骨髓间充质干细胞的分离鉴定及向血管内皮细胞诱导分化过程中microRNA126 的表达

目的 建立分离大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)及将其定向诱导分化为血管内皮细胞(endothelial cells,EDCs )的方法,并通过观察microRNA126的表达规律初步探索其在定向分化中的调控作用。方法 通过反复贴壁及人工分选方法进行大鼠骨髓MSCs的体外分离纯化及扩增,运用免疫荧光法检测MSCs中CD34、CD105和CD73 的表达。应用MEF诱导分化培养液将MSCs定向诱导分化为EDCs,采用qRT-PCR法检测细胞诱导分化过程不同时间点EDCs特征基因CD34、血管内皮钙黏蛋白(VE-cadherin)及调控小RNA分子microRNA126的表达。结果 经分离纯化的骨髓MSCs CD34呈阴性表达,CD105呈强阳性表达,CD73呈阳性表达,证实骨髓MSCs分离成功,且细胞均一性较好,占细胞总数的90%以上。MSCs定向诱导为EDCs早期分化过程中,在诱导前期第1～4天,VE-cadherin和CD34基因呈阴性表达；第5～6天 VE-cadherin和CD34表达显著；第7～9天,VE-cadherin呈持续阳性表达,而CD34于第7天下降,第8～9天又呈阳性表达。microRNA126在分化过程中的表达趋势与CD34一致。结论 成功建立了骨髓MSCs的分离方法及定向诱导分化为EDCs的方法；microRNA126在MSCs向EDCs的定向分化过程中可能起一定的调控作用。

Purification and identification of bone marrow mesenchymal stem cells and microRNA126 expression during their differentiation into vascular endothelial cells

Objective To establish a method for isolation, purification of bone marrow mesenchymal stem cells (MSCs) and for their differentiation into vascular endothelial cells, and to observe the regulatory role of microRNA126 expression during the differentiation. Methods MSCs were isolated from normal rat bone marrow using gradient density centrifugation and repeated attachment method. CD34, CD105 and CD73 expressions in MSCs were detected with immunofluorescent staining. MSCs were cultured with MEF medium for differentiating into endothelial cells; CD34, VE-cadherin and microRNA126 expressions were examined by qRT-PCR at different time points. Results Immunofluorescent detection demonstrated that MSCs have been isolated and purified successfully, with MSCs negative for CD34, strongly positive for CD105 and positive for CD73. The purified MSCs had a good uniformity and a purity above 90%. qRT-PCR examination revealed that CD34 and VE-cadherin expressions were not detected on day 1-4 of induction, strongly positive on day 5-6, and on day 7-9, VE-cadherin was still positive, CD34 decreased on day 7, and increased again on day 8-9. Interestingly, the expression of CD34 and microRNA126 mRNA was consistent during the differentiation. Conclusion We have successfully established a method for MSCs isolation and differentiation into endothelial cells; microRNA126 may play a regulatory role in MSCs differentiation into endothelial cells.