SOCS3在小鼠肝脏纤维化进展和逆转过程中对巨噬细胞极化的影响
发布时间：2018-10-31 13:47
【摘要】：近些年,肝脏疾病的发病率在逐年增高,众多损伤因素如酒精、病毒、化学物质等存在下,肝脏将发生不同程度的损伤,如病毒性肝炎、酒精肝、肝纤维化、肝硬化、肝癌等肝脏疾病的发生。而肝脏作为人体重要的代谢器官,其自身器官不同程度的损伤直接影响着机体其他疾病的发生发展。因此,研究肝脏疾病的发生发展和寻找其治愈肝脏疾病的方法方式已刻不容缓。Kupffer细胞(KCs)是存在于肝脏窦状隙内的一类非实质细胞,属于定居肝脏内的固有细胞,KCs即能够非特异的吞噬和清除血液中的细菌、外源性异物等抗原性物质,还具有特异性的免疫应答、抗肿瘤免疫反应、内毒素解毒、抗感染、调节微循环及物质代谢等方面的作用。在肝脏不同的病理生理状态下,KCs能够极化成不同的亚型,即M1(classically activated macrophages)型和M2(alternatively activated macrophages)型,M1型巨噬细胞主要能够促进炎症的发生发展,分泌产生TNF-α(tumor necrosis factor alpha)、IL-1(interleukin 1)、IL-6(interleukin 6)等促炎细胞因子;M2型巨噬细胞其主要功能是抑制炎症的发生发展和对组织的修复产生作用,通过合成和分泌抗炎细胞因子IL-10(interleukin 10)以及通过Arg-1(arginase 1)将精氨酸分解成聚胺,来参与合成和稳定细胞外基质。巨噬细胞通过M1型和M2型的动态调控来影响肝脏的病理生理状况,因此,通过上述现象来调控肝脏巨细胞的极化从而来达到肝脏疾病的恢复这一研究思路将为肝脏疾病的治疗寻找新的靶点和策略。已有研究表明SOCS3(Suppressor of cytokine signaling 3)参与肝脏疾病的发生发展,通过影响多种细胞因子的产生和信号传导进行负性调控,如通过免疫调控来影响炎症因子和趋化因子进而去调控炎症反应,而肝脏疾病的发生发展有巨噬细胞的参与,巨噬细胞与炎症的发生发展密切相关,设想是否SOCS3参与肝纤维化的发生发展,此外,另有报道显示,肝纤维化在去除损伤因素后能够发生逆转[1],进而,通过调控SOCS3蛋白的表达去影响肝纤维化的逆转过程和肝脏巨噬细胞极化分型过程从而探索其对肝纤维化的影响。且课题组前期研究发现SOCS3在动物肝纤维化和逆转过程中存在着表达的变化,且在肝脏巨噬细胞中表达,但SOCS3在肝纤维化中的调控作用,尤其是对肝巨噬细胞极化分型相关作用尚未报道,于是本实验室重点研究通过调控SOCS3的表达观察对KCs极化分型的影响进而为肝纤维化的防治提供新的思路。主要研究内容概括如下:1.SOCS3在小鼠肝脏纤维化进展和逆转过程中的表达。取肝脏组织,利用免疫荧光双染和免疫组织化学的方法观察SOCS3在肝脏组织的的表达情况。结果发现:SOCS3在小鼠肝脏巨噬细胞中表达,并且,随着小鼠肝纤维化的形成,SOCS3的表达逐渐增多,随着小鼠肝纤维化的逆转的进行,SOCS3的表达逐渐减少。2.小鼠肝纤维化进展和逆转过程中肝脏巨噬细胞表型的变化。通过原位灌流技术提取不同时期小鼠肝脏巨噬细胞,利用流式细胞技术对提取的肝脏巨噬细胞进行分型研究。结果发现:随着小鼠肝纤维化的形成,CD86标记的M1型巨噬细胞表达逐渐增多,随着小鼠肝纤维化的逆转,CD206标记的M2型巨噬细胞逐渐增多。3.SOCS3在体外RAW264.7细胞中通过刺激因子作用在巨噬细胞分型中的表达。体外通过RAW264.7细胞通过LPS和IFN-γ刺激形成M1型,IL-4刺激形成M2型,观察SOCS3在不同分型中的表达。结果发现:SOCS3相对于M2型,在M1型表达量显著增高。4.干扰SOCS3的表达对RAW264.7细胞极化分型的影响。LipofectamineTM2000转染SOCS3-si RNA到RAW264.7细胞内,可使LPS(Lipopolysaccharides)和IFN-γ(Interferonγ)刺激形成M1型表达显著增多,LipofectamineTM2000转LV3-SOCS3-mus-1225到RAW264.7细胞内,可使IL-4(interleukin 4)刺激形成M2型表达显著增多。
[Abstract]:In recent years, the incidence of liver disease is increasing year by year. In the presence of many damage factors such as alcohol, virus, chemical substance and so on, the liver will have different degrees of damage, such as viral hepatitis, alcohol liver, liver fibrosis, liver cirrhosis, liver cancer and other liver diseases. Liver, as an important metabolic organ of human body, directly affects the development of other diseases in the body. Therefore, it is urgent to study the development of liver diseases and to find ways to cure liver diseases. Kruper cell (KCs) is a kind of non-essential cell which exists in the liver allogenic gap, and belongs to the intrinsic cell in the liver of the sedentary liver. KCs is an antigenic substance capable of non-specific phagocytizing and clearing bacteria in blood, exogenous foreign matter and the like, and also has specific immune response and anti-tumor immune response. Endotoxins detoxification, anti-infection, regulation of microcirculation and substance metabolism. In different pathophysiological states of the liver, KCs can be polarized into different subtypes, M1 (classically activated macimages) and M2 (alternate activated macimages), and M1-type macrophages can promote the development of inflammation, and produce pro-inflammatory cytokines such as TNF-CoV (tubetrosis factor alpha), IL-1 (interleukin1) and IL-6 (interleukin 6); The main function of M2-type macrophages is to inhibit the development of inflammation and to play a role in the repair of tissues, and to participate in the synthesis and stabilization of extracellular matrix by synthesizing and secreting anti-inflammatory cytokines IL-10 (interleukin 10) and by decomposing arginine into polyamines by Arg-1 (arginiase 1). Macrophages regulate the pathological and physiological condition of liver by the dynamic regulation of M1 and M2. Therefore, it can regulate the polarization of the giant cell of the liver by the phenomenon, so as to achieve the restoration of liver disease. Previous studies have shown that SOCS3 (Suppressor of Cytokine signaling 3) is involved in the development of liver diseases, and negatively regulates the production and signaling of various cytokines, such as by regulating the immune regulation to affect the