【摘要】：病毒感染機(jī)體后,干擾素(Interferon, IFNs)大量產(chǎn)生,Ⅰ型干擾素的產(chǎn)生在天然免疫中起重要的作用,對宿主的防御起關(guān)鍵作用。MicroRNAs (miRNAs)是近年發(fā)現(xiàn)的一類內(nèi)源性的非編碼RNA,它除了對基因表達(dá)的轉(zhuǎn)錄調(diào)控作用外,今年來發(fā)現(xiàn)miRNAs介導(dǎo)的轉(zhuǎn)錄后調(diào)節(jié)在調(diào)節(jié)基因的表達(dá)也有重要的作用。MicroRNA-223(miR-223)是特異性表達(dá)在髓系細(xì)胞的miRNA。對于miR-223在巨噬細(xì)胞中Ⅰ型干擾素產(chǎn)生的調(diào)控作用,以及靶點都還未有研究。 我們發(fā)現(xiàn),在小鼠巨噬細(xì)胞系RAW264.7或原代培養(yǎng)的巨噬細(xì)胞中,用VSV病毒感染,熒光定量PCR檢測結(jié)果顯示VSV病毒感染后巨噬細(xì)胞中miR-223的mRNA表達(dá)水平發(fā)生明顯下調(diào)。用miR-223mimics進(jìn)行功能性實驗研究發(fā)現(xiàn),miR-223過表達(dá)可明顯促進(jìn)VSV誘導(dǎo)的巨噬細(xì)胞IFN-α、IFN-βmRNA表達(dá)水平,而對TNF-α和IL-6的mRNA表達(dá)沒有影響。在小鼠巨噬細(xì)胞系RAW264.7中轉(zhuǎn)染miR-223mimics,用VSV-GFP病毒感染,運(yùn)用共聚焦熒光顯微鏡和流式細(xì)胞術(shù)檢測,發(fā)現(xiàn)過表達(dá)miR-223的細(xì)胞中熒光減少,說明miR-223在抗病毒感染中可能起重要的調(diào)節(jié)作用。由于miRNA發(fā)揮生物學(xué)功能主要通過調(diào)控其靶基因的表達(dá),通過進(jìn)一步的熒光素酶報告基因試驗和熒光定量PCR等實驗結(jié)果顯示miR-223可以直接靶向抑制NLRP3的表達(dá)。同時,過表達(dá)NLRP3后,熒光素酶報告基因結(jié)果顯示,IRF3和IFN-β的活性降低,說明NLRP3對Ⅰ型干擾素的產(chǎn)生可能有抑制作用。 綜上所述,我們的實驗結(jié)果初步發(fā)現(xiàn),VSV感染后巨噬細(xì)胞中miR-223表達(dá)下調(diào),miR-223可通過靶向抑制NLRP3的表達(dá),促進(jìn)巨噬細(xì)胞IFN-α、IFN-β的分泌。上述研究為揭示非編碼RNA在巨噬細(xì)胞天然免疫功能的精細(xì)調(diào)控中發(fā)揮的作用提供了理論依據(jù),并可能為感染性疾病的診治提供潛在的靶點和思路。
[Abstract]:Interferon (Interferon, IFNs) is produced in large quantities after virus infection. Interferon I plays an important role in innate immunity and plays a key role in host defense. MicroRNAs (miRNAs) is a kind of endogenous non-coding RNA, discovered in recent years. In addition to its transcriptional regulation of gene expression, miRNAs-mediated post-transcriptional regulation is also found to play an important role in regulating gene expression this year. MicroRNA-223 (miR-223) is specifically expressed in the miRNA. of myeloid cells. The regulatory effects and targets of miR-223 on IFN-I production in macrophages have not been studied. We found that the expression level of miR-223 mRNA in murine macrophage cell line RAW264.7 or primary cultured macrophages was down-regulated by fluorescence quantitative PCR (FQ-PCR) after infection with VSV virus. The results showed that the expression level of VSV in macrophages was down-regulated after infection with VSV virus by fluorescence quantitative PCR (FQ-PCR). The results of functional experiment with miR-223mimics showed that the overexpression of miR-223 significantly promoted the expression of IFN- 偽 and IFN- 尾 in macrophages induced by VSV, but had no effect on the expression of mRNA in TNF- 偽 and IL-6. The transfection of miR-223mimics, into mouse macrophage cell line RAW264.7 was infected with VSV-GFP virus, and detected by confocal fluorescence microscope and flow cytometry, it was found that the fluorescence of over-expressed miR-223 cells decreased. These results suggest that miR-223 may play an important role in the regulation of anti-viral infection. Because miRNA plays a biological role by regulating the expression of its target gene, the results of luciferase reporter gene test and fluorescent quantitative PCR show that miR-223 can directly target the expression of NLRP3. At the same time, the results of luciferase reporter gene showed that the activities of IRF3 and IFN- 尾 decreased after overexpression of NLRP3, suggesting that NLRP3 may inhibit the production of interferon 鈪，

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