本文選題：microRNA + IgA腎病　； 參考：《川北醫(yī)學院》2014年碩士論文

【摘要】：目的：IgA腎病（IgAN）是導致終末期腎臟病常見的原因之一，其確切的病因和發(fā)病機制仍然不清楚。研究表明O-糖基化異常的IgA1分子可能是導致IgAN發(fā)病的關(guān)鍵因素，但IgA1分子O-糖基化異常的機制還不清楚。前期有限的研究提示IgAN患者外周單核細胞microRNA表達異常和C1GALT1C1基因甲基化異常，均提示表觀遺傳學改變在IgAN發(fā)病中可能起重要作用。但研究沒有集中在產(chǎn)生IgA1的B淋巴細胞上。本研究的目的是通過檢測人外周血B淋巴細胞microRNA的表達，篩選在正常人和IgAN患者中差異表達的microRNAs分子，進一步驗證其表達差異，并分析其與IgAN臨床病理特點及IgA1分子O-糖基化異常之間的關(guān)系，探索microRNA在IgAN發(fā)病中的可能機制。 方法：1、收集7例IgAN患者及4例正常人外周血5ml，磁珠法分選出CD19+B淋巴細胞，然后提取RNA，采用Exiqon的miRCURY LNAarray表達譜芯片進行雜交檢測，對芯片結(jié)果變化差異在2倍以上進行聚類分析。挑選出差異表達明顯的microRNAs作為候選microRNAs，對它們進行靶基因預測，GO分析，Pathway分析。2、臨床收集29例IgAN患者，16例正常人，5例局灶節(jié)段硬化性腎小球腎炎（FSGS）患者外周血5ml，磁珠法分選出CD19+B淋巴細胞，然后提取RNA。采用ABI基于莖-環(huán)的實時定量PCR檢測篩選出來的候選microRNAs的表達量，，并分析它們與臨床病理的關(guān)系。3、用ELISA檢測方法測定用于驗證的29例IgAN患者，16例正常人，5例FSGS患者IgA1分子半乳糖缺陷（Gd-IgA1）的水平，分析其與microRNAs表達水平的關(guān)系。 結(jié)果：1、Exiqon miRCURY LNA miRNA芯片檢測正常人與IgAN患者microRNAs表達，有115個microRNAs表達差異在2倍以上。在IgAN患者中有85個上調(diào)，30個下調(diào)。5個候選microRNAs，上調(diào)的miR-3189-5p、let-7g-5p、miR-4258、miR-4695-3p及下調(diào)的miR-99b-5p。通過生物信息學方法分析候選microRNAs參與調(diào)控的生物信息功能。2、QRT-PCR結(jié)果顯示5個候選microRNAs與正常組、FSGS組比較差異均無統(tǒng)計學意義。其中只有miR-4258在IgAN組的相對表達量與其余兩組比較有略高的趨勢：正常組為2.22×10-5（0-1.119×10-4），IgAN組為2.59×10-5（8.327×10-6-2.320×10-4），F(xiàn)SGS組為1.89×10-5（3.692×10-6-5.807×10-5）（P=0.77）。5個候選microRNAs表達量與IgAN患者的臨床病理指標沒有明顯的相關(guān)性。3、IgAN組血清IgA1，Gd-IgA1高于正常組與FSGS組（P0.05）,而正常組與FSGS組差異無統(tǒng)計學意義（P0.05）。5個候選microRNAs的水平與IgAN患者IgA1分子O-連接半乳糖基化缺陷之間沒有明顯相關(guān)性。 結(jié)論：1、通過microRNA芯片篩查顯示IgAN患者和正常人外周血CD19+B淋巴細胞microRNA表達譜存在明顯差異。2、在IgAN組和正常組的初步驗證結(jié)果顯示，5個候選microRNAs，miR-3189-5p、let-7g-5p、miR-4258、miR-4695-3p及下調(diào)的miR-99b-5p，表達無明顯差異，且與IgAN的臨床病理特點和IgA1半乳糖基缺陷之間無明顯相關(guān)性，提示這5個microRNAs可能不是影響IgA1分子O-糖基化的重要分子。
[Abstract]:Objective IgA nephropathy is one of the most common causes of end-stage renal disease. The exact etiology and pathogenesis of IgA nephropathy are still unclear.It is suggested that the IgA1 molecule with abnormal Oglycosylation may be the key factor leading to the pathogenesis of IgAN, but the mechanism of Oglycosylation abnormality of IgA1 molecule is not clear.The limited previous studies suggest that the abnormal expression of microRNA in peripheral monocytes and the abnormal methylation of C1GALT1C1 gene in peripheral monocytes of patients with IgAN all suggest that epigenetic changes may play an important role in the pathogenesis of IgAN.But the study did not focus on B lymphocytes that produce IgA1.The purpose of this study was to detect the expression of microRNA in human peripheral blood B lymphocytes and to screen the differentially expressed microRNAs molecules in normal subjects and IgAN patients.The relationship between microRNA and the clinicopathological characteristics of IgAN and the abnormal Oglycosylation of IgA1 molecules was analyzed to explore the possible mechanism of microRNA in the pathogenesis of IgAN.Methods 7 IgAN patients and 4 normal controls were collected from peripheral blood of 7 IgAN patients and 4 normal controls. CD19 B lymphocytes were isolated by magnetic bead method, then CD19 was extracted