IgA肾病患者外周血B淋巴细胞microRNA差异表达及临床意义研究

发布时间：2018-04-17 21:56

  本文选题：microRNA + IgA肾病　； 参考：《川北医学院》2014年硕士论文

【摘要】：目的：IgA肾病（IgAN）是导致终末期肾脏病常见的原因之一，其确切的病因和发病机制仍然不清楚。研究表明O-糖基化异常的IgA1分子可能是导致IgAN发病的关键因素，但IgA1分子O-糖基化异常的机制还不清楚。前期有限的研究提示IgAN患者外周单核细胞microRNA表达异常和C1GALT1C1基因甲基化异常，均提示表观遗传学改变在IgAN发病中可能起重要作用。但研究没有集中在产生IgA1的B淋巴细胞上。本研究的目的是通过检测人外周血B淋巴细胞microRNA的表达，筛选在正常人和IgAN患者中差异表达的microRNAs分子，进一步验证其表达差异，并分析其与IgAN临床病理特点及IgA1分子O-糖基化异常之间的关系，探索microRNA在IgAN发病中的可能机制。 方法：1、收集7例IgAN患者及4例正常人外周血5ml，磁珠法分选出CD19+B淋巴细胞，然后提取RNA，采用Exiqon的miRCURY LNAarray表达谱芯片进行杂交检测，对芯片结果变化差异在2倍以上进行聚类分析。挑选出差异表达明显的microRNAs作为候选microRNAs，对它们进行靶基因预测，GO分析，Pathway分析。2、临床收集29例IgAN患者，16例正常人，5例局灶节段硬化性肾小球肾炎（FSGS）患者外周血5ml，磁珠法分选出CD19+B淋巴细胞，然后提取RNA。采用ABI基于茎-环的实时定量PCR检测筛选出来的候选microRNAs的表达量，，并分析它们与临床病理的关系。3、用ELISA检测方法测定用于验证的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，分析其与microRNAs表达水平的关系。 结果：1、Exiqon miRCURY LNA miRNA芯片检测正常人与IgAN患者microRNAs表达，有115个microRNAs表达差异在2倍以上。在IgAN患者中有85个上调，30个下调。5个候选microRNAs，上调的miR-3189-5p、let-7g-5p、miR-4258、miR-4695-3p及下调的miR-99b-5p。通过生物信息学方法分析候选microRNAs参与调控的生物信息功能。2、QRT-PCR结果显示5个候选microRNAs与正常组、FSGS组比较差异均无统计学意义。其中只有miR-4258在IgAN组的相对表达量与其余两组比较有略高的趋势：正常组为2.22×10-5（0-1.119×10-4），IgAN组为2.59×10-5（8.327×10-6-2.320×10-4），FSGS组为1.89×10-5（3.692×10-6-5.807×10-5）（P=0.77）。5个候选microRNAs表达量与IgAN患者的临床病理指标没有明显的相关性。3、IgAN组血清IgA1，Gd-IgA1高于正常组与FSGS组（P0.05）,而正常组与FSGS组差异无统计学意义（P0.05）。5个候选microRNAs的水平与IgAN患者IgA1分子O-连接半乳糖基化缺陷之间没有明显相关性。 结论：1、通过microRNA芯片筛查显示IgAN患者和正常人外周血CD19+B淋巴细胞microRNA表达谱存在明显差异。2、在IgAN组和正常组的初步验证结果显示，5个候选microRNAs，miR-3189-5p、let-7g-5p、miR-4258、miR-4695-3p及下调的miR-99b-5p，表达无明显差异，且与IgAN的临床病理特点和IgA1半乳糖基缺陷之间无明显相关性，提示这5个microRNAs可能不是影响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.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.3

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