骨形成蛋白-2基因转染人骨髓间质干细胞诱导异位成骨的实验研究

BMP-2 GENE TRANSFECTED HUMAN BONE MARROW MESENCHYMAL STEM CELLS INDUCING IN VIVO ECTOPIC OSTEOGENESIS OF NUDE MICE

目的 评价腺病毒介导的人骨形成蛋白 - 2基因 (Adv- h BMP- 2 )转染人骨髓间质干细胞 (MSCs)的诱导成骨能力。方法 从人骨髓中分离培养获取 MSCs,分为三组 :1Adv- h BMP- 2转染细胞组 ;2 Adv- βgal转染细胞组 ;3未转染细胞组。分别于体外行 Western immunoblot试验、碱性磷酸酶 (AL P)和 Von Kossa染色、AL P定量测定 ,以及裸鼠肌内诱导成骨试验。共 9只裸鼠 ,双侧股后肌群内注射 ,每组 6侧。结果 Adv- h BMP- 2组 MSCs可分泌 BMP- 2蛋白 ;转染后第 9天 AL P染色多数细胞为阳性 ,其它两组 AL P染色阳性细胞少见 ;AL P活性第 3天开始升高 ,12天达高峰为 14 .76单位 ,与其它两组比较有统计学意义 (P<0 .0 5 ) ;于第 2 1天后出现钙结节 ,而其它两组未见。裸鼠肌内注射后 4周 Adv- h BMP- 2组 X线片均有异位成骨 ,Adv- βgal转染细胞组未见明显成骨 ,未转染细胞组仅有少量骨形成。组织学检查发现 :Adv- h BMP- 2组也可见典型的板层骨和骨髓腔形成 ,Adv- βgal组主要为纤维组织 ,未转染组也可见散在骨小梁形成。结论 Adv- h BMP- 2基因转染可诱导人骨髓间质干细胞成骨。

Objective To evaluate the osteogenic potential of human bone marrow mesenchymal stem cells (MSCs) transferred with human bone morphogenetic protein 2(BMP 2) gene by adenovirus. Methods The MSCs were isolated from human bone marrow and cultured in vitro. They were divided into 3 groups: Adv hBMP 2 transduced group; Adv βgal transduced group; untransduced group. Western immunoblot analysis, alkaline phosphatase(ALP) staining, Von Kossa staining, and a quantitative ALP activity assay were performed. Nine nude mice received injection into a thigh muscle to test the osteoinductivity of the three types of cells. Results In the Adv hBMP 2 transduced group, Western immunobolt analysis on tissue culture demonstrated that these cells indeed produced the BMP 2 protein; most MSCs were stained positively for ALP activity 9 day after transduction; the MSCs reached the peak of ALP activity 12 day after transduction; the calcified nodes formed 21 days after transduction. The ectopic bones formed in the thigh muscles of the nude mice. Little bone formation was observed in the other groups 4 weeks after cell injection. Conclusion Adenovirus mediated hBMP 2 gene transfection can induce osteogenesis of human bone marrow mesenchymal stem cells.