全反式维甲酸逆转髓源抑制细胞介导的脓毒症免疫抑制研究

发布时间：2018-06-24 03:48

本文选题：脓毒症 + 免疫抑制　；参考：《华中科技大学》2015年博士论文

【摘要】：研究背景：脓毒症是机体对严重感染作出的一系列复杂的免疫反应,从初期的由细胞因子介导的高炎症反应阶段逐步进展到后期的低炎症反应阶段。脓毒症后期机体处于免疫抑制状态,表现为机体清除侵袭病原体的能力下降及对条件致病菌的易感性增加,目前已成为脓毒症患者的主要死亡原因。髓源抑制细胞(myeloid-derived suppressor cells, MDSCs)是由各类不成熟的髓系细胞构成的异质性细胞群体,脓毒症后MDSCs会发生过度扩增和活化,通过影响T细胞增值和功能,抑制适应性免疫反应,从而诱导免疫抑制状态。体外实验、动物载体实验及临床药物试验均已证实全反式维甲酸((all-trans-retinoic acid,ATRA)能诱导MDSCs分化成熟为巨噬细胞、树突状细胞和粒细胞从而减轻MDSCs对T细胞的抑制作用。但是这些研究均集中在肿瘤领域,缺乏在脓毒症中的证据。本研究拟探究脓毒症时,特别是脓毒症后期发生免疫抑制时,ATRA是如何调控MDSCs的扩增和功能从而影响机体免疫状态和预后的,以及可能参与其中的信号转导通路。方法：本研究采用小鼠盲肠结扎穿孔(cecal ligation and puncture,CLP)脓毒症模型并在其基础上通过绿脓杆菌滴鼻建立了二次感染打击模型。观察了ATRA处理对小鼠CLP+二次细菌打击模型生存情况；CLP小鼠诱导产生的MDSCs的数量、表型、形态学和免疫抑制活性；CD4+T细胞增值、凋亡和迟发型超敏反应(delayed type hypersensitivity, DTH);血清及MDSCs中炎症因子分泌水平：及相关信号传导分子表达的影响。结果：ATRA能明显减少CLP后期小鼠脾脏、外周血和骨髓中MDSCs的绝对数量和所占总细胞的百分比,并降低MDSCs的精氨酸酶活性和NO产生量,恢复其促炎／抗炎细胞因子的分泌平衡,最终减轻其对CD4+T细胞的抑制作用。这些变化带来的是脓毒症小鼠后期机体免疫状态的好转,具体表现为CD4+T细胞增值能力改善、凋亡减少、血清促炎因子水平增加,抗炎因子分泌减少,脾脏内精氨酸酶和一氧化氮合酶蓄积减少。反应到动物整体水平则是相较于CLP组,ATRA治疗组小鼠DTH反应增强,二次细菌感染后生存时间延长、生存率提升。而ATRA对脓毒症后期小鼠二次打击生存预后的有利作用会被过继回输CLP-7d产生的MDSCs所中和,而用抗体耗竭脓毒症后期小鼠体内MDSCs则会达到与使用ATRA相似的效果。在筛选有可能参与ATRA调控MDSCs的信号传导分子时发现STAT-3的激活程度(磷酸化水平)有明显改变,而STAT-6和AKT这两个指标变化不显著。结论：以上研究结果提示ATRA能通过抑制MDSCs的扩增和活性,恢复特异性免疫功能和炎症反应平衡,改善脓毒症后期的免疫状态。从而为脓毒症后免疫抑制的治疗提供新的思路。
[Abstract]:Background: sepsis is a series of complex immune responses to severe infections, from the early stage of high inflammation mediated by cytokines to the later stage of low inflammation. In the late stage of sepsis, the body is in an immunosuppressive state, which is characterized by a decrease in the ability of the body to clear the invading pathogens and an increase in susceptibility to conditional pathogens, which has become the main cause of death in sepsis patients. Myeloid-derived suppressor cells (MDSCs) are heterogeneous cells composed of immature myeloid cells. After sepsis, myeloid-derived suppressor cells will be over-expanded and activated, which can inhibit adaptive immune response by affecting T cell proliferation and function. Thus the immunosuppressive state is induced. In vitro experiments, animal carrier experiments and clinical drug trials have demonstrated that all trans retinoic acid (all-trans-retinoic) can induce differentiation and maturation of MDSCs into macrophages, dendritic cells and granulocytes so as to reduce the inhibitory effect of MDSCs on T cells. But these studies are focused on cancer and lack evidence for sepsis. This study aims to explore how ATRA regulates the expansion and function of MDSCs during sepsis, especially in the late stage of sepsis, and may be involved in signal transduction pathways. Methods: in this study, the sepsis model was established by cecal ligation and perforation of (cecal ligation and puncture. on the basis of this model, a secondary infection attack model was established by Pseudomonas aeruginosa drip nose. The number, phenotypic, morphological and immunosuppressive activity of MDSCs induced by ATRA were observed. Apoptosis and delayed hypersensitivity (delayed type hypersensitivity,), the level of inflammatory factor secretion in serum and MDSCs, and the effect of signal transduction molecules. Results the absolute number and percentage of MDSCs in spleen, peripheral blood and bone marrow were significantly decreased, argininase activity and no production of MDSCs were decreased, and the secretion balance of proinflammatory / anti-inflammatory cytokines was restored. Finally, the inhibition of CD4 T cells was alleviated. These changes bring about the improvement of immune status in the later stage of sepsis mice, which is manifested by the improvement of CD4 T cell proliferation ability, the decrease of apoptosis, the increase of serum pro-inflammatory factor level and the decrease of anti-inflammatory factor secretion. The accumulation of arginase and nitric oxide synthase in spleen decreased. Compared with the CLP group, the DTH response was increased, the survival time was prolonged and the survival rate was increased after secondary bacterial infection. The beneficial effect of ATRA on survival and prognosis of mice after sepsis was neutralized by repeated transfusion of MDSCs produced by CLP-7d, while the effect of using antibody to exhaust MDSCs in mice of late stage of sepsis was similar to that of mice treated with ATRA. In screening signal transduction molecules that may be involved in ATRA regulation of MDSCs, it was found that the activation degree of STAT-3 (phosphorylation level) changed significantly, but STAT-6 and AKT did not change significantly. Conclusion: these results suggest that ATRA can improve the immune state of sepsis by inhibiting the expansion and activity of MDSCs and restoring the balance of specific immune function and inflammatory reaction. This provides a new idea for the treatment of immunosuppression after sepsis.
【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】：R459.7

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