抑制性细胞因子IL-35对急性髓细胞白血病免疫功能的影响及其机制研究

发布时间：2018-07-25 07:34

【摘要】：急性髓细胞白血病(Acute Myeloid Leukemia,AML)是最常见的成人血液系统恶性肿瘤,其发病机制极为复杂,大多数AML的发生为自身遗传因素与外界环境因素共同作用的结果,且具体发病过程涉及多个致病因素在多个步骤的相互作用。有关AML发病机制的学说众多,“二次打击”学说具有代表性,该学说认为AML的发生一般来说至少有二个阶段:即各种原因所致的单个细胞内基因的决定性突变,激活某种信号通路,导致了克隆性异常造血细胞生成和强势增殖,凋亡受阻;进一步遗传学改变再涉及到某些转录因子,导致克隆性增殖的异常造血细胞分化阻滞或紊乱,从而引起白血病。现代免疫学观点认为,各种免疫细胞及其相关免疫分子表达和功能的动态平衡是机体维持各项免疫功能的重要保障。故大多数疾病的发生除了自身特殊的发病机理外,几乎均涉及到机体免疫平衡紊乱而导致的功能缺陷。我们前期的研究提示免疫功能缺陷同样是AML发生发展的重要原因。CD4+CD25+调节性T细胞(CD4+CD25+Regulatory T Cells,Tregs)是一群免疫负调控细胞,具有下调免疫应答的免疫抑制功能,可通过细胞-细胞接触或分泌抑制性细胞因子来抑制免疫效应细胞的活性。在免疫调节、维持外周免疫耐受和防止自身免疫性疾病等方面发挥着重要的作用。多项研究表明,Tregs是诱导保护AML细胞逃逸机体抗肿瘤免疫应答的关键因素之一。IL-35是近年来新发现的一种细胞因子,由IL-12α亚基p35和IL-27β亚基EB病毒诱导的基因3(Epstein-Barr Virus Induced Gene 3,EBI3)以异二聚体的形式组成。研究发现小鼠的IL-35仅特异性、组成性地来源于Tregs,但在人体内,IL-35的来源和表达尚不完全明确。目前普遍认为IL-35是一种新的抑制性细胞因子,它可以显著促进Tregs的增殖,抑制CD4+CD25-效应性T细胞(CD4+CD25-Effector T Cells,Teffs)增殖及其分泌的细胞因子,此外,它还可以诱导初始T细胞分化成一种被称为“i Tr35”(IL-35 Induced Regulatory T Cells)的Tregs新亚群,从而级联放大Tregs的免疫调节作用。鉴于IL-35在Tregs分化发育和功能活性中的关键作用,我们推测IL-35可以促进AML患者体内Tregs积累和功能增强,最终导致机体内Tregs和Teffs之间的平衡向抑制性偏倚,有利于AML细胞逃逸机体的抗肿瘤免疫应答。此外,衰老基因SENEX可能通过诱导应力性衰老(Stress Induced Premature Senescence,SIPS)的发生而保护Tregs,参与AML的发生发展。我们前期收集了AML患者的外周血标本,对IL-35在AML中的表达及诱导AML细胞免疫逃逸的作用和机制进行了初步的研究,结果显示,AML患者体内IL-35的表达升高且与疾病的发生发展密切相关,IL-35可以促进Tregs和i Tr35的表达和功能、抑制Teffs的功能,从而介导AML细胞的免疫逃逸。本课题进一步研究抑制性细胞因子IL-35在AML患者骨髓中的表达及其对AML细胞的直接作用,结合前期相关研究及本次研究结果探讨IL-35对AML免疫功能的影响及其机制。本研究共选取20例成人初诊AML患者作为研究对象,并进行为期2年的追踪随访,其中17人在治疗后获得了完全缓解,5人其后又复发,同时按照年龄、性别相匹配的原则,选取了7例缺铁性贫血患者作为对照组,然后对IL-35在AML患者骨髓中的表达及其对AML细胞的作用进行了分析。具体实验方法和实验结果如下:(1)实验采用多种方法(PCR、ELISA、FCM、IHC)检测AML初诊组和对照组骨髓标本中IL-35的表达情况,结果表明,与对照组相比,初诊AML患者骨髓中IL-35的表达显著升高,提示,IL-35与AML的发生有关。(2)实验采用ELISA的方法检测AML进展的不同阶段IL-35蛋白的表达,结果显示,在初诊AML患者骨髓中IL-35蛋白的表达显著增高,在获得完全缓解后其表达降低,而复发时又再度升高。提示,IL-35与AML的发展密切相关。(3)实验采用FCM的方法检测AML进展的不同阶段Tregs的表达情况,结果表明,初诊AML患者骨髓中Tregs的比例升高,获得完全缓解后其比例降低,而复发时又再度升高,且Person相关性分析表明,初诊AML患者骨髓中IL-35的表达水平与其各自的Tregs和叉头样转录因子p3(Forkhead Box p3,Foxp3)表达水平显著正相关,初步提示,在AML中Tregs可能是IL-35的来源之一,或者其他来源的IL-35可以显著促进AML患者体内Tregs的表达。(4)实验对AML细胞株进行培养和干预,通过对IL-35刺激的AML细胞株增殖和凋亡情况进行分析发现,IL-35显著上调AML细胞株中IL-35R的表达,同时与PBS对照组相比IL-35还明显促进AML细胞株的增殖,进一步研究发现,IL-35预刺激的AML细胞株能显著抵抗阿糖胞苷(Ara-C)诱导的凋亡。(5)实验对FCM分选的患者骨髓中AML原始细胞进行培养和干预,通过对IL-35刺激的AML细胞增殖情况进行分析,同样发现IL-35显著上调AML细胞中IL-35R的表达,同时显著促进AML细胞的增殖,而凋亡实验也发现IL-35预刺激的AML细胞能显著抵抗阿糖胞苷(Ara-C)诱导的凋亡。提示,IL-35可以通过与其受体结合作用于AML细胞,并显著促进AML细胞的增殖、抑制AML细胞的凋亡。(6)实验采用Real-time PCR的方法检测AML进展不同阶段SENEX基因的表达情况,结果显示,在初诊AML患者骨髓中SENEX基因的表达显著增高,获得完全缓解后其表达降低,复发时又再度升高,提示,SENEX基因与AML的发生发展密切相关。综上,AML是一种发病机制与免疫因素密切相关的常见血液肿瘤,一般认为抑制性细胞及细胞因子在AML患者体内的积聚,使得免疫系统的平衡被打破免疫功能受到抑制,从而有利于AML细胞逃逸机体的抗肿瘤免疫应答,最终导致AML的发生发展。本研究发现,抑制性细胞因子IL-35显著高表达于AML,且可通过促进Tregs、抑制Teffs的表达和功能以及直接促进AML细胞增殖和抑制AML细胞的凋亡来参与AML的发生发展。同时,衰老基因SENEX诱导的SIPS具有抗凋亡和免疫抑制效应,故推测衰老基因SENEX是促进AML免疫逃逸的机制之一。
[Abstract]:Acute Myeloid Leukemia (AML) is the most common malignant tumor of the adult blood system. Its pathogenesis is very complex. Most of the occurrence of AML is the result of the interaction of its own genetic factors and the external environmental factors, and the specific pathogenesis involves the interaction of multiple pathogenic factors in multiple steps. The related AML hair is related to the pathogenesis of AML. The theory of the disease mechanism is numerous, and the theory of "two times" is representative. The theory holds that the occurrence of AML is generally at least two stages: the decisive mutation of the gene in a single cell caused by various reasons, activating a certain signal pathway, resulting in the formation of cloned abnormal hematopoietic cells and strong proliferation, and the inhibition of apoptosis; further remains. In modern immunological view, the dynamic balance between the expression and function of various immune cells and