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

【摘要】： 在SLE患者體內(nèi)樹(shù)突狀細(xì)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(dá)MHC分子與T細(xì)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(duì)細(xì)胞因子介導(dǎo)的活化敏感,SLE患者血清中異常升高的成份可以誘導(dǎo)DC及其前體細(xì)胞產(chǎn)生促炎性因子,活化T/B細(xì)胞,促使T/B細(xì)胞引起自身免疫的發(fā)生,導(dǎo)致了SLE患者對(duì)自身抗原的反應(yīng)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(duì)產(chǎn)生DC的影響上來(lái)部分闡述SLE的發(fā)病機(jī)制。本實(shí)驗(yàn)主要是針對(duì)高IL-10 SLE患者血清對(duì)單核細(xì)胞和造血干細(xì)胞來(lái)源的DC的影響進(jìn)行研究。 本實(shí)驗(yàn)用ELISA法檢測(cè)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(shí)檢測(cè)部分病人的抗dsDNA抗體、補(bǔ)體(C3、C4)和24小時(shí)尿蛋白。應(yīng)用GM-CSF+IL-4+TNF-α體系誘導(dǎo)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(cè)到的IL-10、IL-6、IFN-α濃度,選擇單獨(dú)IL-10增高的SLE患者血清參與誘導(dǎo)臍血CD34~+造血干細(xì)胞分化為DC。用流式細(xì)胞術(shù)檢測(cè)DC的表型和DC刺激的活化T細(xì)胞胞內(nèi)細(xì)胞因子,CCK-8法檢測(cè)DC刺激同種異體T淋巴細(xì)胞增殖能力,ELISA法檢測(cè)DC分泌細(xì)胞因子水平、與DC共培養(yǎng)的T細(xì)胞分泌細(xì)胞因子的水平。 結(jié)果發(fā)現(xiàn),以超過(guò)正常人IL-10濃度95%參考值范圍為IL-10異常增高標(biāo)準(zhǔn)(＞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(gè)SLE患者的53.20%。將這50例血清的濃度進(jìn)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(shí)驗(yàn)濃度為30pg/ml。當(dāng)應(yīng)用GM-CSF+IL-4+TNF-α誘導(dǎo)培養(yǎng)DC時(shí),如加入外源性IL-10 30pg/ml作為實(shí)驗(yàn)觀察因素,與正常誘導(dǎo)DC相比,所誘導(dǎo)的DC HLA-DR、CD86、CD80、CD83的表達(dá)陽(yáng)性率均下降并具有統(tǒng)計(jì)學(xué)意義;對(duì)其刺激同種異體淋巴細(xì)胞增殖能力的研究結(jié)果顯示,刺激細(xì)胞:效應(yīng)細(xì)胞為1:10比例組中IL-10濃度為30pg/ml的誘導(dǎo)組誘導(dǎo)的DC刺激淋巴細(xì)胞增殖的能力顯著降低;該種DC分泌細(xì)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(shì)。然而,在高IL-10的SLE患者血清參與誘導(dǎo)干細(xì)胞來(lái)源的DC的實(shí)驗(yàn)中,DC的表型、刺激同種異體T淋巴細(xì)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(dǎo)培養(yǎng)的DC統(tǒng)計(jì)學(xué)的差異。 以上實(shí)驗(yàn)結(jié)果證實(shí),30pg/ml的外源性IL-10可使單核細(xì)胞來(lái)源的DC的MHC-Ⅱ類分子和共刺激分子的表達(dá)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(xì)胞增殖能力下降。但是IL-10異常增高的SLE患者血清在誘導(dǎo)CD34~+造血干細(xì)胞分化發(fā)育為DC時(shí)由于作用的細(xì)胞不同、SLE患者血清中存在IL-6、IFN-α以外的其它復(fù)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(jī)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(xì)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，

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