瘦素对单核—巨噬细胞免疫功能的影响及其机制的研究

发布时间：2018-04-05 23:07

本文选题：瘦素　切入点：单核巨噬细胞　出处：《南方医科大学》2011年硕士论文

【摘要】：瘦素是一种由167个氨基酸组成的激素样细胞因子,主要由脂肪细胞分泌。人编码瘦素的肥胖基因定位于人类第7号染色体,而鼠的编码基因则位于第6号染色体。人们对瘦素最初的研究表明,瘦素主要是通过作用于下丘脑的神经核团,调节食物摄取与能量消耗之间的平衡。随着研究的不断深入,人们发现,瘦素受体不仅表达于神经系统,也表达于许多外周组织,如肝、肺、睾丸。因此,近年来,瘦素对免疫应答和自身免疫性疾病的影响受到越来越多的关注。人类的单核巨噬细胞是一类非常重要的免疫细胞,无论在获得性免疫的初始阶段还是在天然免疫中都发挥着举足轻重的功能。在天然免疫中,单核巨噬细胞可以依靠表面的模式识别受体迅速识别入侵的病原体,并由此触发后续的炎症反应和杀菌效应。它还能依靠表面的甘露糖受体(MR)、Fc受体和C3b受体等介导对病原体的吞噬,同时可以分泌趋化因子募集炎症细胞,控制感染。在获得性免疫的初始阶段,单核巨噬细胞作为重要的抗原递呈细胞可以递呈抗原给淋巴细胞,从而促使淋巴细胞激活或分化。我们首先以人THP-1细胞作为研究对象,加入PMA诱导其分化成巨噬细胞,并在此基础上评价了瘦素对其吞噬功能、增殖活性、杀菌效应、趋化能力以及共刺激分子表达的影响,同时对造成这些变化的可能机制做了初步的探讨。杀菌效应是单核巨噬细胞的一个重要功能,我们利用金黄色葡萄球菌为对象研究了不同剂量瘦素处理的THP-1及其来源的巨噬细胞对它们的杀伤作用。结果表明,足够浓度的瘦素能明显增强THP-1及其来源的巨噬细胞对细菌的杀伤作用,但1ng/ml的瘦素无此作用。这种杀菌作用的诱导可能有赖于肿瘤坏死因子(TNF-α)和活性氧(ROS)的产生增加而与一氧化氮(NO)的生成之间关系不明显。对吞噬功能的评价,我们采用了流式细胞术。我们用荧光素FITC对白色念珠菌进行了标记,然后用流式细胞术评价了瘦素处理对THP-1及其来源的巨噬细胞对细菌的吞噬作用。结果显示,足够浓度的瘦素能明显增强THP-1及其来源的巨噬细胞对白色念珠菌的吞噬能力。虽然MR是与吞噬作用密切相关的受体,但我们发现瘦素的这种作用并不是通过上调MR表达来实现的。此外我们还发现,瘦素可以刺激THP-1及其来源的巨噬细胞的增殖。趋化功能是单核巨噬细胞的另外一种重要功能,我们用Boyden小室测定了瘦素对THP-1及其来源的巨噬细胞的趋化功能的影响。结果表明,足够浓度的瘦素显著增强了对HL-60粒细胞系和自身的趋化作用,这种作用可能和IL-8以及MCP-1分泌的增强有关。我们还对THP-1及其来源的巨噬细胞和人外周血单核细胞表面的共刺激分子的表达进行了评价。结果显示,瘦素可以上调这些细胞表面共刺激分子的表达,但结果同时也发现,对于不同类型的细胞,瘦素所需的有效刺激浓度存在差异。第一章瘦素对THP-1细胞及其来源的巨噬细胞杀菌活性的影响及其机制的研究单核巨噬细胞有很强的杀伤能力,可非特异性杀伤多种病原微生物,是机体非特异性免疫防御中的重要细胞。单核巨噬细胞的这种杀伤作用可被补体的免疫黏附作用、特异性免疫中抗体的调理作用及细胞因子加强。单核巨噬细胞也能利用此功能清除体内衰老损伤细胞,参与免疫自稳作用。我们首先参照相关文献用PMA对人THP-1细胞进行了诱导使其成功分化成巨噬细胞样细胞。结果表明,PMA诱导24h后可见细胞表面伸出伪足,细胞体积增大,胞浆内出现空泡,说明细胞向巨噬细胞转化。我们利用金黄色葡萄球菌为对象研究了不同剂量瘦素处理的THP-1及其来源的巨噬细胞对它们的杀伤作用。结果表明,10ng/ml以上组瘦素能明显增强THP-1及其来源的巨噬细胞对细菌的杀伤作用(F=73.79,P=0.000；F=25.01,P=0.000),但1ng/ml的瘦素无此作用。由于单核巨噬细胞激活后主要通过活性氧簇(ROS)、NO的生成来实现其对细菌的杀伤。因此,我们分别测定了瘦素处理后,单核巨噬细胞分泌这些生物活性分子的能力。结果发现,10、50、100ng/ml的瘦素均可以显著刺激ROS的产生(F=168.95,P=0.000；F=113.00,P=0.000),其量可以达到对照的数倍甚至数十倍。然而,各个剂量组却对NO的生成无明显的刺激作用(F=1.179,P=0.377;F=1.978,P=0.174)。而且1ng/ml组对这几种活性物质均无诱导作用。TNF-α是单核巨噬细胞分泌的促炎症因子,在感染的控制中也具有重要作用。我们通过ELISA的方法测定了瘦素处理的THP-1细胞及其来源的巨噬细胞产生这种细胞因子的能力。结果显示,lOng/ml以上组瘦素可以刺激细胞产生TNF-α(F=502.48,P=0.000;F=2979.01,P=0.000)。这些结果提示,足够浓度的瘦素可以显著刺激THP-1细胞及其来源的巨噬细胞杀菌活性物质的分泌并显著增强其杀菌活性。