and hybridized by Exiqon miRCURY LNAarray expression microarray. The difference of the results of the microarray was more than 2 times.The differentially expressed microRNAs were selected as candidates for microRNAs.The target gene predictor go analysis Pathway analysis was used to analyze them. Clinical data were collected from 29 IgAN patients and 16 normal controls (5 ml in peripheral blood and 5 ml in magnetic beads) from 5 patients with focal segmental sclerosing glomerulonephritis (FSGs).CD19 B lymphocytes were isolated by Elisa.Then the RNA was extracted.The expression of candidate microRNAs was detected by ABI real-time quantitative PCR based on stem loop.The relationship between Gd-IgA1 and clinicopathology was analyzed. The level of IgA1 Gd-IgA1 was determined by ELISA assay in 29 IgAN patients and 5 FSGS patients. The relationship between Gd-IgA1 and microRNAs expression was analyzed.Results the expression of microRNAs in normal subjects and IgAN patients was detected by 1: 1Exiqon miRCURY LNA miRNA chip. The difference of microRNAs expression in 115 cases was more than 2 times.In patients with IgAN, there were 85 upregulation, 30 down-regulation, 5 candidate miR-3189-5pNas, up-regulated miR-3189-7g-5pnmiR-4258miR-4695-3p and down-regulated miR-99b-5p.The bioinformatics method was used to analyze the bioinformatics function of candidate microRNAs. The results of QRT-PCR showed that there was no significant difference between the five candidate microRNAs and the control group.Only the relative expression of miR-4258 in the IgAN group was slightly higher than that in the other two groups: 2.22 脳 10-5 ~ (0-1.119 脳 10 ~ (-4)) in the normal group was 2.59 脳 10 ~ (-5) ~ 8.327 脳 10 ~ (-6) to 2.320 脳 10 ~ (-4) 脳 10 ~ (-4) IgAN group, 1.89 脳 10 ~ (-5) -5.807 脳 10 ~ (-5) microRNAs expression level was 1.89 脳 10 ~ (-5) ~ (-5.807) 脳 10 ~ (-5) microRNAs 0.770.There was no significant correlation between the expression of 5 candidate microRNAs and the clinicopathological index of IgAN patients.It was higher than that of normal group and FSGS group (P 0.05), but there was no significant difference between normal group and FSGS group. There was no significant correlation between the level of 5 candidate microRNAs and the defect of IgA1 molecule O-junction galactosylation in IgAN patients.Conclusion microRNA microarray screening showed that there was a significant difference in microRNA expression profiles between peripheral blood CD19 B lymphocytes of IgAN patients and normal controls. The results of preliminary verification in IgAN group and normal group showed that there was no significant difference in the expression of 5 candidate microRNAsmiR-3189-5plet-7g-5pmiR-4258 miR-4695-3p and down-regulated miR-99b-5p.There was no significant correlation between the clinicopathological features of IgAN and IgA1 galactosyl defects, suggesting that these five microRNAs may not be important molecules affecting the Oglycosylation of IgA1 molecules.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692.3

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