视黄酸核受体α的siRNA重组腺病毒构建及干扰效果初步鉴定  

Construction and identification of small interfering RNAs against rat retinoic acid receptor a by recombinant adenovirus ~

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

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400014 [2]重庆医科大学附属儿童医院干细胞实验室,重庆400014 [3]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400014

出　　处：《解剖学杂志》2012年第5期562-566,共5页Chinese Journal of Anatomy

基　　金：国家自然科学基金（30830106,81100309）;重庆市自然科学基金（CSTC,2010BB5097）;重庆市医学科研计划项目（2010-2-217）;中国博士后基金（20100480667）

摘　　要：目的：构建视黄酸核受体a（RARa）的siRNA重组腺病毒，为反向研究RARa受体在全反式视黄酸（ATRA）抗凋亡过程中的作用提供实用的技术手段。方法：设计3对大鼠RA风的siRNA序列，将其分别克隆到pSES-HUS穿梭质粒；进而将重组穿梭质粒pSES-HUS-siRARa与pAdEasml在BJ5183感受态大肠埃希菌内同源重组以得到pAd-siRARa，PacI酶切线性化后转染HEK293进行包装扩增以得到重组腺病毒Ad-siRAR^Ad-siRARa感染大鼠PCI2细胞48h，提取各组mRNA和总蛋白，Real-timePCR和免疫印迹检测Ad-siRAR^-I、一2、一3对RARQ的抑制效率。结果：PCR、酶切及测序均证实RARa的siRNA序列正确克隆至腺病毒质粒载体，经过包装扩增得到大量高滴度重组腺病毒Ad-siRARa，而且对RARa的表达抑制效率接近70％。结论：成功构建视黄酸核受体RARⅡ的siRNA重组腺病毒，并具有下调PCI2细胞RARa基因和蛋白表达的功能。Objective： To construct small interfering RNAs against rat retinoic acid receptor α（RARα） by recombinant adenovirus, with the purpose to provide practical technique and material for researching the function of RARα in the process of anti-apoptosis a- roused by all-trans retinoic acid （ATRA）. Methods： Three pairs of double-stranded DNA fragments for silencing rat RARa were de- signed and cloned into pSES-HUS vector to obtain pSES-HUS-siRARα The recombinant vector pAd-siRARa was gained by homol- ogous recombination between pSES-HUS-siRARa and backbone vector pAdEasy-1 in E. coli BJ5183. pAd-siRARa was linearized by Pac I and transfected into HEN 293 cell lines to package recombinant adenovirus Ad-siRARα Rat PC12 cells were infected by Ad- siRARtrl, -2, -3 for 48 h, and the expressions α RARa were detected by real-time PCR and Western blotting. Results： All results from PCR, endonuclease cutting and gene sequanceing confirmed that double-stranded DNA fragments with RNA interference function were cloned into adenovirus vector correctly. A large number of high-titer Ad-siRARα-1, -2, -3 were obtained by pack- aged and amplified in HEK 293 cell lines. Both the RARα mRNA and protein expression level of PC12 cells were down-regu- lated by Ad-siRARα infection. And the inhibition efficiency was about 70%. Conclusion; The recombinant adenovirus Ad- siRARα-1, -2, -3 were created successfully, and the adenovirus has the function of silencing RARαexpression in PC12 cells.

关 键 词：视黄酸受体α 小干扰RNA 重组腺病毒 RNA干扰 PC12细胞 

分 类 号：R977.21[医药卫生—药品]