酒精对神经前体细胞损伤及其机制研究

发布时间：2018-02-08 22:12

本文关键词： 神经前体细胞酒精增殖毒蕈碱乙酰胆碱受体卡巴可　出处：《山东大学》2007年硕士论文　论文类型：学位论文

【摘要】： 研究背景：胎儿酒精综合症(fetal alcohol syndrome，FAS)是由于父母双方饮酒尤其是妇女孕前、孕期饮酒造成的子代出生缺陷，大量研究己证实孕期酒精暴露会影响胎儿的生长和发育，主要表现为包括中枢神经系统在内的各种器官畸形。在神经系统发育早期，神经前体细胞(neural precursor cells)对酒精非常敏感，特别是胎儿出生前3个月，短暂的酒精接触就能引起神经元的大量死亡，而此时也是神经元突触形成的时期。对酒精大鼠模型的研究证明，胎儿期接触酒精，会使发育中的大脑皮质层神经上皮细胞增殖能力降低，减少神经细胞数量。孕13天胎鼠神经上皮主要由神经干细胞和前体细胞组成，统一地称之为神经前体细胞。同时，对神经细胞、神经元类似细胞系及星形胶质细胞的研究表明，酒精能显著抑制上述细胞的增殖、降低细胞存活率。最近的体外研究发现，酒精可以导致大鼠大脑皮层前体细胞和小脑前体细胞周期延迟和大量细胞死亡。目前为止，酒精对神经系统致畸的机制了解很少，特别是酒精对神经前体细胞的影响仅见国外几篇文献报导，国内尚未见有文献报道。研究目的：建立稳定的贴壁培养神经前体细胞方法和体外研究酒精损伤神经前体细胞实验模型；进一步研究酒精对神经前体细胞增殖的影响，研究M型乙酰胆碱受体(mAChR)介导的信号通路在神经前体细胞增殖过程中的作用及酒精对其影响；为揭示酒精对脑发育过程中神经前体细胞的毒性作用机制及其防治提供实验依据，对控制胎儿酒精综合症发生、降低智力低下儿童出生率，提高我国人口素质具有重要意义。实验方法：从孕13天胎鼠端脑分离神经前体细胞，用本实验室建立的贴壁培养神经前体细胞方法培养神经前体细胞进行实验。 1．神经前体细胞的鉴定及纯度分析： (1)原代贴壁培养神经前体细胞5天后，用Nestin抗体进行免疫荧光染色标记神经前体细胞，DAPI荧光染料复染，荧光显微镜下观察，拍照，计算神经前体细胞纯度。 (2)将BrdU掺入神经前体细胞，用抗Nestin、BrdU荧光抗体进行双标记，DAPI复染方法，分析贴壁培养的神经前体细胞增殖能力。 (3)神经前体细胞培养5天后，去除bFGF和EGF，继续培养5天，分别进行MAP_2和GFAP免疫荧光染色，检测神经前体细胞向神经元、神经胶质细胞分化的能力。 (4)采用神经前体悬浮培养方法培养神经前体细胞，培养5天后，对克隆球细胞进行Nestin、DAPI免疫荧光双染，与贴壁培养5天的神经前体细胞纯度进行分析对比。 2、研究酒精对神经前体细胞增殖、凋亡和坏死的影响： (1)神经前体细胞培养5天后，加入不同浓度酒精作用24h，0.125％胰蛋白酶消化细胞，台盼兰染色计数，分析不同浓度的酒精对神经前体细胞坏死的影响。(2)贴壁培养5天的神经前体细胞，酒精作用24h，在作用结束前4h加入BrdU。掺入到细胞中的BrdU用Cy3连接的BrdU抗体标记、DAPI复染，荧光显微镜观察，，计算细胞增殖指数。 (3)用MTT检测法分析不同浓度酒精(0、25、50、100mmol／L)对神经前体细胞存活能力的影响。 (4)使用荧光探针碘化丙啶(PI)和Ho.33342双染色方法从形态学观察分析酒精诱导的神经前体细胞凋亡和坏死情况。 (6)用MTT检测方法分析不同浓度的酒精代谢产物乙醛(0、0.5、2、8mmol／L)对神经前体细胞活性的影响。 3、mAchRs介导的信号通路在神经前体细胞增殖过程的作用及酒精对其影响： (1)用MTT检测方法分析不同浓度的mAchRs激动剂卡巴可(Carbachol，CCh)对神经前体细胞增殖的影响。 (2)用形态学观察和MTT检测的方法观察分析酒精(100mM)和阿托品(50μM)对卡巴可诱导的神经前体细胞增殖的影响。 (3)采用磷酸化Erk1／2抗体标记、流式细胞仪检测的方法定量分析酒精对卡巴可诱导的MAPK磷酸化表达的影响。实验结果： 1．贴壁培养5天的神经前体细胞纯度达97％，绝大多数细胞保持未分化状态，并有增殖能力，增殖指数达51.8％；去除生长因子后，神经前体细胞能够分化成神经元和星形胶质细胞； 2．中、低等浓度的酒精(25～50 mmol／L)能抑制神经前体细胞增殖。高浓度的酒精(100 mmol／L)能显著降低神经前体细胞数目，细胞存活率降至68.1％，细胞增殖指数仅为16.1％，并呈剂量依赖效应。 3．0.5～2 mmol／L的乙醛对神经前体细胞影响并不明显，高浓度乙醛(8mmol／L)可以显著降低细胞存活率至59.7％。 4．低、中浓度(25～50mM)的酒精短时间作用对神经前体细胞的影响并不明显，高浓度的酒精(100mM)能显著诱导神经前体细胞凋亡和死亡。 5．体外培养的神经前体细胞加入不同浓度的卡巴可(mAchRs激动剂)作用48h后，神经前体细胞增殖迅速，以100μmol／L卡巴可最为明显。 6．100mM酒精和50μM阿托品(mAchRs非特异性阻断剂)能显著抑制卡巴可的促神经前体细胞增殖作用。 7．卡巴可激活mAchRs后，使用流式细胞仪检测发现细胞内的磷酸化ERK1／2增加；而100mM的酒精和MEK抑制剂PD98059能显著抑制这种增强的荧光信号。实验结论： 1．成功建立了贴壁培养神经前体细胞的方法和研究酒精对神经前体细胞增殖影响的体外实验模型。 2．酒精能显著抑制神经前体细胞增殖。 3．酒精能够诱导神经前体细胞凋亡和坏死。 4．酒精代谢产物乙醛能降低神经前体细胞存活率。 5．卡巴可明显刺激神经前体细胞增殖，酒精和阿托品能显著抑制卡巴可的促神经前体细胞增殖作用。 6．酒精能够抑制卡巴可诱导的细胞内蛋白质激酶磷酸化。 7．mAchRs及其介导的MAPK信号通路在神经前体细胞增殖过程起重要作用。酒精对神经前体细胞增殖的抑制作用是通过阻断mAchRs介导的信号通路来实现的。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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