酒精对胎鼠脑新皮质神经干细胞自我更新和分化的影响研究

发布时间：2018-04-22 18:08

本文选题：酒精 + 神经干细胞　；参考：《复旦大学》2010年博士论文

【摘要】：孕期饮洒可导致多器官系统生长发育异常和生长发育迟缓以及功能障碍,其中脑是酒精作用的最主要靶器官之一。以往临床病例显示孕期饮酒所导致的脑结构和功能发育异常主要表现为神经元的形态和功能发育异常,而神经胶质细胞对神经元具有营养和支持作用,故研究多侧重于酒精对神经元和神经胶质细胞的影响。神经干细胞的发现和分离培养为毒理学研究提供了有利工具。神经干细胞具有自我更新和多向分化能力,通过自我更新补充干细胞库,通过多向分化能力产生神经元和神经胶质细胞。外源性化学物干扰神经干细胞自我更新和分化将影响神经系统的正常发育和功能。本课题从胎鼠脑皮质分离纯化神经干细胞,研究酒精对神经干细胞自我更新和分化的影响和可能机制,以期为探讨其作用机制提供科学依据。 1大鼠胚胎脑新皮质神经干细胞的体外分离培养方法研究本研究从孕14天SD大鼠胚胎脑分离新皮质,通过机械吹打改善细胞分散状态、优化无血清培养基条件,从而改进神经干细胞的培养方法。利用神经干细胞标志蛋白(nestin和SOX2)、增殖和克隆成球能力以及多向分化能力测试三方面对神经干细胞鉴定。以该法培养的神经干细胞nestin蛋白呈强阳性表达,SOX2阳性表达率达99%以上,BrdU掺入阳性,培养3天后细胞量为接种量的10.55倍,6日克隆成球率为(33.004±4.40)%。经相应特异性标志蛋白检测证实神经干细胞经诱导分化后可形成神经元(MAP2)、星形胶质细胞(GFAP)和少突胶质细胞(04)。本法获得的神经干细胞纯度和细胞产出率高,具有强自我更新和多向分化能力,可为毒理学研究提供良好的模型。 2酒精对胎鼠脑新皮质神经干细胞超微结构的影响利用透射电镜和扫描电镜技术观察神经干细胞的超微结构。神经球结构较松散,细胞主要为核质比大的原始细胞。神经球呈现异质性,由电子密度高的暗细胞和电子密度低的明细胞组成。可观察到不同分裂时相的细胞。存在细胞凋亡,一些细胞吞噬功能旺盛。细胞表面有很多突起和微绒毛,可能是神经球细胞维持球体、物质和信息交换的重要结构基础。酒精造成神经干细胞线粒体损伤,损伤程度呈剂量效应关系。25mM酒精引起线粒体轻度肿胀,50mM酒精引起细胞线粒体中度肿胀,嵴断裂缺失。100mM剂量组可见细胞线粒体损伤程度进一步加剧。扫描电镜下酒精使神经球结构更为松散。酒精导致的线粒体损伤可能引起细胞凋亡。 3酒精对胎鼠脑新皮质神经干细胞自我更新的影响及作用机制研究 3.1酒精对胎鼠脑新皮质神经干细胞自我更新的影响利用台盼蓝染色计数存活细胞、神经干细胞成球率实验(成球个数和球径)、BrdU掺入率检测神经干细胞的自我更新能力。结果表明,随酒精剂量增加活细胞数下降,100mM酒精组活细胞数是对照组的78%。神经干细胞成球率随酒精剂量增加而下降,神经球直径也随酒精剂量增加而降低,呈剂量依赖关系。BrdU掺入率反映DNA合成状况,随酒精剂量增加而下降,呈剂量效应关系。该结果表明酒精能够抑制神经干细胞的自我更新。 3.2酒精对胎鼠脑新皮质神经干细胞细胞周期和相关调控基因的影响采用流式细胞术分析了神经干细胞的细胞周期。酒精使S期和G2/M期细胞数下降,G0/G1期细胞数增加。未观察到凋亡峰。上述结果表明酒精能够引起神经干细胞G0/G1期阻滞,延缓细胞周期进程。利用荧光定量PCR技术检测细胞周期抑制基因p53、p16、p21和p27mRNA的表达。酒精能够诱导神经干细胞p16和p21mRNA表达的增加,p53和p27mRNA表达未发生显著性改变(P0.05)。表明酒精可通过细胞周期抑制基因p16和p21抑制G1/S细胞周期检验点的转换。细胞周期阻滞可能预示着神经干细胞命运的转变,向更为成熟的前体细胞分化的启动。 3.3酒精对胎鼠脑新皮质神经干细胞标志物的影响利用荧光定量PCR技术检测神经干细胞标志物Nestin、Sox2和CD133mRNA的表达,利用流式细胞术检测SOX2蛋白的表达。神经干细胞Nestin mRNA表达随酒精剂量增加而下降,Sox2和CD133mRNA表达未受影响,SOX2阳性细胞数未受影响,但丰度增加。酒精抑制Nestin和促进SOX2表达可能与分化的启动有关。 3.4酒精对胎鼠脑新皮质神经干细胞Egfr和Fgfr1 mRNA表达的影响利用荧光定量PCR技术检测神经干细胞生长因子受体Egfr和Fgfr1 mRNA的表达。酒精能促进两者的表达,可能与酒精诱导神经干细胞向更为成熟的神经前体细胞分化有关。 3.5酒精对胎鼠脑新皮质神经干细胞Wnt通路相关基因表达的影响利用荧光定量PCR技术检测神经干细胞Wnt通路相关基因β-catenin、c-myc和CyclinD1mRNA的表达。酒精能诱导c-myc mRNA表达上调,β-catenin和CyclinD1mRNA表达未发生改变。C-myc除了促细胞增殖作用外,还有诱导凋亡的作用。结果表明酒精可能通过上调c-myc引发凋亡。 4酒精对胎鼠脑新皮质神经干细胞凋亡的影响及作用机制研究 4.1酒精对胎鼠脑新皮质神经干细胞凋亡的影响利用Annexin V/PI法检测酒精染毒3d和4d的细胞凋亡率。50mM和100mM酒精染毒3d和4d均能诱导神经干细胞早期凋亡的增加。100mM酒精染毒3d和4d均能导致神经干细胞膜电位的显著性下降。晚期凋亡率无显著性变化。 4.2酒精对神经干细胞凋亡相关基因表达的影响利用荧光定量PCR技术检测神经干细胞凋亡相关基因Bcl-2、Survivin、Bax、Bad和Caspase3 mRNA的表达。酒精染毒2d,50mM和100mM酒精抑制凋亡抑制基因Survivin的表达,酒精染毒3d,25-100mM酒精使促凋亡基因Bax表达增加。酒精染毒4d,各基因表达没有变化。利用分光光度法试剂盒检测Caspase3蛋白活性,酒精染毒2-4d均未引起Caspase 3蛋白活性的显著增加。结合前文中发现酒精引起神经干细胞c-myc表达增加,可认为25-100mM酒精染毒2-4d,神经干细胞出现早期凋亡事件,凋亡抑制基因survivin下调,促凋亡基因c-myc上调,bax随之上调,bcl-2/bax比值下降。线粒体途径参与了酒精诱导的神经干细胞早期凋亡。 5酒精对胎鼠脑新皮质神经干细胞分化的影响利用荧光定量PCR技术检测神经干细胞分化后神经元、星形胶质细胞、少突胶质细胞标志物mRNA的表达。酒精染毒4d促进神经元前体细胞标志物Tuj1、星形胶质细胞Gfap、少突胶质细胞Mbp的表达,酒精染毒8d未引起分化细胞标志物的改变。结果表明,酒精主要在神经干细胞培养早期影响了神经干细胞向神经元、星形胶质细胞和少突胶质细胞分化时相应标志蛋白基因表达水平的改变。
[Abstract]:In previous clinical cases , the abnormal brain structure and function development caused by drinking during pregnancy showed abnormal morphology and function of neurons , while glial cells had the nutrition and support effect on neurons , so the study focused on the effects of alcohol on neurons and glial cells .

