应用5-氮胞苷体外诱导P19细胞分化为心肌细胞

THE INDUCTION OF P19 CELLS INTO MYOCARDIUM CELLS USING 5-AZA CYTIDINE IN VITRO

目的探讨P19细胞体外经5-氮胞苷(5-aza)诱导向心肌细胞分化的特征。方法将P19细胞接种于铺有琼脂的培养皿中,用含不同浓度5-aza的培养基诱导培养7d,细胞悬浮生长形成类胚体(EBs)。将EBs用生长培养基黏附培养,观察细胞收缩情况;免疫细胞荧光检测α-横纹肌肌动蛋白(α-sarcomeric actin)、心肌肌钙蛋白(cTnT)的表达;RT-PCR检测GATA-4、α-MHC基因表达,鉴定细胞分化。结果将5-aza诱导形成的EBs黏附培养后,在EBs周围的生长晕中出现自发性节律收缩的细胞团片,α-sarcomeric actin及cTnT染色阳性,表达GATA-4、α-MHC基因。10μmol/L 5-aza处理组心肌细胞分化率较高。结论5-aza暴露结合悬浮培养可诱导P19细胞向心肌细胞分化,其分化与5-aza浓度有关。

Objective To analyze the features of P19 cells differentiating into cardiomyocyte after induced by 5- azacytidine（5-aza） in vitro. Methods The P19 cells were cultivated in the medium containing different concentrations（1,5 and 10μmol/L） of 5-aza in dishes laid with a thin layer of agar to form embryonic bodies（EBs）. On the 7th day, EBs were transferred to coverslips in 24 well plates, allowed to adhere and be cultured in the medium without 5-aza. The beating of cells was observed. u-sarcomeric actin and cardiac Troponin T（cTnT） immunofluorescence stain and GATA-4, α-MHC gene RT-PCR analysis were used to identify cell differentiation. Results After the 5-aza induced EBs were cohesively cultured, spontaneously and rhythmically beating cells were found within the EBs outgrowths, which were u-sarcomeric actin-positive and cTnT-positive, and expressed GATA-4, u-MHC gene simultaneously. The treatment with 10μmol/L 5-aza can increase the rate of cardiomyocyte differentiation. Conclusion The combination of 5-aza exposure and suspension culture could induce cardiomyocyte differentiation, and the differentiation was related to the concentration of 5-aza.