Foxa2调控P19胚胎癌细胞心肌分化过程的分子机制

摘要

目的:研究转录因子forkhead box A2(Foxa2)在P19胚胎癌细胞心肌分化过程中的作用及其分子机制.方法:运用在P19细胞胚胎小体(embryoid bodies,EBs)培养基中加入二甲亚砜(dimethyl sulfoxide,DMSO)的方法将P19细胞诱导分化成心肌细胞,运用反转录PCR (reverse transcription PCR,RT-PCR)方法检测诱导分化过程中不同时间点细胞样本中胚胎性干细胞多能性相关基因(Nanog)、心肌分化相关基因[Cerberus1 (Cer1)和Sonic Hedgehog (Shh)]及Foxa2的mRNA水平,用免疫荧光染色的方法检测分化成熟的心肌细胞.同时分别运用转染真核表达质粒pCMV-rFoxa2(大鼠Foxa2)和构建稳定表达绿色荧光蛋白(green fluorescence protein,GFP)-rFoxa2 P19细胞株的方法以提高P19细胞中Foxa2的表达水平,采用RT-PCR和Western印迹的方法检测上述细胞样本中多能性相关基因和心肌分化相关基因的mRNA和蛋白水平,及其EBs分化体系中心肌分化相关基因的mRNA水平.结果:P19细胞经DMSO诱导后分化形成心肌细胞,并伴随有Foxa2的表达激活.转染pCMV-rFoxa2质粒能在P19细胞中瞬时高量表达rFoxa2并激活其下游Cer1的转录.运用稳定表达GFP-rFoxa2的P19细胞株增高Foxa2的表达可以抑制Nanog的表达,并激活Shh的表达,促进P19细胞EBs心肌分化,表达Cer1和心脏α肌动蛋白(actin,alpha cardiac muscle 1,Actc1).结论:Foxa2参与P19细胞心肌分化过程,Foxa2在DMSO诱导P19细胞心肌分化过程中的作用机制可能为直接抑制Nanog的表达,并激活Cer1和Shh的表达.%Objective:To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism.Methods:P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs).The mRNA levels of pluripotency markers of embryonic pluripotent stem cells,cardiac differentiation related genes,and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR).Differentiated and mature cardiomyocytes were identified by immunofluorescence.Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P 19 cells,and clonal populations of P19 ceils that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells.The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot.The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined.Results:P19 cells differentiated into cardiomyocytes in the presence of DMSO,accompanied by stimulated expression of Foxa2.Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cerl).The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells.The expression of Cerl and cardiac muscle marker actin,alpha cardiac muscle 1 (Actc 1) was upregulated in EBs of GFP-rFoxa2 P19 cells.Conclusion:Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells.Foxa2 may inhibit Nanog expression and stimulate the expression of Cerl and Shh directly during cardiac differentiation in P 19 cells in the presence of DMSO.