本文選題：APAP + miR-223�。� 參考：《安徽醫(yī)科大學(xué)》2016年博士論文

【摘要】：對(duì)乙酰氨基酚(acetaminophen, APAP)過量使用是世界各地急性肝臟衰竭的主要誘因之一,其與肝臟內(nèi)大量中性粒細(xì)胞的浸潤密切相關(guān)。然而,有關(guān)肝臟內(nèi)中性粒細(xì)胞浸潤與炎癥的具體機(jī)制仍不是很清楚。近年來研究發(fā)現(xiàn)微小RNA(microRNA, miRNA)可參與調(diào)控細(xì)胞增殖、分化、凋亡等多種重要生物學(xué)過程。髓系特異表達(dá)的miRNA-223被報(bào)道可以調(diào)節(jié)中性粒細(xì)胞的生成和功能,其是否參與調(diào)控APAP誘導(dǎo)的急性肝損傷及相關(guān)中性粒細(xì)胞的功能目前還未見文獻(xiàn)報(bào)道。本研究構(gòu)建miR-223敲除小鼠,以此深入探討其對(duì)APAP誘導(dǎo)急性肝損傷及中性粒細(xì)胞功能的影響及其作用的分子機(jī)制。本實(shí)驗(yàn)分別對(duì)空腹過夜饑餓或正常飲食條件下野生型小鼠和miR-223敲除小鼠進(jìn)行APAP腹腔注射以建立小鼠急性肝損傷模型。6小時(shí)或者24小時(shí)后,取小鼠血清進(jìn)行肝臟轉(zhuǎn)氨酶和miR-223檢測,取肝組織進(jìn)行病理組織學(xué)及相關(guān)免疫組化檢測。采用磁珠分選方法分離肝臟,骨髓及外周血中中性粒細(xì)胞進(jìn)行相關(guān)基因?qū)崟r(shí)定量PCR或者western blot檢測。結(jié)果發(fā)現(xiàn),過量APAP注射能明顯升高小鼠肝臟和血清中miR-223水平。更重要的是,相比于野生型小鼠,miR-223敲除小鼠血清中具有更高水平的ALT,AST,及更加嚴(yán)重的肝臟壞死,這提示miR-223敲除小鼠更易于APAP誘導(dǎo)的肝損傷。正如預(yù)期的一樣,miR-223敲除小鼠肝臟中有更多的中性粒細(xì)胞和巨噬細(xì)胞的浸潤。相對(duì)應(yīng)的,miR-223敲除小鼠肝臟中炎癥反應(yīng)及氧化性損傷標(biāo)記4-羥基壬烯酸(4-Hydroxynonenal,4-HNE)和N-硝基酪氨酸(N-nitrotyrosine)的表達(dá)明顯高于野生型小鼠。有趣的是,腹腔注射壞死細(xì)胞可以引起小鼠腹腔中性粒細(xì)胞的大量浸潤,而這種反應(yīng)在miR-223敲除小鼠中更加明顯。更有甚者,在體內(nèi)或者體外,Toll樣受體9(toll-like receptor 9, TLR9)配體或者壞死肝臟細(xì)胞釋放的自由DNA通過TLR9/NF-κB通路誘導(dǎo)中性粒細(xì)胞miR-223的表達(dá),而TLR9阻斷劑可以抑制APAP誘導(dǎo)的中性粒細(xì)胞miR-223表達(dá)。此外,體外用TLR9配體刺激中性粒細(xì)胞后,缺乏miR-223的中性粒細(xì)胞產(chǎn)生更多的炎癥細(xì)胞因子和趨化因子。機(jī)制上,miR-223被發(fā)現(xiàn)可以部分調(diào)控IKKa的表達(dá)負(fù)反饋控制TLR9/NF-κB介導(dǎo)的炎癥反應(yīng)。以上研究表明,miR-223的缺失增加肝臟中性粒細(xì)胞炎癥反應(yīng)和氧化應(yīng)激反應(yīng),隨后加重APAP誘導(dǎo)的肝毒性。這些發(fā)現(xiàn)提示,miR-223是控制急性中性粒細(xì)胞浸潤和功能的重要調(diào)節(jié)分子,其可能作為治療APAP誘導(dǎo)肝損傷的一個(gè)治療靶點(diǎn)。
[Abstract]:Excessive use of acetaminophen (APAP) is one of the main causes of acute liver failure all over the world. It is closely related to the infiltration of a large number of neutrophils in the liver. However, the specific mechanisms of neutrophils infiltration and inflammation in the liver are still not clear. In recent years, small RNA (microRNA, miRNA) has been found. There are many important biological processes involved in regulating cell proliferation, differentiation, and apoptosis. Myeloid specific expression of miRNA-223 is reported to regulate the formation and function of neutrophils. Whether it participates in the regulation of APAP induced acute liver injury and related neutrophils has not yet been reported. This study constructs miR-223 knockout small In this study, the effect and the molecular mechanism of APAP induced acute liver injury and neutrophils function were investigated in this experiment. In this experiment, the mice were intraperitoneally injected with APAP in the wild and miR-223 knockout mice under empty stomach starvation or normal diet to establish a mouse model of acute liver injury for.6 hours or 24 hours. The liver transaminase and miR-223 were detected in the mice serum. The liver tissue was examined by histopathology and related immunohistochemical detection. The liver was separated by magnetic bead separation, and the neutrophils in the bone marrow and peripheral blood were detected in real-time quantitative PCR or Western blot. The results showed that excessive APAP injection could significantly increase the liver of mice. And miR-223 levels in the serum. More importantly, compared to the wild type mice, the miR-223 knockout mice had a higher level of ALT, AST, and more severe liver necrosis, suggesting that miR-223 knockout mice were more prone to APAP induced liver damage. As expected, there were more neutrophils and giant cells in the miR-223 knockout mice liver. The expression of inflammatory reaction and oxidative damage in the liver of miR-223 knockout mice, the expression of 4- hydroxyl NONYLIC acid (4-Hydroxynonenal, 4-HNE) and N- nitro tyrosine (N-nitrotyrosine) was significantly higher than that of wild type mice. The reaction is more obvious in miR-223 knockout mice. Furthermore, the free DNA released by Toll like receptor 9 (Toll-like receptor 9, TLR9) ligand or necrotic liver cells in vivo or in vitro can induce the expression of miR-223 in neutrophils through the TLR9/NF- kappa B pathway, and TLR9 blockers can inhibit APAP induced neutrophils. MiR-223 expression. In addition, after the stimulation of neutrophils with TLR9 ligands in vitro, more inflammatory cytokines and chemokines are produced by the neutrophils lacking miR-223. On the mechanism, miR-223 is found to partially regulate the negative feedback of IKKa to control the inflammatory response mediated by TLR9/NF- kappa B. The above study suggests that the absence of miR-223 increases the liver. The inflammatory and oxidative stress responses of neutrophils then aggravate the hepatotoxicity induced by APAP. These findings suggest that miR-223 is an important regulator to control the infiltration and function of acute neutrophils, and may be a therapeutic target for the treatment of APAP induced liver injury.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96

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本文編號(hào)：1947710