【摘要】：研究背景與實(shí)驗(yàn)?zāi)康模涸l(fā)性膽汁性肝硬化(Primary biliary cirrhosis, PBC)是一種自身免疫性肝臟疾病。好發(fā)于中年婦女,組織學(xué)上以小葉間膽管進(jìn)行性損傷表現(xiàn)為主；血清中出現(xiàn)堿性磷酸酶(Alkaline phosphatase, ALP)和免疫球蛋白M(Immunoglobulin M, IgM)升高和多種自身抗體陽性,其中以M2型抗線粒體抗體最為特異。調(diào)節(jié)性T細(xì)胞(Regulatory T cell, Treg)是一類重要的免疫調(diào)節(jié)細(xì)胞,控制著體內(nèi)自身免疫反應(yīng),在免疫耐受和動(dòng)態(tài)平衡中起中心作用。機(jī)體在健康的情況下,功能完好的Treg可以使肝臟中的免疫反應(yīng)處于抑制狀態(tài)。一旦Treg數(shù)量和功能出現(xiàn)受損,免疫耐受就被打破。CD3+細(xì)胞可以包括經(jīng)典T細(xì)胞(CD3+CD56-)和自然殺傷T(Natural killer T, NKT)細(xì)胞(CD3+CD56+)。 NKT細(xì)胞被報(bào)道可促進(jìn)自身免疫反應(yīng),其作用機(jī)制可能與破壞Th1/Th2類型反應(yīng)平衡,調(diào)節(jié)其分泌細(xì)胞因子有關(guān)。本課題通過對(duì)臨床患者外周血中細(xì)胞因子和淋巴細(xì)胞分布進(jìn)行檢測(cè),體外實(shí)驗(yàn)研究其數(shù)量和功能上的改變,發(fā)現(xiàn)這兩種細(xì)胞在PBC發(fā)病中的作用和相互關(guān)系,旨在找到一條影響PBC發(fā)病的免疫機(jī)制。研究方法：通過收集,整理和分析55例PBC患者的臨床資料,并用流式細(xì)胞術(shù)檢測(cè)外周血中Treg和NKT細(xì)胞的比例,數(shù)量和酶聯(lián)免疫實(shí)驗(yàn)(Enzyme-linked immunosorbent assay, ELISA)檢測(cè)相關(guān)細(xì)胞因子水平。分離出樣本中的單個(gè)核細(xì)胞,分選培養(yǎng)以通過體外實(shí)驗(yàn)檢測(cè)其功能。研究結(jié)果：第一部分：結(jié)果表明PBC患者的Treg數(shù)量和產(chǎn)生白介素-10(Interleukin-10, IL-10)的能力都顯著下降；PBC患者的Treg難以有效抑制T細(xì)胞的增殖和干擾素(interferon-γ, IFN-γ)的產(chǎn)生,Treg具有抑制T細(xì)胞對(duì)膽管上皮細(xì)胞的殺傷能力的作用,PBC患者的Treg細(xì)胞的這種抑制作用低于健康對(duì)照組；另外,PBC患者DC表面的CD80/CD86共刺激分子顯著增高并且在自身抗體刺激后上升更為顯著：PBC患者PBMC抑制肝星形細(xì)胞表達(dá)的平滑肌動(dòng)蛋白(α-smooth muscle actin, α-SMA)能力減弱；PBC患者的Treg抑制DC表面CD86的表達(dá)能力顯著減弱。PBC患者PBMC抑制肝星形細(xì)胞表達(dá)的α-SMA能力減弱。第二部分：結(jié)果發(fā)現(xiàn),在PBC患者外周血中CD3+CD56+NKT細(xì)胞數(shù)量顯著上升；而經(jīng)典T細(xì)胞(CD3+CD56-)的數(shù)量與健康成人無明顯差異。PBC患者外周血血漿中,IL-17及其相關(guān)上下游細(xì)胞因子水平均上升：胞內(nèi)染色發(fā)現(xiàn)CD3+CD56+NKT細(xì)胞是產(chǎn)生IL-17最多的一群細(xì)胞；用分離的自身抗體刺激PBMC后,上清中IL-17水平顯著上升,并且上升最多的來源即為CD3+CD56+NKT細(xì)胞：1L-17可以使肝星形細(xì)胞活化產(chǎn)生轉(zhuǎn)化生長因子(Transforming growth factor-β, TGF-β)和表達(dá)α-SMA,且呈劑量依賴,PBC患者的PBMC可使肝星形細(xì)胞活化產(chǎn)生更高水平的TGF-β。結(jié)論：在PBC發(fā)病過程中Treg數(shù)量,比例和功能的下降,導(dǎo)致其抑制樹突細(xì)胞活化和炎癥發(fā)生的能力顯著降低；與此同時(shí),NKT細(xì)胞在自身抗原等刺激下產(chǎn)生IL-17,促進(jìn)肝星形細(xì)胞活化。Treg的抑制作用不足以抑制NKT細(xì)胞的促炎作用,使纖維化進(jìn)一步發(fā)展。
[Abstract]:Background and Objective: Primary biliary cirrhosis (PBC) is an autoimmune liver disease. In the middle-aged women, the progressive injury of the interlobular bile duct was mainly seen in the middle-aged women. The level of alkaline phosphatase (ALP) and immunoglobulin M (IgM) in the serum increased and the autoantibodies were positive, among which the anti-mitochondrial antibodies were most specific. Regulatory T cell (Treg) is an important immunomodulatory cell, which controls autoimmunity in vivo and plays a central role in immune tolerance and dynamic balance. In the case of health, the well-functioning Treg allows the immune response in the liver to be in a suppressed state. The immune tolerance is broken once the number and function of Treg is impaired. CD3 + cells can include classical T cells (CD3 + CD56-) and natural killer T (NKT) cells (CD3 + CD56 +). NKT cells are reported to promote autoimmunity, and the mechanism of action may be related to the disruption of the Th1/ Th2 type of reaction and the regulation of the secretion of cytokines. In this paper, the distribution of cytokines and lymphocytes in peripheral blood of clinical patients was detected, and the number and function of