Treg细胞亚群参与强直性脊柱炎发病机制的研究

发布时间：2018-06-02 07:43

  本文选题：强直性脊柱炎(AS) + 调节性T细胞(Treg　； 参考：《第四军医大学》2017年博士论文

【摘要】：目的强直性脊柱炎(ankylosing spondylitis,AS)是一种主要累及脊柱、骶髂关节及外周关节的慢性免疫性炎性疾病,其根本发病机制尚不清楚。近年来的研究表明,CD4+T细胞亚群调节性T细胞(regulatory T cells,Treg cells)、辅助性T细胞17(T-helper cells 17,Th17 cells)以及辅助性T细胞1(T-helper cells 1,Th1 cells)等均可能参与AS发病。其中CD4+CD25+FoxP3+Treg细胞是体内重要的抑制性T细胞,它们可以在体内和体外抑制多种免疫细胞功能,从而维持自身免疫耐受及免疫平衡。如果实验动物Treg细胞发育异常,会发生严重的自身免疫性疾病。既往文献表明Treg细胞参与多种自身免疫性疾病的发生,如多发性硬化(multiplesclerosis,MS)与1型糖尿病(type1 diabetes,T1D)等。目前各CD4+T细胞亚群,尤其是Treg细胞与AS发生发展的关系尚未明确。为了研究Treg细胞等CD4+T细胞亚群参与AS发病的分子机制,我们进行了本研究。方法收集活动性及稳定性AS患者外周血,分离外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),应用流式细胞术从PBMCs中分选SYTOX Green-CD4+CD25-CD45RA+幼稚T细胞(na?ve T cells,Tn cells)和SYTOX Green-CD4+CD25high Treg细胞。第一部分检测Tn细胞的增殖、凋亡及向CD4+T细胞亚群Th17细胞诱导分化的能力。取分选的Tn细胞,用5(6)-羧基二乙酸荧光素琥珀酰亚胺酯(5,6-carboxyfluorescein succinimidyl ester,CFSE)标记后,加入抗-CD3/CD28磁珠培养,于细胞培养第5天收集细胞,用流式细胞仪检测并分析Tn细胞增殖;取分选的Tn细胞,加入抗-CD3/CD28磁珠培养,于细胞培养24小时和72小时分别收集细胞,用AnnexinⅤ和PI将细胞染色后,15分钟内用流式细胞仪检测并分析早期与晚期凋亡的Tn细胞比例;取PBMCs,用流式细胞仪检测IL-23R表达情况,同时取分选的Tn细胞,加入抗-CD3/CD28磁珠与Th17细胞诱导分化细胞因子重组人白细胞介素6(recombinant human interleukin 6,rhIL-6),重组人转化生长因子β1(recombinant human transforming growth factorβ1,rhTGF-β1),重组人白细胞介素1β(recombinant human interleukin 1β,rhIL-1β),重组人白细胞介素23(recombinant human interleukin23,rhIL-23)培养,于细胞培养第三天和第七天检测Th17细胞诱导分化比例。第二部分应用细胞流式染色法分析PBMCs中Treg细胞等CD4+T细胞亚群比例及其相应的平均荧光强度(mean fluorescence intensity,MFI)。取PBMCs,加入适量相应的细胞外、细胞内以及细胞核内流式抗体染色后,用流式细胞仪检测并分析Treg细胞,Th17/Th1细胞比例及相应MFI。第三部分检测Treg细胞对Tn细胞增殖的抑制功能。取分选的Tn细胞,用CFSE标记后单独或者与分选的Treg细胞共培养,同时加入抗-CD3/CD28磁珠,于细胞培养第5天收集细胞,用流式细胞仪检测并分析Treg细胞对Tn细胞增殖的抑制功能;同时用酶联免疫分析法(enzyme-linked immunosorbent assay,ELISA)法检测Treg细胞与Tn细胞共培养上清液中转化生子因子β(transforming growth factorβ,TGF-β)、颗粒酶B(Granzyme B)、白细胞介素10(interleukin 10,IL-10)和白细胞介素2(interleukin 2,IL-2)等细胞因子的浓度。第四部分检测Treg细胞IL-2信号通路(包括IL-2使用及其下游信号通路)是否异常。取分选的Tn细胞单独或者与Treg细胞共培养,同时加入抗-CD3/CD28磁珠,细胞培养第5天收集细胞,提取mRNA并反转录为cDNA,应用实时定量PCR(Real-Time quantitative polymerase chain reaction,qPCR)检测Tn细胞IL-2 mRNA表达及Treg细胞相关细胞因子m RNA表达。同时取分选的Treg细胞,在IL-2刺激后不同时间点检测其信号转导与转录激活因子5(signal transducer and activator of transcription 5,STAT5)的磷酸化水平。第五部分检测Treg细胞FoxP3基因中保守的非编码序列(the conserved non-coding DNA sequence,CNS)2表观遗传学调控是否异常。取分选的Treg细胞,提取基因组DNA,应用亚硫酸氢钠测序分析其FoxP3基因CNS2区域10个Cp G岛甲基化水平。数据采用GraphPad Prism 7.6.1软件进行统计描述和分析。p0.05为差异具有统计学意义。结果1.与健康对照者相比,活动性AS患者Tn细胞增殖、凋亡及向CD4+T细胞亚群Th17细胞诱导分化功能均没有明显改变。2.与健康对照者相比,稳定性AS患者与活动性AS患者PBMCs中Treg细胞,Th17/Th1细胞等CD4+T细胞亚群比例均没有明显改变。但活动性AS患者Treg细胞FoxP3 MFI显著下降,提示Treg细胞功能异常。3.与健康对照者相比,活动性AS患者Treg细胞不能有效抑制Tn细胞增殖,说明活动性AS患者Treg细胞功能有缺陷。4.与健康对照者相比,活动性AS患者Treg细胞与Tn细胞共培养上清液中IL-2浓度增高,同时qPCR检测结果表明AS患者Tn细胞并没有比健康对照者Tn细胞分泌更多IL-2,在共培养体系中的Tn细胞利用IL-2也并未减少,这些结果表明活动性AS患者Treg细胞可能存在IL-2利用缺陷,从而导致其使用IL-2减少。进一步检测IL-2刺激后不同时间点Treg细胞STAT5磷酸化水平,发现活动性AS患者在IL-2刺激后Treg细胞STAT5表达增加,但是其磷酸化水平却没有相应增加,这说明Treg细胞存在STAT5磷酸化不足或者不稳定。以上结果表明活动性AS患者Treg细胞IL-2信号通路确实存在异常,这可能会影响Treg细胞功能。5.活动性AS患者Treg细胞FoxP3基因表观遗传学调控异常,其CNS2区域Cp G岛甲基化水平升高。结论本课题首先研究了Treg等CD4+T细胞亚群是否参与AS发病。在这部分中我们检测了活动性AS患者CD4+T细胞亚群分化前Tn细胞的增殖,凋亡以及向CD4+T细胞亚群Th17细胞诱导分化的能力,并没有发现异常。我们进一步分析了AS患者PBMCs中CD4+T细胞亚群Treg细胞以及Th17/Th1细胞的比例及相应MFI,首次发现活动性AS患者Treg细胞FoxP3 MFI明显下降,提示Treg细胞功能异常。因此我们进一步检测了Treg细胞对Tn细胞的增殖抑制功能,结果发现活动性AS患者Treg细胞不能有效抑制Tn细胞增殖。