P38MAPK信号通路参与BMP-13诱导C3H10T1/2细胞向心肌样细胞分化

P38 MAPK signaling pathway is involved in BMP-13-induced cardiomyocyte-like differentiation from C3H10T1/2 cells

目的探讨P38 MAPK对BMP-13诱导C3H10T1/2细胞向心肌样细胞分化的影响。方法实验共4个部分,分组如下:1)BMP-13腺病毒(Ad-BMP-13)对P38 MAPK的作用:Ad-BMP-13转染组、Ad-GFP转染组和C3H10空白组。Western blot检测磷酸化P38 MAPK(p-P38 MAPK)和总P38 MAPK(t-P38 MAPK)的表达变化,免疫荧光技术定位p-P38 MAPK;2)P38 MAPK干扰腺病毒(Ad-si-P38)对P38 MAPK的作用:si-P38干扰组、si-NC干扰对照组和C3H10空白组。Western blot检测t-P38 MAPK的表达;3)Ad-si-P38阻断P38 MAPK后对BMP-13诱导分化的影响:si-P38+Ad-BMP-13转染组、si-NC+Ad-BMP-13转染组、si-NC+Ad-GFP转染组和C3H10空白组。Western blot检测cTnT和Cx43的表达,荧光定量PCR检测GATA-4和MEF-2C的mRNA表达;4)SB203580阻断P38 MAPK后对BMP-13诱导分化的影响:DMSO+Ad-BMP-13转染组、SB203580(2、5和10μmol/L)+Ad-BMP-13转染组。荧光定量PCR检测GATA-4和MEF-2C的mRNA表达。结果 BMP-13促进P38 MAPK的磷酸化。Ad-si-P38可以有效降低P38 MAPK表达水平。Ad-si-P38阻断P38 MAPK后BMP-13诱导组cTnT、Cx43表达有明显降低(P<0.05),GATA-4和MEF-2C的表达也有显著降低(P<0.05)。随P38 MAPK特异性抑制剂SB203580浓度增加,BMP-13诱导组GATA-4和MEF-2C的表达降低(P<0.05)。结论 Ad-BMP-13可以通过激活P38 MAPK信号通路来调控C3H10T1/2细胞向心肌样细胞分化。

Objective To investigate the role of P38 MAPK in BMP-13-induced differentiation of C3H10T1/2 cells into eardiomyocyte-like cells. Methods The four parts of experiment are grouped as follows ： 1 ） BMP-13 adenovi- ms （Ad-BMP-13） on the role of P38 MAPK： Ad-BMP-13 transfeetion group, Ad-GFP transfection group and C3H10 blank group. The phosphorylated P38 MAPK （p-P38 MAPK） and total P38 MAPK （t-P38 MAPK） were detected by Western blot. The positioning of p-P38 MAPK was detected by immunofluorescence technique ;2）P38MAPK interference adenovirus （Ad-si-P38） on the role of P38 MAPK： si-P38 interference group,si-NC control in- terference group and C3H10 blank group. The t-P38 MAPK was detected by Western blot. 3 ）The influence of BMP-13 induced differentiation after Ad-si-P38 blocking P38 MAPK signal pathway：si-P38 ＋ Ad-BMP-13 transfec- tion group,si-NC ＋ Ad-BMP-13 transfection group, si-NC ＋ Ad-GFP transfection group and C3H10 blank group. cTnT and Cxg3 were detected by Western blot and the GATA-4 and MEF-2C were detected by fluorescent quantita- tive PCR. 4）The influence of BMP-13 induced differentiation after SB203580 blocking P38 MAPK signal pathway： DMSO ＋ Ad-BMP-13 transfection group, SB203580 （2,5 and 10 μmol/L） ＋ Ad-BMP-13 transfection group. The GATA-4 and MEF-2C were detected by by fluorescent quantitative PCR. Results BMP-13 promoted P38 MAPK phosphorylation. Ad-si-P38 effectively inhibited the P38 MAPK expression. Ad-si-P38 which blocked P38 MAPK signal pathway significantly inhibited the BMP-13-induced expression of cTnT, Cx43 （ P 〈 0. 05 ） and GATA4, MEF-2C（P 〈0. 05）. With the increased concentration of P38 MAPK specific inhibitor SB203580, expression of GATA-4,MEF-2C was significantly reduced. Conclusion P38 MAPK signal pathway can be activated by Ad-BMP- 13 to promote cardiomyocyte-like cells differentiation from C3H10T1/2 ceils.