【作者】 徐进；

【导师】 戴显伟；

【作者基本信息】 中国医科大学 ， 普通外科学， 2009， 博士

【摘要】 目的急性胰腺炎导致的肺脏水肿主要是表现为肺泡腔内的液体量增多。现代观点认为肺脏水肿主要是肺泡表面的水清除能力的发生障碍,当肺泡清除液体的能力下降,导致肺脏水肿的发生。在低血容量休克,感染性休克等外科病理过程中验证了这一规律。因此本研究大胆提出假设:在急性重症胰腺炎中肺脏存在肺泡液体清除能力的异常改变。为了验证这个假设,通过对急性重症胰腺炎（SAP）Sprague-Dawley大鼠模型的肺脏进行观察研究,以期发现SAP中的肺脏液体清除能力（AFC）的变化规律,及与肺泡细胞表面Na-K-ATP酶和Na离子通道（ENaC）相关性。解决以下问题:1.急性重症胰腺炎大鼠的肺脏是否发生肺脏水清除能力（AFC）的改变?2.AFC的改变与哪种肺泡表面的肾上腺能受体相关?AFC受哪些因素调节?3.SAP肺脏中ENaC和Na-K-ATP酶的数目是否发生变化?材料和方法一、诱发急性重症胰腺炎（SAP）的SD大鼠模型选取健康的SD大鼠,随机分为造模组（SAP组）,假手术组（sham组）和对照组（control组）。造模组大鼠麻醉后开腹经十二指肠壁行胰胆管插管后,用5%牛磺胆酸0.2ml向胰胆管内逆行注射的方法制作急性重症胰腺炎（SAP）大鼠模型;假手术组大鼠仅开腹翻动十二指肠;另建立对照组无任何处置。观察SAP术后4小时,24小时,48小时的大鼠症状,胰腺形态和腹腔情况,血清中淀粉酶和脂肪酶水平的测定,胰腺和肺脏的病理改变,绘制SAP时间-生存曲线。二、观察SAP肺脏水清除能力（AFC）在急性重症胰腺炎中的变化将手术组（SAP组）大鼠,对照组和假手术组麻醉后,气管切开法插管,再分别放血法处死后,将双侧肺脏取出注入已经预热（37℃）的5%的蛋白溶液4ml。肺脏立即放入37℃水浴箱内,通100%纯氧1小时,全部吸出肺脏中的蛋白溶液,分别测定通气前和通气后的蛋白溶液浓度,带入公式计算出不同时间段SAP组,对照组和假手术组中肺泡主动清除液体能力（AFC）值。三、观察SAP肺泡肾上腺素受体对肺脏水清除能力的影响在AFC升高最明显的4h时间段,分别在注入肺脏内蛋白溶液中预先加入prazosin（α₁肾上腺受体阻滞剂）、yohimbine（α₂肾上腺受体阻滞剂）、prazosin+yohimbine、propranolol（β肾上腺受体阻滞剂）,然后再将肺脏放入37℃水浴箱内,通100%纯氧1小时,全部吸出肺脏中的蛋白溶液,分别测定通气前和通气后的蛋白溶液浓度,带入公式计算处各组的肺泡主动清除液体能力（AFC）值。观察不同的肺泡表面的肾上腺受体与升高的肺泡主动液体清除能力的关系。四、观察SAP内源性儿茶酚胺对肺脏水清除能力的的影响先行手术将大鼠双侧肾上腺切除,后立即进行牛黄胆酸的逆行性注射,制造出SAP。测定4小时后的AFC的变化情况。五观察SAP肺脏内Na-K-ATP酶和ENaC的变化用定量real-time PCR的方法测定在SAP4h组中肺脏的不同亚型的Na-K-ATP酶和ENaC mRNA的变化规律。统计学方法:采用SPSS13.0版本软件对资料进行统计学分析。测得结果用（?）±s表示,采用区组方差分析、t检验统计方法。P＜0.05有统计学意义。结果一向大鼠的胰胆管内逆行注入牛磺胆酸可以诱发急性重症胰腺炎,症状明显,腹腔内产生大量血性腹水,皂化斑大量形成。血清中淀粉酶和脂肪酶明显升高,脂肪酶升高更为明显比对照组高30倍以上,淀粉酶比对照组升高3倍以上。病理切片中胰腺有间质水肿和白细胞的浸润,肺泡水肿和白细胞的浸润。时间-生存曲线中SAP大鼠的生存率随时间明显下降,本组72小时内全部死亡。SAP动物模拟成功。二在SAP大鼠的动物模型中,AFC测量值明显高于对照组和假手术组的。4小时AFC升值最高,是对照组的180%（p＜0.001）。之后逐渐下降。24小时为对照组的120%（p＜0.05）。48小时接近对照组水平。对照组和假手术组两组AFC值无明显差别（p＞0.05）。三在SAP-4h大鼠组中,升高的AFC值被不同亚型的肾上腺素受体阻滞剂不同的程度的抑制。其中表现最为明显的为α₁+α₂肾上腺受体抑制剂,几乎完全抑制了AFC的升高。β肾上腺受体抑制剂仅轻度抑制AFC。四切除双侧肾上腺的大鼠即使发生SAP4小时后,AFC测定值对比SAP4h组仅轻度升高。五在SAP大鼠的肺脏4小时组中,发现α,γ-ENaC mRNA升高明显,分别升高了3倍和2倍,而Na-K-ATP酶的仅α亚体升高3倍以上,β亚体几乎不升高。结论在急性重症胰腺炎的大鼠模型中,肺脏出现了一过性的肺泡液体清除能力的升高。这种升高在早期达到高峰（4小时内）,之后随着时间而逐渐减弱,导48小时后逐渐接近正常。这种升高的调节途径主要是通过机体的肾上腺产生内源性的肾上腺素通过血液循环激动肺泡表面的α₁,α₂肾上腺受体,增加肺泡细胞表面的α,γ-ENaC和底部的αNa-K-ATP酶的数目,增加Na离子的主动转运来实现的肺泡腔内液体的转运。本实验结果不同以往的其他的动物病理模型的研究结果,说明在急性重症胰腺炎中存在着新的肺泡液体调节途径。为治疗SAP导致的肺脏水肿提供了新的理论依据和新的治疗思路。更多还原

