【作者】 张馨木；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 急性胰腺炎(Acute pancretitis,AP)是一种炎症性疾病,基本病理改变为胰腺腺泡的急性损害和胰腺的急性炎症,炎症不仅涉及胰腺局部,而且会发生全身性炎症反应。急性胰腺炎发病急、病情凶险、病死率高,早期容易并发多脏器功能衰竭,尤其是急性肺损伤(ALI)。急性肺损伤是一种肺泡毛细血管膜弥散性损伤,导致肺水肿和肺不张,进一步发展即为急性呼吸窘迫综合征(ARDS)。ALI和ARDS是AP最常见和最严重的全身并发症之一,也是致死的主要原因。AP和AP并发ALI发病机制复杂。Elafin称弹性蛋白酶特异性抑制因子,是支气管Clara细胞、II型肺泡上皮细胞及肺泡巨噬细胞等在炎性刺激诱导下表达。Elafin为中性粒细胞弹性蛋白酶、胰弹性蛋白酶、蛋白酶3等丝氨酸蛋白酶特异性抑制剂。炎性反应是机体对损伤的一种保护性反应,其目的是将损伤源破坏、稀释并清除,与此同时也会引起不同程度的自身损伤。中性粒细胞弹性蛋白酶被认为是炎性反应的主要终效应因子,Elafin是内源性的弹性蛋白酶抑制剂,具有抗蛋白酶活性、抗炎活性和直接的抗微生物活性等重要作用。本研究首先利用牛磺胆酸钠逆行胆胰管注射法建立大鼠急性胰腺炎和胰腺炎合并肺损伤模型,之后给与重组人Elafin,通过急性胰腺炎大鼠血清生化指标检测,观察胰腺组织和肺组织病理学变化,考察重组人Elafin对大鼠急性胰腺炎及合并肺损伤的作用,对其作用机制进行探讨。本论文的创新之处在于:1)证实了Elafin对实验性大鼠急性胰腺炎的治疗作用,并对其作用机制进行探讨。2)证实了Elafin对实验性大鼠急性胰腺炎合并肺损伤的治疗作用,并对其作用机制进行探讨。国内外尚未见Elafin对大鼠急性胰腺炎和胰腺炎合并肺损伤作用的报道。更多还原

【Abstract】 Acute necrosis pancreatitis is one of the common severe clinical diseases with many complications, high mortality, the complicated causes and pathophysiological changes and the unclear mechanism. Leukocytes over-activity hypothesis consider that Cascade Effect induced by cytokines and the whole body leukocytes activation mediated by cytokines lead to the over-activity of Systemic Inflammatory Response Syndrome (SIRS),and this cause the damage of distant organs, the multiple organ dysfunction syndrome(MODS),and even the multiple organ system failure(MSOF).Acute lung injury (ALI) is one of the common complications. Most of the patients died combined with lung injury or died of severe lung injury. The mechanism of pancreatitis-associated lung injury is complicated. In recent years, most researches focus on the activation pathway of the inflammatory cells and mediators and consider that lung injury is the local manifestation of SIRS in lung, and the inflammatory injury is easy to occur in lung for its tissue specificity.Recombinant human elafin is an elastase inhibitor with the Mr of 6kDa, and its gene located on chromosome 20. Elafin is the specific inhibitor of neutrophil elastase (NE), pancreatic elastase and proteinase 3.Elafin inhibits the activity of serine proteases, and then plays its role in adjusting the release of inflammatory meditors, inhibiting the transendothelial migration of leukocytes and the activation of hemacytes, reducing the infiltration of inflammatory cells and the release of tissue toxic substances, etc. From this we can see that elafin display its multi-layered anti-inflammatory effect. Elafin has attracted clinic widespread attention by its protective effect through anti-inflammatory.This suggests that elafin inhibits the pancreatic exocrine and pancreatin activity, such as the activity of pancreatic elastase, thus prevent the pancreatic tissue from the direct damage and blood vessel injury of actived elatase. Moreover, elafin inhibits the release of inflammatory cells and mediators, thus exert its function on defending or curing the pancreatitis.This paper established the model of rat acute pancreatitis by using retrograde injection of Sodium Taurocholate into cholangiopancreatic duct, and then we observed the protective effects of elafin on rats acute pancreatitis by biochemical and pathological changes of rats after elafin treatment. In the last we will discuss the mechanism of pancreatitis.Test 1 the effects of recombinant human elafin on experimental acute pancreatitis in ratsRats were done with abdominal operation after anesthesia of 3% pentobarbital sodium, and then infused slowly 3.5% sodium taurocholate 0.1mg/100g in cholangiopancreatic duct t, whereas control group were infused the same amount physiological saline. Rats were randomly divided into six groups: the blank group (NS group), the model group (ANP+NS group) and the high-dose elafin group, the medium-dose group, the low-dose group, the positive control group, with 10 rats in each group. In addition of tosodium taurocholate, the high,medium,low-dose elafin groups received intravenous infusion of (5IU/kg、10IU /kg、20IU/kg)elafin, while the control and model group received the same amount of physiological saline, with the period of every 8 hours one infusion , continuously 6 times . 48 hours after the last injection, the blood samples from the abdominal aorta were obtained, then the serum NO, SOD,LPO, content were detected; After the pancreatic tissue homogenate, the tissue protein, amylase, lipase, SOD,LPO content were detected; pathological changes of rats pancreatic tissue were observed and its morphological structure were observed by electron microscope.Test 1 Results1 The changes of Amylase and Lipase of serum and pancreatic tissue: After treatment, serum and pancreatic tissue amylase and lipase in model group increased significantly(p<0.001), Compared with the model group,serum amylase and lipase of elafin(low dose, medium dose, high dose) groups decreased significantly (p<0.05, p<0.01 or p<0.001). The effect of high dose group is almost equal to aprotinin group.2 The changes of SOD and LPO of serum and pancreatic tissue and the serum NO:The serum and pancreatic tissue SOD in model group decreased significantly(p<0.001 ) . while the content of LPO and NO increased significantly(p<0.001). The