MK-801在谷氨酸-Shh信号通路中的阻断作用

发布时间：2018-02-25 20:18

本文关键词： 神经干细胞信号通路谷氨酸 Shh MK-801　出处：《新乡医学院》2014年硕士论文　论文类型：学位论文

【摘要】：背景神经干细胞(neural stem cells)原位激活是指脑组织内的成体NSCs在受到某种活性物质的激活后而出现增殖、迁移、分化的现象,这种内源性NSCs原位激活为干细胞来源以及神经修复开创了新途径。研究发现脑损伤后NSCs原位激活、增殖、迁移和分化与谷氨酸受体NMDA受体的表达有密切相关性。NSCs的增殖和分化受多种因素调控,而非竞争性NMDA受体拮抗剂MK-801(地佐环平/地卓西平)对大鼠内源性NSC的增殖与分化有抑制作用。探索成体NSCs增殖激活信号通路分子机制具有重要意义。本课题分两部分：第一部分神经干细胞培养及鉴定目的将SD (Sprague-Dawley)大鼠神经干细胞进行体外原代培养以及传代培养后,对其增殖的神经干细胞和分化的神经元、星形胶质细胞给予鉴定。为下一步研究神经干细胞增殖分化信号传导通路提供干细胞。方法从出生2天内SD新生大鼠海马中分离出神经干细胞进行体外原代培养,细胞分离液accutase消化后进行传代培养,各代神经干细胞增殖情况用CCK-8试剂检测；取第3代神经干细胞以及诱导分化培养的细胞进行鉴定,采用免疫荧光共聚焦技术检测神经干细胞特异性标记物nestin(巢蛋白),神经元标记物βⅢ-tubulin和星形胶质细胞标记物GFAP,从而对神经干细胞和分化后的神经元、胶质细胞给予鉴定。结果体外培养的神经干细胞分裂生长后形成干细胞球,且传代生长情况良好,选取第3代、5代、7代神经干细胞进行CCK-8细胞活性检测,各代神经干细胞的增殖结果示：24h各代神经干细胞数分别为38675±2480,45105±2405,41259±3419；48h各代细胞数分别为67597±3965,70148+2000,68001±1125；72h各代细胞数分别为139641±3456,140890±3092,140314±2884；96h第各细胞数分别为139164±4497,156602±708,156159±4253。对各代神经干细胞进行免疫荧光检测,结果显示神经干细胞特异性标志物巢蛋白nestin呈阳性表达；利用5%血清诱导分化培养后,免疫荧光共聚焦技术检测到神经干细胞分化为神经元的标志物βⅢ-tubulin和星形胶质细胞的标志物GFAP呈阳性表达。结论经免疫荧光及共聚焦技术鉴定,成功从成体SD大鼠海马齿状回分离并培养出神经干细胞。第二部分MK-801在Shh-谷氨酸信号传导通路中的阻断作用目的利用MK-801对大鼠NSCs谷氨酸受体的阻断作用,观察NSCs中的Shh(sonic hedgehog)及相关标志物的mRNA和蛋白表达的情况,探讨内源性神经干细胞原位激活机制。方法取第3代神经干细胞,消化为单细胞悬液后,随机分成7组,使MK-801的浓度为0μM、5μM、10μM、20μM、40μM、80μM、160μM,各浓度MK-801作用下连续培养7天,采用倒置显微镜下细胞计数和cck-8试剂检测活性细胞数分别检测0-160μM浓度的MK-801作用下神经干细胞增殖情况；将单细胞悬液随机分成空白对照组,谷氨酸组,MK-801组以及谷氨酸、MK-801共同处理组,各组神经干细胞经过相应处理后进行培养,通过western blot、RT-PCR以及实时荧光定量PCR技术分别在蛋白表达水平、基因表达水平了解神经干细胞增殖以及分化过程中信号通路的shh、Nestin、βⅢ-tubulin、GFAP的表达情况。结果神经干细胞在0-160μ M浓度MK-801组中连续悬浮培养7天后,倒置显微镜下进行细胞计数,结果示10μ M以上浓度组细胞数量明显减少,5u M组细胞数量比接种数量多,但比空白对照组细胞数量少；CCK-8连续测量各浓度细胞活性,结果示第2天后各组活性细胞数(单位为105个/ml)分别为1.516±0.084,1.384±0.154,0.937±0.035,0.837±0.020,0.824±0.008,0.866±0.008,0.842±0.008,7.207±0.317,随着时间延长10-160μ M浓度组神经干细胞数量持续减少,5μ M组细胞细胞数量持续增加,生长趋势与空白对照组类似；空白对照组,谷氨酸组,共同处理组以及MK-801组,western blot结果显示：四组Shh/GAPDH灰度值之比分别为空白对照0.115,谷氨酸组0.1239,共同处理组0.047,MK-801组0.03629；Nestin/GAPDH空白对照0.7625,谷氨酸组0.9915,共同处理组0.01871,MK-801组0.29050.115;(FAP/GAPDH灰度值之比分别为空白对照0.7625,谷氨酸组0.9915,共同处理组0.01871,MK-801组0.2905；βⅢ-tubulin/GAPDH灰度值之比分别为空白对照0.598,谷氨酸组0.8834,共同处理组0.0246,MK-801组0.0308。RT-PCR结果示在阻断剂MK-801作用后,MK-801组及联合处理组shh、Nestin、βⅢ-tubulin、 GFAP基因和蛋白表达明显抑制,且两组表达基本一致；谷氨酸组表达明显高于正常对照组。实时荧光相对定量结果显示：四组中ShhmRNA相对于空白对照组的表达水平分别为MK-801处理组0.00381,共同处理组0.00106,谷氨酸组shh基因表达量为空白对照组的5.195倍；基因nestin在谷氨酸组aestin表达量是空白对照组的7.61倍,在共同处理组及MK-801组分别为空白对照组的0.0067、0.0042倍；GFAPmRNA表达量,谷氨酸组是空白对照组的4.6913倍,而共同处理组和MK-801组的表达量分别为于空白对照组；MK-80I组βⅢ-tubulin的基因表达量是空白对照组的0.00165倍。共同处理组是对照组的0.0102374倍。谷氨酸组βⅢ-tubulin的基因表达量是相对于空白对照组的2.615倍。结论MK-801在10μ M及以上浓度可有效抑制神经干细胞增殖；在蛋白表达、基因表达层面上,MK-801能阻断NSCs经谷氨酸/]NMDAR-Shh信号传导的增殖分化通路。
