携带siRbpj重组腺病毒载体的构建及其对BMP9诱导骨髓间充质干细胞成骨分化作用影响

Construction of small interfering RNAs against mouse nuclear transcription factor Rbpj by recombinant adenovious and its inhibitory during osteogenetics induced by BMP9 in C3H10T1/2

摘要:

目的：构建并筛选特异性干扰Notch信号通路核转录因子Rbpj基因的重组腺病毒，探讨通过Rbpj的经典Notch信号通路在骨形态发生蛋白（bone morphogenetic protein，BMP）9诱导骨髓间充质干细胞（mesenchymal stem cells，ＭＳＣs）成骨分化中的作用。方法：设计3对针对小鼠Rbpj的siRNA序列，将其分别亚克隆至pSES-HUS穿梭质粒，再与骨架质粒pAdEasy-1在大肠杆菌BJ5183内同源重组获得重组质粒，经脂质体转染至HEK293细胞中包装扩增腺病毒，利用RT-PCR及Western blot进行筛选、验证有效的干扰腺病毒。该重组腺病毒与AdBMP9处理ＭＳＣs细胞株C3H10T1/2，采用组织化学和RT-PCR检测早期成骨指标碱性磷酸酶（alkaline phosphatase，ALP）和晚期成骨指标骨桥蛋白（osteopontin，OPN），骨钙蛋白（osteocalcin，OCN）及BMP下游靶基因Id 1、Runx 2等的表达。结果：成功构建了3个重组腺病毒，并筛选出干扰效率最好的AdsiRbpj-2重组腺病毒，转染C3H10T1/2，AdsiRbpj-2可以明显下调BMP9诱导的早期成骨指标ALP的活性及晚期成骨指标OPN、OCN的表达，成骨相关基因Runx 2等的表达也有下降。结论：成功构建了特异性阻断Notch经典信号通路腺病毒载体AdsiRbpj-2，该重组腺病毒能够抑制BMP9诱导的C3H10T1/2细胞的成骨分化进程，从而进一步证明Notch信号通路在BMP9诱导的MSCs成骨分化过程中有着重要的作用。

Abstract:

Objective：To construct recombinant adenovirus against mouse nuclear transcription factor Rbpj，downstream of Notch，with the purpose to provide a powerful molecular tool for exploring the role of bone morphogenetic protein（BMP） ９ signaling in osteogene－sis of mesenchymal stem cells. Methods：Three pairs of double-stranded DNA sequence targeted mouse Rbpj were designed and sub－cloned to pSES-HUS vector to obtain pSES-HUS-siRbpj using AdEasy system，then reorganized with pAdEasy-1 plasmid in E.coli BJ5183. pAd-siRbpj was transfected into HEK293 to package recombinant adenovirus AdsiRbpj after being linearized by PacⅠand screened by RT-PCR and Western blot. C3H10T1/2 were co-treated with recombinant adenovirus and BMP9 proteins，then the early osteogenetic maker alkaline phosphatase（ALP） was detected at the 7th d. Osteopontin（OPN） and osteocalcin（OCN），BMP downstream target genes Id 1 and Runx 2 expression were also detected by RT-PCR. Results：Three interference recombinant adenoviruses were successfully constructed，of which AdsiRbpj-2 had the best interference ability and can significantly decrease BMP9-induced os－teogenic indexes，including the early osteogenetic index ALP，late osteoblasic differention indicators OPN，OCN and osteogenic gene Runx 2. Conclusions：The siRNA adenovious vector AdsiRbpj-2，with the ability to specifically block mouse Notch signaling，is suc－cessfully constructed，future indicating that Notch signaling pathway plays an important role in osteogenic differention of BMP9 induced bone marrow mesenchymal stem cells.