巨细胞病毒增强子提高人甲胎蛋白启动子效率的研究  

Improvement of Human AFP Promoter Efficiency by CMV Early Enhancer

作　　者：叶景佳[1] 陈萍[2] 贾振宇[3] 李春春[1] 陈丽红[1] 曹江[1] 

机构地区：[1]浙江大学医学院附属第二医院临床研究中心,杭州310009 [2]浙江大学医学院附属邵逸夫医院中心实验室,杭州310016 [3]浙江省医学科学院,杭州310013

出　　处：《中国细胞生物学学报》2013年第4期486-493,共8页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金重点项目(批准号:LZ12H16003);浙江省教育厅项目(批准号:Y200804137)资助的课题~~

摘　　要：人巨细胞病毒(CMV)早期转录增强子能够提高其他基因启动子的转录启动效率。CMV早期转录增强子对人甲胎蛋白(AFP)启动子的转录增强的作用及特异性的影响,将决定其是否适用于肝癌靶向性基因治疗。作者利用PCR法分别克隆了人AFP增强子、启动子和CMV早期转录增强子,构建了相应的pGL4.10荧光素酶报告基因载体,与内参照pGL4.74质粒共转染人肝癌细胞Hep3B、HepG2、SMMC7721和人宫颈癌细胞HeLa、乳腺癌细胞Bcap37,利用双荧光素酶检测系统检测分析了这些细胞中AFP增强子—启动子的效率。结果表明,CMV早期转录增强子能够明显增强AFP增强子—启动子在肝癌细胞中的效率(在Hep3B、HepG2和SMMC7721细胞中分别提高33.07、134.22和465.18倍),但是也能提高AFP增强子—启动子在非肝癌细胞中的效率(在HeLa和Bcap37细胞中分别提高335.73和1096.81倍)。因此,CMV增强子虽然可以大大提高AFP增强子—启动子的效率,但无特异性,直接将其用于靶向AFP的肝癌基因治疗时可能会产生由于治疗基因在正常组织中的非特异表达而引起的副作用。CMV early enhancer can be used to improve the efficiency of promoters of other genes. The impact of CMV early enhancer on the efficiency and specificity of AFP promoter determines whether it is suitable for AFP promoter in targeted gene therapy of hepatocyte carcinoma. In this study, human AFP enhancer/promoter and CMV early enhancer were cloned by PCR, and luciferase reporter vectors were constructed and transfected into hepatocyte carcinoma Hep3B, HepG2 and SMMC7721 cells, cervical carcinoma HeLa cells and breast cancer Bcap37 cells, with pGL4.74 vector as transfection control. Dual luciferase reporter assay was performed to analyze the efficiency of AFP promoter in these cells. The results showed that CMV early enhancer can markedly improve the efficiency of AFP enhancer/promoter in hepatocyte carcinoma cells （33.07-, 134.22-and 465.18-fold in Hep3B, HepG2 and SMMC7721 cells respectively）, and can also improve the efficiency of AFP enhancer/promoter in non- hepatocyte cells significantly （335.73- and 1096.81-fold in HeLa and Bcap37 cells respectively）. Therefore, the CMV early enhancer improves the efficiency of AFP enhancer/promoter without cell-specificity. Side-effects may arise when applying CMV enhancer directly in AFP-targeted gene therapy of hepatocyte carcinoma due to nonspe- cific expression of therapeutic genes in normal tissues.

关 键 词：细胞特异性启动子 甲胎蛋白 巨病毒早期转录增强子 基因治疗 

分 类 号：Q78[生物学—分子生物学]