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目的观察细胞凋亡因子核酸内切酶G(endonuclease G,Endo G)基因过表达在镉诱导人胚胎肾细胞HEK-293凋亡中的影响。方法从人肝癌细胞系(human hepatocarcinoma cells,HepG 2)中提取RNA,经逆转录获取互补DNA cDNA),通过聚合酶链反应(PCR)扩增Endo G基因,与pcDNA 3.0相连,构建pcDNA 3.0-Endo G真核表达载体,转染HEK-293细胞,并结合不同浓度氯化镉处理细胞;通过蛋白印迹实验(Western blot)验证基因过表达,吖啶橙/溴乙锭(AO/EB)双染法及流式细胞术(FCM)检测Endo G基因过表达对镉诱导HEK-293细胞凋亡的影响,噻唑蓝法(MTT)检测细胞存活率。结果成功构建pcDNA 3.0-EndoG真核表达载体,Endo G基因扩增片段大小约1 100 bp;该载体转染细胞后,Endo G成功过表达,使HEK-293细胞凋亡率>37%,且随氯化镉浓度增加,Endo G表达量先上升后下降。结论 Endo G以诱导细胞早期凋亡和晚期凋亡为主要形式。
Objective To investigate the effect of endogenetic G (Endo G) gene overexpression on the apoptosis of human embryonic kidney cell line HEK-293 induced by cadmium. Methods RNA was extracted from human hepatocarcinoma cells (HepG 2) and obtained by reverse transcription. The Endo G gene was amplified by polymerase chain reaction (PCR) and ligated with pcDNA 3.0 to construct pcDNA 3.0- Endo G eukaryotic expression vector was transfected into HEK-293 cells and treated with different concentrations of cadmium chloride. Western Blot was used to verify the gene overexpression. Acridine orange / ethidium bromide (AO / EB) The effect of Endo G gene overexpression on cadmium-induced apoptosis in HEK-293 cells was detected by flow cytometry (FCM) and cell viability was detected by MTT assay. Results The pcDNA3.0-EndoG eukaryotic expression vector was successfully constructed. The size of the amplified fragment of Endo G gene was about 100 bp. Endo G was overexpressed successfully and the apoptosis rate of HEK-293 cells was over 37% With the increase of cadmium chloride concentration, Endo G expression increased first and then decreased. Conclusion Endo G is the main form of inducing early apoptosis and late apoptosis.