【作者】 李玉萍；

【导师】 陶国才；

【作者基本信息】 第三军医大学 ， 麻醉学， 2008， 硕士

【摘要】 研究背景芬太尼是临床肝移植手术中最常用的强效麻醉性镇痛药,主要在肝脏代谢,代谢产物与约10%的原形药由肾脏排出。肝移植手术中在无肝期失去肝脏的主要代谢作用后,芬太尼的代谢会受到怎样的影响,影响其代谢的因素是什麽,通过哪种途径产生影响,目前对这方面的报道较少。细胞色素P450(cytochromeP450)属于血红素蛋白基因超家族,编码一系列代谢酶系统,参与各类不同结构亲脂性化合物的生物转化,增强代谢物水溶性,利于其排出体外,从而降低外源化合物对机体靶器官的毒性效应。在CYP超家族中,涉及芬太尼代谢的CYP3A亚家族成员为CYP3A4(人),其在大鼠体内的同源蛋白为CYP3A1/2。肠内CYP450酶主要为CYP3A4亚族,分布于上皮细胞。已有的研究证实,肠内CYP3A与肝脏中的CYP3A酶cDNA序列一样。将芬太尼代谢酶表达活性的改变与药代动力学特征相结合有助于深入了解无肝期芬太尼代谢的特征,为临床用药提供参考。目的本实验以大鼠为动物模型,小肠为研究对象,解剖分离并阻断肝门,模拟临床肝移植无肝期,观察入肝血流阻断前后芬太尼血药浓度的变化,计算其代谢的相关参数;观察细胞色素P450酶系中芬太尼代谢相关CYP3A1酶活性的变化,并通过RT-PCR和Western-blot技术观察CYP3A1基因、蛋白的表达和变化,为初步探讨芬太尼无肝状态下的代谢途径及作用机制提供依据。材料和方法实验分为三部分:1.无肝状态下大鼠芬太尼血药浓度的变化。采用芬太尼标准品(1 g/ml,纯度99.9 %),通过高效液相色谱—质谱联用(LC-MS/MS)分析仪,制备大鼠芬太尼血药浓度标准曲线,建立微量血样检测芬太尼浓度的实验方法。SPF级雄性大鼠25只,5只用于建立芬太尼标准曲线,其余随机分为对照组(A1)和处理组(A2)(每组10只),大鼠麻醉后,处理组阻断入肝血流,夹闭肝门,两组均由右侧颈深静脉置入24#静脉留置针,用于采血;右股静脉切开,置入24#静脉留置针,A1组直接,A2组于夹闭肝门后静脉注射芬太尼(20μg/kg),分别于注药后1、2、3、5、10、15、20、30、45、60、70、90 min等时间点由颈静脉各采血0.5 ml,同时回输等量乳酸林格液。采用LC-MS/MS方法检测各时相点血药浓度,并应用DAS2.0药代动力学软件测算芬太尼的消除半衰期、清除率、表观分布容积和药-时曲线下面积。2.芬太尼代谢相关CYP3A1酶活性的研究。30只SPF级雄性大鼠随机分为3组,每组10只,B1为对照组,B2为阻断肝门30 min组,B3为阻断肝门60 min组。大鼠麻醉后,B1组直接,B2、B3组分别在相应时间点纵行开腹,截取距胃幽门4 cm处小肠组织,采用免疫荧光比色法定量检测各实验组酶活性的变化。3.阻断肝门后芬太尼代谢相关CYP3A1在小肠表达和变化的研究。将大鼠分为三组,正常对照(C1)组、阻断肝门30min(C2)组、阻断肝门60min(C3)组,n=10,大鼠小肠组织采用逆转录聚合酶链式反应(RT-PCR)检测各实验组CYP3A1 mRNA的表达;免疫印迹(Western-blot)技术检测各实验组CYP3A1蛋白的表达。结果1.在选定的色谱条件下,测得芬太尼色谱图被测物与内标物两者峰形良好,分离完全,无杂质峰干扰,芬太尼及内标的保留时间良好;芬太尼标准曲线方程为y = 0.0156 x + 0.0072(r = 0. 9997),大鼠体内芬太尼最低检测浓度为0.5 ng/ml,检测方法精密优良。2.在阻断肝门的情况下,单次剂量注射芬太尼后其血药浓度仍呈下降趋势,但下降速度减缓,尽管消除半衰期明显延长,药-时曲线下面积明显增大,但清除率、表观分布容积无显著变化。3.阻断肝门后小肠组织CYP3A1酶活性比阻断肝门前增强,差异具有统计学意义(P < 0.05),阻断肝门30 min和阻断肝门60 min相比较差异无统计学意义(P > 0. 05)。4. CYP3A1酶mRNA和蛋白的表达水平阻断肝门后30 min和60 min均高于阻断肝门前,具有统计学差异(P < 0.05),阻断肝门30 min和阻断肝门60 min相比较差异无统计学意义(P > 0. 05)。结论1.本实验建立的微量血样检测芬太尼血药浓度的方法特异性强,日间日内变异小,线性范围广,简便准确。2.无肝期芬太尼的代谢特点为血药浓度下降缓慢,Vd、CL无明显改变,T1/2β延长,AUC明显增大。3.肝脏是芬太尼代谢的主要器官,无肝状态下芬太尼可能存在有其他代谢途径。4.小肠组织芬太尼代谢相关CYP3A1酶活性在无肝期明显增强,可能为无肝状态下,芬太尼肝外代谢的机制之一。5.小肠组织芬太尼代谢相关CYP3A1酶mRNA的表达和蛋白的表达在无肝期均增强,可能是使CYP3A1酶含量增多,促进芬太尼在小肠代谢的分子机制之一。同时酶含量增多,活性增强可能是芬太尼无肝状态代谢特点形成的原因之一。更多还原

