滤泡辅助性T细胞及其亚型在IgG4相关性疾病中的研究

发布时间：2018-03-20 23:15

本文选题：IgG4-相关性疾病　切入点：滤泡辅助性T细胞　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：目的:研究IgG4相关性疾病(IgG4-related disease,IgG4-RD)中滤泡辅助性T(Tfh)细胞及其亚型的表达及功能。方法:收集初治IgG4-RD患者的外周血及新鲜受累组织细胞悬液,同时收集性别、年龄相匹配的健康人作为对照,运用流式染色技术研究IgG4-RD患者外周血和组织中Tfh细胞及其亚型的表达;运用RT-PCR技术比较IgG4-RD患者和健康对照组中与Tfh表达密切相关的重要转录因子Bcl-6和Blimp-1以及与其功能密切相关的重要细胞因子IL-21在外周血中转录水平上表达的差异;运用免疫组化和免疫荧光技术定位IL-21和(CD4+CXCR5+)Tfh细胞在IgG4-RD受累组织中的分布;进一步通过将IgG4-RD患者的循环Tfh(cTfh)及其亚型分别与健康正常人的B细胞共培养,探索Tfh及其亚型在IgG4-RD中对B细胞增殖、细胞损伤、凋亡及分化分泌抗体的影响。结果:IgG4-RD患者外周血和受累组织中的(CD4+CXCR5+)T、(CD4+CXCR5+ICOS+)T、(CD4+CXCR5+PD-1+)T、(CD4+CXCR5+ICOS+PD-1+)T细胞群均较正常人比明显高表达,且这些细胞群在组织中的表达水平远远高于其在外周血中的表达,其中,(CD4+CXCR5+ICOS+PD-1+)T以及PD-1在(CD4+CXCR5+ICOS+)T细胞群中的表达水平与IgG4-RD患者的血清IgG、IgG4、IgG4/IgG水平及受累器官数目均呈显著正相关性,后者还与高分泌IgG4的浆母细胞(CD19+CD24-CD38hi)显著正相关。决定Tfh细胞表达的重要转录因子Bcl-6在IgG4-RD外周血CD4+T细胞中的转录水平较正常人无差异,而与Tfh功能相关的重要细胞因子IL-21的mRNA转录水平在IgG4-RD外周血中明显增高,且与IgG4、IgG、IgG4/IgG的水平呈显著正相关性。在IgG4-RD患者受累组织中,(CD4+CXCR5+)Tfh细胞和IL-21均显示为高表达,主要定位于腺体细胞或异位生发中心周围,部分为弥漫分布;较正常人而言,IgG4-RD的cTfh细胞更能促进B细胞增殖和加重细胞损伤,并抑制其凋亡,并且能更多地促使naive B细胞向(CD19+CD27+IgD-)switched(S)memory B细胞和(CD19+CD24-CD38hi)B亚群分化,从而产生分泌更多的IgG4抗体。进一步通过对cTfh细胞亚型的研究,我们发现,三种cTfh亚型在IgG4-RD中均具有很强的促进B细胞增殖并向S memory B细胞分化,而抑制其凋亡的功能,但主为cTfh1和cTfh2而非cTfh17在IgG4-RD中高表达,cTfh1和cTfh2均能促进B细胞向(CD19+CD24-CD38hi)B细胞亚群分化并产生分泌更多的IgG4抗体,以cTfh2细胞的能力更为显著。结论:Tfh在IgG4-RD的发病中起重要作用,(CD4+CXCR5+ICOS+)T细胞中PD-1的表达水平在促进GC中B细胞向分泌IgG4为主的浆细胞分化中起着关键的作用。主因cTfh细胞分泌的IL-21在IgG4-RD的发病机制中可能扮演着重要的角色。此外,IgG4-RD中的Tfh细胞功能异常活化,可显著促进B细胞的增殖、加重细胞损伤,抑制其凋亡,促进naive B细胞向switched(S)memory B细胞和(CD19+CD24-CD38hi)B 细胞亚群分化。其中,cTfh1 和 cTfh2 细胞在 IgG4-RD中高表达,具有促进IgG4-RD中B细胞向浆细胞分化及分泌IgG4抗体而抑制其凋亡的功能,且以cTfh2细胞最为显著。
[Abstract]:Objective: to study the expression and function of follicular helper TFH cells and their subtypes in IgG4-related disease (IgG4-RD) of patients with IgG4. Methods: peripheral blood and fresh tissue cell suspensions were collected from patients with IgG4-RD. The expression of Tfh cells and their subtypes in peripheral blood and tissues of IgG4-RD patients was studied by flow cytometry in age-matched healthy controls. RT-PCR technique was used to compare the expression of Bcl-6 and Blimp-1, which were closely related to the expression of Tfh, and the important cytokines IL-21, which were closely related to the expression of Tfh, in the peripheral blood of patients with IgG4-RD and healthy controls. Immunohistochemical and immunofluorescence techniques were used to locate the distribution of IL-21 and CD4 CXCR5 tfh cells in the affected tissues of IgG4-RD, and to further co-culture the circulating TfhcTfh cells of IgG4-RD patients and their subtypes with B cells of healthy people. To explore the effects of Tfh and its subtypes on the proliferation and damage of B cells in IgG4-RD. Results the expression of CD4 CXCR5 in peripheral blood and