Smad信号通路在骨髓源性心肌干细胞向心肌分化中的作用

目的研究Smad信号通路在BMP-2诱导的骨髓源性心肌干细胞（MCSCs）向心肌分化中的作用，探讨MCSCs向心肌分化的信号转导机制。方法从SD大鼠骨髓中筛选MCSCs，用骨形态发生蛋白-2（BMP-2）诱导向心肌定向分化。Western blotting和免疫细胞化学法检测诱导后细胞内磷酸化的Smadl／5／8的表达及其随诱导时间在细胞内分布的变化。RT-PCR检测诱导前后细胞内GATA-4和心肌特异性肌钙蛋白T（cTnT）mRNA的表达。结果BMP-2诱导后15min，细胞内可检测到磷酸化的Smadl／5／8的表达，30min-1h表达明显增加，1h后开始降低，4h呈低表达。BMP-2诱导后15min，磷酸化的Smadl／5／8仅见于胞质中，30min胞质和胞核均见表达，1h主要在胞核内表达。BMP-2诱导后2周，细胞明显表达GATA-4和cTnT mRNA。诱导后4周细胞呈现成熟心肌细胞样形态。用SB203580抑制Smad信号后，GATA-4和cTnTmRNA的表达显著降低，细胞形态变化不明显。结论Smad信号通路在BMP-2诱导MCSCs向心肌分化中发挥重要的介导作用。

EFFECTS OF THE Smad SIGNALING PATHWAY ON DIFFERENTIATION OF MARROW-DERIVED CARDIAC STEM CELLS TOWARD CARDIOMYOCYTES

Objective To investigate the effects of Smad signaling pathway on differentiation of the marrow-derived cardiac stem cells(MCSCs) induced with BMP-2 toward cardiomyocytes and to explore the molecular mechanisms in signal transduction on differentiation of MCSCs toward cardiomyocytes. Methods MCSCs were selected from the marrow of SD rats, differentiation of the cells toward cardiomyocytes was induced with BMP-2. Changes of expression and distribution of p-Smad1/5/8 in MCSCs in different times of induction were determined with Western blotting and immunocytoehemistry. Expression of GATA-4 and cTnT mRNA of MCSCs before and after induction were examined with RT-PCR. Results Expression of p-Smad1/5/8 of MCSCs could be observed at 15 min after induction with BMP-2, increased obviously from 30 min to 1 hour, decreased from 1 hour, and was weak at 4 hours. p-Smad1/5/8 was expressed only in the cytoplasm at 15 min after induction, expressed both in the cytoplasm and nuclei at 30 min, and expressed mainly at the nuclei at 1 hour. The cells expressed GATA-4 and cTnT obviously at 2 weeks after induction. The cells showed the shape of the mature cardiomyocyte and formed myotube-like structures at 4 weeks after induction. After Smad signaling was inhibited with SB203580, expression of GATA-4 and cTnT reduced obviously, the change in the shape of the cells was not obvious.Conclusion Smad signaling pathway plays important roles in mediating differentiation of MCSCs induced with BMP-2 toward cardiomyocytes.