Hsp70在缺糖损伤的HeLa细胞中对Bax与Bcl-2表达及Bax构象改变的影响

发布时间：2018-02-03 05:41

本文关键词： Hsp70 缺糖损伤 Bax Bcl-2　出处：《复旦大学》2008年硕士论文　论文类型：学位论文

【摘要】： 缺血性疾病常常对人体造成不可逆的损伤,其发生机制也日益受到人们的关注。葡萄糖的匮乏是缺血损伤的主要致病因素,由于葡萄糖的摄取是细胞内能量代谢的中心环节,所以缺糖损伤的直接严重后果就是细胞死亡,这一论点已经得到了充分的证实。近年的研究还表明缺糖损伤还会导致细胞内许多凋亡因子的产生,在众多凋亡通道上左右着凋亡的进程。因此了解细胞在缺糖损伤过程中细胞水平,亚细胞水平及分子水平的变化对于缺糖损伤致病机制的阐明有重要的意义。人类宫颈癌HeLa细胞是分子生物实验常用的细胞模型。以往很多研究表明,热激蛋白Hsp70在很多应激情况下可以抑制细胞的凋亡,所以本文中主要检测缺糖引起的HeLa细胞损伤情况以及Hsp70对其的影响。本文用获赠的人类正义Hsp70重组质粒[pcDNA3.1(+)-Hsp70]转染正常HeLa细胞,用G418筛选并经Western Blot法鉴定获得了Hsp70过表达HeLa细胞。用无糖培养建立缺糖损伤细胞模型;用MTT法测细胞活率;用Hoechst染色法和Giemsa染色法从形态上观察并计算细胞凋亡率。结果表明,在缺糖48h内,Hsp70明显抑制了缺糖诱导的HeLa细胞凋亡并增强其活力。以往研究证实,在细胞凋亡过程中,Hsp70主要通过与其它蛋白的相互作用来调控凋亡,包括Bcl-2家族。Bcl-2家族蛋白是在细胞凋亡过程中起关键性作用的一类蛋白质,在细胞发生凋亡时,Bcl-2家族中的促凋亡蛋白成员能够发生活化,移位到线粒体的外膜上,进而引起其他促凋亡因子的释放,导致细胞凋亡,但同时此现象能够被该家族的抗凋亡蛋白阻止,所以Bcl-2家族成员的构成比例是凋亡调控的重要因素,尤其Bax/Bcl-2比值是启动细胞凋亡的关键所在。为了进一步探讨Hsp70抑制缺糖凋亡的机制,本文用RT-PCR法和Western-Blot法分别在mRNA和蛋白水平测Bax和Bcl-2的表达,并计算Bax/Bcl-2比值;用细胞免疫化学法和荧光免疫法测Bax构象改变情况。结果表明,HeLa细胞在缺糖48h内,Hsp70能够抑制细胞中Bax/Bcl-2的增高以及Bax构象的改变。根据以上结果我们初步推断,在缺糖诱导的HeLa细胞中,Hsp70能够通过与Bcl-2家族成员作用而抑制凋亡的发生。本研究采用Hsp70正常表达和过表达的两组细胞,拟通过建立缺糖应激损伤的模型,检测并比较两组模型在细胞水平及分子水平的损伤指标和相关凋亡因子的表达水平,来研究Hsp70对凋亡的影响及其可能机制。旨在为一些重要的应激蛋白在能量代谢损伤过程中的作用位点,作用对象及作用方式的研究提供一个适宜的研究平台,从而为相关蛋白的功能研究奠定一定基础。
[Abstract]:Ischemic diseases often cause irreversible damage to human body, the mechanism of which has been paid more and more attention. The lack of glucose is the main cause of ischemic injury. Because glucose uptake is the central link of intracellular energy metabolism, the direct and serious consequence of glucose deficiency injury is cell death. This argument has been fully confirmed. Recent studies have also shown that glucose deficiency can also lead to the production of many apoptosis factors in cells. Therefore, it is important to understand the changes of cell level, subcellular level and molecular level in the process of glucose deficiency injury in order to elucidate the pathogenetic mechanism of glucose deficiency injury. Human cervical cancer HeLa cells are commonly used as cell models in molecular biological experiments. Many previous studies have shown that heat shock protein Hsp70 can inhibit cell apoptosis under many stress conditions. Therefore, the damage of HeLa cells caused by glucose deficiency and the effect of Hsp70 on it were detected in this paper. The purpose of this study was to use the donated recombinant plasmid of human justice Hsp70. [PcDNA3.1 (hsp70) was transfected into normal HeLa cells. Hsp70 overexpression HeLa cells were obtained by G418 screening and identified by Western Blot method. Cell viability was measured by MTT method. The apoptosis rate was observed and calculated by Hoechst staining and Giemsa staining. The results showed that the rate of apoptosis was less than 48 hours after glucose deficiency. Hsp70 significantly inhibited the apoptosis and enhanced the activity of HeLa cells induced by glucose deficiency. Previous studies have confirmed that Hsp70 regulates apoptosis mainly through interaction with other proteins. Including the Bcl-2 family. Bcl-2 family proteins are a class of proteins that play a key role in the process of cell apoptosis. Apoptosis-promoting protein members of Bcl-2 family can be activated and translocated to the outer membrane of mitochondria, which leads to the release of other pro-apoptotic factors, leading to apoptosis. But at the same time, this phenomenon can be blocked by the anti-apoptotic protein, so the proportion of members of Bcl-2 family is an important factor in the regulation of apoptosis. In particular, Bax/Bcl-2 ratio is the key to initiate apoptosis. In order to further explore the mechanism of Hsp70 inhibiting the apoptosis of glucose deficiency. The expression of Bax and Bcl-2 were measured by RT-PCR and Western-Blot at mRNA and protein levels, respectively, and the Bax/Bcl-2 ratio was calculated. The conformation changes of Bax were measured by cell immunocytochemistry and fluorescence immunoassay. The results showed that Hela cells were deficient in glucose within 48 hours. Hsp70 can inhibit the increase of Bax/Bcl-2 and the conformation of Bax in cells. Based on the above results, we infer that in HeLa cells induced by glucose deficiency. Hsp70 can inhibit apoptosis by interacting with Bcl-2 family members. In this study, two groups of cells with normal expression and overexpression of Hsp70 were used to establish the model of glucose deficiency stress injury. The damage index and the expression level of apoptosis factor were detected and compared between the two groups at the cell level and molecular level. To study the effect of Hsp70 on apoptosis and its possible mechanism. The aim of this study is to provide sites for some important stress proteins in the process of energy metabolism damage. The research of action object and action mode provide a suitable research platform, thus lay a certain foundation for the study of the function of related protein.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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