【作者】 范娟；

【导师】 王百忍； 王曦；

【作者基本信息】 第四军医大学 ， 神经生物学， 2009， 博士

【摘要】 颈动脉体(carotid body,CB)位于颈动脉分叉处,是重要的外周化学感受器,其功能主要是感受动脉血氧分压(PaO2)、二氧化碳分压(PaCO2)和pH值的变化,从而调节呼吸和循环反射。CB的实质细胞包括两类特殊类型的细胞:I型细胞(或称主细胞,球细胞)和II型细胞(或称支持细胞,卫星细胞)。I型细胞聚集成团,细胞呈椭圆形,有大量囊泡,其囊泡内含乙酰胆碱、儿茶酚胺(多巴胺和去甲肾上腺素)、脑啡肽、P物质等神经递质和神经活性肽。II型细胞薄而细长,以鞘膜包囊的方式围绕着球细胞。CB的感觉传入神经为窦神经(carotid sinus nerve,CSN)。CSN为舌咽神经的一个分支,其胞体位于舌咽神经的岩神经节。另外,CB还接受从邻近的的颈上节发出的交感神经和从迷走神经发出的副交感神经的支配。本研究主要包括两方面内容:(1)促炎性细胞因子白细胞介素(interleukin)-6对正常成年大鼠CB球细胞胞内钙和递质分泌的影响;(2)在以前工作的基础上,进一步研究了在体缺氧刺激诱导的大鼠CB中儿茶酚胺(catecholamines,CAs)的分泌。一、促炎性细胞因子IL-6对大鼠CB球细胞胞内钙和儿茶酚胺分泌的影响在免疫系统和神经系统之间存在广泛的联系,在机体发生感染的情况下,两者之间产生双向调节作用。外来致病因素(如细菌等)的侵入触发免疫细胞(如巨噬细胞)合成和释放一系列化学物质,包括IL-1β、IL-6和肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)。大量研究表明,当机体发生感染时,外周细胞因子通过多种途径作用于脑。其中一条重要途径是,外周细胞因子直接刺激迷走神经末梢或间接通过迷走旁神经节,形成神经冲动,完成免疫炎症信号向脑的传递。CB作为机体最大的旁神经节,也可能发挥同样的作用。本实验室以前的研究发现,在正常大鼠CB主细胞有白细胞介素-1受体(IL-1RI)和白细胞介素6受体(IL-6Rα)的高表达,提示CB具有感受促炎性细胞因子刺激的物质基础。电生理研究证实,外源性IL-1β刺激抑制了球细胞的外向钾电流,引起球细胞胞内钙离子浓度([Ca2+]i)水平升高和CSN放电频率增加。以上研究均证明了CB除了传统的化学感受功能外,还可以感受外周免疫刺激(如IL-1),并通过其传入神经CSN的放电频率改变向中枢神经系统传递信息。而作为机体感染时产生的一个重要细胞因子,IL-6也可能在免疫-脑途径中发挥重要作用。前面提到,CB上有IL-6Rα的表达,提示CB可以直接感受IL-6的刺激。但目前关于IL-6对CB的影响还不清楚。在这一部分研究中,我们以CB为研究对象,采用免疫组织化学染色、Western Blotting、细胞培养、钙成像技术和电化学分析检测(Amperometry)等技术,就IL-6对正常大鼠CB球细胞[Ca2+]i和递质分泌的影响做了初步探讨。同时,我们采用免疫组织化学染色,观察了多巴胺D1受体(D1R)和D2受体(D2R)在CB中的表达。主要结果有:1.在大鼠CB球细胞上有IL-6Rα和gp130的高表达,提示CB球细胞可以感受促炎性细胞因子IL-6的刺激。2.在培养的CB球细胞,胞外给予IL-6刺激引起球细胞[Ca2+]i迅速升高。外源性给予TNF-α也可引起球细胞[Ca2+]i迅速升高。3.在培养的CB球细胞及细胞簇,胞外给予IL-6刺激引起球细胞释放儿茶酚胺类递质。这种释放是剂量依赖性的,且与胞外Ca2+内流有关。4.免疫荧光染色实验结果显示,在正常SD大鼠的CB上有D1R和D2R免疫阳性产物的表达。D1R仅在CB的球细胞上有表达。而D2R除了在CB的球细胞上表达外,在CB的神经纤维上也有表达。这一部分研究证实:大鼠CB球细胞上有感受IL-6刺激的受体,并发现IL-6和TNF-α刺激引起球细胞[Ca2+]i迅速升高,IL-6刺激还引起球细胞释放儿茶酚胺类递质。这些研究进一步完善了我们关于CB是外周免疫信息感受器的学说,引领我们从一个新的角度认识和研究CB的功能。二、在体缺氧刺激诱导的大鼠CB中儿茶酚胺的分泌在CB感受外界刺激释放的递质中,CAs是其中一种非常重要的神经递质。缺氧可引起CB球细胞释放CAs,主要是多巴胺(dopamine,DA)。但是以往关于缺氧诱导CB释放CAs的实验都是在离体的CB上完成的,并不能真实的反映在体情况下CB对缺氧等刺激的反应。为了解在真实在体情况下,CB对缺氧刺激的感受。在本实验中,利用在体Amperometry测定技术,实时监测CB中DA随缺氧刺激的分泌动力学特征,并与离体时的反应作对比,为我们进一步理解DA在CB缺氧感受过程中的作用提供在体的证据。这一部分研究是对我们实验室树海峰以前工作的延续。在他的研究中发现,缺氧刺激麻醉状态下的大鼠,碳纤电极(carbon fiber electrode,CFE)在CB中可以记录到一个明显下降的氧化电流(Iamp)。此结果提示,在体情况下急性缺氧引起CB内CAs释放急剧降低,这与以往所有的离体实验结果相反。为了进一步证实,在体情况下,缺氧刺激大鼠时,CFE在CB中记录的氧化电流的下降是否真正代表了CAs释放的降低,我们采用离体和在体的电化学分析检测等技术,对此问题进行了研究。结果概括如下:1.与本实验室以前预实验结果一致,在in vivo条件下,给予大鼠急性缺氧刺激,CFE在CB中可以记录到一个明显下降的氧化电流。在in vitro条件下,离体CB标本经缺氧外液灌流刺激后,CFE在CB中可以记录到一个明显升高的氧化电流。这个结果提示,急性缺氧刺激在体引起CB释放CAs的急剧降低,而离体引起CB释放CAs的增加。2.CB的去神经支配不能逆转在体缺氧诱导的氧化电流的下降。说明在体和离体情况下,缺氧诱导的CB释放CAs的不同,不是由于神经支配的反馈作用导致的。3.进一步研究表明,在in vivo条件下,多巴胺转运体抑制剂(可卡因和诺米芬新)对缺氧诱导的CB中下降的氧化电流没有影响,这说明在体记录到的下降的氧化电流不是DA氧化引起的。给予不同的控制电压也发现,在in vivo条件下,CFE记录的缺氧引起的CB中下降的氧化电流不是DA氧化引起的,甚至也不是CAs氧化引起的。这两个实验说明了在体条件下,缺氧刺激大鼠,CFE在CB中记录到的氧化电流下降并不是由CAs释放减少引起的。4.CFE在大鼠CB中和CB外PBS溶液中记录到的Iamp基线相同,说明CFE在CB中记录的Iamp基线并不代表CAs基础水平。并且发现,缺氧刺激大鼠,CFE在CB中记录的Iamp下降并不是CB所特有的,在CB外附近组织也可以记录到。5.另外,在实验中还观察到,在in vivo条件下,给予大鼠急性CO2刺激,CFE在CB中可以记录到一个明显升高的氧化电流,这提示与以前报导的离体结果一致,急性CO2刺激在体也引起CAs释放增加。还观察到急性CO2刺激引起CSN放电增加。这一部分研究否定了本实验室以前预实验中的结果,认为在体条件下,缺氧刺激大鼠,CFE在CB中记录到的氧化电流下降并不代表CAs释放减少。在体CO2刺激引起的氧化电流升高是否代表CAs释放增加,还需进一步研究。更多还原

