目的:研究5拷贝缺氧反应元件（5HRE)和甲胎蛋白启动子（AFPp)联合调控的自杀基因系统硝基还原酶/CB1954(nitroreductase/CB1954, NTR/CB1954)在缺氧环境下对人肝癌细胞株HepG2生长的抑制作用。方法： 利用5HRE作为增强子和AFPp构成联合调控元件，与自杀基因NTR构成真核表达载体。将载体转染AFP阳性的人肝癌细胞株HepG2及AFP阴性的人胃癌细胞株MKN45，利用G418筛选稳定转染的细胞。用RTPCR和Western blotting检测NTR的表达。将前体药物CB1954加入稳定表达NTR基因的细胞，用MTT法观察其毒性代谢产物4羟胺还原产物对肿瘤细胞生长的抑制作用。结果:成功筛选出稳定表达NTR的单克隆HepG2细胞和MKN45细胞。RTPCR和Western blotting证实单克隆HepG2细胞中NTR在mRNA和蛋白水平的表达，且缺氧环境中的表达量更高；单克隆MKN45细胞在常氧和缺氧环境中均无NTR表达。MTT法检测发现，缺氧环境下NTR基因能有效地活化前体药物CB1954，剂量依赖性地抑制NTR阳性的HepG2细胞的生长（P＜0.05）；但对MKN45细胞和野生型HepG2细胞无抑制作用。结论： 5HRE和AFPp双重调控的NTR/CB1954自杀基因系统在体外缺氧环境下可促进对人肝癌细胞株HepG2靶向性杀伤作用。

Objective:To evaluate the inhibitory effect of Nitroreductase/CB1954 (NTR/CB1954) suicide gene system，which contains 5 copies of hypoxiaresponsive element (5HRE) and promoter of alphafetoprotein gene (AFPp), against human hepatocellular carcinoma cell line HepG2 under hypoxia condition in vitro. Methods: 5HRE and AFPpregulated nitroreductase (NTR) gene eukaryotic expression vector was transfected into AFPpositive human hepatocellular carcinoma cell line HepG2 and AFPnegative human gastric carcinoma cell line MKN45 by Lipofectamine 2000. Stably transfected cell lines were selected by G418. RTPCR and Western blotting were employed to examine the expression of NTR mRNA and NTR protein, respectively. Prodrug CB1954 was added into stablytransfected cell lines; inhibitory activity of its derivative product (4hydroxylamine) was observed by MTT. Results: Monoclonal HepG2 cells stably expressing NTR were successfully obtained. Expression of NTR mRNA and NTR protein in monoclonal HepG2 cells under hypoxia condition was significantly higher than those under normoxia condition as confirmed by RTPCR and Western blotting, respectively. Monoclonal MKN45 cells did not express NTR protein under either hypoxia condition or normoxia condition. NTR effectively activated CB1954 under hypoxia condition, resulting in dosedependent inhibition on the proliferation of HepG2 cells as shown by MTT assay (P<0.05). But CB1954 did not inhibit proliferation of MKN45 cells and wild type HepG2 cells. Conclusion: 5HRE and AFPpregulated NTR/CB1954 suicide gene system can specifically kill human hepatocellular carcinoma cell line HepG2 under hypoxia condition.

国家自然科学基金资助项目（No.30672070, No.30400430）