TIGAR基因真核表达载体的构建及在HepG_2细胞中的作用初探

Construction of TIGAR gene eukaryotic expression vector and effect of TIGAR expression in HepG_2 cells

目的:构建TP53诱导的糖酵解和凋亡调控子(TIGAR)全长cDNA的真核表达载体pEGFP-TIGAR,观察TIGAR在人肝细胞HepG2中的作用。方法:RT-PCR技术扩增获得TIGAR全长cDNA,将扩增的产物克隆,经酶切、DNA测序鉴定正确,构成pEGFP-TIGAR的真核表达重组质粒。利用脂质体将重组质粒转染入HepG2细胞,流式细胞仪和TUNEL法检测细胞凋亡、流式细胞仪检测细胞周期、MIB-1(Ki-67抗原测定)法检测细胞增殖、实时荧光PCR检测mRNA表达和Western-blot检测蛋白质的表达。结果:构建完成的真核表达重组质粒pEGFP-TIGAR,经DNA测序与GenBank中的人TIGARcDNA序列一致。荧光显微镜观察到转染重组质粒的细胞中有绿色荧光蛋白的表达。检测结果显示,转染pEGFP-TIGAR质粒的HepG2晚期凋亡细胞明显减少(P<0.05);S期细胞百分数则明显增加(P<0.05),增殖能力明显增高(P<0.05)。结论:本实验成功构建了pEGFP-TIGAR真核表达质粒,过表达的TIGAR可降低HepG2细胞发生晚期凋亡和促进细胞增殖,为进一步研究TIGAR基因在肿瘤细胞中作用提供了基础。

Objective To construct TP53-induced glycolysis and apoptosis regulator （TIGAR）gene full-length eDNA eukaryotie expression vector pEGFP-TIGAR and to assess its functioning in human HepG2 cell. Methods Fulllength eDNA encoding TIGAR gene was obtained by reverse transcription polymerase chain reaction （RT-PCR） and cloned to eukalyotic expression vector pEGFP-N1, the recombinant pEGFP-TIGAR was identified by double digestion with restriction endonuelease and sequencing, The recombinant plasmids were transfeeted into HepG2 cells by lipofectamine method. Expression of TIGAR and TIGAR mRNA in HepG2 cells was determined by Western blot and real time fluorescence PCR, respectively. Cell apoptosis and cell cycle were detected by flow cytometry and TUNEL, cell proliferation capacity was assessed by MIB-1（deteetion of Ki-67 antigen） assay. Results DNA sequencing showed that the constructed eukaryotic expression recombinant plasmid pEGFP-TIGAR was consistent with human TIGER cDNA in GenBank. Flow cytometry and TUNEL test showed that late apoptotic cells were greatly reduced in pEGFP-TIGAR transfected HepG2 cells （P〈0.05）and the percentage of cells in S stage increased significantly （P〈0,05）; MIB-1 assay showed that cell proliferation increased markedly （P〈0.05）. Conclusions In this study, pEGFP-TIGAR eukaryotic expression plasmid was successfully constructed. Overexpression of TIGAR can reduce late stage apoptosis and promote cell proliferation in HepG2 cells, which provides a basis for further study of the role of TIGAR gene in tumor cell biology.