【Abstract】 The carotid body (CB) is a peripheral arterial chemoreceptor organ located the bifurcation of the common carotid artery. The carotid body chemoreceptor can sense PaO2, PaCO2 and pH in blood, and generate preventive respiratory and cardiovascular reflexes. The carotid body chemoreceptors consist of groups of specialized sensory glomus (type I) cells and sustenacular (type II) cells surrounded by a plexus of fenestrated capillaries and sinusoids. The most distinguishing feature of type I cell is the presence of numerous dense-core vesicles in their cytoplasm, which are known to contain ACh, catecholamines (CAs, including DA and NA), enkephalin, substance P and other putative neurotransmitters. Type II cells are thin and elongate in shape and surround clusters of type I cell in an ensheathing, capsule-like fashion.The carotid body is innervated by afferent terminals of the carotid sinus nerve (CSN), a branch of the ninth cranial nerve, whose neurons located in the petrosal ganglion. The carotid body is innervated by adrenergic nerve and parasympathetic nerve, too.This study includes two sets of experiments: (1) The effect of proinflammatory cytokine IL-6 on intracellular Ca2+ ([Ca2+]i) and neurotransmitters release in rat CB glomus cells.(2) Based on the previous work, hypoxia-induced CAs secretion in rat CB in vivo was further explored.1 The effect of IL-6 on [Ca2+]i and neurotransmitters release in rat CB glomus cellsCommunication pathways exist between the immune system and brain, allowing bidirectional regulation of immune and brain responses to infection. Detection of pathogens (e.g., bacteria) by the cells of the immune system (e.g., macrophages) triggers these immune cells to synthesize and release a series of chemical messengers, including the cytokines tumor necrosis factor-α(TNF-α), interleukin-β(IL-1β) and IL-6. A lot of evidence showed the cytokines signal the brain through several routes when infection has occurred. One important route is that cytokines stimulate vagal afferents terminals or paraganglia which form afferent synapses with vagal fibers, increase vagal afferent nerve activity, which carry these signals directly to the brain. CB, considered the largest paraganglion in the body, might play the common role with abdominal paraganglia.In a previous study, we found that interleukin 1 receptor type I (IL-1RI) and interleukin 6 receptor alpha (IL-6Rα) was strongly expressed in type I cells of the carotid body in SD rats, and this indicated there is a substance basis in the carotid body which could senses the stimulation of proinflammatory cytokine. Electrophysiologic studies confirmed