重组腺相关病毒介导黑色素瘤分化相关基因7对人肝癌细胞体内外抑制效应的研究

Anti-tumor effect of adeno-associated viral vector-mediated systemic delivery of MDA-7 on human hepatocellular carcinoma cells

目的观察PEG启动子调控腺相关病毒介导的黑色素瘤分化相关基因7（MDA-7）在体内外抑制肝癌细胞的生物学活性，探讨重组腺相关病毒介导MDA-7基因用于肝癌基因治疗的应用前景。方法构建重组腺相关病毒rAAV-PEG-MDA-7表达系统，体外感染人肝癌细胞株HepG2细胞。Western印迹检测转染细胞内MDA-7蛋白；MTT法检测细胞增殖抑制率；流式细胞术分析细胞周期和细胞凋亡变化。构建肝癌细胞HepG2裸鼠皮下移植瘤模型，尾静脉注射rAAV-PEG-MDA-7，观察其对肝癌生长的抑制作用；ELISA方法检测血浆MDA-7蛋白浓度；TUNEL法分析MDA-7对肿瘤细胞的凋亡诱导情况；免疫组织化学分析MDA-7在肿瘤组织中的表达。结果重组腺相关病毒rAAV-PEG-MDA-7可特异性转染HepG2细胞，MDA-7蛋白在HepG2细胞中高效表达，并呈时间依赖性。重组腺相关病毒rAAV-PEG-MDA-7可抑制HepG2细胞增殖并诱导其凋亡，G0／G1期细胞百分比明显增多，G2／M期的细胞显著减少（P〈0、05）。全身系统性给予rAAV-PEG-MDA-7后，血清中可持续检测到MDA-7蛋白，且注射后2周浓度达高峰（200ng／ml）；肿瘤生长受抑制，抑瘤率为62％（P〈0、05）；免疫组化结果显示MDA-7在肿瘤组织中表达；TUNEL结果显示rAAV-PEG-MDA-7可诱导肿瘤细胞凋亡。结论构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统具有良好的肿瘤靶向性，通过抑制肝癌细胞增殖和诱导其凋亡发挥抗肝癌作用。

Objective To investigate the anti-tumor effect of the recombined adeno-associated virus （AAV） encoding melanoma differentiation-associated gene-7 （ MDA-7 ） regulated by PEG promotor on human hepatocellular carcinoma（HCC） in vitro and in vivo. Methods In vitro studies, rAAV-PEG-MDA-7 was transfected into human hepatocellular carcinoma cell line HepG2 and normal human hepatocytes LO2. Cell growth inhibition, cell cycle, and apoptosis were assessed. MDA-7 protein was detected by Western Blot. Cell growth-inhibiting rate was evaluated by MTT assay. Cell cycle was examined by flow cytometry analysis. In vivo studies, the model of subcutaneous tumor was generated by injection of HCC cells HepG2 into the dorsum of nude mice. AAV-PEG-MDA-7 was injected from the tail vain of the tumor model mice after tumor cell innoculation. The concentration of plasma MDA-7 was detected by ELISA assay. Tumor growth was observed. Immunohistochemistry was employed to detect MDA-7 expression in tumor tissues. Tumor cell apoptosis was measured by TUNEL. Results rAAV-PEG-MDA-7 was effectively transfected into HepG2 cells. MDA-7 protein was highly expressed in HepG2 cells in a time-dependent manner, rAAV-PEG-MDA-7 suppressed HepG2 cell growth. Cells accumulated in G0/G1 phases, while reduced in G2/M phases of the cell cycle（ P 〈 0.05）. ELISA assay showed that the concentration of plasma MDA-7 was gradually increased to reach the plateau（200 ng/ml）. Tumor growth was significantly inhibited in mice injected with rAAV-PEG-MDA-7, and the tumor growth-inhibiting rate was 62% （P 〈 0. 05, vs control AAV-PEG and control PBS）. Immunohistochemistry showed MDA-7 protein was strongly expressed in tumor tissue. TUNEL demonstrated significant induction of tumor cell specific apoptosis. Conclusions rAAV-PEG-MDA-7 exhibits tumor-specific cytotoxicity, inhibits HCC proliferation and induces apoptosis, which may be an ideal candidate for new gene therapy for hepatocellular carcinoma.