逆转录病毒介导人肿瘤坏死因子-α基因抑制胆管癌细胞的生长

Effects of recombinant retro virus tumor necrosis factor alpha gene transduction on proliferation and apoptosis of human cholangiocarcinoma cells

关键词:  逆转录病毒  胆管癌  基因治疗  凋亡  肿瘤坏死因子

Key words:  reverse transcript virus  cholangiocarcinoma  gene therapy  apoptosis  tumor necrosis factor alpha

摘要:

研究逆转录病毒介导的人肿瘤坏死因子α(TNFα)基因转染对人胆管癌细胞生长的抑制作用。［方法］构建逆转录病毒-TNFα(pLNCX-TNFα)复合体，转染人胆管癌细胞得到稳定表达，通过聚合酶链式反应（PCR）、流式细胞仪（FCM）、免疫组织化学等方法检测基因表达、细胞生长增殖和细胞凋亡等情况。［结果］pLNCX-TNFα基因转染人胆管癌细胞后，pLNCX空质粒转染组、pLNCX-TNFα基因转染组的细胞均能扩增出708 bp的基因片断，而未转染组则无；pLNCX-TNFα基因转染组的细胞克隆抑制率为64%，与对照组和pLNCX空质粒转染组相比，差异性非常显著(P＜0.01)；转染的人胆管癌细胞G1期比例从29.07% 增加到 54.24 %，G2-M期和S期比例分别从52.03%下降到2.02%，18.32%下降到10.34%；凋亡细胞数从5.83% 增加到34.76%。［结论］TNFα基因通过诱导胆管癌细胞发生凋亡并导致其发生G1期阻滞抑制胆管癌细胞的生长。

Abstract:

To evaluate the inhibitory effect of tumor necrosis factor alpha (TNFα) on proliferation and apoptosis of human cholangiocarcinoma cells. ［Methods］ Recombinant retro virus TNFα gene was transfected into the cells of human cholangiocarcinoma in vitro,which stably overexpressed TNFα gene.The transfected cells were detected by PCR and immunocytochemical methods respectively. The cycle pattern and apoptosis were assayed by flow cytometry. ［Result］ After transfection of recombinant retro virus TNFα gene, cells growth rate were obviously inhibited. The proportion of the cells in G1 phase was obviously increased from 29.07 % to 54.24 %, and that of the cells in S and G2-M phases obviously decreased from 18.32 % to 10.34 % and from 52.03 % to 2.02 %, respectively, as compared with group. Colony formation in these pLNCX-TNFα-infected cells greatly decreased, compared with pLNCX-TNFα-uninfected cells. The apoptotic index was increased in pLNCX-TNFα-infected cells from 5.83 % to 34.76 %. ［Conclusion］ TNFα gene could inhibit the growth of human cholangiocarcinoma cells and induce cells apoptosis