摘要
目的研究白细胞介素-12(Interleukin-12,IL-12)在肝癌细胞靶向性表达,探索肝癌基因治疗新方法。方法将白介素-12分别克隆到带有人甲胎蛋白启动子和增强子的Epstein-Barr(EB)病毒载体中,得到重组表达载体pEBAF/P35和pEBAF/P40,分别在4种细胞系中作共转染,用逆转录-聚合酶链反应(RT-PCR)、酶联免疫吸附试验(ELISA)和Westernblot检测各细胞系中IL-12的表达情况。结果用pEBAF/P35和pEBAF/P40共转染4种细胞后IL-12基因只在产生甲胎蛋白的肝癌细胞(HepG2)中表达,活性检测证明,表达的IL-12有诱使IFN-γ产生的生物学活性。结论本研究在肝癌细胞中靶向性和组织特异性的表达了有活性的IL-12,为既能杀死肝癌细胞,又不影响正常肝细胞功能的新型基因治疗方案做了有益的探索。
EB virus vector pEBAF, which contains human α-fetoprotein gene promoter and enhancer was used as expression vector. Two recombinant expression vectors, pEBAF/P35 and pEBAF/P40, were constructed and co-transfected into 4 cell lines. RT-PCR suggested that both P35 and P40 mRNA are expresssed in HepG2 cell secreting α-fetoprotein, in contrast, neither P35 mRNA nor P40m RNA was expressed in non-secreting α-fetoprotein cell lines. This proved that the pEBAF may lead to target-expression of IL-12 in hepatoma and has tissue specificity and exclusiveness for hepatoma. The cotransfected Hyg resistent clone of HepG2 cell was selected. Northern blot indicated that the HepG2 cell clone had transcription of P35 mRNA and P40 mRNA. Expression of IL-12 in the supernatant of HepG2 cell culture had been testified by ELISA and Western blot. IL-12 biological activity test indicated that the expressed IL-12 had activity of inducing IFN-γ production. This research laid the foundation to develop an in vivo gene therapy strategy pointing to the treatment of hepatocellular carcinoma, with selective killing of the tumor cells and no effect on the normal hepatic cells.
出处
《中华实验和临床病毒学杂志》
CSCD
1998年第4期306-309,共4页
Chinese Journal of Experimental and Clinical Virology
关键词
IL-12
基因治疗
基因表达
肝癌
IL-12
Gene expression
Gene therapy Hepatocellular carcinoma