NKX2-5诱导P19细胞向心肌分化的研究

Cardiomyocyte Differentiation from P19 Cells Induced by NKX2-5

目的探索NKX2-5在心肌分化中的作用和机制。方法实验分转染组和未转染组,转染组为稳定表达NKX2-5的P19细胞,未转染组为P19细胞。两组细胞悬浮培养4d,形成聚集体后贴壁培养,于贴壁培养的4d、8d、12d、16d,免疫荧光双标和Western blot检测横纹肌α肌动蛋白(α-SA)和心肌肌钙蛋白T(cTnT)的表达;取贴壁培养16d的细胞透射电镜观察细胞超微结构变化。结果转染组α-SA和cTnT两种蛋白在聚集体贴壁4d时没有表达,8d、12d、16d出现表达和共表达。转染组部分细胞出现幼稚的细胞连接和肌丝样结构;未转染组没有发现两种蛋白的表达,电镜观察未发现分化的细胞。结论稳定表达NKX2-5基因的P19细胞可向心肌分化,但不适于作为心脏发育的体外模型。

Objective To identify the role of NKX2-5 gene in cardiomyocyte differentiation and its mechanism. Methods P19 cells were divided into transfected and non-transfected groups. In the transfected group,P19 cells were with stable expression of NKX2-5 gene. The P19 cells were cultured in suspension for 4 days,and the formed aggregates were transferred to Petri dish for adherent culture. On days 4,8,12, and 16 of the adherent culture,the expressions of α-sarcomeric actin（α-SA）and cardiac troponin T（cTnT）were detected with double-labeling immunofluorescence and Western blot. The ultrastructural changes were observed on day 16. Results In the transfected group,no expression of α-SA and cTnT was found on day 4,and the expression of these 2 proteins or co-expression existed on days 8,12,and 16. There were early cell junction and myofilament-like structure in the cytoplasm of some cells in the transfected group. In the non-transfected group,these 2 proteins were negative,and no differentiated cell was found. Conclusion Stable expression of NKX2-5 gene can induce cardiomyocyte differentiation from P19 cells,but the P19 cells with stable expression of NKX2-5 gene is not suitable to be an in vitro model of cardiac development.