【作者】 黎冬暄；

【导师】 田伏洲；

【作者基本信息】 第三军医大学 ， 外科学， 2004， 博士

【摘要】 目的：以急性胰腺炎（AP）的肺损伤作为研究对象，分别通过动物实验和原代细胞培养对肝枯否细胞在其中的作用进行观察，以期对AP病程中胰外器官损伤发生率高、病死率高的原因，及其与全身炎症反应综合征（SIRS）之间的相互关系进行初步探讨，为临床最终达到提高AP的疗效、减少SAP的发生率、降低其病死率提供理论和实验依据。 方法：1、实验（1）将健康雄性Wistar大鼠随机分为3组：A组向胰管内逆行注入5％牛磺胆酸钠造成急性坏死性胰腺炎（ANP组）；B组预先以氯化钆抑制枯否细胞后再造成急性坏死性胰腺炎（Kupffer细胞抑制组）；C组提前以氯化钆抑制枯否细胞，然后向胰管内逆行注入生理盐水作为对照（对照组）。检测指标：血清淀粉酶、胰腺形态学改变、血清TNF-α及IL-1β、肺形态学改变、肺湿重／干重比值（W／D）和动脉血氧分压（PaO₂）的变化情况。2、对大鼠的肝细胞、枯否细胞及肺微血管内皮细胞进行分离、培养及鉴定，以获得纯度高、活力强的细胞，满足实验的要求。3、实验（2）将分离获得的枯否细胞（KC）分为4组：A组培养基上清中加入生理盐水作为对照（对照组）；B组上清中加入脂多糖（LPS组）；C组上清中加入胰弹性蛋白酶（elastase组）；D组上清中同时加入脂多糖和胰弹性蛋白酶（LPS+elastase组）。检测指标：RT-PCR检测KC的TNF-α、IL-1β及TLR4mRNA表达水平、ELISA法测定培养上清的TNF-α、IL-1β蛋白含量、Westem blot测定KC的TLR4蛋白表达情况。4、实验（3）将培养的肺微血管内皮细胞（PMVEC）分为3组：A组加入被LPS和胰弹性蛋白酶干预的KC上清进行培养（KC组）：B组加入被LPS和胰弹性蛋白酶干预的肝细胞上清进行培养（肝细胞对照组）；C组加入未被LPS和胰弹性蛋白酶干预的KC上清进行培养（对照组）。检测指标：PMVEC的镜下形态学改变、细胞内F-肌动蛋白的改变、细胞内[Ca²⁺]ⅰ的变化。 结果：1、实验（1）：A组（ANP组）与B组（Kupffer细胞抑制组）大鼠的血清淀粉酶水平无显著差异，但显著高于C组：A组和B组大鼠胰腺形态学变化符合急性出血坏死性胰腺炎改变，但C组未见明显的急性胰腺炎改变。与胰腺的病理改变不一致的是：A组的血清TNF-α和IL-1β高于B组：A组肺组织病理改变严重，病理形态学积分显著高于B组（P＜0.01）；A组的W／D值显著高于B组（P＜0.01）；A组的PaO₂第三军医大学博士学位论文值显著低于B组（P<0.01）。结果表明，用氯化礼对枯否细胞功能进行抑制后，虽然胰腺炎的程度无显著改变，但血清炎症介质水平显著下降、肺损伤的程度显著减轻。2、分离、培养获得的肝细胞形态典型、贴壁生长良好、分裂增殖活跃;KC形态较典型，有较强的吞噬活性，吞噬实验证实分离所得细胞为KC;PMVEC细胞形态典型、传至4一5代细胞仍生长良好，与FITC标记的植物血凝素B4结合呈黄绿色荧光，符合PMVEC特征。3、实验（2）的Rl飞PCR产物电泳结果显示，LPS（B组）或胰弹性蛋白酶（C组）均可造成TNF一a、工L一1 p mRNA的表达显著增高，当两者同时（D组）刺激KC时，TNF一a、工L一1 p mRNA的表达增高更加显著，并显著高于B组和C组;但两者同时（D组）刺激KC时，TLR4 mRNA的表达增高不显著，与B组无显著差异。EL工SA结果显示，D组大鼠KC培养上清的TNF一a、工L一lp蛋白显著高于B组和C组。W亡stern blot结果显示，B组、D组杂交带蛋白含量较A组、C组显著增高（尸<0.01）;但B组与D组杂交带的蛋白含量比较无显著差异（尸>0.05）。4、实验（3）结果显示，被LPS和胰弹性蛋白酶干预的KC上清（A组），使PMVEC产生显著的损伤表现;细胞F一actin发生解聚、密度减低;细胞内[C扩+]i显著升高。而被LPs和胰弹性蛋白酶干预的肝细胞上清（B组），使PMVEC产生的相应改变则不显著。 结论:1、AP的病程中，KC在炎症放大、胰外器官的损害中起着重要作用。2.AP的病情严重程度、胰外器官的损害程度等与胰腺本身的病变程度并不完全一致，而与全身炎症反应的程度相一致。3.抑制KC的功能虽不能减轻胰腺的病变，但可以减轻急性胰腺炎的病情。4.KC受到胰弹性蛋白酶等胰酶及LPS的刺激后对炎症因子的大量表达，可能是AP发生S工RS和高MODS发生率、高病死率的原因。5.胰弹性蛋白酶等胰酶刺激KC后，州F一a、IL一lp等炎症因子的表达显著增加，这表明AP病程中炎症反应较其它疾病更易被异常放大的原因可能与胰弹性蛋白酶等胰酶的存在有关。6.KC受到胰弹性蛋白酶刺激后，对LPS跨膜信号转导受体的表达不产生影响，表明胰弹性蛋白酶在引起炎症反应异常放大时，并不增加KC对LPS刺激的反应性;胰弹性蛋白酶通过LPS信号转导通路以外的其它途径引发炎症反应的异常放大。7.KC受到刺激后产生的有害物质，对肺PMVEC有显著损害作用，在细胞水平提示肺损伤与KC的激活有关。更多还原

