衰老小鼠巨噬细胞microRNA表达及其调控机制研究

发布时间：2018-01-03 06:24

本文关键词：衰老小鼠巨噬细胞microRNA表达及其调控机制研究　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

更多相关文章： miRNA 免疫衰老 巨噬细胞 炎症 miR-146a

【摘要】：衰老是一个多方面衰退并呈现疾病倾向的生理过程,尽管有越来越多的证据表明miRNA (microRNA)参与对衰老的调节,但是对它在巨噬细胞中与年龄相关的表达变化及其调控机制还知之甚少。与年龄相关的免疫紊乱表现为细胞因子如TNFa和IL-6等的表达水平升高和对感染的敏感性增加。因此,miRNA作为在转录后水平上基因表达的重要调节因子,对其深入研究有助于我们更好地了解免疫衰老的机制,并可为延缓衰老提供新的策略。本论文分析了年轻及自然衰老小鼠巨噬细胞在炎症应答过程中miRNA的差异表达谱,使用LNA标记的芯片技术,鉴定出各自的差异表达miRNA,在衰老小鼠巨噬细胞中,16个miRNA的表达与LPS刺激无关,但在年轻组中却表现为上调或下调；对其中miR-146a、miR-223、miR-28和miR-lOla进行了q-PCR验证,结果与芯片结果基本一致,提示这些差异miRNA的表达可能与巨噬细胞的免疫衰老密切相关；利用生物信息学软件对这些差异miRNA的靶基因进行了预测,并通过GO (gene ontology)和KEGG (Kyoto Encyclopedia of Genes and Genomes)生物信息学分析,对这些靶基因进行了生物过程、分子功能、细胞组分以及信号通路方面的注释,并建立起免疫、凋亡以及转录因子和表观遗传学三个方面的miRNA-gene相互作用网络图,结果表明,这些靶基因参与了17条重要信号通路的调节。通过芯片和real time PCR的鉴定,我们发现衰老小鼠巨噬细胞中miR-146a具有较高的表达水平,且在体内及体外均对LPS刺激缺乏免疫应答。由于在衰老小鼠巨噬细胞中]miR-146a对LPS和促炎因子刺激均无反应,我们推测miR-146a的表达调控有其他调节机制,且其表达异常与炎症细胞因子表达异常有关；对miR-146a的调控机制研究发现,NF-κB与pre-miR-146a(primary miR-146a)启动子区结合异常导致了miR-146a在小鼠腹腔巨噬细胞中的表达异常；表观遗传学研究显示,衰老小鼠巨噬细胞中,DNA甲基化及组蛋白乙酰化均参与miR-146a的表达调控。组蛋白去乙酰化酶(HDAC)抑制剂TSA (trichostatinA)可显著提高衰老小鼠的巨噬细胞中NF-κB的活性和NF-κB p65与miR-146a启动子的结合能力,且HDAC表达水平比年轻小鼠巨噬细胞高。Real time PCR检测发现LPS处理小鼠腹腔巨噬细胞24小时的过程中,衰老小鼠细胞中HDAC 1-11个亚型的mRNA表达相对于年轻小鼠表现出更明显的变化趋势,但各个时间点的整体表达均比年轻小鼠高,说明高表达的HDAC显著抑制了衰老小鼠巨噬细胞miR-146a的表达。我们进一步探讨了miR-146a对巨噬细胞Th1/Th2类细胞因子表达的影响,以miR-146a mimics、阴性对照mimics (NC mimics)、抑制性miR-146a (miR-146a inhibitor)和抑制性miR-146a阴性对照(NC inhibitor)分别瞬时转染体外培养的小鼠单核-巨噬细胞RAW264.7以及腹腔新鲜分离的原代巨噬细胞,利用real time PCR定量检测IL-18、IL-5和IL-10的表达情况,结果证实,miR-146a能够负向调控Thl类细胞因子IL-18的表达,但是不能调控Th2类细胞因子IL-5及IL-10的表达。结论：本文鉴定了衰老小鼠巨噬细胞差异表达的miRNA,分析了其靶基因及其功能,证实miR-146a等的表达异常可导致衰老小鼠巨噬细胞功能异常,转录因子NF-κB和表观遗传学均参与了衰老小鼠巨噬细胞miR-146a的表达调控,为进一步理解巨噬细胞在小鼠衰老中的作用及其干预和延缓衰老提供了新的资料。
[Abstract]:Aging is a physiological process in many aspects and presents the tendency of decline disease, although there is growing evidence that miRNA (microRNA) is involved in the regulation of aging, but it correlated with age in macrophages of the expression and regulation mechanism is still poorly understood. Age related immune disorders as the expression level of cytokines such as TNFa and IL-6, and increased susceptibility to infection. Therefore, miRNA is in the level of post transcriptional gene expression of important regulatory factors, the research helps us to better understand the mechanism of immune senescence, and may provide a new strategy for anti-aging.
This paper analyzes the difference between the young and aged mice macrophages in the inflammatory response in the expression of miRNA spectrum, using the LNA tag chip technology, identified miRNA expression of their differences in aging mice macrophages, the expression of miRNA 16 and LPS stimulation has nothing to do, but in the young group is up or down; the miR-146a, miR-223, miR-28 and miR-lOla were verified by q-PCR, the results are basically consistent with the microarray results, suggesting that these different expression of miRNA may be associated with macrophage immune senescence; target genes by using bioinformatics software for these differences in miRNA were predicted by GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) bioinformatics analysis of these target genes for molecular function, biological process, cellular components and the signaling pathway and the construction of Party notes The miRNA-gene interaction network maps of three aspects, including immunity, apoptosis, transcription factor and epigenetics, are established. These results indicate that these target genes are involved in the regulation of 17 important signaling pathways.
Through the identification chip and real time PCR, we found that miR-146a aging mice macrophages with higher levels of expression, and in vitro and in vivo stimulation of LPS immune response in aging. Due to lack of LPS and]miR-146a in mouse macrophage proinflammatory cytokine stimulation had no reaction, we speculate that the regulation of miR-146a expression with other regulatory mechanisms, and the abnormal expression of inflammatory cytokines and abnormal expression; regulation mechanism of miR-146a, NF- and pre-miR-146a (primary miR-146a B) promoter region with abnormal expression of miR-146a in mouse peritoneal macrophages in abnormal; epigenetic studies show that senescence of mouse macrophages, DNA methylation and histone acetylation were involved in the regulation of miR-146a expression. The histone deacetylase (HDAC) inhibitor TSA (trichostatinA) can significantly improve the aging of mice With the ability of macrophages in NF- kappa B activity and NF- kappa B p65 and miR-146a promoter, and the expression level of HDAC than the young mouse macrophage PCR detected high.Real time LPS process of mouse peritoneal macrophages 24 hours, HDAC cells in aging mice and 1-11 subtype mRNA expression in young mice showed a trend of more obviously compared to, but overall each time point expression than in young mice, indicating the high expression of HDAC significantly inhibited the expression of macrophage miR-146a in aging mice.
We further investigated the effect of miR-146a on expression of Th1/Th2 cytokines in miR-146a, mimics, negative control mimics (NC mimics), miR-146a (miR-146a inhibitor) inhibitory and inhibitory miR-146a negative control (NC inhibitor) were transfected into cultured mouse monocyte macrophage RAW264.7 and freshly isolated primary peritoneal macrophages using real, time quantitative detection of PCR IL-18, the expression of IL-5 and IL-10, the results show that miR-146a can negatively regulate the expression of Thl cytokines IL-18 expression, but not the regulation of Th2 cytokines IL-5 and IL-10.
Conclusion: the identification of the aging mice macrophage expression of miRNA, analyzed its target gene and its function, confirmed the expression of miR-146a can be caused by the abnormal function of aging mice macrophage abnormalities, regulation of transcription factor NF- kappa B expression and epigenetics are involved in miR-146a macrophage cells of aging mice, provide new data for further understanding the role of macrophages in aging mice and the intervention and delaying senility.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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