肝细胞核因子4α对肝肿瘤细胞功能基因表达的影响  

Construction of recombinant adenoviruses carrying hepatocyte nuclear factor 4αand evaluation of its effect on hepatoma gene expression.

作　　者：尹川[1] 林勇[1] 沈健伟[1] 张新 王要军[1] 岳海燕[1] 谢渭芬[1] 曾欣[1] 

机构地区：[1]上海长征医院消化科,200003 [2]上海市国家人类基因组南方研究中心

出　　处：《中华消化杂志》2007年第5期318-321,共4页Chinese Journal of Digestion

基　　金：国家自然科学基金(30400205)

摘　　要：目的利用AdEasy系统构建共同表达人肝细胞核因子4α(HNF4α)和绿色荧光蛋白(GFP)的复制缺陷型重组腺病毒——AdHNF4α,体外感染人肝肿瘤细胞株,观察HNF4α表达上调对肝肿瘤细胞的肝细胞功能基因表达的影响。方法通过逆转录(RT)-PCR方法获取目的基因HNF4αcDNA片段,利用腺病毒载体AdEasy系统构建腺病毒质粒pAdHNF4α,经293细胞包装后得到复制缺陷型重组腺病毒AdHNF4α;将AdHNF4α体外感染人肝肿瘤细胞株HepG2和Hep3B,通过RT-PCR、Western印迹法检测HNF4α在上述细胞中的表达;同时观察外源导入HNF4α对肝肿瘤细胞的肝细胞功能基因表达的影响。结果连接、重组后通过酶切和测序法筛选出病毒质粒pAdHNF4α;经293细胞包装,4 d后观察到GFP明显表达,反复扩增最终获得约1×10^(10)efu/ml滴度的重组腺病毒AdHNF4α;AdHNF4α体外感染人肝肿瘤细胞72 h后,有约90%以上的细胞表达GFP,HNF4αmRNA表达明显上调,蛋白表达分别增加3.4倍(HepG2)和5.2倍(Hep3B);同时肝细胞相关功能基因包括载脂蛋白、细胞色素P450家族、谷氨酰胺合成酶、磷酸烯醇丙酮酸羧激酶及葡萄糖-6-磷酸酶mRNA表达均明显上调,且肿瘤细胞凋亡率也明显增加。结论利用重组腺病毒载体可将外源HNF4α基因导入肝肿瘤细胞并维持高效表达,同时正常肝细胞重要功能基因表达亦增强,肿瘤细胞生物学特性明显改善,这将为基因修饰移植肝细胞研究提供新思路。Objective To construct replication-deficient recombinant adenoviruses AdHNF4α that co-expresse human hepatocyte nuclear factor 4α（HNF4α） and green fluorescent protein（GFP） gene, and to evaluate the effect of HNF4α up-regulation on hepatocyte gene expression. Methods The HNF4α cDNA was obtained through RT-PCR from human hepatocyte. The recombinant adenoviral plasmid-- pAdHNF4α was established using AdEasy system and packed in 293 cells. After transfection of human hepatoma ceil lines HepG2 and Hep3B with AdHNF4α, the expression of HNF4α and other liver-associated functional genes were evaluated by RT-PCR and Western blot. Results The recombinant plasmid pAdHNF4α was confirmed by restriction endonuclease digestion and sequencing. GFP expression was observed on the fourth day after packing the linearized pAdHNF4α in 293 cells. Stable transfection of AdHNF4α with a titer of 1 × 10^10 efu/ml was obtained after repeated amplification. More than 90% of human hepatoma cells had GFP expression in 72 hours after transfection of AdHNF4α. The expression of HNF4α mRNA and protein were significantly up-regulated compared with the control group（3.4 folds in HepG2 infected with AdHNF4α and 5.2 folds in Hep3B infected with AdHNF4α）. Furthermore, the transcriptional expressions of some liver-associated functional genes such as apolipoprotein, cytochrome P450 families, glutamine synthetase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase also increased after transfection of the virus, and the apoptosis ratio of the cells increased. Conclusions Up- regulating the expression of HNF4a in human hepatoma cells with AdHNF4a could enhance normal liverspecific function. Our study would provide a new idea for the researches on gene regulation of transplanted hepatocytes.

关 键 词：肝细胞核因子 肝肿瘤 基因 

分 类 号：R735.7[医药卫生—肿瘤]