中文摘要：

目的研究磷脂酰肌醇-3激酶/丝氨酸/苏氨酸蛋白激酶（PI3-K/Akt）信号通路在骨形态发生蛋白-2（BMP-2）诱导骨髓源性心肌干细胞（MCSCs）向心肌细胞分化中的调控作用,探讨MCSCs向心肌细胞分化的信号转导机制。方法利用单克隆培养技术从SD大鼠骨髓中筛选MCSCs,用BMP-2诱导其向心肌细胞定向分化,通过免疫细胞化学标记和Western blotting检测BMP-2诱导后细胞内p-Akt水平的变化,并通过RT-PCR检测心肌早期转录因子和心肌特异基因的表达。结果BMP-2诱导前MCSCs内p-Akt水平较低,诱导后渐增强,20~30min达高峰,1h后逐渐降低。用PI3-K抑制剂Ly294002预处理细胞后,p-Akt水平明显降低。RT-PCR检测显示,诱导前MCSCs的Nkx2.5和GATA-4呈低表达,诱导后2周表达增强,4周表达更加明显。cTnT和Cx-43 mRNA在诱导前未见表达,诱导后2周表达明显,4周表达增强。用Ly294002处理后,细胞的Nkx2.5、GATA-4和cTnT、Cx-43 mRNA的表达显著降低,细胞形态变化不明显。结论BMP-2能诱导MCSCs向心肌细胞分化,PI3-K/Akt信号通路在BMP-2诱导MCSCs向心肌细胞分化中发挥重要的调控作用。

英文摘要：

Objective To investigate the regulating effects of PI3-FUAkt signaling pathway on the differentiation of the marrow-derived cardiac stem cells （MCSCs） induced with BMP-2 toward cardiomyocytes and to determine the possible mechanisms in signal transduction on differentiation of MCSCs toward cardiomyocytes. Methods MCSCs were selected from the bone marrow of SD rats with single-cell cloning culture. The differentiation of MCSCs toward cardiomyocytes was induced with 10 μg/L BMP-2, and changes in p-Akt levels in MCSCs were examined with Western blotting and immunocytochemistry using several time points after induced with the BMP-2. Effect of PI3-K was detected with the PI3-K inhibitor Ly294002. Expression of Nkx2.5 ,GATA-4 and cTnT,Cx-43 mRNA of MCSCs before and after induction were determined with RT-PCR. After induction with BMP-2, the morphological alterations to the cells were examined under a phase contrast microscope. Results MCSCs express c-kit and the early cardiac transcription factors Nkx2.5. Before induction with BMP-2, the p-Akt level in MCSCs was low. After induction with BMP-2, the p-Akt level in MCSCs was increased and was to reach a peak at 20 to 30 min and subsequently decreased at 1 hour. After treatment with the PI3-K inhibitor Ly294002, p-Akt level of the cells was decreased significantly. RT-PCR detection showed that expression of Nkx2.5 and GATA-4 were weak before induction; however expression of Nkx2.5 and GATA-4 was increased at two weeks and conspicuous at four weeks after induction. Expression of cTnT and Cx-43 mRNA was not observed before induction,and was obvious at two weeks and even stronger at four weeks after induction. After treatment with Ly294002, the expression of Nkx2.5, GATA-4, cTnT and Cx-43 mRNA was reduced significandy, although no morphological change to the ceils was obvious. Conclusion BMP-2 may induces differentiation of MCSCs toward cardiomyocytes, and the PI3-K/Akt signaling pathway plays an important role in mediating the differentiation of MCSCs induced with BMP-