基因沉默解离素-金属蛋白酶17(ADAM17)对高糖环境下人视网膜微血管内皮细胞通透性、增殖、迁移以及蛋白表达的影响  

Effects of ADAM17 on high-glucose mediated permeability,proliferation,migration and proteins expression in human retinal microvascular endothelial cells

作　　者：吕伯昌 刘蓓 刘静[1] 杨新光[1] 赵曦泉 朱忠桥 LV Bo-Chang LIU Bei LIU Jing YANG Xin-Guang ZHAO Xi-Quan ZHU Zhong-Qiao

机构地区：[1]陕西省眼科诊疗中心西安市第四医院眼科西安交通大学医学院附属广仁医院眼科,陕西省西安市710004

出　　处：《眼科新进展》2017年第3期205-209,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81273902)~~

摘　　要：目的探讨基因沉默解离素-金属蛋白酶17(a disintegrin and metalloproteinase 17,ADAM17)对高糖环境下人视网膜微血管内皮细胞(human retinal microvascular endothelial cells,HRMECs)通透性、增殖、迁移以及蛋白表达的影响。方法将HRMECs分为4组:正常组(5 mmol·L^(-1)D-葡萄糖)、高糖组(25 mmol·L^(-1)D-葡萄糖)、NC(siRNA阴性对照)+高糖组和siADAM17(ADAM17 siRNA)+高糖组。ADAM17表达采用实时定量PCR和蛋白印迹法检测;辣根过氧化酶法检测HRMECs通透性;CCK-8和Brd U检测细胞增殖;Transwell检测细胞迁移;蛋白印迹法检测p-EGFR、p-ERK和MMP9表达水平。结果与正常组相比,高糖组ADAM17蛋白表达水平显著升高(P<0.01),NC+高糖组与高糖组相比无明显差异(P>0.05);与NC+高糖组比较,siADAM17+高糖组ADAM17蛋白表达水平显著下调(P<0.05)。沉默ADAM17可降低高糖环境下HRMECs的通透性。沉默ADAM17可抑制高糖介导的细胞增殖。Transwell实验结果显示,各组相对迁移的细胞数分别为高糖组(157.00±7.93)个、NC+高糖组(169.00±10.12)个、siADAM17+高糖组(121.00±9.28)个、正常组(110.00±8.25)个。沉默ADAM17可抑制高糖介导的细胞迁移。siADAM17+高糖组较NC+高糖组p-EGFR、p-ERK和MMP9蛋白表达显著减少(均为P<0.01)。结论基因沉默ADAM17可能通过EGFR、ERK、MMP9通路降低高糖诱导的细胞通透性并抑制细胞增殖和迁移。Objective To investigate the effects and mechanisms of a disintegrin and metalloproteinase 17 （ADAM17） on high-glucose mediated permeability,proliferation and migration in human retinal microvascular endothelial cells （HRMECs）.Methods HRMECs were divided into 4 groups：normal group （5 mmol·L-1 glucose）,high glucose group （25 mmol·L-1 glucose）,NC （Negative control for siRNA）＋high glucose group and siADAM17 （ADAM17 siRNA）＋high glucose group.The expression of ADAM17 was detected using real time PCR and Western blot.Horseradish Peroxidase （HRP） was used to detect the permeability of HRMECs.Cell Counting Kit-8 （CCK-8） and BrdU were used to evaluate cell proliferation.Cell migration was determined using Transwell assay.In addition,the expression of p-EGFR,p-ERK and MMP9 was assayed using Western blot.Results Compared with normal group,the mRNA and protein levels of ADAM17 were increased in high glucose group （P〈0.01）.ADAM17 expression of siADAM17＋high glucose group was markedly reduced compared with NC＋high glucose group.High glucose increased the permeability of HRP comparison to normal group,whereas in siADAM17＋high glucose group the permeability of HRP was reduced compared with NC＋high glucose group.The optical density of HRMECs was decreased in siADAM17＋high glucose group 1.53±0.29 in comparison with NC＋high glucose group 2.43±0.25,as well as the content of BrdU-incorporation（P〈0.05）.The number of migrated cells in high glucose group,NC＋high glucose group,siADAM17＋high glucose group and normal group were 157.00±7.93,169.00±10.12,121.00±9.28,110.00±8.25,respectively.Moreover,the expression of p-EGFR,p-ERK and MMP9 in siADAM17＋high glucose group was decreased compared with NC＋high glucose group （all P〈0.01）.Conclusion SiADAM17 can reduce the cell permeability,suppressed and migration induced by high glucose via EGFR/ERK/MMP9 signaling pathway.

关 键 词：解离素-金属蛋白酶17 细胞通透性 细胞迁移 

分 类 号：R774[医药卫生—眼科]