【作者】 段国辰；

【导师】 凌亦凌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2003， 博士

【摘要】 胆囊收缩素(cholecystokinin，CCK)是一种脑—肠肽, 参与了多种生理及病理生理过程。已知CCK的作用主要由两种受体介导，即CCK-A受体（CCK-AR）和CCK-B受体(CCK-BR)。硫酸化八肽CCK（CCK-8）是具有CCK完全生物学功能的最小活性片段。以往对CCK的研究多偏重于其在消化系统、神经系统及内分泌功能活动中的调节作用。上世纪80年代末Riepl 和Guarini等相继报道CCK有改善失血性休克的作用,90年代初Holmgren报道CCK-8引起肺血流量增加。1996年我室首次发现CCK-8有明显抗内毒素性休克(ES)作用:可保护细胞、减轻脏器病理损伤，降低死亡率；ES时肺对CCK的清除作用降低，血液中CCK含量增加。CCK-8抗ES作用主要表现在改善ES时肺循环、体循环紊乱，即逆转ES时平均动脉压（MAP）的下降及ES早期的肺动脉压（PAP）增高，但其机制尚未完全阐明。脂多糖（LPS）是革兰氏阴性细菌产生的内毒素的主要成分，静脉注射LPS,可引起ES，而各种休克晚期又多合并肠源性内毒素血症。本研究在我室以往系列研究的基础上，进一步同时观察CCK-8对ES时家兔主动脉、肺动脉反应性变化的调节作用,并对其机制进行探讨，拟从以下几方面进行研究：⑴八肽胆囊收缩素改善家兔内毒素休克时血液动力学紊乱的实验研究；⑵CCK-8对ES家兔离体动脉反应性的调节；⑶ES时主动脉和肺动脉组织及LPS诱导的主动脉平滑肌细胞(ASMC)和肺动脉平滑肌细胞(PASMC)CCK受体基因表达的变化；⑷CCK-8抑制LPS诱导的ASMC iNOS和HO-1 mRNA的表达及其意义初探。1CCK-8对ES时MAP和PAP变化的调节作用本实验通过静脉注入LPS复制ES模型，同步观察了CCK-8<WP=6>对家兔ES时主动脉压、肺动脉压变化的不同调节作用。雄性家兔40只（2~2.1㎏）随机分为5组（每组8只）：（1）对照组：静脉注入生理盐水（0.8 ml/kg）；（2）LPS组：经静脉导管缓慢（10min内）注射等容量的LPS（8mg· 0.8ml-1·㎏-1）；（3）CCK-8+LPS组：静脉注入CCK-8(15μg·0.8ml-1·㎏-1)后30min再注入LPS；（4）丙谷胺（proglumide，Pro）+LPS组：静脉注入Pro(CCK受体阻断剂，1mg·0.8ml-1·㎏-1)后30min再注入LPS；（5）CCK-8组：注入同剂量的CCK-8。耳缘静脉注射25%乌拉坦（1g/㎏），麻醉动物，手术分离左侧颈总动脉和右侧颈静脉,血液肝素化（1250u/㎏ iv）后，分别插入聚乙烯导管至升主动脉弓处和经右心室至肺动脉入口处，导管经压力传感器与八导生理记录仪相连，用于监测给药后5h平均动脉压（MAP）、肺动脉压（PAP）的动态变化。分别取给药前（0h）及给药后1h、3h和5h的动脉血，测定血浆中一氧化氮（NO）、丙二醛（MDA）含量，一氧化氮合酶（NOS）和超氧化物歧化酶（SOD）活性。整体给药5h后放血处死家兔，迅速摘取主动脉和肺动脉，用4℃生理盐水冲洗，一部分动脉组织用10%中性福尔马林固定，制备石蜡切片，HE染色后，光镜下观察。另一部分动脉组织经2.5%或4%戊二醛固定后，制备扫描和透射电镜标本，观察血管内皮细胞的超微结构变化。结果：①LPS入血后，MAP呈进行性持续降低，0.5h MAP明显下降，2h接近最低水平，一直维持到5h，均明显低于同时间点的对照组MAP值（均P <0.01）；而LPS却使PAP呈进行性持续升高，0.5h达高峰，然后一直维持在较高水平（P﹤0.05或0.01）。②CCK-8+LPS组MAP在1.5h、2h、3h、4h和5h均显著高于同时间点的LPS组MAP（P<0.05, P<0.01）,而相应时间点的PAP则较LPS组明显降低（P<0.05）。③预注入Pro，导致LPS引起的MAP降低程度更加明显（P<0.01），MAP显著降低提前至0.5h；Pro使LPS引起的PAP进一步升高（P<0.05）。④单纯注入CCK，对正常家兔MAP、PAP无明显影响，与对照组相比无明显差异。⑤NOS和SOD活性，NO、MDA含量各组有较大变化。静脉注入LPS后1h、3h和5h，血液NOS<WP=7>活性、NO和MDA含量均显著增高，SOD活性均显著降低，而此时MAP明显降低，PAP升高；预先静脉注入CCK-8，可明显降低LPS所致家兔血液中NOS活性、NO和MDA含量的升高，提高抗氧化酶SOD的活性，而此时的MAP、PAP变化显著轻于LPS组。⑥动脉组织（主动脉、肺动脉组织）光镜检查，发现LPS组内皮细胞受损，部分内皮细胞脱落；CCK-8+LPS组血管壁各层的形态学改变明显减轻；而Pro+LPS组血管的形态学改变明显重于LPS组。扫描电镜下见LPS组内皮细胞形态不规则，大小不一，有些内皮细胞脱落缺失，内皮细胞下胶原暴露；CCK-8+LPS组内皮形态接近对照组，排列较整齐，细胞间隙稍增宽，未见细胞明显缺失。透射电镜下见正常血管内皮细胞器完整，形态轮廓清晰。LPS组、Pro+LPS组血管内皮细胞器水肿，胞浆空泡较多，线粒体肿胀、嵴减少或消失，部分线粒体膜不完整。CCK-8+LPS组血管内皮病变减轻,胞浆空泡减少，线粒体结构较完整。本实验初步探讨了ES时CCK-8对主动脉压、肺动脉压变化的调节作用，结果表明LPS可诱导家兔ES的发生，表现为MAP降低，PAP增高及主动脉、肺动脉血管内皮细胞的损伤，CCK-8对此具有调节作用，既能改善ES时家兔肺循环、体循环紊乱，又能减轻血管内皮细胞结构损伤，ES时循环紊乱以及CCK-8的保护作用机制可能与NO有关。但在动物整体实验中，影响血压的因素众多，其中血管反应性改变是ES时循环紊乱的主要因?更多还原

