【摘要】：細胞衰老定義為不可逆的細胞周期阻滯。研究發(fā)現(xiàn)細胞衰老在多種生物學過程如腫瘤發(fā)生,機體衰老和胚胎發(fā)育中起重要作用。參與調控細胞衰老的因素復雜多樣,越來越多的證據(jù)表明衰老細胞發(fā)生了顯著的代謝改變以及細胞代謝的中間產(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系依然大部分未知。本課題以人成纖維細胞為實驗對象,分別通過阿霉素給藥模擬腫瘤抑制治療藥物引起的細胞衰老和細胞連續(xù)傳代導致的復制性衰老為體外模型研究細胞衰老。實驗發(fā)現(xiàn)在以上兩種細胞衰老過程中,衰老細胞胞內和線粒體內的氧化應激增強,并伴隨發(fā)生線粒體形態(tài)改變和功能亢進,而細胞的代謝率也隨之升高。隨著細胞衰老進入晚期,我們發(fā)現(xiàn)衰老細胞的線粒體膜電位發(fā)生去極化,細胞隨之發(fā)生凋亡。我們的結果表明,細胞衰老過程中,伴發(fā)胞內和線粒體內高氧化應激,同時線粒體發(fā)生形態(tài)改變和功能亢進,且細胞代謝率升高。而隨著衰老進入晚期,線粒體也在推動細胞命運從衰老到凋亡的轉變中起到了關鍵作用。為進一步研究代謝與細胞衰老之間的關系,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)轉變?yōu)楸岬拇呋?丙酮酸激酶與人成纖維細胞衰老的關系。我們發(fā)現(xiàn),敲降丙酮酸激酶會引起人成纖維細胞發(fā)生明顯衰老,同時伴有代謝率升高的現(xiàn)象。在人成纖維細胞中過表達小鼠M1或M2型丙酮酸激酶能夠逆轉細胞的高代謝,但并不能減輕細胞的衰老表型。據(jù)此我們認為,細胞衰老過程中伴隨著代謝改變,但代謝可能并不一定是細胞衰老發(fā)生的決定性因素。細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胞阻礙我們對其發(fā)生機制的精確了解。利用細胞衰老發(fā)生的細胞周期阻滯原理,我們利用聯(lián)合熒光泛素化細胞周期指示技術(fluorescent ubiquitination-based cell cycle indicator, FUCCI)分選活的衰老細胞。HIFF細胞經(jīng)阿霉素處理后阻滯在S/G2/M期,攜帶了mAG-hGeminin報告質粒的HFF細胞可以使處于S/G2/M期的細胞呈現(xiàn)GFP陽性。 通過分選GFP+和GFP-細胞后做進一步的細胞衰老鑒定發(fā)現(xiàn)GFP+細胞富集更多的衰老細胞。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胞,為研究細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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q255

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