these two cells were studied in vitro, and the role and relationship of these two cells in the pathogenesis of PBC were found, and the purpose of this study was to find an immune mechanism that affected the pathogenesis of PBC. Methods: The clinical data of 55 PBC patients were collected, sorted and analyzed, and the proportion, quantity and enzyme-linked immunosorbent assay (ELISA) of Treg and NKT cells in peripheral blood were detected by flow cytometry. The individual nuclear cells in the sample were isolated and cultured for its function by in vitro experiments. Results: The first part: The results showed that the number of Treg in PBC patients and the ability to produce interleukinkin-10 (IL-10) decreased significantly; Treg in PBC patients was difficult to effectively inhibit the proliferation of T cells and the production of interferon-10 (IFN-1). Treg has the effect of inhibiting the anti-killing ability of T cells to the bile duct epithelial cells, and the inhibitory effect of the Treg cells in the PBC patients is lower than that of the healthy control group; in addition, the CD80/ CD86 costimulatory molecules of the DC surface of the PBC patients are significantly increased and the increase is more significant after the autoantibody stimulation: In PBMCs, the ability of PBMC to inhibit the expression of hepatic stellate cells was reduced, and the ability of Tregs in PBC patients to inhibit the expression of CD86 was significantly reduced. In PBMCs, the ability of PBMC to inhibit the expression of hepatic stellate cells was reduced. The second part: The results showed that the number of CD3 + CD56 + NKT cells increased significantly in the peripheral blood of the PBC patients, and the number of the classical T cells (CD3 + CD56-) was not significantly different from that of healthy adults. In the peripheral blood plasma of PBC patients, the levels of IL-17 and related upstream and downstream cytokines increased: the intracellular staining found that the CD3 + CD56 + NKT cells were a group of cells that produced the most IL-17; after the PBMCs were stimulated with isolated autoantibodies, the level of IL-17 in the supernatant was significantly increased, and the most ascending source was CD3 + CD56 + NKT cells: 1L-17 can cause the activation of the hepatic stellate cells to produce a transformation growth factor-1 (TGF-1) and the expression of the antigen-SMA, and is in a dose-dependent manner, and the PBMCs of the PBC patients can enable the activation of the hepatic stellate cells to produce a higher level of TGF-1. Conclusion: The decrease of the number, proportion and function of Treg in the pathogenesis of PBC leads to a significant decrease in the ability to inhibit the activation and inflammation of the dendritic cells. At the same time, the NKT cells generate IL-17 under the stimulation of the autoantigen, and promote the activation of the hepatic stellate cells. The inhibition of Treg is not sufficient to inhibit the pro-inflammatory action of NKT cells and to further develop fibrosis.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Primary biliary cirrhosis:What do autoantibodies tell us?[J];World Journal of Gastroenterology;2010年29期

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本文編號(hào)：2447396