本文選題：實(shí)驗(yàn)性自身免疫性神經(jīng)炎　切入點(diǎn)：吉蘭-巴雷綜合征　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景及目的:實(shí)驗(yàn)性自身免疫性神經(jīng)炎(EAN)為探索研究吉蘭-巴雷綜合征(GBS)應(yīng)用最廣范、最經(jīng)典動(dòng)物模型,可以模仿GBS疾病的發(fā)生、疾病的發(fā)展過程、神經(jīng)電生理和組織病理學(xué)等特征。EAN的病理特征是在外周神經(jīng)系統(tǒng)聚集反應(yīng)性T細(xì)胞和巨噬細(xì)胞,攻擊血神經(jīng)屏障,從而使周圍神經(jīng)脫髓鞘導(dǎo)致疾病。各種免疫反應(yīng)對于EAN疾病的發(fā)生發(fā)展都起作用,但細(xì)胞免疫所起的作用無可比擬。程序性死亡受體-1(PD-1,CD279)是經(jīng)典的CD28家族成員之一,為細(xì)胞表面的一種抑制性受體,最常見表達(dá)于T細(xì)胞的細(xì)胞膜上,該受體在感染性疾病和癌癥中激活后可使T細(xì)胞功能失調(diào)、抑制免疫反應(yīng),從而使病毒或癌細(xì)胞發(fā)生免疫逃逸。PD-1的配體PD-L1(也稱為B7-H1,CD274)是B7家族的成員。研究發(fā)現(xiàn)PD-1結(jié)合其配體PD-L1,可以激活PD-1通路,阻止免疫反應(yīng)的發(fā)生及發(fā)展,這一過程主要通過抑制T細(xì)胞膜表面的受體(TCR)/CD3介導(dǎo)的細(xì)胞的激活、增殖,影響T細(xì)胞的分化及細(xì)胞因子的產(chǎn)生。目前有研究發(fā)現(xiàn),PD-L1與PD-1結(jié)合后,激活PD-1途徑,可以抑制T細(xì)胞增殖并且減少干擾素-γ(IFN-γ)和腫瘤壞死因子α(TNF-α)的分泌。目前,已證明通過激活PD-1通路能夠緩解的自身免疫疾病(如I型糖尿病、多發(fā)性硬化癥、系統(tǒng)性紅斑狼瘡等)、炎癥感染性疾病和器官移植后免疫排斥的報(bào)道屢見不鮮。而且各種免疫抑制劑、單克隆抗體等藥物作為治療GBS的選擇均在在EAN動(dòng)物模型上進(jìn)行實(shí)驗(yàn)驗(yàn)證其有效性,但有關(guān)PD-L1對GBS的治療潛力及確切的機(jī)制尚無報(bào)道。根據(jù)以往關(guān)于PD-1/PD-L1對自身免疫性疾病作用的研究,我們提出假設(shè)PD-L1阻止免疫反應(yīng)的過度激活,主要通過抑制T細(xì)胞增殖、影響T細(xì)胞的分化及細(xì)胞因子的分泌從而發(fā)揮對EAN疾病的抗炎及神經(jīng)保護(hù)作用。在本研究中,通過觀察EAN大鼠的神經(jīng)病學(xué)評(píng)分、EAN大鼠坐骨神經(jīng)的病理及電生理變化,以評(píng)價(jià)PD-L1對EAN大鼠的治療效果,并且追溯了其可能的細(xì)胞內(nèi)機(jī)制。方法:EAN大鼠成模后,根據(jù)其神經(jīng)癥狀進(jìn)行功能評(píng)分。然后根據(jù)分組情況腹腔注射給藥。在EAN發(fā)病的高峰期,評(píng)價(jià)坐骨神經(jīng)中單個(gè)核細(xì)胞(MNC)浸潤程度及脫髓鞘病變的嚴(yán)重程度。進(jìn)行神經(jīng)肌電圖檢測觀察坐骨神經(jīng)的復(fù)合肌肉的動(dòng)作電位(CMAP)的波幅、潛伏期及運(yùn)動(dòng)神經(jīng)傳導(dǎo)速度(MCV)。分別通過EDU細(xì)胞實(shí)驗(yàn)及淋巴細(xì)胞增殖實(shí)驗(yàn)來篩選PD-L1治療后大鼠體內(nèi)發(fā)生變化的免疫細(xì)胞。