本文選題：抗中性粒細胞胞漿抗體相關(guān)性血管炎 + 中性粒細胞胞外誘捕網(wǎng)　； 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：背景與目的:抗中性粒細胞胞漿抗體(anti-neutrophil cytoplasmic antibodies,ANCA)相關(guān)血管炎(ANCA associated vasculitis,AAV)屬于自身免疫系統(tǒng)性小血管炎,病變常累及腎臟,主要表現(xiàn)為寡免疫復合物局灶節(jié)段壞死性腎小球腎炎(pauci-immune focal segmental necrotizing glomerulonephritis,PiFSGN),可伴新月體形成。在現(xiàn)有免疫抑制劑治療基礎(chǔ)上病情反復進展,最終發(fā)展至終末期腎衰竭。研究者發(fā)現(xiàn)針對B淋巴細胞的藥物治療仍有很高復發(fā)率,尤其PR3-ANCA(+)患者高達50%,說明其發(fā)病機制并沒完全弄清楚。因此,需更加深入地研究AAV的發(fā)病機理,以尋找更有效的治療方案。單核細胞(Monocyte,Mo)來源于骨髓造血干細胞,浸潤于組織后可分化為巨噬細胞和樹突狀細胞,它參與病原微生物及內(nèi)源性物質(zhì)的吞噬、抗原提呈、T淋巴細胞功能調(diào)節(jié)等重要過程,單核巨噬細胞具有強大吞噬功能,吞噬異常物質(zhì)后可分泌促炎因子或抑炎因子,在維持機體穩(wěn)態(tài)扮演重要作用。研究表明單核細胞異常激活分泌大量炎癥介質(zhì)參與多種疾病病理生理過程,包括AAV。在AAV節(jié)段壞死性腎小球腎炎早期階段以單核細胞浸潤為主,表明單核細胞異常激活可能在AAV腎損害扮演了啟動作用。ANCA相關(guān)性血管炎最顯著血清學特征是患者血清可檢測出抗中性粒細胞胞漿抗體,它可激活中性粒細胞脫顆粒及產(chǎn)生活性氧,釋放中性粒細胞胞外誘捕網(wǎng)(neutrophil extracellular traps,NETs)。NETs的形成釋放了胞漿自身抗原DNA、髓過氧化物酶(myeloperoxidase,MPO)、蛋白水解酶3(Protease3,PR3)及抗微生物肽LL37(Cathelicidin LL37,LL37)等物質(zhì),這些自身抗原暴露于免疫監(jiān)視中誘導自身抗體形成及自身免疫性疾病。研究表明:異常形成的NETs參與多種自身免疫性疾病,包括誘發(fā)ANCA相關(guān)性血管炎。異常分泌的炎癥介質(zhì)一直被證實參與AAV病理損傷,如白細胞介素1β(interleukin-1β,IL-1β)、干擾素α(interferon-α,IFN-α)、白細胞介素6(interleukin-6,IL-6)等。但NETs對外周血單核細胞激活分泌炎癥介質(zhì)的影響以及意義尚不完全清楚。本實驗擬觀察NETs對單核細胞分泌炎癥介質(zhì)IL-1β、IFN-α、TNF-α、IL-6、MCP-1、白細胞介素10(interleukin-10,IL-10)、白細胞介素12(interleukin-12,IL-12)的影響,探討NETs對單核細胞活化的影響。方法:1、分別抽取6名健康志愿者(研究生同學)外周血各55ml,共后續(xù)實驗。2、每人取5ml外周血,采用Polymorphprep分離液密度梯度離心法分離中性粒細胞,DAPI染色觀察細胞核形態(tài),Beckman流式細胞儀檢測中性粒細胞提取純度。純化的中性粒細胞調(diào)整密度一致后分為對照組和PMA組兩組,對照組采用等量PBS與中性粒細胞孵育,PMA組采用等量佛波脂(PMA,終濃度30ng/ml)與中性粒細胞孵育4h誘導NETs形成,吸棄含PMA的培養(yǎng)上清并洗棄殘留PMA后留存于培養(yǎng)板底的即為NETs,DAPI熒光染色,Leica激光共聚焦顯微鏡觀察NETs形成效率。