【摘要】：急性髓細胞白血病(Acute Myeloid Leukemia,AML)是最常見的成人血液系統(tǒng)惡性腫瘤,其發(fā)病機制極為復雜,大多數(shù)AML的發(fā)生為自身遺傳因素與外界環(huán)境因素共同作用的結(jié)果,且具體發(fā)病過程涉及多個致病因素在多個步驟的相互作用。有關(guān)AML發(fā)病機制的學說眾多,“二次打擊”學說具有代表性,該學說認為AML的發(fā)生一般來說至少有二個階段:即各種原因所致的單個細胞內(nèi)基因的決定性突變,激活某種信號通路,導致了克隆性異常造血細胞生成和強勢增殖,凋亡受阻;進一步遺傳學改變再涉及到某些轉(zhuǎn)錄因子,導致克隆性增殖的異常造血細胞分化阻滯或紊亂,從而引起白血病。現(xiàn)代免疫學觀點認為,各種免疫細胞及其相關(guān)免疫分子表達和功能的動態(tài)平衡是機體維持各項免疫功能的重要保障。故大多數(shù)疾病的發(fā)生除了自身特殊的發(fā)病機理外,幾乎均涉及到機體免疫平衡紊亂而導致的功能缺陷。我們前期的研究提示免疫功能缺陷同樣是AML發(fā)生發(fā)展的重要原因。CD4+CD25+調(diào)節(jié)性T細胞(CD4+CD25+Regulatory T Cells,Tregs)是一群免疫負調(diào)控細胞,具有下調(diào)免疫應答的免疫抑制功能,可通過細胞-細胞接觸或分泌抑制性細胞因子來抑制免疫效應細胞的活性。在免疫調(diào)節(jié)、維持外周免疫耐受和防止自身免疫性疾病等方面發(fā)揮著重要的作用。多項研究表明,Tregs是誘導保護AML細胞逃逸機體抗腫瘤免疫應答的關(guān)鍵因素之一。IL-35是近年來新發(fā)現(xiàn)的一種細胞因子,由IL-12α亞基p35和IL-27β亞基EB病毒誘導的基因3(Epstein-Barr Virus Induced Gene 3,EBI3)以異二聚體的形式組成。研究發(fā)現(xiàn)小鼠的IL-35僅特異性、組成性地來源于Tregs,但在人體內(nèi),IL-35的來源和表達尚不完全明確。目前普遍認為IL-35是一種新的抑制性細胞因子,它可以顯著促進Tregs的增殖,抑制CD4+CD25-效應性T細胞(CD4+CD25-Effector T Cells,Teffs)增殖及其分泌的細胞因子,此外,它還可以誘導初始T細胞分化成一種被稱為“i Tr35”(IL-35 Induced Regulatory T Cells)的Tregs新亞群,從而級聯(lián)放大Tregs的免疫調(diào)節(jié)作用。鑒于IL-35在Tregs分化發(fā)育和功能活性中的關(guān)鍵作用,我們推測IL-35可以促進AML患者體內(nèi)Tregs積累和功能增強,最終導致機體內(nèi)Tregs和Teffs之間的平衡向抑制性偏倚,有利于AML細胞逃逸機體的抗腫瘤免疫應答。此外,衰老基因SENEX可能通過誘導應力性衰老(Stress Induced Premature Senescence,SIPS)的發(fā)生而保護Tregs,參與AML的發(fā)生發(fā)展。我們前期收集了AML患者的外周血標本,對IL-35在AML中的表達及誘導AML細胞免疫逃逸的作用和機制進行了初步的研究,結(jié)果顯示,AML患者體內(nèi)IL-35的表達升高且與疾病的發(fā)生發(fā)展密切相關(guān),IL-35可以促進Tregs和i Tr35的表達和功能、抑制Teffs的功能,從而介導AML細胞的免疫逃逸。本課題進一步研究抑制性細胞因子IL-35在AML患者骨髓中的表達及其對AML細胞的直接作用,結(jié)合前期相關(guān)研究及本次研究結(jié)果探討IL-35對AML免疫功能的影響及其機制。