【作者】 黄俊龙；

【导师】 蒋春雷； 沈兴华； 王云霞；

【作者基本信息】 第二军医大学 ， 航空、航天和航海医学， 2008， 博士

【摘要】 应激相关激素去甲肾上腺素（NE）对机体免疫功能的影响已受到学者们的广泛关注。以往研究结果提示，NE能促进或加剧内毒素休克、过敏性疾病、慢性炎症性疾病以及肿瘤等，但对其作用机制尚不很清楚。长期反复应激导致NE释放从而致使机体炎症和免疫功能发生改变，甚而影响到个体健康状态的研究，对其进行研究具有重要的理论和实践意义。众所周知，巨噬细胞广泛分布于机体全身，被刺激后能释放种类繁杂的细胞因子等物质，参与调节免疫系统功能。除分泌炎症因子等之外，巨噬细胞在某些刺激物如LPS等作用下会合成分泌相当数量的神经肽类物质以及NE等。与此同时，交感神经末梢细密的分布在全身各个脏器，而且在那些存在相当数量巨噬细胞的免疫器官中，也广泛分布有交感神经末梢。如淋巴结、脾脏和胸腺等免疫器官，交感神经末梢极其丰富，甚至存在着类似于“突触”样的结构基础，而且在免疫系统，巨噬细胞、树突状细胞和淋巴细胞等都有NE受体表达，这些结构可能为其在局部发挥作用提供结构基础，也提示交感神经系统与免疫系统间相互作用的紧密性和直接性。除下丘脑-垂体-肾上腺皮质轴外，交感神经系统是另一个重要的应激反应系统。在应激时交感神经末梢兴奋，其释放的NE是血液中NE的主要来源。无论是交感神经释放NE，还是自身受到LPS刺激后分泌NE，巨噬细胞很容易处身于一个富于NE的微环境。近年来，模式识别受体TLR受到免疫学家的高度重视，被认为是连接天然免疫和获得性免疫的桥梁。有文献报道，NE能够调节心血管细胞的氧化应激，而活性氧（ROS）参与调节toll样受体4（TLR4）表达。那么，NE能否调节TLR4呢?NE是否可能通过调节巨噬细胞氧化应激进而调节TLR4?为此，我们设计并进行了以下实验：（1）从整体水平，检验NE是否能够调节模式识别受体TLR4的表达。应用束缚应激模型，通过免疫组化观察模型小鼠脾脏TLR4表达变化，并观察束缚应激小鼠LPS所致休克后72 h存活率。（2）Western blot及Quantitative real time PCR技术检测NE对体外巨噬细胞TLR4表达的影响。（3）Western blot检验ROS是否能够调节TLR4表达。观察不同剂量的活性氧对TLR4表达的调节作用，应用细胞色素C还原法及活性氧荧光探针法检测NE是否能够影响巨噬细胞氧化应激状态。（4）NE或ROS处理过的巨噬细胞，ELISA检测在LPS刺激下TNFα变化，化学发光法检测NO生成变化。（5）探讨NE调节巨噬细胞氧化应激状态的具体机制，Western blot检测NE可能是通过影响PKC进而影响NADPH氧化酶等。主要结果如下：（1）免疫组化结果发现慢性束缚应激后的模型小鼠脾脏组织TLR4表达增高，与注射6-OHDA后束缚应激小鼠相比，TLR4表达量也显著增加。（2）NE能够促进体外巨噬细胞TLR4表达。不同剂量的NE处理巨噬细胞12 h和24 h后，小鼠巨噬细胞TLR4蛋白表达量及mRNA转录显著增加，β受体阻断剂Propranolol及α受体阻断剂Phtentolamine都能部分阻断其上述作用，还原剂N乙酰半胱氨酸能部分阻断NE对TLR4表达的促进作用。（3）NE能够促进巨噬细胞产生ROS，后者能够调节TLR4。不同剂量的NE能够促进巨噬细胞产生ROS，而且具有剂量依赖性。ROS调节TLR4表达具有双向性。高剂量（>100μM）的H₂O₂抑制TLR4表达，而较低剂量（10μM）则促进TLR4表达。（4）NE或者H₂O₂处理巨噬细胞24 h后，再用LPS刺激巨噬细胞后产生的TNFα和NO比LPS单纯刺激组显著增加。（5）NE能够促进PKC磷酸化，β受体阻断剂Propranolol及α受体阻断剂Phentolamine都能部分阻断其作用。更多还原