这些结果证明活动性AS患者Treg细胞确实存在功能缺陷。在发现活动性AS患者Treg细胞存在功能缺陷的基础上,本课题进一步重点研究了Treg细胞功能缺陷的可能机制。一方面我们检测了活动性AS患者Treg细胞IL-2利用及IL-2下游信号通路是否异常,结果发现Treg细胞利用IL-2能力下降,在IL-2充足时也不能很好的利用环境中的IL-2,同时IL-2下游重要信号分子STAT5磷酸化水平相对不足或者不稳定。另一方面我们检测了活动性AS患者Treg细胞FoxP3基因表观遗传学调控,发现其CNS2区域CpG岛甲基化水平异常升高。IL-2信号通路异常与CpG岛甲基化水平升高共同引起Treg细胞抑制功能异常,最终导致AS发生。目前,靶向Treg细胞治疗自身免疫性疾病和炎性疾病方兴未艾,已取得突破性进展,目前应用不同方法恢复Treg细胞数量以及功能而治疗系统性红斑狼疮(systemic lupus erythematosus,SLE)、MS、银屑病和溃疡性结肠炎(ulcerative colitis,UC)等疾病均有报道。本研究的发现为临床上靶向恢复Treg细胞功能治疗AS提供了理论依据,也为从根本机制上治疗AS提供了更多方向与选择。
[Abstract]:Objective ankylosing spondylitis (AS) is a chronic inflammatory inflammatory disease involving the spinal, sacroiliac and peripheral joints. Its underlying pathogenesis is still unclear. Recent studies have shown that the CD4+T cell subgroup (regulatory T cells, Treg cells), auxiliary T cell 17 (T-helper 17) H17 cells) and auxiliary T cells 1 (T-helper cells 1, Th1 cells) may all participate in the pathogenesis of AS, and CD4+CD25+FoxP3+Treg cells are important inhibitory T cells in the body. They can inhibit a variety of immune cell functions in vivo and in vitro so as to maintain their immune tolerance and immune balance. If experimental animals Treg cell development Often, there will be a serious autoimmune disease. Previous literature shows that Treg cells are involved in a variety of autoimmune diseases, such as multiple sclerosis (multiplesclerosis, MS) and type 1 diabetes (type1 diabetes, T1D). At present, the relationship between the CD4+T cell subsets, especially the Treg cells and the development of AS is not clear. In order to study Treg fine The molecular mechanism of cell like CD4+T cell subsets involved in the pathogenesis of AS. We conducted this study. Methods to collect peripheral blood of active and stable AS patients, separate peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs), and to separate SYTOX Green-CD4+CD25-CD45RA+ T cells from PBMCs by flow cytometry. Cells) and SYTOX Green-CD4+CD25high Treg cells. The first part detected the proliferation, apoptosis and the ability to induce the differentiation of Tn cells to CD4+T cell subgroup Th17 cells. The selected Tn cells were labeled with 5 (6) - carboxyl fluorescein succinimide (5,6-carboxyfluorescein succinimidyl ester, CFSE) and added to the magnetic bead culture. Cultured cells were collected for fifth days in cell culture and Tn cells were detected and analyzed by flow cytometry. Tn cells were selected and cultured with anti -CD3/CD28 magnetic beads. Cells were collected for 24 hours and 72 hours in cell culture. Cells were stained with Annexin V and PI. The early and late apoptotic Tn were detected and analyzed by flow cytometry in 15 minutes. Cell ratio, PBMCs, flow cytometry was used to detect the expression of IL-23R, and the selected Tn cells were selected, and the recombinant human interleukin 6 (recombinant human interleukin 6, rhIL-6) was induced by the anti -CD3/CD28 magnetic beads and Th17 cells, and the recombinant human factor beta 1 (recombinant human transforming) was transformed. HTGF- beta 1), recombinant human interleukin 1 beta (recombinant human interleukin 1 beta, rhIL-1 beta), recombinant human interleukin 23 (recombinant human interleukin23, rhIL-23) culture, and cell culture third days and seventh days to detect the induction of differentiation ratio of Th17 cells. Second part used cell flow staining method to analyze Treg cells in PBMCs. The proportion of cell subgroups and their corresponding average fluorescence intensity (mean fluorescence intensity, MFI). Taking PBMCs, adding appropriate amount of corresponding cells, intracellular and nuclear flow antibody staining, Treg cells were detected and analyzed by flow cytometry, the proportion of Th17/Th1 cells and the corresponding MFI. third parts were used to detect the proliferation of Treg cells to Tn cells. Inhibition function. The selected Tn cells were selected and cultured separately or with the selected Treg cells, at the same time, the -CD3/CD28 magnetic beads were added to the cells. The cells were collected for fifth days in cell culture. The inhibitory function of Treg cells to the proliferation of Tn cells was detected by flow cytometry, and the enzyme linked immunoassay (enzyme-linked immunosorbent Assa) was used. Y, ELISA) method was used to detect the transformation of cell factor beta (transforming growth factor beta, TGF- beta), B (Granzyme B), interleukin 10 (interleukin 10), and interleukin 2 (2, 2) in co culture supernatant of Treg cells and Tn cells. Tn cells were selected separately or co cultured with Treg cells, and -CD3/CD28 beads were added to the cells. Cells were collected for fifth days to collect cells, and mRNA was extracted and reverse transcribed to cDNA. The real-time quantitative PCR (Real-Time quantitative polymerase chain reaction, qPCR) was used to detect the expression. And the expression of Treg cell related cytokine m RNA. Meanwhile, the selected Treg cells were selected to detect the phosphorylation level of the signal transduction and transcription activator 5 (signal transducer and activator of transcription 5, STAT5) at different time points after IL-2 stimulation. Rved non-coding DNA sequence, CNS) 2 epigenetic regulation is abnormal. Select the selected Treg cells, extract genomic DNA, and use Sodium Bisulfite sequencing to analyze the level of the 10 Cp G island methylation of the CNS2 region of the FoxP3 gene. Results 1. compared with healthy controls, the proliferation of Tn cells in active AS patients, apoptosis and the differentiation function to CD4+T cell subgroup Th17 cells were not significantly altered by.2. and healthy controls. There was no significant change in the proportion of Treg cells, Th17/Th1 cells and CD4 +T cell subgroups in PBMCs and Th17/Th1 cells in both stable AS patients and active AS patients. The Treg cell FoxP3 MFI decreased significantly in the patients with sex AS, suggesting that the Treg cell dysfunction.3. was not effective in inhibiting the proliferation of Tn cells in the active AS patients, indicating that the Treg cell function of the active AS patients was less than that of the healthy controls. At the same time, the qPCR test showed that the Tn cells in AS patients did not produce more IL-2 than the healthy control Tn cells, and the use of IL-2 in the co culture system did not decrease. These results suggest