膀胱平滑肌细胞条件培养液诱导脐带MSCs向平滑肌细胞分化的实验研究

EXPERIMENTAL STUDY OF DIFFERENTIATION OF UMBILICAL CORD MESENCHYMAL STEM CELLS INTO SMOOTH MUSCLE CELLS INDUCED BY BLADDER SMOOTH MUSCLE CELLS CONDITIONED MEDIUM

目的探讨膀胱平滑肌细胞（bladdersmoothmusclecells，BSMCs）条件培养液能否诱导脐带MSCs（umbilicalcordMSCs，UCMSCs）向平滑肌细胞（smoothmusclecells，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、14d，采用免疫荧光染色和Westernblot检测各组细胞中a一平滑肌肌动蛋白（d—smoothmuscleactin，a．SMA）、Calponin、平滑肌肌球蛋白重链（smoothmusclemyosinheavychain，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Q．SMA、Calponin和SM．MHC均呈阳性表达。培养7d，A、B组可见a-SMA呈阳性表达；14d时，A组q．SMA阳性表达逐渐增多，B组无明显变化。培养7d，A组可见Calponin阳性表达，14d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。Westernblot检测示各组细胞a-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 observe whether umbilical cord mesenchymal stem cells （UCMSCs） can differentiate into the smooth muscle cells （SMCs） induced by bladder SMCs （BSMCs） conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system. Methods UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1 ： 1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium （induced group, group A） and complete medium （control group, group B）, respectively; simple BSMCs served as positive control group （group C）. The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of a-smooth muscle actin （α-SMA）, Calponin, and smooth muscle myosin heavy chain （SM-MHC） of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days. Results The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of α-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of α-SMA could be observed in groups A and B; at 14 days, the positive expression of α-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of a-SMA,Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining. Conclusion UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.