The discovery and isolation of neural stem cells provides an advantageous tool for toxicology research . The neural stem cells have self - renewal and multi - directional differentiation ability . The self - renewal and differentiation of neural stem cells can influence the normal development and function of the nervous system . The problem of self - renewal and differentiation of exogenous chemical interfering neural stem cells will influence the normal development and function of the nervous system . This task studies the influence and possible mechanism of alcohol on the self - renewal and differentiation of neural stem cells .

In vitro isolation and culture of neural stem cells from rat embryonic brain

In this study , neural stem cells were isolated from the embryonic brain of SD rats from 14 days of gestation . The neural stem cells were identified by means of mechanical blowing to improve cell dispersion state , optimize serum - free medium conditions .

the effect of alcohol on the ultrastructure of neural stem cells in the neocortex of fetal rat

The ultrastructural changes of neural stem cells were observed by transmission electron microscopy and scanning electron microscopy ( SEM ) .

Alcohol causes mitochondrial damage to neural stem cells , and the degree of injury is dose - dependent . 25 mM alcohol causes mild swelling of mitochondria , 50 mM alcohol causes moderate swelling of mitochondria , and the absence of crest fracture . The damage degree of mitochondria in 100 mM dose group is further increased . Alcohol - induced mitochondrial damage may cause apoptosis .

Effect of alcohol on self - renewal of neural stem cells in fetal rat brain and its mechanism of action

3.1 Effect of alcohol on self - renewal of neural stem cells in fetal rat brain

The results showed that the number of viable cells decreased with the increase of alcohol dosage , and the diameter of neural stem decreased with the increase of alcohol dosage . The results showed that alcohol could inhibit the self - renewal of neural stem cells . The results showed that alcohol could inhibit the self - renewal of neural stem cells .

3.2 Effect of alcohol on cell cycle and related regulatory genes of neocortex neural stem cells in fetal rat brain

Cell cycle of neural stem cells was analyzed by flow cytometry . The number of cells in S phase and G2 / M phase decreased and the number of G0 / G1 phase cells increased . No apoptotic peak was observed . The results indicated that alcohol could induce G0 / G1 arrest of neural stem cells and delay cell cycle progression .

The expression of p53 , p16 , p21 and p27mRNA was detected by fluorescence quantitative PCR . There was no significant change in the expression of p16 and p21 mRNA in neural stem cells induced by alcohol ( P0.05 ) .

3.3 Effect of alcohol on neural stem cell markers in fetal brain neocortex neural stem cells

The expression of Nestin , Sox2 and CD133 mRNA in neural stem cells was detected by fluorescence quantitative PCR . The expression of SOX2 protein was detected by flow cytometry . The expression of Nestin mRNA in neural stem cells decreased with the increase of alcohol dosage , and the expression of Sox2 and CD133 mRNA was not affected . The number of SOX2 positive cells was not affected , but the abundance of SOX2 positive cells was increased . Alcohol inhibited Nestin and promoted SOX2 expression might be related to the initiation of differentiation .

The effect of alcohol on the expression of the mRNA of the neural stem cells of the new cortical neural stem cells in the fetal rat brain

The expression of IGF - 1 mRNA in neural stem cell growth factor receptor ( Fgfr1 ) was detected by fluorescence quantitative PCR . Alcohol can promote the expression of both , which may be related to the differentiation of neural stem cells into more mature neural progenitor cells .

Effects of alcohol on Wnt pathway - related gene expression in neocortex neural stem cells of fetal mice

The expression of 尾 - catenin , c - myc and Cyclin D1 mRNA in the Wnt pathway of neural stem cells was detected by fluorescence quantitative PCR . The expression of c - myc mRNA was up - regulated by alcohol .

Effect of alcohol on apoptosis of neural stem cells in brain neocortex of fetal rat and its mechanism

Effects of alcohol on apoptosis of neocortex neural stem cells in fetal rat brain

The apoptosis rate of neural stem cells could be induced by 50 mM and 100 mM alcohol in both days and 4 days , and the apoptosis rate of neural stem cells decreased significantly after 3 days and 4 days in 100 mM alcohol .

4.2 Effect of alcohol on expression of apoptosis - related genes in neural stem cells

The expression of apoptosis - related genes Bcl - 2 , Survivin , Bax , Bad and Caspase3 mRNA in neural stem cells was detected by fluorescence quantitative PCR . The expression of Survivin was inhibited by alcohol exposure to 2 d , 50 mM and 100 mM alcohol . The expression of apoptotic gene Bax was increased by alcohol exposure to 3 days , 25 - 100 mM alcohol .

It was found that the expression of c - myc in neural stem cells induced by alcohol increased in 25 - 100mM alcohol . Apoptosis was downregulated in neural stem cells , the expression of apoptosis - inhibiting gene survivin was downregulated , the expression of bax was up - regulated , and the ratio of bcl - 2 / bax decreased . Mitochondrial pathway was involved in early apoptosis of neural stem cells induced by alcohol .

Effect of alcohol on the differentiation of neural stem cells in the neocortex of fetal rat

The expression of mRNA of neural stem cells after differentiation of neural stem cells was detected by fluorescence quantitative polymerase chain reaction ( PCR ) .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【参考文献】