【作者】 胡屹屹；

【导师】 江善祥；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 目前肝微粒体水平的药物代谢相互作用研究日趋增多，但用食品动物猪肝微粒体进行药物代谢研究在国内尚未见报道。本科题研究了不同年龄和性别猪肝CYP450酶系的检测方法、四种临床常用药物对猪肝CYP450酶系的作用和探针药物法评价新兽药氟尼辛葡甲铵对猪肝CYP450酶系的影响。具体研究内容如下： 1．用生物化学法和紫外分光光度法检测猪肝微粒体蛋白含量和P450酶系活性。 取新鲜猪肝样品匀浆，差速离心法制备肝微粒体，Bradford法测定蛋白浓度，分光光度计法检测6种肝细胞色素P450酶含量及活性。测定一月龄至八月龄不同性别的猪，肝微粒体蛋白含量、CYP450含量、CYPb5含量、NADPH-细胞色素C还原酶活性、氨基比林-N-脱甲基酶活性、红霉素-N-脱甲基酶活性和苯胺-4-羟化酶活性。结果表明，猪一到八月龄肝微粒体蛋白含量变化范围为6.646±0.457.13.596±1.077mg／ml，细胞色素P450含量变化范围为0.270±0.044-0.587±0.043 nmol／mg protein，细胞色素b5含量变化范围为0.105±0.019-0.251±0.018 nmol／mg protein，NADPH-细胞色素C还原酶活性变化范围为1.632±0.255.3.043±0.229 nmol／min／mg protein，氨基比林-N-脱甲基酶(AND)活性变化范围为0.625±0.071-2.990±0.144 nmol／min／mgprotein，红霉素-N-脱甲基酶(ERND)活性变化范围为0.562±0.095-2.540±0.177nmol／min／mg protein，苯胺-4-羟化酶(AH)活性变化范围为0.090±0.016-0.231±0.021nmol／min／mg protein。建立的猪P450酶系检测方法灵敏可靠，简单易行，重复性较好。猪肝微粒体蛋白含量和P450酶系活性随着年龄增长，含量增加，活性增强。同时肝微粒体蛋白含量、CYP450含量和红霉素-N-脱甲基酶活性呈现有一定的性别差异。 2．几种临床常用药物对猪肝微粒体细胞色素P450酶系的影响。 四种药物红霉素、环丙沙星、地塞米松和乙醇，每天按正常治疗剂量的5倍进行肌肉注射，连续给药10天。差速离心法制备猪肝微粒体，Bradford法测定蛋白浓度，分光光度法检测6种肝细胞色素P450酶含量及活性，结果进行统计学分析处理。研究表明，四种药物(除环丙沙星外)对P450酶的总活性均产生显著影响，同时地塞米松组的微粒体蛋白含量和红霉素-N-脱甲基酶活性，乙醇组的苯胺-4-羟化酶活性和红霉素组的红霉素-N-脱甲基酶活性，与对照组相比均差异显著。说明环丙沙星和红霉素对猪肝细胞色素P450酶系有选择性抑制作用，而地塞米松和乙醇对其起选择性诱导效应。 3．用Cocktail探针药物法研究氟尼辛葡甲铵对猪肝细胞色素P450酶系的影响。更多还原

【Abstract】 The drug interactions at microsome level are studied more and more recently, but research on medicines metabolizability in food animals are seldom reported. Microsomal cytochrome P450 enzymes in pigs were studied in this paper. The method was used to determine the CYP450 in pigs, the effects of several clinical medicines and flunixin meglumine on CYP450 in pigs were evaluated. The details as follow:Pig liver microsome protein content and P450 enzymes activities were studied by biochemical and ultraviolet spectrophotometry method. The fresh pig liver samples were refined, and liver microsome was distilled by different velocity centrifugation. The Bradford method was used to measure the concentration of protein in liver microsome, and activities of cytochrome P450 enzymes were determined by the spectrophotometer method. Value of liver microsome protein content, CYP450 content, CYPb5 content, NADPH-CytochromeC reductase activity, aminopyrine-N-demethylase activity, erythromycin-N-demethylase activity and aniline-4-hydroxylase activity were measured in different sex pigs within one month to eight month old. Result suggested that protein concentration in hepatic microsome of pigs with age from one month to eight month was 6.646 ± 0.457-13.596 ± 1.077 mg/ml, content of cytochrome P450 was 0.270 ± 0.044-0.587 ± 0.043 nmol/mg protein, content of cytochrome b5 was 0.105 ± 0.019-0.251 ± 0.018 nmol/mg protein, the activity of NADPH-CytochromeC reductase was 1.632 ± 0.255-3.043 ±0.229 nmol/min/mg protein, the activity of AND was 0.625 ± 0.071-2.990 ± 0.144 nmol/min/mg protein, the activity of ERND was 0.562 ± 0.095-2.540 ± 0.177 nmol/min/mg protein, and the activity of AH was 0.090 ± 0.016-0.231 ± 0.021 nmol/min/mg protein. The established method for determining contents and activities of hepatic microsomal cytochrome P450s in pigs was sensitive, simple, credibility with good reproducibility. The protein content of the pig liver microsome and P450 enzymes, growing along with the ages, was increasing and strengthened. And the liver microsome protein content, CYP450 content and erythromycin-N-demethylase activity appear some gender differences.Effects of several clinical medicines on cytochrome P450 enzymes in pigs were studied. Erythromycin, ciprofloxacin, dexamethasone and ethanol were given to pigs by intramuscular injection at a single dose of 5 times of the normal dosage continuously for 10 days, respectively. Liver microsome was distilled by different velocity centrifugation. The更多还原