本文選題：原發(fā)性免疫性血小板減少癥 + 地塞米松��； 參考：《第二軍醫(yī)大學(xué)》2016年碩士論文

【摘要】：研究目的明確白介素-23(interleukin-23,IL-23)在原發(fā)性免疫性血小板減少癥(primary immune thrombocytopenia,ITP)患者中的表達情況,初步探討IL-23對ITP患者中Th17細胞亞群的調(diào)控機制,并探討地塞米松(dexamethasone,DXM)對ITP患者中IL-23表達的影響及其可能的機制。研究方法分別收集ITP患者和健康對照組的外周血標本,以EDTA抗凝,離心后收集血漿;剩余血細胞標本利用Ficoll密度梯度離心法分離獲得外周血單個核細胞(peripheral blood mononuclear cell,PBMC),分離得到的 PBMC 和血漿均分裝到若干EP管中保存以備不同實驗需求。抽提PBMC中總RNA,并反轉(zhuǎn)錄為cDNA,利用實時熒光定量 PCR(TaqMan-MGB 法)檢測 IL-23p19、I]L-12p40、IL-12p35、IL-23R、IL-12Rβ1及IL-12Rβ2的mRNA表達水平。利用實時熒光定量PCR(SYBR Green法)檢測IL-17A、IL-17F及RORC的mRNA相對表達量。利用ELISA法檢測血漿中IL-23和IL-17蛋白的表達量,western blot法檢測IL-23受體的表達量,流式細胞術(shù)檢測ITP患者Th17細胞亞群百分比,全自動血細胞計數(shù)儀檢測納入研究對象的外周血血小板計數(shù)(plateletcount,PLT)。分析ITP患者血漿中IL-23水平與Th17細胞亞群百分比,IL-17以及PLT的相關(guān)性;體外應(yīng)用不同濃度的rhIL-23和培養(yǎng)基刺激ITP患者PBMC,檢測IL-6、IL-17、IL-21蛋白水平,及RORC、STATsmRNA的表達量,并分析STATs的磷酸化水平。隨訪15例新診斷的ITP患者,利用ELISA方法,檢測經(jīng)高劑量DXM(40mg/day×4天)治療后,血漿中IL-23和IL-17的變化情況,在體外應(yīng)用不同濃度的DXM處理這組患者PBMC,檢測IL-23的變化趨勢,p38 MAPKmRNA的表達及其磷酸化水平,進一步研究加入P38MAPK的特異性激活劑(anisomycin)后,DXM對IL-23表達的影響。結(jié)果與健康對照組相比,ITP患者中IL-23p19、p40亞基,IL-23R和IL-23Rβ1的表達均顯著增高,IL-12p35亞基和IL-12Rβ2也有增高,但其差異無統(tǒng)計學(xué)意義。ITP患者中IL-17A、IL-17F及調(diào)控Th17細胞發(fā)育的特異性轉(zhuǎn)錄因子RORC mRNA的表達量亦顯著增高。ITP患者血漿中IL-23和IL-17的表達量顯著上升,且在PLT≤20×109/L的患者中增高得更為顯著。與健康對照組相比,ITP患者外周血IL-23受體的表達及Th17細胞亞群百分比均顯著增高。ITP患者組血漿IL-23的表達量與外周血Th17細胞亞群百分比以及IL-17的表達量呈顯著正相關(guān),而與PLT呈顯著負相關(guān)。與培養(yǎng)基刺激組相比,體外20ng/ml和10ng/ml的rhIL-23刺激組培養(yǎng)上清液中IL-6、IL-17、IL-21蛋白水平和RORC mRNA的表達量均顯著上升,且IL-17、IL-21蛋白水平和RORC mRNA在20ng/ml rhIL-23刺激組中的增高更為顯著,STATs信號通路mRNA表達在rhIL-23的刺激后均有上調(diào)的趨勢,但STAT3的增高幅度最大,并且其磷酸化水平顯著增高。通過隨訪15例新診斷的ITP患者,發(fā)現(xiàn)在經(jīng)高劑量DXM治療后,其血漿中IL-23和IL-17的表達均顯著下降,在體外用不同濃度DXM處理該組患者PBMC后發(fā)現(xiàn),隨著DXM濃度增高,IL-23的表達呈下降趨勢,且p38 MAPK mRNA的表達及其磷酸化水平亦呈下降趨勢,呈現(xiàn)接近劑量依賴性的模式,進一步加入anisomycin后,DXM對IL-23的抑制作用顯著減弱。結(jié)論ITP患者中IL-23 mRNA和蛋白質(zhì)含量均顯著增高,且與疾病嚴重程度相關(guān),IL-23在ITP的疾病進展中具有重要作用。ITP患者中高表達的IL-23可能通過介導(dǎo)STAT3的活化并激活RORC的表達,進一步刺激Th17細胞的分化增殖,促進疾病的發(fā)展。高劑量DXM治療可以抑制IL-23的表達,這種抑制作用可能是通過p38 MAPK信號通路介導(dǎo)的,并呈劑量依賴性的趨勢。
[Abstract]:Objective to investigate the expression of interleukin-23 (IL-23) in patients with primary immune thrombocytopenia (primary immune thrombocytopenia, ITP) and to explore the regulatory mechanism of IL-23 on Th17 cell subsets in ITP patients and to explore the effect of dexamethasone (dexamethasone, DXM) on the expression of primary immune -23 and its expression in patients with primary immune thrombocytopenia (ITP). The possible mechanism. The study methods collected peripheral blood samples from the ITP patients and the healthy control group and collected the plasma with EDTA anticoagulant and centrifugation. The residual blood cell specimens were separated by Ficoll density gradient centrifugation to obtain the peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC). The dry EP tube was preserved for different experimental requirements. The total RNA in PBMC was extracted, and the reverse transcription was cDNA. The expression level of IL-23p19, I]L-12p40, IL-12p35, IL-23R, IL-12R beta 1 and IL-12R beta 2 were detected by real-time fluorescent quantitative PCR (TaqMan-MGB method). The expression of IL-23 and IL-17 protein in plasma was detected by ELISA, the expression of IL-23 receptor was detected by Western blot, the percentage of Th17 cell subsets in ITP patients was detected by flow cytometry, and the peripheral blood platelet count (plateletcount, PLT) of the subjects was detected by the full automatic blood cell counting instrument (plateletcount, PLT). The level of IL-23 in the plasma of ITP patients was analyzed and the level of IL-23 was analyzed. The correlation between the percentage of Th17 cell subsets, IL-17 and PLT, and the use of different concentrations of rhIL-23 and culture medium to stimulate ITP patients PBMC, detect IL-6, IL-17, IL-21 protein level, RORC, STATsmRNA expression, and analyze the level of phosphorylation of STATs. 