【Abstract】 It has been well documented that cholecystokinin (CCK), a kind of brain-gut peptide, is involved in many physiological and pathophysiological processes. The actions of CCK are mainly mediated by two distinct receptors, referred to as CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR). Sulfated cholecystokinin-octapeptide (CCK-8) is the minimum sequence for biological activity. The actions of CCK have been studied widely in nervous system, digestive system, and endocrine system in the past years. Effect of CCK-8 on reversing hemorrhagic shock in rats was reported by Riepl and Guarini respectively in the late of 1980s. In the early of 1990s, Holmgren reported that CCK-8 increased pulmonary blood flow. In 1996，it was found that CCK-8 had the effect of anti-endotoxic shock (ES) for the first time in our laboratory, CCK could protect cells function, attenuate the pathomorphological changes of main organs , decrease the mortality . During ES, the clearance rate of CCK-8 was decreased in lung and the CCK-8 content in blood was increased. The main action of CCK-8 was to improve the circulation state of ES, CCK-8 could partly reverse the fall of mean artery pressure (MAP) and the rise of pulmonary artery pressure (PAP), but the mechanism was not clear. Lipopolysaccharide (LPS), a main component of bacterial endotoxin, can induce ES. Intestinal bacterial endotoxemia occur frequently in the late stages of all kinds of shock. The purposes of the present study were to investigate the effects of CCK-8 on aortic hypo-reactivity and pulmonary arterial hyper-reactivity, and its<WP=15>mechanisms in ES rabbits or LPS-treated aortic smooth muscle cell (ASMC) and pulmonary artery smooth muscle cell (PASMC) of rats. Our studies were as following: (1) Regulatory effects of CCK-8 on changes in MAP and PAP in ES rabbits; (2) Effects of CCK-8 on changes in vascular (thoracic aorta, pulmonary artery) reactivity; (3) LPS-induced expression of CCK-R mRNA in vascular tissues, ASMC and PASMC; (4) CCK-8 inhibiting the expression of iNOS mRNA and HO-1 mRNA in ASMC induced by LPS.1 Regulatory effects of CCK-8 on changes in MAP and PAP in ES rabbitsThe aim of this study was to simultaneously observe the effects of CCK-8 on changes in MAP, PAP in ES rabbits. Forty male rabbits were divided into five groups randomly (n=8 in each group):⑴ Control group, saline(0.8ml/kg, iv) was injected ⑵ LPS group, LPS (8mg·0.8ml-1·㎏-1, iv) was injected slowly within 10 min. ⑶ CCK-8+LPS group, CCK-8 (15μg·0.8ml-1·㎏-1, iv) was injected 30 min before LPS administration.⑷ Proglumide (Pro) +LPS group, Pro (an antagonist for CCK recptor, 1mg·0.8ml-1·㎏-1, iv) was injected 30 min before LPS administration.⑸CCK-8 group, CCK-8(15μg·0.8ml-1·㎏-1)was injected. The rabbits were anesthetized with 25% urethane（iv）. The left common carotid artery was exposed, and a catheter was inserted and advanced into thoracic aorta. Another catheter was inserted into pulmonary artery through the right carotid vein. The changes in MAP and PAP were measured by physiological record instrument in these five groups for 5 hours. The contents of nitric oxide (NO) and malondialdehyde（MDA）, the activity of nitric oxide synthase (NOS) and superoxide dismutase（SOD）in plasma were measured before drug delivery(0h) and 1h, 3h or 5h after drug delivery, respectively. The rabbits were killed after 5h.The thoracic aorta and pulmonary artery were excised. A part of arterial sample was fixed with<WP=16>paraformaldehyde and chopped into slices, then stained by HE. The pathomorphological changes were observed under light microscope. The other part of the sample was fixed with 2.5% or 4% glutaral to observe the cellular ultrastructure changes of vascular endothelium by electron microscope. The experimental results were as follows：①The MAP began to reduce at 0.5h after LPS injection, reached to the lowest point at 2h, and continued to 5h. The MAP significantly reduced, compared with control group (P<0.01) in the whole experimental course. Whereas the PAP increased significan更多还原