国家自然科学基金(30500302) 和国家重点基础研究发展计划 (973) (2004CB518800) 资助项目.
This work was supported by grants from The National Natural Science Foundation of China (30500302) and National Basic Research Program of China (2004CB518800).
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;24 h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Hoechst 33258、 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;Hoechst 33258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-7402 were 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.
薛志刚,李剑,尹彪,张雅坤,刘雄昊,潘乾,龙志高,戴和平,夏昆,邬玲仟,梁德生,夏家辉.人源基因载体pHrn 介导p53基因在Bel-7402中的抗癌研究[J].生物化学与生物物理进展,2007,34(5):465-470
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