發(fā)布時(shí)間：2018-02-03 05:41

  本文關(guān)鍵詞： Hsp70 缺糖損傷 Bax Bcl-2　出處：《復(fù)旦大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 缺血性疾病常常對(duì)人體造成不可逆的損傷,其發(fā)生機(jī)制也日益受到人們的關(guān)注。葡萄糖的匱乏是缺血損傷的主要致病因素,由于葡萄糖的攝取是細(xì)胞內(nèi)能量代謝的中心環(huán)節(jié),所以缺糖損傷的直接嚴(yán)重后果就是細(xì)胞死亡,這一論點(diǎn)已經(jīng)得到了充分的證實(shí)。近年的研究還表明缺糖損傷還會(huì)導(dǎo)致細(xì)胞內(nèi)許多凋亡因子的產(chǎn)生,在眾多凋亡通道上左右著凋亡的進(jìn)程。因此了解細(xì)胞在缺糖損傷過程中細(xì)胞水平,亞細(xì)胞水平及分子水平的變化對(duì)于缺糖損傷致病機(jī)制的闡明有重要的意義。 人類宮頸癌HeLa細(xì)胞是分子生物實(shí)驗(yàn)常用的細(xì)胞模型。以往很多研究表明,熱激蛋白Hsp70在很多應(yīng)激情況下可以抑制細(xì)胞的凋亡,所以本文中主要檢測(cè)缺糖引起的HeLa細(xì)胞損傷情況以及Hsp70對(duì)其的影響。本文用獲贈(zèng)的人類正義Hsp70重組質(zhì)粒[pcDNA3.1(+)-Hsp70]轉(zhuǎn)染正常HeLa細(xì)胞,用G418篩選并經(jīng)Western Blot法鑒定獲得了Hsp70過表達(dá)HeLa細(xì)胞。用無(wú)糖培養(yǎng)建立缺糖損傷細(xì)胞模型;用MTT法測(cè)細(xì)胞活率;用Hoechst染色法和Giemsa染色法從形態(tài)上觀察并計(jì)算細(xì)胞凋亡率。結(jié)果表明,在缺糖48h內(nèi),Hsp70明顯抑制了缺糖誘導(dǎo)的HeLa細(xì)胞凋亡并增強(qiáng)其活力。 以往研究證實(shí),在細(xì)胞凋亡過程中,Hsp70主要通過與其它蛋白的相互作用來(lái)調(diào)控凋亡,包括Bcl-2家族。Bcl-2家族蛋白是在細(xì)胞凋亡過程中起關(guān)鍵性作用的一類蛋白質(zhì),在細(xì)胞發(fā)生凋亡時(shí),Bcl-2家族中的促凋亡蛋白成員能夠發(fā)生活化,移位到線粒體的外膜上,進(jìn)而引起其他促凋亡因子的釋放,導(dǎo)致細(xì)胞凋亡,但同時(shí)此現(xiàn)象能夠被該家族的抗凋亡蛋白阻止,所以Bcl-2家族成員的構(gòu)成比例是凋亡調(diào)控的重要因素,尤其Bax/Bcl-2比值是啟動(dòng)細(xì)胞凋亡的關(guān)鍵所在。 為了進(jìn)一步探討Hsp70抑制缺糖凋亡的機(jī)制,本文用RT-PCR法和Western-Blot法分別在mRNA和蛋白水平測(cè)Bax和Bcl-2的表達(dá),并計(jì)算Bax/Bcl-2比值;用細(xì)胞免疫化學(xué)法和熒光免疫法測(cè)Bax構(gòu)象改變情況。結(jié)果表明,HeLa細(xì)胞在缺糖48h內(nèi),Hsp70能夠抑制細(xì)胞中Bax/Bcl-2的增高以及Bax構(gòu)象的改變。根據(jù)以上結(jié)果我們初步推斷,在缺糖誘導(dǎo)的HeLa細(xì)胞中,Hsp70能夠通過與Bcl-2家族成員作用而抑制凋亡的發(fā)生。 本研究采用Hsp70正常表達(dá)和過表達(dá)的兩組細(xì)胞,擬通過建立缺糖應(yīng)激損傷的模型,檢測(cè)并比較兩組模型在細(xì)胞水平及分子水平的損傷指標(biāo)和相關(guān)凋亡因子的表達(dá)水平,來(lái)研究Hsp70對(duì)凋亡的影響及其可能機(jī)制。旨在為一些重要的應(yīng)激蛋白在能量代謝損傷過程中的作用位點(diǎn),作用對(duì)象及作用方式的研究提供一個(gè)適宜的研究平臺(tái),從而為相關(guān)蛋白的功能研究奠定一定基礎(chǔ)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

【參考文獻(xiàn)】

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1 賀芳;鄔力祥;劉發(fā)益;楊麗娟;張琰;張海福;周p，

本文編號(hào)：1486582