inflammatory factors and chemokine, and then to regulate the inflammatory response. The development of liver disease has the involvement of macrophages. Macrophages are closely related to the development of inflammation. It is envisaged that SOCS3 is involved in the development of liver fibrosis. In addition, another report shows that liver fibrosis can be reversed after removing the damage factors[1], and further, The effect of SOCS3 protein on hepatic fibrosis was studied by regulating the expression of SOCS3 protein to affect the reverse course of hepatic fibrosis and the polarization classification of liver macrophages. The results showed that SOCS3 was expressed in liver fibrosis and liver fibrosis, but the regulation of SOCS3 in liver fibrosis, especially the effect of SOCS3 in hepatic fibrosis, was not reported. Therefore, this laboratory focused on the influence of the expression of SOCS3 on the polarization typing of KCs, thus providing a new idea for the prevention and treatment of hepatic fibrosis. The main contents of this study were as follows: 1. SOCS3 was expressed in the progression and reversal of liver fibrosis in mice. Liver tissue was taken to observe the expression of SOCS3 in liver tissue by immunofluorescent double staining and immunohistochemistry. The results showed that SOCS3 was expressed in mouse liver macrophages, and the expression of SOCS3 increased with the formation of liver fibrosis in mice. The expression of SOCS3 decreased gradually with the reversal of liver fibrosis in mice. Changes of liver macrophage phenotype during the progression and reversal of liver fibrosis in mice. Rat liver macrophages were extracted by in situ perfusion technique, and the extracted liver macrophages were classified by flow cytometry. Results: With the formation of liver fibrosis in mice, the expression of CD86-labeled M1-type macrophages was gradually increased. With the reversal of liver fibrosis in mice, the M2-type macrophages labeled with CD206 were gradually increased. 3. SOCS3 was expressed in RAW264.7 cells in vitro by stimulating factor. The expression of SOCS3 in different types was observed by means of RAW264.7 cells stimulated by LPS and IFN-OVA to form M1 type, IL-4 stimulated to form M2 type. The results showed that the expression level of SOCS3 was significantly higher than that of M2 type. The effect of interfering SOCS3 expression on the polarization typing of RAW264.7 cells. With the transfection of SOCS3-si RNA into RAW264.7 cells by Lipofectinine TM2000, the expression of LPS (LPS) and IFN-jun (Interfering RNAs) could be increased significantly, and the expression of IL-4 (interleukin4) could be increased significantly in the cells of LV3-SOCS3-mus-1225 to RAW264.7. The expression of IL-4 (interleukin4) could be increased significantly.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
本文编号：2302422
[Abstract]:In recent years, the incidence of liver disease is increasing year by year. In the presence of many damage factors such as alcohol, virus, chemical substance and so on, the liver will have different degrees of damage, such as viral hepatitis, alcohol liver, liver fibrosis, liver cirrhosis, liver cancer and other liver diseases. Liver, as an important metabolic organ of human body, directly affects the development of other diseases in the body. Therefore, it is urgent to study the development of liver diseases and to find ways to cure liver diseases. Kruper cell (KCs) is a kind of non-essential cell which exists in the liver allogenic gap, and belongs to the intrinsic cell in the liver of the sedentary liver. KCs is an antigenic substance capable of non-specific phagocytizing and clearing bacteria in blood, exogenous foreign matter and the like, and also has specific immune response and anti-tumor immune response. Endotoxins detoxification, anti-infection, regulation of microcirculation and substance metabolism. In different pathophysiological states of the liver, KCs can be polarized into different subtypes, M1 (classically activated macimages) and M2 (alternate activated macimages), and M1-type macrophages can