细胞外钾离子对体外培养神经干细胞凋亡和分化的影响及其机制的研究

发布时间：2018-01-07 07:42

本文关键词：细胞外钾离子对体外培养神经干细胞凋亡和分化的影响及其机制的研究　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【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,P0.01),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)%,P0.01],20 mM K+组较对照组相比降低[(4.8±1.2)% VS (7±1.4)%,P0.05],40 mmol/L K+组无显著变化[(5.4±1.5)% VS (7±1.4)%,P0.05],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)%,P0.01]。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,P0.01),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)%,P0.05]。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)%,P0.01)],分化7 d后NF-200表达升高[(65.1±5.5)%, (62.2±6.1) % VS (39.4±4.3)%, P0.01)],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。
[Abstract]:The previous view and exists only in the embryonic and early postnatal formation of the mammalian brain nerve, and adult central nerve degeneration or injury due to lack of nerve cells cannot regenerate. In recent years, in the embryonic and adult brain found one terminal at the end of the differentiation of cells, called neural stem cells "these cells maintain life-long self-renewal capacity, and could differentiate into astrocytes, oligodendrocytes and neurons potential. Under physiological condition, the adult mammalian brain persistent nerve regeneration mainly exists in the subventricular zone and the subgranular zone of the hippocampus under two area, and in the state of cerebral ischemia or cerebral injury and other pathological conditions, enhance the regeneration of Central nerve regeneration, these nerve cells in the brain and neural circuits can be integrated in the network remodeling and nerve repair, but The central nervous system itself regeneration ability is limited, so neural stem cell transplantation in the treatment of diseases of the nervous system has broad prospects. The study found that due to the pathological state of micro environment changes after transplantation, cell survival rate decreased and neuronal differentiation rate is low, so the study on the regulation of neural stem cell proliferation, differentiation and apoptosis signaling mechanism, has important significance the use of neural stem cells in treatment of nervous system diseases.
Potassium is the major intracellular cation, plays an important role in the maintenance of cell osmotic pressure and membrane normal electrical activity. Recent studies have found that K+ and K+ channels play an important function in the regulation of cell proliferation differentiation and apoptosis, but there are still some controversy about the effect of K+ on cell differentiation and apoptosis of neural stem and its mechanism, the domestic study is less. This study culture model to obtain stable fetal rat telencephalon neural stem cells in vitro, and on the basis of research on K+ and its concentration in the environment of culture differences on stem cell apoptosis in vitro proliferation and differentiation of nerve, the relationship between K+ and proliferation of neural stem cells differentiation gene Hes1 was discussed. The aim of this study was to further explore the extracellular K+ on neural stem cell differentiation and apoptosis effect and its possible mechanism, to provide for the application of neural stem cells Theoretical basis.
Primary culture of neural stem cells in I mice and identification of the effects of different concentrations of potassium ions on the apoptosis of neural stem cells in vitro
In vitro culture and identification of mouse neural stem cells in vitro
Objective to cultivate a stable and reliable model of the neural stem cells in vitro, to obtain a large number of neural stem cells in fetal rat brain. The separation method of the microscopic end, the medium was in serum-free medium containing epidermal growth factor and basic fibroblast growth factor, cultured and passaged by trypsin digestion method. Cell morphology was observed by microscope, and the detection of cell culture nestin by immunofluorescence (Nestin), and 5% serum induced differentiation in cells after 7 d neurofilament 200 (NF-200) and glial fibrillary acidic protein (GFAP) expression. Results in serum-free medium conditions, cultured cells continue to proliferate and form neural cells, immunofluorescence showed that cultured cells were Nestin positive after induction can differentiate into NF-200 positive or GFAP positive mature nerve cells. Conclusion the primary cultured cells into neural stem cells.
