骨形成蛋白-2结合纤维连接蛋白体外诱导骨髓间充质干细胞分化为成骨细胞的观察

Research of marrow mesenchymal stem cells differentiating into osteoblast cells induced by fibronectin and bone morphogenetic protein-2 in vitro

目的:研究骨形成蛋白-2(bone morphogenetic protein,BMP-2)和纤维连接蛋白(fibronectin,FN)在体外联合和单独应用促进骨髓间充质干细胞(marrowmesenchymal stem cells,MSCs)向成骨细胞分化的能力。方法:SD大鼠的MSCs分离培养后,分为4组:对照组;A组(加入100μg/ml BMP-2);B组(加入10 ng/ml FN);C组(加入100μg/ml BMP-2和10 ng/ml FN),通过检测不同时间点(3,6,9,12天)各组细胞数量?细胞碱性磷酸酶(ALP)活性?成骨细胞表型以及扫描电镜观察细胞在材料上的生长状况,比较分析各组成骨诱导能力的差异。结果:A组、B组和C组细胞形态,成骨细胞表型检测都发生了变化;各组的细胞数和ALP活性随着培养时间的延长而增加,但C组变化最明显,与相同时间点其他各组相比,差异有统计学意义(P<0.05)。电镜观察发现各组材料上均有细胞生长,但C组细胞的黏附生长状况明显好于其他组。结论:BMP-2和FN在体外能明显促进MSCs向成骨细胞分化,两者具有明显的协同效应,可以作为理想的成骨诱导剂应用于组织工程。

Objective： To study the single and combined effect of bone morphogenetic protein-2 （BMP- 2 ） and fibronectin （FN） on the differentiation of the marrow mesenchymal stem cells（MSCs） into osteoblast cells in vitro. Methods： MSCs obtained from SD rats were cultured,the experiments were investigated into four groups ： control normal group ; （A） 100 μg/ml BMP-2 ; （ B ） 10 ng/ml FN ; （ C ） 100 μg/ml BMP-2 and 10 ng/ml FN. At different time points（3, 6, 9,12 d） ,the numbers of the cells were counted and the levels of alkaline phosphatase（ALP） activity were detected; the expression of osteoblastic phenotypes were demonstrated and the speed of cells growth on n-HAC scaffold were also observed by scanning electron micrography （SEM）. The differentiation of bone among groups were analysed. Results： After differentiation of MSCs obtained from SD rats. The cells possessed osteoblast morphological properties ;the cell numbers and the expression of the ALP increased with cultivating time of the four groups were all changed between group C and others, and group C were most obviously demonstrated. At the same time point compared group C with other groups, the difference had statistical significance （P 〈 0.05 ）. Cells could be observed on every by SEM, the properties of cells in group C had significant change. Conclusion： FN combined BMP-2 can effectively induce BMSCs to differentiation and proliferation into osteoblast cells in vitro, It is suggested that interaction between BMP-2 and FN can be used as a better inducer for bone tissue engineering.