本文選題：長鏈非編碼RNA + 腸巨噬細胞��； 參考：《江蘇大學》2017年碩士論文

【摘要】：目的本課題旨在分析長鏈非編碼RNA(long noncoding RNA,lncRNA)在脂多糖(Lipopolysaccharide,LPS)誘導大鼠的腸巨噬細胞組與未處理組之間差異表達譜,并對差異表達的mRNA進行生物信息學分析,為初步探討lncRNA的調控機制奠定基礎。方法1.提取六只大鼠的腸巨噬細胞進行培養(yǎng),隨機分成兩組,實驗組加脂多糖(濃度為1 mg/L)誘導大鼠的腸巨噬細胞,對照組則未加脂多糖。應用高通量基因芯片技術(Affymetrix Gene Chip Mouse Transcriptome Array 1.0)檢測兩組之間差異表達的lncRNA和mRNA,并用分層聚類的方法分析兩組間差異表達的lncRNA和mRNA。2.對芯片篩選出差異表達的mRNA進行基因功能分析(GO-Analysis)和信號通路分析(Pathway-Analysis)探索其參與的生物學功能,并構建差異表達lncRNA和mRNA的共表達網絡,從中發(fā)現(xiàn)處于核心調控地位的lncRNA。3.結合共表達網絡分析結果和基因芯片數(shù)據,挑選出差異表達的lncRNA和mRNA采用實時熒光定量PCR技術,對芯片結果進行驗證。結果1.將差異表達的納入標準設為:差異表達倍數(shù)(Flod-change)1.5,P值0.05,與對照組相比,LPS處理的腸巨噬細胞組lncRNA和mRNA表達譜發(fā)生明顯變化,差異表達的lncRNA共有357個,其中上調的有245個,下調的有112個。差異表達的mRNA共有542個,其中上調的有187個,下調的有355個。2.基因功能分析顯示表達上調的m RNA主要涉及炎癥反應、固有免疫應答、代謝過程、信號轉導等生物學功能;表達下調的mRNA主要涉及代謝過程、細胞周期、免疫應答以及凋亡過程等生物學功能。通路分析顯示與上調基因相關的顯著性通路主要包括NF-kappa B信號通路、B細胞受體信號通路、細胞凋亡信號通路等;與下調基因相關的顯著性通路主要有代謝通路、磷脂酰肌醇信號通路、細胞因子-細胞因子受體相互作用以及Toll樣受體信號通路等。構建lncRNA和mRNA共表達網絡,其中l(wèi)ncRNA NONMMUT062258、NONMMUT046238、NONMMUT024673、NONMMUT062258處于核心地位。3.實時熒光定量PCR結果顯示差異表達的lncRNA和mRNA與基因芯片數(shù)據一致。結論本研究首次通過基因芯片技術對LPS處理的大鼠腸巨噬細胞中l(wèi)ncRNA的表達譜進行了檢測。芯片結果顯示與無LPS處理的對照組相比,lncRNA在LPS處理的腸巨噬細胞中呈差異表達。實時定量PCR驗證結果證實芯片數(shù)據穩(wěn)定而可靠。因此差異表達的lncRNA可能參與調控LPS誘導腸巨噬細胞引起的炎癥反應。此外用生物信息學分析探討了潛在的調控機制。盡管需要更多的研究來證明這些lncRNA的確切調控機制,但我們鑒定的基因組數(shù)據可以為深入研究腸屏障功能障礙時腸巨噬細胞調控炎癥反應的分子機制提供新的線索和思路。
[Abstract]:Objective to analyze the differential expression profiles of long-chain noncoding RNA(long noncoding RNAs in lipopolysaccharide polysaccharide (LPS) -induced intestinal macrophage group and untreated group, and to analyze the differentially expressed mRNA by bioinformatics. It lays a foundation for the preliminary study of the regulatory mechanism of lncRNA. Method 1. The intestinal macrophages of six rats were cultured and randomly divided into two groups. The experimental group was induced by lipopolysaccharide (1 mg / L), while the control group was not lipopolysaccharide. The differentially expressed lncRNA and mRNAs were detected by affymetrix Gene Chip Mouse Transcriptome Array 1.0, and the lncRNA and mRNA.2differentially expressed between the two groups were analyzed by hierarchical clustering. Gene function analysis (GO-analysis) and signal pathway analysis (Pathway-Analysis) of differentially expressed mRNA were carried out to explore its biological function, and to construct coexpression network of differentially expressed lncRNA and mRNA, from which we found lncRNA.3in the core regulatory position. Based on the results of coexpression network analysis and gene chip data, the differentially expressed lncRNA and mRNA were selected and verified by real-time fluorescence quantitative PCR. Result 1. The inclusion criteria of differential expression were as follows: the differential expression multiple of lncRNA and mRNA in LPS-treated intestinal macrophages was 0.05. The expression profiles of lncRNA and mRNA in LPS-treated intestinal macrophages were significantly changed, and there were 357 differentially expressed lncRNA, 245 of which were up-regulated. There were 112 downgrades. Of the 542 differentially expressed mRNA, 187 were up-regulated and 355. 2. Gene function analysis showed that the up-regulated expression of m RNA was mainly related to inflammatory response, innate immune response, metabolic process, signal transduction and other biological functions, while down-regulated mRNA was mainly involved in metabolic process, cell cycle, and so on. Immune response and apoptosis process and other biological functions. Pathway analysis showed that significant pathway related to up-regulation gene mainly included NF-kappa B signal pathway, cell apoptosis signal pathway and so on, and the significant pathway related to down-regulation gene was metabolic pathway. Phosphatidylinositol signaling pathway, cytokine-cytokine receptor interaction and Toll-like receptor signaling pathway. The coexpression network of lncRNA and mRNA was constructed, in which lncRNA NONMMUT062258, NONMMUT046238, NONMMUT024673, NONMMUT062258 was at the core. The results of real-time fluorescence quantitative PCR showed that the differentially expressed lncRNA and mRNA were consistent with the gene chip data. Conclusion for the first time, the expression profile of lncRNA in LPS treated rat intestinal macrophages was detected by gene chip technique. The microarray results showed that there was a difference in the expression of lncRNA in LPS treated intestinal macrophages compared with the control group without LPS treatment. Real-time quantitative PCR verification results show that the chip data is stable and reliable. Therefore, differentially expressed lncRNA may be involved in regulating the inflammatory response induced by LPS in intestinal macrophages. In addition, the potential regulation mechanism was analyzed by bioinformatics. Although more studies are needed to prove the exact regulatory mechanisms of these lncRNA, the genomic data we have identified can provide new clues and ideas for the further study of the molecular mechanisms of intestinal macrophages regulating inflammatory response in the context of intestinal barrier dysfunction.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R57

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本文編號：1795780