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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: Tumor suppressor gene p16 is one of the most commonly used therapeutic genes in gene therapy of tumors. Adenovirus vector-mediated p16 gene expression causes tumor cell apoptosis in a variety of tumor cell lines inactivated by p16 gene . Studies show that p16 has high frequency of inactivation in human primary hepatocellular carcinoma. Therefore, in this study, we observed the effect of exogenous p16 gene on the AFP positive hepatoma cells after AFP promoter-driven expression to explore the feasibility of p16 gene in hepatocellular carcinoma-targeted gene therapy. Methods: The exogenous p16 gene was placed in the downstream of human AFP core promoter AF0.3. The recombinant replication-defective adenovirus Ad-AFP-p16 carrying p16 gene was constructed and transfected into hepatoma cells. MTT assay, Western blot , DNA fragmentation analysis, flow cytometry and other methods in vitro study exogenous p16 gene expression on cell growth. The mice were subcutaneously inoculated with H22 cells infected with the recombinant adenovirus to observe the effect of the virus on the tumorigenicity of the hepatoma cells in vivo. Results: The P16 protein was highly expressed in hepatoma cells infected with Ad-AFP-p16. The MTT assay showed that the growth of cells was significantly inhibited by Ad-AFP-p16. The growth inhibition rates of Bel-7402 and HepG2 were (94.42 ± 11.70)% and (94.99 ± 6.74)% respectively. DNA fragmentation analysis and flow cytometry analysis confirmed that p16 could induce significant apoptosis in hepatocellular carcinoma cells. The apoptosis rates of Bel-7402 and HepG2 cells were 39.57% and 39.75 %. The tumorigenicity of Ad-AFP-p16-infected hepatoma cells was significantly inhibited in vivo. The tumor volumes in control, Ad-GFP, Ad-AFP-p16 and Ad-CMV-p16 groups were (1.54 ± 0.65) 1.71 ± 1.01) cm3, (0.25 ± 0.39) cm3 and (0.25 ± 0.45) cm3. Conclusion: The AFP promoter can induce the apoptosis of hepatoma cells induced by p16 gene expression.