【作者】 孙威；

【导师】 戴显伟；

【作者基本信息】 中国医科大学 ， 外科学， 2003， 博士

【摘要】 目的 急性坏死性胰腺炎(acute necrotizing pancreatitis，ANP)临床上常见，其疾病经过凶险，死亡率很高，有报道高达40％。但其发病机理至今仍不十分很清楚。20世纪50年代以来，应用激素治疗急性胰腺炎的利弊一直是学者们争论的内容。我们应用免疫组织化学LSAB法、Western印迹杂交和流式细胞术检测了选择素家族细胞粘附分子(P-选择素，P-selectin)、免疫球蛋白超家族细胞粘附分子(细胞间粘附分子-1，intercellular adhesion molecules-1，ICAM-1)及其配体整合素家族细胞粘附分子(LFA-1、Mac-1)在大鼠ANP合并多器官损伤模型中的表达，以探讨上述细胞粘附分子在ANP合并多器官损伤中的作用及其机制。并观察皮质醇对上述细胞粘附分子在ANP合并多器官损伤中表达的影响，探讨皮质醇在ANP合并多器官损伤中的作用及机制。 方法 1．动物模型及实验方法：SD大鼠雌雄不拘，体重250-300克。随机分成对照组、ANP组和皮质醇处理组，各25只。模型制作前12h禁食，自由饮水，吸入麻醉(氟烷、氧气、笑气混合吸入)，流量2L／min。皮质醇给药方式采用Osman等的方法，分别于右侧股静脉及皮下注射皮质醇剂量各5mg／Kg(溶于200uL注射用生理盐水中)，ANP组则分别注射同剂量的生理盐水。注射后即开始制作ANP模型，ANP模型采用谷俊朝等的方法。对照组仅行开腹手术，轻轻翻动胰腺和十二指肠后关腹。 2．主要试剂及仪器：磷酸氢化考的松(万有制药株式会社)；牛磺胆酸钠(和光纯药工业株式会社)；小鼠抗大鼠ICAM-1单克隆抗体和小鼠抗大鼠P-selectin单克隆抗体均购自Santa Cruz生物制品公司；FITC标记的小鼠抗大鼠LFA-1单克隆抗体和FITC标记的小鼠抗大鼠Mac-1单克隆抗体均购自美国BD PharMingen公司；LSABⅡ免疫组织化学试剂盒为Dako公司产品；Coulter Epics XL型流式细胞仪(美国)。 3．观察指标及方法：模型制作成功后分1h、3h、6h、12h及24h五个时相点，每个时相点5只动物，麻醉下剖腹抽取腹水后经下腔静脉取血Zd，IInl用2％EDTA抗凝，另外IM行血生化检查，测量腹水量，并取胰腺、肺和肾组织常规石蜡切片对E染色，观察病理改变。 4．免疫组织化学方法实验步骤（hAB法卜按照 DAKO公司 MAB 2System试剂说明，石蜡组织切片厚4 Urn。ICAM－1单克隆抗体稀释比例为1：800，P－S单克隆抗体稀释比例为人1000。 5·Western blot检测步骤：采用 Werner等的方法。 6．流式细胞仪检测步骤：LFA－l、Mao－1在血白细胞上的表达采用流式细胞仪检测。抗凝血经离心＼溶血素溶血后，加人单克隆抗体，4℃避光反应30ndn，流式细胞仪上样检测。启动Coulter流式细胞仪专用Elite分析软件，分别测定白细胞前向散射光及侧向散射光，并测定其平均荧光密度（mean fi见比scence i咖nsity，MFI）。 7．免疫组织化学结果判定：染色结果以均匀一致的浅黄色为阴性（一人以稍深的点线状着色为可疑（。X散在棕色点线状着色折旋光性较弱为弱阳性（十八棕色点线状着色、折光强、镜下多为阳性（＋＋＊广泛棕褐色短棒状着色、颗粒样着色、折旋光性强为强阳性（＋＋十八 8．数据处理：应用NIH Image．61勺Pc软件对免疫组织化学染色阳性表达区域及可疑阳性区进行量值化处理，每张切片选10个视野，测定阳性区密度分数。Weste。blot免疫反应条带分析采用 NIH Image．61巾pC软件测定密度分数。结果以均值上标准差＊。s）表示，应用 Microsof Excel软件行t检验。 结 果 1．胰腺病理改变及ICAM－l在胰腺中的表达：ANP组和皮质醇组两组胰腺周围均有皂化斑形成，胰腺瘀血呈灰褐色。镜下所见：胰腺呈片状坏死，红细胞外渗，有大量白细胞浸润，腺胞水肿呈岛状。ICAM－1阳性表达区多分布在微小血管内皮层、毛细血管内皮层，常伴有大量中性粒细胞浸润。在皮质醇治疗组中ICAM－l表达在模型制作后3h和6h均明显低于＊＊P组（P＜o．05）。 2．肺脏病理改变及 ICAM－l在肺脏中的表达：ANP组和皮质醇组两组肺瘀血明显，部分萎缩；镜下见：肺间质水肿，肺泡上皮细胞肿胀有些出现 ·2·坏死，并伴有白细胞浸润。皮质醇处理组肺组织每高倍视野炎症细胞浸润数从 3h至 12h均明显低于 ANP组讨＜0．05或 p＜0．01人皮质醇处理组肺组织病损分级从 3h至 12h均明显低于 ANP组h＜0．05或 p＜0．of人ICAM－1在肺组织阳性表达区多分布在微小血管内皮层、毛细血管内皮及肺泡内皮，常伴有大量中性粒细胞浸润。应用Western blot方法对肺脏ICAMJ表达进行定量测定，结果显示皮质醇组ICAM一表达在模型制作后 3h即明显低于 ANP组，并一直持续至 12hh＜0．05或 P＜0．01人 3．P＊electin在肺组织中表达：在对照组多为一或、在炎症组中多为十至十十十不等，且主要表达在微小血管内皮层、毛细血管内皮及肺泡内皮，且伴有大量炎性细胞浸润。P－selectin在ANP组肺组织表达从模型制作后 3 h开始明显高于对照组，并逐渐增强h＜0．05或 p更多还原