【Abstract】 Background:Fentanyl is an intravenous narcotic analgesics with high efficiency and most commonly used in liver transplantation. As an opiate receptor agonist, it takes effect very rapidly, lasts a very short time in blood, does not release histamine, and has little effect on cardiovascular function. It is metabolized mainly in the liver, and its metabolites and about 10%original drug are discharged by the kidneys. In liver transplant operation, there is no liver metabolism during anhepatic phase. How is fentanyl metabolized? And what will influence this proscess?These questions remain unclear. Cytochrome P450 (CYP), a member of the hemoprotein gene superfamily, encodes a series of metabolic enzymes, participates biological transformation of lipophilc compounds in various structures, and enhances the water-solubility of the metabolic products which will be prone to be discharged so as to reduce the toxicity of these exogenous compounds. The liver has the highest contents of CYP3A1 among other tissues and organs. Its expression is modulated in an immediate, tissue-specific and environment-related manner. In the anhepatic phase of liver transplantation, internal milieu changes vigorously, and the levels of some hormones may affect extrahepatic expression of CYP3A1, thus influencing extrahepatic metabolism of fentanyl. In small intestine, CYP3A4 is the main form and distributed in the epithelial cells.Objective:In this study, fentanyl metabolism in anhepatic rats was studied, and the changes in CYP3A1 enzymatic activity, gene expression and protein expression in small intestine before and after the anhepatic phase were also investigated. This study was aimed to preliminarily elucidate how the anhepatic state influences the CYP3A1 expression and what fentanyl metabolism is in the anhepatic phase.Materials and Methods: This study is composed of three parts:1. Fentanyl plasma cocentration changes in the anhepatic rats.Five SPF male SD rats were used to plot the standard curve of blood fentanyl concentration, and another 20 were equally divided into control and treatment group (anhepatic group). After all rats were etherized, a 24# vein detaining-pin was inserted into their right deep cervical vein to collect blood sample, and another detaining-pin was set to the right femoral vein to add fentanyl. In treatment group, fentanyl (20μg/kg) was infused after hepatic hilum blocking, and blood sample (0.5 ml) was colleted in 1, 2, 3, 5, 10, 15, 30, 45, 60, 70 and 90 min after the infusion. Rats of control group did not receive hepatic hilum blocking. The fentanyl concentration was measured with LC-MS/MS and analyzed with DAS2.0 Pharmacokinetics Program. The standard curve of rat blood fentanyl concentration was plotted by high performance liquid chromatograph-mass spectrogram using standard fentanyl (1μg/ml, purity 99.9%), and the method for measuring fentanyl concentration with small volume of blood sample was established.2. Extrahepatic expression of fentanyl metabolism related CYP3A1 in the anhepatic phase.Totally 30 SPF male SD rats were equally and randomly divided into the control groups (group B1, as before the anhepatic phase), rats with hepatic portal blocking for 30 min (group B2, as 30 min anhepatic phase), and rats with 60 min blocking (groupB3, as 60 min anhepatic phase). All rats were treated as done in part 1 for drug injection and infusion. The intestinal specimen 4 cm from the stomachus pyloricus was taken out, and the activity of CYP3A1 in the specimens was detected by colorimetry.3. Extrahepatic expression of CYP3A1 gene and protein in the anhepatic phase. The rats were randomly divided into (n=10 in each group): control group (group C1, before the anhepatic phase), group C2 (30 min anhepatic phase) and group C3 (60 min anhepatic phase).After etherization, small intestine were harvested, and CYP3A1 mRNA and protein expression was detected by RT-PCR and Western blotting respectively.Results:1. Under the selected chromatographic conditions, the samples and the internal standard of fentanyl showed satisfactory and completely-separated peaks and had no interference of impurity peak, and maintained in a sound time period. The standard curve equation of fentanyl was y = 0.0156 x+0.0072 (r=0.9997, P<0.05). The minimal detectable concentration of fentanyl was 0.5 ng/ml, indicating the detection method is highly precise.2. Before and after the anhepatic phase, single dose of fentanyl did not change significantly with CL and Vd (P>0.05), AUC and T1/2βchanged obviously.(P<0.05).3. CYP3A1 activity in small intestine was significantly higher after 30 min or 60 min anhepatic phase than before the anhepatic phase (P<0.05), but no significant difference was seen between 30 min and 60 min of the anhepatic phase (P>0.05).4. The expression of CYP3A1 gene and protein in rat small intestine was significantly higher after 30 min or 60 min anhepatic phase than before the anhepatic phase (P<0.05), but there is no significant difference between 30 min and 60 min anhepatic phase(P>0.05).Conclusions:1. The method of measuring blood fentanyl concentration in small volume of blood sample is specific, simple and accurate, with little time variation and wide linear range. It can be used as a conventional assay measuring blood fentanyl concentration.2. Melabolism of fentanyl during the anhepatic phase is characterized with no significantly changed CL and Vd, prolonged T1/2βand obviously increased AUC.3. Liver is the main organ to metabolize fentanyl, but there maybe other ways for fentanyl metabolization.4. CYP3A1 activity in small intestine is significantly higher in the anhepatic phase, indicating it maybe one of the mechanism for frntanyl metabolized in small intestine.5. The expression of CYP3A1 gene and protein in rat small intestine is significantly higher in the anhepatic phase, which maybe one of the mechanism about the elevated enzymic activity and extrahepatic metabolism of fentanyl. Simultaneously, it maybe one of the reasons that generate the metabolic characters in the rat small intestine about fentanyl.更多还原