PD-L1对实验性自身免疫性神经炎保护作用的实验研究

发布时间：2018-03-04 16:38

本文选题：实验性自身免疫性神经炎　切入点：吉兰-巴雷综合征　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景及目的:实验性自身免疫性神经炎(EAN)为探索研究吉兰-巴雷综合征(GBS)应用最广范、最经典动物模型,可以模仿GBS疾病的发生、疾病的发展过程、神经电生理和组织病理学等特征。EAN的病理特征是在外周神经系统聚集反应性T细胞和巨噬细胞,攻击血神经屏障,从而使周围神经脱髓鞘导致疾病。各种免疫反应对于EAN疾病的发生发展都起作用,但细胞免疫所起的作用无可比拟。程序性死亡受体-1(PD-1,CD279)是经典的CD28家族成员之一,为细胞表面的一种抑制性受体,最常见表达于T细胞的细胞膜上,该受体在感染性疾病和癌症中激活后可使T细胞功能失调、抑制免疫反应,从而使病毒或癌细胞发生免疫逃逸。PD-1的配体PD-L1(也称为B7-H1,CD274)是B7家族的成员。研究发现PD-1结合其配体PD-L1,可以激活PD-1通路,阻止免疫反应的发生及发展,这一过程主要通过抑制T细胞膜表面的受体(TCR)/CD3介导的细胞的激活、增殖,影响T细胞的分化及细胞因子的产生。目前有研究发现,PD-L1与PD-1结合后,激活PD-1途径,可以抑制T细胞增殖并且减少干扰素-γ(IFN-γ)和肿瘤坏死因子α(TNF-α)的分泌。目前,已证明通过激活PD-1通路能够缓解的自身免疫疾病(如I型糖尿病、多发性硬化症、系统性红斑狼疮等)、炎症感染性疾病和器官移植后免疫排斥的报道屡见不鲜。而且各种免疫抑制剂、单克隆抗体等药物作为治疗GBS的选择均在在EAN动物模型上进行实验验证其有效性,但有关PD-L1对GBS的治疗潜力及确切的机制尚无报道。根据以往关于PD-1/PD-L1对自身免疫性疾病作用的研究,我们提出假设PD-L1阻止免疫反应的过度激活,主要通过抑制T细胞增殖、影响T细胞的分化及细胞因子的分泌从而发挥对EAN疾病的抗炎及神经保护作用。在本研究中,通过观察EAN大鼠的神经病学评分、EAN大鼠坐骨神经的病理及电生理变化,以评价PD-L1对EAN大鼠的治疗效果,并且追溯了其可能的细胞内机制。方法:EAN大鼠成模后,根据其神经症状进行功能评分。然后根据分组情况腹腔注射给药。在EAN发病的高峰期,评价坐骨神经中单个核细胞(MNC)浸润程度及脱髓鞘病变的严重程度。进行神经肌电图检测观察坐骨神经的复合肌肉的动作电位(CMAP)的波幅、潜伏期及运动神经传导速度(MCV)。分别通过EDU细胞实验及淋巴细胞增殖实验来筛选PD-L1治疗后大鼠体内发生变化的免疫细胞。用流式细胞术及荧光染色观察CD4+T细胞在大鼠脾脏及坐骨神经的分化情况。实时聚合酶链式反应(RT-PCR)测定EAN大鼠脾脏单个核细胞(MNCs)中细胞因子表达水平变化,通过酶联免疫吸附试验(ELISA)测定大鼠MNCs培养上清中多种细胞因子分泌水平情况。通过Western blot和PCR方法观察通路相关蛋白的表达并进一步探讨PD-L1可能的作用机制。结果:首先,该实验发现PD-L1延迟了EAN疾病的发生,减轻EAN大鼠临床表现。PD-L1治疗能够减轻EAN大鼠坐骨神经中各种炎性细胞的浸润,并且可以减轻坐骨神经髓鞘脱失的严重程度。PD-L1治疗后的大鼠坐骨神经的神经传导速度得到改善,提高了CMAPs的振幅且缩短神经传导潜伏期。另外,在EAN大鼠脾的MNCs中,该实验发现PD-L1能够选择性的抑制CD4+T细胞的增殖,对于巨噬细胞的增殖变化没有意义。更重要的是,PD-L1治疗可以影响CD4+T细胞的分化,降低了促炎性的Th1细胞和Th17细胞的比例,而增加了抗炎性的Th2细胞和Treg细胞的比例;但是对于巨噬细胞各型的分化三组之间没有差别。在坐骨神经中,PD-L1抑制Th17细胞浸润,促进Treg细胞的增殖,从而发挥了对外周神经的保护性作用。此外,PD-L1还可改善EAN大鼠的免疫炎症微环境,影响各种细胞因子的分泌,其中IFN-γ、TNF-α等促炎性细胞因子表达明显下调。关于机制的研究,该实验发现PD-L1治疗的大鼠体内PI3K/Akt/mTOR通路相关蛋白p-PI3K、p-AKT、p-mTOR、p-P70S6K、p-4EBP1及其mRNA的表达量下调,而PD-1受体的蛋白和基因的表达量上调。结合以上实验结果,总结出PD-L1对EAN的保护性作用主要是通过调节CD4+T细胞的增殖分化,而其机制主要与阻碍PI3K/Akt/mTOR细胞传导通路有关。结论:综上所述,该研究表明PD-L1在EAN大鼠中发挥抗炎症反应及保护神经的作用主要是通过PI3K/Akt/mTOR通路实现的。作为治疗EAN或GBS的一种新型的可选择的药物,PD-L1可以抑制CD4+T细胞增殖,影响T细胞的分化及促炎性细胞因子的表达量,从而抑制EAN大鼠体内炎症反应。
[Abstract]:Background and purpose: experimental autoimmune neuritis (EAN) for the study of Guillain Barre syndrome (GBS) is the most widely used fan, the most classic animal model can imitate the occurrence of GBS disease, disease progression, pathological characteristics of electrophysiological and histological features of.EAN in peripheral nerve system aggregation of reactive T cells and macrophages, attack the blood nerve barrier, so that the peripheral nerve demyelination leads to illness. Various immune responses play a role in the occurrence and development of EAN's disease, but the role of immune cells. There is nothing comparable to this programmed death receptor -1 (PD-1, CD279) is a member of the CD28 family is a classic. Inhibitory receptors on the surface of the cell, the most common cell membrane expression in T cells, activation of this receptor in infectious diseases and cancers that after T cell dysfunction, suppression of the immune response, so that the virus or cancer cell Cell immune escape.PD-1 ligand PD-L1 (also known as B7-H1, CD274) is a member of the B7 family. The study found that PD-1 combined with its ligand PD-L1, can activate PD-1 pathway and prevent the occurrence and development of immune response, which is mainly through the inhibition of T cell surface receptor (TCR) activation mediated by /CD3 cells the effect of proliferation, differentiation and cytokine production of T cells. The present study found that PD-L1 combined with PD-1 after activation of the PD-1 pathway, can inhibit the proliferation of T cells and reduce the interferon gamma (IFN- gamma) and tumor necrosis factor alpha (TNF- alpha) secretion. At present, has been shown to activate the PD-1 pathway remission of autoimmune diseases (such as type I diabetes, multiple sclerosis, systemic lupus erythematosus), inflammatory infectious disease and organ transplantation rejection and immunosuppression. It is often seen. reported agents, monoclonal antibody drugs such as The treatment of GBS was tested for its effectiveness in EAN in animal models, but the