视黄酸核受体γ重组腺病毒构建及其鉴定  

Construction and Identification of Recombinant Adenoviral Vector Carrying Rat Retinoic Acid Receptor γ Gene

作　　者：龚敏[1,2] 毕杨[1,2,3] 张赟[1,2] 江伟[1,2] 陈洁[1,2] 魏小平[1,2] 刘友学[1,2] 李廷玉[1,2] 

机构地区：[1]重庆医科大学附属儿童医院儿童营养研究中心,重庆市400014 [2]重庆医科大学儿童发育疾病研究省部共建教育部重点实验室儿科学重庆市重点实验室,重庆市(儿童发育重大疾病诊治与预防)国际科技合作基地,重庆市400014 [3]重庆医科大学附属儿童医院干细胞实验室,重庆市400014

出　　处：《医学分子生物学杂志》2011年第4期283-288,共6页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30830106,No.30972461）,重庆市自然科学基金（No.CSTC,2010BB5097）,中国博士后基金（No.20100800667）

摘　　要：目的构建携带大鼠视黄酸核受体γ（retinoic acid receptor γ，RARγ）的重组腺病毒，为研究RAR3,在骨髓间充质干细胞（mesenchymal stem cells，MSCs）成神经分化中的作用奠定基础。方法体外扩增大鼠础研基因，将其定向克隆至腺病毒穿梭质粒pAd Trace—TOX构建重组质粒pAdTrace—RARγ，并在BJ5183菌中与骨架质粒pAd Easy-1重组获得腺病毒载体pad—RARγ，Pac I酶切后转染HEK293细胞包装腺病毒。腺病毒Ad—RARγ感染大鼠MSCs，Real—time PCR和Western印迹检测RARγ的表达。结果PCR，酶切及测序均证实础研正确克隆至腺病毒质粒载体中，Ad—RARγ对MSCs感染率达60％～70％，并明显增强础研基因和蛋白的表达。结论成功构建携带RARγ的重组腺病毒，并具有上调大鼠MSCsRA脚基因和蛋白表达的功能。Objective To construct a recombinant adenovirus carrying rat retinoic acid receptor γ （RARγ） gene, and research the function of RARγ in the process of differentiation of mesenchy- real stem cells （MSCs） into neuronal-like cells. Methods Rat RARe/ gene was amplified and cloned into pAdTrace-TOX vector. The recombinant vector pAd-RARe/was transfected into HEK 293 cell lines to package recombinant adenovirus Ad-RARγ. Rat MSCs were infected by Ad-RARγ endoviruses. Expression of RARγ was detected by Real-time PCR and Western blot. Results The ade novirus vector containing RARγ gene was correctly constructed, as confirmed by sequencing. Infection efficiency of Ad-RARγ in MSCs reached to 60-70%. Both RARγ mRNA and protein expression levels in of MSCs were increased significantly by Ad-RARγ infection. Conclusion The recombinant adenovirus carrying rat RARγ gene is capable of upregulating RARγ expression in MSCs.

关 键 词：视黄酸核受体γ 重组腺病毒 视黄酸信号转导通路 骨髓间充质干细胞 

分 类 号：Q782[生物学—分子生物学]