FOXP3甲基化介导的Treg细胞功能受损在急性冠脉综合征发生中的作用

发布时间：2018-06-01 04:21

本文选题：急性冠脉综合征 + 调节性T细胞　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分急性冠脉综合征患者调节性T细胞免疫抑制功能的研究目的：动脉粥样硬化是一种免疫失调的慢性炎症过程,是导致急性冠脉综合征(acute coronary syndrome, ACS)的主要原因。特异性表达叉头状转录因子(Forkhead transcription factor P3, FOXP3)的胸腺生成的CD4+CD25+调节性T细胞(CD4+CD25+regulatory T cells, Tregs)是CD4+T细胞的一个亚群,在限制机体对自身抗原或外来抗原过度的免疫反应中发挥必不可少的免疫调节作用。Tregs的数量减少或免疫抑制功能受损均可诱发自身免疫性疾病或者使炎症性疾病加重,因此,本实验旨在探讨Tregs的免疫调节功能在急性冠脉综合征发生中的作用。方法：收集62例ACS患者及52例健康对照者的外周血标本,采用密度梯度法分离出淋巴细胞后,用磁珠分选出CD4+CD25+调节性T细胞及CD4+CD25效应性T细胞。将Tregs与羧基荧光素二醋酸盐琥珀酰亚胺酯(Carboxyfluorescein succinimidyl este,CFSE)标记的自体及异体的Teffs以不同的比例混合培养,用流式细胞仪检测Teffs的增殖指数,得出ACS患者及对照者Tregs的抑制率,比较ACS组与对照组Tregs抑制功能的差异。而且,将ACS患者与对照者CFSE标记的Teffs分别培养3天,用流式细胞仪检测ACS组与对照组Teffs增殖功能的差异。结果:①ACS患者与对照者的Teffs增殖功能未见差异(.P0.05)；②ACS患者及对照者的Tregs分别与自身Teffs以不同的比例混合培养后,与对照组相比,ACS患者Tregs的免疫抑制功能降低(P0.05)；③同样地,ACS患者及对照者的Tregs分别与来自同一个体的Teffs以不同的比例混合培养后,与对照组相比,ACS患者Tregs的免疫抑制功能降低(P0.05)。结论：急性冠脉综合征患者[regs的免疫抑制功能降低可能是ACS发生的一种因素,干预Tregs可能会成为预防和治疗ACS的一种策略。第二部分急性冠脉综合征患者调节性T细胞FOXP3甲基化水平的研究目的：表观遗传学修饰在T细胞系分化中起着重要作用,FOXP3基因中存在一段保守序列即调节性T细胞特异性去甲基化区域,这段去甲基化的保守序列对于Tregs形成及保持Tregs免疫抑制功能极为重要。本研究旨在探索调节性T细胞FOXP3增强子甲基化水平在ACS发病中的作用。方法：入选60例ACS患者和56例对照者,采用密度梯度法从抗凝外周血中分离出淋巴细胞,应用流式细胞仪检测CD4+CD25+FOXP3+Treg s的表达水平。用磁珠从入选者外周血中分选出CD4+CD25+调节性T细胞及CD4+CD25效应性T细胞,用焦磷酸测序法检测分别检测Tregs与Teffs的FOXP3增强子甲基化水平。采用实时定量PCR检测入选者淋巴细胞中的FOXP3mRNA含量。用ELISA检测入选者血浆中IL-10,TGF-p和INF-γ含量。结果：①应用流式细胞术未发现ACS患者的CD4+CD25+FOXP3+Tregs的表达水平降低(P0.05)。②ACS患者与对照者Teffs的FOXP3增强子位点均呈高度甲基化水平,两组间Teffs的FOXP3增强子甲基化水平无差异(P0.05)；而ACS患者与对照者Tregs的FOXP3增强子位点均呈低甲基化状态,两组患者Tregs的FOXP3增强子甲基化水平均显著低于Teffs的FOXP3增强子甲基化水平(P0.05); ACS患者Tregs的FOXP3增强子位点甲基化水平显著高于对照组Tregs的FOXP3增强子甲基化水平(P0.05)。③ACS患者淋巴细胞中的FOXP3mRN A表达水平低于对照组(P0.05);与培养前比较,培养5天后,ACS患者及对照者淋巴细胞中的FOXP3mRNA表达水平均减低(P0.05),并且,ACS患者淋巴细胞中的FOXP3mRNA表达水减少比例显著高于对照者(P0.05)。④各组间血浆IL-10的含量未见显著差异(P0.05)；与对照组比较,ACS患者血浆中的TGF-β含量减少(P0.05),而INF-γ含量增加(P0.05)。结论：急性冠脉综合征患者调节性T细胞FOXP3增强子甲基化水平升高可能是造成Tregs免疫抑制功能受损的原因,进而Tregs免疫抑制功能受损参与ACS的发生,因此,降低FOXP3增强子甲基化水平可能成为治疗ACS的潜在策略。
[Abstract]:Part one study of regulatory T cell immunosuppression in patients with acute coronary syndrome
Objective: atherosclerosis is a chronic inflammatory process of immune disorders and is the main cause of acute coronary syndrome (ACS). CD4+CD25+ regulatory T cells specifically express the thymic gland of Forkhead transcription factor P3 (FOXP3). GS) is a subgroup of CD4+T cells. The decrease in the immune response of the body to the excessive immune response to the autoantigen or external antigen, the decrease in the number of.Tregs or the impairment of the immunosuppressive function can induce autoimmune diseases or aggravate the inflammatory disease. Therefore, the aim of this experiment is to explore the immunological modulation of Tregs. The role of joint function in the occurrence of acute coronary syndrome.
Methods: the peripheral blood samples from 62 ACS patients and 52 healthy controls were collected and the lymphocytes were separated by density gradient method. The CD4+CD25+ regulatory T cells and CD4+CD25 effector T cells were selected by magnetic beads. Tregs and carboxyl fluorescein two acetate succinimide (Carboxyfluorescein succinimidyl Este, CFSE) were labeled from Tregs. The body and allogenic Teffs were mixed in different proportions, and the proliferation index of Teffs was detected by flow cytometry. The inhibition rate of Tregs in ACS patients and controls was obtained. The difference of Tregs inhibitory function between the ACS group and the control group was compared. Moreover, the Teffs of the CFSE markers in the ACS patients and the control group was cultured for 3 days respectively, and the ACS group and the control were detected by flow cytometry. The difference in the proliferative function of group Teffs.
