超低频磁场基于ERK信号通路介导促进骨髓间充质干细胞向神经元样细胞分化  

Extremely low-frequency electromagnetic fields promote neuronal differentiation of ratmesenchymal stem cells via ERK signaling pathways

作　　者：林产勇 石海杉[1] 刘巍[1] 吴文[1] 

机构地区：[1]南方医科大学珠江医院康复理疗科,广州510280

出　　处：《中华神经医学杂志》2016年第2期109-116,共8页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81473769）

摘　　要：目的探讨10Hz超低频磁场（ELF．EMF）对离体培养大鼠骨髓间充质于细胞（BMSCs）向神经元样细胞分化的影响及其作用机制。方法应用全骨髓贴壁法分离培养大鼠BMSCs，流式细胞仪检测P3代细胞表面标记物。将P3代BMSCs分为ELF—EMF组、ELF．EMF＋抑制剂组、抑制剂组及对照组，给予诱导培养基（含2％二甲基亚砜和200ixmol／L丁基羟基茴香醚1诱导培养5h，诱导过程中ELF—EMF组、ELF-EMF＋抑制剂组给予10Hz、500GSELF．EMF干预，ELF—EMF＋抑制剂组、抑制剂组在诱导前预先加入50ixmol／LU0126共培养30min，使用倒置显微镜观察细胞形态学变化。应用免疫荧光染色及Westernblotting检测各组细胞巢蛋白表达，应用Westernblotting检测各组细胞内磷酸化细胞外信号调节激酶1／2（p-ERK1／2）蛋白表达水平。结果P3代BMSCs呈长梭形，漩涡样排列，CD90表达率为97．9％，CD45表达率为4．7％。诱导结束后细胞均逐渐出现典型的神经元样细胞形态学特征。免疫荧光染色及Westernblotting检测结果显示，与对照组、ELF-EMF＋抑制剂组及抑制剂组比较．ELF—EMF组巢蛋白、P-ERK1／2表达水平均明显增高，差异均有统计学意义fP〈0．05）。ELF—EMF＋抑制剂组巢蛋白表达水平与对照组及抑制剂组比较差异均无统计学意义（p0．05）。结论ELF-EMF可通过活化ERK1／2信号转导通路，促进BMSCs向神经元样细胞分化。Objective To investigate the effect of extremely low frequency-electromagnetic field （ELF-EMF） on bone mesenchymal stem cells （BMSCs） differentiating into neuron like cells in vitro and research its mechanism. Methods BMSCs were collected from rats by means of whole bone marrow adherent. Flow cytometry was used to assay cell surface marker at passage 3. And then, BMSCs were assigned into four groups： ELF-EMF group, ELF-EMF＋U0126 （inhibitor） group, U0126 group and control group; cells were induced by medium （2% DMSO and 200 μmol/L BHA） for 5 h. In the process of neural induction, ELF-EMF group and ELF-EMF＋U0126 group were received 10 Hz, 500 GS ELF-EMF stimulation. Besides, ELF-EMF＋U0126 group and U0126 group were pretreated with 50 μmol/L extracellular signal-regulated kinase （ERK）I/2 inhibitor U0126. The morphology of BMSCs was observed under inverted microscope. The expression of nestin was detected by immunofluorescent staining and Western blotting to identify and determine the differentiation. Western blotting was appliedto detect the preotein level of phosphorylase-ERK1/2 after ELF-EMF exposure. Results BMSCs presented a single long spindle morphology, growing with close whirlpoor-like arrangement; CD90 expression rate was up to 97.9%, while that of CD45 only 4.7%. After induction, each group of cells showed similar shape with neuron-like cells gradually. As compared with the other three groups, ELF-EMF group had significantly higher expression levels of nestin and phosphorylatd ERK1/2 detected by immunofluorescent staining and Western blotting, respectively （P〈0.05）. Meanwhile, the expression levels of nestin among the ELF-EMF＋U0126 group, U0126 group and control group were not statistically significant （P〉0.05）. Conclusion ELF-EMF could promote neuronal differentiation of mesenchymal stem cells via activation of ERK1/2 signaling pathways.

关 键 词：超低频磁场 骨髓间充质干细胞 神经元样细胞 细胞分化 细胞外信号调节激酶1/2 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]