本研究共選取20例成人初診AML患者作為研究對象,并進行為期2年的追蹤隨訪,其中17人在治療后獲得了完全緩解,5人其后又復發(fā),同時按照年齡、性別相匹配的原則,選取了7例缺鐵性貧血患者作為對照組,然后對IL-35在AML患者骨髓中的表達及其對AML細胞的作用進行了分析。具體實驗方法和實驗結(jié)果如下:(1)實驗采用多種方法(PCR、ELISA、FCM、IHC)檢測AML初診組和對照組骨髓標本中IL-35的表達情況,結(jié)果表明,與對照組相比,初診AML患者骨髓中IL-35的表達顯著升高,提示,IL-35與AML的發(fā)生有關(guān)。(2)實驗采用ELISA的方法檢測AML進展的不同階段IL-35蛋白的表達,結(jié)果顯示,在初診AML患者骨髓中IL-35蛋白的表達顯著增高,在獲得完全緩解后其表達降低,而復發(fā)時又再度升高。提示,IL-35與AML的發(fā)展密切相關(guān)。(3)實驗采用FCM的方法檢測AML進展的不同階段Tregs的表達情況,結(jié)果表明,初診AML患者骨髓中Tregs的比例升高,獲得完全緩解后其比例降低,而復發(fā)時又再度升高,且Person相關(guān)性分析表明,初診AML患者骨髓中IL-35的表達水平與其各自的Tregs和叉頭樣轉(zhuǎn)錄因子p3(Forkhead Box p3,Foxp3)表達水平顯著正相關(guān),初步提示,在AML中Tregs可能是IL-35的來源之一,或者其他來源的IL-35可以顯著促進AML患者體內(nèi)Tregs的表達。(4)實驗對AML細胞株進行培養(yǎng)和干預,通過對IL-35刺激的AML細胞株增殖和凋亡情況進行分析發(fā)現(xiàn),IL-35顯著上調(diào)AML細胞株中IL-35R的表達,同時與PBS對照組相比IL-35還明顯促進AML細胞株的增殖,進一步研究發(fā)現(xiàn),IL-35預刺激的AML細胞株能顯著抵抗阿糖胞苷(Ara-C)誘導的凋亡。(5)實驗對FCM分選的患者骨髓中AML原始細胞進行培養(yǎng)和干預,通過對IL-35刺激的AML細胞增殖情況進行分析,同樣發(fā)現(xiàn)IL-35顯著上調(diào)AML細胞中IL-35R的表達,同時顯著促進AML細胞的增殖,而凋亡實驗也發(fā)現(xiàn)IL-35預刺激的AML細胞能顯著抵抗阿糖胞苷(Ara-C)誘導的凋亡。提示,IL-35可以通過與其受體結(jié)合作用于AML細胞,并顯著促進AML細胞的增殖、抑制AML細胞的凋亡。(6)實驗采用Real-time PCR的方法檢測AML進展不同階段SENEX基因的表達情況,結(jié)果顯示,在初診AML患者骨髓中SENEX基因的表達顯著增高,獲得完全緩解后其表達降低,復發(fā)時又再度升高,提示,SENEX基因與AML的發(fā)生發(fā)展密切相關(guān)。綜上,AML是一種發(fā)病機制與免疫因素密切相關(guān)的常見血液腫瘤,一般認為抑制性細胞及細胞因子在AML患者體內(nèi)的積聚,使得免疫系統(tǒng)的平衡被打破免疫功能受到抑制,從而有利于AML細胞逃逸機體的抗腫瘤免疫應答,最終導致AML的發(fā)生發(fā)展。本研究發(fā)現(xiàn),抑制性細胞因子IL-35顯著高表達于AML,且可通過促進Tregs、抑制Teffs的表達和功能以及直接促進AML細胞增殖和抑制AML細胞的凋亡來參與AML的發(fā)生發(fā)展。同時,衰老基因SENEX誘導的SIPS具有抗凋亡和免疫抑制效應,故推測衰老基因SENEX是促進AML免疫逃逸的機制之一。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.71

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