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周凤云, 潘杨滨, 何志娟, 毛伟平, 李文秀, 刘宣宣, 徐翀. Endo G基因过表达对镉诱导HEK-293细胞凋亡影响[J]. 中国公共卫生, 2012, 28(10): 1322-1326. DOI: 10.11847/zgggws2012-28-10-21
引用本文: 周凤云, 潘杨滨, 何志娟, 毛伟平, 李文秀, 刘宣宣, 徐翀. Endo G基因过表达对镉诱导HEK-293细胞凋亡影响[J]. 中国公共卫生, 2012, 28(10): 1322-1326. DOI: 10.11847/zgggws2012-28-10-21
ZHOU Feng-yun, PAN Yang-bin, HE Zhi-juan, . Effect of Endo G over-expression on cadmium induced apoptosis of HEK-293 cells[J]. Chinese Journal of Public Health, 2012, 28(10): 1322-1326. DOI: 10.11847/zgggws2012-28-10-21
Citation: ZHOU Feng-yun, PAN Yang-bin, HE Zhi-juan, . Effect of Endo G over-expression on cadmium induced apoptosis of HEK-293 cells[J]. Chinese Journal of Public Health, 2012, 28(10): 1322-1326. DOI: 10.11847/zgggws2012-28-10-21

Endo G基因过表达对镉诱导HEK-293细胞凋亡影响

Effect of Endo G over-expression on cadmium induced apoptosis of HEK-293 cells

  • 摘要: 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。

     

    Abstract: ObjectiveTo examine the effects of over-expression of endonuclease G(Endo G)gene on cadmiuminduced apoptosis of embryonic kidney cell HEK-293 in vitro.MethodsTotal RNA was obtained from human hepatocarcinoma cell line(HepG 2)and cDNA was obtained by reverse transcription.The Endo G gene was amplified by PCR.The Endo G was inserted reversely into pcDNA 3.0,and the recombinant plasmid pcDNA 3.0-Endo G was selected and then transferred into DH5αstrain.The pcDNA 3.0-Endo G was transfected into HEK-293 cells.Western blot was used to confirm the expression of Endo G gene in HEK-293 cells.Acridine orange/ethidium bromide(AO/EB)and flow cytometry were used to determine the effect of Endo G gene on cadmium-induced apoptosis of HEK-293 cells.The cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)method.Results The size of Endo G gene fragment is about 1100 bp.The eukaryotic expressing plasmid of pcDNA 3.0-Endo G was constructed successfully.The expression of Endo G was first increased and then decreased in HEK-293 cells with the increase of concentration of cadmium chloride.The study with AO/EB and flow cytometry showed that the apoptotic rate of HEK-293 cells over-expressing Endo G gene was over 37% and significantly increased.ConclusionEndo G induces apoptosis of HEK-293 cells with the main form of early and late apoptosis.

     

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