低频电刺激诱导外周血干细胞增殖并向施万细胞分化的实验研究

Experimental study of proliferation and differentiation of peripheral blood stem cells into Schwann cells induced by low frequency electrical stimulation

目的探讨体外培养下低频电刺激诱导外周血干细胞增殖并向施万细胞（SC）分化的可能机制。方法原代培养SD大鼠外周血干细胞，将传至第3代的SD大鼠外周血干细胞分为低频电刺激组、细胞外信号凋节激酶（ERK）组、联合应用组、对照组。4组细胞均采用含2％胎牛血清的达尔伯改良伊格尔培养基（DMEM）进行培养，加入SC上清液后，低频电刺激组给予1h持续低频电刺激，ERK组在DMEM中加入浓度为50mmol／L的抑制剂PD98059，联合应用组在ERK组基础上给予1h持续低频电刺激，对照组不行特殊干预处理。诱导前、后，利用噻唑蓝比色法检测4组细胞在570nm处的吸光度A值，并采用Western blot法对诱导后各组细胞的增殖周期蛋白D1（cyclin D1）及细胞周期蛋白依赖性激酶（CDK4）含量进行测定。结果干预前，各组外周血干细胞的A750值无明显差异（P〉0．05）；干预后，低频电刺激组、ERK组、联合应用组、对照组A750值分别为（1．051±0．058）、（0．363±0．343）、（0．894±0．343）、（0．758±0．047），除ERK组外，其它各组A750值均较干预前升高（P〈0．05）；各组A750值组间比较，差异均有统计学意义（P〈0．05）。诱导后，低频电刺激组S-100、神经胶质纤维酸性蛋白（GFAP）及P75的表达率均高于其它各组（P〈0．05），ERK组各蛋白表达率则均低于其它各组（P〈0．05），联合应用组S-100、GFAP及P75的蛋白表达率介于低频电刺激组与ERK组之间，高于ERK组，低于低频电刺激组，差异有统计学意义（P〈0．05）。各组组间ERK表达量比较，差异无统计学意义（P〉0．05）；与对照组比较，低频电刺激组和联合应用组磷酸化细胞外信号调节激酶1／2（p-ERK1／2）、cyelin D1及CDK4蛋白表达水平均较高（P〈0．05），ERK组则较低（P〈0．05）；与联合应用组比较，低频电刺激组p-ERK1／2、cyclin D1及CDK4蛋白表达水平较高（P〈0．05），ERK组较低（P〈0．05）。结论体外培养条件下，低频电刺激可促进SD大鼠外周血干细胞增殖并诱导其向SC分化，且ERK信号传导通路是促进SC增殖分化的途径之一。

Objective To investigate in vitro possible mechanisms by which low frequency electrical stim- ulation may stimulate peripheral blood stem cells＇ proliferation and differentiation into Schwann cells （SCs）. Methods The original generation of peripheral blood stem cells was cultured using Sprague-Dawley （SD） rats. The third passage stem cells were divided into a low frequency stimulation group, an extracellular signal-regulated kinase （ERK） group, a combination group and a control group. All 4 groups were cultured in DMEM containing 2% fetal bovine serum by adding the SC supernatant. The low frequency electrical stimulation group was given 1 h of continuous low frequency stimulation. For the ERK group 50 mmol/L of the inhibitor PD98059 was added. The combination group was given the inhibitor plus 1h of sustained low frequency electrical stimulation. The control group received no special intervention. Before and after induction the 3-（4, 5-dimethyl-thiazol-2 ）-2, 5-diphenyl tetrazolium bromide （MTT） assay was used to detect the absorbance value A of 4 cells at 570 nm （ A750 ） , and after induction Western blotting was used to determine cyclin D1 （ cyclin D1 ） and cyclin-dependent kinases （ CDK4）. Results Before the intervention peripheral blood stem cells in each group had no significant differences in their A750 values. After the intervention, the A750 values of the low frequency electrical stimulation, the ERK group, the combination group and the control group were （1.051 ±0.058）, （0.363 ±0.343）, （0.894 ±0. 343） and （0.758±0. 047）, respectively. This showed a statistically significant increase for all groups except the ERK group. The differences among the groups were also statistically significant. The expression of S-100, glial fibrillary acidic protein （GFAP） and P75 was highest in the low frequency electrical stimulation group, and in the ERK group they were the lowest. S-100, GFAP and P75 protein expression also was highest in the low frequency electrical stimulation group and lowest in the ERK group, and the inter-group differences were statistically significant. ERK expression showed no significant difference among the groups. Compared with the control group, the expres- sion of phosphorylated extracellular signal-regulated kinase 1/2 （p-ERK1/2） , eyclin D1 and CDK4 protein in the low frequency electrical stimulation group and the combination group were all significantly higher and the protein expression in the ERK group was significantly lower. The p-ERK1/2, cyclin D1 and CDK4 protein levels in the combination group were significantly lower than in the low frequency electrical stimulation group and higher than in the ERK group. Conclusions Low frequency electrical stimulation can promote peripheral blood stem cell proliferation and induce cell differentiation into Schwann cells, at least in vitro. The ERK signaling pathways are part of the signaling pathways of the proliferation and differentiation.