[Abstract]:The background of neural stem cells (neural stem cells) refers to the in situ activation in the brain of adult NSCs by some active substances after activation and proliferation, migration, differentiation, the endogenous NSCs in situ activation as sources of stem cells and nerve repair and create a new path. The proliferation of NSCs was found in situ activation, injury after the brain, migration and differentiation and expression of glutamate receptor NMDA receptor is closely related to the proliferation and differentiation of.NSCs are regulated by many factors and non NMDA receptor antagonist competition agent MK-801 (dizocilpine / Zhuo Xiping) inhibited the proliferation and differentiation of endogenous NSC in rats. To explore the NSCs proliferation activation signal pathway has important significance.
This topic is divided into two parts:
The first part of the culture and identification of neural stem cells
The purpose of the SD (Sprague-Dawley) of rat neural stem cells were cultured and passaged cells and differentiation of stem on the proliferation of neural neurons, astrocytes identified stem cells. Cell proliferation and differentiation signaling the next step of the research of neural stem.
Methods from birth isolated neural stem cells were cultured in vitro within 2 days of SD in the hippocampus of newborn rats, cell separation after accutase digestion were cultured. CCK-8 reagent was used to detect cell proliferation in each generation nerve; the third generation of neural stem cells and differentiation of the cultured cells were identified by immunofluorescence. Focus on the detection of neural stem cell specific marker nestin (nestin), neuronal marker III beta -tubulin and astrocyte marker GFAP, and thus the neural stem cells and differentiated neurons, to identify to glial cells.
Stem cell formation results in vitro cultured neural stem cell division and growth, and growth were in good condition, select the third generation, 5 generation, 7 generation of neural stem cells were detected in CCK-8 cells, the proliferation of neural stem cells showed that the 24h generation of neural stem cell numbers were 38675 + 248045105 + 240541259 + 3419; 48h cells were 67597 + 396570148+200068001 + 1125; 72h cells were 139641 + 3456140890 + 3092140314 + 2884; 96h cell numbers were 139164 + 4497156602 + 708156159 + 4253. for each generation of neural stem cells for immunofluorescence detection showed that neural stem cell specific markers were nestin positive expression of nestin; induction and differentiation culture with 5% serum, immunofluorescence confocal microscopy to detect the differentiation of neural stem cells into neuronal marker beta III -tubulin and astrocytes The marker of the stromal cells, GFAP, was positive.
