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目的：探讨转化生长因子β2(TGF-β2)在低氧条件下诱导骨髓基质干细胞(BMSCs)向软骨细胞分化的作用。方法：无菌条件下分离Wistar 大鼠股骨骨髓，采用全贴壁培养法纯化BMSCs。传6 代后，将细胞随机分为3 组，A组加入25 ng/mL TGF-β2在1%氧浓度条件下培养；B 组加入25 ng/mL TGF-β2在21%氧浓度条件下培养；C 组仅加入含10%胎牛血清的DMEM-α培养液在1%氧浓度条件下培养。3 周后，通过甲苯胺蓝染色检测细胞糖胺多糖，聚合酶链反应检测Ⅱ型胶原和蛋白聚糖(Aggrecan)的表达水平。结果：骨髓细胞经换液后贴壁聚集生长，形态均一，连续传代后形态无明显改变。分组培养第1 周，A、C 组生长速度低于B 组；第 2 周各组均出现不规则形态细胞，A、C 组细胞形态小于B 组；第3 周各组均可见透明样基质，以A组最明显。3 周后行甲苯胺蓝染色，A 组细胞内外均可见丰富的蓝染颗粒，B、C 组染色较A 组略浅。A 组Ⅱ型胶原的表达相对量(1.246± 0.287) 高于B 组 (0.973± 0.365)、C组(0.802± 0.196)，差异有统计学意义(P＜0.05)；B、C 组比较无明显差异(P＞0.05)。A 组Aggrecan 的表达相对量 (0.833± 0.375)高于B组(0.724± 0.173)、C 组(0.602± 0.091)，差异有统计学意义(P＜0.05)；B、C 组比较无明显差异(P＞0.05)。结论：TGF-β2联合低氧环境可明显促进骨髓基质干细胞分化为软骨细胞。

英文摘要:

Objective: To study the effect of TGF-β2 induced bone mesenchymal stemcells (BMSCs) differentiation into chondrocyte in hypoxia condition.Methods:The thighbone marrow of Wistar rat was separated under sterile condition, and the BMSCs was purified by whole bone marrow adherent culture. After 6 passage, the cells were divided into three groups randomly. Cells of Group A were added 25 ng/ml TGF-β2 cultured in 1% oxygen concentration. Group B was joined 25 ng/ml TGF-β2 cultured in 21% oxygen concentration. Group C was just added DMEM-αwith 10%fetal bovine serum cultured in 1%oxygen concentration. Three weeks later, all the groups were stained by toluidine blue to detect the glycosaminoglycan, then PCR was used to test the expression level of Collagen Ⅱ and Aggrecan.Results:The BMSCs grew in colonies with uniform shape, and the morphology was no significant change after serial passage. At the 1st week, the growth rate of Group A and C were lower than Group B. At the 2ed week, all the groups appeared irregular cells, and the cells shape of Group A and C were less than Group B. At the 3rd week, hyaline matrix was visible in all the three groups, and the group A was more than the other two. After 3 weeks, toluidine blue staining showed abundant blue granules in the Group A, which was much more than those of Group B and C. The CollagenⅡexpression level of GroupA (1.246± 0.287) was higher than those of GroupB (0.973± 0.365) and GroupC (0.802± 0.196), and the difference was statistically significant (P＜0.05). There was no difference between Group B and C(P＞0.05). Aggrecan expression level of Group A(0.833± 0.375)was higher than Group B (0.724± 0.173)and Group C (0.602± 0.091), and the difference was statistically significant(P＜0.05). There was no difference between Group B and C(P＞0.05).Conclusion:TGF-茁2 combined hypoxia condition could promote the BMSCs differentiation into chondrocyte.

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