构建携带hIL-10基因的腺病毒载体及其对大鼠肝细胞损伤的保护作用
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江苏省卫生职业技术教育研究课题(J201017)


Construction of a recombinant adenovirus carrying human interleukin-10 gene and its protection effect on rat hepatocytes during liver injury
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    目的:构建携带人白细胞介素(human interleukin,hIL)-10基因的腺病毒载体,细胞学及动物模型实验鉴定其介导的目的基因表达,并验证其对大鼠肝细胞损伤的保护作用-方法:以pORF-hIL10质粒为模板,PCR扩增hIL-10基因,插入腺病毒质粒pDC315,构建pDC315-hIL10-将质粒pDC315-hIL10与腺病毒包装质粒共转染至293细胞,产生复制缺陷型重组腺病毒Ad5-hIL10;感染大鼠肝细胞,免疫学方法鉴定目的基因的表达;建立体内外大鼠肝细胞损伤模型,验证Ad5-hIL10对肝细胞损伤的保护作用-结果:经PCR扩增-鉴定,证明已正确构建重组腺病毒Ad5-hIL10-大鼠肝细胞感染Ad5-hIL10后,随着病毒感染强度的升高,hIL-10表达明显上升-大鼠肝细胞体外培养模型预先感染病毒Ad5-hIL10,经四氯化碳诱导损伤,则细胞存活率随感染强度(multiplicity of infection,MOI)值升高而增加,至MOI=50时,细胞存活率恢复至92%;大鼠体内肝损伤模型显示,与损伤对照组和空白病毒对照组比较,Ad5-hIL10处理的大鼠肝细胞水样变性和间质炎症反应均减轻,肝功能的各项指标均有明显改善(P < 0.05)-结论:成功构建了重组腺病毒质粒Ad5-hIL10,且能明显提高肝细胞的存活能力,对肝功能有明显保护作用-

    Abstract:

    Objective:To construct a recombinant adenovirus carrying human interleukin (hIL)-10 gene and identify its expression and protection effect on rat hepatocytes during liver injury. Methods:The hIL-10 gene was amplified by PCR from template plasmid pORF-hIL10,then digested with endonuclease and inserted into pDC315 to recombine adenovirus Ad5-hIL10. Ad5-hIL10 was used to infect rat hepatocytes,and the hIL-10 expression was identified by immunology method. Rat hepatocyte injury model was established,and protection effect of the Ad5-hIL10 was investigated both in hepatocytes in vitro and rat models in vivo. Results:The recombinant adenovirus Ad5-hIL10 was recombined successfully. After infected with Ad5-hIL10,the expression level of hIL-10 in hepatocytes was increased with viral MOIs. The cell viability of virus-infected hepatocytes increased along with viral MOIs under carbon tetrachloride induction,being 92% when MOI=50. In rat model,compared with the control group and blank virus group,the hepatocyte hydropic degeneration and stroma inflammation in Ad5-hIL10-treated group were alleviated,and the liver function was improved(P < 0.05). Conclusion:The recombinant adenovirus Ad5-hIL10 has been successfully constructed,which was demonstrated to have activity of improving rat hepatocyte viability and protecting liver function.

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程 剑,林 彬,沈晓洁,刘 艳.构建携带hIL-10基因的腺病毒载体及其对大鼠肝细胞损伤的保护作用[J].南京医科大学学报(自然科学版),2012,(11):1516-1521

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  • 收稿日期:2012-08-31
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