生长分化因子5诱导人骨髓间充质干细胞微团成软骨分化的研究

An experimental study on growth differentiation factor-5 promoting chondrogenesis of pellets generated from hMSCs

[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 explore the feasibility and effect of GDF-5 on promoting chondrogenesis of pellets generated from human bone marrow mcenchymal stem cells. [Methods]hMSCs were isolated and cultured in vitro,and the cells from passage 3 were used in this study.hMSCs were randomly divided into five groups according to the kinds of induction medium-serum-free DMEM-H,chondrogenic medium supplemented with 10,20,50,and 100 ng/ml GDF-5.Cell morphology was observed under inverted phase contrast microscope.The proliferation of hMSCs was investigated by MTT assay.At two weeks of culture,cells were re-suspended.Pellets were constructed by centrifugation at a density of 5×106/ml,and then cultured for another three weeks.RT-PCR was performed to detect the expression of mRNA of type Ⅱ collagen.Immunohistochemistry and toluidine blue staining were performed to detect the expression of type Ⅱ collagen and proteoglycan. [Results]Most hMSCs exhibited a long fusiform shape.The hMSCs induced with 100 ng/ml GDF-5 showed a round or polygonal shape.After another two-weeks induction,mRNA of type Ⅱ collagen was not expressed in group A,type Ⅱ collagen and proteoglycan,and the expression of mRNA of type Ⅱ collagen,type Ⅱ collagen and proteoglycan in other four groups kept increasing in concentration-dependent manner.There were significant differences between them（P0.05）. [Conclusion] Treated with a certain concentration of GDF-5,expression of type Ⅱ collagen and proteoglycan in the pellets was increased in concentration-dependent manner.And the pellets prossessed some biological functions similar to normal cartilage.