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目的探讨膀胱平滑肌细胞(bladder smooth muscle cells,BSMCs)条件培养液能否诱导脐带MSCs(umbilical cord MSCs,UCMSCs)向平滑肌细胞(smooth muscle cells,SMCs)分化,为组织工程技术应用于泌尿系统修复重建寻找可供选择的种子细胞。方法取足月新生儿脐带和行膀胱全切术患者捐赠的正常膀胱组织,分别分离培养UCMSCs和BSMCs。收集第1~5代BSMCs的培养液,与完全培养基以1∶1比例配制成BSMCs条件培养液。取第3代UCMSCs作为诱导细胞,使用BSMCs条件培养液培养为诱导组(A组),完全培养基培养为对照组(B组),倒置显微镜观察两组细胞形态变化;另设单纯BSMCs为阳性对照组(C组)。培养7、14 d,采用免疫荧光染色和Western blot检测各组细胞中α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Calpon in、平滑肌肌球蛋白重链(smooth muscle myosin heavy chain,SM-MHC)的表达情况。结果诱导培养后,A组细胞逐渐变长,由短棒状、多个突起逐渐转变为长梭形,与BSMCs形状相似;B组细胞形态未见明显变化。免疫荧光染色示,C组BSMCs中α-SMA、Calponin和SM-MHC均呈阳性表达。培养7 d,A、B组可见α-SMA呈阳性表达;14 d时,A组α-SMA阳性表达逐渐增多,B组无明显变化。培养7 d,A组可见Calponin阳性表达,14 d时阳性表达明显增多;B组各时间点均未见Calponin阳性表达。各时间点A、B组均未见SM-MHC阳性表达。Western blot检测示各组细胞α-SMA、Calponin和SM-MHC蛋白表达情况与免疫荧光染色结果基本一致。结论 BSMCs条件培养液能诱导UCMSCs向SMCs分化,UCMSCs有望成为泌尿系统修复重建可供选择的种子细胞之一。
Objective To investigate whether bladder transitional cell (BSMCs) conditioned medium can induce the differentiation of umbilical cord MSCs into smooth muscle cells (SMCs), and to apply tissue engineering techniques to urinary system repair and reconstruction Find alternative seed cells. Methods The normal bladder tissue donated by neonates with umbilical cord and total hysterectomy was obtained. UCMSCs and BSMCs were isolated and cultured respectively. The first to fifth generation of BSMCs culture medium was collected, and the complete medium to 1: 1 ratio formulated BSMCs conditioned medium. The third generation of UCMSCs was taken as induced cells, and the conditioned medium of BSMCs was used as induction group (group A). The complete medium was cultured as control group (group B). The morphological changes of the two groups were observed under inverted microscope. Control group (C group). After cultured for 7 and 14 days, the expression of α-smooth muscle actin (α-SMA), Calpon in, smooth muscle myosin heavy chain chain, SM-MHC) expression. Results After induction, the number of cells in group A gradually became longer and gradually changed from short rod to multiple fusiform to long fusiform, which was similar to that of BSMCs. No significant change was observed in group B. Immunofluorescence staining showed that α-SMA, Calponin and SM-MHC were positive in group C BSMCs. After cultured for 7 days, the positive expression of α-SMA was observed in group A and B. On the 14th day, the positive expression of α-SMA in group A gradually increased, while in group B there was no significant change. After 7 days of culture, Calponin positive expression was observed in group A, and the positive expression was significantly increased on day 14; no positive expression of Calponin was found in group B at all time points. At each time point, no positive expression of SM-MHC was found in A and B groups. The results of Western blot showed that the expressions of α-SMA, Calponin and SM-MHC in each group were consistent with those of immunofluorescence staining. Conclusion BSMCs conditioned medium can induce the differentiation of UCMSCs into SMCs. UCMSCs are expected to be one of the available seed cells for urinary system repair and reconstruction.