3-I,Beclin-1,Atg3 and VEGF among the three groups were compared.Then,the supernatant of ARPE-19 cells in the above three groups was respectively added into RF/6 A cell culture medium and RF/6 A cells were also grouped by the above method.The migration and tube formation of RF/6 A cells were detected by transwell and matrigel assay,respectively.The number of migrated cells and tube formation of the RF/6 A cells were compared.Results In the control group,high-glucose group and 3-MA+high-glucose group,the LC3-II/LC3-I ratio in ARPE-19 cells was 0.405±0.095,0.932±0.024 and 0.635±0.048,respectively;the relative expression of Beclin-1 protein in the cells was 0.205±0.035,0.590±0.120 and 0.425±0.082,respectively;and the relative expression of Atg3 protein in the cells was 0.277±0.035,0.539±0.071 and 0.389±0.019,respectively.The protein expression of VEGF in the supernatant of ARPE-19 cells was(44.03±9.08)ng·L^-1,(205.70±17.90)ng·L^-1 a

关 键 词：自噬 血管生成 高糖 血管内皮生长因子 视网膜新生血管 糖尿病视网膜病变 细胞培养 

分 类 号：R774.1[医药卫生—眼科]