【作者】 王轩；

【导师】 刘卫平； 姬西团；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 硕士

【摘要】 以往观点认为哺乳动物大脑的神经形成和发生只存在于胚胎期和出生早期,而成体中枢中因神经变性或损伤而缺失的神经细胞则无法再生。近年来,在胚胎和成体大脑中发现了一类终末未分化细胞,称为“神经干细胞”,这类细胞终生保有自我更新能力,并具有分化成为星形胶质细胞、少突胶质细胞和神经元的潜能。在生理状态下,成年哺乳动物脑内持续性神经再生主要存在于侧脑室的室管膜下区和海马的颗粒细胞下层两个的区域,而在脑缺血或脑外伤等病理状态下,中枢神经系统神经再生能力增强,这些再生的神经细胞能够与脑内神经回路进行整合,参与神经网络重塑和修复,但中枢神经系统本身再生能力有限,因此神经干细胞移植治疗神经系统疾病具有广阔前景。研究发现,由于病理状态下微环境的改变,移植后细胞存活率下降并且神经元分化率较低,所以研究调控神经干细胞增殖、分化及凋亡的信号机制,对利用神经干细胞治疗神经系统疾病具有重要意义。钾离子是细胞内主要的阳离子,对维持细胞渗透压和细胞膜的正常电活动等具有重要的意义。近年来的研究发现,K+及K+通道在调节细胞增殖分化和凋亡上发挥着重要的功能,但目前关于K+对神经干细胞分化及凋亡的影响及其机制还存在一定的争议,国内研究也较少。本研究利用体外培养模型获得稳定的胎鼠端脑神经干细胞,并以此为基础研究培养环境中K+及其浓度差异对体外神经干细胞凋亡、增殖及分化的影响,初步探讨了K+与神经干细胞增殖分化相关基因Hes1的关系。通过本研究旨在进一步探讨细胞外K+对神经干细胞的凋亡及分化的影响和其可能的机制,为神经干细胞应用提供理论基础。I小鼠神经干细胞的原代培养、鉴定及不同浓度钾离子对体外培养神经干细胞凋亡的影响第一部分小鼠神经干细胞的体外培养及鉴定目的通过稳定可靠的神经干细胞体外培养模型,获得大量神经干细胞。方法显微下分离胎鼠端脑组织,培养液为无血清培养基中添加表皮生长因子和碱性成纤维生长因子,利用胰酶消化法培养并传代。显微镜观察细胞形态,并利用免疫荧光法检测培养细胞中巢蛋白(Nestin),及5%血清诱导分化7 d后细胞中神经丝蛋白200(NF-200)和胶质纤维酸性蛋白(GFAP)的表达。结果在无血清条件培养基条件下,分离培养的细胞持续增殖并形成神经细胞球,免疫荧光显示,培养细胞Nestin阳性,诱导后,可以分化为NF-200阳性或GFAP阳性的成熟神经细胞。结论实验中原代培养细胞为神经干细胞。第二部分不同浓度钾离子对神经干细胞凋亡的影响及机制目的研究不同浓度K+对神经干细胞凋亡的影响及其机制的初步探讨。方法体外培养获得原代神经干细胞,取第3～5代培养细胞,分为对照组(Control组),20 mM K+组,40 mM K+组和80 mM K+组,硝苯地平组(Nifedipin组)。对照组为基础培养液(0.1% N2 +DMEM/F12),20 mM K+组、40 mM K+、80 mM K+为基础培养液中添加KCL配制成相应浓度,Nifedipin组为在80 mM K+组培养液基础上添加硝苯地平配制20μmmol/L Nifedipin +80 mmol/L KCL培养液。利用MTT法检测细胞活性,TUNEL染色分析各组细胞凋亡,Western blot法检测Caspase-3活性片段表达,Hoechst33342染色观察凋亡细胞核变化。结果20 mM K+组、40 mM K+组和80 mM K+组细胞增殖活性与对照组相比均降低(0.455±0.006、0.44±0.007、0.226±0.017 VS 0.61±0.012,P<0.01),其中80 mM K+组活性最低。TUNEL阳性细胞数统计分析结果显示,80 mM K+组明显高于对照组[(27.3±5.3)% VS (7±1.4)%,P<0.01],20 mM K+组较对照组相比降低[(4.8±1.2)% VS (7±1.4)%,P<0.05],40 mmol/L K+组无显著变化[(5.4±1.5)% VS (7±1.4)%,P>0.05],80 mmol/L K+组Caspase-3活性片段表达高于其余各组。硝苯地平组TUNEL阳性细胞率与80 mmol/L K+组相比显著降低[(10.4±2.1)% VS (26.2±5.7)%,P<0.01]。结论升高细胞外浓度K+可以抑制神经干细胞活性,且过高浓度K+可以诱导神经干细胞凋亡,而膜Ca2+通道的开放和细胞内Ca2+的增加可能是诱导凋亡发生的机制之一。II细胞外钾离子对神经干细胞增殖分化的影响目的探讨细胞外K+浓度对神经干细胞增殖分化的影响,并分析Hes1基因表达的差异。方法体外培养获得原代神经干细胞,取第3～5代培养细胞,分为正常培养组(Normal组),普通诱导分化组(Control组),20mM K+组和40mM K+组。正常对照组为基础培养液(DMEM/F12、0.1% N2),普通诱导分化组为普通诱导分化液(DMEM/F12、5% FBS、0.1% N2),20mM K+和40mM K+组培养液分别为20 mM /40 mM KCL、0.1% N2、DMEM/F12。MTT法和台盼兰排除实验检测细胞活性,免疫荧光法检测细胞Nestin及NF-200表达,RT-PCR法检测Hes1基因mRNA表达水平。结果与正常培养组相比,36 h后20 mM K+组和40 mM K+组细胞活性均降低(0.379±0.006,0.372±0.007 VS 0.435±0.012,P<0.01),而死亡细胞数检测无明显差异[(8.2±1.3)%, (9.7±2.0)% VS (9.0±1.5)%,P>0.05]。与普通诱导分化组相比,干预2 d后20 mM K+组和40 mM K+组Nestin阳性细胞百分比降低[(51.4±7.2)%, (49.1±5.6)% VS (75.7±8.2)%,P<0.01)],分化7 d后NF-200表达升高[(65.1±5.5)%, (62.2±6.1) % VS (39.4±4.3)%, P<0.01)],RT-PCR结果显示,与普通分化组相比20 mM K+组和40 mM K+组细胞Hes1mRNA水平上表达降低。结论细胞外K+抑制神经干细胞增殖并促进神经干细胞向神经元分化,对Hes1基因表达的抑制可能是其影响分化的机制之一。更多还原

【Abstract】 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.更多还原