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目的通过两种细胞的共培养探讨细胞间接触对间充质干细胞(mesenchymal stem cells,MSCs)分化的影响。方法分别采用密度梯度离心法和胶原酶消化法分离培养了骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)和膀胱平滑肌细胞(bladder smooth muscle cells,BSMCs)。并对其进行了鉴定。在此基础上以单独培养的BMSCs和BSMCs分别作为阴性和阳性对照组,将获得的BMSCs和BSMCs在体外通过接触与非接触共培养作为实验组,对诱导后的BMSCs是否向平滑肌(smooth muscle cells,SMCs)分化通过免疫荧光和Western blot进行鉴定。结果对BMSCs进行流式细胞术检测结果显示CD90、CD44表达阳性率分别为99.78%,60.27%,CD45表达阴性,阳性率为0.21%。在BMSCs与BMSCs共同培养的实验中,Western blot结果显示:BMSCs本身表达少量calponin蛋白,接触组calponin的表达随培养时间的延长逐渐增多,非接触组无明显变化;免疫荧光显示:接触组在共培养第8天部分细胞抗calponin阳性,非接触组与诱导前无明显变化。结论初步认为BMSCs与BMSCs接触共同培养能诱导BMSCs向平滑肌分化。
Objective To investigate the effect of cell-cell contact on the differentiation of mesenchymal stem cells (MSCs) by co-culture of two kinds of cells. Methods Bone marrow mesenchymal stem cells (BMSCs) and bladder smooth muscle cells (BSMCs) were isolated and cultured by density gradient centrifugation and collagenase digestion. And its identification. On this basis, BMSCs and BSMCs cultured separately were taken as negative and positive control groups, respectively. BMSCs and BSMCs obtained in vitro were used as experimental group by contact and non-contact co-culture in vitro, and whether BMSCs were induced to smooth muscle cells , SMCs) were differentiated by immunofluorescence and Western blot. Results The results of flow cytometry showed that the positive rates of CD90 and CD44 were 99.78% and 60.27%, respectively. The positive rate of CD45 was 0.21%. In BMSCs and BMSCs co-culture experiments, Western blot results showed that: BMSCs themselves express a small amount of calponin protein, contact group calponin expression gradually increased with the incubation time, no significant change in non-contact group; immunofluorescence showed: contact group in a total of On the 8th day, some cells were positive for calponin. There was no significant change in the non-contact group before induction. Conclusion It is preliminarily believed that BMSCs can induce the differentiation of BMSCs to smooth muscle by contact culture of BMSCs with BMSCs.