米贝地尔对大鼠侧脑室下区神经干细胞体外增殖的影响

Suppression of mibefradil on proliferation of neural stem cell separated from subventricular zone of mice in vitro

目的探讨体外条件下T型钙通道阻滞剂米贝地尔对神经干细胞增殖的影响,间接阐明T型钙通道在神经干细胞增殖中的作用。方法构建成年大鼠脑损伤模型,分离侧脑室壁的脑室下层(subventricular zone,SVZ)细胞及新生大乳鼠SVZ细胞,以神经干细胞悬浮培养方法培养得到的细胞,观察细胞扩增及生长形态,并行Nestin染色;对培养的细胞传代培养及诱导分化,并对分化的细胞行NSE、GFAP免疫荧光染色。在神经干细胞培养基中加入米贝地尔,通过计数神经干细胞球,噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]法分析米贝地尔对神经干细胞增殖的影响,计算半抑制浓度,在培养基中加入半抑制浓度的米贝地尔,将细胞分为实验组和对照组,Western blot法检测细胞周期蛋白(Cylin A)的表达。结果米贝地尔能够明显抑制神经干细胞球的形成,MTT法显示加入米贝地尔的浓度>5μmol/L时可显著抑制细胞增殖,5μmol/L组、10μmol/L组和20μmol/L组的OD值与对照组差异有统计学意义。米贝地尔对神经干细胞的半抑制浓度为8.93μmol/L。Western blot法提示加入半抑制浓度的米贝地尔后,Cyclin A蛋白的表达显著降低。结论米贝地尔能够抑制神经干细胞的增殖,其机制可能与抑制细胞周期相关。

Objective To investigate the suppression of mibefradil（a kind of T-type calcium channels retardant） on the proliferation of neural stem cell in vitro and clarify the role of T-type calcium channels in the proliferation of neural stem cells indirectly. Methods Adult mice brain injury model was established and neural stem cell was separated from subventricular zone（SVZ）. The proliferation and growth after adding 0, 1.25, 2.5, 5, 10 and 20μmol/L of mibefradil in culture medium were observed and nestin staining was performed. The cells were subcultured and differentiated, and NSE and GFAP immunofluorescence staining were performed in differentiated cells. The effect of mibefradil on cell proliferation was tested by 3-（4, 5-dimethyl-2-thiazolyl）-2, 5-diphenyl-2-Htetrazolium bromide（MTT） and neural stem cell sphere counting. Then 50% inhibiting concentration（IC50） was calculated and IC50 of mibefradil was added in culture medium and neural stem cells were divided into two groups： control group and experimental group. The expression of Cyclin A was detected by western blot. Results The number of neural stem cell sphere decreased after adding mibefradil. MTT showed that the proliferation of cells could be inhibited significantly when mibefradil was added with the concentration over 5μmol/L. The OD value in 5μmol/L group, 10μmol/L group and 20μmol/L was significantly lower than that in control group. The IC50 rate of mibefradil to neural stem cells was 8.93μmol/L. The expression of Cyclin A protein in experimental group decreased significantly compared to control group. Conclusion Mibefradil can suppress the proliferation of neural stem cell. The mechanism is related to inhibit cell cycle progression.