1-磷酸鞘氨醇促进间充质干细胞向心肌分化

Stimulation of sphingosine-1-phosphate on cardiomyogenic differentiation of mesenchymal stem cells

摘要:

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0.5 μmol/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。

Abstract:

To study the effect of sphingosine-1-phosphate (S1P) on the cardiomyogenic differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) and human adipose-derived mesenchymal stem cells (AD-MSCs), we seeded the cells in the culture plates and used cardiomyocyte culture medium (CMCM) combining with different concentration of S1P to induce UC-MSCs and AD-MSCs in vitro for 7, 14 and 28 days. Cardiomyogenic differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. The effects of S1P and CMCM on cell activity were evaluated by the methyl thiazolyl tetrazolium assay. The functional characteristic similar to cardiomyocytes was evaluated through detecting calcium transient. Our results showed that cardiomyogenic differentiation of UC-MSCs or AD-MSCs were enhanced with S1P concentration increasing, but cell activities declined. Results showed that the suitable differentiation time was 14 days, and the optimal concentration of S1P was 0.5 μmol/L. When working together with CMCM, S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes, giving rise to specific electrophysiological properties (the calcium transient). Taken together, our results suggested that S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes when being cultured in CMCM.