本文關(guān)鍵詞： 神經(jīng)前體細(xì)胞 酒精 增殖 毒蕈堿乙酰膽堿受體 卡巴可　出處：《山東大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 研究背景： 胎兒酒精綜合癥(fetal alcohol syndrome，F(xiàn)AS)是由于父母雙方飲酒尤其是婦女孕前、孕期飲酒造成的子代出生缺陷，大量研究己證實(shí)孕期酒精暴露會(huì)影響胎兒的生長(zhǎng)和發(fā)育，主要表現(xiàn)為包括中樞神經(jīng)系統(tǒng)在內(nèi)的各種器官畸形。在神經(jīng)系統(tǒng)發(fā)育早期，神經(jīng)前體細(xì)胞(neural precursor cells)對(duì)酒精非常敏感，特別是胎兒出生前3個(gè)月，短暫的酒精接觸就能引起神經(jīng)元的大量死亡，而此時(shí)也是神經(jīng)元突觸形成的時(shí)期。對(duì)酒精大鼠模型的研究證明，胎兒期接觸酒精，會(huì)使發(fā)育中的大腦皮質(zhì)層神經(jīng)上皮細(xì)胞增殖能力降低，減少神經(jīng)細(xì)胞數(shù)量。孕13天胎鼠神經(jīng)上皮主要由神經(jīng)干細(xì)胞和前體細(xì)胞組成，統(tǒng)一地稱之為神經(jīng)前體細(xì)胞。同時(shí)，對(duì)神經(jīng)細(xì)胞、神經(jīng)元類似細(xì)胞系及星形膠質(zhì)細(xì)胞的研究表明，酒精能顯著抑制上述細(xì)胞的增殖、降低細(xì)胞存活率。最近的體外研究發(fā)現(xiàn)，酒精可以導(dǎo)致大鼠大腦皮層前體細(xì)胞和小腦前體細(xì)胞周期延遲和大量細(xì)胞死亡。目前為止，酒精對(duì)神經(jīng)系統(tǒng)致畸的機(jī)制了解很少，特別是酒精對(duì)神經(jīng)前體細(xì)胞的影響僅見(jiàn)國(guó)外幾篇文獻(xiàn)報(bào)導(dǎo)，國(guó)內(nèi)尚未見(jiàn)有文獻(xiàn)報(bào)道。 研究目的： 建立穩(wěn)定的貼壁培養(yǎng)神經(jīng)前體細(xì)胞方法和體外研究酒精損傷神經(jīng)前體細(xì)胞實(shí)驗(yàn)?zāi)Ｐ�；進(jìn)一步研究酒精對(duì)神經(jīng)前體細(xì)胞增殖的影響，研究M型乙酰膽堿受體(mAChR)介導(dǎo)的信號(hào)通路在神經(jīng)前體細(xì)胞增殖過(guò)程中的作用及酒精對(duì)其影響；為揭示酒精對(duì)腦發(fā)育過(guò)程中神經(jīng)前體細(xì)胞的毒性作用機(jī)制及其防治提供實(shí)驗(yàn)依據(jù)，對(duì)控制胎兒酒精綜合癥發(fā)生、降低智力低下兒童出生率，提高我國(guó)人口素質(zhì)具有重要意義。 實(shí)驗(yàn)方法： 從孕13天胎鼠端腦分離神經(jīng)前體細(xì)胞，用本實(shí)驗(yàn)室建立的貼壁培養(yǎng)神經(jīng)前體細(xì)胞方法培養(yǎng)神經(jīng)前體細(xì)胞進(jìn)行實(shí)驗(yàn)。 1．神經(jīng)前體細(xì)胞的鑒定及純度分析： (1)原代貼壁培養(yǎng)神經(jīng)前體細(xì)胞5天后，用Nestin抗體進(jìn)行免疫熒光染色標(biāo)記神經(jīng)前體細(xì)胞，DAPI熒光染料復(fù)染，熒光顯微鏡下觀察，拍照，計(jì)算神經(jīng)前體細(xì)胞純度。 (2)將BrdU摻入神經(jīng)前體細(xì)胞，用抗Nestin、BrdU熒光抗體進(jìn)行雙標(biāo)記，DAPI復(fù)染方法，分析貼壁培養(yǎng)的神經(jīng)前體細(xì)胞增殖能力。 (3)神經(jīng)前體細(xì)胞培養(yǎng)5天后，去除bFGF和EGF，繼續(xù)培養(yǎng)5天，分別進(jìn)行MAP_2和GFAP免疫熒光染色，檢測(cè)神經(jīng)前體細(xì)胞向神經(jīng)元、神經(jīng)膠質(zhì)細(xì)胞分化的能力。 (4)采用神經(jīng)前體懸浮培養(yǎng)方法培養(yǎng)神經(jīng)前體細(xì)胞，培養(yǎng)5天后，對(duì)克隆球細(xì)胞進(jìn)行Nestin、DAPI免疫熒光雙染，與貼壁培養(yǎng)5天的神經(jīng)前體細(xì)胞純度進(jìn)行分析對(duì)比。 2、研究酒精對(duì)神經(jīng)前體細(xì)胞增殖、凋亡和壞死的影響： (1)神經(jīng)前體細(xì)胞培養(yǎng)5天后，加入不同濃度酒精作用24h，0.125％胰蛋白酶消化細(xì)胞，臺(tái)盼蘭染色計(jì)數(shù)，分析不同濃度的酒精對(duì)神經(jīng)前體細(xì)胞壞死的影響。(2)貼壁培養(yǎng)5天的神經(jīng)前體細(xì)胞，酒精作用24h，在作用結(jié)束前4h加入BrdU。摻入到細(xì)胞中的BrdU用Cy3連接的BrdU抗體標(biāo)記、DAPI復(fù)染，熒光顯微鏡觀察，，計(jì)算細(xì)胞增殖指數(shù)。 (3)用MTT檢測(cè)法分析不同濃度酒精(0、25、50、100mmol／L)對(duì)神經(jīng)前體細(xì)胞存活能力的影響。 (4)使用熒光探針碘化丙啶(PI)和Ho.33342雙染色方法從形態(tài)學(xué)觀察分析酒精誘導(dǎo)的神經(jīng)前體細(xì)胞凋亡和壞死情況。 (6)用MTT檢測(cè)方法分析不同濃度的酒精代謝產(chǎn)物乙醛(0、0.5、2、8mmol／L)對(duì)神經(jīng)前體細(xì)胞活性的影響。 3、mAchRs介導(dǎo)的信號(hào)通路在神經(jīng)前體細(xì)胞增殖過(guò)程的作用及酒精對(duì)其影響： (1)用MTT檢測(cè)方法分析不同濃度的mAchRs激動(dòng)劑卡巴可(Carbachol，CCh)對(duì)神經(jīng)前體細(xì)胞增殖的影響。 (2)用形態(tài)學(xué)觀察和MTT檢測(cè)的方法觀察分析酒精(100mM)和阿托品(50μM)對(duì)卡巴可誘導(dǎo)的神經(jīng)前體細(xì)胞增殖的影響。 (3)采用磷酸化Erk1／2抗體標(biāo)記、流式細(xì)胞儀檢測(cè)的方法定量分析酒精對(duì)卡巴可誘導(dǎo)的MAPK磷酸化表達(dá)的影響。 實(shí)驗(yàn)結(jié)果： 1．貼壁培養(yǎng)5天的神經(jīng)前體細(xì)胞純度達(dá)97％，絕大多數(shù)細(xì)胞保持未分化狀態(tài)，并有增殖能力，增殖指數(shù)達(dá)51.8％；去除生長(zhǎng)因子后，神經(jīng)前體細(xì)胞能夠分化成神經(jīng)元和星形膠質(zhì)細(xì)胞； 2．中、低等濃度的酒精(25～50 mmol／L)能抑制神經(jīng)前體細(xì)胞增殖。高濃度的酒精(100 mmol／L)能顯著降低神經(jīng)前體細(xì)胞數(shù)目，細(xì)胞存活率降至68.1％，細(xì)胞增殖指數(shù)僅為16.1％，并呈劑量依賴效應(yīng)。 3．0.5～2 mmol／L的乙醛對(duì)神經(jīng)前體細(xì)胞影響并不明顯，高濃度乙醛(8mmol／L)可以顯著降低細(xì)胞存活率至59.7％。 4．低、中濃度(25～50mM)的酒精短時(shí)間作用對(duì)神經(jīng)前體細(xì)胞的影響并不明顯，高濃度的酒精(100mM)能顯著誘導(dǎo)神經(jīng)前體細(xì)胞凋亡和死亡。 5．體外培養(yǎng)的神經(jīng)前體細(xì)胞加入不同濃度的卡巴可(mAchRs激動(dòng)劑)作用48h后，神經(jīng)前體細(xì)胞增殖迅速，以100μmol／L卡巴可最為明顯。 6．100mM酒精和50μM阿托品(mAchRs非特異性阻斷劑)能顯著抑制卡巴可的促神經(jīng)前體細(xì)胞增殖作用。 7．卡巴可激活mAchRs后，使用流式細(xì)胞儀檢測(cè)發(fā)現(xiàn)細(xì)胞內(nèi)的磷酸化ERK1／2增加；而100mM的酒精和MEK抑制劑PD98059能顯著抑制這種增強(qiáng)的熒光信號(hào)。 實(shí)驗(yàn)結(jié)論： 1．成功建立了貼壁培養(yǎng)神經(jīng)前體細(xì)胞的方法和研究酒精對(duì)神經(jīng)前體細(xì)胞增殖影響的體外實(shí)驗(yàn)?zāi)Ｐ汀?2．酒精能顯著抑制神經(jīng)前體細(xì)胞增殖。 3．酒精能夠誘導(dǎo)神經(jīng)前體細(xì)胞凋亡和壞死。 4．酒精代謝產(chǎn)物乙醛能降低神經(jīng)前體細(xì)胞存活率。 5．卡巴可明顯刺激神經(jīng)前體細(xì)胞增殖，酒精和阿托品能顯著抑制卡巴可的促神經(jīng)前體細(xì)胞增殖作用。 6．酒精能夠抑制卡巴可誘導(dǎo)的細(xì)胞內(nèi)蛋白質(zhì)激酶磷酸化。 7．mAchRs及其介導(dǎo)的MAPK信號(hào)通路在神經(jīng)前體細(xì)胞增殖過(guò)程起重要作用。酒精對(duì)神經(jīng)前體細(xì)胞增殖的抑制作用是通過(guò)阻斷mAchRs介導(dǎo)的信號(hào)通路來(lái)實(shí)現(xiàn)的。
[Abstract]:Research background:
Fetal alcohol syndrome (fetal alcohol, syndrome, FAS) is due to both parents, especially in women before drinking, drinking during pregnancy caused offspring birth defects, a large number of studies have confirmed that prenatal alcohol exposure may affect fetal growth and development, mainly for various organs including the central nervous system malformations. In the early development of the nervous system. Neural precursor cells (neural precursor cells) is very sensitive to alcohol, especially fetal 3 months before birth, alcohol can cause transient contact the death of a large number of neurons, while also synapse formation period. Research on the model of alcohol in rats demonstrated that prenatal alcohol