野生型及跨膜型肿瘤坏死因子真核表达载体构建及其对心肌细胞凋亡的影响  

Construction of Eukaryotic Expression Vector of Wild-type/Transmembrane TNF and The Effect on Cardiomyocyte Apoptosis

作　　者：苗琨[1] 周玲[1] 李卓娅[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院高血压病研究所,武汉市430030 [2]华中科技大学同济医学院基础医学院免疫学系,武汉市430030

出　　处：《中国分子心脏病学杂志》2013年第5期677-681,共5页Molecular Cardiology of China

基　　金：国家自然科学基金重大研究计划(91029709)

摘　　要：目的构建鼠野生型肿瘤坏死因子(Wild type tumor necrosis factor-alpha,wtTNF-α)和跨膜型肿瘤坏死因子(Transmembrane tumor necrosis factor-alpha,tmTNF-α)基因真核表达载体,转染心肌细胞,比较两型TNF-α对心肌细胞凋亡的影响。方法应用RT-PCR方法从小鼠腹腔巨噬细胞中扩增wtTNF-α基因编码区,根据小鼠TNF氨基酸序列设计缺失肿瘤坏死因子转化酶(TNF alpha converting enzyme,TACE)作用位点(缺失1-12位氨基酸)的突变引物,通过重叠PCR得到构建不被TACE剪切的TNF-α突变体,即跨膜型肿瘤坏死因子tm TNF-α,扩增得到的wt TNF-α,tm TNF-αPCR产物经EcoR I+BanH I双酶切后克隆到pIRES2-EGFP真核表达载体中并转染入大鼠H9c2(2-1)心肌细胞,用RT-PCR及Western Blot方法鉴定转染效率及不同表达载体对心肌细胞凋亡的影响。结果成功构建pIRES2-eGFP-wtTNF和pIRES2-eGFP-tmTNF真核表达载体并转染大鼠H9c2(2-1)心肌细胞,tmTNF-α对心肌细胞无明显促凋亡作用,而wtTNF-α可以诱导心肌细胞凋亡。结论由于wtTNF-α可被酶解产生sTNF-α,提示sTNF-α,而并非跨膜型TNF-α对该心肌细胞株具有诱导凋亡的作用。Objective Construction of eukaryotic expression vector for mouse Wild-type tumor necrosis factor-alpha （wtTNF〈0 and transmembrane tumor necrosis factor-α（tmTNF-α. The influence of both forms of TNF-α on apoptosis was compared in H9c2 （2-1） cell line. Methods RT-PCR was used to amplify the coding region of wtTNF-α gene from murine macrophages. Non-cleavable tmTNF-α mutant was made by deletion of cleave site of TACE （No. 1-12 amino acids） using overlap PCR, The PCR products were cloned into the eukaryotic expression vector pIRES2-EGFP. The plasmids were transfected into H9c2 （2-1） cardiac cell lines. The transfection efficiency and the impact of both forms of TNF-α on the apoptosis in H9c2 （2-1） cardiac ceU lines were analyze by RT-PCR and Western Blot. Results We successfully constructed pIRES2-eGFP-wtTNF and pIRES2- eGFP-tmTNF eukaryotic expression vector and transfected into H9c2 （2-1） cardiac cell lines. There was a significant pro-apoptotic effect in wtTNF〈q but not in tmTNF-expressing cardiac cells. Conclusion As sTNF-α can be released by tmTNF-α, our results indicate that it is sTNF-α, not tmTNF-α, leading to apoptosis in H9c2 （2-1） cardiac cell line.

关 键 词：跨膜型肿瘤坏死因子 载体构建 转染 心肌细胞 凋亡 

分 类 号：R541[医药卫生—心血管疾病]