携带大鼠白细胞介素-10基因重组腺病毒载体的构建及其在肝星状细胞中的表达

Construction of adenoviral vector carrying Rat IL-10 and its expression in hepatic stellate cell

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。

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.