本文選題：miRNA　切入點：血清　出處：《南京醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：過敏性紫癜（HSP）是兒童最常見的系統(tǒng)性小血管炎之一，六個月之內(nèi)極易出現(xiàn)血尿和（或）蛋白尿形成紫癜性腎炎（HSPN）。目前對該疾病的發(fā)病機制尚未完全明確。腎活檢術(shù)是診斷腎臟疾病的金標準，但其有創(chuàng)傷性，并具有一定的風(fēng)險，不易被患者接受，更不易通過重復(fù)腎活檢來動態(tài)判斷治療效果和評估預(yù)后。因此，尋找方便、有效、無創(chuàng)傷性的生物學(xué)標志，將極大有利于疾病的診斷和治療。 微小RNA（microRNAs，miRNAs）是一組長約18-24nt的內(nèi)源性非編碼RNA，其表達具有組織細胞特異性、發(fā)育時序性和進化保守性，在轉(zhuǎn)錄后水平調(diào)節(jié)靶基因的表達，參與細胞增殖、分化、凋亡、脂肪代謝、氧化應(yīng)激和人類疾病形成等生理和病理過程。 眾多研究證實血清中存在穩(wěn)定表達的miRNA，可抵制RNase的降解，且受極端環(huán)境（如高溫、極酸或極堿、10次凍融循環(huán)）的影響較小，可作為疾病診斷和檢測的小侵襲性的生物學(xué)標志。本研究旨在應(yīng)用miRNA基因芯片技術(shù)，從血清標本中篩選在過敏性紫癜和紫癜性腎炎中差異表達顯著的miRNA，并應(yīng)用生物信息學(xué)預(yù)測差異表達miRNA的靶基因，初步探討miRNA在該疾病中發(fā)揮的作用，并為將來的臨床應(yīng)用奠定基礎(chǔ)。 研究材料和方法： 1.從正常對照組、過敏性紫癜組和紫癜性腎炎組中各抽出10例患兒血清標本用于芯片篩選實驗。 2.采用miRNeasy Mini Ki（tQiagen）提取分離總RNA，使用NanoDrop1000(Nanodrop, Wilmington, Delaware, USA)檢測RNA濃度。 3.利用AB TaqMan Human MicroRNA Array（TLDL低密度芯片）進行miRNA的篩選（北京博奧科技公司提供），應(yīng)用DataAssist2.0軟件進行數(shù)據(jù)分析，找出有差異表達的miRNA。 4.通過逆轉(zhuǎn)錄和實時定量PCR技術(shù)驗證芯片結(jié)果。 5.應(yīng)用TargetScan、PicTar、miRanda數(shù)據(jù)庫對差異表達miRNA進行交集靶基因預(yù)測，之后采用Cytoscape v2.8.2軟件和DAVID在線數(shù)據(jù)庫進行GO富集分析和KEGG通路分析。 實驗結(jié)果： 1.所提取的總RNA經(jīng)過檢測符合芯片實驗的要求。 2.以基因表達相差2倍為閾值，通過比較過敏性紫癜組和對照組芯片研究結(jié)果，發(fā)現(xiàn)96個miRNAs差異表達顯著，其中7個表達顯著上調(diào)，89個顯著下調(diào)；比較紫癜性腎炎組和對照組miRNA的芯片表達結(jié)果，發(fā)現(xiàn)有68個miRNAs表達呈顯著差異，其中11個表達顯著上調(diào)，57個表達顯著下調(diào)；進一步利用芯片比較紫癜性腎炎組和過敏性紫癜組miRNA的表達，發(fā)現(xiàn)53個miRNAs差異表達顯著，其中36個表達顯著上調(diào)，，17個表達顯著下調(diào)。 3.對過敏性紫癜組和紫癜性腎炎組中差異表達顯著的hsa-miR-15b，hsa-let-7d進行相對定量分析，結(jié)果顯示這些miRNAs的表達與芯片結(jié)果表達趨勢一致。 4.應(yīng)用TargetScan、PicTar、miRanda軟件對HSP/NC和HSPN/NC中有顯著表達差異的miRNA進行靶基因預(yù)測，發(fā)現(xiàn)有47個miRNAs存在交集靶基因，預(yù)測到的靶基因數(shù)為617個，而HSPN/HSP中有19個miRNAs存在交集靶基因，預(yù)測到的靶基因數(shù)為435個。之后，對這兩組交集靶基因進行GO富集分析，發(fā)現(xiàn)這些靶基因分別富集于224和219個群中（p0.05），與代謝、生長發(fā)育、表達調(diào)控等過程有關(guān)。KEGG通路分析顯示這兩組靶基因分別富集于13和11個通路中（p0.05），與細胞循環(huán)分裂、腫瘤形成和信號通路等密切相關(guān)。 實驗結(jié)論： 1.使用芯片從過敏性紫癜中篩選出96個顯著表達差異的miRNAs；從紫癜性腎炎中篩選出68個顯著表達差異的miRNAs；將紫癜性腎炎和過敏性紫癜比較時，篩選出53個差異表達顯著的miRNAs，證實miRNA在過敏性紫癜和紫癜性腎炎中均存在差異性表達。 2.應(yīng)用RT-PCR對其中2個差異表達顯著的miRNAs（hsa-miR-15b、hsa-let-7d）進行相對定量分析，其結(jié)果與芯片表達趨勢一致。 3.差異表達顯著miRNA預(yù)測交集靶基因參與細胞增殖分化、信號通路和轉(zhuǎn)錄調(diào)控等過程，miRNA有可能在過敏性紫癜、紫癜性腎炎的發(fā)病中發(fā)揮重要的作用，作為疾病新的生物學(xué)標志。
[Abstract]:Henoch Schonlein purpura (HSP) is one of the most common systemic vasculitis in children six months, prone to hematuria and (or) the formation of proteinuria of Henoch Schonlein purpura nephritis (HSPN). The pathogenesis of the disease has not yet entirely clear. Renal biopsy is the gold standard for the diagnosis of kidney disease, but its traumatic, and has a certain risk, is not easy to be accepted, it is not easy to judge the dynamic effect of treatment and prognosis through repeated renal biopsy. Therefore, looking for convenient, effective, noninvasive biomarker, will greatly benefit the diagnosis and treatment of disease.
