本文選題：紅斑狼瘡 + 系統(tǒng)性　； 參考：《南京醫(yī)科大學》2008年碩士論文

【摘要】： 在SLE患者體內(nèi)樹突狀細胞(dendritic cell,DC)的研究中發(fā)現(xiàn),非成熟DC更加有效的提呈抗原,成熟DC高表達MHC分子與T細胞相互作用,表現(xiàn)了不同的作用和在誘發(fā)疾病中的協(xié)同配合。DC對細胞因子介導的活化敏感,SLE患者血清中異常升高的成份可以誘導DC及其前體細胞產(chǎn)生促炎性因子,活化T/B細胞,促使T/B細胞引起自身免疫的發(fā)生,導致了SLE患者對自身抗原的反應和持續(xù)產(chǎn)生大量自身抗體。本課題希望從SLE患者血清及其對產(chǎn)生DC的影響上來部分闡述SLE的發(fā)病機制。本實驗主要是針對高IL-10 SLE患者血清對單核細胞和造血干細胞來源的DC的影響進行研究。 本實驗用ELISA法檢測SLE患者和正常人血清中的IL-10、IL-6、IFN-α,以篩選高IL-10 SLE患者血清。同時檢測部分病人的抗dsDNA抗體、補體(C3、C4)和24小時尿蛋白。應用GM-CSF+IL-4+TNF-α體系誘導培養(yǎng)DC,在該體系中加入外源性IL-10(30pg/ml)。根據(jù)ELISA法檢測到的IL-10、IL-6、IFN-α濃度,選擇單獨IL-10增高的SLE患者血清參與誘導臍血CD34~+造血干細胞分化為DC。用流式細胞術(shù)檢測DC的表型和DC刺激的活化T細胞胞內(nèi)細胞因子,CCK-8法檢測DC刺激同種異體T淋巴細胞增殖能力,ELISA法檢測DC分泌細胞因子水平、與DC共培養(yǎng)的T細胞分泌細胞因子的水平。 結(jié)果發(fā)現(xiàn),以超過正常人IL-10濃度95%參考值范圍為IL-10異常增高標準(＞8.12pg/ml),則在收集的94例SLE患者中50例IL-10濃度增高,占整個SLE患者的53.20%。將這50例血清的濃度進行分析得到IL-10平均濃度為33.77±41.94pg/ml,其中有16份血清僅表現(xiàn)IL-10異常增高,IL-6、IFN-α均在正常范圍內(nèi)。故選擇外源性IL-10的實驗濃度為30pg/ml。當應用GM-CSF+IL-4+TNF-α誘導培養(yǎng)DC時,如加入外源性IL-10 30pg/ml作為實驗觀察因素,與正常誘導DC相比,所誘導的DC HLA-DR、CD86、CD80、CD83的表達陽性率均下降并具有統(tǒng)計學意義;對其刺激同種異體淋巴細胞增殖能力的研究結(jié)果顯示,刺激細胞:效應細胞為1:10比例組中IL-10濃度為30pg/ml的誘導組誘導的DC刺激淋巴細胞增殖的能力顯著降低;該種DC分泌細胞因子的水平有變化,表現(xiàn)有高分泌IL-10,低分泌IL-12p40和IFN-γ的趨勢。然而,在高IL-10的SLE患者血清參與誘導干細胞來源的DC的實驗中,DC的表型、刺激同種異體T淋巴細胞增殖和分化的能力、DC分泌IL-12p40、IL-10、IFN-γ水平,并未顯示出與常規(guī)誘導培養(yǎng)的DC統(tǒng)計學的差異。 以上實驗結(jié)果證實,30pg/ml的外源性IL-10可使單核細胞來源的DC的MHC-Ⅱ類分子和共刺激分子的表達以及JL-12p40和IFN-γ的分泌水平受到抑制,并使其刺激同種異體T細胞增殖能力下降。但是IL-10異常增高的SLE患者血清在誘導CD34~+造血干細胞分化發(fā)育為DC時由于作用的細胞不同、SLE患者血清中存在IL-6、IFN-α以外的其它復雜因素的影響,并不能相同地呈現(xiàn)明顯抑制作用。 總之,IL-10在SLE發(fā)病機制中起到了重要作用,但是如協(xié)同作用的因素不同及作用的細胞不同,可能得到不同的結(jié)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- 偽 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- 偽 system, and exogenous IL-10g / ml was added into the system. According to the concentration of IL-10 and IL-6 IFN- 偽 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 鹵41.94 PG / ml, 16 of which only showed abnormal increase of IL-6 and IFN- 偽 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- 偽, 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- 緯. However, the ability of DC to stimulate the proliferation and differentiation of allogeneic T lymphocytes and the secretion of IL-12p40 / IL-10IFN- 緯 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，

本文編號：1843630