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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。
OBJECTIVE: To establish a method for the isolation of mesenchymal stem cells (MSCs) from rat and to induce them to differentiate into endothelial cells (EDCs). The expression of microRNA126 was initially explored in directional differentiation The regulatory role. Methods The bone marrow MSCs were isolated, purified and expanded by repeated adherent and artificial sorting methods. The expression of CD34, CD105 and CD73 in MSCs was detected by immunofluorescence. MSCs were induced to differentiate into EDCs by using MEF-induced differentiation medium. The expression of CD34, VE-cadherin and microRNA126 in EDCs were detected by qRT-PCR at different time points. . Results The purified MSCs were negative for CD34, strong positive for CD105 and positive for CD73. The results showed that MSCs were separated successfully and their cell homogeneity was good, accounting for more than 90% of the total cells. VE-cadherin and CD34 were negatively expressed on the first to fourth day of preadministration, while the expression of VE-cadherin and CD34 was significant on the 5th to 6th day. On the 7th to 9th day, VE- Cadherin was continuously positive expression, while CD34 decreased on the 7th day, again on the 8th to 9th day again positive expression. The trend of microRNA126 expression during differentiation is consistent with that of CD34. Conclusion The isolation method of bone marrow MSCs and the method of inducing differentiation into EDCs were established successfully. MicroRNA126 may play a regulatory role in the directional differentiation of MSCs into EDCs.