promote the development of inflammation, and produce pro-inflammatory cytokines such as TNF-CoV (tubetrosis factor alpha), IL-1 (interleukin1) and IL-6 (interleukin 6); The main function of M2-type macrophages is to inhibit the development of inflammation and to play a role in the repair of tissues, and to participate in the synthesis and stabilization of extracellular matrix by synthesizing and secreting anti-inflammatory cytokines IL-10 (interleukin 10) and by decomposing arginine into polyamines by Arg-1 (arginiase 1). Macrophages regulate the pathological and physiological condition of liver by the dynamic regulation of M1 and M2. Therefore, it can regulate the polarization of the giant cell of the liver by the phenomenon, so as to achieve the restoration of liver disease. Previous studies have shown that SOCS3 (Suppressor of Cytokine signaling 3) is involved in the development of liver diseases, and negatively regulates the production and signaling of various cytokines, such as by regulating the immune regulation to affect the inflammatory factors and chemokine, and then to regulate the inflammatory response. The development of liver disease has the involvement of macrophages. Macrophages are closely related to the development of inflammation. It is envisaged that SOCS3 is involved in the development of liver fibrosis. In addition, another report shows that liver fibrosis can be reversed after removing the damage factors[1], and further, The effect of SOCS3 protein on hepatic fibrosis was studied by regulating the expression of SOCS3 protein to affect the reverse course of hepatic fibrosis and the polarization classification of liver macrophages. The results showed that SOCS3 was expressed in liver fibrosis and liver fibrosis, but the regulation of SOCS3 in liver fibrosis, especially the effect of SOCS3 in hepatic fibrosis, was not reported. Therefore, this laboratory focused on the influence of the expression of SOCS3 on the polarization typing of KCs, thus providing a new idea for the prevention and treatment of hepatic fibrosis. The main contents of this study were as follows: 1. SOCS3 was expressed in the progression and reversal of liver fibrosis in mice. Liver tissue was taken to observe the expression of SOCS3 in liver tissue by immunofluorescent double staining and immunohistochemistry. The results showed that SOCS3 was expressed in mouse liver macrophages, and the expression of SOCS3 increased with the formation of liver fibrosis in mice. The expression of SOCS3 decreased gradually with the reversal of liver fibrosis in mice. Changes of liver macrophage phenotype during the progression and reversal of liver fibrosis in mice. Rat liver macrophages were extracted by in situ perfusion technique, and the extracted liver macrophages were classified by flow cytometry. Results: With the formation of liver fibrosis in mice, the expression of CD86-labeled M1-type macrophages was gradually increased. With the reversal of liver fibrosis in mice, the M2-type macrophages labeled with CD206 were gradually increased. 3. SOCS3 was expressed in RAW264.7 cells in vitro by stimulating factor. The expression of SOCS3 in different types was observed by means of RAW264.7 cells stimulated by LPS and IFN-OVA to form M1 type, IL-4 stimulated to form M2 type. The results showed that the expression level of SOCS3 was significantly higher than that of M2 type. The effect of interfering SOCS3 expression on the polarization typing of RAW264.7 cells. With the transfection of SOCS3-si RNA into RAW264.7 cells by Lipofectinine TM2000, the expression of LPS (LPS) and IFN-jun (Interfering RNAs) could be increased significantly, and the expression of IL-4 (interleukin4) could be increased significantly in the cells of LV3-SOCS3-mus-1225 to RAW264.7. The expression of IL-4 (interleukin4) could be increased significantly.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
【参考文献】
相关期刊论文 前2条
1 沈竹夏;段胜仲;;巨噬细胞极化在2型糖尿病和心血管疾病中的作用[J];生命科学;2013年02期
2 孔德松;郑仕中;陆茵;王爱云;;肝内肌成纤维细胞的来源及其在肝纤维化中作用的研究[J];中国药理学通报;2011年03期
，本文编号：2302422
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