【摘要】 目的探讨CCCTC结合因子(CTCF)蛋白对人肝癌细胞(HepG2)的肿瘤干细胞的影响以及对HepG2和CNE1(鼻咽癌细胞系)细胞生存能力的影响。方法构建pEGFP-N1/CTCF、CTCFshRNA和GFPshRNA质粒,转染至HepG2和CNE1细胞后,以RT-PCR及Western blot检测CTCF mRNA和蛋白的表达。采用流式细胞术检测转染CTCF-shRNA质粒后48h的HepG2细胞中携带表面抗原CD90肿瘤干细胞的比例,以转染GFP-shRNA的HepG2细胞和野生型HepG2细胞为对照。采用MTT方法分别检测转染CTCF过表达重组质粒及CTCF-shRNA质粒后24、48、72hHepG2和CNE1细胞的生存能力。结果 pEGFP-N1/CTCF转染后增加HepG2和CNE1细胞中CTCF mRNA和蛋白的表达(与pEGFP-N1相比,P<0.05),CTCF-shRNA转染则降低表达(与GFP-shRNA相比,P<0.05)。流式细胞仪检测结果显示,CD90+细胞的检出率在转染CTCF-shRNA质粒细胞〔(1.733 0±0.417 7)%〕高于野生型HepG2细胞〔(0.575 0±0.062 9)%〕及转染对照GFP-shRNA质粒细胞〔(0.350 0±0.086 6)%〕(P<0.05);MTT实验结果显示CTCF的表达改变对HepG2和CNE1细胞的生存能力的影响不明显(P>0.05)。结论 CTCF可抑制人肝癌干细胞生成,对HepG2和CNE1细胞生长无明显影响。更多还原

【Abstract】 Objective To explore the effects of CCCTC-binding factor(CTCF)on human liver cancer stem cells(HepG2)and cell proliferation of HepG2and Nasopharyngeal carcinoma cell line(CNE1).Methods The pEGFP-N1/CTCF、CTCF-shRNA and GFP-shRNA plasmids were constructed and transfected into HepG2and CNE1cells,and RT-PCR or Western blot were performed to detect the mRNA or protein levels of CTCF.The subpopulation of CD90+cancer stem cells in HepG2cells transfected with CTCF-shRNA plasmid or GFP-shRNA plasmid(as transfection control)were assayed by flow cytometry with the wild type HepG2cells as control. Proliferation of cells transfected with CTCF-overexpression or CTCF-shRNA plasmid was evaluated by MTT assay. Results The levels of both mRNA and protein of CTCF were increased in pEGFP-N1/CTCFtransfected HepG2 and CNE1cells compared to that in pEGFP-N1transfected cells(P<0.05),and decreased in CTCF-shRNA transfected cells compared to that in cells transfected with GFP-shRNA(P<0.05).The results of flow cytometry demonstrated that,detection rate of CD90+cells in cells transfected with CTCF-shRNA plasmid〔(1.733 0± 0.417 7)%〕was obviously higher than that of wild-type HepG2cells〔(0.575 0±0.062 9)%〕and cells transfected with GFP-shRNA plasmid〔(0.350 0±0.086 6)%〕(P<0.05).The results of MTT analysis showed that, alteration of CTCF had no effect on cancer cell proliferation(P>0.05).Conclusion CTCF inhibits human liver cancer stem cells but no effect on cell proliferation.更多还原