【作者】 高维娟；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 炎症反应是机体对抗外来致病因素侵袭的保护性应激反应。但若过分强烈，则体内的炎症细胞被过度激活，持续大量地表达、产生和释放多种促炎因子，如TNF-α、IL-1、IL-6等，这些促炎因子又以“自分泌”或“旁分泌”的方式诱生多种炎症介质的分泌，由此激发炎症连锁反应，以致失去控制，形成全身炎症反应综合征（SIRS），并进而发展为多器官功能障碍综合征（MODS）。感染性因素和非感染性因素均可刺激宿主炎症细胞，产生大量细胞因子和炎症介质。其中，G-菌菌壁的主要活性成分脂多糖（LPS）是诱导巨噬细胞产生和释放炎症介质最强烈、最有效的激活物，在G-菌感染和内毒素休克发病中发挥重要作用。在SIRS和MODS的发病过程中，肺脏是公认的最易受损的靶器官。同时也是MODS重要的动力器官，因为肺是炎症细胞激活和聚积的重要场所，它可诱发或加重局部和全身炎症反应，介导组织损伤，促进SIRS的发生和发展。巨噬细胞是体内产生和释放多种炎症介质、诱导炎症反应的关键效应细胞。肺巨噬细胞包括肺泡巨噬细胞（AMs）、肺间质巨噬细胞（PIMs）、肺血管内巨噬细胞、胸膜巨噬细胞、血管壁巨噬细胞、支气管壁巨噬细胞和树突状细胞，其中，PIMs在炎症反应中的作用日益受到人们的关注。与AMs一样，PIMs也具有吞噬能力和释放各种炎症介质的功能，尤其具有较强的分泌IL-1、IL-6的能力；在内毒素血症时，PIMs较AMs受到LPS攻击更早，且其吞噬功能、对补体的趋化作用以及产生氧自由基的能力明显强于AMs，在肺组织的炎症反应中发挥着重要作用。胆囊收缩素（cholecystokinin, CCK）是一种典型的脑肠肽，它不但具有胃肠激素的功能，还以神经递质和神经调质的形式发挥着重要的作用。近年的研究表明，八肽胆囊收缩素（CCK-8）具有一定的抗炎作用，它可减轻LPS引起的大鼠心肌组织、肺脏、脾脏和肾脏白细胞浸润、毛细血管血液淤积等征象，抑制体内TNF-α、IL-1、IL-6等促炎因子的升高；体外实验证实，CCK-8可抑制LPS诱导的大鼠PIMs的NF-κB活性和<WP=6>mCD14的表达，导致PIMs对LPS的敏感性降低，从而抑制炎症反应。以上研究结果显示了CCK-8抗炎作用的良好前景。目前，有关CCK-8抗炎作用的受体及信号转导机制尚不清楚。CCK-8的抗炎作用是通过其受体介导的。既往发现使细胞内cAMP增加的物质可抑制NF-κB活性，减少巨噬细胞产生TNF-α，可见cAMP-PKA信号通路的活化可抑制炎症反应。在消化系统, CCK可激活cAMP-PKA通路和DAG-PKC通路，并且CCK可引起腹腔巨噬细胞cAMP含量增加而PKC活性降低，表明cAMP-PKA、DAG-PKC两条途径之间存在着互相抑制的cross-talk。在肺组织，有CCK-A和CCK-B两种受体亚型表达，但在PIMs，CCK受体的表达亚型是什么？其结合特性怎样？在信号传递过程中，CCK-8在NF-κB上游是通过哪条通路起作用的？目前未见相关报道。本课题从CCK受体入手，研究CCK受体在PIMs上的表达亚型和结合特性，并探讨CCK受体-cAMP-PKA-NF-κB这条信号通路及其与DAG-PKC通路间的cross-talk，以期阐明CCK-8的抗炎作用机制。1 大鼠PIMs CCK受体的表达亚型及结合特性CCK具有一定的抗炎作用，其发挥生理功能是通过受体介导的。根据其对内源性CCK-8和胃泌素亲和力的不同，CCK受体主要分为CCK-A和CCK-B两种亚型，在体内分布十分广泛，但在大鼠PIMs，CCK受体的表达亚型和结合特性尚未见报道，本实验对此进行了探讨。SD大鼠10只，随机分为两组。实验组舌静脉注射5g·L-1的LPS 5 mg·kg-1，对照组注射等量生理盐水。用酶消化法结合肺泡耗竭灌洗和肺循环灌洗技术分离纯化大鼠PIMs，超速离心法提取细胞膜，与3H标记的硫酸化CCK-8（3H-CCK-8S）进行放射配基结合实验，观察正常大鼠和LPS诱导48h后的大鼠PIMs膜与3H-CCK-8S的结合情况，观察孵育时间和温度对特异性结合的影响。并在最佳条件下制备膜受体与配基结合的饱和曲线，观察CCK受体的结合特性。用非标记的CCK-8S、CCK-A受体特异性拮抗剂CR-1409及CCK-B受体特异性拮抗剂CR-2945进行竞争抑制实验，观察CCK受体表达亚型。实验数据用均数±标准差(±s)表示。3H-CCK-8S与膜结合的平衡解离常数（KD）和最大结合容量（Bmax）由Scatchard作图法经直线回归方程求得。CCK-8S和CR-1409及 CR-2945的IC50值由对数-几率单位法求得。结果显示：正常大鼠PIMs未能检出特异性结合，<WP=7>静脉注射LPS 48h出现特异性结合，且对孵育时间和温度有依赖性，在37℃和21℃孵育条件下，3H-CCK-8S能够与膜迅速结合，30min可达平衡，但解离速度较快；而4℃孵育时，3H-CCK-8S与膜的特异性结合稳步升高，于3h达到平衡状态且不易解离，4℃孵育12h特异性结合仍然维持在平衡状态。经Scatchard分析，平衡解离常数KD值为：0.68±0.28nmol·L-1，最大结合容量Bmax值为32.50±2.70fmol·mg-1蛋白。在竞争抑制实验中，3H-CCK-8S与大鼠PIMs膜的结合可被CCK-8S、CR-1409和CR-2945所抑制，当拮抗剂浓度由10-12mol·L-1逐渐增加至10-6mol·L-1时，特异性结合进行性下降，呈剂量依赖关系。其IC50值分别为：2.30±0.80nmol·L-1、3.20±1.13nmol·L-1和0.19±0.06μmol·L-1。在所有的结合实验中，非特异性结合量占总结合量的更多还原

【Abstract】 It is well known that inflammatory response is a kind of protective stress for organism to resist the attack of external etiological factors. Excessive activated immune cells produce and release large quantity of various pro-inflammatory cytokines including TNF-α, IL-1, IL-6 and so on. Overproduction of the cytokines can induce cascade reaction of inflammatory mediators by autocrine and paracrine, which result in uncontrolled disturbance of organisms, systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS).Among infectious and non-infectious factors, which stimulate the host inflammatory cells to produce large quantity of cytokines and inflammatory mediators, endotoxin (lipololysaccharide, LPS), the main outer membrane component of Gram-negative bacteria, is the most potent and plays a key role during severe Gram-negative infection, sepsis and septic shock.In the pathological process of SIRS and MODS, the lung is a high sensitive target organ to pathogenic factors to be dysfunctional, and an important drive organ of MODS. The inflammatory cells are activated and accumulated in the lung to produce cytokines, which induce or facilitate local and systemic inflammatory response, mediate tissue damage, promote the MODS generation and development. Macrophages are the critical effector cells, which produce and release many kinds of inflammatory mediators and induce inflammatory response. Pulmonary macrophages consist of alveolar macrophages (AMs), pulmonary interstitial macrophages (PIMs), pulmonary intravascular macrophages, pleural macrophages, dendritic cell and so on. Among them, PIMs, to which are paid close attention recently, play a pivotal role in inducing inflammatory response. The same as AMs, PIMs also have the ability of phagocytosis and release various inflammatory mediators, especially <WP=14>have the strong ability to secret IL-1 and IL-6. It was reported that there was a significant increase in production of oxygen free radicals from PIMs, but not from AMs in the rats treated with LPS. The treatment also markedly enhanced phagocytosis only in PIMs and caused a significant increase in chemotaxis toward C5a in PIMs. These data demonstrate that PIMs play an important role in the inflammatory response of the lungs in endotoxemia. Cholecyetokinin (CCK), a typical braingut peptide, is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuromodulator to play a pivotal role. Recent years, a series of studies showed that CCK-8 had the effect of anti-inflammation. A lot of data demonstrate that CCK-8 causes an in vitro inhibition of LPS-induced NF-κB activity and mCD14 expression in rat PIMs. Consistently, the production of pro- inflammatory cytokines including TNF-α, IL-1β and IL-6 in endotoxin shock (ES) rat was also inhibited by CCK-8 in vivo. These results suggested that CCK-8 has anti-inflammatory effect to some extent, which was also confirmed by a morphological observation that CCK-8 clearly lessened the leukocytic infiltrate and capillary silt in lung, spleen, kidney and myocardium tissues in ES rat. Series studies show the fine prospects of the CCK-8’s anti-inflammation effects. However, the mechanisms of receptor and anti-inflammatory signal transduction activated by CCK-8 remain unclear. As we all know, CCK-8 exerts anti-inflammation effect through its receptors in target cell. More recently, some studies of our laboratory suggested the presence of CCK-AR and CCK-BR mRNA expression in rat spleen cells, lung and heart tissues, and the expression could be up-regulated by LPS obviously, indicating that CCK-8 might bind to CCK receptors and interfere with the over activation of immune cells induced by LPS to exert its anti-inflammatory effect during endotoximia. The expression subtypes and binding characteristics of CCK receptors in PIMs and the upstream signaling mechanisms of更多还原