伴胸腺瘤重症肌无力T辅助17细胞变化及其调控机制研究

发布时间：2018-06-17 21:47

  本文选题：重症肌无力 + 胸腺瘤　； 参考：《中国人民解放军医学院》2017年博士论文

【摘要】：正文:第一部分:伴胸腺瘤重症肌无力患者外周血Th17细胞比例及细胞因子表达量变化目的:重症肌无力(myasthenia gravis, MG)是T细胞辅助、乙酰胆碱受体抗体(AchR-Abs)介导的神经肌肉接头传递障碍的自身免疫性疾病。Th17细胞在MG发病机制中的作用仍不明确。本研究目的为探索伴胸腺瘤MG患者外周血中Th17细胞比例及其相关炎性细胞因子表达是否发生变化。方法:共分为三组:伴MG胸腺瘤组(TM) 35例、伴MG胸腺增生组(TH) 22例、健康对照组(HC ) 22例。使用流式细胞术实验检测MG患者及健康对照外周血Th17细胞比例;采用实时定量PCR和ELISA检测Th17细胞相关细胞因子mRNA、和蛋白表达量。结果:TM组外周血Th17细胞比例显著高于HC (P0. 05 ) , TH组与HC组Th17细胞比例无统计学差异(P0.05); TM组IL-17、IL-1β,IL-6及IL-23mRNA及蛋白表达量显著高于对照组(P0. 05 ),然而TH组与HC组IL-17、IL-6及IL-23mRNA及蛋白表达量差异无统计学意义(P0. 05 ),TH组IL-1 β蛋白表达量显著高于HC组(P0. 05 )。结论:MG胸腺瘤中Th17细胞增多,相关炎性细胞因子表达增多,可能参与MG发病。第二部分:microRNA-19b在伴胸腺瘤重症肌无力发病中表观遗传调控机制研究目的第一部分研究结果发现伴胸腺瘤MG患者的外周血Th 17细胞升高,然而Th17细胞的上游调控机制仍不清楚。胸腺间质淋巴细胞生成素(TSLP )是Th17细胞分化发育的上游调控分子。microRNA通过转录后调控机制调控蛋白表达水平和生物学功能,然而microRNA在胸腺瘤细胞发育和分化一直未见报道。本研究着眼于探索伴MG胸腺瘤组织中TSLPmRNA及蛋白的表达水平以及证实microRNA-19b-TSLP-Th17细胞信号通路参与MG发病的分子机制。方法分为三组:胸腺瘤伴MG组(MG-T) 52例、单纯胸腺瘤不伴MG组(NMG-T )12例和健康对照组(HC ) 11例。使用RT-PCR和Western blot测定各组胸腺瘤组织中microRNA-19b及TSLP的mRNA和蛋白表达水平;体外实验采用荧光素酶报告基因实验验证microRNA-19b对于TSLPmRNA的靶向调控关系;使用Spearman相关分析分析microRNA-19b与TSLPmRNA以及蛋白表达量的相关性。结果MG-T组和NMG-T组TSLPmRNA转录量及蛋白表达量显著低于正常对照;同时MG-T组TSLP蛋白表达量低于NMG-T组,差异有统计学差异(P0. 05 );各种不同类型胸腺瘤组织TSLPmRNA转录量存在显著差异,B2组TSLP、B3组TSLP低于正常胸腺。荧光素酶报告基因实验证实microRNA19b-5p通过与TSLP3 'UTR的相互作用靶向调控TSLPmRNA表达。胸腺瘤microRNA19b-5p成熟体、前体、初级转录体转录量显著高于正常胸腺。microRNA19b-5p成熟体、初级转录体、前体与TSLP蛋白表达呈负相关。结论伴MG胸腺瘤组织中microRNA19b-5p表达上调,负向靶向调控TSLPmRNA表达,下调TSLP蛋白表达,进而可能影响Th17细胞的分化发育,microRNA-19b-TSLP-Th17细胞信号通路可能与MG发病相关。
[Abstract]:Text: part I: changes of Th17 cells and cytokines expression in peripheral blood of patients with thymoma myasthenia gravis objective: myasthenia gravis (MGG) is a T cell helper. The role of acetylcholine receptor antibody AchR-Abs-mediated autoimmune disease. Th17 cells in the pathogenesis of MG is still unclear. The aim of this study was to investigate the changes of Th17 cell ratio and the expression of inflammatory cytokines in peripheral blood of MG patients with thymoma. Methods: a total of 35 patients with MG thymoma, 22 patients with Thymoma with MG thymus hyperplasia and 22 patients with HC in healthy control group were divided into three groups: 35 cases with MG thymoma, 22 cases with TH with MG thymus hyperplasia and 22 cases with HC. The proportion of Th17 cells in peripheral blood of MG patients and healthy controls was detected by flow cytometry, and the expression of Th17 cytokine mRNAs and protein was detected by real-time quantitative PCR and Elisa. Results the percentage of Th17 cells was significantly higher in the group of 1: TM than that in the group of HC P0. There was no significant difference in the proportion of Th17 cells between th group and HC group (P 0.05), while the expression of IL 17 尾 IL 6 and IL 23 mRNA and protein in TM group was significantly higher than that in control group (P 0. 