發(fā)布時(shí)間：2018-03-05 11:36

  本文選題：銅鋅超氧化物歧化酶　切入點(diǎn)：家族性肌萎縮側(cè)索硬化癥　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：肌萎縮側(cè)索硬化癥（ALS）是一種致命的漸進(jìn)型神經(jīng)退行性疾病。它是由遺傳缺陷或者非遺傳性因素造成的。發(fā)病年齡通常在45歲至60歲，發(fā)病后的生存時(shí)間一般為1-5年，目前還沒有可治愈的藥物。已有多個(gè)基因缺陷被發(fā)現(xiàn)引起ALS，稱作家族性肌萎縮側(cè)索硬化癥（fALS）。其中，最為著名的是由Cu,ZnSOD1的突變而造成的fALS。Cu,ZnSOD1因催化細(xì)胞中自由基的歧化反應(yīng)而著稱。它在細(xì)胞抵抗超氧化合物對(duì)細(xì)胞的損傷過程中起關(guān)鍵作用。目前已發(fā)現(xiàn)近150個(gè)Cu,ZnSOD1的突變能導(dǎo)致fALS。轉(zhuǎn)基因小鼠研究結(jié)果表明fALS并不是因?yàn)橥蛔兪笴u,ZnSOD1失去活性，而是使Cu,ZnSOD1獲得了一個(gè)新的有毒功能導(dǎo)致的。同時(shí)，也認(rèn)為不同的突變是通過相同的機(jī)理引起fALS。但是，這一假說還未在實(shí)驗(yàn)上被完全證明。至于突變使Cu,ZnSOD1獲得了什么性質(zhì)的有毒新功能還有待被發(fā)現(xiàn)。 本論文選擇在酵母細(xì)胞中研究在人體中引起fALS的Cu,ZnSOD1突變體A4V，G41D，G41S，G85R，G93C和I113T。我們實(shí)驗(yàn)室的研究結(jié)果表明酵母細(xì)胞也是研究fALS的理想模型。我們發(fā)現(xiàn)，當(dāng)酵母細(xì)胞的H+-ATPase Pma1被過度表達(dá)時(shí)，酵母Cu,ZnSod1的G85R突變使其獲得一個(gè)能使酵母細(xì)胞產(chǎn)生急劇形態(tài)變化和死亡的有毒新功能。酵母細(xì)胞和人體的Cu,ZnSod1相互間具有高度的同源性。因此，，本論文在酵母細(xì)胞中研究人體Cu,ZnSod1的這些突變體是否獲得相同的有毒新功能。所得的實(shí)驗(yàn)結(jié)果表明人體的Cu,ZnSod1完全能夠替代酵母細(xì)胞的Cu,ZnSod1抵抗H2O2的毒性，使酵母細(xì)胞能夠在39℃溫度下生長(zhǎng)。人體Cu,ZnSod1也能夠像酵母的Cu,ZnSod1那樣使Pma1正確定位于細(xì)胞膜。突變體A4V和I113T也能夠使sod1細(xì)胞抵抗H2O2的毒性和在39℃溫度下生長(zhǎng)。相比之下，其它G41D，G41S，G85R和G93C均在抗氧化和耐高溫方面顯示了不同程度的缺陷。盡管像A4V，G93C和I113T這樣的突變體具有正常的催化歧化反應(yīng)活性，然而在這些突變體細(xì)胞中Pma1-GFP仍然發(fā)生了定位錯(cuò)誤。在G41D和G41S突變體細(xì)胞中，Pma1-GFP有正確顯示細(xì)胞膜定位，也有發(fā)生了與A4V，G85R，G93C和I113T突體類似的定位錯(cuò)誤。除了Pma1定位錯(cuò)誤外，A4V和I113T突變體引起酵母細(xì)胞的體積發(fā)生了明顯增大的變化。和酵母Cu,ZnSod1的G85R突變體細(xì)胞一樣，人體的I113T突變體使酵母在YPD培養(yǎng)基上呈現(xiàn)棕黑色的皺紋形態(tài)，突變體細(xì)胞呈現(xiàn)細(xì)絲狀形態(tài)。如此急劇的形態(tài)變化表明人體Cu,ZnSod1的I113T突變和酵母Cu,ZnSod1的G85R突變一樣嚴(yán)重?cái)_亂了細(xì)胞周期這一關(guān)鍵的生物過程。本論文所得實(shí)驗(yàn)結(jié)果首次利用酵母細(xì)胞模型證明在人體中能引起fALS疾病的I113T突變確實(shí)使Cu,ZnSod1獲得了一個(gè)新的有毒功能，使酵母細(xì)胞產(chǎn)生了急劇的形態(tài)變化和死亡。這結(jié)果從而在酵母模型中證明了有關(guān)fALS的假設(shè)，也就是a toxic gain-of-function;進(jìn)一步表明酵母細(xì)胞是研究人體fALS疾病的一個(gè)重要模型。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease. It is caused by genetic defects or non-genetic factors. There is no cure yet. Several genetic defects have been found to cause ALS, known as familial amyotrophic lateral sclerosis. The most famous one is fALS.CuOZnSOD1, which is caused by the mutation of CuOZnSOD1. It plays a key role in cell resistance to the damage caused by superoxide compounds. Nearly 150 CuOZnSOD1 compounds have been found to catalyze the disproportionation of free radicals in cells. The results showed that fALS was not due to the inactivation of CuOZnSOD1, However, it is believed that different mutations cause fALSs by the same mechanism. This hypothesis has not yet been fully proved experimentally. The nature of the toxic new function obtained by the mutation of CuPZnSOD1 has yet to be discovered. In this paper, we chose to study in yeast cells the mutant A4VG41DG41SG85RG93C and I113T. our laboratory results showed that yeast cells were also ideal models for studying fALS. We found that when the H -ATPase Pma1 of yeast cells was overexpressed, The G85R mutation of yeast CuPZnSod1 enables it to obtain a new toxic function which can cause sharp morphological changes and death of yeast cells. There is a high homology between yeast cells and human Cu-ZnSod1. In this paper, we studied whether these mutants of human CuPZnSod1 have the same toxic new function in yeast cells. The experimental results show that the human CuPZnSod1 can completely replace the CuOZnSod1 of yeast cells to resist the toxicity of H _ 2O _ 2. The yeast cells were able to grow at 39 鈩

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