【作者】 赵军；

【导师】 李玉民；

【作者基本信息】 兰州大学 ， 外科学， 2009， 硕士

【摘要】 目的:探讨环氧合酶-2(cyclooxygenase-2、COX-2)抑制剂塞来昔布和5-脂氧合酶(5-lipoxygenase,5-LOX)抑制剂MK-886对大鼠肝脏缺血再灌注损伤的影响。方法:成年雄性健康SD大鼠50只,随机数字表法分为5组:正常组(B1)、缺血再灌注组(B2)、COX-2抑制剂治疗组(B3)、5-LOX抑制剂治疗组(B4)、联合治疗组(B5),每组10只。制作大鼠肝脏缺血再灌注模型,夹闭肝脏血管30分钟致缺血,再灌注后不同时点(术后30分钟,术后60分钟)处死大鼠,抽取下腔静脉血并留取肝脏组织(肝左叶)保存待检测。一步法逆转录-聚合酶链式反应RT-PCR检测各组肝组织中COX-2 mRNA及5-LOX mRNA的表达;流式细胞术检测肝细胞凋亡率,;肝组织切片HE染色光镜下观察肝细胞变性、坏死等改变的情况,确定组织损伤程度并统计:血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移(aspartate aminotransferase,AST)检测肝功能变化。结果:1.RT-PCR:再灌注后各时点B2组中COX-2 mRNA及5-LOX mRNA的表达水平均明显高于B1组(P＜0.05);再灌注后30min,B2,B3,B4及B5组间数据无统计学差异(P＞0.05);再灌注后1h,塞来昔布干预组(B3)COX-2 mRNA的表达水平较B2下调,而5-LOX mRNA的表达量较B2组明显升高(P＜0.05);B4组给予MK-886后5-LOX的mRNA表达与B2组比较明显下调(P＜0.05),而COX-2的mRNA表达与B2比较无统计学差异(P＞0.05);两种药物联合作用后COX-2和5-LOX的mRNA表达较B2组均下调(P＜0.05)2.凋亡:通过流式细胞术分析肝细胞凋亡率显示,与B2组相比,各时点B3,B4,B5组的肝细胞凋亡率显著下降(P＜0.05);B5组凋亡率较B3,B4组明显下降(P＜0.05);而B3组与B4组之间肝细胞凋亡率无显著性差异(P＞0.05)。3.血清AST、ALT:再灌注后各时点B2组中血清AST、ALT明显高于B1组(P＜0.05);B5组数据明显低于B2组(P＜0.05);B3,B4组血清AST、ALT水平低于B2组,但无统计学差异(P＞0.05)。4.HE染色:与B1组相比,B2组肝组织改变明显;B3,B4及B5组较B2组肝组织改变减轻;B5组病理学损伤较B3,B4更轻。结论:COX-2抑制剂塞来昔布及5-LOX抑制剂MK-886减轻肝细胞凋亡,对大鼠肝脏缺血再灌注起保护作用;单独作用肝脏缺血再灌注后,两药之间比较,作用无明显差异;两种药物联合作用COX-2与5-LOX两条通路能更有效的减轻大鼠肝脏缺血再灌注损伤。更多还原

【Abstract】 Objective:Test the efficacy of the selective COX-2 inhibitor celecoxib、5-LOX inhibitor MK-886 or their combination on hepatic ischemia-reperfusion injury in rat,and to investigate whether the dual inhibition of COX-2 and 5-LOX has a more power protective effect than single inhibition of either COX-2 or 5-LOX alone on hepatic ischemia-reperfusion injury in rat.Method:Fifty male SD rats were randomly divided into 5 groups:sham-operated control group(B1)、liver ischemia-reperfusion group(B2)、Celecoxib and liver IR group(B3)、MK-886 and liver IR group(B4)、Celecoxib+ MK-886 and liver IR group(B5).Establish a rat model of hepatic ischemia-reperfusion,30 min of total hepatic vascular occlusion and then 30min or 60 min reperfusion were performed to animals in groups B2,B3,B4 and B5.The left hepatic lobes and blood sample which were obtained from the inferior cava vena were taken at 0.5h and 1h after reperfusion.;One step-RT-PCR was used to detect the expressions of COX-2 and 5-LOX;The apoptosis rate of the hepatocytes were detected by flow cytometry.; Hematoxylin Eosin staining was used to classify the injury extent of every samples; Alanine aminotransferase(ALT),aspartate aminotransferase(AST) as well as hepatic histopathological changes were measured;Result:1.RT-PCR:The expressions of COX-2 mRNA and 5-LOX mRNA in B2 group was significantly higher compared with the B1 group in different time after reperfusion(P＜0.05);The difference did not reach a statistically significant level in the B3,B4 and B5 groups compared with the B2 group at 30min after reperfusion (P＞0.05);The expressions of COX-2 mRNA was lower in COX-2 inhibitor preconditioning in B3 group than the B2 group at 1h after reperfusion,meanwhile,the expressions of 5-LOX mRNA was significantly higher compared with the B2 group (P＜0.05);The expression of 5-LOX mRNA in the B4 group which was treated with MK-886 was significantly lower than in the B2 group at 1h after reperfusion(P＜0.05), meanwhile,MK-886 had not effect on the expression of the Cox-2(p＞0.05);The expressions of Cox-2 mRNA and 5-lox mRNA in the B5 which were treated with the combination of celecoxib and MK-886 were lower than B2 group at 1h after reperfusion(p＜0.01)2.Apoptosistic:Analysis of apoptosis by the flow cytometry method showed that the apoptosis rate of the hepatocytes decreased significantly in the B3,B4 and B5 groups compared with the B2 group(P＜0.05);The data of the B5 group was significantly lower than the B3 and B4 groups(P＜0.05);There was no difference in the decline in the apoptosis rate of the hepatocytes between the B3 and B4 groups(P＞0.05).3.The levels of AST and ALT in blood serum:The datas of B2 group was significantly higher compared with B1 group in different time after reperfusion (P＜0.05);The dates of the B5 group was significantly lower than the B2 group(P＜0.05);The dates of the B3 and B4 groups was lower than the B2 group, which had no significant difference(P＞0.05);The dates of the B5 group was lower than the B3 and B4 groups,but the difference did not reach a statistically significant level(P＞0.05).4.HE stain:Compared with B1 group,B2 group showed great changes.The pathological lesions were lighter in B3,B4 and B5groups than in B2 group.The pathological lesions were lighter in B5 group than in B3 and B4 group.Conclusion:COX-2 inhibitor Celecoxib and 5-LOX inhibitor MK-886 were found to be effective in prevention of liver injury by reducing apoptosis in a hepatic I/R model. The efficacy was no different between the celecoxib and MK-886 pretreated groupson hepatic ischemia-reperfusion injury in rat.The dual inhibition of COX-2 and 5-LOX may present a superior protective effect than single inhibition of either COX-2 or 5-LOX alone.更多还原