【作者】 曲金荣；

【导师】 冯子坛；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:胰腺和胆道的某些疾病常同时或先后发生于同一个体,这种“胰胆同病”现象越来越受到广泛的重视。目前许多动物实验和临床资料证实胰腺与胆系疾病之间存在着内在联系,这与胰管、胆管联合部形成共同通道开口于十二指肠壶腹部有关,当共同通道过长形成胰胆合流异常(pancreaticobiliary maljunction PBM)或其开口处因结石、肿物、瘢痕或壶腹括约肌痉挛而阻塞时,胰液、胆汁之间则会发生相互混流和反流。近年来对由于存在胰胆合流异常胰液反流所致的胆系慢性病变及胆道梗阻后致胰液、胆汁排泄不畅所引起的胆源性胰腺炎作了系统研究与报道。且有大量动物实验及临床研究对胆道梗阻时肝脏损伤的机制、病理生理改变作了比较系统详尽的论述,同时也证实了胆道梗阻时反映肝功能的多项血清学指标及肝脏病理形态有不同程度的变化。而对于因胰胆共同通道阻塞后胰液反流入胆道引起急性胆囊炎同时发生的急性肝损伤少见详尽报道。本实验采用向家兔胆管内注入胰酶的方式模拟制备胰胆共同通道阻塞致胰液反流时发生急性胆囊炎和肝损伤的动物模型,观察胰液反流入胆道引发急性胆囊炎同时血清丙氨酸转氨酶(ALT)、门冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)、r-谷氨酰转肽酶(GGT)、总胆红素(TBIL)等反映肝功能的各项指标,胆囊胆汁淀粉酶(AMY),血清、肝组织一氧化氮(NO)、一氧化氮合酶(NOS)及肝脏病理形态的变化,进一步研究胰液反流对急性肝损伤的影响以及NO在急性肝损伤中的作用,为研究发生胆道梗阻胰液反流时引起的急性肝损伤机制提供理论依据。方法:选择健康家兔,雌雄不限共45只,体重2.0-2.5kg/只(购自河北医科大学实验动物中心),随机分为3组,假手术组(SO),单纯胆道梗阻组(BDL),胆道梗阻并注入胰酶组(M),所有家兔手术前禁食过夜,自由饮水。3%戊巴比妥钠按35mg/kg耳缘静脉麻醉,无菌条件下取腹部正中切口(3cm)开腹,观察肝脏及胆囊大体形态,寻找胆总管。胆道梗阻并注入胰酶组仔细钝性剥离胆总管,结扎近肠端,以儿科静脉留置针穿刺成功后,见黄绿色胆汁流出,以1ml注射器抽出0.2ml胆汁,用微量枪抽取50U/ml的胰液0.2ml注入1ml的注射器中,以每分钟0.1ml的速度注入胆总管,观察无胆汁流出后,穿刺点近肝端结扎胆总管;单纯胆道梗阻组仔细钝性剥离胆总管并予以结扎;假手术组只仔细剥离胆总管。所有实验动物术后3天处死,观察肝脏、胆囊大体形态,留取胆囊胆汁2ml进行淀粉酶检测,并取血标本检测ALT、AST、ALP、GGT、TBIL、NO、NOS等指标,各组均取适量肝脏、胆囊组织,秤取肝脏组织100mg研磨成匀浆,取上清液于-20℃保存用于测NO、NOS,其余肝脏组织以及胆囊组织置于10%中性甲醛中以备制石蜡切片,行HE染色,光镜下(放大400倍)观察。所有数据均采用SPSS FOR WINDOWS 13.0软件包进行统计学处理,所得结果,计量资料均采用均数±标准差(X|-±S)表示,各组间均数比较采用单因素方差分析,P<0.05认为有统计学意义。结果:1肝脏、胆囊组织病理学变化1.1假手术组肉眼观肝脏红润无肿胀,被膜光滑;胆囊色泽稍暗,粘膜完整,与周围组织无粘连。光镜下观察肝脏形态结构正常,无炎细胞浸润,细胞无肿胀、变性和坏死;胆囊粘膜上皮完好,各层无充血、水肿及炎细胞浸润。1.2单纯胆道梗阻组肉眼观肝脏呈暗红色,明显肿胀,被膜光滑;胆囊色泽暗,体积增大,表面无溃疡、脓苔。光镜下观察肝脏组织变化表现为肝细胞浊肿、空泡变性、散在小灶性或片状坏死、炎细胞浸润;胆囊粘膜上皮轻度脱落,固有膜、肌层、浆膜充血、水肿,并有少量炎细胞浸润。1.3胆道梗阻并注入胰酶组肉眼观肝脏呈暗红色,明显肿胀,少数动物被膜可见散在的脓白苔;胆囊色泽晦暗,较梗阻组增大,胆囊底部有大小不一脓苔。光镜下观察肝脏组织表现为肝细胞浊肿、空泡变性、灶性或片状坏死、炎细胞浸润,少数动物肝脏组织空泡变性、坏死面积较大;胆囊粘膜上皮重度脱落,甚至消失,粘膜、固有膜、肌层、浆膜均可见高度水肿、充血,可见大量炎细胞浸润。2血清ALT、AST、ALP、TBIL、GGT的变化2.1 ALT胆道梗阻并注入胰酶组、单纯胆道梗阻组较假手术组血清ALT明显升高,各组间比较P<0.05,有统计学意义。2.2 AST胆道梗阻并注入胰酶组、单纯胆道梗阻组较假手术组血清AST明显升高,各组间比较P<0.05,有统计学意义。2.3 ALP、TBIL、GGT胆道梗阻并注入胰酶组与单纯胆道梗阻组血清ALP、TBIL、GGT均明显升高,以胆道梗阻并注入胰酶组升高较为明显,但两组间比较P值均大于0.05,差异无统计学意义;与假手术组比较有显著差异(p<0.05)。3血清、肝组织NO、NOS的变化胆道梗阻并注入胰酶组及单纯胆道梗阻组血清及肝组织NO、NOS均明显升高,两组间比较无统计学意义(p>0.05);与假手术组比较有显著性差异(p<0.05)。4胆囊胆汁淀粉酶的变化胆道梗阻并注入胰酶组及单纯胆道梗阻组与假手术组比较胆囊胆汁淀粉酶明显升高,有显著差异(p<0.05);单纯胆道梗阻组及假手术组之间比较,无统计学意义(p>0.05)。结论:1胆道梗阻并注入胰酶组血清ALT、AST较单纯胆道梗阻组升高,表明胰液反流可加重肝损伤。2胆道梗阻后引起急性胆囊炎的同时发生肝损伤。但胆道梗阻并注入胰酶组与单纯胆道梗阻组比较血清ALP、GGT、TBIL各指标变化无显著差异,说明在发病早期胆道梗阻、胆汁淤积可能是引起肝损伤的主要原因,尽早解除胆道梗阻是防治肝损伤的主要治疗手段。随着胰液反流时间的增加是否会使肝损伤明显加重尚待进一步研究。3胆道梗阻并注入胰酶组及单纯胆道梗阻组肝组织、血清中NO、NOS与假手术组比较明显升高,说明NO、NOS参与肝损伤过程,并起到重要作用。更多还原

【Abstract】 Objective: Some diseases about the pancreas and gallbladder often occur at the same person, The phenomenon of“the pancreas and gallbladder disease come on at the same time”has been taken more and more attention. Many experiments and clinical datum have approved that the pancreas and gallbladder diseases have some internal relationship. All of this attributed to pancreatcobiliary, when it was obstructed with small stone、foreign matter and the dysfunction of Oddi spincter that the pancreatic juice and bile could mutual reflux ,and the abnormal flux of pancreatic juice and bile could cause the injury of gallbladder and pancreas. Recently, many animal experiments and clinical researches have detailedly discussed the injury of hepatic when the bile duct obstructed, and it approved that the obstruction of bile duct causes the changes of hepatic function index and the changes of the liver tissue. However ,it is rarely reported on the hepatic injury caused by the pancreatic juice reflux to the bile duct after pancreatcobiliary duct has been obstructed .In our experiment, we methoded of injecting pancreatic juice to rabbit