靶向慢性活動性EB病毒感染細胞治療的探索性研究
發(fā)布時間：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胞性DNA病毒,同時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性疾病。其中,慢性活動性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。目前針對該病的有效治療手段仍無金標準指南。大量臨床研究提示CAEBV患者并沒有免疫缺陷病的基礎,而患者體內(nèi)EBV表現(xiàn)為II/III型潛伏感染(高表達LMP-1),高表達TNF-α、IL-1α和IL-1β。同時,由于該病是由EBV引起的淋巴細胞增殖,這些都提示我們該病可能存在核因子-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。為此,本研究通過對高通量芯片數(shù)據(jù)分析、蛋白印跡和免疫熒光發(fā)現(xiàn)T細胞亞型CAEBV細胞的確存在NF-κB激活。同時利用新型NF-κB抑制劑DHMEQ進行靶向研究,發(fā)現(xiàn)NF-κB抑制能夠特異性殺傷T細胞亞型CAEBV細胞,而對正常造血細胞沒有毒性作用。機制研究提示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CAEBV發(fā)病機制的認識,同時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胞性DNA病毒,同時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性疾病。其中,慢性活動性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。目前針對改變的有效治療手段仍無金標準指南。Connectivity Map(C-MAP)是由Todd Golub與Eric Lander所領導的精英,利用小分子化合物處理人類細胞后的基因表達差異,建立的一個關聯(lián)小分子化合物、基因表達譜以及疾病的生物應用數(shù)據(jù)庫。該數(shù)據(jù)庫已經(jīng)被成功應用到靶向白血病干細胞、結腸癌、前列腺癌等藥物篩選中。為此,我們利用C-MAP數(shù)據(jù)庫進行CAEBV的靶向化合物預測,以便讓我們從人類基因組學的角度出發(fā)來探討如何逆轉(zhuǎn)CAEBV的治療預后。通過預測,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)合增強特異性針對EB病毒的抗病毒藥物更昔洛韋(GCV)殺傷CAEBV的作用。白血病干細胞(Leukemia stem cells,LSCs)在白血病發(fā)生、發(fā)展、復發(fā)中起了重要的作用,因而是白血病治療潛在的重要靶點。然而,目前針對LSCs的治療方案少有報道,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驗和Nod/scid小鼠移植實驗的結果,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的同時不影響正常的造血干祖細胞。Fenretinide對于原代AML CD34+細胞,特別是富集LSCs的CD34+CD38-細胞亞群具有選擇性的細胞殺傷作用,而對于正常的該群細胞則沒有觀察到顯著效應。甲基纖維素集落形成實驗進一步顯示,fenretinide顯著抑制了AML CD34+細胞的集落形成能力,但對正常CD34+細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白血病干細胞在Nod/scid小鼠中白血病的重建能力,但是正常造血則沒有產(chǎn)生影響。深入機制研究表明:fenretinide誘導的AML白血病干細胞凋亡與活性氧迅速增加、應激反應和凋亡相關基因的激活、NF-κB和Wnt通路相關基因的抑制相關。通過基因集富集分析(Geneset Enrichment Analysis,GSEA)進一步對fenretinide可能與臨床預后的關聯(lián)性進行生物信息學,結果顯示:被fenretinide下調(diào)的基因與AML患者的不良預后高度相關�；谶@些發(fā)現(xiàn),我們推斷fenretinide是一個極具選擇性清除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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R725.1
本文編號：2244136
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R725.1
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