颈动脉体对IL-1β和急性缺氧刺激的反应

发布时间：2018-05-23 22:13

本文选题：颈动脉体 + 窦神经　；参考：《第四军医大学》2007年硕士论文

【摘要】： 应激是机体在面对环境中不利因素时,实际或认知上的要求与适应和应付能力之间的不平衡所导致的身心紧张状态及其反应。对应激环路和调控机制的研究对阐明多种疾病的发病机理及寻找针对应激(包括军事应激)的应对措施有重要的意义。应激反应实质上是神经-免疫-内分泌网络中各系统共同反应的综合体现。随着研究的不断深入,人们逐渐认识到中枢神经系统(central nervous system, CNS)、免疫系统、内分泌系统并不是各自为政、独立存在的系统,它们之间存在着密切的相互联系和调控。一般认为,CNS通过下丘脑-垂体-肾上腺轴、下丘脑-垂体-性腺轴以及植物性神经系统等传出通路调控和影响免疫、内分泌系统的功能1-4。内分泌系统也通过一些靶腺激素的反馈机制影响CNS的功能。但是,免疫系统是通过何种途径影响CNS功能的?外周血液中的细胞因子是通过怎样的方式将“信息”传递给CNS的?一直是个未解之谜。在机体遭受病原体刺激早期,外周免疫系统中的细胞即迅速作出反应,释放出多种促炎性细胞因子,如:IL-1β、TNFα、IL-6、IFNγ等。一些研究发现,在免疫应激或者感染的情况下,IL-1β是中枢性发热反应的始动因子。因此,IL-1β可能是在外周免疫系统和CNS之间发挥重要“信使”作用的分子之一3,5。但由于IL-1β分子量较大,且具有亲水性的特点,因而几乎不能通过血脑屏障(blood brain barrier,BBB)6。有人认为,在脑室周围脑组织中存在一些BBB缺如的区域,外周循环中的IL-1β可能通过这些区域进入CNS;还有人认为细胞因子可能在与脑血管内皮细胞上的受体结合后,产生一些下游分子(例如前列腺素E2等),将外周免疫信号传递入脑4,7。然而,资料表明通过这些途径入脑的细胞因子量很少8。最近,外周感觉神经,尤其是迷走神经的感觉传入纤维在“免疫-脑”通信传入途径中的作用,逐渐引起人们广泛的关注9,10。一些研究者发现,迷走神经一些分支的终末或者神经末梢旁的多巴胺能细胞(这些细胞常聚集成团称为迷走神经旁神经节,受迷走神经传入纤维支配)具有IL-1β等细胞因子的结合位点11;推测细胞因子可能通过直接刺激迷走神经末梢,或通过旁神经节细胞的换能,引起迷走神经传入纤维放电频率的改变,向CNS传递外周免疫刺激信号12。哺乳动物的颈动脉体(carotid body, CB)是一个位于颈动脉血管分叉处的血运极其丰富的器官。传统观念认为,CB是重要的外周化学感受器,可以非常精确、灵敏地感受动脉血中的氧分压(PO2)、二氧化碳分压(PCO2)、酸碱度(pH)和渗透压等的变化13。最新的研究发现,CB还可以感受外周循环中葡萄糖浓度的变化14。这些发现不断地揭示了CB化学感受功能的多样性和复杂性。根据解剖学的分类,CB也属于旁神经节。同迷走神经旁神经节一样15,CB也是由主细胞(球细胞,I型细胞)和支持细胞(II型细胞)组成;其球细胞是多巴胺能细胞,可以合成、储存、分泌儿茶酚胺类递质。我们实验室前期的工作发现,在正常大鼠的球细胞上有强烈的IL-1受体I型(IL-1RI)16和IL-6受体17表达。因此,我们推测:CB除了传统的化学感受功能外,还可以感受外周循环中的免疫刺激信号(如:IL-1、IL-6等),并通过其传入神经—窦神经(carotid sinus nerve, CSN)的放电频率改变向中枢神经系统传递信息。本研究的主要目的是验证上述假说。我们先在细胞特性和CB球细胞非常相似的PC12细胞进行了初步观察。PC12细胞是源自于大鼠肾上腺嗜铬细胞肿瘤的细胞系。同嗜铬细胞一样,PC12细胞也可以合成、储存、分泌儿茶酚胺类递质。另外,PC12细胞在NGF诱导下可以分化成具有交感神经元特性的细胞18,因此,PC12细胞作为一种细胞模型,被广泛用于神经元的药理、生理学研究19-21。近年来的研究发现, PC12细胞同CB的球细胞具有很多类似之处。例如:PC12细胞和CB的球细胞膜上都有氧敏感的钾通道22,23,PC12细胞也可以感受低氧、低pH等刺激。在组织学上,肾上腺嗜铬细胞和CB细胞都起源于神经嵴,它们都可以合成并分泌多巴胺(dopamine, DA)、乙酰胆碱(acetylcholine, ACh)等神经递质13,24。大量的实验证据发现,PC12细胞和CB球细胞具有很多生理学、药理学的共性25-27。因此,PC12细胞也被广泛用作外周化学感受性细胞(如CB球细胞)功能研究的细胞模型和工具细胞。在本研究中,我们综合应用免疫细胞化学技术、膜片钳技术、钙成像技术、在体胞外记录技术以及离体和在体的电化学分析检测等技术,首先用PC12细胞模型研究了外源性IL-1β刺激对PC12细胞电生理特性以及细胞内游离钙离子浓度([Ca2+]i)的影响。然后,从培养细胞、组织切片以及在体水平,分别研究了外源性IL-1β刺激对大鼠CB球细胞的电生理特性及其传入神经—CSN放电频率的影响。