PD-1调节自身免疫性肝炎肝硬化患者脾切除后免疫耐受功能的研究

发布时间：2018-03-03 03:05

本文选题：自身免疫性肝炎　切入点：脾切除　出处：《天津医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:自身免疫性肝炎(Autoimmune hepatitis,AIH)的发病与免疫系统功能紊乱有关,AIH肝硬化患者预后差,由于存在诸多禁忌症而不能使用免疫抑制进行有效的治疗。AIH肝硬化患者常常伴随脾功能亢进,脾切除治疗后可以有效缓解门静脉高压症状。我们以往的研究证实了脾切除可以减少患者脾功能亢进的症状,改变机体的免疫状态,本研究将在AIH肝硬化患者体内研究脾切除对其免疫细胞分化和免疫环境的影响,从而阐明其影响AIH肝硬化患者免疫功能的机制。本研究将在AIH肝硬化患者和动物模型中,进一步研究脾切除对其免疫细胞分化和免疫环境产生影响的通路,寻找可干预的潜在靶点。研究对象:本研究纳入从2005年1月份至2016年12月份,在天津医科大学总医院消化科和普通外科经过临床及病理检查,确诊自身免疫性肝炎肝硬化患者20例,脾切除后8例患者,另外慢性肝炎(乙肝)肝硬化患者30例,脾切除后15例患者作为对照。收集患者的外周血检测血常规,肝功能和免疫学指标;检测外周血中趋化因子和细胞因子的表达变化;利用流式细胞术检测外周血CD4+,CD8+T细胞亚群的比例变化;进一步研究与免疫耐受相关的巨噬细胞亚群的分化情况。利用刀豆蛋白(Con A)建立模型,研究脾切除对于CD8+T细胞表面PD-1(Programmed death-1)等分子表达的影响,以及对于糖皮质激素诱导肿瘤坏死因子受体GITR(glucocorticoid—induced tumor necrosis factor receptor)和关键转录因子NF-κB表达的影响,讨论T细胞耗竭与AIH发生发展的关系。研究结果:我们的研究发现,AIH肝硬化患者切脾随访6个月后其临床和生化指标显著改善,脾功能亢进相关症状好转。脾切除可以抑制免疫趋化因子CXCL9和IP-10的表达,抑制CD4+T细胞的过度活化,增加外周血CD8+T细胞的比例。和对照组患者相比,AIH肝硬化患者以M1型巨噬细胞活化为主,脾切除促进免疫耐受相关的CD14+CD16+CD206+CD163+M2c型巨噬细胞增多,促使AIH肝硬化患者中M1型巨噬细胞向M2型转化,可能与促进组织的修复,减少自身免疫反应对肝细胞的破坏有关。进一步我们发现,AIH肝硬化患者肝脏内CD8+T细胞增多,利用外周血进一步分析,脾切除患者外周血中CD8+T细胞表达PD-1上调,同时伴随CD160上调和4-1BB表达抑制,而且,与激素调节有关的GITR信号也在脾切除后患者的外周血中上调。进一步研究与控制上述分子表达的核内转录因子NF-κB的二聚体组分,发现脾切除可以促进p50-p50二聚体的DNA结合活性,这有助于抑制过度的免疫反应。研究结论:脾切除可以改善AIH肝硬化患者的临床和生化指标,脾功能亢进的缓解有助于增加患者使用免疫抑制剂使用的机会。脾切除不仅可以增加免疫抑制相关的M2c型巨噬细胞的比例,还可以促进CD8+T细胞表达PD-1分子上调,诱导T细胞耗竭表型表达,为AIH肝硬化患者未来治疗提供了新的思路。
[Abstract]:Background: the pathogenesis of autoimmune hepatitis autoimmune hepatitis (AIH) is associated with the dysfunction of the immune system. The prognosis of patients with AIH cirrhosis is poor. Because of many contraindications, immunosuppression can not be used to effectively treat. AIH cirrhosis patients are often accompanied by hypersplenism. Splenectomy can effectively relieve the symptoms of portal hypertension after splenectomy. Our previous studies have confirmed that splenectomy can reduce the symptoms of hypersplenism and change the immune state of the body. In this study, the effects of splenectomy on immune cell differentiation and immune environment in patients with AIH cirrhosis were studied to elucidate the mechanisms that affect the immune function of patients with AIH cirrhosis. This study will be carried out in AIH cirrhosis patients and animal models. To further study the pathway of splenectomy on its immune cell differentiation and immune environment, and to find potential targets for intervention. After clinical and pathological examination, 20 cases of autoimmune hepatitis cirrhosis, 8 cases of splenectomy and 30 cases of chronic hepatitis (hepatitis B) cirrhosis were confirmed in the Department of Digestive and General surgery, Tianjin Medical University General Hospital. After splenectomy, 15 patients were used as controls. The peripheral blood samples were collected to detect the blood routine, liver function and immunological indexes, and the expression of chemokines and cytokines in peripheral blood were detected. Flow cytometry was used to detect the percentage of CD8 T cell subsets in peripheral blood of CD4, to further study the differentiation of macrophage subsets related to immune tolerance, and to establish a model by using concanavalin (Con A). To investigate the effects of splenectomy on the expression of PD-1(Programmed death-1 and other molecules on the surface of CD8 T cells, and on glucocorticoid-induced expression of tumor necrosis factor receptor GITR(glucocorticoid-induced tumor necrosis factor receptor and NF- 魏 B, To discuss the relationship between T cell depletion and the occurrence and development of AIH. Splenectomy can inhibit the expression of immune chemokine CXCL9 and IP-10 and inhibit the excessive activation of CD4 T cells. Compared with the control group, M 1 macrophages were the main type of macrophages in the patients with liver cirrhosis, and the number of CD14 CD16 CD206 CD163 M2c macrophages associated with splenectomy and immune tolerance was increased. Promoting the transformation of M1 type macrophages to M2 type in patients with AIH cirrhosis may be related to promoting the repair of tissues and reducing the damage of liver cells caused by autoimmune reaction. Further, we found that the number of CD8 T cells in liver of patients with AIH cirrhosis increased. By further analysis of peripheral blood, the expression of PD-1 was up-regulated by CD8 T cells in peripheral blood of patients with splenectomy, accompanied by the up-regulation of CD160 and the inhibition of 4-1BB expression. The GITR signal associated with hormone regulation was also up-regulated in the peripheral blood of patients with splenectomy. Further studies on the dimer components of NF- 魏 B, a transcription factor expressed by these molecules, showed that splenectomy could promote the DNA binding activity of p50-p50 dimer. Conclusion: splenectomy can improve the clinical and biochemical parameters of patients with AIH cirrhosis. The remission of hypersplenism may increase the chance of using immunosuppressive agents. Splenectomy not only increases the proportion of immunosuppressive M2c macrophages, but also promotes the up-regulation of PD-1 expression by CD8 T cells. The induction of T cell depletion phenotypic expression provides a new approach for the future treatment of AIH cirrhosis patients.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575

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