本部分实验主要评价了瘦素对THP-1细胞及其来源的巨噬细胞的杀菌功能的影响。实验中首先通过杀菌试验和平板计数的方法检测了瘦素诱导的杀菌效应。瘦素诱导了TNF-α和ROS的产生,但对NO的合成影响不显著,这在一定程度上说明了瘦素诱导的杀菌机制。第二章瘦素对THP-1细胞及其来源的巨噬细胞吞噬功能的影响及其机制的研究吞噬功能是单核巨噬细胞的一个重要功能,我们希望通过本部分实验评价瘦素对这种功能的作用。我们利用流式细胞术检测了瘦素刺激对THP-1细胞及其来源的巨噬细胞吞噬功能的影响。流式结果表明,THP-1细胞及其来源的巨噬细胞经过10ng/ml以上浓度的瘦素处理后,其对白色念珠菌的吞噬活性可以达到30%以上,说明其吞噬功能有所增强。50ng/ml以上组的瘦素处理后的THP-1细胞增殖活性显著增高(F=64.39,P=0.000)。但和其他实验结果不同,10ng/ml以下组增殖不明显。对于THP-1来源的巨噬细胞也有类似作用(F=93.95,P=0.000)。MR是单核巨噬细胞表面的一种受体,与其吞噬功能密切相关。不同剂量的瘦素刺激THP-1细胞及其来源的巨噬细胞后,我们用real-time PCR的方法检测了MR的表达。结果显示,不同剂量瘦素处理后,MR表达无显著改变(F=0.343,P=0.843；F=0.644,P=0.644)。本部分实验主要评价了瘦素对THP-1细胞及其来源的巨噬细胞的增殖和吞噬功能的影响。结果显示足够剂量的瘦素可以诱导THP-1细胞及其来源的巨噬细胞的增殖,并增强其吞噬功能。但这种作用并不依赖MR的表达变化,这说明瘦素诱导的吞噬功能的变化可能依赖其他受体的作用。第三章瘦素对THP-1细胞及其来源的巨噬细胞趋化功能的影响及其机制的研究趋化功能在控制炎症感染中具有重要意义。单核巨噬细胞可以依靠这种功能募集炎症细胞向炎症感染的部位聚集,从而有效控制感染。我们希望通过本部分实验观察瘦素对单核巨噬细胞趋化能力的影响。我们采用了两种细胞系一—-THP-1和HL-60来分别代表单核细胞和粒细胞。我们的研究结果表明,和未刺激对照相比,lOng/ml以上组瘦素可以将THP-1细胞及其来源的巨噬细胞的趋化指数提高数倍(F=72.59,P=0.000；F=220.78,P=0.000),说明其趋化功能显著增强。IL-8和MCP-1是单核巨噬细胞分泌的两种主要的趋化因子,ELISA结果显示,瘦素刺激的细胞能够分泌更多的IL-8和MCP-1。本部分实验主要评价了瘦素对THP-1细胞及其来源的巨噬细胞的趋化功能的影响。结果显示瘦素可以增强THP-1细胞及其来源的巨噬细胞的趋化功能,这种作用可能是通过诱导IL-8和MCP-1的产生来实现的,这在一定程度上说明了瘦素诱导的趋化机制。第四章瘦素对人单核巨噬细胞共刺激分子表达的影响单核巨噬细胞是最重要的一类抗原呈递细胞。外来抗原经单核吞噬细胞处理后呈递给T细胞,这是诱发免疫应答的先决条件。此外,在抗原呈递过程中单核巨噬细胞产生的IL-1也是TH活化不可缺少的刺激信号。这种抗原呈递功能与其表面共刺激分子表达的强弱密切相关。因此我们通过流式细胞术对它们表面的CD86、HLA-DR分子进行了检测。我们发现,和未刺激对照相比,lOng/ml以上组的瘦素可以明显上调及THP-1细胞表面这些共刺激分子的表达(F=449.32,P=0.000；F=436.55 P=0.000)。而对于人外周血单核细胞而言,这种有效浓度则需要达到100ng/ml。这说明对于不同的细胞类型,瘦素的作用强度存在着差异。本部分实验主要评价了瘦素对人单核巨噬细胞共刺激分子表达的影响。通过流式检测,我们发现,瘦素可以明显上调CD86和HLA-DR的表达。这提示瘦素可能具有增强单核巨噬细胞抗原呈递的能力。
[Abstract]:Leptin is a hormone - like cytokine composed of 167 amino acids , mainly secreted by fat cells . Human - coded leptin is located on chromosome 7 , while the mouse ' s coding gene is located on chromosome 6 . As the study progresses , leptin receptors are found not only in the nervous system , but also in many peripheral tissues , such as liver , lung , and testis . Therefore , leptin has been more and more concerned with immune responses and autoimmune diseases in recent years .

