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目的探索人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)在胰岛细胞微环境中向胰岛样细胞的分化潜能及功能变化趋势。方法将分离的第3代hUC-MSCs用低糖达尔伯克必需基本培养基(L-DMEM)重悬,接种单纯培养组及与大鼠胰岛细胞共培养组(共培养组)。倒置显微镜观察hUC-MSCs的形态变化;流式细胞仪鉴定其细胞表面标志;免疫细胞化学鉴定胰十二指肠同源框-1基因(PDX-1)的表达;放免法检测hUC-MSCs胰岛素和C肽的分泌及对糖刺激的反应。结果 hUC-MSCs呈均一纺锤形平行排列或漩涡状排列生长,不表达CD34、CD45和CD14,高表达CD29、CD44和CD105;共培养组诱导后3d及7d经细胞逐渐变圆并聚集成团,PDX-1呈阳性表达,至10d及14dPDX-1阳性细胞罕见。单纯培养组细胞形态未发生变化,PDX-1表达阴性。共培养组培养上清中检测到较高水平的胰岛素及C肽,单纯培养组检测到少量的胰岛素,检测不到C肽,两组培养3、7、10和14d的胰岛素分泌水平比较差异均有统计学意义(P<0.01)。共培养组胰岛素及C肽水平以7d最高,10d次之,与3d和14d比较差异均有统计学意义(P<0.01),共培养组糖刺激指数为1.41。结论 hUC-MSCs在胰岛细胞微环境中具有向胰岛样细胞分化的潜能,其内分泌功能随体外诱导时间的延长呈现先递增后递减的趋势。
Objective To explore the differentiation potential and function of human umbilical cord mesenchymal stem cells (hUC-MSCs) into islet-like cells in islet microenvironment. Methods The isolated third generation hUC-MSCs were resuspended in low-Dulbecco’s essential basic medium (L-DMEM), inoculated alone and co-cultured with rat islet cells (co-culture group). The morphological changes of hUC-MSCs were observed by inverted microscope, the cell surface markers were identified by flow cytometry, the expression of PDX-1 gene was identified by immunocytochemistry, and the expression of hUC-MSCs insulin And C peptide secretion and response to sugar stimulation. Results The hUC-MSCs were uniformly spindle-shaped or spirally arranged, but not expressed in CD34, CD45 and CD14. CD29, CD44 and CD105 were highly expressed in hUC-MSCs. The cells were rounded and aggregated into clusters after 3d and 7d, PDX-1 positive expression, to 10d and 14d PDX-1 positive cells rare. The cell morphology of pure culture group did not change, PDX-1 expression was negative. In the co-culture group, a higher level of insulin and C-peptide were detected in the culture supernatant. A small amount of insulin was detected in the culture group and no C-peptide was detected. The levels of insulin secretion at 3, 7, 10 and 14 d There was statistical significance (P <0.01). The levels of insulin and C-peptide in co-culture group were the highest at 7d and the lowest at 10d, which were significantly different from those at 3d and 14d (P <0.01). The co-culture group had a stimulation index of 1.41. Conclusion The hUC-MSCs have the potential to differentiate into pancreatic islet-like cells in the microenvironment of islet cells. The endocrine function of hUC-MSCs increases with the extension of in vitro induction time and then decreases gradually.