BIGH3基因突变子R124C的过表达及其对人角膜上皮细胞的影响  

Construction of a site-directed mutant of R124C-BIGH3 and its influence on human corneal epithelial cells

作　　者：殷秀丽[1] 刘平[1] 金迪[1] 赵秀梅[1] 张丽娟[1] 葛红岩[1] 

机构地区：[1]哈尔滨医科大学第一临床医学院眼科医院,黑龙江哈尔滨150001

出　　处：《哈尔滨医科大学学报》2010年第6期535-539,共5页Journal of Harbin Medical University

基　　金：黑龙江省教育厅资助项目(11531177)

摘　　要：目的对我国常见的BIGH3基因相关性人角膜营养不良的热点突变R124C进行体外定点诱变研究,并构建其真核表达载体,探讨其过表达对人角膜上皮细胞的影响。方法以pMD18-T/BIGH3载体为模板扩增野生型BIGH3基因全长编码序列,克隆至真核表达载体pIRES2-EGFP上,采用快速体外定点诱变技术获得R124C突变体。瞬时转染体外培养的人角膜上皮细胞,免疫印迹检测野生与突变型BIGH3基因的表达,分别检测细胞培养基中TNF-α的含量,并收集细胞,分析caspase-3活性。结果 PCR成功扩增了野生型和R124C突变型的BIGH3基因,两种重组表达质粒转染人角膜上皮细胞后,免疫印迹检测结果证实其均在细胞中表达。细胞因子TNF-α分泌量增加,同时R124C型突变也可导致caspase-3酶活性增高。结论应用快速体外定点诱变技术,成功获得突变型R124C-BIGH3基因,转染人角膜上皮细胞后,通过caspase-3途径引起细胞凋亡,并上调分泌因子TNF-α。Objective To perform PCR-based site-directed mutagenesis of BIGH3 common mutations R124C identified in a Chinese family with corneal dystrophies.To construct the eukaryotic express vector of wild type and R124C mutated transforming growth factor β induced（BIGH3） gene,and to determine the effects of overexpression of R124C mutated BIGH3 in human corneal epithelial（HCE） cells.Methods The full coding domain sequence of BIGH3 gene was cloned from human corneal tissue from operative spaceman with RT-PCR.Mutagenesis of R124C-BIGH3 was performed in rapid site-directed mutagenesis technique in the expression vector pIRES2-EGFP.The two types of BIGH3 gene were transient transfected to HCE cells.Immunoblotting was used to detect the expression of BIGH3.The cytokine content of TNF-α in the culture medium was tested quantitatively with ELISA kit.The viability of caspase-3 was examined.Results Wild type and mutated BIGH3 gene were successfully amplified by PCR.After HCE cells transfected with two types eukaryotic expression plasmind,the BIGH3 were detected in the HCE cells.The overexpression of R124C increased the level of TNF-α and upregulate the apoptosis rate of HCE cells.The viability of caspase-3 was increased in the R124C mutated type.Conclusion The R124C mutated BIGH3 is successfully obtained by rapid site-directed mutagenesis technique.Overexpression of R124C mutated BIGH3 induces apoptosis in HCE cells through activation of caspase-3 and upregulate TNF-α secretion of HCE cells.

关 键 词：BIGH3 定点诱变 人角膜上皮细胞 瞬时转染 凋亡 TNF-α 

分 类 号：R768[医药卫生—耳鼻咽喉科]