3、每人取50ml外周血,采用Ficoll密度梯度離心法分離單個核細胞,免疫磁珠法(Stem Cells人CD14正選試劑盒)從外周血單個核細胞分離提取單核細胞,Beckman流式細胞儀檢測單核細胞提取純度。純化的單核細胞調(diào)整密度一致后分為對照組及NETs組,對照組采用等量細胞培養(yǎng)基與單核細胞培養(yǎng)體系,NETs組為等量上訴實驗誘導的培養(yǎng)板底部NETs與單核細胞共培養(yǎng)體系,孵育12小時后,收集培養(yǎng)上清存于-80oC冰箱。LEGENDplex人炎癥因子試劑盒檢測各組培養(yǎng)上清IL-1β、IFN-α、TNF-α、IL-6、MCP-1、IL-10、IL-12的濃度。4、數(shù)據(jù)分析:數(shù)據(jù)以均值±標準差表示,數(shù)據(jù)分析采用SPSS 18.0統(tǒng)計分析軟件進行統(tǒng)計學處理。兩樣本均數(shù)比較采用t檢驗,P0.05表示差異有統(tǒng)計學意義。結(jié)果:1、Polymorphprep分離液密度梯度離心法分離提取健康志愿者外周血中性粒細胞,DAPI染色,細胞核呈分葉核,流式細胞儀鑒定純度為98%。純化的中性粒細胞分為對照組和PMA組兩組,孵育4小時后,DAPI染色行激光共聚焦顯微鏡觀察,對照組中性粒細胞核仍保持分葉核結(jié)構(gòu),而PMA組分葉核結(jié)構(gòu)破壞,可見絲狀DNA即NETs形成。2、CD14正選試劑盒提取單核細胞純度為95.3%。3、NETs對單核細胞分泌IL-1β的影響:NETs組與對照組IL-1β濃度分別為(97.60±60.11)pg/m L、1.17pg/m L,與對照組相比,NETs組單核細胞分泌IL-1β水平顯著升高(P0.05)。4、NETs對單核細胞分泌IFN-α的影響:NETs組與對照組IFN-α濃度分別為(208.90±77.38)pg/m L、1.17 pg/mL,與對照組相比,NETs組單核細胞分泌IFN-α水平顯著升高(P0.01)。5、NETs對單核細胞分泌TNF-α的影響:NETs組與對照組TNF-α濃度分別為(218.88±86.27)pg/mL、0.17 pg/m L,與對照組相比,NETs組單核細胞分泌TNF-α的水平顯著升高(P0.01)。6、NETs對單核細胞分泌MCP-1的影響:NETs組與對照組MCP-1濃度分別為(1063.25±740.01)pg/mL、(157.16±3.86)pg/m L,與對照組相比,NETs組單核細胞分泌MCP-1水平顯著升高(P0.05)。7、NETs對單核細胞分泌IL-6的影響:NETs組與對照組IL-6濃度分別為(67.76±15.51)pg/mL、(2.76±0.47)pg/m L,與對照組相比,NETs組單核細胞分泌IL-6的水平顯著升高(P0.01)。8、NETs對單核細胞分泌IL-10的影響:NETs組與對照組IL-10濃度分別為1.68±0.58 pg/mL、1.15 pg/mL,兩組IL-10水平無顯著差異。9、NETs對單核細胞分泌IL-12的影響:NETs組與對照組IL-12濃度分別為1.30pg/m L、1.30 pg/mL,兩組IL-12水平無顯著差異。結(jié)論NETs能激活單核細胞,誘導IFN-α、IL-1β、IL-6、TNF-α、MCP-1炎癥介質(zhì)生成,可能參與AAV發(fā)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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R593.2;R692

【參考文獻】

相關(guān)期刊論文 前1條

1 牟翠萍;毛慧娟;;ANCA相關(guān)性小血管炎發(fā)病機制研究進展[J];中國中西醫(yī)結(jié)合腎病雜志;2012年01期

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