TGF-β_2在低氧条件下诱导骨髓基质干细胞向软骨细胞分化的研究

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。

Study on the TGF-β_2 induced the bone Mesenchymal Stem Cells Differentiation into Chondrocyte in Hypoxia Condition

Objective： To study the effect of TGF-β2induced bone mesenchymal stem cells（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-β2cultured in 1% oxygen concentration. Group B was joined 25 ng/ml TGF-β2cultured in 21% oxygen concentration. Group C was just added DMEM-α with 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 Group A（1.246±0.287） was higher than those of Group B（0.973 ±0.365） and Group C（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-β2combined hypoxia condition could promote the BMSCs differentiation into chondrocyte.