特异性启动子LEP503介导的自杀基因系统对晶状体上皮细胞增殖的抑制作用  

Selective cytotoxicity of LEP503 promoter-mediated suicide gene system on human lens epithelial cells

作　　者：杨晋[1] 卢奕[1] 刘天津 蒋永祥[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海市200031 [2]中科院上海生命科学院细胞研究所,上海市200031

出　　处：《眼科新进展》2011年第10期912-916,共5页Recent Advances in Ophthalmology

基　　金：上海市自然科学基金资助(编号:11ZR1406100);上海市卫生局青年科研基金(编号:2007Y06)~~

摘　　要：目的构建晶状体上皮细胞(lens epithelial cells,LEC)特异性启动子LEP503(lens epithelium gene product503)调控的Lenti-LEP503-HSV-tk-EGFP载体,观察慢病毒介导的单纯疱疹病毒胸苷激酶/丙环鸟苷系统(herps simplex virus thymidine kinase/ganciclov-ir,HSV-tk/GCV)自杀基因系统对人晶状体上皮细胞(HLEC)增殖的靶向性抑制作用。方法用RT-PCR方法从HLEC基因组中克隆LEP503启动子序列,构建单纯LEP503启动子调控HSV-tk基因表达的慢病毒载体Lenti-LEP503-HSV-tk-EGFP,同时构建广谱启动子巨细胞病毒(cytomegalo virus,CMV)调控的HSV-tk载体Lenti-CMV-HSV-tk-EGFP。将特异性表达载体Lenti-LEP503-HSV-tk-EGFP分别转入HLEC和Hela细胞,用荧光显微镜观察荧光强度,用RT-PCR方法评估相关蛋白的组织特异性表达。将Lenti-LEP503-HSV-tk-EGFP和Lenti-CMV-HSV-tk-EGFP分别转入HLEC,通过荧光显微镜观察EGFP的表达,用流式细胞仪及RT-PCR比较CMV与LEP503启动子介导下游靶基因HSV-tk的表达差异。结果 Lenti-LEP503-HSV-tk-EGFP能在HLEC中特异性表达,但Lenti-LEP503-HSV-tk-EGFP的表达效率明显低于Lenti-CMV-HSV-tk-EGFP。转染Lenti-LEP503-HSV-tk-EGFP的HLEC在20mg·L-1GCV作用48h后EGFP阳性细胞的形态发生明显变化;GCV作用24h后Lenti-LEP503-HSV-tk-EGFP转染HLEC较正常HLEC和Lenti-LEP503-HSV-tk-EGFP转染Hela细胞活性有明显抑制,且随时间的延长抑制效果逐渐增强。结论 LEP503启动子能介导HSV-tk基因在HLEC中特异表达,并介导HSV-tk/GCV自杀基因系统靶向性抑制HLEC增殖,但该启动子表达效率较低,所介导的HSV-tk/GCV系统对HLECs增殖的抑制效率低于CMV启动子。Objective To construct human lens epithelial cells（HLEC） specific promoter lens epithelium gene product 503（EP503）-mediated Lenti-HSV-tk-EGFP vector and investigate the selective cytotoxicity of lentivirus mediated suicide system of herps simplen virus thymidine kinase/ganciolovir（HSV-tk/GCV） on HLEC.Methods LEP503 promoter was cloned from the genome of HLEC,and Lenti-LEP503-HSV-tk-EGFP vector was constructed by LEP503 promoter replacing cytomegalo virus（CMV） promoter from Lenti-CMV-HSV-tk-EGFP vector.The lentiviral vectors were produced by transient transfection of transfering vector,packaging vector and enveloping vector into 293T cells.The HLEC and Hela cells were transfected with Lenti-LEP503-HSV-tk-EGFP,the specific expression of EGFP and HSV-tk proteins were detected by fluorescence microscopy or RT-PCR,respectively.After the HLEC were infected with Lenti-LEP503-HSV-tk-EGFP or Lenti-CMV-HSV-tk-EGFP,the cytotoxicity of two groups were assayed and compared by CCK-8 kit（Cell Counting kit-8 Cell Proliferation Assay）.Results Lenti-LEP 503-HSV-tk-EGEP can be specifically expressed in HLEC,but the expressional efficiency is less than Lenti-CMV-HSV-kt-EGFP.After transfected cell were cultured with 20 mg·L-1 GCV for 48 hours,the morphologic changes of apoptosis or necrosis were seen in EGFP positive cells.The viability of Lenti-LEP503-HSV-tk-EGFP transfected cells were inhibited less than that of CMV-HSV-tk transfected cells.The inhibitive effect increased with the time.Conclusions LEP503 promoter-mediated HSV-tk gene can be specifically expressed in HLEC,and mediate HSV-tk/GCV suicide system to target inhibit proliferation of HLEC.The inhibitory effect of the LEP503-mediated HSV-tk/GCV suicide system on HLEC is less than that of CMV-mediated the suicide system.

关 键 词：慢病毒 单纯疱疹病毒酶 胸苷激酶 晶状体上皮细胞 特异性启动子 LEP503 自杀基因系统 

分 类 号：R776[医药卫生—眼科]