复制缺陷型腺病毒Ad-p14ARF治疗肝细胞癌的实验研究及作用机制的探讨  

The feasibility and mechanism of recombinant adenovirus Ad-p14 ARF in gene therapy of liver cancer

作　　者：宋海峰[1] 钱海利[1] 张雪艳[1] 梁萧[1] 付明[1] 林晨[1] 

机构地区：[1]中国医学科学院中国协和医科大学肿瘤医院肿瘤研究所分子肿瘤学国家重点实验室,北京100021

出　　处：《中华肿瘤杂志》2006年第9期641-645,共5页Chinese Journal of Oncology

摘　　要：目的研究复制缺陷型重组腺病毒Ad-p14ARF用于肿瘤基因治疗的可行性及其作用机制。方法利用Ad-Easy系统构建并扩增了Ad-p14ARF复制缺陷型腺病毒;应用台盼蓝细胞计数法和倒置相差显微镜观察Ad-p14ARF对肝癌细胞的生长抑制作用;AnnexinV／PI双染法结合流式细胞仪检测不同滴度的Ad-p14ARF作用后肝癌细胞的凋亡情况;Western blot法检测相关蛋白的表达情况,构建皮下肝癌移植瘤裸鼠模型,研究Ad-p14ARF在体内的抑瘤活性。结果Ad-p14ARF可以明显抑制野生型p53和突变型p53表型的肝癌细胞增殖、生长。Annexin V／PI双染色法显示,Ad- p14ARF能促进肝癌细胞株HepG2和BEL7402的细胞凋亡,凋亡细胞比例与腺病毒滴度呈现一定的剂量相关性。凋亡相关蛋白检测提示,Ad-p14ARF能上调p53下游基因Bax、p21的表达。体内抑瘤试验显示,Ad-ECRG2可以明显抑制肝癌细胞BEL7402裸鼠移植瘤的生长。结论p14ARF基因的导入可以通过p53依赖和非依赖途径抑制肿瘤细胞增殖,促进肿瘤细胞凋亡,有望在恶性肿瘤的基因治疗中发挥重要作用。Objective To explore the feasibility and mechanism of recombinant adenovirus Ad-p14ARF in cancer gene therapy. Methods The proliferation of different liver cancer cells was assessed by morphology and trypan blue assay. Cell apoptosis was confirmed by detecting phosphatidylserine （PS） externalization with Annexin V/PI double staining. The expression of related proteins was analyzed by Western bloting. Nude mouse model bearing subcutaneous transplanted BEL7402 tumor was established to study the therapeutic ability of Ad-p14ARF. Results Ad-p14ARF suppressed cell growth and proliferation, and promoted cell apoptosis of cancer cell lines with different genetic background. Ad-p14ARF inhibited growth of liver cancer cells （HepG2, BEL7402 ） in a dose-dependent manner. Ad-p14ARF lead to overexpression of Bax and p21, the downstream regulating genes of p53. In the experimental therapy on nude mice bearing subcutaneous transplanted BEL7402 tumor, Ad-p14ARF suppressed tumor growth significantly. Conclusion p14ARF is a short gene and with powerful function, which are consistent with the requirements for tumor suppressor genes used in gene therapy. It may play an important role in gene therapy against malignancies in the future.

关 键 词：复制缺陷型腺病毒 P14ARF 肝癌细胞 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤]