發(fā)布時間：2018-04-22 18:08

  本文選題：酒精 + 神經(jīng)干細胞�。� 參考：《復(fù)旦大學(xué)》2010年博士論文

【摘要】：孕期飲灑可導(dǎo)致多器官系統(tǒng)生長發(fā)育異常和生長發(fā)育遲緩以及功能障礙,其中腦是酒精作用的最主要靶器官之一。以往臨床病例顯示孕期飲酒所導(dǎo)致的腦結(jié)構(gòu)和功能發(fā)育異常主要表現(xiàn)為神經(jīng)元的形態(tài)和功能發(fā)育異常,而神經(jīng)膠質(zhì)細胞對神經(jīng)元具有營養(yǎng)和支持作用,故研究多側(cè)重于酒精對神經(jīng)元和神經(jīng)膠質(zhì)細胞的影響。 神經(jīng)干細胞的發(fā)現(xiàn)和分離培養(yǎng)為毒理學(xué)研究提供了有利工具。神經(jīng)干細胞具有自我更新和多向分化能力,通過自我更新補充干細胞庫,通過多向分化能力產(chǎn)生神經(jīng)元和神經(jīng)膠質(zhì)細胞。外源性化學(xué)物干擾神經(jīng)干細胞自我更新和分化將影響神經(jīng)系統(tǒng)的正常發(fā)育和功能。本課題從胎鼠腦皮質(zhì)分離純化神經(jīng)干細胞,研究酒精對神經(jīng)干細胞自我更新和分化的影響和可能機制,以期為探討其作用機制提供科學(xué)依據(jù)。 1大鼠胚胎腦新皮質(zhì)神經(jīng)干細胞的體外分離培養(yǎng)方法研究 本研究從孕14天SD大鼠胚胎腦分離新皮質(zhì),通過機械吹打改善細胞分散狀態(tài)、優(yōu)化無血清培養(yǎng)基條件,從而改進神經(jīng)干細胞的培養(yǎng)方法。利用神經(jīng)干細胞標(biāo)志蛋白(nestin和SOX2)、增殖和克隆成球能力以及多向分化能力測試三方面對神經(jīng)干細胞鑒定。以該法培養(yǎng)的神經(jīng)干細胞nestin蛋白呈強陽性表達,SOX2陽性表達率達99%以上,BrdU摻入陽性,培養(yǎng)3天后細胞量為接種量的10.55倍,6日克隆成球率為(33.004±4.40)%。經(jīng)相應(yīng)特異性標(biāo)志蛋白檢測證實神經(jīng)干細胞經(jīng)誘導(dǎo)分化后可形成神經(jīng)元(MAP2)、星形膠質(zhì)細胞(GFAP)和少突膠質(zhì)細胞(04)。本法獲得的神經(jīng)干細胞純度和細胞產(chǎn)出率高,具有強自我更新和多向分化能力,可為毒理學(xué)研究提供良好的模型。 2酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞超微結(jié)構(gòu)的影響 利用透射電鏡和掃描電鏡技術(shù)觀察神經(jīng)干細胞的超微結(jié)構(gòu)。神經(jīng)球結(jié)構(gòu)較松散,細胞主要為核質(zhì)比大的原始細胞。神經(jīng)球呈現(xiàn)異質(zhì)性,由電子密度高的暗細胞和電子密度低的明細胞組成�？捎^察到不同分裂時相的細胞。存在細胞凋亡,一些細胞吞噬功能旺盛。細胞表面有很多突起和微絨毛,可能是神經(jīng)球細胞維持球體、物質(zhì)和信息交換的重要結(jié)構(gòu)基礎(chǔ)。 酒精造成神經(jīng)干細胞線粒體損傷,損傷程度呈劑量效應(yīng)關(guān)系。25mM酒精引起線粒體輕度腫脹,50mM酒精引起細胞線粒體中度腫脹,嵴斷裂缺失。100mM劑量組可見細胞線粒體損傷程度進一步加劇。掃描電鏡下酒精使神經(jīng)球結(jié)構(gòu)更為松散。酒精導(dǎo)致的線粒體損傷可能引起細胞凋亡。 3酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞自我更新的影響及作用機制研究 3.1酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞自我更新的影響 利用臺盼藍染色計數(shù)存活細胞、神經(jīng)干細胞成球率實驗(成球個數(shù)和球徑)、BrdU摻入率檢測神經(jīng)干細胞的自我更新能力。結(jié)果表明,隨酒精劑量增加活細胞數(shù)下降,100mM酒精組活細胞數(shù)是對照組的78%。神經(jīng)干細胞成球率隨酒精劑量增加而下降,神經(jīng)球直徑也隨酒精劑量增加而降低,呈劑量依賴關(guān)系。BrdU摻入率反映DNA合成狀況,隨酒精劑量增加而下降,呈劑量效應(yīng)關(guān)系。該結(jié)果表明酒精能夠抑制神經(jīng)干細胞的自我更新。 3.2酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞細胞周期和相關(guān)調(diào)控基因的影響 采用流式細胞術(shù)分析了神經(jīng)干細胞的細胞周期。酒精使S期和G2/M期細胞數(shù)下降,G0/G1期細胞數(shù)增加。未觀察到凋亡峰。上述結(jié)果表明酒精能夠引起神經(jīng)干細胞G0/G1期阻滯,延緩細胞周期進程。 