The effect and mechanism of different concentrations of potassium ion on neural stem cell apoptosis in the second part
Objective to study the effect of different concentration of K+ on apoptosis of neural stem cells and its mechanism. The primary neural stem cells cultured in vitro cell culture, from third to 5, divided into control group (Control group), 20 mM K+ 40 mM group, K+ group and 80 mM K+ group, nifedipine group (Nifedipin group). The control group culture fluid based (0.1% N2 +DMEM/F12), mM 20 K+ group, 40 mM K+, 80 mM K+ based medium supplemented with KCL prepared by the corresponding concentration, Nifedipin group as the culture medium added with 20 mmol/L Nifedipin +80 nifedipine mmol/L KCL cultured in 80 mM K+ group. Cell viability was measured by MTT assay, cell apoptosis was analyzed by TUNEL staining, detect the expression of Caspase-3 fragment Western blot method, to observe the apoptotic changes of Hoechst33342 staining. Results of the 20 mM K+ 40 mM group, K+ group and 80 mM group K+ cell proliferation activity was decreased compared with the control group Low (0.455 + 0.006,0.44 + 0.007,0.226 + 0.017 VS 0.61 + 0.012, P0.01), of which 80 mM K+ group was the lowest number of.TUNEL positive cells in statistics, 80 mM in group K+ was significantly higher than the control group [(27.3 + 5.3)% VS (7 + 1.4)%, P0.01], mM 20 K+ group than in the control group lower than [(4.8 + 1.2)% VS (7 + 1.4)%, P0.05] 40, mmol/L K+ group had no significant change [(5.4 + 1.5)% VS (7 + 1.4)%, P0.05] 80, mmol/L K+ in Caspase-3 group was higher than that of the other groups. The expression of TUNEL positive cells compared with nifedipine group with the 80 mmol/L K+ group was significantly reduced [(10.4 + 2.1)% VS (26.2 + 5.7)%, P0.01]. conclusion elevated extracellular concentration of K+ can inhibit the activity of neural stem cells, and high concentration of K+ can induce apoptosis of neural stem cells, and the cell membrane and open Ca2+ channels in the increase of Ca2+ may be one of the induction machine for the occurrence of apoptosis.
Effect of II extracellular potassium on the proliferation and differentiation of neural stem cells
Objective to investigate the effects of extracellular K+ concentration on the proliferation and differentiation of neural stem cells, and analyze the difference of Hes1 gene expression. The primary neural stem cells cultured in vitro cell culture, from third to 5, divided into the normal group (Normal group), normal differentiation group (Control group), 20mM group and K+ the 40mM K+ group. Normal control group based medium (DMEM/F12,0.1% N2), the common differentiation group is a common differentiation medium (DMEM/F12,5% FBS, 0.1% N2), 20mM K+ and 40mM K+ were cultured respectively 20 mM /40 mM KCL, 0.1% N2, DMEM/F12.MTT assay and trypan blue exclusion cell activity assay. The expression of Nestin cells were detected by immunofluorescence and NF-200 to detect the expression of Hes1 gene mRNA RT-PCR method. Results compared with the normal culture group, after 36 h mM 20 K+ group and 40 mM group K+ cells activity were significantly decreased (0.379 + 0.006,0.372 + 0.007 VS 0.435 + 0.012, P0.01), and the death of fine No significant differences in cell number detection [(8.2 + 1.3)% and (9.7 + 2)% VS (9 + 1.5)%, compared with ordinary P0.05]. induced group, intervention after 2 D 20 mM 40 mM K+ group and K+ group the percentage of Nestin positive cells decreased [(51.4 + 7.2)%, (49.1 + 5.6)% VS (75.7 + 8.2)%, P0.01), 7 d after differentiation increased expression of NF-200 [(65.1 + 5.5)% and (62.2 + 6.1)% VS (39.4 + 4.3)%, P0.01), RT-PCR results showed that compared with the ordinary differentiation group 20 K+ group and 40 mM mM K+ cells Hes1mRNA expression level decreased. Conclusion K+ inhibits neural stem cells and neural stem cells into neurons and promote cell proliferation and differentiation, inhibit the expression of Hes1 gene may be one of the mechanism of the differentiation of its effects.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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