本文關(guān)鍵詞： 神經(jīng)干細(xì)胞 信號(hào)通路 谷氨酸 Shh MK-801　出處：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景神經(jīng)干細(xì)胞(neural stem cells)原位激活是指腦組織內(nèi)的成體NSCs在受到某種活性物質(zhì)的激活后而出現(xiàn)增殖、遷移、分化的現(xiàn)象,這種內(nèi)源性NSCs原位激活為干細(xì)胞來(lái)源以及神經(jīng)修復(fù)開創(chuàng)了新途徑。研究發(fā)現(xiàn)腦損傷后NSCs原位激活、增殖、遷移和分化與谷氨酸受體NMDA受體的表達(dá)有密切相關(guān)性。NSCs的增殖和分化受多種因素調(diào)控,而非競(jìng)爭(zhēng)性NMDA受體拮抗劑MK-801(地佐環(huán)平/地卓西平)對(duì)大鼠內(nèi)源性NSC的增殖與分化有抑制作用。探索成體NSCs增殖激活信號(hào)通路分子機(jī)制具有重要意義。 本課題分兩部分： 第一部分神經(jīng)干細(xì)胞培養(yǎng)及鑒定 目的將SD (Sprague-Dawley)大鼠神經(jīng)干細(xì)胞進(jìn)行體外原代培養(yǎng)以及傳代培養(yǎng)后,對(duì)其增殖的神經(jīng)干細(xì)胞和分化的神經(jīng)元、星形膠質(zhì)細(xì)胞給予鑒定。為下一步研究神經(jīng)干細(xì)胞增殖分化信號(hào)傳導(dǎo)通路提供干細(xì)胞。 方法從出生2天內(nèi)SD新生大鼠海馬中分離出神經(jīng)干細(xì)胞進(jìn)行體外原代培養(yǎng),細(xì)胞分離液accutase消化后進(jìn)行傳代培養(yǎng),各代神經(jīng)干細(xì)胞增殖情況用CCK-8試劑檢測(cè)；取第3代神經(jīng)干細(xì)胞以及誘導(dǎo)分化培養(yǎng)的細(xì)胞進(jìn)行鑒定,采用免疫熒光共聚焦技術(shù)檢測(cè)神經(jīng)干細(xì)胞特異性標(biāo)記物nestin(巢蛋白),神經(jīng)元標(biāo)記物βⅢ-tubulin和星形膠質(zhì)細(xì)胞標(biāo)記物GFAP,從而對(duì)神經(jīng)干細(xì)胞和分化后的神經(jīng)元、膠質(zhì)細(xì)胞給予鑒定。 結(jié)果體外培養(yǎng)的神經(jīng)干細(xì)胞分裂生長(zhǎng)后形成干細(xì)胞球,且傳代生長(zhǎng)情況良好,選取第3代、5代、7代神經(jīng)干細(xì)胞進(jìn)行CCK-8細(xì)胞活性檢測(cè),各代神經(jīng)干細(xì)胞的增殖結(jié)果示：24h各代神經(jīng)干細(xì)胞數(shù)分別為38675±2480,45105±2405,41259±3419；48h各代細(xì)胞數(shù)分別為67597±3965,70148+2000,68001±1125；72h各代細(xì)胞數(shù)分別為139641±3456,140890±3092,140314±2884；96h第各細(xì)胞數(shù)分別為139164±4497,156602±708,156159±4253。對(duì)各代神經(jīng)干細(xì)胞進(jìn)行免疫熒光檢測(cè),結(jié)果顯示神經(jīng)干細(xì)胞特異性標(biāo)志物巢蛋白nestin呈陽(yáng)性表達(dá)；利用5%血清誘導(dǎo)分化培養(yǎng)后,免疫熒光共聚焦技術(shù)檢測(cè)到神經(jīng)干細(xì)胞分化為神經(jīng)元的標(biāo)志物βⅢ-tubulin和星形膠質(zhì)細(xì)胞的標(biāo)志物GFAP呈陽(yáng)性表達(dá)。 結(jié)論經(jīng)免疫熒光及共聚焦技術(shù)鑒定,成功從成體SD大鼠海馬齒狀回分離并培養(yǎng)出神經(jīng)干細(xì)胞。 第二部分MK-801在Shh-谷氨酸信號(hào)傳導(dǎo)通路中的阻斷作用 目的利用MK-801對(duì)大鼠NSCs谷氨酸受體的阻斷作用,觀察NSCs中的Shh(sonic hedgehog)及相關(guān)標(biāo)志物的mRNA和蛋白表達(dá)的情況,探討內(nèi)源性神經(jīng)干細(xì)胞原位激活機(jī)制。 