Results: (1) there was no difference in the proliferation of Teffs between the ACS patients and the control group (.P0.05); after the Tregs of the ACS patients and the controls was mixed with their own Teffs in a different proportion, the immunosuppressive function of Tregs in ACS patients was lower than that of the control group (P0.05); similarly, the Tregs of the ACS patients and the controls was from the same individual. After mixed culture of Teffs in different proportions, the immunosuppressive function of Tregs in ACS patients was lower than that in the control group (P0.05).
Conclusion: the decrease of immunosuppressive function of [regs in patients with acute coronary syndrome may be a factor in the occurrence of ACS, and intervention of Tregs may be a strategy for the prevention and treatment of ACS.
The second part is the FOXP3 methylation level of regulatory T cells in patients with acute coronary syndrome.
Objective: epigenetic modification plays an important role in the differentiation of T cell lines. There is a conservative sequence in the FOXP3 gene, the regulatory T cell specific demethylation region. The conservative sequence of this demethylation is very important for the formation of Tregs and the preservation of Tregs immunity. The aim of this study is to explore the FOXP3 enhancement of regulatory T cells. The role of the level of methylation in the pathogenesis of ACS.
Methods: 60 ACS patients and 56 controls were selected to separate the lymphocytes from the anticoagulant peripheral blood by the density gradient method, and the expression level of CD4+CD25+FOXP3+Treg s was detected by flow cytometry. The CD4+CD25+ regulatory T cells and CD4+ CD25 effect T cells were selected from the peripheral blood of the selected subjects by magnetic beads and detected by pyrosequencing method. The level of FOXP3 enhancer methylation of Tregs and Teffs was detected respectively. The content of FOXP3mRNA in the lymphocytes of the selected participants was detected by real-time quantitative PCR, and the content of IL-10, TGF-p and INF- in the plasma of the selected participants was detected by ELISA.
Results: (1) the expression level of CD4+CD25+FOXP3+Tregs in ACS patients was not detected by flow cytometry (P0.05). (2) the FOXP3 enhancer loci of Teffs in ACS patients and controls were highly methylation levels, and the level of methylation of FOXP3 enhancers in the two groups was no difference (P0.05), while ACS patients and control Tregs FOXP3 enhancers The methylation level of the Tregs FOXP3 enhancers in the two groups was significantly lower than that of the FOXP3 enhancer methylation level of Teffs (P0.05), and the level of methylation of the FOXP3 enhancer loci of Tregs in ACS patients was significantly higher than the FOXP3 enhancer methylation level (P0.05) of Tregs in the control group (P0.05). Compared with the control group (P0.05), the level of FOXP3mRNA expression in the lymphocytes of ACS patients and controls decreased (P0.05) after 5 days of culture, and the proportion of FOXP3mRNA expression in lymphocyte of ACS patients was significantly higher than that of the control (P0.05). In the control group, the TGF- beta content in plasma of ACS patients decreased (P0.05), while the INF- gamma content increased (P0.05).
Conclusion: the elevated level of FOXP3 enhancer methylation in T cells in patients with acute coronary syndrome may be the cause of the impairment of Tregs immunosuppressive function, and the damage of Tregs immunosuppressive function is involved in the occurrence of ACS. Therefore, reducing the level of FOXP3 enhancer methylation may be a potential strategy for the treatment of ACS.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541.4

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