involved tissues was significantly higher than that in normal controls. And the expression level of these cell groups in tissues is much higher than that in peripheral blood. The expression of CD4 CXCR5 ICOS PD-1 T and PD-1 in the CD4 CXCR5 ICOS T cell group were positively correlated with the serum IgG4 / IgG4 / IgG4 / IgG4 / IgG4 / IgG4 / IgG4 and the number of affected organs in patients with IgG4-RD. The latter was also positively correlated with CD19 CD24-CD38hi. the transcription level of Bcl-6, an important transcription factor that determines the expression of Tfh cells, in IgG4-RD peripheral blood CD4 T cells was not different from that in normal controls. However, the mRNA transcription level of IL-21, an important cytokine related to the function of Tfh, was significantly increased in the peripheral blood of IgG4-RD, and positively correlated with the level of IgG4 / IgG4 / IgG4.In the involved tissues of IgG4-RD patients, both the CD4 CXCR5 Tfh cells and IL-21 were highly expressed. The cTfh cells located mainly around glandular cells or ectopic germinal centers, and some of them were diffuse, and the cTfh cells of IgG4-RD could promote the proliferation of B cells, aggravate the damage of B cells and inhibit the apoptosis of B cells. And it can induce naive B cells to differentiate into CD19 CD27 IgD-)switched(S)memory B cells and CD19 CD24-CD38hi)B subsets, thus producing more IgG4 antibodies. Further studies on cTfh cell subtypes show that, All of the three cTfh subtypes have the function of promoting the proliferation of B cells and differentiating into S memory B cells in IgG4-RD, while inhibiting the apoptosis of B cells. However, the high expression of cTfh1 and cTfh2 in IgG4-RD was mainly composed of cTfh1 and cTfh2 instead of cTfh17, which promoted the differentiation of B cells into subsets of CD19 CD24-CD38hi)B cells and produced more IgG4 antibodies. Conclusion cTfh2 plays an important role in the pathogenesis of IgG4-RD. The expression of PD-1 in CD4 CXCR5 ICOS T cells plays a key role in promoting the differentiation of B cells into plasmacytes mainly secreting IgG4 in GC. IL-21 secreted by cTfh cells may play an important role in the pathogenesis of IgG4-RD. In addition, the function of Tfh cells in IgG4-RD is abnormal. It can significantly promote the proliferation of B cells, aggravate the injury of B cells, inhibit the apoptosis of B cells, promote the differentiation of naive B cells into switched(S)memory B cells and the subsets of CD19 CD24-CD38hi)B cells, in which the expression of Tfh1 and cTfh2 cells in IgG4-RD is high. It can inhibit the apoptosis of IgG4-RD by promoting the differentiation of B cells into plasma cells and secreting IgG4 antibody, especially cTfh2 cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.2

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