靶向慢性活动性EB病毒感染细胞治疗的探索性研究

发布时间：2018-09-15 07:09

【摘要】：EB病毒(Epstein-Barr virus,EBV)既是一种特异性嗜淋巴细胞性DNA病毒,同时也是一种肿瘤源性病毒,并在人群中广泛传播。EBV的致病普极为广,涵盖恶性肿瘤、良性增殖以及炎症反应性疾病。其中,慢性活动性EB病毒感染(chronic active Epstein-Barr virus infection,CAEBV)是一种新确定的EBV感染性疾病,以其生存率低、恶性转化率高为转归特征。目前针对该病的有效治疗手段仍无金标准指南。大量临床研究提示CAEBV患者并没有免疫缺陷病的基础,而患者体内EBV表现为II/III型潜伏感染(高表达LMP-1),高表达TNF-α、IL-1α和IL-1β。同时,由于该病是由EBV引起的淋巴细胞增殖,这些都提示我们该病可能存在核因子-κB(nuclear factor kappa B,NF-κB)组成性活化。如果的确如此,那么这将成为理想的治疗靶标。为此,本研究通过对高通量芯片数据分析、蛋白印迹和免疫荧光发现T细胞亚型CAEBV细胞的确存在NF-κB激活。同时利用新型NF-κB抑制剂DHMEQ进行靶向研究,发现NF-κB抑制能够特异性杀伤T细胞亚型CAEBV细胞,而对正常造血细胞没有毒性作用。机制研究提示DHMEQ通过整合NF-κB抑制、ROS和p53激活产生其靶向作用。本研究拓展了我们对CAEBV发病机制的认识,同时也为靶向治疗做出探索性的尝试。EB病毒(Epstein-Barr virus,EBV)既是一种特异性嗜淋巴细胞性DNA病毒,同时也是一种肿瘤源性病毒,并在人群中广泛传播。EBV的致病普极为广,涵盖恶性肿瘤、良性增殖以及炎症反应性疾病。其中,慢性活动性EB病毒感染(chronic active Epstein-Barr virus infection,CAEBV)是一种新确定的EBV感染性疾病,以其生存率低、恶性转化率高为转归特征。目前针对改变的有效治疗手段仍无金标准指南。Connectivity Map(C-MAP)是由Todd Golub与Eric Lander所领导的精英,利用小分子化合物处理人类细胞后的基因表达差异,建立的一个关联小分子化合物、基因表达谱以及疾病的生物应用数据库。该数据库已经被成功应用到靶向白血病干细胞、结肠癌、前列腺癌等药物筛选中。为此,我们利用C-MAP数据库进行CAEBV的靶向化合物预测,以便让我们从人类基因组学的角度出发来探讨如何逆转CAEBV的治疗预后。通过预测,我们发现蛋白去乙酰化酶抑制剂曲古菌素A(TSA)是一极具潜质的化合物,能联合增强特异性针对EB病毒的抗病毒药物更昔洛韦(GCV)杀伤CAEBV的作用。白血病干细胞(Leukemia stem cells,LSCs)在白血病发生、发展、复发中起了重要的作用,因而是白血病治疗潜在的重要靶点。然而,目前针对LSCs的治疗方案少有报道,使得急性髓系白血病(acute myelogenous leukemia,AML)的治疗停滞不前。在本文中,我们通过一系列体外实验和Nod/scid小鼠移植实验的结果,证明fenretinide,这一可低毒性、易被人体耐受的维生素A衍生物,在临床常规剂量使用的情况能够在杀伤LSCs的同时不影响正常的造血干祖细胞。Fenretinide对于原代AML CD34+细胞,特别是富集LSCs的CD34+CD38-细胞亚群具有选择性的细胞杀伤作用,而对于正常的该群细胞则没有观察到显著效应。甲基纤维素集落形成实验进一步显示,fenretinide显著抑制了AML CD34+细胞的集落形成能力,但对正常CD34+细胞的集落形成能力没有明显影响。更让人感兴趣的是,fenretinide能够显著影响AML白血病干细胞在Nod/scid小鼠中白血病的重建能力,但是正常造血则没有产生影响。深入机制研究表明:fenretinide诱导的AML白血病干细胞凋亡与活性氧迅速增加、应激反应和凋亡相关基因的激活、NF-κB和Wnt通路相关基因的抑制相关。通过基因集富集分析(Geneset Enrichment Analysis,GSEA)进一步对fenretinide可能与临床预后的关联性进行生物信息学,结果显示:被fenretinide下调的基因与AML患者的不良预后高度相关。基于这些发现,我们推断fenretinide是一个极具选择性清除LSCs潜质的临床候选靶向药物。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.1

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