人源基因载体pHrn 介导p53基因在Bel-7402中的抗癌研究
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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.


p53 Anti-tumor Research in Bel-7402 by Using Human-derived Vector
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This work was supported by grants from The National Natural Science Foundation of China (30500302) and National Basic Research Program of China (2004CB518800).

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    摘要:

    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.

    Abstract:

    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.

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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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  • 收稿日期:2006-11-14
  • 最后修改日期:2006-12-13
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  • 在线发布日期: 2007-04-16
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