成人骨髓间充质干细胞分化为成骨细胞的研究

Study on the adult human bone marrow mesenchymal stem cells differentiating into osteoblast

目的:通过成人骨髓间充质干细胞(mesenchymalstemcells,MSC)体外定向诱导为成骨细胞,探讨理想而有临床实用价值的成人骨髓MSC体外诱导培养体系。方法:体外分离、扩增成人骨髓MSC,流式细胞仪检测细胞表面抗原的表达。采用基础诱导培养液[地塞米松、β-甘油磷酸钠、L-抗坏血酸加不同浓度的重组人转化生长因子-β1(recombinanthumantransforminggrowthfactor-beta1,rhTGF-β1)]诱导成人骨髓MSC体外分化为成骨细胞。利用倒置光学显微镜、透射电镜、四甲基偶氮唑盐(MTT)比色、碱性磷酸酶(ALP)染色、ALP活性测定等方法研究成人骨髓MSC增殖和分化情况。结果:成人骨髓MSC的增殖和分化作用和rhTGF-β1有剂量依赖关系,低浓度时促进增殖,高浓度抑制增殖,5μg/L浓度达到高峰,其浓度升高促进MSC分化。结论:含有rhTGF-β15μg/L的基础诱导培养液是理想且有临床实用价值的成人骨髓间充质干细胞体外诱导为成骨细胞的培养体系。

Objective:To establish an ideal culture method on the differentiation of the adult human bone marrow mesenchymal stem cells(MSC)to osteoblasts in vitro. Method:Separate and expand the adult human bone marrow MSC in vitro. The cell surface antigens were detected by flow cytometry. And then the different concentrations of rhTGF-β1 were added into the basic induction medium including β-glycerophosphate, Vitamin C, Dexamethasone to induce the adult human bone marrow MSC to differentiate into osteoblasts in vitro. The proliferation and differentiation of the adult human bone marrow MSC were examined by phase-contrast microscope, transmission electron microscope, MTT, the determination of alkaline phosphatase (ALP) activities and ALP staining. Result:The low concentration of the rhTGF-β1 promoted the proliferation of the adult human bone marrow MSC, which reached a peak when the rhTGF-β1 was 5 μg/L. Over this point, it inhibited the proliferation of the adult human bone marrow MSC with the increase of concentrations of rhTGF-β1 in a dose-dependent manner. The differentiation of the adult human bone marrow MSC was enhanced with the increase of concentrations of rhTGF-β1 in a dose-dependent manner. Conclusion:The basic induction medium combined the 5 μg/L concentrations of rhTGF-β1 was the more valid culture environment on the differentiation of the adult human bone marrow MSC to osteoblasts in vitro.