發(fā)布時(shí)間：2018-09-15 07:09

【摘要】：EB病毒(Epstein-Barr virus,EBV)既是一種特異性嗜淋巴細(xì)胞性DNA病毒,同時(shí)也是一種腫瘤源性病毒,并在人群中廣泛傳播。EBV的致病普極為廣,涵蓋惡性腫瘤、良性增殖以及炎癥反應(yīng)性疾病。其中,慢性活動(dòng)性EB病毒感染(chronic active Epstein-Barr virus infection,CAEBV)是一種新確定的EBV感染性疾病,以其生存率低、惡性轉(zhuǎn)化率高為轉(zhuǎn)歸特征。目前針對(duì)該病的有效治療手段仍無金標(biāo)準(zhǔn)指南。大量臨床研究提示CAEBV患者并沒有免疫缺陷病的基礎(chǔ),而患者體內(nèi)EBV表現(xiàn)為II/III型潛伏感染(高表達(dá)LMP-1),高表達(dá)TNF-α、IL-1α和IL-1β。同時(shí),由于該病是由EBV引起的淋巴細(xì)胞增殖,這些都提示我們?cè)摬】赡艽嬖诤艘蜃?κB(nuclear factor kappa B,NF-κB)組成性活化。如果的確如此,那么這將成為理想的治療靶標(biāo)。為此,本研究通過對(duì)高通量芯片數(shù)據(jù)分析、蛋白印跡和免疫熒光發(fā)現(xiàn)T細(xì)胞亞型CAEBV細(xì)胞的確存在NF-κB激活。同時(shí)利用新型NF-κB抑制劑DHMEQ進(jìn)行靶向研究,發(fā)現(xiàn)NF-κB抑制能夠特異性殺傷T細(xì)胞亞型CAEBV細(xì)胞,而對(duì)正常造血細(xì)胞沒有毒性作用。機(jī)制研究提示DHMEQ通過整合NF-κB抑制、ROS和p53激活產(chǎn)生其靶向作用。本研究拓展了我們對(duì)CAEBV發(fā)病機(jī)制的認(rèn)識(shí),同時(shí)也為靶向治療做出探索性的嘗試。EB病毒(Epstein-Barr virus,EBV)既是一種特異性嗜淋巴細(xì)胞性DNA病毒,同時(shí)也是一種腫瘤源性病毒,并在人群中廣泛傳播。EBV的致病普極為廣,涵蓋惡性腫瘤、良性增殖以及炎癥反應(yīng)性疾病。其中,慢性活動(dòng)性EB病毒感染(chronic active Epstein-Barr virus infection,CAEBV)是一種新確定的EBV感染性疾病,以其生存率低、惡性轉(zhuǎn)化率高為轉(zhuǎn)歸特征。目前針對(duì)改變的有效治療手段仍無金標(biāo)準(zhǔn)指南。Connectivity Map(C-MAP)是由Todd Golub與Eric Lander所領(lǐng)導(dǎo)的精英,利用小分子化合物處理人類細(xì)胞后的基因表達(dá)差異,建立的一個(gè)關(guān)聯(lián)小分子化合物、基因表達(dá)譜以及疾病的生物應(yīng)用數(shù)據(jù)庫。該數(shù)據(jù)庫已經(jīng)被成功應(yīng)用到靶向白血病干細(xì)胞、結(jié)腸癌、前列腺癌等藥物篩選中。為此,我們利用C-MAP數(shù)據(jù)庫進(jìn)行CAEBV的靶向化合物預(yù)測,以便讓我們從人類基因組學(xué)的角度出發(fā)來探討如何逆轉(zhuǎn)CAEBV的治療預(yù)后。通過預(yù)測,我們發(fā)現(xiàn)蛋白去乙�；敢种苿┣啪谹(TSA)是一極具潛質(zhì)的化合物,能聯(lián)合增強(qiáng)特異性針對(duì)EB病毒的抗病毒藥物更昔洛韋(GCV)殺傷CAEBV的作用。白血病干細(xì)胞(Leukemia stem cells,LSCs)在白血病發(fā)生、發(fā)展、復(fù)發(fā)中起了重要的作用,因而是白血病治療潛在的重要靶點(diǎn)。然而,目前針對(duì)LSCs的治療方案少有報(bào)道,使得急性髓系白血病(acute myelogenous leukemia,AML)的治療停滯不前。在本文中,我們通過一系列體外實(shí)驗(yàn)和Nod/scid小鼠移植實(shí)驗(yàn)的結(jié)果,證明fenretinide,這一可低毒性、易被人體耐受的維生素A衍生物,在臨床常規(guī)劑量使用的情況能夠在殺傷LSCs的同時(shí)不影響正常的造血干祖細(xì)胞。Fenretinide對(duì)于原代AML CD34+細(xì)胞,特別是富集LSCs的CD34+CD38-細(xì)胞亞群具有選擇性的細(xì)胞殺傷作用,而對(duì)于正常的該群細(xì)胞則沒有觀察到顯著效應(yīng)。甲基纖維素集落形成實(shí)驗(yàn)進(jìn)一步顯示,fenretinide顯著抑制了AML CD34+細(xì)胞的集落形成能力,但對(duì)正常CD34+細(xì)胞的集落形成能力沒有明顯影響。更讓人感興趣的是,fenretinide能夠顯著影響AML白血病干細(xì)胞在Nod/scid小鼠中白血病的重建能力,但是正常造血?jiǎng)t沒有產(chǎn)生影響。深入機(jī)制研究表明:fenretinide誘導(dǎo)的AML白血病干細(xì)胞凋亡與活性氧迅速增加、應(yīng)激反應(yīng)和凋亡相關(guān)基因的激活、NF-κB和Wnt通路相關(guān)基因的抑制相關(guān)。通過基因集富集分析(Geneset Enrichment Analysis,GSEA)進(jìn)一步對(duì)fenretinide可能與臨床預(yù)后的關(guān)聯(lián)性進(jìn)行生物信息學(xué),結(jié)果顯示:被fenretinide下調(diào)的基因與AML患者的不良預(yù)后高度相關(guān)�；谶@些發(fā)現(xiàn),我們推斷fenretinide是一個(gè)極具選擇性清除LSCs潛質(zhì)的臨床候選靶向藥物。
[Abstract]:Epstein-Barr virus (EBV) is not only a specific lymphocytic DNA virus, but also a tumor-borne virus, which is widely spread in the population. EBV is a widespread disease, covering malignant tumors, benign proliferation and inflammatory diseases. Among them, chronic active Epstein-Barr virus (EBV) infection CAEBV is a newly identified EBV infectious disease characterized by low survival rate and high malignant transformation rate. There is no gold standard guide for effective treatment of the disease. LMP-1, high expression of TNF-alpha, IL-1alpha and IL-1beta, as well as lymphocyte proliferation induced by EBV, suggest