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[目的]探讨应用生长分化因子5(growth differentiation factor 5,GDF-5)诱导人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hMSCs)微团成软骨分化的可行性及效果。[方法]体外分离培养hMSCs,取第3代细胞实验。将hMSCs分别用无血清H-DMEM和含10、20、50、100 ng/ml GDF-5的软骨诱导液(chondrogenic medium,CM)诱导,显微镜下观察细胞形态变化,MTT法检测各组细胞的增殖情况。诱导2周后重悬细胞,以5×106/ml的细胞密度离心构建微团,继续诱导2周。RT-PCR检测两组细胞Ⅱ型胶原mRNA的表达。免疫组化、甲苯胺蓝染色检测Ⅱ型胶原和蛋白多糖表达。[结果]hMSCs呈梭形、漩涡状生长。100 ng/ml GDF-5诱导的hMSCs呈圆形或多角形。五组微团分别由无血清H-DMEM、含10,20,50,100 ng/ml GDF-5的软骨诱导液诱导2周后,除H-DMEM组无Ⅱ型胶原mRNA、Ⅱ型胶原蛋白和蛋白多糖的表达外,其他各组Ⅱ型胶原mRNA、Ⅱ型胶原蛋白和蛋白多糖的表达呈浓度依赖性增加,各组差异均有统计学意义(P<0.05)。[结论]经一定浓度GDF-5诱导的hMSCs微团,Ⅱ型胶原和蛋白多糖表达呈浓度依赖性增加,具有软骨的部分生物学功能。
[Objective] To investigate the feasibility and effect of using GDF-5 to induce chondrocyte differentiation of human bone marrow mesenchymal stem cells (hMSCs). [Method] The hMSCs were isolated and cultured in vitro, and the third generation cells were selected for experiment. The hMSCs were induced by serum-free H-DMEM and chondrogenic medium (CM) containing 10, 20, 50 and 100 ng / ml GDF-5 respectively. The morphological changes of the cells were observed under the microscope. Proliferation. After 2 weeks of induction, the cells were resuspended and the micelles were centrifuged at a density of 5 × 10 6 / ml for 2 weeks. The mRNA expression of type Ⅱ collagen was detected by RT-PCR. Immunohistochemistry and toluidine blue staining were used to detect the expression of type Ⅱ collagen and proteoglycan. [Results] hMSCs were spindle-shaped and swirling. HMSCs induced by 100 ng / ml GDF-5 were round or polygonal. Five groups of microdomains were induced by chondrocytes containing serum-free H-DMEM, 10, 20, 50 and 100 ng / ml GDF-5 respectively for 2 weeks. Except H-DMEM group, no collagen type II mRNA, The expression of type Ⅱ collagen mRNA, type Ⅱ collagen protein and proteoglycan increased in a concentration-dependent manner in all the other groups, with statistical significance (P <0.05). [Conclusion] The expression of type Ⅱ collagen and proteoglycan of hMSCs induced by certain concentration of GDF-5 increased in a concentration-dependent manner, and had some biological functions of cartilage.