人源基因载体pHrn介导p53基因在Bel-7402中的抗癌研究(英文)

p53 Anti-tumor Research in Bel-7402 by Using Human-derived Vector

利用人源基因载体结合人巨细胞病毒(CMV)增强子,人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子及肿瘤抑制基因p53构建肿瘤抑制载体,研究它们在肝癌细胞株Bel-7402中的功能.将CMV增强子(CMVe)与肿瘤特异性启动子hTERTp组合,分别结合GFP及精氨酸型和脯氨酸型肿瘤抑制基因p53,构建GFP表达载体pchEGFP以及p53表达载体pchp53Arg和pchp53Pro;脂质体2000转染肝癌细胞株Bel-7402;24h后利用荧光显微镜以及流式细胞仪检测GFP的表达;RT-PCR、蛋白质印迹检测p53基因的表达;利用Hoechst33258、Annexin V/PI染色,检测p53促使肿瘤细胞的促凋亡情况.结果表明:GFP荧光表达载体在Bel-7402细胞中能有效表达;RT-PCR及蛋白质印迹检测到精氨酸型和脯氨酸型p53基因在Bel-7402细胞内表达;Hoechst33258染色及Annexin V/PI染色检测细胞凋亡实验没有得到明显的细胞凋亡结果.结果表明:实验所构建的肿瘤抑制载体在肝癌细胞Bel-7402能有表达,并能形成外源性p53蛋白,但没有检测到明显的p53促使肝癌细胞调亡的结果.这可能与p53对hTERT的负调控作用有关.

In order to study the tumor suppression effect of p53 with CMV enhancer and hTERT promoter mediated by human-derived vector pHrn in liver cancer cell Bel-7402, report plasmid pchEGFP, tumor suppressor plasmids pchp53Arg and pchp53Pro were constructed by inserting expression cassette CMVe＋hTERTp＋EGFP, CMVe＋hTERTp＋p53Arg and CMVe＋hTERTp＋p53Pro into pHrn respectively. 24 h after cell transfection by lipofectamine 2000, GFP expression pattern was analyzed through fluorescence microscope and flow cytometry; RT-PCR and Western blot were taken to study the p53 expression pattern. The cell apoptosis by Hoechst 33258 and Annexin V-FITC/PI staining was also studied. Results show that the expression of GFP and p53 protein in Bel-7402were detected, but apparent cell apoptosis could not be found. The recombinant p53 mediated by human-derived vector could express in Bel-7402, but no significant tumor suppression effect was detected, which might result from the down regulation effect of the wild type p53 on hTERT promoter.