KLF2和KLF15基因在hBMSCs定向分化中的动态表达

Dynamic expression of KLF2 and KLF15 during the lineage commitment process of human bone marrow mesenchymal stem cells

目的研究KLF2和KLF15在人骨髓间充质干细胞(hBMSCs)成脂、成骨和成肌分化过程中的表达水平及变化趋势,探讨KLF2和KLF15在hBMSCs定向分化过程中可能的作用方式。方法密度梯度离心法分离hBMSCs,流式细胞仪检测细胞表面标志物,取第4代细胞分别进行成脂、成骨和成肌诱导并以油红O、茜素红及免疫荧光染色进行鉴定。通过实时定量PCR和免疫荧光分别从mRNA水平和蛋白水平检测相关标志物、KLF2、KLF15和GLUT4的表达。结果体外培养的hBMSCs表达间充质干细胞表面标志CD29、CD90,并在特定诱导剂作用下能够定向分化为脂肪细胞、成骨细胞和成肌细胞,染色鉴定结果为阳性,并分别检测到相关标志基因的表达;hBMSCs成脂、成骨和成肌过程中KLF2、KLF15和GLUT4的表达均呈动态变化。结论 KLF2和KLF15与hBMSCs成脂、成骨和成肌分化的启动和维持有关;KLF2和KLF15可能通过GLUT4调节能量代谢从而影响hBMSCs定向分化。

Objective To find out whether there process of induced adipogenic, osteogenic, and is any alteration of KLF2 and KLF15 gene expression during the myogenic differentiation from human bone marrow mesenchymal stem cells （hBMSCs） in vitro, and to discuss the possible roles of KLF2 and KLF15 in these processes. Methods Cells were isolated by density gradient centrifugation from human bone marrow. Surface markers were analyzed by flow cytometry. Cultures in the 4th passage were treated with established lineage-specific agents in vitro and confirmed by Oil red O, Alizarin red S, myosin immunofluorescent staining, and several differentiation marker genes expression. Meanwhile, the expression of KLF2, KLF15 and GLUT4 were detected by quantitative RT-PCR and im- munofluorescent staining. Results The hBMSCs expressed markers of mesenchymal stem cells, CD29 and CD90. Cultures in the 4th passage gained the ability to undergo lineage commitment after exposure to adipogenic, osteogenic, and myogenic stimuli, and expressed specific markers. Both KLF2 and KLF15 showed characteristic timecourse expression during adipogenesis, osteogenesis, and myogenesis. Conclusions KLF2 and KLF15 are possibly related to the initiation and maintenance of hBMSCs adipogenesis, osteogenesis, and myogenesis ; KLF2 and KLFI5may affect hBMSCs differentiation through GLUT4 mediated energy metabolism.