B细胞免疫紊乱在原发性胆汁性胆管炎中的研究

发布时间：2018-05-22 18:25

本文选题：原发性胆汁性胆管炎 + B细胞　；参考：《南方医科大学》2017年博士论文

【摘要】：背景原发性胆汁性胆管炎(primary biliary cholangitis,PBC)的血清学特点是存在高滴度和高特异性的的抗线粒体抗体(antimitochondrial antibodies,AMA),抗线粒体抗体的靶抗原位于丙酮酸脱氢酶复合体E2亚基的硫辛酰区。抗线粒体抗体阳性是罹患PBC的征兆,目前普遍认为在PBC患者出现临床症状或生化学检查异常之前数年即可在血清中检测到AMA。根据目前的检测方法,超过95%的PBC患者AMA检测阳性,而在普通人群中其阳性率较低,约为0.16%-1%。自身抗体在PBC中的致病和临床意义尚不完全清楚。熊去氧胆酸(ursodeoxycholic acid,UDCA)被批准用于PBC治疗,UDCA治疗1或2年后的血清碱性磷酸酶、谷草转氨酶、胆红素和白蛋白水平可以预测无肝移植生存,而巴黎-Ⅰ标准具有很好的预测作用。虽然PBC患者不存在整体的免疫缺陷,但其B细胞功能紊乱,血清中存在大量免疫球蛋白M,且B细胞对胞嘧啶磷酸鸟甘的应答增强。B细胞免疫紊乱的机制尚不清楚。目的和意义本研究以尚未接受UDCA治疗的横向以及接受UDCA治疗的纵向PBC患者为研究对象,分析B细胞免疫紊乱的特点,通过研究不同亚型AMA滴度的变化、B细胞亚群频数、细胞因子微环境的作用及重要的辅助性T细胞的功能解析B细胞免疫紊乱在PBC病理机制中的作用,挖掘对疾病预测或治疗具有重要价值的生物标志物,可对未来PBC的免疫治疗提供新的线索。方法纳入66例PBC患者,其中25例接受UDCA治疗,并纳入52例健康对照和41例慢性乙型病毒性肝炎患者作疾病对照。通过酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测血清中 IgG-AMA、IgM-AMA和IgA-AMA滴度以及IL-4、IL-6、IL-10和IL-21、CXCL13等细胞因子水平,采用流式细胞术检测CD19+B淋巴细胞亚群和CXCR5+CD4+T淋巴细胞频数,通过共培养实验、胞内因子染色和增殖实验等分析B淋巴细胞和CXCR5+CD4+T淋巴细胞以及IL-21等细胞因子的功能,通过免疫组化染色检测肝内浸润淋巴细胞的定位和IL-21、CXCL13的表达。将不同亚型AMA的滴度、淋巴细胞频数和细胞因子水平与UDCA应答和血清碱性磷酸酶、谷草转氨酶、胆红素水平进行相关性分析。结果对UDCA治疗产生生化学应答的患者血清IgG-AMA滴度显著下降(P=0.005)。与健康对照相比,PBC患者外周血总CD19+B细胞和浆母细胞的频数显著升高(P=0.001;P=0.001)。PBC患者血清和肝内IL-21表达均增多(P0.001;P0.001),其能显著促进B细胞增殖、STAT3磷酸化和AMA产生。值得注意的是,在重组线粒体抗原PDC-E2刺激下,PBC患者外周血中扩增和活化的CXCR5+CD4+T细胞能分泌大量IL-21(P=0.001),而分选的CXCR5+CD4+T细胞与自身CD19+B细胞共培养能促进B细胞分泌AMA。另外,发现CXCR5+细胞的趋化因子CXCL13在PBC患者肝内汇管区周围表达增多,同时伴有CD4+、CXCR5+、CD19+和CD38+细胞浸润。结论趋化因子CXCL13促进CD19+B细胞和CXCR5+CD4+T细胞在PBC患者肝内聚集,CXCR5+CD4+T细胞通过分泌IL-21促进CD19+B细胞分泌大量的AMA。本研究的发现反映了 PBC患者体液免疫紊乱的特点,也有助于开发潜在的治疗策略。
[Abstract]:Background the serological characteristics of primary biliary cholangitis (PBC) are anti mitochondrial antibodies (antimitochondrial antibodies, AMA) with high titer and high specificity. The target antigen of anti mitochondrial antibody lies in the sulfonyl region of the pyruvate dehydrogenase complex E2 subunit. The anti mitochondrial antibody positive is PBC. The symptoms of AMA. are generally believed to be detected in serum for several years before PBC patients have clinical symptoms or abnormal chemical tests. According to the current detection methods, more than 95% of PBC patients are positive for AMA detection, while the positive rate is low in the general population, which is about the pathogenicity and clinical significance of 0.16%-1%. autoantibodies in PBC. It is clear that ursodeoxycholic acid (UDCA) is approved for PBC treatment. Serum alkaline phosphatase (ALP), gluten transaminase, bilirubin and albumin levels can predict no liver transplantation for 1 or 2 years after UDCA treatment, while the Paris - I standard has good predictive use. Although PBC patients do not have overall immune deficiency, but their B Cell dysfunction, a large number of immunoglobulin M exists in serum, and the mechanism of B cell response to cytosine phosphate Gump response to.B cell