that there may be a constitutive activation of nuclear factor kappa B (NF-kappa B). If so, this would be an ideal therapeutic target. Western blot and immunofluorescence assay showed that NF-kappa B activation did exist in T cell subtype CAEBV cells. At the same time, a novel inhibitor of NF-kappa B, DHMEQ, was used for targeted study. It was found that NF-kappa B inhibition could specifically kill T cell subtype CAEBV cells, but had no toxic effect on normal hematopoietic cells. This study broadened our understanding of the pathogenesis of CAEBV and also made exploratory attempts to target therapy. Epstein-Barr virus (EBV) is not only a specific lymphotropic DNA virus, but also a tumor-derived virus, which is widely spread in the population. Chronic active Epstein-Barr virus infection (CAEBV) is a newly identified EBV infectious disease characterized by low survival rate and high malignant transformation rate. Connectivity Map (C-MAP) is an elite led by Todd Golub and Eric Lander. It uses small molecule compounds to treat gene expression differences in human cells. It has been successfully applied to a database of small molecule compounds, gene expression profiles, and biological applications for diseases. For this reason, we use the C-MAP database to predict the target compounds of CAEBV, so that we can explore how to reverse the prognosis of CAEBV from the perspective of human genomics. Potential compounds can be combined to enhance the antiviral effect of ganciclovir (GCV), a specific antiviral drug against EBV, on CAEBV. Leukemia stem cells (LSCs) play an important role in the occurrence, development and recurrence of leukemia, and are therefore potentially important targets for the treatment of leukemia. Fenretinide, a low toxic and easily tolerated derivative of vitamin A, has been shown to be effective in the treatment of acute myelogenous leukemia (AML) by a series of in vitro experiments and Nod/scid mice transplantation experiments. Fenretinide has selective cytotoxicity to primary AML CD34 + cells, especially to the CD34 + CD38 - cell subsets enriched with LSCs, but no significant effect on normal cells. The methylcellulose colony formation assay further showed that fenretinide had selective cytotoxicity to primary AML CD34 + cells, especially to the CD34 + CD38 - cell subsets enriched with LSCs. It is interesting to note that fenretinide can significantly inhibit the ability of AML CD34 + cells to reconstitute leukemia in Nod/scid mice, but has no effect on normal hematopoiesis. These results indicate that the apoptosis of AML stem cells induced by fenretinide is related to the rapid increase of reactive oxygen species (ROS), the activation of stress response and apoptosis-related genes, and the inhibition of NF-kappa B and Wnt pathway-related genes. Informatics results show that the genes down-regulated by fenretinide are highly correlated with the poor prognosis of AML patients. Based on these findings, we conclude that fenretinide is a highly selective clinical candidate for LSCs.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.1

【共引文獻(xiàn)】

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本文編號(hào)：2244136