电磁刺激对脊髓神经干细胞增殖与分化作用研究

摘要

目的 探讨电磁刺激对大鼠脊髓神经干细胞(NSCs)向功能性神经元增殖分化的作用及可能的机制.方法 体外分离培养妊娠第14天(E14 d)的Wistar大鼠脊髓NSCs,无血清培养14 d后,分为对照组和电磁刺激组.电磁刺激组置于脉冲强磁场条件下用促增殖参数(0.1 Hz、4T、8次)进行干预,每次脉冲放电20 ms.对照组不做干预处理.干预后第1天,10％胎牛血清诱导贴壁分化;诱导贴壁后第7天,蛋白免疫印迹法(Western Blot)检测β-catenin蛋白表达情况;NCSs分化后第14天,免疫荧光染色检测神经元MAP2表达情况,红外可视膜片钳技术记录神经元自发放电情况.结果 干预后,免疫荧光染色显示电磁刺激组MAP2阳性细胞数45.4％±3.1％,较对照组38.3％±6.0％明显增多(P＜0.05).Western Blot检测结果显示干预后第7天,电磁刺激组神经干细胞β-catenin蛋白量较对照组增高.膜片钳实验结果显示,诱导后第14天,可以记录到神经元自发放电,是有生理功能的.结论 强电磁刺激能够促进大鼠脊髓神经干细胞增殖分化为功能性神经元.%Objective The study aims to investigate the effects of electromagnetic stimulation on proliferation and differentiation of rat spinal cord neural stem cells (NSCs) and the possible mechanisms.Methods NSCs were isolated from the spinal cord of embryonic 14 d (E14 d) Wistar rats,and cultured in serum-free medium for two weeks.Then they were divided into the experiment and control groups.NSCs of experimental group were exposed in 0.1 Hz,4T high-intensity pulsed electromagnetic field for 8 times,each 20 ms pulse discharge,and NSCs of control group were given sham stimulation.On the firt day after intervention,10％ fetal bovine serum was used to induce adherence and differentiation;protein immunoblotting (Western Blot) was used to detect β-catenin protein expression one week after adherence,and NCSs could differentiate into MAP2 positive neurons by immunofluorescence staining two weeks later.Neuronal spontaneous discharge case was recorded by inflated visual patch clamp after two weeks.Results After the intervention,immunofluorescence staining revealed that the MAP2 positive cells of electromagnetic stimulation group was 45.4％ ± 3.1％,which was significantly increased compared with 38.3％ ± 6.0％ of the control group (P ＜ 0.05).Western Blot showed that 7 days after the intervention the β-catenin protein level in electromagnetic stimulation group was higher than that of the control group.Spontaneous neuronal discharge which was recorded by patch clamp at 14th day after induction showed its physiological function.Conclusion High-intensity pulsed electromagnetic stimulation can promote the rat spinal cord neural stem cells to proliferate and differentiate into functional neurons.