本文選題：微小RNA　切入點(diǎn)：B細(xì)胞趨化因子13　出處：《廣西醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：通過(guò)采用微小RNA基因芯片技術(shù)、生物信息學(xué)方法、實(shí)時(shí)熒光定量PCR（QRT-PCR）和雙熒光報(bào)告等方法，探討重癥肌無(wú)力伴胸腺增生（Myasthenia Gravis with thymic Hyperplasia，MGH）患者胸腺組織內(nèi)B細(xì)胞趨化因子13（CXCL13）的微小RNA調(diào)控機(jī)制。 方法：重癥肌無(wú)力的胸腺組織來(lái)源于2012年3月至2013年5月在廣西醫(yī)科大學(xué)第一附屬醫(yī)院心胸外科行胸腺切除手術(shù)的13例MGH患者，對(duì)照組的胸腺組織來(lái)源于同期在廣西醫(yī)科大學(xué)心胸外科行心臟手術(shù)的13例心臟病患者，對(duì)照組患者的性別和年齡與MGH組相匹配，并排除自身免疫性疾�。挥肨rizol裂解液提取胸腺組織中的總RNA；用微小RNA基因芯片技術(shù)篩選MGH組胸腺組織和對(duì)照組胸腺組織之間差異表達(dá)的微小RNA；用生物信息學(xué)預(yù)測(cè)方法并結(jié)合微小RNA基因芯片結(jié)果尋找靶向CXCL13的微小RNA；對(duì)CXCL13mRNA和尋找到的微小RNA在MGH組胸腺組織內(nèi)和對(duì)照組胸腺組織內(nèi)的表達(dá)進(jìn)行實(shí)時(shí)熒光定量PCR（QRT-PCR）定量分析；構(gòu)建CXCL13基因3’-非編碼區(qū)（3’-untranslated region，3’-UTR）雙熒光報(bào)告載體，然后進(jìn)行雙熒光報(bào)告分析。 結(jié)果：1、芯片結(jié)果顯示，，相比對(duì)照組，MGH組胸腺組織內(nèi)顯著差異表達(dá)的微小RNA有33個(gè)，微小RNA-548k是下調(diào)最顯著中的一個(gè)（1.982倍，p＜0.01）。2、生物信息學(xué)預(yù)測(cè)顯示，微小RNA-548k與CXCL13基因的3′UTR有明顯的相互作用（P＜0.05）。3、QRT-PCR定量分析發(fā)現(xiàn)，MGH組胸腺組織內(nèi)微小RNA-548k的表達(dá)量比對(duì)照組胸腺組織內(nèi)的表達(dá)量[（0.5486±0.20234）比(1.3338±0.36488) P＜0.01）]顯著下調(diào)，與芯片結(jié)果相一致；MGH組胸腺組織內(nèi)CXCL13的表達(dá)量比對(duì)照組胸腺組織內(nèi)的表達(dá)量[（4.9304±1.95019）比(1.0378±0.19667) P＜0.01）]顯著上調(diào)；MGH組胸腺組織內(nèi)微小RNA-548k與CXCL13表達(dá)呈明顯負(fù)相關(guān)（r=-0.919,P0.01）。4、經(jīng)酶切及基因測(cè)序驗(yàn)證，成功構(gòu)建含有CXCL13基因3’-UTR段雙熒光素酶基因報(bào)告載體。5、雙熒光報(bào)告顯示，微小RNA-548k mimics組的CXCL13基因3’-UTR雙熒光報(bào)告載體熒光素酶活性比空白對(duì)照組[（0.385±0.0156）比（1±0.0501） P0.01）]下降61.5％，微小RNA-548k對(duì)CXCL13有明顯負(fù)調(diào)控作用。 結(jié)論：1、本實(shí)驗(yàn)首次發(fā)現(xiàn)MGH患者胸腺組織內(nèi)有其特異的微小RNA表達(dá)譜。2、MGH患者胸腺組織內(nèi)微小RNA-548k的低表達(dá)很可能通過(guò)轉(zhuǎn)錄后基因沉默機(jī)制使其靶基因CXCL13過(guò)表達(dá)，從而參與重癥肌無(wú)力的發(fā)生發(fā)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 鹵0.20234 vs 1.3338 鹵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 鹵1.95019 vs 1.0378 鹵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 鹵0.0156 vs 1 鹵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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R746.1

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