Human mononuclear phagocytes are very important immune cells , play an important role in the initial stage of acquired immunity or in innate immunity . In natural immunity , mononuclear phagocytes can quickly identify invading pathogens by means of pattern recognition receptors on the surface , and trigger subsequent inflammatory responses and bactericidal effects . It can also secrete chemokine to recruit inflammatory cells and control infection . In the initial stage of acquired immunity , mononuclear phagocytes can be presented to lymphocytes as important antigen presenting cells , thus promoting the activation or differentiation of lymphocytes .

The effects of leptin on the phagocytic function , proliferation activity , bactericidal effect , chemoattractant ability and co - stimulatory molecule expression of THP - 1 and its origin were evaluated . The results showed that leptin could significantly enhance the killing effect of THP - 1 and its derived macrophages on bacteria by using Staphylococcus aureus as the subject .

Flow cytometry was used to evaluate the phagocytic function of THP - 1 and its origin . The results showed that leptin could significantly enhance the phagocytosis of THP - 1 and its derived macrophages against Candida albicans . The results showed that leptin could significantly enhance the phagocytosis of THP - 1 and its derived macrophages against Candida albicans .

Chemotaxis function is another important function of mononuclear phagocytes . We measured the effects of leptin on THP - 1 and its origin by Boyden chamber . The results showed that leptin significantly enhanced the effect of leptin on HL - 60 granulocyte and its own , which could be related to the enhancement of IL - 8 and MCP - 1 secretion .

We also evaluated the expression of co - stimulatory molecules on the surface of macrophages and human peripheral blood mononuclear cells from THP - 1 and its origin . The results showed that leptin could up - regulate the expression of co - stimulatory molecules on these cell surfaces , but also found that there was a difference in the effective stimulation concentrations required for leptin for different types of cells .

The effect and mechanism of leptin on the bactericidal activity of THP - 1 cell and its origin

Monocyte macrophages have a strong killing ability , and can kill various pathogenic microorganisms in a non - specific manner , which is an important cell in nonspecific immune defense of the organism . The killing effect of mononuclear phagocytes can be enhanced by the immune adherence of the complement , the regulating action of the antibody in specific immunity and the enhancement of the cytokines .