【Abstract】 ObjectiveAcute pancreatitis (AP) is a disease of variable severity. Approximately 80% of patients have a relatively mild attack that resolves with little of no complications, the mortality rate is low. However, the 20% of patients in whom pancreatic necrosis develops may incur systemic complications and a mortality rate as high as 40%. Recent investigations have established that one of the earliest pathophysiologic events in pancreatitis is the colocalization of acinar cell or-ganelles containing digestive and lysosomal enzymes, resulting in premature in-tracellular activation of proteases. The individual pancreatic cell injury becomes magnified and propagated by inducing (through incompletely defined mediators) impaired microcirculation leukocyte adhesion, and leukocyte infiltration. These become central events in the pathogenesis of pancreatic necrosis and its ex-trapancreatic complications. Organ dysfunction occurs in one in four patients with acute pancreatitis, and 60% who die from pulmonary damage.Intercellular adhesion molecule-1(ICAM-1) and P - selectin are expressed on endothelial cells and are responsive to numerous inflammatory mediators. Both of them mediate leukocyte adhesion and migration through the endo-thelium into tissues. LFA - 1 and Mac - 1 are the counter receptors of ICAM -1 , both of them are expressed on leukocyte and are concerned with mediating both leukocyte adhesion and migration through the endothelium into tissues, too.Corticosteroids have well - known immunosuppressive effects including inhibition of the release of various cytokines. The role corticosteroid in the treatment of acute pancreatitis has been a matter of dispute. Earlier reports claimed beneficial effects both in human and experimental AP. By contrast, experimen-tal studies in rabbits and rats, and a number of case reports, have demonstrated an association of pancreatitis with steroid administration, although the basis for steroid - induced pancreatitis still remains unproven.It is our hypothesis that corticosteroids inhibit not only cytokines but also adhesion molecules and thereby improve the physiological derangement observed during AP. Therefor, the aim of the present study was to investigate the patho-physiological role of adhesion molecules, namely P - selectin, ICAM -1 and its counter receptors ( LFA-1 and Mac-1) , on mediating the systemic manifestations associated with severe necrotizing pancreatitis through neutralization of their effects by a well - known immunosuppressive drug like hydrocortisone.MethodAnimals, Anesthesia and Surgical ProceduresSprague - Dawley rats (250 to 300g) were fasted 12 hours before the experiment but were allowed free access to water. Fluothane , oxygen and nitrons oxide were used in surgical anesthesia ( 2 L/min). All rats underwent a 2 - cm -long midline laparotomy, and the pancreatic duct was ligated followed by injection of 5% chenodeoxycholic bile acid (1 ml/kg, Wako Pure Chemical Industries, Ltd. , Osaka, Japan) into the pancreas at multiple points.Experimental designA total of 75 rats was allocated to Hydrocortisone group, AP group and control group ( respectively 25 rats). The rats of AP group were pretreated with a subcutaneous bolus injection of 200 ul of 0.9% saline 30 min before induction of AP followed by injection of 200 ul of 0.9% saline intravenously for about 5 min. The rats of Hydrocortisone group were pretreated with hydrocortisone by a subcutaneous bolus injection ( 5mg/kg) followed by injection of hydrocortisone ( 5mg/ kg diluted in 200 ul 0.9% saline) intravenously for about 5 min before induction of AP, as mentioned previously. The rats of control group only received the laparotomy. Blood samples(2ml) were drawn by v. cava inferior, pancreatic and lung tissues were removed and processed as indicated below before the rats were killed at lh,3h,6h,12h and 24h after induction of pancreatitis(n =5 per group). Ascitic Fluid was quantified by weighing towels after absorption of the ascitic fluid from the abdominal cavity更多还原