CD24分子在刀豆蛋白A诱导小鼠急性肝损伤中的作用及机制研究

发布时间：2018-12-10 11:14

【摘要】：背景与目的：急性肝损伤(Acute liver injury)是临床较为常见、严重影响人类健康的全球危害性疾病。外界有毒物质、病原微生物以及药物等均可能造成急性肝损伤,该疾病表现为：短时间内肝细胞出现坏死、出血,血清谷丙/草转氨酶及细胞因子急剧升高,累积多脏器功能障碍,严重至肝功能衰竭。目前对于急性肝损伤的机制仍然不明确。越来越多的证据表明肝脏由于其双重血供,该器官其实也是一个特殊的免疫耐受微环境。研究表明导致肝损伤的原因并非是外界物质的直接作用,而是表达在受感染的肝脏细胞表面针对抗原所产生的免疫应答导致严重的免疫失衡,继而产生大量的炎症因子致使肝损伤发生直至严重的多器官功能障碍。CD24是一种人和鼠中多种细胞表面表达的、具有多种不同生物学功能的低分子量的细胞表面膜蛋白。CD24分子被证实调控了自身反应性T细胞和抑制抗原递呈及T细胞活化作用。刀豆蛋白A诱导的肝损伤模型较为真实的模拟急性免疫性肝损伤的体内过程。然而CD24分子在刀豆蛋白A诱导的急性肝损伤中的作用尚未报道。本论文的主要研究内容概括如下：一、CD24缺陷小鼠与同窝野生型小鼠在刀豆蛋白A诱导的急性肝损伤模型中肝损伤程度(包括肝脏病理、血清ALT水平和细胞因子表达水平)的差别。二、研究造成差别的免疫机制(包括肝脏细胞亚群和相关细胞信号通路)。方法：通过尾静脉注射刀豆蛋白A建立小鼠急性肝损伤模型,比较CD24缺陷小鼠及同窝野生型小鼠在刀豆蛋白A诱导的急性肝损伤程度(包括肝脏病理、血清ALT水平和细胞因子表达水平)的差异。检测肝脏及脾脏中CD4+T及CD8+T细胞表面CD24表达的表达水平。检测CD24缺陷小鼠和对照组小鼠T细胞的成熟情况。确认CD24缺陷小鼠导致急性肝损伤差异的细胞来源(胞内染色确定CD4+、CD8+T和NKT细胞释放IFN-γ)。再通过过继转输实验体内确定急性肝损伤差异的免疫细胞来源。通过刀豆蛋白A、抗CD3/CD28抗体及PMA/Ionomycin体外刺激CD24缺陷小鼠及对照小鼠T细胞,确认产生IFN-γ差异的T细胞。通过Western blot方法分析CD24缺陷小鼠及对照小鼠在刀豆蛋白A作用后,肝脏及脾脏T细胞相关信号通路的差异,寻找相关的信号分子。结果：(1)刀豆蛋白A刺激后使T细胞上CD24分子表达明显上调;(2)体内实验表明刀豆蛋白A对CD24缺陷小鼠所造成的肝损伤程度明显低于野生型小鼠;(3)体内实验和体外实验均证实,在刀豆蛋白A造成肝损伤情况下,CD24缺陷的CD4+T细胞所分泌的IFN-γ较少；(4)在刀豆蛋白A诱导的肝损伤后,CD24缺陷小鼠肝脏STAT1磷酸化明显降低。结论：我们通过构建T细胞介导的刀豆蛋白A肝损伤模型,证实CD24缺陷小鼠在刀豆蛋白A肝损伤模型中的保护肝脏损伤的效应及机制,证实CD24缺陷小鼠通过降低肝脏细胞STAT1磷酸化来降低CD4+T分泌IFN-γ介导刀豆蛋白A作用后的肝损伤减轻作用,为临床治疗急性肝损伤提供潜在的治疗靶点。
[Abstract]:BACKGROUND & OBJECTIVE: Acute liver injury (acute liver injury) is a kind of global harmful disease which is more common in clinic and has a serious effect on human health. Acute liver injury can be caused by external toxic substances, pathogenic micro-organisms and drugs, and the disease is manifested as the occurrence of necrosis of the liver cells in a short time, the bleeding of the liver cells, the rapid increase of the serum glutamic acid/ grass transaminases and the cytokines, the accumulation of the multi-organ dysfunction, and the serious to the liver failure. The mechanism for acute liver injury is still unclear. An increasing number of evidence suggests that the liver is a special immune tolerance microenvironment due to its double blood supply. Studies have shown that the cause of liver injury is not a direct effect of external substances, but that the expression of an immune response to an antigen on the surface of an infected liver cell leads to a serious immune imbalance, In turn, a large number of inflammatory factors are produced that cause liver injury to occur until severe multiple organ dysfunction occurs. CD24 is a low-molecular-weight cell surface membrane protein that is expressed in a variety of cell surfaces in a human and a mouse and has a variety of different biological functions. The CD24 molecules were confirmed to regulate the self-reactive T cells and to inhibit the antigen-presenting and T-cell activation. The model of liver injury induced by bean protein A is a real model for simulating the in-vivo process of acute immune liver injury. However, the role of CD24 in acute liver injury induced by bean protein A has not been reported. The main contents