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

发布时间：2018-03-03 00:15

本文选题：Rbpj(CBF)　切入点：重组腺病毒　出处：《重庆医科大学学报》2014年05期 　论文类型：期刊论文

【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 and screen recombinant adenovirus that specifically interferes with nuclear transcription factor Rbpj gene of Notch signaling pathway. To investigate the role of classical Notch signaling pathway of Rbpj in osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) induced by bone morphogenetic protein (BMP) 9. Methods: three pairs of siRNA sequences targeting mouse Rbpj were designed and subcloned into pSES-HUS shuttle plasmid. The recombinant plasmid was obtained by homologous recombination with skeleton plasmid pAdEasy-1 in Escherichia coli BJ5183. The recombinant plasmid was transfected into HEK293 cells by liposome. The adenovirus was amplified by RT-PCR and Western blot. The recombinant adenovirus treated with AdBMP9 and MSCs cell line C3H10T1 / 2 was used to detect the early osteogenic index, alkaline phosphatase (ALP), osteopontin (osteopontinin), osteocalcin (OCN) and downstream BMP by histochemistry and RT-PCR. Results: three recombinant adenoviruses were successfully constructed. The recombinant adenovirus of AdsiRbpj-2 with the best interference efficiency was screened. Transfection of C3H10T1 / 2 adsiRbpj-2 could significantly down-regulate the activity of ALP, an early osteogenic marker induced by BMP9, and the expression of OCN in late osteogenesis. Conclusion: adenovirus vector AdsiRbpj-2 was successfully constructed to specifically block the classical Notch signaling pathway. The recombinant adenovirus can inhibit the osteogenic differentiation of C3H10T1 / 2 cells induced by BMP9. It is further demonstrated that Notch signaling pathway plays an important role in the process of MSCs osteogenic differentiation induced by BMP9.
【作者单位】：重庆医科大学检验医学院临床检验诊断学教育部重点实验室;
【基金】：重庆市自然科学基金资助项目(编号:CSTC2012jjA10004) 重庆市教委科学技术研究资助项目(编号:KJ130305)
【分类号】：R322.71

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