15 newly diagnosed patients were followed up. After 4 days of treatment, the changes of IL-23 and IL-17 in plasma and PBMC in different concentrations of DXM in vitro were used in vitro to detect the change trend of IL-23, the expression of p38 MAPKmRNA and the level of phosphorylation, and the effect of DXM on IL-23 expression after adding P38MAPK specific activator (anisomycin). The results were compared with the healthy control group. In ITP patients, the expression of IL-23p19, P40 subunit, IL-23R and IL-23R beta 1 increased significantly, and IL-12p35 subunits and IL-12R beta 2 also increased, but the difference was not statistically significant in IL-17A, IL-17F and specific transcription factors for Th17 cell development. Compared with the healthy control group, the expression of IL-23 receptor and the percentage of Th17 cell subsets in the peripheral blood of the patients with ITP were significantly higher than those in the healthy control group. The expression of IL-23 in the plasma of the patients with.ITP was significantly correlated with the percentage of Th17 cell subsets in peripheral blood and the expression of IL-17 in the peripheral blood, but in the group of ITP patients, the expression of IL-23 receptor was significantly higher than that of the healthy control group. The expression of IL-6, IL-17, IL-21 protein and RORC mRNA in the culture supernatant of 20ng/ml and 10ng/ml in vitro was significantly increased, and IL-17, IL-21 protein level and RORC rhIL-23 increased significantly. There was a trend of up-regulation after rhIL-23 stimulation, but STAT3 increased significantly and its phosphorylation level increased significantly. After follow-up of 15 newly diagnosed ITP patients, the expression of IL-23 and IL-17 in plasma was significantly decreased after high dose DXM treatment. After the treatment of PBMC in the group of patients with different concentrations of DXM in vitro, it was found with the presence of PBMC in this group. The expression of DXM increased, and the expression of IL-23 decreased, and the expression of p38 MAPK mRNA and its phosphorylation level also declined, showing a close dose dependence model. After adding anisomycin, the inhibitory effect of DXM on IL-23 decreased significantly. Conclusion IL-23 mRNA and protein content in ITP patients were significantly increased, and the severity of the disease was serious. Degree related, IL-23 plays an important role in the progression of ITP disease. The high expression of IL-23 in.ITP patients may stimulate the activation of STAT3 and activate the expression of RORC to further stimulate the differentiation and proliferation of Th17 cells and promote the development of the disease. High dose DXM therapy can inhibit the expression of IL-23, which may be through p38 MAPK signal. Pathway mediated, and in a dose-dependent manner.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R558.2

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