本文關(guān)鍵詞：衰老小鼠巨噬細(xì)胞microRNA表達(dá)及其調(diào)控機(jī)制研究　出處：《北京協(xié)和醫(yī)學(xué)院》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： miRNA 免疫衰老 巨噬細(xì)胞 炎癥 miR-146a

【摘要】：衰老是一個(gè)多方面衰退并呈現(xiàn)疾病傾向的生理過程,盡管有越來越多的證據(jù)表明miRNA (microRNA)參與對(duì)衰老的調(diào)節(jié),但是對(duì)它在巨噬細(xì)胞中與年齡相關(guān)的表達(dá)變化及其調(diào)控機(jī)制還知之甚少。與年齡相關(guān)的免疫紊亂表現(xiàn)為細(xì)胞因子如TNFa和IL-6等的表達(dá)水平升高和對(duì)感染的敏感性增加。因此,miRNA作為在轉(zhuǎn)錄后水平上基因表達(dá)的重要調(diào)節(jié)因子,對(duì)其深入研究有助于我們更好地了解免疫衰老的機(jī)制,并可為延緩衰老提供新的策略。 本論文分析了年輕及自然衰老小鼠巨噬細(xì)胞在炎癥應(yīng)答過程中miRNA的差異表達(dá)譜,使用LNA標(biāo)記的芯片技術(shù),鑒定出各自的差異表達(dá)miRNA,在衰老小鼠巨噬細(xì)胞中,16個(gè)miRNA的表達(dá)與LPS刺激無關(guān),但在年輕組中卻表現(xiàn)為上調(diào)或下調(diào)；對(duì)其中miR-146a、miR-223、miR-28和miR-lOla進(jìn)行了q-PCR驗(yàn)證,結(jié)果與芯片結(jié)果基本一致,提示這些差異miRNA的表達(dá)可能與巨噬細(xì)胞的免疫衰老密切相關(guān)；利用生物信息學(xué)軟件對(duì)這些差異miRNA的靶基因進(jìn)行了預(yù)測(cè),并通過GO (gene ontology)和KEGG (Kyoto Encyclopedia of Genes and Genomes)生物信息學(xué)分析,對(duì)這些靶基因進(jìn)行了生物過程、分子功能、細(xì)胞組分以及信號(hào)通路方面的注釋,并建立起免疫、凋亡以及轉(zhuǎn)錄因子和表觀遺傳學(xué)三個(gè)方面的miRNA-gene相互作用網(wǎng)絡(luò)圖,結(jié)果表明,這些靶基因參與了17條重要信號(hào)通路的調(diào)節(jié)。 通過芯片和real time PCR的鑒定,我們發(fā)現(xiàn)衰老小鼠巨噬細(xì)胞中miR-146a具有較高的表達(dá)水平,且在體內(nèi)及體外均對(duì)LPS刺激缺乏免疫應(yīng)答。由于在衰老小鼠巨噬細(xì)胞中]miR-146a對(duì)LPS和促炎因子刺激均無反應(yīng),我們推測(cè)miR-146a的表達(dá)調(diào)控有其他調(diào)節(jié)機(jī)制,且其表達(dá)異常與炎癥細(xì)胞因子表達(dá)異常有關(guān)；對(duì)miR-146a的調(diào)控機(jī)制研究發(fā)現(xiàn),NF-κB與pre-miR-146a(primary miR-146a)啟動(dòng)子區(qū)結(jié)合異常導(dǎo)致了miR-146a在小鼠腹腔巨噬細(xì)胞中的表達(dá)異常；表觀遺傳學(xué)研究顯示,衰老小鼠巨噬細(xì)胞中,DNA甲基化及組蛋白乙�；鶇⑴cmiR-146a的表達(dá)調(diào)控。組蛋白去乙酰化酶(HDAC)抑制劑TSA (trichostatinA)可顯著提高衰老小鼠的巨噬細(xì)胞中NF-κB的活性和NF-κB p65與miR-146a啟動(dòng)子的結(jié)合能力,且HDAC表達(dá)水平比年輕小鼠巨噬細(xì)胞高。Real time PCR檢測(cè)發(fā)現(xiàn)LPS處理小鼠腹腔巨噬細(xì)胞24小時(shí)的過程中,衰老小鼠細(xì)胞中HDAC 1-11個(gè)亞型的mRNA表達(dá)相對(duì)于年輕小鼠表現(xiàn)出更明顯的變化趨勢(shì),但各個(gè)時(shí)間點(diǎn)的整體表達(dá)均比年輕小鼠高,說明高表達(dá)的HDAC顯著抑制了衰老小鼠巨噬細(xì)胞miR-146a的表達(dá)。 我們進(jìn)一步探討了miR-146a對(duì)巨噬細(xì)胞Th1/Th2類細(xì)胞因子表達(dá)的影響,以miR-146a mimics、陰性對(duì)照mimics (NC mimics)、抑制性miR-146a (miR-146a inhibitor)和抑制性miR-146a陰性對(duì)照(NC inhibitor)分別瞬時(shí)轉(zhuǎn)染體外培養(yǎng)的小鼠單核-巨噬細(xì)胞RAW264.7以及腹腔新鮮分離的原代巨噬細(xì)胞,利用real time PCR定量檢測(cè)IL-18、IL-5和IL-10的表達(dá)情況,結(jié)果證實(shí),miR-146a能夠負(fù)向調(diào)控Thl類細(xì)胞因子IL-18的表達(dá),但是不能調(diào)控Th2類細(xì)胞因子IL-5及IL-10的表達(dá)。 結(jié)論：本文鑒定了衰老小鼠巨噬細(xì)胞差異表達(dá)的miRNA,分析了其靶基因及其功能,證實(shí)miR-146a等的表達(dá)異�？蓪�(dǎo)致衰老小鼠巨噬細(xì)胞功能異常,轉(zhuǎn)錄因子NF-κB和表觀遺傳學(xué)均參與了衰老小鼠巨噬細(xì)胞miR-146a的表達(dá)調(diào)控,為進(jìn)一步理解巨噬細(xì)胞在小鼠衰老中的作用及其干預(yù)和延緩衰老提供了新的資料。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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1 王磊;單良;;microRNA與宮頸癌關(guān)系的研究[J];科技信息;2011年21期

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本文編號(hào)：1372760