体外诱导兔骨髓间充质干细胞向软骨细胞分化的实验研究

目的探讨兔骨髓间充质干细胞（bone marrow derived mesenchymal stem cells，BMSCs）在特定培养条件下向软骨细胞定向分化的情况。方法密度梯度离心法分离兔BMSCs，在体外培养扩增后用含有骨形态发生蛋白（bone morphogenetic protein,BMP)-2的条件培养液诱导其在体外单层培养模式下向软骨细胞分化。采用甲苯胺蓝染色、Masson染色、免疫组织化学染色、阿利新蓝比色法测定糖胺聚糖（glycosaminoglycan,GAG）的含量检测BMSCs分化情况。统计学分析比较第2、4、7代（P₂、P₄、P₇）BMSCs诱导后增殖能力及向软骨细胞分化能力的变化。结果定向诱导后BMSCs表现出软骨细胞的特性。P₂、P₄间的细胞增殖能力和成软骨细胞分化能力比较差异无统计学意义（P＞0.05）；P₇与P₂、P₄相比，细胞增殖能力和成软骨细胞分化能力均降低，差异有统计学意义（P＜0.01）。结论BMP-2能诱导兔BMSCs在体外单层培养模式下向软骨细胞分化；细胞传代对BMSCs向软骨细胞分化有重要影响。

In vitro differentiation of rabbit bone marrow derived mesenchymal stem cells into chondrocytes

Objective To explore chondrogenic differentiation of rabbit bone marrow derived mesenchymal stem cells(BMSCs)in experimental culture medium.Methods BMSCs,isolated from rabbit bone marrow aspirates by density gradient centrifugation,were cultured,expanded and induced into chondrocytes in defined culture medium containing bone morphogenetic protein(BMP)-2 in monolayer culture in vitro.Toliudine blue staining,Masson staining,immunohistochemical staining, and Alcian blue chromatomery of glycosaminoglycan(GAG) were used to determine the differentiation.Statistical analysis of the proliferation and chondrogenic differentiation abilities of the induced BMSCs was deployed among the 2nd,4th and 7th passages.Results Induced BMSCs had characteristics of the chondrocyte.There was no statistical difference in the proliferation and chondrogenic differentiation abilities between the P2 and P4 BMSCs(P>0.05).Compared with the P2 and P4 BMSCs,the proliferation and chondrogenic differentiation abilities of the P7 BMSCs was decreased(P<0.01).Conclusion BMP-2 can induce BMSCs to differentiate into chondrocytes in monolayer culture in vitro.Cell passage is an important factor in the chondrogenesis differentiation of BMSCs.