免疫性血小板减少症患者miRNAs表达谱研究及miR-148a功能初探

发布时间：2018-04-15 22:08

  本文选题：免疫性血小板减少症 + micro　； 参考：《第二军医大学》2017年硕士论文

【摘要】：第一部分免疫性血小板减少症患者外周血PBMCs中miRNAs表达谱检测本部分筛选出与免疫性血小板减少症(Immune Trombocytopenia,ITP)发病相关是的差异性表达的micro RNA(miRNAs)。首先,我们采用密度梯度离心法分离外周血单个核细胞,再通过miRNAs芯片分析出不同分期ITP患者外周血单个核细胞中的miRNAs和mRNA表达谱。选取其中与ITP发病相关有明显差异性表达的miRNAs作为进一步的研究对象,并应用RT-PCR对选取的miRNAs表达情况进行验证。芯片结果显示,慢性组与健康对照组相比有216个表达上调的mi RNAs和150个表达下调的mi RNAs,慢性组与初诊组相比有55个表达上调的mi RNAs和45个表达下调的miRNAs。我们选择芯片结果中在ITP患者PBMCs内明显表达异常的miRNAs和文献报道的与免疫相关的miRNAs进行交叉检测,筛选出芯片中差异性表达升高的miR-148a作为研究对象。纳入在上海长海医院和解放军100医院确诊为初诊ITP的患者(n=21)及慢性ITP的患者(n=24),用同期入院体检人员作为健康对照(n=22),分离外周血单个核细胞,经RT-PCR检测miR-148a表达水平,结果显示,初诊、慢性ITP患者PBMCs中miR-148a的表达较健康对照组明显上调,且初诊与慢性ITP患者PBMCs中miR-148a之间的表达亦有差异,与mi RNA芯片检测的结果一致,表明芯片的结果可信。同时,我们运用酶联免疫吸附法(ELISA)检测不同分期ITP患者血浆中Th1及Th2相关细胞因子的表达。本部分结果提示,ITP患者存在特异性的miRNAs表达谱,且不同分期的ITP患者miRNAs表达亦存在差异,表明miRNAs可能参与了ITP发病进程,miR-148a在不同的分期ITP患者中表达不同,可以作为潜在的生物学标记物。同时我们的研究提示ITP患者外周血中存在Th1细胞极化状态。第二部分Mi R-148a在免疫性血小板减少症发病中的功能初探在第一部分研究中,我们筛选并验证了与ITP发病相关的miR-148a。在这一部分,我们将进一步阐明miR-148a在ITP发病中的可能作用机制。首先,我们通过生物信息学分析预测,结合第一部分的mRNA芯片数据,同时结合文献筛选出与免疫相关的mi R-148a的靶基因。其次,我们通过miR-148a mimics/inhibitors转染健康人原代PBMCs,采用RT-PCR验证miR-148a靶基因的表达情况。最后,运用miR-148a mimics/inhibitors转染ITP患者原代PBMCs后,行LPS刺激,通过RT-PCR验证Th1及Th2相关细胞因子表达情况。结果显示,内源性mi R-148a功能增强后,RelA的表达明显被抑制。相反,转染miR-148a inhibitors使miR-148a功能抑制后RelA的表达则显著上调。同时,miR-148a功能增强的PBMCs再经LPS刺激后,Th1相关细胞因子IL-8、IL-1b、TNF-a的水平均稍上调,而Th2相关细胞因子IL-4、IL-5、IL-10的水平均有明显的下降。mi R-148a功能受抑制的PBMCs再经LPS刺激后,Th1相关细胞因子IL-8、IL-1b、TNF-a的表达均轻微上调了,而Th2相关细胞因子IL-4、IL-5、IL-10的水平均有明显的升高。本部分结果提示,miR-148a可能通过RelA参与NF-κB信号途径,抑制Th2相关细胞因子的分泌,促进T细胞向Th1极化偏移,进一步参与ITP的发病进程。
[Abstract]:This part of spectrum detection screening and immune thrombocytopenia miRNAs expression in peripheral blood of PBMCs patients in the first part of immune thrombocytopenia (Immune Trombocytopenia ITP) is the pathogenesis related differential expression of micro RNA (miRNAs). First, we use the density gradient centrifugation of peripheral blood mononuclear cells. Through the analysis of the different stages of miRNAs chip ITP in peripheral blood mononuclear cells in miRNAs and mRNA expression. The expression of ITP related with significant difference miRNAs for further study, and the application of RT-PCR to the selection of the expression of miRNAs was verified. The results showed that chronic group 216 expression of MI RNAs and MI RNAs 150 expression decreased compared with healthy control group, chronic group and untreated group compared with 55 up-regulated and 45 down regulated expression of MI RNAs miRNAs. us Select the chip result in patients with ITP PBMCs were abnormal expression of miRNAs and the reported immune related miRNAs cross detection, screening out the elevated expression of miR-148a as the research object. The difference of the chip included in the 100 hospital of PLA and Changhai Hospital of Shanghai diagnosed for newly diagnosed patients with ITP (n=21) and chronic ITP patients (n=24), with the medical staff admitted in the same period as healthy control (n=22), peripheral blood mononuclear cells were isolated and detected by RT-PCR showed that