通过细菌内同源重组 ,制备含绿荧光 (GFP)基因的CD腺病毒 ,初步观察其体外杀瘤作用。方法 :先构建含CD基因的腺病毒穿梭载体pAdTrack CMV CD ,与骨架载体pAdEasy 1在细菌内重组为pAd CD ,经 2 93细胞包装为增殖缺陷性腺病毒 ,体外感染人结肠癌细胞株LoVo,并给予前药 5 FC ,观察其体外杀瘤效果。结果 :pAdTrack CMV CD和同源重组的pAd CD经酶切鉴定正确 ,pAd CD转染 2 93细胞 ,包装扩增腺病毒后 ,感染LoVo细胞 ,予 5 FC治疗 ,4d后在荧光显微镜下观察到转染的LoVo和未转染细胞胞均被杀死 ,并且这种旁观者效应不依赖于细胞间接触。结论 :细菌内质粒同源重组法可以高效制备含绿荧光 (GFP)基因的CD腺病毒 ,体外腺病毒介导CD/ 5 FC可以杀伤肿瘤细胞 ,其旁观者效应不依赖于细胞间接触。

Objective: To generate an adenovirus, Ad-CD, expressing green fluorescence protein and bacterial cytosine deaminase, and use it for tumor suicide gene therapy. Methods: CD gene was cloned into pAdTrack-CMV, pAdTrack-CMV-CD and pAdEasy-1 were homologous recominated in bacteria. 293 cells were transfected by the newly-recombinated plasmid pAd-CD and generated adenoviruses. LoVo cells were infected with the adenovirus, and the tumor-killing effect after 5-FC was administered.Results: pAdTrack-CMV-CD and the newly-recombinated plasmid pAd-CD were tested by restriction endonuclease digestions. Followed by GFP, Ad-CD were produced in 293 and LoVo cells infected by Ad-CD were killed after 5-FC was administered.Conclusion: Adenovirus plasmid homologous recombinant in bacteria is a simple method for adenovirus generation. In vitro adenoviral carried CD gene is efficient for tumor cell suicide gene therapy and has a bystand-effect, which is not depended on cell direct contact

R737.31

本课题受国家自然基金（39600142）和上海长海医院123人才基金资助