【摘要】 目的　构建载有白细胞介素 10 (IL 10 )基因的 ,能在肝星状细胞 (HSC)稳定表达的重组腺病毒载体 ,为将来的肝纤维化基因治疗提供基础。方法　将IL 10cDNA克隆于腺病毒穿梭质粒PAdTrack CMV ,得到重组质粒PadTrack CMV IL 10。采用电穿孔法将质粒PadTrack CMV IL 10与腺病毒骨架质粒PadEasy 1共转化E .coliBJ5 183细胞 ,通过在细胞内同源重组 ,生成带有IL 10基因的重组腺病毒载体。重组腺病毒质粒经过 2 93细胞的扩增及CsCl的纯化 ,感染HSC细胞。结果　经形态学、病毒DNA酶切、聚合酶链反应 (PCR)和逆转录 (RT ) PCR等方法的鉴定 ,证实了载体构建的正确性 ,经测定 ,病毒滴度为 2 .5× 10 10 efu/ml。结论　成功构建带有IL 10cDNA的重组腺病毒载体 ,所携带的IL 10载体能够转染大鼠HSC并在HSC中稳定表达 ,为进一步的研究打下了基础。更多还原

【Abstract】 Objective Interlukin-10 is a inflammatory inhibitor factor,It act as a important function in decreasing the liver injury and fibrosis.But in the hepatic stellate cell (HSC),The ingenerate IL-10 production is too low to detected.There for We aimed to develop an adenoviral vector for gene transfer of IL-10 and transfect into SD Rat HSC,so we can enhance the IL-10?mRNA production stably.Methods Rat IL-10cDNA was inserted into adenovirus shuttle plasmid -PAdTrack CMV to generate a recombinant plasmid PAdTrack-Il-10,then homologeous recombination was carried out in E.coli BJ5183 bacteria by contransforming linearized shuttle vector with adenovirus backbone plasmid PadEasy-1.The advenovirus vector was then packaged and amplified in 293 cells.After purificated by cscl,we transfected the Adenoviral into SD rat HSC.The expression of IL-10mRNA in infectd rat HSC was detected by RT-PCR.Result The morphology,restriction analysis and RT-PCR were verified the successful construction and the titer of Adenovial was 2.5×10 10 efu/ml,the IL-10mRNA was detected in the infected rat HSC.Conclusion The recombinant adenoviral vector carrying IL-10 was successfully constructed and transfected into rat HSC,It can enhance the production of IL-10mRNA stably.更多还原