CCN1siRNA对视网膜血管内皮细胞活性的抑制及其机制  

Inhibition of CCN1 siRNA on retinal endothelial ceils

作　　者：底煜[1] 张轶欧[2] 王爱媛[1] 陈晓隆[1] 

机构地区：[1]中国医科大学附属盛京医院眼科,沈阳110004 [2]中国医科大学人事处,沈阳110122

出　　处：《中华实验眼科杂志》2016年第1期24-29,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：基金项目：国家自然科学基金项目（81371045）;辽宁省科技计划基金项目（2010225034）;辽宁省博士启动基金项目（201501020）

摘　　要：背景富含半胱氨酸蛋白61（Cyr61）/CCN1在早产儿视网膜病变（ROP）的视网膜新生血管（RNV）形成中发挥重要作用，但目前国内外关于CCN1小干扰RNA（CCN1 siRNA）对ROP有无延缓和治疗作用的研究较少。 目的探讨特异性抑制CCN1的表达对视网膜血管内皮细胞的调控作用。 方法恒河猴脉络膜－视网膜内皮细胞株（RF/6A）分别于常氧（正常对照组）和低氧环境（体积分数1%O2、5%CO2和94%N2的混合气体）中培养，低氧环境培养的细胞分别采用脂质体Lipofectamine？2000介导转染空载体质粒（低氧对照组）和CCN1 siRNA表达质粒（CCN1 siRNA转染组），于细胞转染后24 h采用逆转录PCR法检测细胞中CCN1 siRNA重组质粒的表达情况；分别于培养后0、24、48、72和96 h采用细胞计数试剂盒-8（CCK-8）法测定各组细胞活性并绘制细胞生长曲线；于培养后24 h采用流式细胞术检测各组培养细胞的增生和凋亡情况；于培养后24 h采用免疫荧光技术和Western blot法检测各组RF/6A细胞中CCN1和血管内皮生长因子（VEGF）蛋白的表达。 结果细胞转染后24 h用逆转录法检测到细胞中CCN1 siRNA的表达条带。CCK-8检测显示随着培养时间的延长，RF/6A细胞增生值（吸光度，A值）均明显增加，但CCN1 siRNA转染组各时间点细胞增生值均明显低于正常对照组和低氧对照组，总体比较差异均有统计学意义（F组别＝198.45，P〈0.05；F时间＝39.26，P〈0.05）；CCN1 siRNA转染组、正常对照组和低氧对照组细胞的凋亡率分别为（68.9±1.1）%、（18.9±1.3）%和（39.6±1.8）%，其中CCN1 siRNA转染组细胞的凋亡率明显高于正常对照组和低氧对照组，差异均有统计学意义（t＝2.93、2.56，均P〈0.05）；CCN1和VEGF蛋白在正常对照组细胞中呈弱表达，在低氧对照组细胞中表达均明显增强，CCN1 siRNA转染组细胞中CCN1和VEGF的相对表达量较低氧对照组均明显BackgroundCysteine-rich 61（Cyr61）/CCN1 has been reported to stimulate retinal neovascularization （RNV） in retinopathy of prematurity （ROP）. However, whether CCN1 small interfering RNA （CCN1 siRNA） can inhibit or cure ROP has not been extensively investigated. ObjectiveThis study was to investigate the regulation effect of CCN1 specific siRNA expression vector on retinal endothelial cells. MethodsRhesus choroid-retinal vascular endothelial cells （RF/6A） were cultured under the normoxic （normoxia control group） and hypoxic condition （1%O2, 5%CO2 with 94%N2） in vitro, and then lipofectamine？2000 （LF2000） vector plasmid with or without CCN1 siRNA was transiently transfected in the hypoxic-cultured cells as the CCN1 siRNA transfected group and hypoxic control group, respectively.Reverse transcription PCR was employed to detect the expression of CCN1 siRNA plasmid 24 hours after transfection.The vatality of the cells was assayed by cell counting kit-8（CCK-8） 0, 24, 48, 72 and 96 hours after cultured.Twenty-four hours after cultured, the apoptosis of the cells was evaluated by flow cytometry, and the expressions of CCN1 and vascular endothelial growth factor （VEGF） proteins were detected by immunofluorescence technique and Western blot assay. ResultsThe expression band of CCN1 siRNA was detected in the cells 24 hours after transfection of CCN1 siRNA.CCK-8 assay showed that RF/6A cells were significantly increased over time, and the proliferating value （absorbancy） of the cells was significantly reduced in the CCN1 siRNA transfected group compared with in the normoxia control group and hypoxic control group （Fgroup=198.45, P〈0.05; Ftime=39.26, P〈0.05）. The apoptosis rates of the cells were （68.9±1.1）%, （18.9±1.3）% and （39.6±1.8）% in the CCN1 siRNA transfected group, normoxia control group and hypoxic control group, and the apoptosis rates of the CCN1 siRNA transfected group were evidently higher than those of the normoxia control group and hypo

关 键 词：富含半胱氨酸蛋白61/生理 视网膜新生血管/预防及控制 早产儿视网膜病变/治疗 缺 氧 RNA干扰 血管内皮生长因子 恒河猴脉络膜一视网膜内皮细胞 

分 类 号：R774.1[医药卫生—眼科]