衰老细胞的代谢表型及基于FUCCI体系的分选研究

发布时间：2019-05-11 10:15

【摘要】：细胞衰老定义为不可逆的细胞周期阻滞。研究发现细胞衰老在多种生物学过程如肿瘤发生,机体衰老和胚胎发育中起重要作用。参与调控细胞衰老的因素复杂多样,越来越多的证据表明衰老细胞发生了显著的代谢改变以及细胞代谢的中间产物在衰老过程中发挥重要作用,但是线粒体功能及代谢与衰老之间的关系依然大部分未知。本课题以人成纤维细胞为实验对象,分别通过阿霉素给药模拟肿瘤抑制治疗药物引起的细胞衰老和细胞连续传代导致的复制性衰老为体外模型研究细胞衰老。实验发现在以上两种细胞衰老过程中,衰老细胞胞内和线粒体内的氧化应激增强,并伴随发生线粒体形态改变和功能亢进,而细胞的代谢率也随之升高。随着细胞衰老进入晚期,我们发现衰老细胞的线粒体膜电位发生去极化,细胞随之发生凋亡。我们的结果表明,细胞衰老过程中,伴发胞内和线粒体内高氧化应激,同时线粒体发生形态改变和功能亢进,且细胞代谢率升高。而随着衰老进入晚期,线粒体也在推动细胞命运从衰老到凋亡的转变中起到了关键作用。为进一步研究代谢与细胞衰老之间的关系,我们集中研究了糖酵解通路最终步骤：使磷酸烯醇丙酮酸(Phosphoenolpyruvate, PEP)转变为丙酮酸的催化酶-丙酮酸激酶与人成纤维细胞衰老的关系。我们发现,敲降丙酮酸激酶会引起人成纤维细胞发生明显衰老,同时伴有代谢率升高的现象。在人成纤维细胞中过表达小鼠M1或M2型丙酮酸激酶能够逆转细胞的高代谢,但并不能减轻细胞的衰老表型。据此我们认为,细胞衰老过程中伴随着代谢改变,但代谢可能并不一定是细胞衰老发生的决定性因素。细胞衰老在多种生物过程中起着重要的作用。但是,缺乏特异性的方法来鉴定和分离活的衰老细胞阻碍我们对其发生机制的精确了解。利用细胞衰老发生的细胞周期阻滞原理,我们利用联合荧光泛素化细胞周期指示技术(fluorescent ubiquitination-based cell cycle indicator, FUCCI)分选活的衰老细胞。HIFF细胞经阿霉素处理后阻滞在S/G2/M期,携带了mAG-hGeminin报告质粒的HFF细胞可以使处于S/G2/M期的细胞呈现GFP阳性。通过分选GFP+和GFP-细胞后做进一步的细胞衰老鉴定发现GFP+细胞富集更多的衰老细胞。我们的研究开发了一种新的方法来鉴定和分离活的衰老细胞,为研究细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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q255

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