尿源性干细胞多向诱导分化及诱导后平滑肌复层细胞膜片的构建

Muitipotent differentiation of urine-derived stem cells and construction of multi-layer cell sheet with induced smooth muscle cells from urine-derived stem cells

目的 通过对尿道下裂患儿尿源性干细胞进行多向诱导分化，探讨其诱导分化潜能；利用诱导后的平滑肌样细胞进行复层细胞膜片构建，评价其在无外源性支架情况下获得自体组织工程补片的可行性。方法 程序性添加细胞因子将尿源性干细胞向平滑肌细胞、内皮细胞和神经细胞方向诱导分化，通过实时定量聚合酶链反应、细胞免疫荧光染色鉴定其诱导效率。诱导后的平滑肌样细胞转移入温度敏感型培养皿（NuncUpcell）中，降低温度获得单层细胞膜片，将单层细胞膜片多次叠加，获得复层平滑肌细胞膜片。结果 ①实验中采集到的尿源性干细胞均表达不同程度的Oct4、Sox2、Nanog等胚胎干细胞特异性标志物，且随诱导时间延长，其表达逐渐减低。②在不同方向的诱导过程中，平滑肌、内皮细胞特异性均逐渐增强。平滑肌细胞特异性标志物α-SMA和Desmin的阳性率在诱导前分别为9.350 0%和16.736 7%，在诱导后分别为91.543 3%和89.093 3%，前后比较，差异均有统计学意义（P〈0.0001）。内皮细胞特异性标志物CD31和vWF的阳性率在诱导前分别为2.613 3%和29.396 7%，在诱导后分别为66.860 0%和74.780 0%，前后比较，差异均有统计学意义（P〈0.0001及P＝0.0001）。神经特异性标志物Nestin的阳性率在诱导前、后分别为49.856 7%和90.990 0%，前后比较，差异有统计学意义（P＝0.000 4）。但神经细胞方向诱导形态变化不明显，未形成神经祖细胞标志性＂花环样＂结构。③诱导后的平滑肌样细胞结合温度敏感型培养皿可获得复层平滑肌细胞膜片。结论 尿源性干细胞向平滑肌和内皮细胞方向诱导效果较好，而神经细胞方向诱导效果欠佳；结合温度敏感型培养皿可获得诱导后平滑肌复层细胞膜片，为泌尿系统组织工程修复提供了新的思路。

Objective To explore the differentiation potential of urine-derived stem cells of hypospadiacs and estimate the feasibility of obtaining autologous tissue engineering patch by using smooth muscle cells derived from urine-derived stem cells without exogenous stents.Methods Urine-derived stem cells were induced into smooth muscle, endothelial and neurogenic cells by adding cytokines procedurally. Then quantitative real-time polymerase chain reaction and cell immunofluorescent staining were used for measuring inducing efficiency. Transferring induced smooth muscle cells into NuncUpcell dishes, single-layer cell sheet was harvested by lowered temperature. Then multi-layer cell sheet was generated by overlaying single-layer cell sheet thrice.Results Urine-derived stem cells expressed specific markers of embryonic stem cells. During induced differentiation of urine-derived stem cells, the expression of pluripotent genes declined relatively. Specific markers of smooth muscle cells and endothelial cells increased significantly after induced differentiation. The pre- and post-induction differences of positive rates of α-SMA and Desmin in smooth muscle cells were statistically significant （9.350 0% vs. 91.543 3% and 16.736 7% vs. 89.093 3%, P〈0.0001）; the pre- and post-induction differences of positive rate of CD31 and vWF in endothelial cells statistically significant （2.613 3% vs. 66.860 0% and 29.396 7% vs. 74.780 0%, P〈0.0001 ＆ P=0.0001）; the pre- and post-induction differences of positive rate of Nestin in neural cells statistically significant （49.856 7% vs. 90.990 0%, P=0.000 4）. However, neurogenic cells could not. Multi-layer cell sheet was formed by using smooth muscle cells induced from urine-derived stem cells with NuncUpcell dishes.Conclusions The induction effect of urine-derived stem cells into smooth muscle and endothelial cells is better than that of neurogenic cells.