白介素-23在原发性免疫性血小板减少症患者中的表达及临床意义

发布时间：2018-04-20 15:02

本文选题：原发性免疫性血小板减少症 + 地塞米松　；参考：《第二军医大学》2016年硕士论文

【摘要】：研究目的明确白介素-23(interleukin-23,IL-23)在原发性免疫性血小板减少症(primary immune thrombocytopenia,ITP)患者中的表达情况,初步探讨IL-23对ITP患者中Th17细胞亚群的调控机制,并探讨地塞米松(dexamethasone,DXM)对ITP患者中IL-23表达的影响及其可能的机制。研究方法分别收集ITP患者和健康对照组的外周血标本,以EDTA抗凝,离心后收集血浆;剩余血细胞标本利用Ficoll密度梯度离心法分离获得外周血单个核细胞(peripheral blood mononuclear cell,PBMC),分离得到的 PBMC 和血浆均分装到若干EP管中保存以备不同实验需求。抽提PBMC中总RNA,并反转录为cDNA,利用实时荧光定量 PCR(TaqMan-MGB 法)检测 IL-23p19、I]L-12p40、IL-12p35、IL-23R、IL-12Rβ1及IL-12Rβ2的mRNA表达水平。利用实时荧光定量PCR(SYBR Green法)检测IL-17A、IL-17F及RORC的mRNA相对表达量。利用ELISA法检测血浆中IL-23和IL-17蛋白的表达量,western blot法检测IL-23受体的表达量,流式细胞术检测ITP患者Th17细胞亚群百分比,全自动血细胞计数仪检测纳入研究对象的外周血血小板计数(plateletcount,PLT)。分析ITP患者血浆中IL-23水平与Th17细胞亚群百分比,IL-17以及PLT的相关性;体外应用不同浓度的rhIL-23和培养基刺激ITP患者PBMC,检测IL-6、IL-17、IL-21蛋白水平,及RORC、STATsmRNA的表达量,并分析STATs的磷酸化水平。随访15例新诊断的ITP患者,利用ELISA方法,检测经高剂量DXM(40mg/day×4天)治疗后,血浆中IL-23和IL-17的变化情况,在体外应用不同浓度的DXM处理这组患者PBMC,检测IL-23的变化趋势,p38 MAPKmRNA的表达及其磷酸化水平,进一步研究加入P38MAPK的特异性激活剂(anisomycin)后,DXM对IL-23表达的影响。结果与健康对照组相比,ITP患者中IL-23p19、p40亚基,IL-23R和IL-23Rβ1的表达均显著增高,IL-12p35亚基和IL-12Rβ2也有增高,但其差异无统计学意义。ITP患者中IL-17A、IL-17F及调控Th17细胞发育的特异性转录因子RORC mRNA的表达量亦显著增高。ITP患者血浆中IL-23和IL-17的表达量显著上升,且在PLT≤20×109/L的患者中增高得更为显著。与健康对照组相比,ITP患者外周血IL-23受体的表达及Th17细胞亚群百分比均显著增高。ITP患者组血浆IL-23的表达量与外周血Th17细胞亚群百分比以及IL-17的表达量呈显著正相关,而与PLT呈显著负相关。与培养基刺激组相比,体外20ng/ml和10ng/ml的rhIL-23刺激组培养上清液中IL-6、IL-17、IL-21蛋白水平和RORC mRNA的表达量均显著上升,且IL-17、IL-21蛋白水平和RORC mRNA在20ng/ml rhIL-23刺激组中的增高更为显著,STATs信号通路mRNA表达在rhIL-23的刺激后均有上调的趋势,但STAT3的增高幅度最大,并且其磷酸化水平显著增高。通过随访15例新诊断的ITP患者,发现在经高剂量DXM治疗后,其血浆中IL-23和IL-17的表达均显著下降,在体外用不同浓度DXM处理该组患者PBMC后发现,随着DXM浓度增高,IL-23的表达呈下降趋势,且p38 MAPK mRNA的表达及其磷酸化水平亦呈下降趋势,呈现接近剂量依赖性的模式,进一步加入anisomycin后,DXM对IL-23的抑制作用显著减弱。结论ITP患者中IL-23 mRNA和蛋白质含量均显著增高,且与疾病严重程度相关,IL-23在ITP的疾病进展中具有重要作用。ITP患者中高表达的IL-23可能通过介导STAT3的活化并激活RORC的表达,进一步刺激Th17细胞的分化增殖,促进疾病的发展。高剂量DXM治疗可以抑制IL-23的表达,这种抑制作用可能是通过p38 MAPK信号通路介导的,并呈剂量依赖性的趋势。
[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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R558.2

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