重症肌无力伴胸腺增生患者胸腺组织内微小RNA-548k调控CXCL13的研究

发布时间：2018-03-19 16:22

本文选题：微小RNA　切入点：B细胞趋化因子13　出处：《广西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：通过采用微小RNA基因芯片技术、生物信息学方法、实时荧光定量PCR（QRT-PCR）和双荧光报告等方法，探讨重症肌无力伴胸腺增生（Myasthenia Gravis with thymic Hyperplasia，MGH）患者胸腺组织内B细胞趋化因子13（CXCL13）的微小RNA调控机制。方法：重症肌无力的胸腺组织来源于2012年3月至2013年5月在广西医科大学第一附属医院心胸外科行胸腺切除手术的13例MGH患者，对照组的胸腺组织来源于同期在广西医科大学心胸外科行心脏手术的13例心脏病患者，对照组患者的性别和年龄与MGH组相匹配，并排除自身免疫性疾病；用Trizol裂解液提取胸腺组织中的总RNA；用微小RNA基因芯片技术筛选MGH组胸腺组织和对照组胸腺组织之间差异表达的微小RNA；用生物信息学预测方法并结合微小RNA基因芯片结果寻找靶向CXCL13的微小RNA；对CXCL13mRNA和寻找到的微小RNA在MGH组胸腺组织内和对照组胸腺组织内的表达进行实时荧光定量PCR（QRT-PCR）定量分析；构建CXCL13基因3’-非编码区（3’-untranslated region，3’-UTR）双荧光报告载体，然后进行双荧光报告分析。结果：1、芯片结果显示，，相比对照组，MGH组胸腺组织内显著差异表达的微小RNA有33个，微小RNA-548k是下调最显著中的一个（1.982倍，p＜0.01）。2、生物信息学预测显示，微小RNA-548k与CXCL13基因的3′UTR有明显的相互作用（P＜0.05）。3、QRT-PCR定量分析发现，MGH组胸腺组织内微小RNA-548k的表达量比对照组胸腺组织内的表达量[（0.5486±0.20234）比(1.3338±0.36488) P＜0.01）]显著下调，与芯片结果相一致；MGH组胸腺组织内CXCL13的表达量比对照组胸腺组织内的表达量[（4.9304±1.95019）比(1.0378±0.19667) P＜0.01）]显著上调；MGH组胸腺组织内微小RNA-548k与CXCL13表达呈明显负相关（r=-0.919,P0.01）。4、经酶切及基因测序验证，成功构建含有CXCL13基因3’-UTR段双荧光素酶基因报告载体。5、双荧光报告显示，微小RNA-548k mimics组的CXCL13基因3’-UTR双荧光报告载体荧光素酶活性比空白对照组[（0.385±0.0156）比（1±0.0501） P0.01）]下降61.5％，微小RNA-548k对CXCL13有明显负调控作用。结论：1、本实验首次发现MGH患者胸腺组织内有其特异的微小RNA表达谱。2、MGH患者胸腺组织内微小RNA-548k的低表达很可能通过转录后基因沉默机制使其靶基因CXCL13过表达，从而参与重症肌无力的发生发展。
[Abstract]:Objective: to use microarray RNA technique, bioinformatics method, real-time fluorescence quantitative PCRQRT-PCRand double fluorescence report, etc. To investigate the mechanism of minimal RNA regulation of B cell chemokine 13 (CXCL13) in thymus tissues of patients with myasthenia gravis and thymic hyperplasia (Myasthenia Gravis with thymic Hyperplasia MGH). Methods: from March 2012 to May 2013, the thymus tissue of myasthenia gravis was obtained from 13 MGH patients who underwent thymectomy in cardiothoracic surgery, the first affiliated Hospital of Guangxi Medical University. The thymus tissue of the control group was derived from 13 patients undergoing cardiac surgery in cardiothoracic surgery of Guangxi Medical University at the same time. The sex and age of the control group matched with that of the MGH group and the autoimmune diseases were excluded. Total RNAs were extracted from thymus tissue by Trizol cleavage solution; microRNAs differentially expressed between thymus tissues of MGH group and control group were screened by microarray technique; bioinformatics method was used to predict the difference between MGH group thymus tissue and control group thymus tissue. Bioinformatics prediction method combined with tiny RNA gene core was used. The expression of CXCL13mRNA and RNA in the thymus tissues of MGH group and control group were analyzed quantitatively by real-time fluorescence quantitative quantitation. A double fluorescent report vector of CXCL13 gene was constructed, and then double fluorescence report analysis was carried out. Results: the microarray results showed that there were 33 micro#en0# in thymus tissues with significant difference compared with the control group. The micro#en1# was one of the most down-regulated significantly (P < 0.01), and the bioinformatics prediction showed that the expression of micro#en0# in thymus tissues was significantly lower than that in control group (P < 0.01). There was a significant interaction between micro#en0# and CXCL13 gene. The quantitative analysis of RNA-548k in thymus tissues of MGH group showed that the expression of RNA-548k in thymus tissue was significantly lower than that in control group [0.5486 卤0.20234 vs 1.3338 卤0.36488, P < 0.01]. In accordance with the microarray results, the expression of CXCL13 in thymus tissue of MGH group was significantly higher than that in control group (4.9304 卤1.95019 vs 1.0378 卤0.19667) P < 0.01). The double luciferase gene report vector. 5 containing CXCL13 gene 3na-UTR was successfully constructed. The luciferase activity of the CXCL13 gene 3H-UTR double fluorescent report vector in the small RNA-548k mimics group was 61.5% lower than that in the blank control group [0.385 卤0.0156 vs 1 卤0.0501) P 0.01]. The minimal RNA-548k had a negative effect on CXCL13. Conclusion: in this study, we first found that there is a specific expression profile of tiny RNA in thymus of MGH patients. 2 the low expression of RNA-548k in thymus tissue of MGH patients may make its target gene CXCL13 overexpression through post-transcriptional gene silencing mechanism. Thus participate in the occurrence and development of myasthenia gravis.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.1

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