利用熒光定量PCR技術(shù)檢測細胞周期抑制基因p53、p16、p21和p27mRNA的表達。酒精能夠誘導(dǎo)神經(jīng)干細胞p16和p21mRNA表達的增加,p53和p27mRNA表達未發(fā)生顯著性改變(P0.05)。表明酒精可通過細胞周期抑制基因p16和p21抑制G1/S細胞周期檢驗點的轉(zhuǎn)換。細胞周期阻滯可能預(yù)示著神經(jīng)干細胞命運的轉(zhuǎn)變,向更為成熟的前體細胞分化的啟動。 3.3酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞標(biāo)志物的影響 利用熒光定量PCR技術(shù)檢測神經(jīng)干細胞標(biāo)志物Nestin、Sox2和CD133mRNA的表達,利用流式細胞術(shù)檢測SOX2蛋白的表達。神經(jīng)干細胞Nestin mRNA表達隨酒精劑量增加而下降,Sox2和CD133mRNA表達未受影響,SOX2陽性細胞數(shù)未受影響,但豐度增加。酒精抑制Nestin和促進SOX2表達可能與分化的啟動有關(guān)。 3.4酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞Egfr和Fgfr1 mRNA表達的影響 利用熒光定量PCR技術(shù)檢測神經(jīng)干細胞生長因子受體Egfr和Fgfr1 mRNA的表達。酒精能促進兩者的表達,可能與酒精誘導(dǎo)神經(jīng)干細胞向更為成熟的神經(jīng)前體細胞分化有關(guān)。 3.5酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞Wnt通路相關(guān)基因表達的影響 利用熒光定量PCR技術(shù)檢測神經(jīng)干細胞Wnt通路相關(guān)基因β-catenin、c-myc和CyclinD1mRNA的表達。酒精能誘導(dǎo)c-myc mRNA表達上調(diào),β-catenin和CyclinD1mRNA表達未發(fā)生改變。C-myc除了促細胞增殖作用外,還有誘導(dǎo)凋亡的作用。結(jié)果表明酒精可能通過上調(diào)c-myc引發(fā)凋亡。 4酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞凋亡的影響及作用機制研究 4.1酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞凋亡的影響 利用Annexin V/PI法檢測酒精染毒3d和4d的細胞凋亡率。50mM和100mM酒精染毒3d和4d均能誘導(dǎo)神經(jīng)干細胞早期凋亡的增加。100mM酒精染毒3d和4d均能導(dǎo)致神經(jīng)干細胞膜電位的顯著性下降。晚期凋亡率無顯著性變化。 4.2酒精對神經(jīng)干細胞凋亡相關(guān)基因表達的影響 利用熒光定量PCR技術(shù)檢測神經(jīng)干細胞凋亡相關(guān)基因Bcl-2、Survivin、Bax、Bad和Caspase3 mRNA的表達。酒精染毒2d,50mM和100mM酒精抑制凋亡抑制基因Survivin的表達,酒精染毒3d,25-100mM酒精使促凋亡基因Bax表達增加。酒精染毒4d,各基因表達沒有變化。利用分光光度法試劑盒檢測Caspase3蛋白活性,酒精染毒2-4d均未引起Caspase 3蛋白活性的顯著增加。 結(jié)合前文中發(fā)現(xiàn)酒精引起神經(jīng)干細胞c-myc表達增加,可認為25-100mM酒精染毒2-4d,神經(jīng)干細胞出現(xiàn)早期凋亡事件,凋亡抑制基因survivin下調(diào),促凋亡基因c-myc上調(diào),bax隨之上調(diào),bcl-2/bax比值下降。線粒體途徑參與了酒精誘導(dǎo)的神經(jīng)干細胞早期凋亡。 5酒精對胎鼠腦新皮質(zhì)神經(jīng)干細胞分化的影響 利用熒光定量PCR技術(shù)檢測神經(jīng)干細胞分化后神經(jīng)元、星形膠質(zhì)細胞、少突膠質(zhì)細胞標(biāo)志物mRNA的表達。酒精染毒4d促進神經(jīng)元前體細胞標(biāo)志物Tuj1、星形膠質(zhì)細胞Gfap、少突膠質(zhì)細胞Mbp的表達,酒精染毒8d未引起分化細胞標(biāo)志物的改變。結(jié)果表明,酒精主要在神經(jīng)干細胞培養(yǎng)早期影響了神經(jīng)干細胞向神經(jīng)元、星形膠質(zhì)細胞和少突膠質(zhì)細胞分化時相應(yīng)標(biāo)志蛋白基因表達水平的改變。
[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 ) .

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329

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