另外为了下一步工作的开展,我们还研究了CB的中最重要的神经递质之一——DA在急性缺氧诱发放电中的作用。结果简要归纳如下: (1)免疫细胞化学和蛋白质印迹分析结果发现,在PC12细胞的胞膜和胞核上均有IL-1RI的分布。电生理结果表明,IL-1β可以通过和胞膜上特异的受体结合,浓度依赖性地抑制PC12细胞上外向性电压依赖的钾电流(IK)的幅度。而且,IL-1β可以引起IK的失活曲线左移,但不影响其激活曲线。另外,IL-1β可以引起PC12细胞膜超极化,并可引起[Ca~(2+)]i迅速升高。 (2)全细胞膜片钳记录和钙成像结果显示,胞外给予IL-1β可以显著地抑制CB球细胞外向型钾电流的幅度,并可导致[Ca~(2+)]i迅速升高。在体的细胞记录结果显示,药理浓度的IL-1β刺激可以使麻醉大鼠的CSN放电活性增强。进一步的研究提示,CB球细胞感受IL-1β刺激的突触传递过程可能有ATP的参与,而DA在这一过程中可能不起主要作用。 (3)与以往的离体实验结果相反,在体情况下实验性急性缺氧引起CB内儿茶酚胺类递质的释放急剧降低,且这种效应可以被切断CB的传入神经CSN所消除或者减弱。进一步的研究发现:①在in vivo记录情况下,CB局部给予DA可以抑制CSN的放电活性,这与以往文献报道的结果一致28-31;②CB局部给予D2受体的阻断剂haloperidol (Hal)可以引起CSN自发放电的频率升高,而局部给予D1受体阻断剂SCH23390则未见CSN放电频率的明显改变;③DA可能通过D1或者D2受体引起CB球细胞内吞作用增强。④腺苷(adenosine)可引起缺氧诱导的离体CB释放CAs减少。主要结论: (1)PC12细胞对胞外IL-1β刺激起反应; (2)CB及其传入神经CSN对外源性IL-1β的直接刺激起反应,提示其可能具有感受外周免疫刺激(如IL-1β)的能力,可能是“免疫-脑”通信的感受器之一; (3)缺氧时, CB中胞外DA量通过腺苷的调节作用而减少,从而增加了CSN的放电频率。推测CB可能利用DA的这种“推-挽”机制而改变CSN的放电,将外周的缺氧信号传向中枢神经系统。
[Abstract]:Stress is the physical and mental state and reaction of the body in the face of adverse factors in the environment, the actual or cognitive requirements and the imbalance between adaptation and coping ability. The study of the stress loop and regulatory mechanism is important to elucidate the pathogenesis of various diseases and to find the response to stress (including military stress). The stress response is essentially a comprehensive reflection of the common reactions of the systems in the neural immuno endocrine network. As the research progressively deepened, people gradually realized that the central nervous system (central nervous system, CNS), the immune system, and the endocrine system were not independent and independent systems, and there was a secret between them. It is generally believed that CNS regulates and affects immunization through the hypothalamus pituitary adrenal axis, the hypothalamus pituitary - gonadal axis and the plant nerve system. The function of the endocrine system of the endocrine system also affects the function of CNS through the feedback mechanism of some target adenoids. However, the immune system is through. Which way to influence the function of CNS? How can cytokines be transferred to CNS in peripheral blood?
