显性负性突变体对AP2α转录因子及其活性的影响  

Construction of Recombinant Adenovirus Vector Containing Dominant Negative Mutant of Transcriptional Factor AP2α and its Expression and Activity in Mesenchymal Stem Cells

作　　者：毕杨[1] 何昀[1] 龚敏[1] 张赟[1] 陈洁[1] 李廷玉[1] 

机构地区：[1]重庆医科大学附属儿童医院营养研究中心儿童发育疾病研究省部共建教育部重点实验室,中国重庆400014

出　　处：《生命科学研究》2011年第5期402-409,共8页Life Science Research

基　　金：国家自然科学基金资助项目(30830106);重庆市卫生局医学科研项目(2010-2-217);重庆市自然科学基金资助项目(CSTC2010BB5097);中国博士后基金资助项目(20100480667)

摘　　要：转录蛋白2α(activator protein-2α,AP2α)的转录活性可能是全反式视黄酸(all-trans-retinoic acid,ATRA)促进骨髓间充质干细胞(mesenchymal stem cells,MSCs)神经分化的关键调控点,但具体机制尚不清楚,显性负性突变是研究基因功能的一个经典手段.以携带AP2α全长基因的pAd-AP2α质粒为模板,分别扩增缺失bHLH及缺失TAD区域的AP2α片段,采用AdEasy腺病毒包装系统获得两种AP2α缺失型显性负性突变体重组腺病毒Ad-dnAP2α-△bHLH及Ad-dnAP2α-△TAD,腺病毒感染ATRA诱导24 h的大鼠MSCs可观察到60%以上RFP阳性细胞.Real-time-PCR、Western blot及免疫荧光结果显示AP2α定位于MSCs细胞核,经A-TRA诱导24 h,AP2α的表达无变化,而其下游基因bcl-2及c-kit表达增高,两种缺失型显性负性突变体腺病毒感染MSCs对野生型AP2α的mRNA及蛋白表达没有影响,却能有效抑制AP2α对下游靶基因bcl-2和c-kit的转录激活作用.为进一步研究AP2α转录活性在ATRA诱导MSCs成神经分化中的作用提供重要的分子手段.Activator protein-2α （AP2α） transcriptional activity might be a key regulatary point of all-transretinoic acid （ATRA） induced neuronal differentiation of mesenchymal stem cells （MSCs）, however, the mechanism is still poorly understood. Dominant negative mutation is a canonical method to study gene function. Taken pAd-AP2α plasmid containing A P2α full-length sequence as template, A P2α sequence deleted bHLH domain and TAD domain were PCR amplified respectively, two adenovirus of dominant negative deletion mutants Ad-dnAP2α-AbHLH and Ad-dnAP2α-ATAD were constructed by using Ad-Easy system. More than 60 percent of RFP-positive MSCs were observed at 24 h after adenovirus infection. Real-time PCR, Western blot, and immunofluorescence results showed that AP2α was located in nucleus, the expression of AP2α in MSCs did not change with 24 h ATRA treatment, however, the expression of downstream target genes bcl-2 and c-kit increased. Two dominant negative deletion mutants did not change the mRNA and protein expression of wild type AP2α, but effectively inhibited the role in activating transcription of A P2α on downstream target genes bcl-2 and c-kit. This study provides an important tool in the further study of transcription activity of AP2α in ATRA induced neuronal differentiation of MSCs.

关 键 词：转录蛋白2α 显性负性突变体 腺病毒 骨髓间充质干细胞 

分 类 号：R34[医药卫生—基础医学]