【Abstract】 Objective: In order to evaluate the role of hepatic Kupffer cell (KC) on the high incidence rate of organ injuries and high mortality rate, and the mutual relations between hepatic Kupffer cell to the degree of SIRS in acute pancreatitis course, animal experiments and primary cell cultures were used respectively to observe the effects of hepatic Kupffer cell on acute lung injury during acute pancreatitis. It is hoped that this study can devote something to improve the treatment effect of acute pancreatitis, reduce the incidence rate of severe acute pancreatitis (SAP) and decrease the death rate in clinic.Methods: 1. In experiment 1, the health male Wistar rats were randomly divided into 3 groups. The animals in group A were retrogradely injected 5% sodium taurocholate into the pancreatic ducts to establish acute necrosis pancreatitis models (ANP group). In group B, the ANP models were established after Kupffer cells were inhibited previously by intravenous gadolinium chloride (inhibited KC group). The rats in group C were given gadolinium previously to inhibit Kupffer cells, and then physiological saline was injected retrogradely into the pancreatic ducts (control group). The observation items were: levels of serum amylase, pancreatic morphological changes, concentrations of serum TNF-a and IL-1 P , pulmonary morphological changes, wet/dry weight ratio of lung tissue (W/D ratio) and changes of partial pressure of oxygen in arterial blood (PaCh). 2. To fit in with the requirement of experiments, we isolated, cultured and identifyed hepatocytes, Kupffer cells and pulmonary microvascular endothelial cells (PMVEC) for getting cells with high purity and activity. 3. In experiment 2, Kupffer cells were divided into 4 groups. In group A, physiological saline was added into the supernatant fluid of media as control (control group). Lipopolysacchride (LPS) was used in group B (LPS group). In group C, pancreatic elastase was added (elastase group). And group D was treated with both lipopolysacchride and pancreatic elastase (LPS+elastase group). The expression of TNF- a , IL-1 3 and TLR4mRNA in Kupffer cells were determined by RT-PCR, concentrations of TNF- a and IL-1 P in the supernatant fluid of media by ELISA. And Western blot was used to observe the expression changes of TLR4 protein in Kupffer cells. 4. In experiment 3, the cultured PMVECs weredivided into 3 groups. The supernatant fluid of cultured Kupffer cells pretreated with LPS and pancreatic elastase was added into group A (KC group). The supernatant fluid of cultured hepatocytes pretreated with LPS and pancreatic elastase was added into group B(hepatocyte control group). The supernatant fluid of cultured Kupffer cells without LPS and pancreatic elastase pretreated was added into group C (control group). The morphological changes of PMVECs under microscope were observed, the changes of F-actin and the concentrations of [Ca2+]i in PMVECs were determined.Results: 1. In experiment 1, no significant differences were observed in serum amylase between the animals of group A (ANP group) and B (inhibited KC group), however, the levels of serum amylase in group A and B were significantly higher than that in group C (control group). The morphological changes of pancreas in group A and B fitted in with the changes of ANP, while no relative changes of pancreatitis were observed in group C. Noteworthily, the concentrations of serum TNF-a and IL-lp in group A were higher than that in group B, the pathological changes were severer and the score of pathological changes of lung tissue in group A were higher significantly than that in group B (PO.01). The W/D ratio in group A was higher than that in group B (P<0.01), accordingly, the PaC>2 level in group A was lower than that in group B (P<0.01). Those results indicated that the inactivation of Kupffer cell did not change the severity of pancreatitis, but reduced significantly the level of serum inflammatory factors and alleviated remarkably the lung injuries. 2. The cultured hepatocytes had typical shape, well viability and a更多还原