大鼠Slit2基因慢病毒干扰载体的构建及其对大鼠RPE细胞中Slit2基因的下调作用  

Construction of combined lentiviral vectors for rat Slit2 gene RNA interfering and its down-regulation effects on Slit2 gene in rat retinal pigment epithelial cells

作　　者：蒋少秋 周希瑗[1] 

机构地区：[1]重庆医科大学附属第二医院眼科重庆市眼科研究所眼科学重庆市市级重点实验室,400010

出　　处：《中华实验眼科杂志》2017年第8期683-689,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81170858）;重庆市科学技术委员会项目（cstc2015shmszx120068）

摘　　要：背景年龄相关性黄斑变性（AMD）是老年人致盲的首要原因。目前，AMD的治疗方式在取得显著疗效的同时亦有一定的不足，寻找新的有效治疗靶点是当前的研究热点。目的构建大鼠Slit2基因慢病毒干扰载体，筛选有效干扰序列以用于大鼠视网膜色素上皮（RPE）细胞中Slit2基因沉默，为大鼠相关体内实验奠定基础。方法设计2条大鼠Slit2基因siRNA序列，退火形成DNA，连接形成慢病毒干扰载体并进行测序鉴定。采用三质粒共转染293T细胞法收获慢病毒（Lv-rSlit2-siRNA），用药物筛选法测定病毒悬液滴度。将大鼠RPE细胞分为空白对照组、空病毒组、Lv—rSlit2-siRNAl组和Lv—rSlit2-siRNA2组，根据分组分别用仅有病毒的载体和不同序列的Lv—rSlit2-siRNA载体转染细胞，分别采用实时荧光定量PCR法和Western blot法测定各组细胞中Slit2mRNA及蛋白的表达以确定Slit2基因的敲减率，筛选有效干扰序列。采用0、100、200和400μmol／LCoCl，孵育大鼠RPE细胞以制备缺氧细胞模型并转染筛选Lv—rSlit2-siRNA2干扰序列，采用实时荧光定量PCR法和ELISA法测定大鼠RPE细胞中血管内皮生长因子A（VEGFA）表达变化及细胞上清液中VEGFA质量浓度。结果成功构建2条大鼠Slit2基因慢病毒干扰载体，病毒滴度分别为5×10^8TU／ml和3×10^8TU／ml，转染病毒后72h于荧光显微镜下观察RPE细胞慢病毒转染率均在70％以上。Lv—rSlit2-siRNAl组和Lv—rSlit2-siRNA2组大鼠RPE细胞中Slit2mRNA相对表达量分别为0．67±0．09和0．23±0．11，明显低于空白对照组的1．03±0．31和空病毒组的0．92±0．07；Lv—rSlit2-siRNAl组和Lv—rSlit2-siRNA2组大鼠RPE细胞中Slit2蛋白相对表达量分别为0．62±0．07和0．49±0．02，明显低于空白对照组的1．00±0．10和空病毒组的0．95±0．11，差异均有统计学意义（均P〈0．01）。0、100、200和400μmol／LCoCl，作用后，空白对照Background Age-related macular degeneration （AMD） is the leading cause of blindness in people over 50 years old, of which 90% cases are caused by choroidal neovascularization （ CNV）. Current treatments on AMD have gained great achievements,but there are still some drawbacks. So we need to search for new targets to cure CNV. Objective This study was to construct two combined lentiviral vectors for rat Slit2 gene RNA interference （RNAi） and verify its interfering effects on Slit2 gene in rat retinal pigment epithelial （RPE） cells. Methods Two specific siRNA sequences targeting towards rat Slit2 gene were designed and were annealed to DNA sequences. The DNA sequences and GV248-enhanced green flourescent protein （EGFP） vectors were combined together as recombinant vectors and then were identified. The GV248-EGFP vector, helper 1.0 and helper 2.0 were transfected together into 293T ceils and the two combined lentiviral vectors for rat Slit2 RNAi were gained from the cell supernatant after 72 hours of transfection. The titers of the combined lentiviral vectors were measured. The cells were divided into blank control group, Lv-EGFP vector group, Lv-rSlit2-siRNA1 group and Lv-rSlit2-siRNA2 group. The interference efficacy of the combined lentiviral vectors targeting to rat Slit2 gene were identified by real-time fluorescence quantitative PCR and Western blot. The sequence with higher interference efficacy was transfected to rat RPE cells again. The transfected and nontransfected rat RPE ceils were treated with 0,100,200 and 400 μmol/L CoCI2 for the preparation of hypoxia models. The expression of vascular endothelial growth factor-A （VEGFA） mRNA in rat RPE ceils was finally measured by real-time fluorescence quantitative PCR and the concentration of VEGFA protein in cell supernatant was assayed by ELISA. Results The recombined lentiviral vectors for rat Slit2 gene RNAi were successfully constructed. The titers of the two recombinant sequences were 5 xl0s TU/ml and 3 ×10^8 TU/ml, with the transfe

关 键 词：慢病毒 转染技术 RNA干扰 视网膜色素上皮/基因 血管内皮生长因子A/代谢 

分 类 号：R774.5[医药卫生—眼科]