中性粒细胞胞外诱捕网激活单核细胞在ANCA相关性血管炎的作用

发布时间：2018-06-17 06:12

本文选题：抗中性粒细胞胞浆抗体相关性血管炎 + 中性粒细胞胞外诱捕网　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景与目的:抗中性粒细胞胞浆抗体(anti-neutrophil cytoplasmic antibodies,ANCA)相关血管炎(ANCA associated vasculitis,AAV)属于自身免疫系统性小血管炎,病变常累及肾脏,主要表现为寡免疫复合物局灶节段坏死性肾小球肾炎(pauci-immune focal segmental necrotizing glomerulonephritis,PiFSGN),可伴新月体形成。在现有免疫抑制剂治疗基础上病情反复进展,最终发展至终末期肾衰竭。研究者发现针对B淋巴细胞的药物治疗仍有很高复发率,尤其PR3-ANCA(+)患者高达50%,说明其发病机制并没完全弄清楚。因此,需更加深入地研究AAV的发病机理,以寻找更有效的治疗方案。单核细胞(Monocyte,Mo)来源于骨髓造血干细胞,浸润于组织后可分化为巨噬细胞和树突状细胞,它参与病原微生物及内源性物质的吞噬、抗原提呈、T淋巴细胞功能调节等重要过程,单核巨噬细胞具有强大吞噬功能,吞噬异常物质后可分泌促炎因子或抑炎因子,在维持机体稳态扮演重要作用。研究表明单核细胞异常激活分泌大量炎症介质参与多种疾病病理生理过程,包括AAV。在AAV节段坏死性肾小球肾炎早期阶段以单核细胞浸润为主,表明单核细胞异常激活可能在AAV肾损害扮演了启动作用。ANCA相关性血管炎最显著血清学特征是患者血清可检测出抗中性粒细胞胞浆抗体,它可激活中性粒细胞脱颗粒及产生活性氧,释放中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)。NETs的形成释放了胞浆自身抗原DNA、髓过氧化物酶(myeloperoxidase,MPO)、蛋白水解酶3(Protease3,PR3)及抗微生物肽LL37(Cathelicidin LL37,LL37)等物质,这些自身抗原暴露于免疫监视中诱导自身抗体形成及自身免疫性疾病。研究表明:异常形成的NETs参与多种自身免疫性疾病,包括诱发ANCA相关性血管炎。异常分泌的炎症介质一直被证实参与AAV病理损伤,如白细胞介素1β(interleukin-1β,IL-1β)、干扰素α(interferon-α,IFN-α)、白细胞介素6(interleukin-6,IL-6)等。但NETs对外周血单核细胞激活分泌炎症介质的影响以及意义尚不完全清楚。本实验拟观察NETs对单核细胞分泌炎症介质IL-1β、IFN-α、TNF-α、IL-6、MCP-1、白细胞介素10(interleukin-10,IL-10)、白细胞介素12(interleukin-12,IL-12)的影响,探讨NETs对单核细胞活化的影响。方法:1、分别抽取6名健康志愿者(研究生同学)外周血各55ml,共后续实验。2、每人取5ml外周血,采用Polymorphprep分离液密度梯度离心法分离中性粒细胞,DAPI染色观察细胞核形态,Beckman流式细胞仪检测中性粒细胞提取纯度。纯化的中性粒细胞调整密度一致后分为对照组和PMA组两组,对照组采用等量PBS与中性粒细胞孵育,PMA组采用等量佛波脂(PMA,终浓度30ng/ml)与中性粒细胞孵育4h诱导NETs形成,吸弃含PMA的培养上清并洗弃残留PMA后留存于培养板底的即为NETs,DAPI荧光染色,Leica激光共聚焦显微镜观察NETs形成效率。3、每人取50ml外周血,采用Ficoll密度梯度离心法分离单个核细胞,免疫磁珠法(Stem Cells人CD14正选试剂盒)从外周血单个核细胞分离提取单核细胞,Beckman流式细胞仪检测单核细胞提取纯度。纯化的单核细胞调整密度一致后分为对照组及NETs组,对照组采用等量细胞培养基与单核细胞培养体系,NETs组为等量上诉实验诱导的培养板底部NETs与单核细胞共培养体系,孵育12小时后,收集培养上清存于-80oC冰箱。LEGENDplex人炎症因子试剂盒检测各组培养上清IL-1β、IFN-α、TNF-α、IL-6、MCP-1、IL-10、IL-12的浓度。4、数据分析:数据以均值±标准差表示,数据分析采用SPSS 18.0统计分析软件进行统计学处理。两样本均数比较采用t检验,P0.05表示差异有统计学意义。结果:1、Polymorphprep分离液密度梯度离心法分离提取健康志愿者外周血中性粒细胞,DAPI染色,细胞核呈分叶核,流式细胞仪鉴定纯度为98%。纯化的中性粒细胞分为对照组和PMA组两组,孵育4小时后,DAPI染色行激光共聚焦显微镜观察,对照组中性粒细胞核仍保持分叶核结构,而PMA组分叶核结构破坏,可见丝状DNA即NETs形成。2、CD14正选试剂盒提取单核细胞纯度为95.3%。3、NETs对单核细胞分泌IL-1β的影响:NETs组与对照组IL-1β浓度分别为(97.60±60.11)pg/m L、1.17pg/m L,与对照组相比,NETs组单核细胞分泌IL-1β水平显著升高(P0.05)。4、NETs对单核细胞分泌IFN-α的影响:NETs组与对照组IFN-α浓度分别为(208.90±77.38)pg/m L、1.17 pg/mL,与对照组相比,NETs组单核细胞分泌IFN-α水平显著升高(P0.01)。5、NETs对单核细胞分泌TNF-α的影响:NETs组与对照组TNF-α浓度分别为(218.88±86.27)pg/mL、0.17 pg/m L,与对照组相比,NETs组单核细胞分泌TNF-α的水平显著升高(P0.01)。6、NETs对单核细胞分泌MCP-1的影响:NETs组与对照组MCP-1浓度分别为(1063.25±740.01)pg/mL、(157.16±3.86)pg/m L,与对照组相比,NETs组单核细胞分泌MCP-1水平显著升高(P0.05)。7、NETs对单核细胞分泌IL-6的影响:NETs组与对照组IL-6浓度分别为(67.76±15.51)pg/mL、(2.76±0.47)pg/m L,与对照组相比,NETs组单核细胞分泌IL-6的水平显著升高(P0.01)。8、NETs对单核细胞分泌IL-10的影响:NETs组与对照组IL-10浓度分别为1.68±0.58 pg/mL、1.15 pg/mL,两组IL-10水平无显著差异。9、NETs对单核细胞分泌IL-12的影响:NETs组与对照组IL-12浓度分别为1.30pg/m L、1.30 pg/mL,两组IL-12水平无显著差异。结论NETs能激活单核细胞,诱导IFN-α、IL-1β、IL-6、TNF-α、MCP-1炎症介质生成,可能参与AAV发病。
[Abstract]:Background and objective: the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis (ANCA associated vasculitis, AAV) belongs to the autoimmune small vasculitis, and the lesions often involve the kidneys. The main manifestations are oligo immune complex focal segmental necrotizing glomerulonephritis (pauci-immune focal segmental). Necrotizing glomerulonephritis, PiFSGN), may be associated with crescent formation. On the basis of existing immunosuppressive therapy, the disease progresses repeatedly and eventually develops to end-stage renal failure. The researchers found that the drug treatment for B lymphocytes still has a high recurrence rate, especially in the