本文選題：免疫性血小板減少癥 + micro　； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：第一部分免疫性血小板減少癥患者外周血PBMCs中miRNAs表達譜檢測本部分篩選出與免疫性血小板減少癥(Immune Trombocytopenia,ITP)發(fā)病相關(guān)是的差異性表達的micro RNA(miRNAs)。首先,我們采用密度梯度離心法分離外周血單個核細胞,再通過miRNAs芯片分析出不同分期ITP患者外周血單個核細胞中的miRNAs和mRNA表達譜。選取其中與ITP發(fā)病相關(guān)有明顯差異性表達的miRNAs作為進一步的研究對象,并應(yīng)用RT-PCR對選取的miRNAs表達情況進行驗證。芯片結(jié)果顯示,慢性組與健康對照組相比有216個表達上調(diào)的mi RNAs和150個表達下調(diào)的mi RNAs,慢性組與初診組相比有55個表達上調(diào)的mi RNAs和45個表達下調(diào)的miRNAs。我們選擇芯片結(jié)果中在ITP患者PBMCs內(nèi)明顯表達異常的miRNAs和文獻報道的與免疫相關(guān)的miRNAs進行交叉檢測,篩選出芯片中差異性表達升高的miR-148a作為研究對象。納入在上海長海醫(yī)院和解放軍100醫(yī)院確診為初診ITP的患者(n=21)及慢性ITP的患者(n=24),用同期入院體檢人員作為健康對照(n=22),分離外周血單個核細胞,經(jīng)RT-PCR檢測miR-148a表達水平,結(jié)果顯示,初診、慢性ITP患者PBMCs中miR-148a的表達較健康對照組明顯上調(diào),且初診與慢性ITP患者PBMCs中miR-148a之間的表達亦有差異,與mi RNA芯片檢測的結(jié)果一致,表明芯片的結(jié)果可信。同時,我們運用酶聯(lián)免疫吸附法(ELISA)檢測不同分期ITP患者血漿中Th1及Th2相關(guān)細胞因子的表達。本部分結(jié)果提示,ITP患者存在特異性的miRNAs表達譜,且不同分期的ITP患者miRNAs表達亦存在差異,表明miRNAs可能參與了ITP發(fā)病進程,miR-148a在不同的分期ITP患者中表達不同,可以作為潛在的生物學(xué)標記物。同時我們的研究提示ITP患者外周血中存在Th1細胞極化狀態(tài)。第二部分Mi R-148a在免疫性血小板減少癥發(fā)病中的功能初探在第一部分研究中,我們篩選并驗證了與ITP發(fā)病相關(guān)的miR-148a。在這一部分,我們將進一步闡明miR-148a在ITP發(fā)病中的可能作用機制。首先,我們通過生物信息學(xué)分析預(yù)測,結(jié)合第一部分的mRNA芯片數(shù)據(jù),同時結(jié)合文獻篩選出與免疫相關(guān)的mi R-148a的靶基因。其次,我們通過miR-148a mimics/inhibitors轉(zhuǎn)染健康人原代PBMCs,采用RT-PCR驗證miR-148a靶基因的表達情況。最后,運用miR-148a mimics/inhibitors轉(zhuǎn)染ITP患者原代PBMCs后,行LPS刺激,通過RT-PCR驗證Th1及Th2相關(guān)細胞因子表達情況。結(jié)果顯示,內(nèi)源性mi R-148a功能增強后,RelA的表達明顯被抑制。相反,轉(zhuǎn)染miR-148a inhibitors使miR-148a功能抑制后RelA的表達則顯著上調(diào)。同時,miR-148a功能增強的PBMCs再經(jīng)LPS刺激后,Th1相關(guān)細胞因子IL-8、IL-1b、TNF-a的水平均稍上調(diào),而Th2相關(guān)細胞因子IL-4、IL-5、IL-10的水平均有明顯的下降。mi R-148a功能受抑制的PBMCs再經(jīng)LPS刺激后,Th1相關(guān)細胞因子IL-8、IL-1b、TNF-a的表達均輕微上調(diào)了,而Th2相關(guān)細胞因子IL-4、IL-5、IL-10的水平均有明顯的升高。本部分結(jié)果提示,miR-148a可能通過RelA參與NF-κB信號途徑,抑制Th2相關(guān)細胞因子的分泌,促進T細胞向Th1極化偏移,進一步參與ITP的發(fā)病進程。
[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.

【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】：2017
【分類號】：R558.2;R440

【參考文獻】

相關(guān)期刊論文 前3條

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 王曉雪;王s，

本文編號：1756003