P38MAPK信号通路在骨形态发生蛋白2诱导骨髓间充质干细胞分化为心肌样细胞中的作用

Effects of the P38MAPK Signaling Pathway on Bone Marrow Mesenchymal Stem Cells Differentiation into Cardiomyocyte-Like Cells Induced by Bone Morphogenetic Proteins-2

目的研究P38MAPK信号通路在骨形态发生蛋白2(BMP-2)诱导骨髓间充质干细胞(BMSC)分化为心肌样细胞中的调控作用,探讨BMSC向心肌样细胞分化的可能信号机制。方法 BMSC分离与体外培养;分3组:对照组、BMP-2组(诱导剂组)和BMP-2+SB203580组(阻断剂组)。倒置相差显微镜观察细胞的形态学变化;免疫细胞化学检测诱导后BMSC心肌特异性肌钙蛋白T(cTnT)和连接蛋白43(Cx43)的表达;Western blot检测诱导后细胞内p-P38MAPK/P38MAPK的变化。结果 BMP-2诱导15 min,p-P38MAPK蛋白呈弱表达,30 min达到高峰,60 min逐渐降低;用P38MAPK阻断剂SB203580预处理细胞后,p-P38MAPK表达明显降低;与诱导剂组比较,阻断剂组的p-P38MAPK蛋白表达减弱,差异有统计学意义(P<0.05)。诱导4周后,对照组cTnT、Cx43呈阴性表达;诱导剂组和阻断剂组呈阳性表达,且阻断剂组Cx43阳性细胞数多于诱导剂组,差异有统计学意义(P<0.05)。结论 BMP-2可诱导BMSC分化为心肌样细胞,P38MAPK信号通路在诱导过程中发挥负性调节作用。

Aim To investigate the regulating effects of P38MAPK signaling pathway on the differentiation of the bone marrow-derived stem cells（BMSC） induced by BMP-2 toward cardiomyocyte-like cells and to determine the possible mechanisms. Methods BMSC were separated and cultured in vitro. The third passage cells were divided into 3 groups： control group, BMP-2 inducer group, BMP-2 ＋ P38MAPK blocker SB203580 group. The morphological observa- tion was performed by the inverted phase contrast microscope, the immunohistochemical technique was used for detecting the expression of cardiac specific troponin（cTnT） and connexin 43 （Cx43）, Western blot was used for examining of the changes in p-P38MAPK/P38MAPK levels in BMSC after induced with the BMP-2. Results Expression of p- P38MAPK of BMSC could be observed at 15 rain after induction with BMP-2, increased obviously at 30 min,increased in- creasingly from 60 min. Compared with inducer group,the expression of p-P38MAPK of blocker group decreased obviously（P 〈 0. 05）. The expression of cardiac specific troponin（cTnT） and Cx43 were negative in control group, and positive in inducer group and blocker group. Compared with inducer group, Cx43 positive cells increased in blocker group （ P 〈 0. 05）. Conclusion BMP-2 could induce differentiation of BMSC toward cardiomyocyte-like cells,and the P38MAPK sinaling pathway plays a negative regulating role during induction.