serum and pancreatic tissue SOD in elafin(low dose, medium dose, high dose) groups increased significantly(p<0.001). while the content of LPO and NO decreased significantly(p<0.001). The effect of high dose group is almost equal to aprotinin group.3 Morphological Changes of the Pancreatic Tissue;In the model group, bloodyascites, necrotic foci in pancreas, and fat necrosis in mesentery and omentum was found grossly. Interstitial lobular inflammatory infiltrations were observed in pancreas microscopically, as well as diffusive bleeding and piecemeal necrosis. In elafin (low dose, medium dose, high dose) groups, ascites and fat necrosis diminished notably compared with those in the model group. Microscopically, slight bleeding, mild acinar degeneration and mild structure damage to lobules were observed together with declining inflammatory infiltration. The results show that the pathological changes significantly become light after elafin treatment.4 The expression of TNF-α、NF-κB in Pancreatic Tissue In the model group, there are many positive cells which can express TNF-α、NF-κB in the pancreatic tissue, while few exists in the elafin (low dose, medium dose, high dose) groups.Test 2 The effects of recombinant human elafin on experimental acute pancreatitis-associated lung injury in rats Rats were done with abdominal operation after anesthesia of 3% pentobarbital sodium, and then infused slowly 3.5% sodium taurocholate 0.1mg/100g in cholangiopancreatic duct t, whereas control group were infused the same amount physiological saline. Rats were randomly divided into six groups: the blank group (NS group), the model group (ANP+NS group) and the high-dose elafin group, the medium-dose group, the low-dose group, the positive control group, with 10 rats in each group. In addition of tosodium taurocholate, the high,medium,low-dose elafin groups received intravenous infusion of (2μg/kg、4μg /kg、8μg /kg)elafin, while the control and model group received the same amount of physiological saline, with the period of every 8 hours one infusion , continuously 6 times . 48 hours after the last injection, the lung W/D ratio was measured; the protein content of BALF was detected with Coomassie Breiliant Blue; after the lung tissue homogenate, the t TNF-α, MPO content were measured; pathological changes of rats lung tissue were observed and its morphological structure were observed by electron microscope.Test 2 Results1 The changes of lung W/D ratio and the protein of BALF Compared with the blank group, the lung W/D ratio and the protein of BALF in the model group increased significantly (p<0.001); Compared with the model group, the lung W/D ratio and the protein of BALF in the elafin (low dose, medium dose, high dose) groups decreased significantly(p<0.05, p<0.01 or p<0.001). The effect of high dose group is almost equal to aprotinin group.2 The changes of TNF-α, MPO content in the lung tissue Compared with the blank group, the lung TNF-α, MPO content in the model group increased significantly (p<0.001); Compared with the model group, the lung TNF-α, MPO content in the elafin (medium dose, high dose) groups increased significantly(p<0.05, p<0.01). The effect of high dose group is almost equal to aprotinin group.3 Morphological Changes of the Lung Tissue;The blank group: the clear lung structure and the slim alveolar wall were observed; and there was no polymorph nuclear neutrophils infiltration. The model group: there were many neutrophils infiltration in the interstitial lung, especially around the vessel and little bronchus. The pulmonary edema became serious; part of the alveolus space was seen to be filled with effusion and neutrophils; alveolar septum significantly became broad. Compared with the model group, alveolar septum of the elafin (low dose, medium dose, high dose) groups significantly became slim; the infiltration of neutrophils significantly became less.The above results show that elafin can significantly reduce serum amylase and lipase in AP group; inhibit the activation of pancreatic elastase and improve the hypersecretion in the pancreatitis. Elafin can reduce LPO of serum and pancreatic tissue rapidly and increase the SOD activity of serum and pancreatic tissue in acute pancreatitis, this suggests that elafin can improve its antioxidant activity by inhibiting lipid peroxide. Elafin can significantly inhibit the release of serum NO and prevent the interaction between inflammatory mediators and leukocytes, thus alleviate the development of acute pancreatitis. In the meantime, elafin can significantly reduce the lung W/D ratio and the protein of BALF and the lung TNF-α, MPO content in the pancreatitis-associated lung injury. According to the pathological observation, elafin can significantly reduce the edema, hemorrhage, and necrosis of pancreatic tissue, this suggest that it has the function of improving the organism repair capacity and protecting organs. Elafin can significantly reduce the infiltration of inflammatory cells in the lung and make the alveolar wall slim.In conclusion, elafin can prevent and cure the acute pancreatitis and the acute pancreatitis-associated lung injury.更多还原