用流式細(xì)胞術(shù)及熒光染色觀察CD4+T細(xì)胞在大鼠脾臟及坐骨神經(jīng)的分化情況。實(shí)時(shí)聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)測定EAN大鼠脾臟單個(gè)核細(xì)胞(MNCs)中細(xì)胞因子表達(dá)水平變化,通過酶聯(lián)免疫吸附試驗(yàn)(ELISA)測定大鼠MNCs培養(yǎng)上清中多種細(xì)胞因子分泌水平情況。通過Western blot和PCR方法觀察通路相關(guān)蛋白的表達(dá)并進(jìn)一步探討PD-L1可能的作用機(jī)制。結(jié)果:首先,該實(shí)驗(yàn)發(fā)現(xiàn)PD-L1延遲了EAN疾病的發(fā)生,減輕EAN大鼠臨床表現(xiàn)。PD-L1治療能夠減輕EAN大鼠坐骨神經(jīng)中各種炎性細(xì)胞的浸潤,并且可以減輕坐骨神經(jīng)髓鞘脫失的嚴(yán)重程度。PD-L1治療后的大鼠坐骨神經(jīng)的神經(jīng)傳導(dǎo)速度得到改善,提高了CMAPs的振幅且縮短神經(jīng)傳導(dǎo)潛伏期。另外,在EAN大鼠脾的MNCs中,該實(shí)驗(yàn)發(fā)現(xiàn)PD-L1能夠選擇性的抑制CD4+T細(xì)胞的增殖,對于巨噬細(xì)胞的增殖變化沒有意義。更重要的是,PD-L1治療可以影響CD4+T細(xì)胞的分化,降低了促炎性的Th1細(xì)胞和Th17細(xì)胞的比例,而增加了抗炎性的Th2細(xì)胞和Treg細(xì)胞的比例;但是對于巨噬細(xì)胞各型的分化三組之間沒有差別。在坐骨神經(jīng)中,PD-L1抑制Th17細(xì)胞浸潤,促進(jìn)Treg細(xì)胞的增殖,從而發(fā)揮了對外周神經(jīng)的保護(hù)性作用。此外,PD-L1還可改善EAN大鼠的免疫炎癥微環(huán)境,影響各種細(xì)胞因子的分泌,其中IFN-γ、TNF-α等促炎性細(xì)胞因子表達(dá)明顯下調(diào)。關(guān)于機(jī)制的研究,該實(shí)驗(yàn)發(fā)現(xiàn)PD-L1治療的大鼠體內(nèi)PI3K/Akt/mTOR通路相關(guān)蛋白p-PI3K、p-AKT、p-mTOR、p-P70S6K、p-4EBP1及其mRNA的表達(dá)量下調(diào),而PD-1受體的蛋白和基因的表達(dá)量上調(diào)。結(jié)合以上實(shí)驗(yàn)結(jié)果,總結(jié)出PD-L1對EAN的保護(hù)性作用主要是通過調(diào)節(jié)CD4+T細(xì)胞的增殖分化,而其機(jī)制主要與阻礙PI3K/Akt/mTOR細(xì)胞傳導(dǎo)通路有關(guān)。結(jié)論:綜上所述,該研究表明PD-L1在EAN大鼠中發(fā)揮抗炎癥反應(yīng)及保護(hù)神經(jīng)的作用主要是通過PI3K/Akt/mTOR通路實(shí)現(xiàn)的。作為治療EAN或GBS的一種新型的可選擇的藥物,PD-L1可以抑制CD4+T細(xì)胞增殖,影響T細(xì)胞的分化及促炎性細(xì)胞因子的表達(dá)量,從而抑制EAN大鼠體內(nèi)炎癥反應(yīng)。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R745.43

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 譚曉冬;段瑞生;時(shí)昌文;曲迅;;脫氫表雄酮對實(shí)驗(yàn)性自身免疫性神經(jīng)炎大鼠細(xì)胞免疫的影響[J];細(xì)胞與分子免疫學(xué)雜志;2008年08期

，

本文編號(hào)：1566473