B.melitensis M5-90及其LPS刺激条件下RAW264.7细胞受CD14影响的miRNAs的作用机制

发布时间：2018-11-04 19:08

【摘要】：白细胞分化抗原14(cluster of differentiation antigen14, CD14)是参与脂多糖(Lipopolysaccharide, LPS)诱导炎症细胞因子释放的重要分子之一,它也是布鲁氏菌细胞壁主要成分LPS的受体。microRNA(miRNA)是一类非编码的RNA,它参与到生命活动的许多进程中,如细胞增殖、凋亡、病毒防御和细菌感染等。研究表明：CD14基因沉默可以有效抑制LPS刺激条件下肿瘤坏死因子a(TNF-a)、趋化因子配体2(MIP-2)、白细胞介素-6(IL-6)和一氧化氮(NO)的产生。该研究以本课题组雷明等人构建的mCD14knockdown RAW264.7(224.3)细胞为基础,运用miRNA基因芯片和qRT-PCR技术分析CD14沉默条件下miRNAs的差异表达；利用B.melitensis M5-90LPS刺激224.3细胞,分析B.melitensis M5-90LPS对miRNAs的影响；利用生物信息学软件和Gene Ontology对差异表达miRNAs的潜在靶基因进行预测并分类；利用qRT-PCR. Western blot和双荧光素酶报告基因技术,对B.melitensis M5-90感染224.3细胞后差异变化miR-199a-3p调控靶基因的作用机制进行初步研究。结果表明：与对照细胞相比,在224.3细胞中,mmu-miR-199a-3p、 mmu-miR-199a-5p和mmu-miR-21-5p表达上调；在B.melitensisM5-90LPS刺激224.3细胞后发现,mmu-miR-199a-3p和mmu-miR-199a-5p表达上调,但与未刺激实验结果相比,mmu-miR-199a-3p的上调水平明显降低；利用四个在线数据库对差异表达miRNAs的靶基因进行生物学预测,并通过Gene Ontology对靶基因进行分类；在B.melitensis M5-90感染224.3细胞后,Cbl-b基因表达下调,在mRNA水平和蛋白水平的验证结果一致；通过荧光素酶实验我们发现,构建含有靶位点的荧光素酶载体与miR-199a-3p模拟物共同转染靶细胞,可以显著抑制萤火虫荧光素酶活性,这些结果初步证实了在B.melitensis M5-90感染224.3细胞内,miR-199a-3p可以靶向调控Cbl-b的表达。这些结论为研究通过抑制上游炎症通路以减弱损伤性炎症反应的作用机理提供了重要信息,同时为探索布鲁氏菌病致病机理提供了重要的理论依据。
[Abstract]:Leukocyte differentiation antigen (14 (cluster of differentiation antigen14, CD14) is one of the important molecules involved in the release of inflammatory cytokines induced by lipopolysaccharide (Lipopolysaccharide, LPS). It is also the receptor. MicroRNA (miRNA) of LPS, the main component of Brucella cell wall, which is a kind of non-coding RNA,. It is involved in many life processes, such as cell proliferation, apoptosis, virus defense and bacterial infection. CD14 gene silencing can effectively inhibit the production of tumor necrosis factor a (TNF-a), chemokine ligand 2 (MIP-2), interleukin-6 (IL-6) and nitric oxide (NO) (NO) stimulated by LPS. On the basis of mCD14knockdown RAW264.7 (224.3) cells constructed by Lei Ming et al., the differential expression of miRNAs under CD14 silencing condition was analyzed by miRNA gene chip and qRT-PCR technique. Using B.melitensis M5-90LPS to stimulate 224.3 cells to analyze the effect of B.melitensis M5-90LPS on miRNAs; using bioinformatics software and Gene Ontology to predict and classify the potential target genes for differentially expressed miRNAs; and using qRT-PCR. Western blot and double luciferase reporter gene technique were used to study the mechanism of differential change of miR-199a-3p regulatory target gene in 224.3 cells infected with B.melitensis M5-90. The results showed that the expression of mmu-miR-199a-3p, mmu-miR-199a-5p and mmu-miR-21-5p was up-regulated in 224.3 cells compared with control cells. The expression of mmu-miR-199a-3p and mmu-miR-199a-5p was up-regulated in 224.3 cells stimulated by B.melitensisM5-90LPS, but the up-regulation level of mmu-miR-199a-3p was significantly lower than that of unstimulated cells. The target genes of differentially expressed miRNAs were predicted by four online databases, and the target genes were classified by Gene Ontology. After B.melitensis M5-90 was infected with 224.3 cells, the expression of Cbl-b gene was down-regulated, and the results were consistent at the level of mRNA and protein. Through luciferase experiment, we found that the construction of luciferase vector containing target sites and miR-199a-3p mimics can significantly inhibit the luciferase activity of firefly. These results suggest that miR-199a-3p can target the expression of Cbl-b in 224.3 cells infected with B.melitensis M5-90. These conclusions provide important information for the study of the mechanism of inhibiting the upstream inflammatory pathway and provide important theoretical basis for exploring the pathogenetic mechanism of brucellosis.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.7

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