that the Treg cells in the active AS patients may have IL-2 utilization defects, which leads to the IL-2 decrease in the use of AS. Further detection of IL-2 stimulation. The level of phosphorylation of STAT5 in Treg cells at different time points showed that STAT5 expression in Treg cells increased after IL-2 stimulation in active AS patients, but the phosphorylation level did not increase correspondingly, which indicated that STAT5 phosphorylation in Treg cells was deficient or unstable. The above results showed that the IL-2 signaling pathway of Treg cells in active AS was indeed different. Often, this may affect the abnormal epigenetic regulation of the FoxP3 gene in the Treg cell of the Treg cell function.5. active AS patients, and the level of methylation in the Cp G island of the CNS2 region is elevated. Cell proliferation, apoptosis and the ability to induce differentiation to CD4+T cell subgroup Th17 cells were not found to be abnormal. We further analyzed the proportion of Treg cells and Th17/Th1 cells in the CD4+T cell subgroup of PBMCs in AS patients and the corresponding MFI, and first found that Treg fine cell FoxP3 MFI decreased significantly in the active AS patients, suggesting the function of the cells. Therefore, we further examined the proliferation inhibition of Treg cells to Tn cells, and found that Treg cells in active AS patients did not effectively inhibit the proliferation of Tn cells. These results showed that Treg cells in active AS patients did have functional defects. On the basis of the discovery of functional defects in Treg fine cells in active AS patients, this topic was introduced. One step is to focus on the possible mechanism of Treg cell dysfunction. On the one hand, we detected the IL-2 utilization of Treg cells in active AS and the abnormal downstream signal pathway of IL-2. It was found that Treg cells use the IL-2 ability to decrease, and the IL-2 in the environment is not well used when IL-2 is sufficient, and the important signal molecules downstream of IL-2 are STAT5. On the other hand, we detected the FoxP3 gene epigenetic regulation of Treg cells in active AS patients, and found that the abnormal elevated level of CpG island methylation in the CNS2 region and the increase of.IL-2 signaling pathway and the elevated level of CpG island methylation resulted in the abnormal inhibitory function of Treg cells, which eventually led to the occurrence of AS. At present, targeted Treg cells are in the ascendant for the treatment of autoimmune diseases and inflammatory diseases, and have made breakthrough progress. At present, different methods have been used to restore the number and function of Treg cells and to treat systemic lupus erythematosus (systemic lupus erythematosus, SLE), MS, psoriasis and ulcerative colitis (ulcerative colitis, UC). The findings of this study provide a theoretical basis for the clinical targeting of the recovery of Treg cell function for the treatment of AS, and also provide more directions and options for the treatment of AS from the underlying mechanism.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.23
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本文编号：1967975