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目的 :探讨截短型bid(truncatedbid ,tbid)基因的表达对Hela细胞的促进凋亡活性。方法 :用RT PCR法克隆人全长bid基因 ,测序正确后 ,通过PCR截去编码N末端 6 0个氨基酸残基的基因 ,而获得tbid基因。将其克隆入含绿色荧光蛋白(GFP)基因的真核表达载体pIRES2 EGFP中 ,用脂质体法转染Hela细胞。通过荧光显微镜、电子显微镜观察和TUNEL检测法 ,检测目的基因的表达对转染细胞的形态及生长状况的影响。结果 :成功地构建了tbid基因的真核表达载体。以其转染Hela细胞后 ,tbid基因在细胞中得到表达 ,随后引起细胞荧光强度下降 ,生长状况不良甚至死亡。电镜观察及TUNEL检测的结果显示 ,许多细胞呈典型的凋亡特征。结论 :tbid基因的表达可促进Hela细胞的凋亡
Objective: To investigate the apoptosis-promoting activity of truncatedbid (tbid) gene on Hela cells. METHODS: Human full-length bid gene was cloned by RT-PCR. After sequencing, the gene encoding 60 amino acid residues at the N-terminal was cloned by PCR to obtain tbid gene. The recombinant plasmid was cloned into eukaryotic expression vector pIRES2 EGFP containing green fluorescent protein (GFP) gene and transfected into Hela cells by liposome. The effects of the expression of the target gene on the morphology and growth of the transfected cells were examined by fluorescence microscopy, electron microscopy and TUNEL assay. Results: The eukaryotic expression vector of tbid gene was successfully constructed. After transfected into Hela cells, the tbid gene is expressed in the cells, then the fluorescence intensity of the cells is decreased, the growth condition is poor and even death occurs. Electron microscopy and TUNEL results showed that many cells showed typical apoptotic features. Conclusion: The expression of tbid gene can promote the apoptosis of Hela cells