缺血再灌注肝一氧化氮合酶活性变化的观察

Study on the changes of nitric oxide synthase in ischemia-reperfusion rat liver

目的探讨大鼠缺血再灌注肝一氧化氮合酶的变化及其意义。方法健康雄性SD大鼠24只,门静脉插管、肝静脉插管、胆道插管,切取肝,建立大鼠离体肝灌注(IPRL)模型。大鼠随机分为4组(每组6只):①热缺血对照组,肝切取后,不作冷保存,立即进行灌注;②冷保存6 h组,肝切取后作冷保存,保存6 h后进行灌注;③冷保存12 h组,肝切取后作冷保存,保存12 h后进行灌注;④冷保存24 h组,肝切取后作冷保存,保存24 h后进行灌注。IPRL采用恒温灌注,经门静脉插管灌注pH 7.4、38℃的Krebs-Blübring buffer液30 min。观察胆汁分泌,检测肝静脉流出液中白蛋白(ALB)含量,测定肝组织内皮素-1(ET-1)、一氧化氮(NO)、一氧化氮合酶(NOS)、ATP酶(ATPase)、超氧化物歧化酶(SOD)、黄嘌呤氧化酶(XOD)、丙二醛(MDA)等指标。结果大鼠肝NOS发生了明显的变化,但肝组织NO、ET-1没有显著变化,ATPase、SOD、XOD、MDA的代偿也是有限的。再灌注过程中,各组大鼠肝均有胆汁分泌。肝静脉引流液中ALB含量无差异。结论NOS的变化是反映缺血再灌注损伤的敏感指标。

Objective To study the level of nitric oxide gynthase（NOS） in ischemia - reperfusion rat liver. Methods 24 healthy male SD rats were anaesthetized and heparinised. The hepatic vein, portal vein and bile duct were cannulated. The liver was then removed to establish a model of isolated porfusion rat liver （IPRL）. The rats were randomly divided into 4 groups （ n = 6 per group）. In Group Ⅰ （warm I/R group）, the IPRL was formed immediately after removal.The livers of Groups Ⅱ to Ⅳ were first stored in cold UW solution for 6, 12 and 24 hours, respectively . The livers were then porfused via the portal vein, with freshly prepared Krebs- Bulbring buffer for 30mins. The temporature of organ bath and porfusate was controlled at 38℃. Bile secretion was noted; albumin （ALB） was determined. Histochemistry was performed to measure the activities of hepatic endothelin - 1 （ET - 1 ）, nitric oxide （ NO ）, nitric oxide synthase （NOS）, ATPase, suporoxide dismutase （SOD）, xanthine oxidase（XOD）and malondiaehyde （MDA） separately. Results The activity of NOS was increased significantly as the period of cold storage wad prolonged （ P 〈 0. 05）, no distinct changes were found in NO and ET - 1, while slight variations were seen in AT- Pase, SOD, XOD and MDA. Bile was secreted during the ischemia- reperfusin process in all of the models. ALB showed no changes. Conclusion NOS is a sensitive index to reflect the liver ischmia/reporfusion injury.