【摘要】：細胞衰老是指細胞喪失增殖能力后進入的一種相對穩(wěn)定的狀態(tài),端粒的縮短、化療藥處理引起的DNA損傷、癌基因的異常活化等都能誘導細胞衰老的發(fā)生。細胞衰老能夠顯著抑制細胞的生長,因此被認為是抑制腫瘤發(fā)生發(fā)展的重要因素之一。然而,越來越多的報道顯示衰老細胞還具有促進腫瘤的作用。更具體地說,衰老的細胞在體內(nèi)積累會誘發(fā)腫瘤,在化療后的實體瘤中積累則有可能促進其周圍細胞的增殖,以及通過SASP (senescence-associated sectroy phenotype)促進癌細胞耐藥性的產(chǎn)生。因此,及時清除化療后積累的衰老細胞或許有利于增強化療的療效。衰老的細胞可以通過向外分泌細胞因子,招募先天免疫細胞如巨噬細胞、中性粒細胞、自然殺傷細胞等將其清除,但是老年個體以及接受化療的患者免疫系統(tǒng)都不完善,無法通過免疫系統(tǒng)達到清除的目的。此外,大多數(shù)細胞衰老后都非常穩(wěn)定,這是由于它們具備抵抗凋亡的能力。因此,我們可以靶向衰老細胞的抗凋亡機制,從而誘導衰老細胞進入凋亡程序而被清除。為探究衰老細胞的抗凋亡機制,本研究建立了低濃度阿霉素(doxorubicin, DOX)誘導的細胞衰老模型。我們以衰老細胞的扁平增大表型為出發(fā)點,發(fā)現(xiàn)Hippo通路上的關鍵效應因子YAP在阿霉素誘導的衰老細胞中有明顯的核累積現(xiàn)象,這很可能是由于衰老細胞與細胞外基質(zhì)的接觸面積增大而誘發(fā)的。YAP是促進腫瘤發(fā)生發(fā)展的癌基因,在許多腫瘤中都異�；罨�。盡管異常表達的癌基因會促進細胞衰老的發(fā)生,但是在本研究中高表達YAP或活化型突變YAPS127A都不能誘導細胞衰老的發(fā)生；敲降YAP后同樣如此,反而促進了更多的細胞凋亡。因此,我們認為在阿霉素誘導的衰老細胞中,YAP的活化是衰老的結果,可以被癌細胞利用來抵抗凋亡。核內(nèi)積累的YAP具有較高的轉(zhuǎn)錄活性,能夠促進下游一系列靶基因的表達,如ANKRD1、CTGF以及CYR61等�？沟蛲龅鞍譻urvivin也是YAP的下游靶基因之一。Survivin作為細胞凋亡蛋白抑制因子家族的成員,在抵抗細胞凋亡過程中發(fā)揮著重要作用。本研究發(fā)現(xiàn),在阿霉素誘導的衰老細胞中,YAP的活化促進了survivin的表達；經(jīng)低濃度阿霉素誘導進入衰老狀態(tài)的細胞,在加入靶向YAP的抑制劑verteporfin或者survivin的抑制劑YM155后,能夠啟動凋亡。綜上,本研究表明,低濃度阿霉素誘導的衰老細胞依賴YAP的核內(nèi)積累及活化來促進survivin的表達,從而抵抗細胞凋亡而維持其存活狀態(tài)；低濃度的阿霉素可與YAP或survivin的抑制劑聯(lián)用,以清除衰老細胞,達到增強化療療效和降低副作用的目的。
[Abstract]:Cell senescence is a relatively stable state in which cells lose their proliferative ability. Telomere shortening, DNA damage induced by chemotherapeutic agents and abnormal activation of oncogenes can induce cell senescence. Cell senescence can significantly inhibit the growth of cells, so it is considered to be one of the important factors to inhibit the development of tumor. However, more and more reports show that aging cells also play a role in tumor promotion. More specifically, the accumulation of aging cells in vivo may induce tumors, while accumulation in solid tumors after chemotherapy may promote the proliferation of peripheral cells and the production of drug resistance in cancer cells through SASP (senescence-associated sectroy phenotype). Therefore, the timely clearance of aging cells accumulated after chemotherapy may be beneficial to enhance the efficacy of chemotherapy. Aging cells can be cleared by secreting cytokines into the body, recruiting innate immune cells such as macrophages, neutrophils, natural killer cells, etc., but neither the elderly nor the patient receiving chemotherapy has a perfect immune system. It is impossible to clear through the immune system. In addition, most cells are very stable after aging, because they have the ability to resist apoptosis. Therefore, we can target the anti-apoptotic mechanism of aging cells, thus inducing aging cells to enter the process of apoptosis and be eliminated. In order to explore the anti-apoptosis mechanism of aging cells, a model of cell senescence induced by low concentration of adriamycin (doxorubicin, DOX) was established. Based on the flattened and enlarged phenotype of aging cells, we found that YAP, a key effector factor in the Hippo pathway, had a significant nuclear accumulation in adriamycin-induced aging cells. This is probably due to the increased contact area between aging cells and extracellular matrix (ECM). YAP is a oncogene that promotes tumorigenesis and development and is activated abnormally in many tumors. Although the abnormal expression of oncogene can promote cell senescence, neither overexpression of YAP nor activated mutant YAPS127A can induce cell senescence in this study. Therefore, we believe that in adriamycin-induced aging cells, the activation of YAP is the result of aging and can be used by cancer cells to resist apoptosis. YAP accumulated in the nucleus has high transcriptional activity and can promote the expression of a series of downstream target genes such as ANKRD1,CTGF and CYR61. Anti-apoptotic protein survivin is also one of the downstream target genes of YAP. As a member of the inhibitor of apoptosis protein family, Survivin plays an important role in the process of resistance to apoptosis. In this study, we found that the activation of YAP promoted the expression of survivin in adriamycin-induced aging cells. Cells in aging state induced by low concentration of doxorubicin could initiate apoptosis after adding verteporfin, an inhibitor of YAP, or YM155, an inhibitor of survivin. In conclusion, the results showed that the aging cells induced by low concentration of adriamycin depended on the accumulation and activation of YAP in nucleus to promote the expression of survivin, so as to resist the apoptosis of the cells and maintain their survival state. Low concentration of doxorubicin can be combined with YAP or survivin inhibitors to remove aging cells, enhance the efficacy of chemotherapy and reduce side effects.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R730.2
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本文編號：2313976