BIGH3基因定点突变体R555W的构建及其对人角膜上皮细胞的影响

摘要

目的 构建野生型BIGH3和突变型R555W-BIGH3基因的真核表达载体,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R555W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,流式细胞仪和透射电镜定量和定性检查细胞凋亡,分析caspase3活性.多组间数据采用单因素方差分析进行统计学处理.结果 PCR成功扩增了野生型和R555W突变型的BIGH3基因.荧光显微镜下可见成功转染BIGH3/R555W-BIGH3-pIRES2-EGFP的CRL-11135细胞发绿色荧光,转染率为30.1%.瞬时转染48 h后的HCE细胞的上清液及其细胞裂解液免疫印迹分析显示,BIGH3/R555W-BIGH3-pIRES2-EGFP两组免疫印迹条带的密度比分别为1.26±0.06和1.27±0.08.正常HCE细胞组、转染pIRES2-EGFP空质粒及BIGH3-pIRES2-EGFP细胞凋亡率分别为(1.49 ±0.02)%、(1.53 ±0.02)%、(1.50 ±0.06)%,转染R555W-BIGH3-pIRES2-EGFP组细胞凋亡率达到19.46%±0.08%.差异有统计学意义(F=175 261.23,p<0.01).透射电镜下可见转染组细胞核内染色质凝集,固缩,分布于核膜下,核仁消失,有的胞核碎裂,细胞呈典型的凋亡改变.转染R555W-BIGH3-pIRES2-EGFP组caspase-3活性为561.03 ±1.05,与其他组相比,差异有统计学意义(F=629 347.38,P<0.01).结论 应用快速体外定点诱变技术,成功获得突变型R555W-BIGH3基因,转染人角膜上皮细胞后,通过caspase3途径引起细胞凋亡.%Objective To construct the eukaryotic express vector of wild type and R555W mutated transforming growth factor β induced ( BIGH3 ) gene, and to determine the effects of overexpression of R555W 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 R555W-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 cell apoptosis rates were observed by flow cytometry.The morphological changes of the cells were examined by electron microscopy.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 cell apoptosis were observed by flow cytometry and electron microscopy.The viability of caspase-3 was increased in the RSSSW mutated type.Conclusions The R555W mutated BIGH3 is successfully obtained by rapid site-directed mutagenesis technique.Overexpression of R555W mutated BIGH3 induces apoptosis in HCE cells through activation of caspase-3.