红景天苷诱导骨髓间充质干细胞向神经元样细胞分化中的Ca^(2+)/CaM信号通路

Salidroside induces the differentiation of mouse bone marrow mesenchymal stem cells into neuron-like cells mediated by calcium/calmodulin signaling pathway

背景:课题组前期研究表明,红景天苷能诱导骨髓间充质干细胞向神经元样细胞定向分化,Ca2+信号是实现其生物学信号传导的重要途径之一目的:探讨Ca2+/CaM信号通路在红景天苷诱导骨髓间充质干细胞向神经细胞定向分化中的作用及机制。方法:实验分为空白对照组和红景天苷诱导组,红景天苷诱导组将不同质量浓度红景天苷(5,10,20,50,100 mg/L)作用骨髓间充质干细胞24 h和100 mg/L红景天苷作用骨髓间充质干细胞12,24,48,72 h。采用Western blot方法分别检测红景天苷诱导骨髓间充质干细胞后神经标志分子MAP2和Ca2+/CaM信号通路中关键蛋白CaM、CaMKⅡ的表达水平。另外实验设阻断剂组,分别加Ca2+信号通路特异性阻断剂:500μmol/L EGTA(细胞外Ca2+螯合剂)、1 mmol/L Nifedipine(L型Ca2+通道阻断剂)、10 mmol/L LY294002(PI3K抑制剂)分别作用细胞30 min后,再加入100 mg/L红景天苷作用细胞24 h,采用Western blot方法检测阻断Ca2+/CaM信号通路后NSE、CaM的表达情况。结果与结论:1红景天苷诱导后,MAP2的表达上调(P<0.01),说明红景天苷可诱导骨髓间充质干细胞分化为神经细胞。2不同质量浓度红景天苷诱导骨髓间充质干细胞24 h后,10 mg/L红景天苷组CaM、CaMKⅡ的表达与其他诱导组比较显著上调(P<0.01);同一质量浓度红景天苷诱导72 h后CaM、CaMKⅡ表达明显下调(P<0.01)。3阻断细胞外Ca2+和PI3K信号通路后,NSE与CaM的表达水平较红景天苷诱导组上调(P<0.05)。结果表明,红景天苷通过抑制Ca2+/CaM信号通路实现骨髓间充质干细胞向神经细胞定向分化。

BACKGROUND：Our previous studies have shown that salidroside can induce bone marrow mesenchymal stem cells directly into neuron-like cells, and Ca2＋signal is one important way to achieve its biological signal transduction. OBJECTIVE：To investigate the role and mechanism of the calcium/calmodulin （Ca 2＋/CaM） signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into nerve cells. METHODS：Bone marrow mesenchymal stem cells were divided into two groups：control groups and salidroside groups. Salidroside groups were induced with different concentrations of salidroside （5, 10, 20, 50 and 100 mg/L） for 24 hours and 100 mg/L salidroside was added to culture cells for different time （12, 24, 48 and 72 hours）. Western blot assay was used to detect the expression levels of neural cellmarker, microtubule-associated protein 2, and the important protein of Ca2＋/CaM signaling pathway：CaM and calmodulin dependent kinase II （CaMK II）. Then Ca2＋/CaM signaling pathway specific blockers were applied to cells respectively for 30 minutes, including 500 μmol/L EGTA （Ca 2＋chelator）, 1 mmol/L Nifedipine（L-type Ca2＋channel blocker） and 10 mmol/L LY294002 （PI3K inhibitor）. Then, 100 mg/L salidroside was added and cultured for 24 hours. Western blot assay was used to detect the expression of neuron-specific enolase and CaM in the Ca2＋/CaM signaling pathway. RESULTS AND CONCLUSION：（1） After inducing with salidroside, the expression of microtubule-associated protein 2 were upregulated （P〈0.01）, indicating that salidrosid can induce the neuronal differentiation of bone marrow mesenchymal stem cells. （2） After different concentrations of salidrosid induced bone marrow mesenchymal stem cells for 24 hours, the expressions of CaM and CaMK II were significantly upregulated in the 10 mg/L group （ P〈0.01）;For the 100 mg/L salidrosid that was added for cellinduction for different time, the expressions of CaM and CaMK II were significantly downregulated in 72-hour group （P〈0.01）. （3） After blocking extracellular Ca2＋and PI3K signaling pathway, the expressions of neuron-specific enolase and CaM were higher than those in salidrosid groups （P〈0.05）. These results suggest that salidrosid can induce bone marrow mesenchymal stem cellto directly differentiate into nerve cells by inhibiting the Ca2＋/CaM signaling pathway.