TNF-α诱导B16细胞凋亡的核蛋白质组研究

发布时间：2018-09-08 16:16

【摘要】：细胞凋亡过程存在着一个复杂的调控网络,许多蛋白质参与细胞凋亡的启动和执行过程。在细胞凋亡的过程中,许多蛋白质在不同的亚细胞器间存在转位现象,包括质膜、内质网、线粒体和细胞核。其中细胞核是一个非常重要的细胞器,细胞凋亡的许多调控因子都是进入细胞核发挥作用的(如caspases、AIF和cytochrome C等)。因此,分析细胞凋亡过程中核蛋白的动态变化,将有可能发现新的细胞凋亡相关蛋白。本课题的研究目的就是分析凋亡细胞和非凋亡细胞核蛋白的变化,寻找新的凋亡相关蛋白,并分析其在细胞凋亡过程中可能的作用。首先,用肿瘤坏死因子α(TNF-α)诱导小鼠黑色素瘤B16细胞凋亡,确定了细胞凋亡的合适的时相,采用细胞荧光染色、流式细胞术、Caspase-3活性测定等手段检测TNF-α作用于B16细胞后不同时间点细胞的凋亡情况。结果表明,TNF-α能够诱导B16细胞凋亡,并具有时效关系。通过流式细胞仪检测TNF-α对细胞周期的影响,结果显示,TNF-α作用于B16细胞后导致细胞发生G1期阻滞。为了寻找新的细胞凋亡相关蛋白,在TNF-α诱导小鼠黑色素瘤B16细胞凋亡36小时后收取细胞,分离了凋亡细胞,提取细胞核总蛋白质,进行二维电泳分离,通过计算机图像分析,发现在凋亡细胞核中有11个蛋白发生了明显的变化(蛋白浓度相差3倍以上),其中6个蛋白下调,5个蛋白上调,对这11个差异蛋白质点分别进行质谱分析,经数据库查询,初步鉴定出这些差异蛋白。其中4个含量增加的蛋白(HSP84,calreticulin,vimentin,GAPDH)均直接或间接地参与细胞凋亡有关,另外一个含量增加的蛋白(plasminogen)尚未见其与细胞凋亡有关的报道。而6个含量降低的蛋白分别属于信号转导相关蛋白(guanine nucleotide binding protein,laminin receptor 1)、转录调控蛋白(heterochromatin protein 1 alpha,heterogeneous nuclear ribonucleoprotein A3,heterogeneous nuclear ribonucleoprotein A2/B1)和未知功能蛋白(nucleolar protein NO38)。细胞凋亡过程中这些变化的核蛋白的发现将有助于深入认识细胞凋亡的分子机制。三磷酸甘油醛脱氢酶(GAPDH)是糖酵解过程中的一个关键酶,与ATP的合成密切相关。近年来越来越多的研究表明,GAPDH存在许多异构体,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 伪 (TNF- 伪) 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- 伪. The results showed that TNF- 伪 could induce apoptosis of B16 cells in a time-dependent manner. The effect of TNF- 伪 on cell cycle was detected by flow cytometry. The results showed that TNF- 伪 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- 伪, 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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R341

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