【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1×10¹⁰efu/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。更多还原

【Abstract】 Objective To construct replication-deficient recombinant adenoviruses AdHNF4αthat co-expresse human hepatocyte nuclear factor 4α（HNF4α） and green fluorescent protein（GFP） gene,and to evaluate the effect of HNF4αup-regulation on hepatocyte gene expression.Methods The HNF4αcDNA was obtained through RT-PCR from human hepatocyte.The recombinant adenoviral plasmid- pAdHNF4αwas established using AdEasy system and packed in 293 cells.After transfection of human hepatoma cell lines HepG2 and Hep3B with AdHNF4α,the expression of HNF4αand other liver-associ- ated functional genes were evaluated by RT-PCR and Western blot.Results The recombinant plasmid pAdHNF4αwas confirmed by restriction endonuclease digestion and sequencing.GFP expression was observed on the fourth day after packing the linearized pAdHNF4αin 293 cells.Stable transfection of AdHNF4αwith a titer of 1×10¹⁰ 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 HNF4αmRNA and protein were significantly up-regulated compared with the control group（3.4 folds in HepG2 infected with AdHNF4a 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 HNF4αin human hepatoma cells with AdHNF4αcould enhance normal liver- specific function.Our study would provide a new idea for the researches on gene regulation of transplan- ted hepatocytes.更多还原