Small RNA (microRNAs, miRNAs) is an endogenous leader about 18-24nt encoding RNA, its expression has tissue specificity, developmental timing and evolutionary conservation, regulating the expression of target genes at the transcriptional level, involved in cell proliferation, differentiation, apoptosis, lipid metabolism, oxidative stress and human disease formation physiology and pathology process.
Many studies have confirmed the existence of the stable expression of miRNA in serum, RNase can resist degradation, and extreme environment (such as high temperature, extremely acid or alkali, 10 times of freeze-thaw cycle) have little effect, can be used as a biological small invasive disease diagnosis and detection of the logo. The purpose of this study is to use miRNA gene chip technology from serum samples, screening of differentially expressed significant miRNA in allergic purpura and purpura nephritis, and application of bioinformatics to predict differential expression of target gene miRNA, preliminary study of miRNA play in the role of the disease, and lay the foundation for future clinical application.
Research materials and methods:
1. from the normal control group, the anaphylactoid purpura group and the purpura nephritis group, 10 serum samples were extracted from each group for the chip screening test.
2. the total RNA was extracted with miRNeasy Mini Ki (tQiagen) and NanoDrop1000 (Nanodrop, Wilmington, Delaware, USA) was used to detect the RNA concentration.
The use of AB TaqMan Human MicroRNA Array 3. (TLDL low density array) screening miRNA (Beijing Boao technology company), DataAssist2.0 software was used for data analysis, to find out the differences in expression of miRNA.
4. the results of the chip were verified by reverse transcription and real-time quantitative PCR technology.
5., we used TargetScan, PicTar and miRanda database to predict the target genes of differentially expressed miRNA. Then we used Cytoscape v2.8.2 software and DAVID online database to do GO enrichment analysis and KEGG pathway analysis.
Experimental results:
The 1. extracted total RNA was detected in accordance with the requirements of the chip experiment.
2. the gene expression difference of 2 times the threshold, by comparing the allergic purpura group and control group chip results showed the expression of 96 miRNAs significantly, of which 7 up-regulated, 89 down regulated significantly; compared with Henoch Schonlein purpura nephritis group and control group miRNA microarray expression results, the expression of miRNAs was found 68 significant differences, of which 11 up-regulated, 57 down regulated expression; the further use of chip comparison purpura nephritis group and allergic purpura group miRNA, found that the expression of 53 miRNAs significantly, of which 36 up-regulated, 17 down regulated.
3., the expression of hsa-miR-15b and hsa-let-7d in Henoch Schonlein purpura group and purpuric nephritis group were analyzed by relative quantitative analysis. The results showed that these miRNAs expressions were consistent with the results of chip expression.
4. application of TargetScan, PicTar, miRanda software has a significant differential expression of miRNA target gene prediction of HSP/NC and HSPN/NC, found that 47 miRNAs overlap target gene and target gene prediction to the number 617, and HSPN/HSP 19 miRNAs in the presence of intersection target gene and target gene prediction to the number of 435. After that, the two group of intersection of target gene GO enrichment analysis found that these target genes were enriched in the 224 and 219 group (P0.05), and metabolism, growth and development, regulation of the.KEGG pathway analysis showed that the two groups of target genes were enriched in the 13 and 11 pathways in expression (P0.05), mitosis and cell cycle, tumor formation and signaling pathways are closely related.
Experimental conclusions:
1. the use of chips from allergic purpura were screened 96 differentially expressed miRNAs; screened from purpura nephritis in 68 differentially expressed miRNAs; comparison of purpura nephritis and allergic purpura, screened 53 differentially expressed miRNAs was confirmed miRNA, there are differences in the expression of allergy purpura and purpura nephritis.
2. the relative quantitative analysis of 2 differentially expressed miRNAs (hsa-miR-15b, hsa-let-7d) was carried out by RT-PCR, and the results were in accordance with the trend of chip expression.
3., differential expression is significant. MiRNA predicts that the target genes are involved in cell proliferation, differentiation, signal transduction and transcriptional regulation. MiRNA may play an important role in the pathogenesis of Henoch Schonlein purpura nephritis, and serve as a new biomarker for diseases.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R725.5

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