extracellular application of IL-1β significantly decreased the outward potassium current, triggered a transient rise of [Ca2+]i in the cultured glomus cells of rat CB, and increased the discharge rate in the carotid sinus nerve. Above studies showed CB is not only a well-known peripheral arterial chemoreceptor organ, but also a organ sensing blood-borne immune stimulus (e.g., IL-1), result in increasing vagal afferent nerve activity, which signal to the brain. L-6 is a polyfunctional proinflammatory cytokine, which has been proposed as a mediator for immune-to-brain communication. As mentioned above, IL-6Rαis expressed in the CB of normal rats, suggesting that the CB might directly sense IL-6 levels. However, the precise effect of IL-6 on CB has not been elucidated.In this part of experiments, regarding the CB as research object, by using immunohistochemistry, western blotting, cell culture, calcium imaging, amperometric technology etc, we investigated the effect of IL-6 on [Ca2+]i and CAs release in rat CB glomus cells. We also observed the expression of dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R) in rat CB by using immunohistochemistry.The results are as follows.(1) In normal rat CB, IL-6Rαand gp130 were expressed in glomus cells, suggesting that the CB might directly sense IL-6 levels.(2) Calcium imaging showed that extracellular application of IL-6 or TNF-αinduced a rise in [Ca2+]i in cultured rat CB glomus cells.(3) Amperometry showed that local application of IL-6 evoked CAs release from isolated glomus cells or the clusters of glomus cell. CAs release from the glomus cells was dose-dependent and mediated by external Ca2+ influx.(4) The results showed D1R and D2R were expressed in normal rat CB. D1R only expressed in CB glomus cells. However, Strong immunoreactivity for D2R is distributed not only in the glomus cells but also in nerve fibers in rat CB.The study shows IL-6 receptor exist in normal rat CB glomus cells, extracellular application of IL-6 or TNF-αinduced a rise in [Ca2+]i in cultured rat CB glomus cells, and IL-6 evoked CAs release from glomus cells. These researches provided more evidence to the hypothesis that CB is a peripheral organ sensing immune information, and may guide us to recognize and research the function of the carotid body in a new view.2 hypoxia-induced CAs secretion in rat CB in vivoIt is well known that CAs is one of transmitters released by CB glomus cells. CAs including DA and NA, DA is predominant in several species. Although glomus cells also contain NA, hypoxia preferentially releases DA rather than NA. However, previous studies on hypoxia induced DA release were based on isolated single glomus cells or intact CBs, these responses in vitro don’t reflect the real responses of the carotid body in vivo. In order to known CB’s response to hypoxia in vivo, the release of