【摘要】：細胞凋亡過程存在著一個復雜的調控網絡,許多蛋白質參與細胞凋亡的啟動和執(zhí)行過程。在細胞凋亡的過程中,許多蛋白質在不同的亞細胞器間存在轉位現象,包括質膜、內質網、線粒體和細胞核。其中細胞核是一個非常重要的細胞器,細胞凋亡的許多調控因子都是進入細胞核發(fā)揮作用的(如caspases、AIF和cytochrome C等)。因此,分析細胞凋亡過程中核蛋白的動態(tài)變化,將有可能發(fā)現新的細胞凋亡相關蛋白。本課題的研究目的就是分析凋亡細胞和非凋亡細胞核蛋白的變化,尋找新的凋亡相關蛋白,并分析其在細胞凋亡過程中可能的作用。 首先,用腫瘤壞死因子α(TNF-α)誘導小鼠黑色素瘤B16細胞凋亡,確定了細胞凋亡的合適的時相,采用細胞熒光染色、流式細胞術、Caspase-3活性測定等手段檢測TNF-α作用于B16細胞后不同時間點細胞的凋亡情況。結果表明,TNF-α能夠誘導B16細胞凋亡,并具有時效關系。通過流式細胞儀檢測TNF-α對細胞周期的影響,結果顯示,TNF-α作用于B16細胞后導致細胞發(fā)生G1期阻滯。 為了尋找新的細胞凋亡相關蛋白,在TNF-α誘導小鼠黑色素瘤B16細胞凋亡36小時后收取細胞,分離了凋亡細胞,提取細胞核總蛋白質,進行二維電泳分離,通過計算機圖像分析,發(fā)現在凋亡細胞核中有11個蛋白發(fā)生了明顯的變化(蛋白濃度相差3倍以上),其中6個蛋白下調,5個蛋白上調,對這11個差異蛋白質點分別進行質譜分析,經數據庫查詢,初步鑒定出這些差異蛋白。 其中4個含量增加的蛋白(HSP84,calreticulin,vimentin,GAPDH)均直接或間接地參與細胞凋亡有關,另外一個含量增加的蛋白(plasminogen)尚未見其與細胞凋亡有關的報道。而6個含量降低的蛋白分別屬于信號轉導相關蛋白(guanine nucleotide binding protein,laminin receptor 1)、轉錄調控蛋白(heterochromatin protein 1 alpha,heterogeneous nuclear ribonucleoprotein A3,heterogeneous nuclear ribonucleoprotein A2/B1)和未知功能蛋白(nucleolar protein NO38)。細胞凋亡過程中這些變化的核蛋白的發(fā)現將有助于深入認識細胞凋亡的分子機制。 三磷酸甘油醛脫氫酶(GAPDH)是糖酵解過程中的一個關鍵酶,與ATP的合成密切相關。近年來越來越多的研究表明,GAPDH存在許多異構體,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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R341

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本文編號：2231041