Conclusion through the identification of immunofluorescence and confocal technique, the neural stem cells were isolated and cultured from the dentate gyrus of adult SD rats.
The blockage of the second part of MK-801 in the Shh- glutamic signal transduction pathway
Objective To observe the blocking effect of MK-801 on NSCs glutamate receptor in rats, observe the Shh (Sonic hedgehog) and mRNA and protein expression of related markers in NSCs, and explore the in situ activation mechanism of endogenous neural stem cells.
Methods third generation of neural stem cells, digested into single cell suspension, were randomly divided into 7 groups, the MK-801 concentration was 0 M, 5 M, 10 M, 20 M, 40 M, 80 M, 160 M, the concentration of MK-801 under continuous cultivation for 7 days using inverted microscope, cell count and CCK-8 reagent test cell number were detected MK-801 activity of 0-160 M concentration of the proliferation of neural stem cells; the single cell suspension were randomly divided into blank control group, glutamate group, MK-801 group and MK-801 treatment group, glutamic acid, neural stem cells of each group after the corresponding treatment after training through the western, blot, RT-PCR and real-time fluorescence quantitative PCR respectively at the protein level, gene expression level understanding of neural stem cell proliferation and differentiation pathways in Shh, Nestin, beta III -tubulin, GFAP expression.
The results of neural stem cells in continuous suspension 0-160 M concentration in the MK-801 group after 7 days of culture, cells were counted under inverted microscope. The results showed that the number of more than 10 mu M group were significantly reduced, the number of 5u cells in M group than the inoculation quantity, but fewer cells than the control group; CCK-8 continuous measurement of the concentration of cell activity the results showed that second days each active cell number (105 /ml units) were 1.516 + 0.084,1.384 + 0.154,0.937 + 0.035,0.837 + 0.020,0.824 + 0.008,0.866 + 0.008,0.842 + 0.008,7.207 + 0.317, with time 10-160 M concentration group continued to reduce the number of neural stem cells, the number of 5 cells in M group cells continued to increase, and the growth trend similar to the blank control group; blank control group, glutamate group, combined treatment group and MK-801 group, Western blot results showed that four groups of Shh/GAPDH gray value than were blank control 0.11 5, glutamate group 0.1239, treatment group 0.047, MK-801 group 0.03629; Nestin/GAPDH control 0.7625, glutamate group 0.9915, treatment group 0.01871, MK-801 group (0.29050.115; FAP/GAPDH gray values were respectively control 0.7625, glutamic acid group 0.9915, treatment group 0.01871, MK-801 group 0.2905; beta III -tubulin/GAPDH gray value of more than 0.598 were control group, glutamic acid 0.8834, treatment group 0.0246, MK-801 group 0.0308.RT-PCR results are shown in the blockade after MK-801, MK-801 group and combined treatment group Shh, Nestin, beta III -tubulin, GFAP gene and protein expression was inhibited, and the two group was basically the same; glutamic acid group was significantly higher than normal control group. Real time fluorescence relative quantitative results showed that: four in the ShhmRNA group compared with the control group the expression levels were 0.00381 MK-801 treatment group, combined treatment group 0.00106, valley The amino acid group of Shh gene expression was 5.195 times higher than that of blank control group; gene nestin in glutamate group aestin expression level was 7.61 times of blank control group, the treatment group and MK-801 group were 0.0067,0.0042 times of blank control group; the expression of GFAPmRNA, glutamic acid group is 4.6913 times of blank control group, the expression of the common treatment group and MK-801 group respectively in control group; group MK-80I beta III -tubulin gene expression is 0.00165 times of blank control group. Treated group was 0.0102374 times higher than the control group. Glutamate group III -tubulin beta gene expression is about 2.615 times of blank control group.
Conclusion MK-801 concentration at 10 M and above can effectively inhibit the proliferation of neural stem cells. At the level of protein expression and gene expression, MK-801 can block the proliferation and differentiation pathway of NSCs through glutamate /]NMDAR-Shh signal transduction.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743

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