全反式维甲酸体外诱导P19细胞向心肌方向分化

目的探讨不同浓度全反式维甲酸(all-trans retinoic acid,atRA)诱导P19细胞向心肌分化的效力。方法细胞分成P19细胞组,2nm/L atRA诱导组,5nm/L atRA诱导组,8nm/L atRA诱导组。各组细胞经过诱导、聚集培养、聚集体贴壁培养10天后,RT-PCR检测GATA-4,α-肌球蛋白重链(α-myosin heavychain,α-MHC)的mRNA表达,免疫荧光双标检测α-sarcomeric actin和cTnT蛋白共表达,Western blot检测cTnT的蛋白表达。结果 atRA可诱导聚集P19细胞表达GA-TA-4、a-MHC mRNA;α-sarcomeric actin和cTnT的表达和共表达增加;5nm/L atRA组,8nm/L atRA组GATA-4、a-MHCmRNA的表达量显著高于P19细胞组;5nm/L atRA组,8nm/L atRA组两种蛋白的表达和共表达量显著高于P19细胞组,以5nm/L atRA组最高,显著高于其它组。结论 atRA可诱导聚集P19细胞向心肌分化,其中5nm/L atRA组效果最好。

Differentiation of cardiomyocytes from P19 cells induced byall-trans retinoic acid in vitro

Objective To investigate the efficiency of P19 cell differentiation into cardiomyocytes induced by different concentrations of all-trans retinoic acid （atRA）. Methods P19 cells were divided into 4 groups： P19 cell group, 2nm/L atRA group, 5nm/L atRA group and 8nm/L atRA group. The cells were cultured and induced by different concentrations of atRA respectively, then aggregation cultured, and the formed aggregates were adherently cultured for 10 days. The expressions of GATA-4, α-myosin heavychain （α-MHC） mRNA were detected with RT-PCR. Double immunofluorescent staining was used to detect the co-expression of α-sarcomeric actin and cTnT protein and Western Blot was used to detect the expression of cTnT protein. Results P19 cells be differentiated towards cardiomyocytes, expressing GA- TA-4 and a--MHC mRNA and co-expressing α-sarcomeric actin and cTnT protein, induced by both atRA treatment and aggregation culture. The expression of GATA-4 and a-MHC mRNA in 5nm/L atRA group 8nm/L atRA groups were significantly higher than that in the P19 cell group. Compared with that of the P19 cell group, the expression and co-expression of the two proteins were significantly increased in 5nm/L atRA and 8nm/L atRA groups. The highest protein expression was in the 5nm/L atRA group, which was significantly higher than in the other groups. Conclusion P19 cells can be differentiated towards cardiomyocytes induced by atRA treatment together with aggregation culture. Among all the groups, the best induc- tion concentration of atRA is 5nm/L.