PR3-ANCA (+) patients with up to 50%, indicating that the pathogenesis is not completely clear. Therefore, it is necessary to study the pathogenesis of AAV more deeply in order to find a more effective treatment scheme. Monocyte (Monocyte, Mo) is derived from bone marrow hematopoietic stem cells, and can differentiate into macrophages and dendritic cells after infiltrating tissue. It participates in the phagocytosis of pathogenic microbes and endogenous substances, antigen presentation, and function regulation of T lymphocytes. In the process, mononuclear macrophages have strong phagocytosis and can secrete proinflammatory or anti inflammatory factors after phagocytosis, and play an important role in maintaining the homeostasis of the body. The study shows that the abnormal activation and secretion of a large number of inflammatory mediators are involved in the pathophysiological process of various diseases, including AAV. in AAV segment necrotizing glomerulonephritis. At the stage of monocyte infiltration, it is suggested that abnormal activation of mononuclear cells may play the most significant serological characteristic of.ANCA associated vasculitis in AAV renal damage, which can detect anti neutrophil cytoplasmic antibodies in the patient's serum, which activates neutrophils and produces living oxygen, releasing neutrophils out of cell induction. The formation of neutrophil extracellular traps (NETs).NETs releases cytoplasmic autoantigens DNA, myeloperoxidase (myeloperoxidase, MPO), protein hydrolase 3 (Protease3, PR3), and antimicrobial peptide LL37 (Cathelicidin), and other substances. These self antigens are exposed to immune surveillance to induce autoantibody formation and autoimmune Disease. Studies have shown that abnormal formation of NETs is involved in a variety of autoimmune diseases, including inducing ANCA associated vasculitis. Abnormal secretion of inflammatory mediators has been proven to be involved in AAV pathological damage, such as interleukin 1 beta (interleukin-1 beta, IL-1 beta), interferon alpha (interferon- a, IFN- a), interleukin 6 (interleukin-6, IL-6), etc. but NETs. The effect and significance of the activation and secretion of inflammatory mediators in peripheral blood mononuclear cells were not completely clear. The effect of NETs on the secretion of IL-1 beta, IFN- a, TNF- a, IL-6, MCP-1, interleukin 10 (interleukin-10, IL-10), interleukin 12 (interleukin-12, IL-12) in monocytes and the activation of mononuclear cells in the mononuclear cells were investigated. Methods: 1, 1, 6 healthy volunteers (postgraduate students) were extracted from the peripheral blood of the peripheral blood respectively, followed by.2, each of them was taken 5ml peripheral blood, Polymorphprep separation liquid density gradient centrifugation was used to separate neutrophils, DAPI staining was used to observe the nucleus morphology, and the purity of neutrophils was detected by Beckman flow cytometry. The purified neutrophils were detected. The control group was divided into two groups: the control group and the PMA group, the control group was incubated with equal amount of PBS and neutrophils. The PMA group adopted the equal amount of PMA (PMA, final concentration 30ng/ml) to incubate 4H with neutrophils to induce NETs formation. The culture supernatant containing PMA and the residue remaining at the bottom of the culture plate were NETs, DAPI fluorescent staining, Leica laser confocal microscope was used to observe the NETs formation efficiency.3, each person took 50ml peripheral blood, Ficoll density