IL-10对树突状细胞分化及其功能的影响

发布时间：2018-05-04 16:00

  本文选题：红斑狼疮 + 系统性　； 参考：《南京医科大学》2008年硕士论文

【摘要】： 在SLE患者体内树突状细胞(dendritic cell,DC)的研究中发现,非成熟DC更加有效的提呈抗原,成熟DC高表达MHC分子与T细胞相互作用,表现了不同的作用和在诱发疾病中的协同配合。DC对细胞因子介导的活化敏感,SLE患者血清中异常升高的成份可以诱导DC及其前体细胞产生促炎性因子,活化T/B细胞,促使T/B细胞引起自身免疫的发生,导致了SLE患者对自身抗原的反应和持续产生大量自身抗体。本课题希望从SLE患者血清及其对产生DC的影响上来部分阐述SLE的发病机制。本实验主要是针对高IL-10 SLE患者血清对单核细胞和造血干细胞来源的DC的影响进行研究。 本实验用ELISA法检测SLE患者和正常人血清中的IL-10、IL-6、IFN-α,以筛选高IL-10 SLE患者血清。同时检测部分病人的抗dsDNA抗体、补体(C3、C4)和24小时尿蛋白。应用GM-CSF+IL-4+TNF-α体系诱导培养DC,在该体系中加入外源性IL-10(30pg/ml)。根据ELISA法检测到的IL-10、IL-6、IFN-α浓度,选择单独IL-10增高的SLE患者血清参与诱导脐血CD34~+造血干细胞分化为DC。用流式细胞术检测DC的表型和DC刺激的活化T细胞胞内细胞因子,CCK-8法检测DC刺激同种异体T淋巴细胞增殖能力,ELISA法检测DC分泌细胞因子水平、与DC共培养的T细胞分泌细胞因子的水平。 结果发现,以超过正常人IL-10浓度95%参考值范围为IL-10异常增高标准(＞8.12pg/ml),则在收集的94例SLE患者中50例IL-10浓度增高,占整个SLE患者的53.20%。将这50例血清的浓度进行分析得到IL-10平均浓度为33.77±41.94pg/ml,其中有16份血清仅表现IL-10异常增高,IL-6、IFN-α均在正常范围内。故选择外源性IL-10的实验浓度为30pg/ml。当应用GM-CSF+IL-4+TNF-α诱导培养DC时,如加入外源性IL-10 30pg/ml作为实验观察因素,与正常诱导DC相比,所诱导的DC HLA-DR、CD86、CD80、CD83的表达阳性率均下降并具有统计学意义;对其刺激同种异体淋巴细胞增殖能力的研究结果显示,刺激细胞:效应细胞为1:10比例组中IL-10浓度为30pg/ml的诱导组诱导的DC刺激淋巴细胞增殖的能力显著降低;该种DC分泌细胞因子的水平有变化,表现有高分泌IL-10,低分泌IL-12p40和IFN-γ的趋势。然而,在高IL-10的SLE患者血清参与诱导干细胞来源的DC的实验中,DC的表型、刺激同种异体T淋巴细胞增殖和分化的能力、DC分泌IL-12p40、IL-10、IFN-γ水平,并未显示出与常规诱导培养的DC统计学的差异。 以上实验结果证实,30pg/ml的外源性IL-10可使单核细胞来源的DC的MHC-Ⅱ类分子和共刺激分子的表达以及JL-12p40和IFN-γ的分泌水平受到抑制,并使其刺激同种异体T细胞增殖能力下降。但是IL-10异常增高的SLE患者血清在诱导CD34~+造血干细胞分化发育为DC时由于作用的细胞不同、SLE患者血清中存在IL-6、IFN-α以外的其它复杂因素的影响,并不能相同地呈现明显抑制作用。 总之,IL-10在SLE发病机制中起到了重要作用,但是如协同作用的因素不同及作用的细胞不同,可能得到不同的结果。
[Abstract]:In the study of dendritic cells (DC) in SLE patients, it was found that immature DCs were more effective in presenting antigens, and mature DCs highly expressed MHC molecules interacting with T cells. Different effects and synergistic effects on cytokine mediated activation of SLE patients with abnormal elevated components of serum can induce DC and its precursor cells to produce pro-inflammatory cytokines and activate T / B cells. It causes T / B cells to cause autoimmunity, which leads to the response of SLE patients to autoantigen and the continuous production of a large number of autoantibodies. This thesis hopes to explain the pathogenesis of SLE from the serum of SLE patients and its effect on DC production. The aim of this study was to investigate the effects of serum from patients with high IL-10 SLE on monocytes and DC derived from hematopoietic stem cells. In order to screen the serum of patients with high IL-10 SLE, IL-10 and IL-6 IFN- 伪 in the serum of SLE patients and normal subjects were detected by ELISA method. At the same time, anti dsDNA antibody, C 3 C 4 and 24 hour urine protein were detected in some patients. DCC was induced by GM-CSF IL-4 TNF- 伪 system, and exogenous IL-10g / ml was added into the system. According to the concentration of IL-10 and IL-6 IFN- 伪 detected by ELISA method, the serum of SLE patients with elevated IL-10 was selected to participate in inducing CD34 ~ hematopoietic stem cell differentiation from cord blood to DC. The phenotypes of DC and the level of cytokines secreted by DC were detected by flow cytometry and the levels of cytokines secreted by DC stimulated by activated T cells were detected by CCK-8 method and the ability of DC to stimulate the proliferation of allogeneic T lymphocytes. Levels of cytokines secreted by T cells co-cultured with DC. The results showed that the reference range of 95% IL-10 concentration was the standard of abnormal increase of IL-10 (> 8.12 PG / ml), then 50 of 94 SLE patients (53.20% of the total SLE patients) had increased IL-10 concentration. The average concentration of IL-10 was 33.77 卤41.94 PG / ml, 16 of which only showed abnormal increase of IL-6 and IFN- 伪 in the normal range. Therefore, the experimental concentration of exogenous IL-10 was 30 PG / ml. When DC was induced by GM-CSF IL-4 TNF- 伪, if exogenous IL-10 30pg/ml was added as experimental observation factor, the positive rate of HLA-DRN CD86 CD80 CD80 CD83 was significantly lower than that of normal DC. The results of the study on the ability of stimulating the proliferation of allogeneic lymphocytes showed that the ability of stimulating cells to stimulate lymphocyte proliferation was significantly decreased in the group with IL-10 concentration of 30pg/ml in the 1:10 proportion of effector cells. The level of cytokines secreted by this kind of DC changed, showing the tendency of high secretion of IL-10 and low secretion of IL-12p40 and IFN- 纬. However, the ability of DC to stimulate the proliferation and differentiation of allogeneic T lymphocytes and the secretion of IL-12p40 / IL-10IFN- 纬 by the serum of SLE patients with high IL-10 were involved in the induction of stem cell-derived DC, and the ability of DC to stimulate the proliferation and differentiation of allogeneic T lymphocytes. There was no statistical difference between DC culture and conventional induction. The results showed that exogenous IL-10 of 30pg / ml inhibited the expression of MHC- 鈪，

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