方法取第3代神經(jīng)干細(xì)胞,消化為單細(xì)胞懸液后,隨機(jī)分成7組,使MK-801的濃度為0μM、5μM、10μM、20μM、40μM、80μM、160μM,各濃度MK-801作用下連續(xù)培養(yǎng)7天,采用倒置顯微鏡下細(xì)胞計(jì)數(shù)和cck-8試劑檢測(cè)活性細(xì)胞數(shù)分別檢測(cè)0-160μM濃度的MK-801作用下神經(jīng)干細(xì)胞增殖情況；將單細(xì)胞懸液隨機(jī)分成空白對(duì)照組,谷氨酸組,MK-801組以及谷氨酸、MK-801共同處理組,各組神經(jīng)干細(xì)胞經(jīng)過(guò)相應(yīng)處理后進(jìn)行培養(yǎng),通過(guò)western blot、RT-PCR以及實(shí)時(shí)熒光定量PCR技術(shù)分別在蛋白表達(dá)水平、基因表達(dá)水平了解神經(jīng)干細(xì)胞增殖以及分化過(guò)程中信號(hào)通路的shh、Nestin、βⅢ-tubulin、GFAP的表達(dá)情況。 結(jié)果神經(jīng)干細(xì)胞在0-160μ M濃度MK-801組中連續(xù)懸浮培養(yǎng)7天后,倒置顯微鏡下進(jìn)行細(xì)胞計(jì)數(shù),結(jié)果示10μ M以上濃度組細(xì)胞數(shù)量明顯減少,5u M組細(xì)胞數(shù)量比接種數(shù)量多,但比空白對(duì)照組細(xì)胞數(shù)量少；CCK-8連續(xù)測(cè)量各濃度細(xì)胞活性,結(jié)果示第2天后各組活性細(xì)胞數(shù)(單位為105個(gè)/ml)分別為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,隨著時(shí)間延長(zhǎng)10-160μ M濃度組神經(jīng)干細(xì)胞數(shù)量持續(xù)減少,5μ M組細(xì)胞細(xì)胞數(shù)量持續(xù)增加,生長(zhǎng)趨勢(shì)與空白對(duì)照組類似；空白對(duì)照組,谷氨酸組,共同處理組以及MK-801組,western blot結(jié)果顯示：四組Shh/GAPDH灰度值之比分別為空白對(duì)照0.115,谷氨酸組0.1239,共同處理組0.047,MK-801組0.03629；Nestin/GAPDH空白對(duì)照0.7625,谷氨酸組0.9915,共同處理組0.01871,MK-801組0.29050.115;(FAP/GAPDH灰度值之比分別為空白對(duì)照0.7625,谷氨酸組0.9915,共同處理組0.01871,MK-801組0.2905；βⅢ-tubulin/GAPDH灰度值之比分別為空白對(duì)照0.598,谷氨酸組0.8834,共同處理組0.0246,MK-801組0.0308。RT-PCR結(jié)果示在阻斷劑MK-801作用后,MK-801組及聯(lián)合處理組shh、Nestin、βⅢ-tubulin、 GFAP基因和蛋白表達(dá)明顯抑制,且兩組表達(dá)基本一致；谷氨酸組表達(dá)明顯高于正常對(duì)照組。實(shí)時(shí)熒光相對(duì)定量結(jié)果顯示：四組中ShhmRNA相對(duì)于空白對(duì)照組的表達(dá)水平分別為MK-801處理組0.00381,共同處理組0.00106,谷氨酸組shh基因表達(dá)量為空白對(duì)照組的5.195倍；基因nestin在谷氨酸組aestin表達(dá)量是空白對(duì)照組的7.61倍,在共同處理組及MK-801組分別為空白對(duì)照組的0.0067、0.0042倍；GFAPmRNA表達(dá)量,谷氨酸組是空白對(duì)照組的4.6913倍,而共同處理組和MK-801組的表達(dá)量分別為于空白對(duì)照組；MK-80I組βⅢ-tubulin的基因表達(dá)量是空白對(duì)照組的0.00165倍。共同處理組是對(duì)照組的0.0102374倍。谷氨酸組βⅢ-tubulin的基因表達(dá)量是相對(duì)于空白對(duì)照組的2.615倍。 結(jié)論MK-801在10μ M及以上濃度可有效抑制神經(jīng)干細(xì)胞增殖；在蛋白表達(dá)、基因表達(dá)層面上,MK-801能阻斷NSCs經(jīng)谷氨酸/]NMDAR-Shh信號(hào)傳導(dǎo)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.

【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743

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