bile duct as model to simulate the pancreatic juice regurgitation , and studyed the changed of ALT、AST、ALP、GGT、TBIL in the serum ,and the NO、NOS in serum and the liver tissue , observed the change of the liver tissue pathologic structure , further to study the effect of pancreatic juice regurgitation on acute liver injury ,and the effect of NO in the process of hepatic injury. And provide the theoretical base for the liver injury when bile duct has been obstructed.Methods: 45 healthy rabbits weighing 2.0-2.5Kg were randomly divided into 3 groups:the sham operation group(SO group)、the simple bile duct ligated group(BDL group),and the group which the bile duct injected with pancreatic enzyme before it ligated (M group),in which male and female were equally and each group including 15 rabbits . All the rabbits were prohibited to eat but not drink before operation. After animal model successfully settled, all the animals were killed after three days, and determined the serum ALT、AST、ALP、GGT、TBIL , the bile AMY,NO、NOS of serum and the liver tissue ,got the liver tissue ,and observed the pathologic changes with light microscope after HE staining. The experimental data expressed as mean standard deviation. The statistic significance is analyzed by SPSS 13.0, a statistic software.Result:1 The change of the liver appearance: The liver of BDL group and the M group become swelling; The change of the liver of part of animals in M group are serious, and sparse pus on the surface could be seen. In the light microscope ,the liver tissue both of the BDL group and M group have the changes as dropsy、vacuole denaturalization and putrescence in small section, the white blood cell invaded. And a few animals in M group are severer than BDL group.2 The serum hepatic function index: the ALT、AST become higher in the M group and BDL group ,when compared with the SO group, have significant difference (p<0.05); Compared the M group with BDL group, have difference (p<0.05); While TBIL、GGT、ALP became higher in the M group and BDL group, when compared with the SO group, have difference (p<0.05) , compared the M group with BDL group, it have no obvious difference(p<0.05).3 The bile amylase: In the M group are obviously higher than the SO group and BDL group , have significant difference(p<0.05).Compared BDL group with SO group, have no difference (p>0.05).4 Compare the NO、NOS of the liver and the serum between the SO group and M group or the BDL group , have difference ( p<0.05); But compare the M group with BDL group, it have no obvious difference (p>0.05).Conclusions:1 After pancreaticobiliary duct has been obstructed, the pancreatic juice and bile could mutual reflux, the levels of ALT and AST in M group increased obviously ,Compared with BDL group have difference. Which approved that when pancreaticobiliary duct has been obstructed, pancreatic juice reflux to bile duct attribute a certain effect on the process of the liver injury.2 After pancreaticobiliary duct has been obstructed and pancreatic juice injected to bile duct caused acute or chronic cholecystitis, and lead to the liver injury. While compared with BDL group, the levels of ALP、GGT and TBIL in M group have no obvious difference . Approved the bile duct obstructed and cholestasis are the important reason of the hepatic injury. It’s the effectively method to resume the function of liver that releasing the obstructing of bile duct as early as possible.3 Compared with the sham operation group the level of NO and NOS in serum and liver tissue increased obviously both in M and BDL group . Which approve NO and NOS play an important role in the course of the liver injury.更多还原