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

Objective Toinvestigate the suppression of mibefradil (a kindof T-type calciumchannels retardant) on the proliferation of neural stemcell in vitro and clarify the role of T-type calciumchannels in the proliferation of neural stemcells indirectly. Methods Adult mice brain injury model was establishedand neural stemcell was separatedfromsubventricular zone (SVZ). The proliferation and growth after adding 0, 1.25, 2.5, 5, 10 and 20μmol/L of mibefradil in culture mediumwere observedand nestin staining was performed. The cells were subculturedand differentiated, and NSE and GFAP immunofluorescence staining were performedin differentiatedcells. The effect of mibefradil on cell proliferation was testedby 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazoliumbromide (MTT) and neural stemcell sphere counting. Then 50% inhibiting concentration (IC50) was calculatedand IC50 of mibefradil was addedin culture mediumand neural stemcells were dividedintotwogroups: control groupand experimental group. The expression of Cyclin A was detectedby western blot. Results The number of neural stemcell sphere decreasedafter adding mibefradil. MTT showedthat the proliferation of cells couldbe inhibitedsignificantly when mibefradil was addedwith the concentration over 5μmol/L. The OD value in 5μmol/L group, 10μmol/L groupand 20μmol/L was significantly lower than that in control group. The IC50 rate of mibefradil toneural stemcells was 8.93μmol/L. The expression of Cyclin A protein in experimental groupdecreasedsignificantly comparedtocontrol group. Conclusion Mibefradil can suppress the proliferation of neural stemcell. The mechanismis relatedtoinhibit cell cycle progression.