P19细胞体外DM SO诱导向心肌样细胞分化

DMSO-induced differentiation in vitro of P19 cells towards cardiomyocytes

目的:体外诱导P19细胞向胚胎心肌样细胞分化.方法:P19细胞经复苏、传代,在体外二甲基亚砜(DMSO)诱导下向心肌细胞分化,分化细胞经HE染色及心肌标志物转录因子(GATA-4),连接蛋白43(connecxin43),α-肌节型肌动蛋白(α-sarcomeric actin)的免疫细胞化学染色后,在光镜、倒置显微镜下观察细胞形态学变化,透射电镜下观察细胞超微结构变化.结果:经DMSO诱导分化后4 d有细胞聚集体形成,6 d有部分细胞GATA-4和connecxin43呈阳性表达,GATA-4阳性表达位于细胞核和细胞质,connecxin43阳性表达呈斑点状分布于心肌细胞胞质内和细胞膜表面.10 d时GA-TA-4和connecxin43阳性表达明显增加,并有部分细胞呈α-sarcomeric actin阳性,阳性表达位于细胞质.电镜观察,未分化细胞为圆形或椭圆形,细胞间连接松散,细胞质较少,细胞器不发达;诱导后6 d,细胞间出现紧密连接和中间连接,细胞质增多,部分细胞表面出现突起,突起内可见微丝.诱导后10d,线粒体、粗面内质网、滑面内质网、高尔基体丰富,并有中心粒形成.结论:P19细胞在DMSO诱导下已有向心肌分化趋向,有类似胚胎心肌细胞形成.

AIM： To induce P19 cells to differentiate towards embryonal cardiomyocytes in vitro. METHODS： P19 cells were induced to differentiate towards cardiomyocytes in the presence of 1% dimethyl sulfoxide （DMSO）. Cell morphological changes were observed under an inverted microscope. The proteins for cardiac markers GATA-4, connecxin43 and α sarcomeric actin were determined by immunocytochemistry. The ultrastructure was observed under transmission electron microscope （ TEM ）. RESULTS： On day 4 after DMSO-induced differentiation, cell aggregation formed. GATA-4 expression was positive on day 6 and obviously positive on day 10 after differentiation. The positive expression was in nuclear and plasma. Connecxin43 expression was positive on day 6. The positive expression was in both plasma and membrane. On day 10 after differentiation, the positive expression was more obvious, α-sarcomeric actin expression was positive on day 10 after differentiation. The positive expression was in plasma. On day 6, plasma and organelle in cells increased, tight junction and intermediate junction were found and there was apophysis in cell surface and microfilament in apophysis. On day 10, mitochondrion, rough endoplasmic reticulum, smooth endoplasmic reticulum and Golgi body were rich in cytoplasm and centriole was found. CONCLUSION： P19 cells exhibit the trend of differentiation towards cardiomyocytes in vitro in the presence of 1% DMSO, which is proved by the emergence of embryonal cardiomyocytes.