In the early stage of the pathogen stimulation, the cells in the peripheral immune system respond quickly and release a variety of proinflammatory cytokines, such as IL-1 beta, TNF alpha, IL-6, IFN gamma, etc. some studies have found that, in the case of immune stress or infection, IL-1 beta is the starting factor of the central fever reaction. Therefore, IL-1 beta may be in the peripheral immune system. 3,5., one of the most important "messenger" molecules between the system and the CNS, has been found to be almost impossible to pass through the blood brain barrier (blood brain barrier, BBB) 6. because of its large molecular weight and hydrophilicity. There are some BBB absent regions in the brain tissue around the ventricle, and the IL-1 beta in the peripheral circulation may pass through this Some regions enter CNS; others think that cytokines may be associated with receptors on cerebral vascular endothelial cells and produce some downstream molecules, such as prostaglandin E2, to transfer peripheral immune signals into the brain 4,7.. The role of nerve sensory afferent fibers in the "immuno brain" communication pathway has gradually aroused widespread concern in 9,10. researchers. Some researchers found that the terminals of some branches of the vagus nerve, or the dopaminergic cells near the nerve endings, are called the paraplastic ganglia, which are afferent fibers of the vagus nerve. The binding site of IL-1 beta and other cytokines is 11. It is speculated that cytokines may directly stimulate the endings of the vagus nerve or through the transfer of paraganglionic cells, causing the change of the frequency of the afferent fibers of the vagus nerve and the transmission of the peripheral immune stimulation signal to CNS by 12..
The carotid body (CB) in mammals is an extremely rich organ in the bifurcation of the carotid artery. The traditional idea is that CB is an important peripheral chemoreceptor and can be very accurate and sensitive to the oxygen partial pressure (PO2), the two carbon oxide pressure (PCO2), the pH (pH) and osmotic pressure in the arterial blood. 13. The latest research has found that CB can also feel the changes in glucose concentration in the peripheral circulation 14.. These findings continue to reveal the diversity and complexity of CB chemoreceptor function.
According to the anatomical classification, CB also belongs to the paraplastic ganglion. Like the paraceal ganglion, 15, CB is also composed of main cells (ball cells, I cells) and support cells (II cells); the ball cells are dopaminergic cells that can synthesize, store and secrete catechol amine transmitters. Our work in the early laboratory found that in normal rats There is a strong expression of IL-1 receptor I (IL-1RI) 16 and IL-6 receptor 17 on the ball cells. Therefore, we speculate that, in addition to the traditional chemoreceptor function, CB can also feel the immune stimulation signals in the peripheral circulation (such as IL-1, IL-6, etc.), and change the frequency of the discharge to the central nerve through the frequency of its afferent nerve - sinus nerve (carotid sinus nerve, CSN). The system passes information.
