人脐带间充质干细胞成软骨诱导过程中软骨标志基因表达的研究

Expression of chondrogenic genes during induction of cartilage differentiation of human umbilical cord mesenchymal stem cells

目的观察脐带间充质干细胞(UCMSCs)体外生物学特征,分析成软骨诱导过程中软骨标志基因的表达,探索分化的最佳时间,为其以后应用于组织工程奠定基础。方法体外培养UCMSCs并使用特定的诱导培养基对第3代UCMSCs进行成脂、成骨、成软骨诱导培养并染色鉴定,流式细胞术检测细胞表面标志物,实时定量PCR检测成软骨诱导0、3、7、14、21 d后SOX9、COL2A1、ACAN的mRNA的表达水平。结果 UCMSCs经过体外培养后,细胞形态均一,呈梭形。UCMSCs经成脂、成骨、成软骨诱导后油红O、茜素红、阿利新蓝染色分别呈阳性反应。流式细胞术检测发现UCMSCs低表达CD34、CD45,高表达CD44、CD105。成软骨标志基因SOX9、COL2A1 mRNA的表达在诱导14 d达到最高水平,ACAN的mRNA的表达在诱导7 d达到最高水平。结论 UCMSCs体外传代培养3代后依然可以保持干细胞的特性,其体外诱导成软骨的最佳时间为14 d。

Objective Characterizing umbilical cord mesenchymal stem cells （UCMSCs） and understanding the changes in chondrogenic gene expression that are involved in the differentiation of UCMSCs to chondrogenic cells is important prior to using this approach for tissue engineering. Methods UCMSCs at passage 3 were tested for adipogenic, osteogenic and chondrogenic capacity and evaluated by flow cytometry to examine the expression of the surface makers. Quantitative polymerase chain reaction analysis determined the expression levels of chondrogenic genes after chondrogenic induction. Results UCMSCs successfully differentiated into adipogenic, osteogenic and chondrogenic lineages and were strongly positive for the mesenchymal marker CD44 and CD105 while negetive for CD34 and CD45. The expression of chondrogenic genes （SOX9, COL2A1） was significantly higher after the fourteenth day of induction. However, a significantly elevated expression of ACAN was observed after seven days of chondrogenic induction. Conclusion UCMSCs retain stem cell characteristics after expansion in culture to passage 3. Chondrogenesis in UCMSCs was most prominent after fourteen days of chondrogenic induction.