gradient centrifugation was used to separate mononuclear cells. The immunomagnetic bead method (Stem Cells CD14 positive Kit) was isolated and extracted from mononuclear cells from peripheral blood, and the purity of mononuclear cells was detected by the Beckman flow cytometry. The control group was divided into the control group and the NETs group, the control group adopted the equal cell culture medium and the monocyte culture system, the NETs group was the co culture system of NETs and mononuclear cells at the bottom of the culture plate, which was induced by the equal quantity appeal experiment. After incubation for 12 hours, the culture supernatant was collected and stored in the.LEGENDplex human inflammatory factor kit of the -80oC refrigerator. The concentration of IL-1 beta, IFN- a, TNF- a, IL-6, MCP-1, IL-10, IL-12 was measured in each group. The data were analyzed with mean mean standard deviation, and the data analysis was statistically processed with SPSS 18 statistical analysis software. The average number of two samples was compared to t test, P0.05 indicated that the difference was statistically significant. Results: 1, Polymorphprep separation liquid density. The peripheral blood neutrophils in healthy volunteers were separated and extracted by gradient centrifugation. DAPI staining, the nucleus was lobular nucleus, and the neutrophils purified by 98%. were divided into two groups, the control group and the PMA group. After 4 hours incubation, the DAPI staining was observed by laser confocal microscope, and the nucleus of the neutrophils still kept the lobular nucleus in the control group. The structure of the PMA group was destroyed, and the.2 was formed by the filamentous DNA, NETs, the purity of the mononuclear cells was 95.3%.3, and the NETs effect on the secretion of IL-1 beta in the mononuclear cells: the concentration of IL-1 beta in the NETs group and the control group was (97.60 + 60.11) pg/m L, respectively, and the level of the secretory beta of the mononuclear cells was significantly higher than that of the control group. The effect of high (P0.05).4 and NETs on the secretion of IFN- alpha by mononuclear cells: the concentration of IFN- alpha in NETs group and control group was (208.90 + 77.38) pg/m L, 1.17 pg/mL respectively. Compared with the control group, the secretion of IFN- alpha level in the NETs group was significantly higher than that in the control group (P0.01). 7) pg/mL, 0.17 pg/m L, compared with the control group, the level of TNF- alpha secreted by mononuclear cells in the NETs group increased significantly (P0.01).6, and NETs had a significant effect on the secretion of MCP-1 in mononuclear cells: the MCP-1 concentration in the NETs group and the control group was (1063.25 + 740.01) pg/mL, (157.16 + 3.86), respectively. 7, the effect of NETs on the secretion of IL-6 by mononuclear cells: the concentration of IL-6 in NETs group and control group was (67.76 + 15.51) pg/mL and (2.76 + 0.47) pg/m L respectively. Compared with the control group, the level of IL-6 in the mononuclear cells of NETs group increased significantly (P0.01).8, and NETs was 1.68 + 0.58, 1.15, respectively. G/mL, there was no significant difference in the level of IL-10 between the two groups of.9, and the effect of NETs on the secretion of IL-12 in mononuclear cells: the concentration of IL-12 in the NETs group and the control group was 1.30pg/m L, 1.30 pg/mL, and IL-12 levels in the two groups.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.2;R692

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