The main purpose of this study is to verify the hypothesis. First, we preliminarily observed that.PC12 cells were derived from rat adrenal chromaffin cells in PC12 cells with very similar cell characteristics and CB cells. Like chromaffin cells, PC12 cells can also be synthesized, stored, and secreted by catecholamine transmitters. In addition, PC12 cells are secreted. NGF induced cells can be differentiated into 18 cells with the characteristics of sympathetic neurons. Therefore, PC12 cells are widely used as a cell model and are widely used in the pharmacological activities of neurons. In recent years, the study of 19-21. found that PC12 cells have many similarities with CB cells. For example, PC12 cells and the membrane of CB are sensitive to oxygen. The potassium channel 22,23, PC12 cells also can feel hypoxic, low pH and other stimuli. Histologically, adrenal chromaffin and CB cells are all derived from the neural crest, and they can synthesize and secrete dopamine (dopamine, DA), acetylcholine (acetylcholine, ACh) and other neurotransmitter 13,24. experimental evidence found, PC12 cells and CB spherical cells. As a result of many physiological and pharmacological generalities, 25-27., PC12 cells are also widely used as cell models and tool cells for the study of peripheral chemoreceptor cells (such as CB spherocytes).
In this study, we used immunocytochemical techniques, patch clamp techniques, calcium imaging techniques, extracellular recording techniques, and electrochemical detection techniques in vitro and in vivo. The electrophysiological characteristics of exogenous IL-1 beta stimulation and intracellular free calcium concentration ([Ca2+]i) in PC12 cells were first studied by PC12 cell model. Then, the effects of exogenous IL-1 beta stimulation on the electrophysiological characteristics of CB cells and the frequency of afferent nerve CSN discharge were studied from cultured cells, tissue sections and at the body level. In addition, in order to carry out the next step, we also studied one of the most important neurotransmitters in CB, DA in acute deficiency. The role of oxygen induced discharge.
The results are summarized as follows:
(1) the results of immunocytochemistry and Western blot analysis showed that there was a distribution of IL-1RI in the cell membrane and nucleus of PC12 cells. Electrophysiological results showed that IL-1 beta could bind to the specific receptors on the membrane and inhibit the extrovert voltage of PC12 cells dependent on the amplitude of the potassium current (IK) of the PC12 cells. Moreover, IL-1 beta could cause IK. The inactivation curve shifted to the left, but did not affect its activation curve. In addition, IL-1 beta could induce hyperpolarization of PC12 cell membrane and cause rapid rise of [Ca~ (2+)]i.
(2) whole cell patch clamp recording and calcium imaging results showed that extracellular given IL-1 beta could significantly inhibit the amplitude of extrovertic potassium current of CB cells and lead to a rapid increase in [Ca~ (2+)]i. The results of cell recording in vivo showed that IL-1 beta stimulation of pharmacological concentration could enhance the activity of CSN discharge in anesthetized rats. Further research suggests CB The involvement of ATP in the synaptic transmission of IL-1 beta stimulated by spheroid cells may not play a major role in this process.
(3) contrary to the previous experimental results, the release of catecholamine transmitters in CB is dramatically reduced in the experimental acute hypoxia, and this effect can be eliminated or weakened by the afferent CSN of the CB. Further studies have found that in the in vivo record, the partial administration of CB to DA can inhibit the discharge of CSN. Sex, this is in agreement with the results reported in the previous literature. (2) haloperidol (Hal), blocking agent haloperidol (Hal) locally given to CB receptor, can cause an increase in the frequency of spontaneous discharge of CSN, while local administration of D1 receptor blocker SCH23390 has not seen a significant change in the frequency of CSN discharge; (3) DA may increase the endocytosis by D1 or D2 receptors. 4 Adenosine (adenosine) can induce hypoxia induced CAs release from CB release in vitro.
The main conclusions are as follows:
(1) PC12 cells responded to extracellular IL-1 beta stimulation.
(2) CB and its afferent nerve CSN react with direct stimulation of external IL-1 beta, suggesting that it may have the ability to feel peripheral immune stimulation (such as IL-1 beta), which may be one of the receptors for "immuno brain" communication.
(3) when hypoxia, the amount of extracellular DA in CB is reduced by the regulation of adenosine, thus increasing the discharge frequency of CSN. It is presumed that CB may use the "push pull" mechanism of DA to change the discharge of CSN and transmit the peripheral anoxic signal to the central nervous system.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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