exposure, will reduce the developing brain the cortex neural epithelial cell proliferation, reduce the number of nerve cells. At day 13 of fetal rat neural epithelium consists of neural stem cells and progenitor cells, called the unified For the neural precursor cells. At the same time, the research on nerve cells, neuron like cells and astrocytes showed that alcohol can significantly inhibit the cell proliferation, reduce cell viability in vitro. Recent study found that alcohol can cause cell cycle delay in rat cortical precursor cells and cerebellum and a large number of cell death. So far, understanding the mechanism of alcohol on the nervous system malformation rarely, especially the effects of alcohol on neural precursor cells was observed in several foreign literatures reported that China has not been reported in the literature.
The purpose of the study is:
To establish a stable adherent nerve before the method body cells and in vitro study of neural precursor cells in alcohol injury experimental model of culture; further study the effect of alcohol on the proliferation of neural precursor cells, M acetylcholine receptor (mAChR) and the effects of alcohol mediated signaling pathway in neural progenitor cell proliferation in the process of action on the; provide experimental basis for revealing alcohol during brain development of neural precursor cell toxicity mechanism and its prevention, control of fetal alcohol syndrome, reduce the birth rate of children with mental retardation, improve the quality of the population in China has important significance.
Experimental methods:
Neural precursor cells were isolated from the end of the fetal rat brain at 13 days of pregnancy, and the neural precursor cells were cultured with the method of adherent culture of neural precursor cells established in our laboratory.
1. identification and purity analysis of neural precursor cells:
(1) after 5 days of primary culture, the neural precursor cells were labeled with Nestin antibody. Immunofluorescence staining was used to label neural precursor cells. DAPI fluorescent dye was used to re stain, and the purity of neural precursor cells was calculated by fluorescence microscope.
(2) BrdU was added into the neural precursor cells, and the anti Nestin, BrdU fluorescent antibody was used for double labeling, and the DAPI replication method was used to analyze the proliferation ability of the cultured neural precursor cells.
