端粒酶逆转录酶TERT通过与NF-kappa B通路的正反馈调控酒精性肝病中巨噬细胞极化的作用机制研究

发布时间：2018-11-05 15:50

【摘要】：酒精性肝病(Alcoholic liver disease,ALD)是一种长期因大量饮酒导致的常见的慢性肝脏疾病之一。其疾病谱从早期的脂肪肝、发展到酒精性肝炎、肝纤维化及肝硬化。ALD发病分子机制复杂,目前尚无有效防治药物。急慢性酒精暴露均可激活枯否细胞(Kupffer cells,KCs),KCs作为肝脏内常驻的巨噬细胞,其活化在ALD发病机理中发挥着主要的作用。具有分泌多种细胞因子、吞噬及免疫调节等功能。由于巨噬细胞本身独特的可塑性和异质性,其可根据外在环境而发生功能和表型的改变。本研究首先建立NIAAA推荐的Lieber-DeCarli液体饮食加一次性大剂量酒精灌胃的方法小鼠ALD模型。病理学分析发现所有慢性酒精喂养的C57BL/6雄性小鼠肝内免疫细胞活化,发生炎症,损伤和脂肪化。免疫组化IHC染色结果显示模型组较对照组相比肝组织内CD68阳性细胞增多;流式细胞术分析原位灌流提取的KCs发现模型组CD45+F4/80+CD11b+细胞数目增多近一倍,且经典caMφ(classically activated macrophages)型表面标记物TNF-α、IL-1β、NOS2和CCL2,以及替代aaMφ(alternatively activated macrophages)型巨噬细胞表面标记物Arg-1、IL-10、Mrc2和CD163水平均升高。这些结果提示慢性酒精喂养加一次性大剂量酒精灌胃可诱导肝内巨噬细胞增多,且caMφ型aaMφ型均增多。由于端粒酶活化(telomerase activation,TA)在髓系和淋巴系细胞活化过程中起着至关重要的作用,因此本研究主要探讨在ALD过程中端粒酶中最主要的限速酶成分,端粒酶逆转录酶(Telomerase reverse transcriptase,TERT)对巨噬细胞活化的潜在作用。免疫荧光双标法检测TERT的表达定位发现肝组织中TERT与CD68共表达,在酒精饲料喂养的老鼠肝组织和原代KCs中TERT表达明显升高,TA定量分析结果显示ALD进展期中TA升高近六倍。而且传代培养的巨噬细胞RAW264.7中乙醇刺激和脂多糖(Lipopolysaccharides,LPS)诱导的caMφ型中TERT的表达水平显著升高。这些结果提示上调的TERT可能在由巨噬细胞介导的ALD进展早期发挥着重要的作用。为了观察TERT在巨噬细胞极化中的潜在作用,分别通过siRNA和质粒体外干预其表达。研究发现沉默TERT,caMφ型巨噬细胞标记物TNF-α、IL-1β、NOS2和CCL2 mRNA水平随之降低,同时与对照组相比,促炎因子TNF-α、IL-1β、IL-6和IL-12分泌减少,反之过表达TERT,相关caMφ型标志物随之升高,然而这种获得性功能研究对aaMφ型巨噬细胞极化没有影响。进一步研究发现NF-κB信号通路在ALD进展期和体外caMφ型巨噬细胞中被激活,更重要的是,TERT对caMφ型巨噬细胞极化的这种调控作用是通过调节NF-κB通路实现的。此外,NF-κB的化学抑制剂PDTC可以显著降低TERT表达和caMφ型巨噬细胞标志物的表达。综上所述,本研究结果提示ALD进展期TERT在巨噬细胞极化中的调节作用以及TERT与p65之间的相互作用,这为乙醇介导的肝细胞炎症反应和caMφ巨噬细胞极化调控机制提供了新思路。
[Abstract]:Alcoholic liver disease (Alcoholic liver disease,ALD) is one of the common chronic liver diseases caused by heavy drinking for a long time. Its disease spectrum has developed from early fatty liver to alcoholic hepatitis, liver fibrosis and cirrhosis. The molecular mechanism of ALD is complex, and there are no effective drugs to prevent and cure it. Acute and chronic alcohol exposure can activate Kupffer cells (Kupffer cells,KCs), KCs as a resident macrophage in the liver, its activation plays a major role in the pathogenesis of ALD. It has the function of secreting many cytokines, phagocytosis and immune regulation. Due to its unique plasticity and heterogeneity, macrophages can undergo functional and phenotypic changes according to the external environment. In this study, we first established the ALD model of mice with Lieber-DeCarli liquid diet recommended by NIAAA and one-off high dose alcohol. Pathological analysis showed that all chronic alcohol-fed C57BL/6 male mice had intrahepatic immune cells activation, inflammation, injury and fat. The results of immunohistochemical IHC staining showed that the number of CD68 positive cells in the model group was higher than that in the control group. Flow cytometry analysis of KCs extracted by in situ perfusion showed that the number of CD45 F4 / 80 CD11b cells in the model group nearly doubled, and the classical caM 蠁 (classically activated macrophages) surface markers TNF- 伪, IL-1 尾, NOS2 and CCL2, The levels of Arg-1,IL-10,Mrc2 and CD163, the surface markers of aaM 蠁 (alternatively activated macrophages) macrophages, were increased. These results suggest that chronic alcohol feeding plus one dose of high dose alcohol can induce the increase of macrophages in the liver and the increase of caM 蠁 type aaM 蠁 type. Because telomerase activation (telomerase activation,TA) plays an important role in the activation of myeloid and lymphoid cells, this study focused on the most important rate-limiting enzyme component, telomerase reverse transcriptase (Telomerase reverse transcriptase, in the process of ALD. The potential role of TERT in the activation of macrophages. The expression of TERT and CD68 in liver tissue was detected by immunofluorescence double labeling method. The expression of TERT in liver tissue and primary KCs of rats fed with alcohol concentrate was significantly increased. The quantitative analysis of TA showed that TA increased nearly six times during ALD progression. The expression of TERT in caM 蠁 type induced by lipopolysaccharide (Lipopolysaccharides,LPS) and ethanol stimulation in cultured macrophages was significantly increased. These results suggest that up-regulated TERT may play an important role in the early stage of ALD progression mediated by macrophages. In order to observe the potential role of TERT in macrophage polarization, the expression of TERT was interfered with by siRNA and plasmids in vitro. It was found that the levels of TNF- 伪, IL-1 尾, NOS2 and CCL2 mRNA, the markers of TERT,caM 蠁 type macrophages, were decreased, and the secretion of TNF- 伪, IL-1 尾, IL-6 and IL-12 was decreased, whereas TERT, was over-expressed. The associated caM 蠁 markers increased, but the acquired function study had no effect on the polarization of aaM 蠁 macrophages. Further studies showed that the NF- 魏 B signaling pathway was activated during the ALD progression and in vitro caM 蠁 macrophages. More importantly, the regulation of TERT on the caM 蠁 type macrophage polarization was achieved by regulating the NF- 魏 B pathway. In addition, PDTC, a chemical inhibitor of NF- 魏 B, could significantly reduce the expression of TERT and caM 蠁 macrophage markers. To sum up, the present study suggests the regulatory role of ALD in macrophage polarization and the interaction between TERT and p65, which provides a new idea for ethanol mediated hepatocyte inflammation and caM 蠁 macrophage polarization regulation.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575

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