【摘要】：細(xì)胞凋亡過(guò)程存在著一個(gè)復(fù)雜的調(diào)控網(wǎng)絡(luò),許多蛋白質(zhì)參與細(xì)胞凋亡的啟動(dòng)和執(zhí)行過(guò)程。在細(xì)胞凋亡的過(guò)程中,許多蛋白質(zhì)在不同的亞細(xì)胞器間存在轉(zhuǎn)位現(xiàn)象,包括質(zhì)膜、內(nèi)質(zhì)網(wǎng)、線粒體和細(xì)胞核。其中細(xì)胞核是一個(gè)非常重要的細(xì)胞器,細(xì)胞凋亡的許多調(diào)控因子都是進(jìn)入細(xì)胞核發(fā)揮作用的(如caspases、AIF和cytochrome C等)。因此,分析細(xì)胞凋亡過(guò)程中核蛋白的動(dòng)態(tài)變化,將有可能發(fā)現(xiàn)新的細(xì)胞凋亡相關(guān)蛋白。本課題的研究目的就是分析凋亡細(xì)胞和非凋亡細(xì)胞核蛋白的變化,尋找新的凋亡相關(guān)蛋白,并分析其在細(xì)胞凋亡過(guò)程中可能的作用。 首先,用腫瘤壞死因子α(TNF-α)誘導(dǎo)小鼠黑色素瘤B16細(xì)胞凋亡,確定了細(xì)胞凋亡的合適的時(shí)相,采用細(xì)胞熒光染色、流式細(xì)胞術(shù)、Caspase-3活性測(cè)定等手段檢測(cè)TNF-α作用于B16細(xì)胞后不同時(shí)間點(diǎn)細(xì)胞的凋亡情況。結(jié)果表明,TNF-α能夠誘導(dǎo)B16細(xì)胞凋亡,并具有時(shí)效關(guān)系。通過(guò)流式細(xì)胞儀檢測(cè)TNF-α對(duì)細(xì)胞周期的影響,結(jié)果顯示,TNF-α作用于B16細(xì)胞后導(dǎo)致細(xì)胞發(fā)生G1期阻滯。 為了尋找新的細(xì)胞凋亡相關(guān)蛋白,在TNF-α誘導(dǎo)小鼠黑色素瘤B16細(xì)胞凋亡36小時(shí)后收取細(xì)胞,分離了凋亡細(xì)胞,提取細(xì)胞核總蛋白質(zhì),進(jìn)行二維電泳分離,通過(guò)計(jì)算機(jī)圖像分析,發(fā)現(xiàn)在凋亡細(xì)胞核中有11個(gè)蛋白發(fā)生了明顯的變化(蛋白濃度相差3倍以上),其中6個(gè)蛋白下調(diào),5個(gè)蛋白上調(diào),對(duì)這11個(gè)差異蛋白質(zhì)點(diǎn)分別進(jìn)行質(zhì)譜分析,經(jīng)數(shù)據(jù)庫(kù)查詢,初步鑒定出這些差異蛋白。 其中4個(gè)含量增加的蛋白(HSP84,calreticulin,vimentin,GAPDH)均直接或間接地參與細(xì)胞凋亡有關(guān),另外一個(gè)含量增加的蛋白(plasminogen)尚未見(jiàn)其與細(xì)胞凋亡有關(guān)的報(bào)道。而6個(gè)含量降低的蛋白分別屬于信號(hào)轉(zhuǎn)導(dǎo)相關(guān)蛋白(guanine nucleotide binding protein,laminin receptor 1)、轉(zhuǎn)錄調(diào)控蛋白(heterochromatin protein 1 alpha,heterogeneous nuclear ribonucleoprotein A3,heterogeneous nuclear ribonucleoprotein A2/B1)和未知功能蛋白(nucleolar protein NO38)。細(xì)胞凋亡過(guò)程中這些變化的核蛋白的發(fā)現(xiàn)將有助于深入認(rèn)識(shí)細(xì)胞凋亡的分子機(jī)制。 三磷酸甘油醛脫氫酶(GAPDH)是糖酵解過(guò)程中的一個(gè)關(guān)鍵酶,與ATP的合成密切相關(guān)。近年來(lái)越來(lái)越多的研究表明,GAPDH存在許多異構(gòu)體,其功能也不
[Abstract]:There is a complex regulatory network in the process of apoptosis, and many proteins are involved in the initiation and execution of apoptosis. During the process of apoptosis, many proteins are translocated among different suborganelles, including plasma membrane, endoplasmic reticulum, mitochondria and nucleus. The nucleus is a very important organelle, and many of the regulatory factors of apoptosis play a role in the cell nucleus (such as caspases,AIF and cytochrome C). Therefore, it is possible to find new apoptosis-related proteins by analyzing the dynamic changes of nuclear proteins during apoptosis. The purpose of this study is to analyze the changes of apoptotic and non-apoptotic nuclear proteins, to find new apoptosis-related proteins and to analyze their possible role in the process of apoptosis. Firstly, tumor necrosis factor 偽 (TNF- 偽) was used to induce the apoptosis of mouse melanoma B16 cells. The apoptosis of B16 cells was detected by flow cytometry and Caspase-3 activity assay at different time points after treated with TNF- 偽. The results showed that TNF- 偽 could induce apoptosis of B16 cells in a time-dependent manner. The effect of TNF- 偽 on cell cycle was detected by flow cytometry. The results showed that TNF- 偽 induced G1 phase arrest in B16 cells. In order to find a new apoptosis-related protein, the apoptotic cells were isolated after 36 hours of apoptosis induced by TNF- 偽, the total nuclear protein was extracted and separated by two-dimensional electrophoresis. It was found that there were 11 proteins in the apoptotic nucleus that had obvious changes (the difference of protein concentration was more than 3 times). Among them, 6 proteins were down-regulated and 5 proteins were up-regulated. The 11 differential protein spots were analyzed by mass spectrometry and queried by database. These differential proteins were preliminarily identified. Four of the increased proteins (HSP84,calreticulin,vimentin,GAPDH) were directly or indirectly involved in apoptosis, while the other one, (plasminogen), was not reported to be related to apoptosis. The six proteins with decreased content belong to signal transduction related protein (guanine nucleotide binding protein,laminin receptor 1, transcription regulatory protein (heterochromatin protein 1 alpha,heterogeneous nuclear ribonucleoprotein A 3 heterogeneous nuclear ribonucleoprotein A2/B1 and unknown function protein (nucleolar protein NO38 respectively. The discovery of these nuclear proteins during apoptosis will be helpful in understanding the molecular mechanism of apoptosis. Triglyceraldehyde triphosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis and is closely related to the synthesis of ATP. In recent years, more and more studies have shown that there are many isomers in GAPDH, and its function is not.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 曹煒，牛建昭;細(xì)胞凋亡的概念及其基因的研究進(jìn)展[J];日本醫(yī)學(xué)介紹;1995年03期

，

本文編號(hào)：2231041