of this thesis are as follows: 1. The difference of the degree of liver injury (including liver pathology, serum ALT level and the level of cytokine expression) in the model of acute liver injury induced by CCD24-deficient mice and the wild-type mice in the same fossa. II. The immune mechanism (including the subpopulation of the liver and the relevant cell signal pathway) of the differential was studied. Methods: The model of acute liver injury in mice was established by tail vein injection of the bean protein A, and the difference of the degree of acute liver injury (including liver pathology, serum ALT level and the level of cytokine expression) induced by CD24 deficient mice and the wild-type mice in the same fossa was compared. The expression level of CD4 + T and CD8 + T cell surface CD24 in the liver and spleen was detected. The maturation of T cells in the CD24-deficient mice and the control group was detected. It was confirmed that the CD24 deficient mice lead to a different cell source for acute liver injury (intracellular staining to determine CD4 +, CD8 + T, and NKT cells to release IFN-1). and the source of the immune cells of the difference of the acute liver injury is determined through the adoptive transfer experiment body. The T cells of the IFN-P difference were confirmed by the in vitro stimulation of the CD24 deficient mice and the control mouse T cells by the knife-bean protein A, the anti-CD3/ CD28 antibody, and the PMA/ Ionone in vitro. Western blot was used to analyze the difference of the signal pathways between the liver and the spleen T cells and to find relevant signal molecules in the mice of CD24 and control mice after the action of the knife-bean protein A. Results: (1) The expression of CD24 on T-cells was up-regulated after the stimulation of the knife-bean protein A; (2) in-vivo experiments, the degree of liver injury of the CD24-deficient mice was significantly lower than that of the wild-type mice; and (3) in vivo and in vitro experiments were confirmed. In the case of the liver injury caused by the bean protein A, the number of IFN-1 secreted by the CD4 + T cells of the CD24 deficiency was less; and (4) after the liver injury induced by the bean protein A, the phosphorylation of the STAT1 in the liver of the CD24 deficient mice was significantly reduced. Conclusion: We have confirmed the effect and mechanism of CD24-deficient mice in the protection of liver injury in the model of liver injury of the bean protein A by constructing a T cell-mediated liver injury model of the bean-bean protein A. It is confirmed that the CD24-deficient mice can reduce the liver injury and reduce the liver injury after the action of the CD4 + T-secreting IFN-1-mediated bean protein A by reducing the phosphorylation of the STAT1 in the liver cells, thereby providing a potential therapeutic target for the clinical treatment of the acute liver injury.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575

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