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为研究1-磷酸鞘氨醇(Sphingosine-1-phosphate,S1P)对脐带间充质干细胞(Umbilical cord mesenchymal stem cells,UC-MSCs)和脂肪间充质干细胞(Adipose derived mesenchymal stem cells,AD-MSCs)向心肌分化的影响,探索其适宜的作用时间和浓度,将UC-MSCs和AD-MSCs接种到培养板,用添加不同浓度S1P的心肌细胞培养液诱导两种干细胞向心肌分化,诱导时间分为7 d、14 d和28 d。采用免疫荧光染色检测心肌特异性蛋白,α-肌动蛋白(α-actin)、缝隙连接蛋白(Connexin-43)以及肌球蛋白重链(MYH-6)的表达,并通过共聚焦显微镜和荧光显微镜进行观察;采用MTT分析细胞的活性;膜片钳检测分化细胞的钙瞬变(此为心肌细胞的功能性指标)。结果表明,S1P与心肌细胞培养液协同作用,能够促进UC-MSCs和AD-MSCs向心肌细胞的分化。并且,随着S1P浓度的增加,促分化作用增强,但细胞活性降低。S1P在心肌细胞培养液中的适宜作用时间为14 d,适宜作用浓度为0.5μmol/L。而且联合心肌细胞培养液可以使UC-MSCs和AD-MSCs的心肌分化细胞产生钙瞬变,具有类似心肌细胞的功能性。S1P能够与心肌细胞培养液协同作用,促进UC-MSCs和AD-MSCs的心肌功能性分化。
To investigate the effect of Sphingosine-1-phosphate (S1P) on the proliferation and differentiation of Umbilical cord mesenchymal stem cells (UC-MSCs) and Adipose derived mesenchymal stem cells (AD-MSCs) ) To differentiate into cardiomyocytes. The suitable time and concentration of UC-MSCs and AD-MSCs were inoculated into the culture plate. The differentiation of two kinds of stem cells into cardiomyocytes was induced by adding different concentrations of S1P cardiomyocytes, For 7 d, 14 d and 28 d. The expression of cardiac-specific proteins, α-actin, Connexin-43 and myosin heavy chain (MYH-6) were detected by immunofluorescence staining and analyzed by confocal microscopy and fluorescence The cells were observed under microscope. The cell viability was analyzed by MTT. The calcium transients of differentiated cells were detected by patch clamp (this is a functional index of cardiomyocytes). The results showed that synergistic effect of S1P and cardiomyocyte culture medium can promote the differentiation of UC-MSCs and AD-MSCs into cardiomyocytes. Further, as the concentration of S1P increased, the differentiation promoting effect increased but the cell activity decreased. The suitable time of S1P in cardiomyocyte culture medium was 14 days and the appropriate concentration was 0.5μmol / L. Moreover, the combination of cardiomyocyte culture medium can make calcium transients in differentiated cardiomyocytes of UC-MSCs and AD-MSCs, which is similar to that of cardiomyocytes. S1P synergizes with cardiomyocyte cultures to promote cardiomyocyte differentiation in UC-MSCs and AD-MSCs.