P38 MAPK通路抑制剂对UC-MSC功能的影响

Effect of P38 MAPK pathway inhibitor on the function of UC-MSC

目的:通过小分子化合物抑制脐带间充质干细胞(UC-MSC)的P38 MAPK信号通路,探究该种抑制对UC-MSC功能特性的影响。方法:选用P38 MAPK通路抑制剂SB202190对第5代UC-MSC进行体外诱导培养48h,显微镜下观察UC-MSC的生长状态;采用流式细胞术检测UC-MSC表面标志物的表达情况,并通过成脂诱导分化实验检测诱导前后UC-MSC的干性;用P38 MAPK通路抑制的UC-MSC干预糖尿病大鼠背部伤口并评价效果。结果:显微镜下,经SB202190处理48h的UC-MSC呈典型的长梭形,细胞的群体倍增时间为36h,与未处理的细胞相比无明显差异;UC-MSC表面标志物的表达均符合阳性标志物CD105、CD73、CD90占比>95%、阴性标志物CD45、CD34、CD14、CD19、HLA-DR占比<2%的标准,体外分化实验中P38 MAPK通路抑制的UC-MSC在诱导的第21天经油红O染色可见红色脂滴;在皮下干预的第3天即可观察到P38 MAPK通路抑制的UC-MSC干预组大鼠伤口愈合率较PBS对照组和UC-MSC干预组高。结论:短时间抑制P38 MAPK通路,对UC-MSC的表型以及分化能力无影响,且能够有效增强其组织修复的能力。

Objective: To explore the effect of small molecule mediated P38 MAPK signaling pathway inhibition on functional characteristics of UC-MSC. Methods: The 5 th generation UC-MSCs were treated with P38 MAPK pathway inhibitor SB202190 in vitro for 48 h, and the cell morphology of UC-MSC was observed by microscope. After 48 hours of culture in vitro, cells were digested with 0.25% trypsin, the expression of UC-MSC surface markers was tested by flow cytometry, and the stemness before and after induction of adipogenic differentiation was detected by adipogenesis induced differentiation experiment. Intervention on the back wounds of diabetic rats was implemented to evaluate the effect of P38 MAPK pathway inhibition in UC-MSCs on improving the repair of back skin lesions in diabetic rats. Results: Under the microscope, UC-MSC treated with SB202190 for 48 h showed a typical long spindle shape. In addition, the population of cells doubling time was 36 h, which had no significant different from that of untreated cells. Flow cytometry showed that positive markers, including CD105, CD73, and CD90, accounted for more than 95% of total UC-MSC surface markers, while the negative markers CD45, CD34, CD14, CD19, and HLA-DR accounted for less than 2%, in both SB202190 treated and untreated cells. In vitro differentiation experiment, inhibition of P38 MAPK pathway in UC-MSC showed red lipid droplets on the 21 th day after induction using oil red O staining. Inhibition of P38 MAPK pathway in UC-MSC effectively promoted wound healing of damaged back skin of diabetic rats. On the third day of subcutaneous intervention, wound healing was better than that of UC-MSC without inhibition of the P38 MAPK pathway and PBS.Conclusion:Short-term inhibition of P38 MAPK pathway can effectively enhance tissue repair ability,while have no effect on the phenotype and differentiation of UC-MSC.