调节性T细胞在原发性胆汁性肝硬化中的免疫调节作用和机制研究
发布时间：2019-03-26 09:14
【摘要】：研究背景与实验目的：原发性胆汁性肝硬化(Primary biliary cirrhosis, PBC)是一种自身免疫性肝脏疾病。好发于中年妇女,组织学上以小叶间胆管进行性损伤表现为主；血清中出现碱性磷酸酶(Alkaline phosphatase, ALP)和免疫球蛋白M(Immunoglobulin M, IgM)升高和多种自身抗体阳性,其中以M2型抗线粒体抗体最为特异。调节性T细胞(Regulatory T cell, Treg)是一类重要的免疫调节细胞,控制着体内自身免疫反应,在免疫耐受和动态平衡中起中心作用。机体在健康的情况下,功能完好的Treg可以使肝脏中的免疫反应处于抑制状态。一旦Treg数量和功能出现受损,免疫耐受就被打破。CD3+细胞可以包括经典T细胞(CD3+CD56-)和自然杀伤T(Natural killer T, NKT)细胞(CD3+CD56+)。 NKT细胞被报道可促进自身免疫反应,其作用机制可能与破坏Th1/Th2类型反应平衡,调节其分泌细胞因子有关。本课题通过对临床患者外周血中细胞因子和淋巴细胞分布进行检测,体外实验研究其数量和功能上的改变,发现这两种细胞在PBC发病中的作用和相互关系,旨在找到一条影响PBC发病的免疫机制。研究方法：通过收集,整理和分析55例PBC患者的临床资料,并用流式细胞术检测外周血中Treg和NKT细胞的比例,数量和酶联免疫实验(Enzyme-linked immunosorbent assay, ELISA)检测相关细胞因子水平。分离出样本中的单个核细胞,分选培养以通过体外实验检测其功能。研究结果：第一部分：结果表明PBC患者的Treg数量和产生白介素-10(Interleukin-10, IL-10)的能力都显著下降；PBC患者的Treg难以有效抑制T细胞的增殖和干扰素(interferon-γ, IFN-γ)的产生,Treg具有抑制T细胞对胆管上皮细胞的杀伤能力的作用,PBC患者的Treg细胞的这种抑制作用低于健康对照组；另外,PBC患者DC表面的CD80/CD86共刺激分子显著增高并且在自身抗体刺激后上升更为显著：PBC患者PBMC抑制肝星形细胞表达的平滑肌动蛋白(α-smooth muscle actin, α-SMA)能力减弱；PBC患者的Treg抑制DC表面CD86的表达能力显著减弱。PBC患者PBMC抑制肝星形细胞表达的α-SMA能力减弱。第二部分：结果发现,在PBC患者外周血中CD3+CD56+NKT细胞数量显著上升；而经典T细胞(CD3+CD56-)的数量与健康成人无明显差异。PBC患者外周血血浆中,IL-17及其相关上下游细胞因子水平均上升：胞内染色发现CD3+CD56+NKT细胞是产生IL-17最多的一群细胞；用分离的自身抗体刺激PBMC后,上清中IL-17水平显著上升,并且上升最多的来源即为CD3+CD56+NKT细胞：1L-17可以使肝星形细胞活化产生转化生长因子(Transforming growth factor-β, TGF-β)和表达α-SMA,且呈剂量依赖,PBC患者的PBMC可使肝星形细胞活化产生更高水平的TGF-β。结论：在PBC发病过程中Treg数量,比例和功能的下降,导致其抑制树突细胞活化和炎症发生的能力显著降低；与此同时,NKT细胞在自身抗原等刺激下产生IL-17,促进肝星形细胞活化。Treg的抑制作用不足以抑制NKT细胞的促炎作用,使纤维化进一步发展。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575.22
本文编号：2447396
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575.22
【参考文献】
相关期刊论文 前1条
1 ;Primary biliary cirrhosis:What do autoantibodies tell us?[J];World Journal of Gastroenterology;2010年29期
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