05). However, there was no significant difference in the expression of IL-6 and IL-23 mRNA and protein between th group and HC group (P 0. 0). The expression of IL-1 尾 protein in th group was significantly higher than that in HC group P0. 0 5. Conclusion Th17 cells and the expression of inflammatory cytokines may be involved in the pathogenesis of MG. The second part: microRNA-19b in the pathogenesis of thymoma myasthenia gravis objective the first part of the study found that the peripheral blood Th 17 cells in patients with thymoma MG increased, but the upstream regulation mechanism of Th17 cells is still unclear. TSLP is the upstream regulatory molecule of Th17 cell differentiation. MicroRNA regulates protein expression and biological function through post-transcriptional regulation mechanism. However, microRNA has not been reported in thymoma cell development and differentiation. The purpose of this study was to explore the expression level of TSLP mRNA and protein in thymoma with MG and to confirm the molecular mechanism of microRNA-19b-TSLP-Th17 cell signaling pathway involved in the pathogenesis of MG. Methods three groups were divided into three groups: 52 cases of thymoma with MG group, 12 cases of simple thymoma without MG group and 11 cases of healthy control group. The mRNA and protein expressions of microRNA-19b and TSLP in thymoma were detected by RT-PCR and Western blot, and the targeting regulation of microRNA-19b on TSLP mRNA was verified by luciferase reporter gene in vitro. Spearman correlation was used to analyze the correlation between microRNA-19b and TSLP mRNA and protein expression. Results the mRNA transcription and protein expression of TSLP in MG-T group and NMG-T group were significantly lower than those in normal control group, while the expression of TSLP protein in MG-T group was lower than that in NMG-T group (P 0). There was significant difference in TSLP mRNA transcription in different types of thymoma. TSLP in TSLP B3 group was lower than that in normal thymus. Luciferase reporter gene experiment confirmed that microRNA19b-5p regulated the expression of TSLP mRNA through the interaction with TSLP3 UTR. MicroRNA19b-5p maturation, precursor and primary transcription of thymoma were significantly higher than those of normal thymus. MicroRNA19b-5p maturation, primary transcripts and precursors were negatively correlated with the expression of TSLP protein. Conclusion the expression of microRNA19b-5p is up-regulated in MG thymoma, the expression of TSLP mRNA is regulated by negative targeting, and the expression of TSLP protein is down-regulated, which may affect the differentiation and development of Th17 cells and the signal pathway of microRNA-19b-TSLP-Th17 cells may be related to the pathogenesis of MG.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R746.1

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