肝癌靶向性腺病毒载体的构建及其作用的研究  

Construction and Study of Replication-Defective Adenovirus Targeting Hepatocarcinoma

作　　者：徐丁尧[1] 杜芝燕[1] 王妍[1] 陈惠华[1] 徐元基[1] 陆应麟[1] 

机构地区：[1]军事医学科学院基础医学研究所病理生物学研究室,北京100850

出　　处：《癌症》2006年第7期798-804,共7页Chinese Journal of Cancer

基　　金：国家重点基础研究发展规划(973)项目(No.2002CB513105)~~

摘　　要：背景与目的:抑癌基因p16是肿瘤基因治疗最常用的治疗基因之一,通过腺病毒载体介导p16基因表达引发肿瘤细胞凋亡在多种p16基因失活的肿瘤细胞系中都得到了证实。研究表明,p16在人原发性肝细胞癌中存在高频率失活。因此,本研究通过观察外源性p16基因经AFP启动子驱动表达后对AFP阳性肝癌细胞的影响,以探讨p16基因在肝癌靶向基因治疗中应用的可行性。方法:将外源性p16基因置于人AFP核心启动子AF0.3下游,构建了携带p16基因的重组复制缺陷型腺病毒Ad-AFP-p16,感染肝癌细胞后,应用MTT比色法、Westernblot、DNA片段化分析、流式细胞术分析等方法在体外研究外源性p16基因表达对细胞生长的影响。小鼠皮下接种感染重组腺病毒的鼠肝癌细胞H22,观察病毒对肝癌细胞体内成瘤性的影响。结果:感染Ad-AFP-p16的肝癌细胞高效表达P16蛋白,MTT结果显示细胞生长受到明显抑制,到第六天Ad-AFP-p16对肝癌细胞Bel-7402和HepG2的生长抑制率分别为(94.42±11.70)%和(94.99±6.74)%,DNA片段化分析和流式细胞术分析证实p16能诱导肝癌细胞发生显著凋亡,感染后48hBel-7402和HepG2的凋亡率分别为39.57%和39.75%。感染Ad-AFP-p16的肝癌细胞体内成瘤受到明显抑制,对照组、Ad-GFP组、Ad-AFP-p16组以及Ad-CMV-p16组瘤体体积分别为(1.54±0.65)cm3、(1.71±1.01)cm3、(0.25±0.39)cm3和(0.25±0.45)cm3。结论:AFP启动子驱动p16基因表达可以诱导肝癌细胞发生凋亡。BACKGROUND ＆ OBJECTIVE： Antioncogene p16 is one of the most important genes used in tumor gene therapy. Apoptosis induced by adenovirus mediated overexpression of p16 in cancer cells has been confirmed in various p16 gene inactive cancers. Studies have indicated that p16 gene is frequently inactive in human primary hepatocarcinoma. Therefore this study was to investigate the effect of exogenous p16 gene driven by alpha fetoprotein （AFP） core promoter on human hepatocellular carcinoma cells and further explore the potentials of p16 in hepatocellular carcinoma gene therapy. METHODS： The recombinant replication-defective adenovirus Ad-AFP-p16 containing p16 gene downstream the AFP core promoter-AF0.3 was constructed and infected hepatocellular carcinoma cells. The expression of p16 was detected by Western blot. The effects of exogenous p16 gene on cell growth and apoptosis were measured by MTT, flow cytometry and DNA ladder in vitro. Subcutaneous injection of mouse hepatocarcinoma cell line H22 infected with Ad-AFP-p16 was applied to observe the effect of Ad-AFP-p16 on tumorigenesis in vivo. RESULTS： Over-expression of exogenous p16 gene was confirmed in hepatocarcinoma cells infected with Ad-AFP-p16. Cell growth was inhibited by （94.42±11.70）% and（94.99±6.74）% in Bel-7402 and HepG2 cells on the 6 day after virus infection; 39.57% and 39.75% apoptotic cells were also induced respectively in these two cell lines on the 2nd day. Moreover, the infection of Ad-AFP-p16 significantly inhibited the tumorigenesis of mouse hepatocarcinoma cell line H22 in vivo. The tumor volumes of control, Ad-GFP, Ad-AFP-p16 and Ad-CMV-p16 groups were （1.54±0.65）cm3, （1.71± 1.01 ）cm^3, （0.25±0.39）cm^3 and （0.25±0.45）cm^3, respectively. CONCLUSION： The expression of p16 gene driven by AFP promoter can induce apoptosis in hepatocarcinoma cells.

关 键 词：肝肿瘤 P16 甲胎蛋白启动子 细胞凋亡 靶向性载体 

分 类 号：R735.7[医药卫生—肿瘤]