【Abstract】 It is generally accepted that stress can affect the immunity of the body notably. More and more researchers have paid so much attention to this. But most of the former research focused on the glucocorticoid （GC） what came from the adrenal cortex. On the other hand, less research work involved another important stress-related hormone NE. It was widely recognized that NE could aggravate many kinds of diseases such as LPS shock, allergic disease, inflammatory disease and tumor and so, yet the mechanism remained absolutely unclear. It would be very instructive and helpful for the human being to study this concretely.It was well known that NE in the circulation of the body mostly came from the sympathetic nerve terminal. Especially when the body was under stress state, the brain excited the sympathetic nerve and a great deal of NE could be released into the circulation. Terminal of sympathetic nerve almost spread all over of the body. Besides the cardiovascular system and nervous system, sympathetic nerve could be found everywhere in each immunity organ. Through releasing NE into the immunity organs, the sympathetic nerve system manipulated the immunity system profoundly. What’s more, between the immunity organs and the sympathetic terminal, there existed an interesting special structure which resembled the"synapse". Macrophage could be found in each immunity organ, and the "synapse" was so close to it definitely. Nevertheless, almost all immune cells expressed adrenergic acceptors. All of these conditions suggested that the sympathetic nerve system and immune system related to each other closely, and it would be very easy to be affected by each other. All of these structures built up a material foundation for the sympathetic nerve system to regulate the function of macrophage. And there was still another interesting thing that when macrophage was treated by LPS, macrophage could release so much NPY and NE and so on. Then when being under stress state or some kind of infection situation, macrophage could be so easily flooded and influenced by NE. But how NE precisely took effect on macrophage still remained unknown, especially when the body was under stress state.Macrophage exerted so many complicated functions in the body though several different ways. It could affect almost all of the diseases such as inflammatory diseases and tumors and so on. There were many toll-like receptors 4 on the membrane of the macrophage, which were the natural receptor of LPS. TLR4 was the key point of the function of macrophage. When TLR4 was stimulated, a great deal of cytokines would be produced and released out of macrophage, such as TNFα, IL-1, IL-6 and IFN and so on. Most of these had important functions on the immunity and inflammation reaction. What’s more, many factors could regulate TLR4 such as MIF, hypooxygen, and O³ and so on. Among these, ROS was the key point of modulating TLR4. By the way, NE could lead to oxidative stress in cardiovascular system evidently. Then maybe NE could affect the oxidative state of the macrophage? And then NE could modulate TLR4 though ROS?Above all, till now there are so many unknown areas about how sympathetic nerve system regulates the function of the immunity, and through which key point it exerts its action. In one word, we still have so long a way to go. What does NE which came from the sympathetic terminal mean to macrophage? And, how does NE take effect on macrophage? Maybe NE implements its function through modulating TLR4 by ROS? In order to prove this hypothesis, we designed a serial of experiments to test it.（1）We applied the immunohistochemistry technique to investigate whether NE could modulate the expression of TLR4 in the spleen after the mice subjected to the restrained stress process. And we also determined the survival rate in 72 hours of the stressed mice when they experienced LPS shock.（2）We applied Western blot and Quantitive real time PCR to test if NE could affect the expression of TLR4 in vitro.（3）We applied Western blot to test whether ROS could take effect on the expression of TLR4 in vitro, and then applied cytochrome C revivification method and fluoresce probe DCFH-DA to test if NE could affect the redox state of macrophage in vitro.（4）After being pretreated with NE or ROS, we applied ELISA and chemiluminescence method to determine the production of TNFa and NO after the macrophage was stimulated by LPS.（5）At last we explored if NE could stimulate the phosphorylation of the PKC of macrophage.The results were listed as follows:（1） NE could affect the expression of TLR4. The immnohistochemistry results showed us that the stressed animal spleens presented much more TLR4 expression than control and 6-OHDA+stess group. The survival rate of stressed animals which were being pretreated with 6-OHDA was higher than that in stressed animals.（2） NE could promote the expression of TLR4. Andβadrenoreceptor inhibitor propranolol, a adrenoreceptor inhibitor phentolamine and N-Acetylcysteine all could partly antagonize NE’s effect.（3） NE could regulate the ROS production of macrophage. Through cytochrome C revivification method and fluorescence probes method, the results showed that NE could stimulate macrophage to produce much ROS. And ROS could modulate the expression of TLR4 in macrophage bidirectionally. An appropriate dose of H₂O₂ could increase the expression of TLR4, but higher level of H₂O₂ could decrease the expression of TLR4, yet much lower level had no effect on it.（4） The result of ELISA and chemiluminescence showed us that macrophage pretreated by NE and H₂O₂ produced more TNFαand NO than that in control group.（5） NE could stimulate the phosphorilation of PKC. Theβadrenoreceptor inhibitor propranolol andαadrenoreceptor inhibitor phentolamine could partly antagonize its effect.更多还原