腺病毒介导骨形态发生蛋白2诱导兔骨髓间充质干细胞的成骨分化

Adenovirus-mediated bone morphogenetic protein 2 induces osteogenic differentiation of rabbit bone marrow mesenchymal stem cells

背景:天然骨形态发生蛋白2在体内无法实现缓释、持续诱导骨髓间充质干细胞向成骨细胞分化,如何获得内源性骨形态发生蛋白2蛋白是目前研究的热点。目的:探讨腺病毒介导人骨形态发生蛋白2转染兔骨髓间充质干细胞的成骨分化能力及其可能机制。方法:采用腺病毒介导人骨形态发生蛋白2基因转染第3代兔骨髓间充质干细胞,转染后48 h采用qRT-PCR、Western blot检测骨髓间充质干细胞中骨形态发生蛋白2的mRNA和蛋白表达,利用Western blot检测Wnt/β-catenin信号通路相关蛋白的表达水平。然后进行成骨诱导培养,第7天检测碱性磷酸酶活性,第14天采用免疫组织化学染色方法观察Ⅰ型胶原蛋白表达,第21天采用茜素红S染色观察钙结节形成情况。结果与结论:①Ad-BMP2-EGFP转染后骨髓间充质干细胞中骨形态发生蛋白2 mRNA和蛋白的表达明显上调;②Ad-BMP2-EGFP转染的骨髓间充质干细胞成骨诱导后碱性磷酸酶活性增强,Ⅰ型胶原蛋白分泌以及钙结节形成增多;β-catenin、cyclin D1、Runx2和c-myc蛋白表达上调,而GSK3β蛋白表达下调;③结果表明,Ad-BMP2-EGFP转染促进了骨髓间充质干细胞向成骨细胞分化,其作用机制可能是通过上调骨形态发生蛋白2蛋白表达水平从而激活Wnt/β-catenin信号通路来实现的。

BACKGROUND:Natural bone morphogenetic protein 2 cannot achieve sustained release in vivo and continuously induce bone marrow mesenchymal stem cells to differentiate into osteoblasts.How to obtain endogenous bone morphogenetic protein 2 protein is the focus of current research.OBJECTIVE:To investigate osteogenic differentiation of rabbit bone marrow mesenchymal stem cells transfected by adenovirus with human bone morphogenetic protein 2 and its possible mechanism.METHODS:The third passage of rabbit bone marrow mesenchymal stem cells were transfected by adenovirus with human bone morphogenetic protein 2 gene.At 48 hours after transfection,expression levels of bone morphogenetic protein 2 mRNA and protein in bone marrow mesenchymal stem cells were detected by qRT-PCR and western blot assay,respectively.The expression levels of proteins related to the Wnt/β-catenin signaling pathway were determined by western blot assay.After an induction with osteogenic medium,the alkaline phosphatase activity at 7 days,the type I collagen at 14 days,and the calcium nodules at 21 days were performed by alkaline phosphatase activity kit,immunohistochemical staining,and alizarin red S staining,respectively.RESULTS AND CONCLUSION:(1)The expression of bone morphogenetic protein 2 mRNA and protein was significantly upregulated in Ad-BMP-2/EGFP transfected bone marrow mesenchymal stem cells.(2)Ad-BMP-2/EGFP induced alkaline phosphatase activity,promoted production of type I collagen and calcium nodules formation in rabbit bone marrow mesenchymal stem cells.The levels ofβ-catenin,cyclin D1,Runx2 and c-myc were upregulated in Ad-hBMP-2/EGFP transfected bone marrow mesenchymal stem cells,while the level of GSK3βsignificantly decreased.(3)Results indicated that Ad-BMP2-EGFP transfection promoted the differentiation of bone marrow mesenchymal stem cells into osteoblasts and its mechanism may be achieved by up-regulating the expression level of bone morphogenetic protein 2 protein to activate the Wnt/β-catenin signaling pathway.