their related immune molecules is an important guarantee for the body to maintain the various immune functions. In addition to its own special pathogenesis, it almost all involves the functional defects caused by the body's immune balance disorder. Our previous study suggests that immune deficiency is also an important cause of the development of AML (CD4+CD25+Regulatory T Cells, Tregs), a group of immune negative regulatory cells, with down regulation. The immunosuppressive function of the immune response can inhibit the activity of immune effector cells by cell cell contact or secretion of inhibitory cytokines. It plays an important role in immune regulation, maintaining peripheral immune tolerance and preventing autoimmune diseases. A number of studies have shown that Tregs is the induction of protection against swelling in AML cells. One of the key factors of the tumor immune response.IL-35 is a newly discovered cytokine in recent years. The gene 3 (Epstein-Barr Virus Induced Gene 3, EBI3), induced by the IL-12 alpha subunit p35 and IL-27 beta subunit EB virus, is composed of a hetero two polymer. The origin and expression of -35 are still not completely clear. It is widely believed that IL-35 is a new inhibitory cytokine that can significantly promote the proliferation of Tregs, inhibit the proliferation and secretion of CD4+CD25- effector T cells (CD4+CD25-Effector T Cells, Teffs), and also induce the differentiation of initial T cells into one kind known as "" The Tregs new subgroup of I Tr35 "(IL-35 Induced Regulatory T Cells) amplifies the immune regulation of Tregs in cascade. In view of the key role of IL-35 in Tregs differentiation and functional activity, we speculate that IL-35 can promote accumulation and function enhancement in the patient's body and ultimately lead to a balance between the body and the body. In addition, the aging gene SENEX may protect Tregs and participate in the occurrence and development of AML by inducing the occurrence of Stress Induced Premature Senescence (SIPS), and we have collected the peripheral blood specimens of AML patients, and the expression and induction of IL-35 in AML, by inducing the occurrence of Stress Induced Premature Senescence (SIPS). The effect and mechanism of AML cell immune escape were preliminarily studied. The results showed that the expression of IL-35 in AML patients was elevated and closely related to the development of the disease. IL-35 could promote the expression and function of Tregs and I Tr35, inhibit the function of Teffs and mediate the immune escape of AML fine cells. This subject further studies the inhibitory fine. The expression of cytokine IL-35 in the bone marrow of patients with AML and its direct effect on AML cells, combined with the previous study and the results of this study to explore the effect and mechanism of IL-35 on the immune function of AML. A total of 20 cases of adult first diagnosed AML patients were selected and followed up for a period of 2 years, of which 17 of them were obtained after treatment. With complete remission, 5 people then relapsed, and 7 cases of iron deficiency anemia were selected as the control group according to the age and sex matching principle. Then the expression of IL-35 in the bone marrow of AML patients and the effect on AML cells were analyzed. The specific experimental methods and the results were as follows: (1) the experiment adopted a variety of methods (PCR, ELISA, F). CM, IHC) detected the expression of IL-35 in the bone marrow specimens of the AML first diagnosis group and the control group. The results showed that the expression of IL-35 in the bone marrow of the first diagnosed AML patients was significantly higher than that of the control group, suggesting