DA was recored from rat CB in real time in vivo by amperometric technique and compared with that in vitro. The study will provide some evidence for us to understand DA’s function during hypoxia processing in rats.The present work was followed SHF’s previous work. In contrast to the increase of catecholamine release from the isolated CB tissue or single glomus cells, he found that hypoxia induced a significant decrease of amperometric current (Iamp) in vivo, it seems to suggest hypoxia induced a rapid decrease in CAs release in vivo. In additional, he observed this evoked rapid CAs decrease signal was reversed by cutting the CSN. By using amperometric technology in vitro and in vivo, we investigated whether the decrease of hypoxia-induced Iamp in vivo reflects the decrease of CAs release.The results are as follows.(1) Consistent with SHF’s recordings, hypoxia induced a significant decrease Iamp recorded by CFE in anaesthetized rats CB in vivo. However, the tests in isolated CBs showed a significant increase in Iamp following perfusion with a hypoxic solution. These results seem to show that hypoxia induced a rapid decrease in CAs release in vivo and a rapid increase in CAs release in vitro.(2) In contrast to SHF’s result, the decrease in Iamp induced by hypoxia in vivo was not reversed by denervation. This result suggests that the difference of hypoxia-induced CAs secretion between in vivo and in vitro was not resulted in by neural feedback.(3) Dopamine transporter inhibitors (cocaine and nomifensine) didn’t affect hypoxia-induced Iamp in CB of rat in vivo. We measured CB response to hypoxia in vivo by CFE at different holding potentials, and found the decreased Iamp induced by hypoxia in vivo was not really due to specific oxidation of CAs. The two experiments showed that hypoxia-induced Iamp decraese in rat CB in vivo was not induced by the reduced CAs secretion.(4) The baseline of Iamp recorded in rat CB by CFE was the same as that in PBS in vivo, it indicates the baseline of Iamp recorded in CB by CFE in vivo didn’t reflect the base level of CAs secretion in CB. We found the decreased Iamp induced by hypoxia in rat CB also was recorded in the area near CB of rat by CFE.(5) In additional, CO2 treatment in anaesthetized rats, a significant increase in Iamp was recorded by CFE in CB in vivo. These results seem to show that CO2 induced a rapid increase in CAs release in viv. However, it needs us to futher explore. We also observed that CO2 significantly increased the discharge rate in the CSN in vivo.This part of experiments denied SHF’s conclusion. We considered that the decreased Iamp induced by hypoxia in anaesthetized rats CB in vivo didn’t reflect CAs secretion decreasing. It needs to be further explored that the increased Iamp induced by CO2 reflect CAs secretion decreasing in anaesthetized rats CB in vivo.更多还原