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目的研究Notch1信号通路对丹参制剂诱导大鼠骨髓基质细胞(BMSCs)向神经元样细胞分化过程的影响。方法取第3代BMSCs,待细胞增殖到60%～70%融合时进行预诱导,预诱导24 h后,更换含100mL/L丹参注射液的DMEM/F12培养液进行诱导分化。待细胞培养0、3、5和7 h,对各组进行形态学观察,RT-PCR和相对定量PCR(qPCR)检测神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)及Notch1的mRNA表达。结果丹参制剂诱导3 h后,细胞形态发生改变,可见少量突起,诱导培养5 h后细胞突起增多相互交织,呈类神经元细胞分化;诱导7 h后,细胞形态和突起生长情况无明显变化。空白对照组细胞数量有所增加,但细胞形态改变不明显。PCR检测发现,丹参制剂组诱导0、3和5 h,NSE和Nestin的mRNA表达随诱导时间的增加,表达量逐渐增加(P<0.05);诱导7 h,NSE和Nestin的mRNA表达量与5 h相比有所降低,但降低不明显(P>0.05)。丹参制剂组诱导0、3、5和7 h,Notch1的mRNA表达随诱导时间的增加,表达量逐渐降低(P<0.05);同一诱导时间(3、5和7 h)Notch1的mRNA表达量与空白对照组比较降低更加明显(P<0.05)。结论丹参制剂可以促进BMSCs向神经元样细胞分化,且能够抑制Notch1信号通路关键因子Notch1的mRNA表达,提示丹参制剂诱导BMSCs向神经元样细胞分化可能与Notch1信号通路有关。
Objective To investigate the effect of Notch1 signaling pathway on the differentiation of rat bone marrow stromal cells (BMSCs) into neuron-like cells induced by Danshen formulation. Methods BMSCs of passage 3 were harvested, and pre-induced when the cells reached 60% -70% confluency. After pre-induction for 24 h, DMEM / F12 medium containing 100 mL / L Salvia miltiorrhiza injection was used for differentiation induction. The cells were cultured for 0, 3, 5 and 7 h. The morphology of each group was observed. The expressions of neuron specific enolase (NSE), nestin and Notch1 were detected by RT-PCR and qPCR. mRNA expression. Results Salvia miltiorrhiza had morphological changes after 3 hours of induction, and a few protrusions were observed. After 5 hours of culture, the protrusions of cells were increased and intertwined, showing neuronal differentiation. After 7 hours of induction, the cell morphology and the growth of the cells did not change significantly. The number of cells in blank control group increased, but the morphological changes of cells were not obvious. The mRNA expression of NSE and Nestin increased with the induction time and increased gradually (P <0.05) at 0, 3 and 5 h after induction by Salvia miltiorrhiza, h decreased, but the decrease was not significant (P> 0.05). At 0, 3, 5 and 7 h, the mRNA expression of Notch1 decreased with the induction time (P <0.05). The expression of Notch1 mRNA at the same induction time (3, 5 and 7 h) The blank control group decreased more obviously (P <0.05). Conclusion Salvia miltiorrhiza can promote the differentiation of BMSCs to neuron-like cells and inhibit the expression of Notch1, a key factor of Notch1 signaling pathway, suggesting that Salvia miltiorrhiza may induce the differentiation of BMSCs into neuron-like cells, which may be related to Notch1 signaling pathway.