骨髓间充质干细胞的旁分泌能影响成骨细胞生物学功能

Paracrine effects of bone marrow mesenchymal stem cells on biological function of osteoblasts

背景:有研究表明骨髓间充质干细胞移植可促进骨缺损的修复,但受损组织的缺血、缺氧和炎症反应限制了其临床应用。目的:观察骨髓间充质干细胞旁分泌作用对成骨细胞MG63增殖、迁徙、分化功能的影响。方法:Ficoll-Paque密度梯度离心法分离骨髓间充质干细胞,制备骨髓间充质干细胞条件培养基培养成骨细胞MG63。CCK-8法检测MG63细胞增殖能力变化;细胞划痕法检测MG63细胞迁徙能力变化;微板检测MG63细胞合成碱性磷酸酶能力的变化;Real-time PCR法检测碱性磷酸酶、Ⅰ型胶原及骨钙素基因表达量的变化;茜素红染色检测其矿化能力的变化。结果与结论:骨髓间充质干细胞表型为CD44、CD73、CD90表达强阳性,CD34表达阴性。与普通培养基(DMEM)相比,在骨髓间充质干细胞条件培养基作用下,MG63细胞的增殖速度明显加快;细胞划痕结果示其迁徙能力明显提高;诱导第4,7天后碱性磷酸酶基因表达量及蛋白合成量明显增多(P<0.01);Ⅰ型胶原及骨钙素基因表达量在诱导第4天后差异无显著性意义,诱导第7天后较对照组明显升高(P<0.05);茜素红染色示骨髓间充质干细胞条件培养基诱导MG63细胞21 d后钙结节形成增多,矿化沉积作用增强。结果证实,骨髓间充质干细胞的旁分泌物质可以显著促进成骨细胞的增殖、迁徙、分化和矿化能力。

OBJECTIVE：To investigate the paracrine effects of bone marrow mesenchymal stem cells on osteoblast biological function. METHODS：Bone marrow mesenchymal stem cells were isolated using standard Ficol-Paquedensity gradient centrifugation. Mesenchymal stem cellconditioned medium was prepared to cultivate osteoblasts, MG63. Proliferation of MG63 cells was analyzed by cellcounting kit-8. Migration of MG63 cells was analyzed by cellscratch method. Alkaline phosphatase activity of MG63 cells was analyzed by microplate test kit. Real-time PCR was performed to evaluate osteoblast differentiation markers, alkaline phosphatase, col agen type I and osteocalcin. Alizarin red staining was performed to evaluate osteoblast mineralization. RESULTS AND CONCLUSION：The cells were strongly positive for CD44, CD73 and CD90, but negative for CD34. MG63 cells cultured in the conditioned medium showed better proliferation and migration than those cultured in the Dulbecco’s modified Eagle’s medium. The activity and mRNA expression of alkaline phosphatase＆amp;nbsp;were much higher after induction of 4, 7 days （P＆lt;0.01）. There was no significant difference in expression of col agen type I and osteocalcin after induction of 4 days, but they were significantly higher than those in the control group after induction of 7 days （P＆lt;0.05）. Alizarin red staining showed that the number of calcium nodules was increased and the mineral apposition was enhanced after induction of 21 days with the conditioned medium. These findings suggest that the paracrine substance of bone marrow mesenchymal stem cells can significantly promote osteoblast proliferation, migration, differentiation and mineralization.