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目的研究细胞外信号调节激酶5(ERK5)和c-Jun N端激酶(JNK)在骨形态发生蛋白9(BMP9)诱导的C3H10T1/2细胞向心肌样细胞分化中的作用。方法利用重组腺病毒将BMP9基因导入C3H10T1/2细胞,Western blot法检测加入BMP9及不同浓度ERK5特异性抑制剂BIX02189或JNK特异性抑制剂SP600125后ERK5和JNK的激活情况;实时定量PCR(qRT-PCR)检测BMP9诱导1周后心肌特异性基因肌细胞增强因子2C(MEF2C)、GATA结合蛋白4(GATA4)表达;Western blot法检测经BMP9诱导3周后心肌特异性蛋白缝隙连接蛋白43(CX43)、心肌肌钙蛋白T(cTnT)表达和免疫荧光技术检测CX43、cTnT在细胞内的表达。结果在转染效率达50%左右的情况下,BMP9可过度激活ERK5和JNK,使其磷酸化水平显著增高(P<0.05);BIX02189抑制ERK5激活后,BMP9诱导的C3H10T1/2细胞的心肌分化标志物MEF2C、GATA4、CX43、cTnT均受到显著抑制(P<0.05),JNK特异性抑制剂SP600125也可抑制MEF2C、GATA4、CX43、cTnT表达,但对MEF2C及GATA4的抑制不如BIX02189显著(P<0.05)。结论 ERK5和JNK的过度激活参与了BMP9诱导的C3H10T1/2细胞向心肌样细胞的分化。
Objective To investigate the role of extracellular signal-regulated kinase 5 (ERK5) and c-Jun N-terminal kinase (JNK) in the differentiation of C3H10T1 / 2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9 (BMP9). Methods BMP9 gene was transfected into C3H10T1 / 2 cells by recombinant adenovirus. The activation of ERK5 and JNK after BMP9 and different concentrations of ERK5-specific inhibitor BIX02189 or JNK-specific inhibitor SP600125 were detected by Western blot. Real-time quantitative PCR (qRT- PCR) was used to detect the expression of cardiomyocyte specific enhancer 2C (MEF2C) and GATA4 (GATA4) 1 week after BMP9 induction. Western blot was used to detect the expression of myocardial specific protein gap junctional protein 43 (CX43 ), Cardiac troponin T (cTnT) expression and immunofluorescence were used to detect the expression of CX43 and cTnT in cells. Results BMP9 overexpressed ERK5 and JNK at a transfection efficiency of about 50% (P <0.05), while BIX02189 inhibited cardiac myogenic differentiation of C3H10T1 / 2 cells induced by BMP9 after ERK5 activation The expression of MEF2C, GATA4, CX43 and cTnT were significantly inhibited (P <0.05). J600 specific inhibitor SP600125 also inhibited the expression of MEF2C, GATA4, CX43 and cTnT, but the inhibition of MEF2C and GATA4 was not as significant as that of BIX02189 (P < 0.05). Conclusion Overexpression of ERK5 and JNK is involved in the differentiation of C3H10T1 / 2 cells into cardiomyocyte induced by BMP9.