the expression level of miR-148a, miR-148a PBMCs ITP, newly diagnosed, patients with chronic expression compared with healthy control group increased significantly, and the expression between miR-148a and PBMCs in newly diagnosed patients with chronic ITP in the there are differences, consistent with the MI RNA chip test results show that the chip results are credible. At the same time, we used enzyme-linked immunosorbent assay (ELISA) detection of ITP patients in different stages of plasma Th1 and Th2 The expression of related cytokines. The results suggest that ITP patients have specific expression profiles of miRNAs and ITP in different stage of patients with miRNAs expression differences, suggests that miRNAs may participate in the progression of ITP, the expression of miR-148a in different stages of ITP were different, can be used as potential biomarkers. At the same time we research suggests that ITP in the peripheral blood of patients with Th1 cell polarization. The second part of the Mi R-148a to reduce the incidence of the function in the first part of the study on immune thrombocytopenic, we screened and verified the correlation with the incidence of ITP miR-148a. in this part, we will further elucidate the possible mechanism of miR-148a in the pathogenesis of ITP first, we analyze the prediction by bioinformatics, combined with mRNA chip data of the first part, combined with the literature screening target substrate and immune related mi R-148a because of its. Again, we through the miR-148a mimics/inhibitors transfection of human primary health PBMCs, the expression of target gene miR-148a RT-PCR verification. Finally, using primary miR-148a mimics/inhibitors transfected ITP PBMCs patients after LPS stimulation, verified by RT-PCR Th1 and Th2 related cytokines expression. The results showed that endogenous Mi enhanced function of R-148a, RelA the expression was significantly inhibited. On the contrary, the expression of miR-148a inhibitors was transfected into miR-148a function after inhibition of RelA was significantly up-regulated. At the same time, the enhanced function of miR-148a PBMCs after LPS stimulation, Th1 related cytokines IL-8, IL-1b, TNF-a levels were slightly increased, while Th2 related cytokines IL-4, IL-5 levels were IL-10 the.Mi R-148a function decreased significantly inhibited PBMCs stimulated by LPS, Th1 related cytokines IL-8, IL-1b, TNF-a expression was slightly up-regulated, while Th2 related cytokines IL- 4, the level of IL-5 and IL-10 increased significantly. Our results suggest that miR-148a may participate in the NF- kappa B signaling pathway through RelA, inhibit the secretion of Th2 related cytokines, promote T cells to Th1 polarization, and further participate in the pathogenesis of ITP.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R558.2;R440

【参考文献】

相关期刊论文 前3条

1 Dong Zheng;Chen-Song Huang;Shao-Bin Huang;Chao-Xu Zheng;;Laparoscopic splenectomy for primary immune thrombocytopenia:Current status and challenges[J];World Journal of Gastrointestinal Endoscopy;2016年17期

2 王晓雪;王s，

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