(3) after 5 days of culture, bFGF and EGF were removed and cultured for 5 days. MAP_2 and GFAP immunofluorescence staining were used to detect the ability of neural precursor cells to differentiate into neurons and glial cells.
(4) using neural precursor suspension culture method to culture neural precursor cells, after 5 days of culture, the Nestin and DAPI immunofluorescence double staining of cloned cells were carried out, and the purity of neural precursor cells for 5 days after adherence culture was analyzed and compared.
2, the effects of alcohol on the proliferation, apoptosis and necrosis of neural precursor cells were studied.
(1) neural precursor cells cultured for 5 days, with different concentrations of alcohol 24h, 0.125% trypsin digestion cells, trypan blue staining to count, analysis of the influence of different concentrations of alcohol on neural precursor cell necrosis. (2) cultured for 5 days of neural precursor cells, alcohol for 24h, in the role of 4h before the end of joining the BrdU. incorporation into BrdU antibody labeled cells in BrdU connected by Cy3, DAPI staining, fluorescence microscope, cell proliferation index was calculated.
(3) the effects of different concentrations of alcohol (0,25,50100mmol / L) on the viability of neural precursor cells were analyzed by MTT assay.
(4) the morphological observation and analysis of apoptosis and necrosis of alcohol induced neural precursor cells were observed by fluorescence probe PI and Ho.33342 double staining.
(6) the effects of acetaldehyde (0,0.5,2,8mmol / L) of alcohol metabolites of different concentrations on the activity of neural precursor cells were analyzed by MTT assay.
3, the role of mAchRs mediated signaling pathway in the proliferation of neural precursor cells and the effect of alcohol on it:
(1) detected by MTT method analysis of different concentrations of mAchRs agonist carbachol (Carbachol, CCh) on the proliferation of neural precursor cells.
(2) the effects of alcohol (100mM) and atropine (50 u M) on the proliferation of Kabako induced neural progenitor cells were observed by morphological observation and MTT detection.
(3) the phosphorylation of Erk1 / 2 antibody labeling, the effects of alcohol on the expression of MAPK phosphorylation induced by the quantitative analysis method of flow cytometry.
Experimental results:
1., the purity of neural precursor cells on the 5 day after culture was 97%. Most of the cells remained undifferentiated and proliferated. The proliferation index reached 51.8%. After removal of growth factors, neural precursor cells could differentiate into neurons and astrocytes.
2., low concentration of alcohol (25~50 mmol / L) could inhibit the proliferation of neural precursor cells. High concentration of alcohol (100 mmol / L) could significantly reduce the number of neural precursor cells, reduce cell survival rate to 68.1%, and cell proliferation index was only 16.1%, and showed a dose-dependent effect.
The effect of acetaldehyde from 3.0.5 to 2 mmol / L on neural precursor cells is not obvious. The high concentration of acetaldehyde (8mmol / L) can significantly reduce the cell survival rate to 59.7%.
4., the effect of alcohol on the neural precursor cells was not obvious in a short time and a moderate concentration (25 ~ 50mM) of alcohol. High concentration of alcohol (100mM) could significantly induce apoptosis and death of neural precursor cells.
5. in vitro, neural precursor cells cultured in different concentrations of Kabako (mAchRs agonist) acted on 48h, and the proliferation of neural precursor cells was rapid, with 100 48h mol L.
6.100mM alcohol and 50 M atropine (mAchRs nonspecific antagonist) inhibited carbachol promoted proliferation of neural precursor cells.
7. by activation of mAchRs, using flow cytometry demonstrated that the phosphorylation of ERK1 / 2 cells increased; the fluorescence signal and 100mM alcohol and MEK inhibitor PD98059 can significantly inhibit the enhancement.
Experimental conclusions:
1. the method of adhering to the cultured neural precursor cells and the study of the effect of alcohol on the proliferation of neural precursor cells were successfully established.
2. alcohol can significantly inhibit the proliferation of neural progenitor cells.
3. alcohol can induce apoptosis and necrosis of neural progenitor cells.
4. alcohol metabolite acetaldehyde can reduce the survival rate of neural precursor cells.
5. carbachol significantly stimulated the proliferation of neural precursor cells, alcohol and atropine can significantly inhibit carbachol promoted proliferation of neural precursor cells.
6. alcohol can inhibit carbachol induced intracellular protein kinase phosphorylation.
7.mAchRs and its mediated MAPK signaling pathway play an important role in the proliferation of neural precursor cells. The inhibitory effect of alcohol on the proliferation of neural precursor cells is achieved by blocking the mAchRs mediated signaling pathway.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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