The effects of THP - 1 and their origin on the killing of THP - 1 and their derived macrophages were studied by PMA . The results showed that leptin could significantly enhance the killing effect of THP - 1 and its derived macrophages on bacteria ( F = 73.79 , P = 0.000 ) .
The results showed that 10 , 50 , 100 ng / ml leptin could significantly stimulate the production of ROS ( F = 168.95 , P = 0.000 ) .
There was no significant irritation to NO production ( F = 1.179 , P = 0.377 , F = 1.978 , P = 0.174 ) . TNF - 伪 is the pro - inflammatory factor secreted by mononuclear phagocytes and plays an important role in the control of infection . The results showed that leptin in leptin treated by leptin could stimulate the production of TNF - 伪 ( F = 502.48 , P = 0.000 ; F = 2979.01 , P = 0.000 ) . These results suggest that leptin with a sufficient concentration can significantly stimulate the secretion of THP - 1 cells and their derived macrophages bactericidal active substances and significantly enhance their bactericidal activity .

In this experiment , the effects of leptin on the bactericidal function of THP - 1 cells and their origin were evaluated . The bactericidal effect of leptin on THP - 1 cells and their origin was first determined by bactericidal test and plate counting . Leptin induced TNF - 伪 and ROS production , but the effect of leptin on NO synthesis was not significant , which indicated a certain extent the mechanism of leptin - induced bactericidal action .

Study on the Effect of the second chapter on the phagocytic function of THP - 1 cell and its origin and its mechanism

The phagocytic function of THP - 1 cells and their origin was evaluated by flow cytometry . The results showed that the phagocytic activity of THP - 1 cells and their origin was 30 % or more , and the proliferation activity of THP - 1 cells increased significantly ( F = 64.39 , P = 0.000 ) . However , unlike other experimental results , the proliferation of the following groups was not significant at 10 ng / ml . Similar effects were observed for macrophages from THP - 1 sources ( F = 93.95 , P = 0.000 ) . MR was one of the receptors on the surface of mononuclear phagocytes and was closely related to its phagocytic function . After stimulation of THP - 1 cells with different doses of leptin and macrophages from their origin , the expression of MR was detected by real - time PCR . The results showed that MR expression did not change significantly after leptin treatment ( F = 0.343 , P = 0.843 ;
F=0.644,P=0.644).

The results showed that leptin could induce the proliferation of THP - 1 cells and their derived macrophages and enhance their phagocytic function . However , this effect did not depend on the expression of leptin , suggesting that leptin - induced changes in the phagocytic function might depend on the role of other receptors .

Study on the Effect and Mechanism of Leptin on the Chemotactic Function of THP - 1 Cells and Their Origin ;

We hope to observe the effect of leptin on the chemoattractant capacity of mononuclear phagocytes by means of this experiment . We hope to observe the effect of leptin on the chemoattractant capacity of mononuclear phagocytes by means of this experiment . We have used two cell lines one - THP - 1 and HL - 60 to respectively represent monocytes and leukocytes . Our results show that the leptin in the above group can increase the chemoattractant index of macrophages from THP - 1 cells and their origin by multiple times ( F = 72.59 , P = 0.000 ) .
The results showed that leptin - stimulated cells could secrete more IL - 8 and MCP - 1 .

In this part , the effects of leptin on THP - 1 cells and their derived macrophages were evaluated . The results showed that leptin could enhance the chemoattractant function of THP - 1 cells and their derived macrophages , which could be achieved by inducing the production of IL - 8 and MCP - 1 .

Effects of leptin on the expression of costimulatory molecules in human mononuclear phagocytes

In addition , IL - 1 produced by mononuclear phagocytes in the process of antigen presentation is a prerequisite for TH activation . In addition , IL - 1 produced by mononuclear phagocytes in the process of antigen presentation is a prerequisite for TH activation .
F=436.55 P=0.000). For human peripheral blood mononuclear cells , this effective concentration needs to reach 100 ng / ml . This suggests that there is a difference in the intensity of action of leptin for different cell types .

In this part , the effects of leptin on the expression of costimulatory molecules of human mononuclear phagocytes were evaluated . By means of flow cytometry , we found that leptin could significantly increase the expression of HLA - DR . This suggests that leptin may have the ability to enhance the antigen presentation of monocytes .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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