Epstein-Barr病毒载体介导的白细胞介素12在肝癌细胞中的靶向表达  

The target-gene expression of interleukin-12 in HepG2 cells

作　　者：王自力[1,2] 梁国栋[1,2] 付士红 施侣元[1,2] 王芝山[1,2] 侯云德[1,2] 

机构地区：[1]武汉同济医科大学 [2]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室

出　　处：《中华实验和临床病毒学杂志》1998年第4期306-309,共4页Chinese Journal of Experimental and Clinical Virology

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.

关 键 词：IL-12 基因治疗 基因表达 肝癌 

分 类 号：R735.705[医药卫生—肿瘤] R373.11[医药卫生—临床医学]