人骨髓间充质干细胞的体外培养、鉴定及成骨分化

In vitro culture,identification and osteogenic differentiation of human bone marrow mesenchymal stem cells

背景:国内外对骨髓间充质干细胞的体外成骨诱导分化研究手段、测定指标均不够全面。目的:建立并完善一整套人骨髓间充质干细胞的分离培养及鉴定方法,探讨其体外成骨分化能力。方法:采用密度梯度离心法分离培养人骨髓间充质干细胞,流式细胞仪鉴定细胞表面表型。传至第3代时更换成骨诱导培养基进行成骨分化诱导。结果与结论:人骨髓间充质干细胞生长旺盛,传代后增殖旺盛,第3代骨髓间充质干细胞表面表型CD44、CD73、CD90表达阳性,CD34表达阴性。诱导后的成骨细胞碱性磷酸酶活性增加,Gomori、Vonkossa、茜素红染色均阳性。RT-PCR检测诱导后细胞有Ⅰ型胶原、碱性磷酸酶、骨钙素、骨唾液酸蛋白、骨桥蛋白及骨连接蛋白基因的表达,证明了人骨髓间充质干细胞成功向成骨方向分化。表明实验建立了一整套稳定、成熟的骨髓间充质干细胞分离、培养、扩增方案。

BACKGROUND：Research approaches and measurement index regarding osteogenic differentiation of bone marrow mesenchymal stem cells in vitro are not fully considered at home and abroad.OBJECTIVE：To establish and consummate a set of methods of isolating,culturing and identifying human bone marrow-derived mesenchymal stem cells（hBMSCs） and investigate their osteogenic differentiation ability in vitro.METHODS：hBMSCs were isolated using the method of density gradient centrifugation.The cell surface phenotypes of BMSCs were identified by flow cytometry.Passage 3 hBMSCs were induced by osteogenic culture medium for 2-3 weeks.RESULTS AND CONCLUSION：The primary cultured hBMSCs grew and proliferated vigorously after subculture.Passage 3 hBMSCs were positive for CD44,CD73,CD90 and were negative for CD34.After osteogenic induction,alkaline phosphatase activity was increased and cells were stained positive by Gomori,Von kossa and alizarin red.RT-PCR results showed the gene expression of collagen Ⅰ,alkaline phosphatase,osteocalcin,bone sialoprotein,osteopotin and osteonectin.This confirms that the primary hBMSCs can differentiate into osteoblasts in vitro successfully.These findings suggest that a stable,mature method of isolating,culturing and amplifying hBMSCs has been established.