immune disorders is unclear. Purpose and significance of this study was to analyze the characteristics of B cell immune disorders in horizontal and longitudinal PBC patients who had not yet received UDCA treatment and received UDCA treatment. By studying the changes in the AMA titer of different subtypes, the frequency of the subsets of B cells, the role of the cytokine microenvironment and the function of the important auxiliary T cells, the role of the B cell immune disorder in the pathological mechanism of PBC is analyzed, and the biomarkers of important value for the prediction or treatment of the disease can be found, which can provide a new line for the immunotherapy of the future PBC. The method was included in 66 patients with PBC, of which 25 were treated with UDCA, and 52 healthy controls and 41 patients with chronic hepatitis B were treated as disease control. The serum IgG-AMA, IgM-AMA, IgA-AMA titer and IL-4, IL-6, IL-10, etc. were detected by enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA). CD19+B lymphocyte subsets and CXCR5+CD4+T lymphocyte frequency were detected by flow cytometry, and the function of B lymphocyte and CXCR5+CD4+T lymphocyte and IL-21 and other cytokines were analyzed by co culture experiment, intracellular factor staining and proliferation test. Immunocytochemical staining was used to detect infiltrating lymphocytes in the liver. Location and expression of IL-21, CXCL13. Correlation analysis of the titer, lymphocyte frequency and cytokine level of different subtypes of AMA with UDCA response and serum alkaline phosphatase, glutamic aminotransferase, bilirubin level. Results the serum IgG-AMA titer of patients with biochemical responses to UDCA decreased significantly (P=0.005). The frequency of total CD19+B cells and plasma mother cells in peripheral blood of PBC patients increased significantly (P=0.001; P=0.001), the expression of IL-21 in serum and liver increased (P0.001; P0.001) in.PBC patients, which could significantly promote B cell proliferation, STAT3 phosphorylation and AMA production. The activated CXCR5+CD4+T cells can secrete a large number of IL-21 (P=0.001), and the co culture of the selected CXCR5+CD4+T cells and their own CD19+B cells can promote the secretion of AMA. in B cells. It is found that the chemokine CXCL13 in CXCR5+ cells is increased around the intrahepatic sinks of the PBC patients, accompanied by CD4+, CXCR5+, and infiltration. SubCXCL13 promotes the aggregation of CD19+B cells and CXCR5+CD4+T cells in the liver of PBC patients. The discovery of CXCR5+CD4+T cells to secrete a large number of AMA. based on the secretion of IL-21 through the secretion of IL-21 reflects the characteristics of the humoral immunity disorder of the PBC patients, and also helps to develop potential therapeutic strategies.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.2

【相似文献】