The Effect And Mechanism Of Extracellular K~+ On Regulating Neural Stem Cell Apoptosis And Differentiation In Vitro

It is traditionally believed that the neurogenesis only occur in embryonic or postnatal period, and neural cells losed with neurodegenerative diseases or injury could not be replaced in the adult mammalian brain. During the recent years, one kind of terminal undifferential cell was found in the embryo and adult's central nervous system(CNS). These cells was termed as neural stem cells (NSCs) which could self-renew and was multipotent to differentiate into neuron, astrocyte and oligodendroglia. It's believed that in the physiological condition the continuous neuranagenesi mainly exist in two regions: subventricular zone (SVZ) and subgranular zone(SGZ). And in the pathologic status such as cerebral ischemia or cerebral trauma, the ability of neuranagenesis enhanced in CNS. These new born neural cells could integrate with the existed neural circuitry and participate into the neural network rebuilt and repair. However, because of limited neuranagenesis potential of the adult mammalian CNS, the neural stem cells transplant was the hope for the treat of nervous system diseases. But some study suggested the lower rate of cell survival and differentiate due to change of niche in pathologic condition, therefore, it's important to study the mechanisms about NSCs apoptosis and differentiation for the therapy of nervous system diseases.Potassium ion is predominant intra-cellular positive ion and plays an important role in the cell normal function include maintenance the osmotic pressure and membrane potential. Recent studies suggested that the K+ and K+ channel is important for regulating cell apoptosis and differentiation. However, these results have not reach agreement and the mechanism is also not clear. In the present study, we explored the effects of extracellular K+ on regulating NSC apoptosis and differentiation in vitro. Moreover, we investigated the relationship between K+ and Hes1. The objective of this study is to discuss mechanisms of the regulation effect of K+ on apoptosis and differentiation of NSCs, which will provide an academic basis for application of neural stem cells.I Primary culture of the neural stem cells in vitro and effect of extracellular K+ on the apoptosis of neural stem cellsPart 1 Primary culture of the neural stem cellsObjective To isolate and culture the neural stem cells. Methods The NSCs was isolated from mouse cortex and cultured in medium containing EGF and bFGF. The morphous was observed by microscope and the expression of Nestin, NF-200 and GFAP were detected by immunofluorescence. Results The isolated culture cells had the ability to consecutively proliferate and form neurosphere in the medium. Nestin positively expressed in these cells which could differentiate into adult neural cells which express NF-200 or GFAP in medium supplemented with fetal bovine serum. Conclusion The study demonstrated that the primary culture cells are NSCs.Part 2 Effect of extracellular K+ on the apoptosis of neural stem cells Objective To explore the effects and mechanism of extracellular K+ on the apoptosis of NSCs. Methods The cells cultured from embryonic mouse brains were randomly divided into 5 groups: control group, 20mM K+ group, 40mM K+ group, 80mM K+ group and nifedipin group. The viability of NSCs was evaluated by MTT method. Apoptosis of NSCs was measured by TUNEL method and the expression of caspase-3 was detected by Western blot. Results Compared to control group, the proliferation activity of K+ in the trial groups decreased significantly (0.455±0.006,0.44±0.007,0.226±0.017 VS 0.61±0.012,P<0.01). Compared with the control group, the percentage of TUNEL-positive cells in the 80mM K+ group increased significantly [(27.3±5.3)% VS (7±1.4)%, P<0.01], the percentage of TUNEL-positive cells in the 20mM K+ group decreased significantly [(4.8±1.2)% VS (7±1.4)%, P<0.05] and no significance differences was found between 40mM K+ group and the control group [(5.4±1.5)% VS (7±1.4)%, P>0.05]. The TUNEL-positive cells was reduced in the nifedipin group contrast to the 80mM K+ group. Conclusion The results suggest the extracellular K+ suppress the proliferation of the NSCs and high concentration of K+ could induce cell apoptosis, Ca2+ as a factor participate the regulation of high concentration K+ inducing apoptosis.II Effect of K+ on the proliferation and differentiation of neural stem cellsObjective To explore the influence of K+ on the proliferation and differentiation of NSCs and effects on Hes1 gene. Methods The primarily culture cells from the embryonic mouse brains were divided into 4 groups: normal group, control group, 20mM K+ group and 40mM K+ group. The viability of NSCs was evaluated by MTT method and trypan blue exclusion test. Immunofluorescent with antibody nestin and NF-200 was taken to calculate the number of NSCs and neuron on the different phases of differentiation. Results Compared with the normal group, the proliferation activity of 20mM K+ and 40mM K+ groups decreased significantly (0.379±0.006, 0.372±0.007 VS 0.045±0.012,P<0.01), but the number of dead cells didn't reach significant level [(8.2±1.3)%, (9.7±2.0)% VS (9.0±1.5)%, P > 0.05]. The percentage of Nestin-positive cells in the 20mM K+ and 40mM K+ groups were lower than the control group[(51.4±7.2)%, (49.1±5.6)% VS (75.7±8.2)%, P<0.01)] after 2 days, and the NF200-positive cells was higher[(65.1±5.5)%, (62.2±6.1) % VS (39.4±4.3)%, P<0.01)] than the control group after 7 days. And compared with the control group the expression Hes1 mRNAs decreased. Conclusion This study demonstrated the extracellular K+ could induce the differentiation of the NSCs and increase the rate of differentiate neurons, and its regulation of the expression of Hes1 gene maybe one of mechanisms.