【Abstract】 ObjectiveAcute pancreatitis causes pulmonary oedema with the accumulation of fluid in the alveolar spaces,possibly due to reduced clearance.This study tested the hypothesis that acute pancreatitis decreases alveolar fluid clearance in a rat model of pulmonary oedema during acute pancreatitis.To investigate the hypothesis,the observation was carried out on the lung of the SAP rat model.Its goal was to find that how to change of alveolar fluid clearence,epithelial sodium channel and Na-K-ATPase.To answer that:1) How to AFC change in SAP?2)Which subtype adrenal receptor on the lung was related with the changed AFC?3)Which subtype epithelial sodium channel and Na-K-ATPase were changed in SAP?Methods1 How to induce SAP rat model.Acute pancreatitis was induced by a retrograde injection of 5%taurocholate sodium（0.2 mL）into the common bile duct.The sham group was only turn over duodenum and pancreas in the operation.The control group was normal without interference.The figure of pancreas and abdomen was recorded,the measurement of amylase and lipase was observed and the pathology of pancreas and lung was observed. The life-time figure was painting.2 to observe the change of alveolar fluid clearance in SAP.The lungs were isolated 4,24 and 48 h after the induction of acute pancreatitis and alveolar fluid clearance was measured in the absence of pulmonary perfusion.The lung was bathed in wet at 37℃and ventilated 100%oxygen for 1h.3 to observe how to be changed the adrenal receptor in alveolar fluid clearance at SAP-4h.Added the prazosin（α₁ adrenal receptor antagonist）,yohimbine（α₂ adrenal receptor antagonist）,prazosin+yohimbine,propranolol（βadrenal receptor antagonist） into the bovine solution and then measured the AFC at SAP-4h.4 to observe whether the endogenous catecholamine affect the alveolar fluid clearance or not?Before making the rat SAP model,we resected the bilateral adrenal grand.The rats were measure AFC5to measure ENaC and Na-K-ATPase mRNA at SAP-4h group.By quantitative real-time PCR we measured ENaC and Na-K-ATPase mRNA of lung in SAP-4h rat.ResultsAlveolar fluid clearance increased to 31.0±3.5%of instilled volume/h in rats with acute pancreatitis for 4 h compared with 17.3±1.0%of instilled volume/h in sham rats （P＜0.01）,then returned to the control level 48 h after acute pancreatitis（16.0±4.1% of instilled volume/h）.The plasma epinephrine levels increased to 25-fold higher in rats with acute pancreatitis for 4 h than in Sham rat group without acute pancreatitis. Prazosin(anα1-adrenergic antagonist,10^-4 mol/L),yohimbine(anα₂-adrenergic antagonist,10^-4 mol/L)or a bilateral adrenalectomy inhibited the increase in part,a combination of prazosin(10^-4 mol/L)and yohimbine(10^-4 mol/L)completely inhibited the increase in alveolar fluid clearance in rats after acute pancreatitis for 4 h,whereas propranolol(aβ-adrenergic antagonist,10^-4 mol/L)had no effect,α,γ-ENaC mRNA increase 3 times and 2 times,separately,but not Psubtype.αNa-K-ATPase mRNA increased 3 times than the normal.ConclusionEndogenous catecholamine stimulatedα-adrenoceptors and increased alveolar fluid clearance in rats with acute pancreatitis.α,γ-ENaC andαNa-K-ATPase mRNA increased.更多还原