that IL-35 was related to the occurrence of AML. (2) the experiment used ELISA method to detect the expression of IL-35 protein in the different stages of AML progress. The results showed that the first diagnosis of AML patients was in the first diagnosis. The expression of IL-35 protein in the bone marrow was significantly increased, and the expression decreased after complete remission, while the recurrence increased again. It suggested that the development of IL-35 was closely related to the development of AML. (3) the expression of Tregs in different stages of AML was detected by FCM. The results showed that the proportion of Tregs in the bone marrow of first diagnosed AML patients was increased, and the results were finished. After full remission, the ratio of IL-35 was increased again, and the Person correlation analysis showed that the expression level of IL-35 in the bone marrow of first diagnosed AML patients was significantly correlated with their respective Tregs and P3 (Forkhead Box P3, Foxp3) expression level. It was suggested that Tregs may be one of the sources of IL-35, or other sources in AML. The source of IL-35 can significantly promote the expression of Tregs in AML patients. (4) the experiment on AML cell line culture and intervention, through the analysis of the proliferation and apoptosis of IL-35 stimulated AML cell lines, IL-35 significantly up-regulated the expression of IL-35R in AML cell lines, while IL-35 also significantly promoted the proliferation of AML cell lines compared with the PBS control group. Further studies have found that the IL-35 pre stimulated AML cell line can significantly resist the apoptosis induced by cytarabine (Ara-C). (5) the experiment on the AML primordial cells in the bone marrow of the FCM separation patients was cultured and intervened, and the proliferation of AML cells stimulated by IL-35 was analyzed. The same discovery that IL-35 significantly up-regulated the expression of IL-35R in AML cells. The proliferation of AML cells was significantly promoted, and the apoptosis experiment also found that IL-35 prestimulated AML cells could significantly resist the apoptosis induced by cytarabine (Ara-C). It is suggested that IL-35 can combine with its receptor in AML cells, promote the proliferation of AML cells and inhibit the apoptosis of AML cells. (6) the experiment used Real-time PCR method to detect AML. The expression of SENEX gene in different stages showed that the expression of SENEX gene in the bone marrow of first diagnosed AML patients was significantly higher, and the expression decreased after complete remission, and the recurrence was increased again. It suggested that the SENEX gene was closely related to the development of AML. To sum up, AML is a common pathogenesis closely related to the immune factors. The accumulation of inhibitory cells and cytokines in AML patients is generally believed to make the balance of the immune system be suppressed by the break of the immune function, which is beneficial to the anti tumor immune response of the AML cells to escape the body and ultimately lead to the development of AML. This study found that the inhibitory cytokine IL-35 is highly expressed in AML. And it can participate in the development of AML by promoting Tregs, inhibiting the expression and function of Teffs and promoting the proliferation of AML cells and inhibiting the apoptosis of AML cells. At the same time, the senescence gene SENEX induced SIPS has the anti apoptosis and immunosuppressive effect. Therefore, the senescence gene SENEX is one of the mechanisms to promote the escape of the AML immune system.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.71

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