therapeutic potential of about PD-L1 to GBS and the exact mechanism has not been reported. According to the previous research on the role of autoimmune disease PD-1/PD-L1, we put forward the hypothesis the excessive activation of PD-L1 prevents the immune response, mainly through inhibit the proliferation of T cells effect of cell differentiation and secretion of T cytokines and anti-inflammatory and neuroprotective effects of EAN disease. In this study, through the observation of EAN rats neurological score, EAN rat sciatic nerve pathological and electrophysiological changes, to evaluate the therapeutic effect of PD-L1 on EAN rats, and its back the possible cellular mechanisms. Methods: the EAN rat model, according to the score of neurological symptoms. Then according to the group after intraperitoneal injection in EAN. The peak incidence, evaluation of sciatic nerve in Mononuclear cells (MNC) severity of infiltration and demyelination. The action potential of sciatic nerve were observed to detect composite muscle electromyogram (CMAP) amplitude, latency and motor nerve conduction velocity (MCV) respectively. By EDU cells and lymphocyte proliferation assay to screen changes of rats after PD-L1 treatment. Immune cells. Flow cytometry and fluorescence staining of CD4+T cells in the spleen and differentiation of rat sciatic nerve. Real time polymerase chain reaction (RT-PCR) determination of nuclear cells in spleen, single EAN rats (MNCs) expression of cytokines by enzyme-linked immunosorbent assay (ELISA) determination of rat MNCs culture the level of cytokine secretion in the supernatant. The expression of Western and PCR by blot method to observe the pathway related proteins and to further explore the possible mechanisms of PD-L1. Results: first The experiment found that, PD-L1 delayed the occurrence of EAN disease, reduce the treatment of rat EAN clinical manifestations of.PD-L1 can reduce the infiltration of inflammatory cells in EAN rat sciatic nerve, the nerve conduction velocity of sciatic nerve in rats treated with.PD-L1 and can reduce the severity of sciatic nerve demyelination after improved, improved CMAPs the amplitude and shortened nerve conduction latency. In addition, EAN in the spleen of rat MNCs, the experimental results show that PD-L1 can selectively inhibit the proliferation of CD4+T cells, no significance for the proliferation of macrophages. More importantly, PD-L1 treatment can influence the differentiation of CD4+T cells, Th1 cells and Th17 cells reduces pro-inflammatory the proportion of increase of Th2 cells and Treg cells of the anti inflammatory ratio; but for each type of macrophage differentiation between the three groups. There was no difference in the sciatic nerve, PD-L1 inhibited Th17 fine Cell infiltration, promote the proliferation of Treg cells, which play a protective role in peripheral nerve. In addition, the immune inflammation in PD-L1 EAN rats can also improve the micro environment, influence the secretion of various cytokines, including IFN- gamma, alpha TNF- expression of proinflammatory cytokines was significantly reduced. Research on the mechanism. The experiment found that PD-L1 treatment of the PI3K/Akt/mTOR pathway in rat associated protein p-PI3K, p-AKT, p-mTOR, p-P70S6K, p-4EBP1 expression and downregulation of mRNA, and the expression of gene and protein of the PD-1 receptor. Combining with the above experimental results, summed up the protective effect of PD-L1 on EAN is mainly through regulating the proliferation and differentiation of CD4+T cells. The mechanism is mainly related with the block PI3K/Akt/mTOR cell pathway. Conclusion: This study suggests that PD-L1 plays an anti-inflammatory and neuroprotective effect in EAN rats is mainly through PI3K/Akt/m As a new alternative drug to treat EAN or GBS, PD-L1 can inhibit the proliferation of CD4+T cells, affect the differentiation of T cells and the expression of proinflammatory cytokines, thereby inhibiting the inflammatory reaction in EAN rats. TOR pathway is a new alternative drug.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R745.43

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