【摘要】：細(xì)胞衰老定義為不可逆的細(xì)胞周期阻滯。研究發(fā)現(xiàn)細(xì)胞衰老在多種生物學(xué)過程如腫瘤發(fā)生,機(jī)體衰老和胚胎發(fā)育中起重要作用。參與調(diào)控細(xì)胞衰老的因素復(fù)雜多樣,越來越多的證據(jù)表明衰老細(xì)胞發(fā)生了顯著的代謝改變以及細(xì)胞代謝的中間產(chǎn)物在衰老過程中發(fā)揮重要作用,但是線粒體功能及代謝與衰老之間的關(guān)系依然大部分未知。本課題以人成纖維細(xì)胞為實(shí)驗(yàn)對(duì)象,分別通過阿霉素給藥模擬腫瘤抑制治療藥物引起的細(xì)胞衰老和細(xì)胞連續(xù)傳代導(dǎo)致的復(fù)制性衰老為體外模型研究細(xì)胞衰老。實(shí)驗(yàn)發(fā)現(xiàn)在以上兩種細(xì)胞衰老過程中,衰老細(xì)胞胞內(nèi)和線粒體內(nèi)的氧化應(yīng)激增強(qiáng),并伴隨發(fā)生線粒體形態(tài)改變和功能亢進(jìn),而細(xì)胞的代謝率也隨之升高。隨著細(xì)胞衰老進(jìn)入晚期,我們發(fā)現(xiàn)衰老細(xì)胞的線粒體膜電位發(fā)生去極化,細(xì)胞隨之發(fā)生凋亡。我們的結(jié)果表明,細(xì)胞衰老過程中,伴發(fā)胞內(nèi)和線粒體內(nèi)高氧化應(yīng)激,同時(shí)線粒體發(fā)生形態(tài)改變和功能亢進(jìn),且細(xì)胞代謝率升高。而隨著衰老進(jìn)入晚期,線粒體也在推動(dòng)細(xì)胞命運(yùn)從衰老到凋亡的轉(zhuǎn)變中起到了關(guān)鍵作用。為進(jìn)一步研究代謝與細(xì)胞衰老之間的關(guān)系,我們集中研究了糖酵解通路最終步驟：使磷酸烯醇丙酮酸(Phosphoenolpyruvate, PEP)轉(zhuǎn)變?yōu)楸岬拇呋?丙酮酸激酶與人成纖維細(xì)胞衰老的關(guān)系。我們發(fā)現(xiàn),敲降丙酮酸激酶會(huì)引起人成纖維細(xì)胞發(fā)生明顯衰老,同時(shí)伴有代謝率升高的現(xiàn)象。在人成纖維細(xì)胞中過表達(dá)小鼠M1或M2型丙酮酸激酶能夠逆轉(zhuǎn)細(xì)胞的高代謝,但并不能減輕細(xì)胞的衰老表型。據(jù)此我們認(rèn)為,細(xì)胞衰老過程中伴隨著代謝改變,但代謝可能并不一定是細(xì)胞衰老發(fā)生的決定性因素。細(xì)胞衰老在多種生物過程中起著重要的作用。但是,缺乏特異性的方法來鑒定和分離活的衰老細(xì)胞阻礙我們對(duì)其發(fā)生機(jī)制的精確了解。利用細(xì)胞衰老發(fā)生的細(xì)胞周期阻滯原理,我們利用聯(lián)合熒光泛素化細(xì)胞周期指示技術(shù)(fluorescent ubiquitination-based cell cycle indicator, FUCCI)分選活的衰老細(xì)胞。HIFF細(xì)胞經(jīng)阿霉素處理后阻滯在S/G2/M期,攜帶了mAG-hGeminin報(bào)告質(zhì)粒的HFF細(xì)胞可以使處于S/G2/M期的細(xì)胞呈現(xiàn)GFP陽性。 通過分選GFP+和GFP-細(xì)胞后做進(jìn)一步的細(xì)胞衰老鑒定發(fā)現(xiàn)GFP+細(xì)胞富集更多的衰老細(xì)胞。我們的研究開發(fā)了一種新的方法來鑒定和分離活的衰老細(xì)胞,為研究細(xì)胞衰老提供了新的工具。
[Abstract]:Cell senescence is defined as irreversible cell cycle arrest. It has been found that cell aging plays an important role in many biological processes such as tumorigenesis, body aging and embryonic development. The factors involved in the regulation of cell senescence are complex and diverse. There is increasing evidence that senescent cells have undergone significant metabolic changes and the intermediate products of cell metabolism play an important role in the process of senescence. However, the relationship between mitochondrial function and metabolism and aging is still largely unknown. In this study, human fibroblasts were used as experimental objects to study cell senescence induced by doxorubicin, which was induced by tumor inhibitory drugs and repeated senescence caused by continuous passage of cells, as in vitro models. It was found that during the senescence of the above two kinds of cells, the oxidative stress in the cells and in the mitochondria increased, accompanied by the morphological changes and hyperfunction of the mitochondria, and the metabolic rate of the cells also increased. As the cells entered the late stage of senescence, we found that the mitochondrial membrane potential of the aging cells was depolarized and the cells were apoptotic. Our results show that during cell senescence, there is high oxidative stress in the cells and mitochondria, at the same time, the morphological changes and hyperfunction of mitochondria occur, and the metabolic rate of the cells increases. As aging enters the late stage, mitochondria also play a key role in promoting the transformation of cell fate from aging to apoptosis. In order to further study the relationship between metabolism and cell senescence, we focused on the final step of glycolysis pathway: making phosphoenolpyruvate (Phosphoenolpyruvate,). PEP) the relationship between pyruvate kinase and aging of human fibroblasts. We found that knockdown pyruvate kinase could cause obvious aging of human fibroblasts, accompanied by an increase in metabolic rate. Overexpression of mouse M1 or M2 pyruvate kinase in human fibroblasts could reverse the hypermetabolism of the cells, but did not reduce the aging phenotype of the cells. Therefore, we believe that metabolism is accompanied by metabolic changes in the process of cell senescence, but metabolism may not be the decisive factor in the occurrence of cell senescence. Cell senescence plays an important role in a variety of biological processes. However, the lack of specific methods to identify and isolate living aging cells hinders our accurate understanding of its pathogenesis. Based on the cell cycle arrest principle of cell senescence, we used the combined fluorescent ubiquitin cell cycle indicator technique (fluorescent ubiquitination-based cell cycle indicator, FUCCI) to isolate the living senescent cells. Hiff cells were blocked in S/G2/M phase after being treated with doxorubicin. HFF cells carrying mAG-hGeminin reporter plasmid could make the cells in S/G2/M phase GFP positive. By sorting GFP and GFP- cells for further cell senescence identification, it was found that GFP cells were enriched in more senescent cells. Our research has developed a new method to identify and isolate living aging cells, which provides a new tool for the study of cell senescence.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q255

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