【作者】 郭喻；

【导师】 汪晖；

【作者基本信息】 武汉大学 ， 药理学， 2005， 硕士

【摘要】 精密肝切片技术作为一种体外研究手段,近年来已广泛应用于异物（包括药物和毒物）代谢转化和毒理学的研究。但是目前尚未见病理状态下尤其是肝纤维化状态下肝切片代谢特征的报道.本文在建立肝纤维化精密肝切片技术,摸索体外最佳培养条件的基础上,研究了肝纤维化切片中Ⅰ、Ⅱ相药物代谢酶的表达及其诱导性,并运用该技术研究和比较了钙拮抗剂维拉帕米在正常和肝纤维化大鼠切片中的代谢情况。 一、肝纤维化精密肝切片方法学的建立及药物代谢酶的诱导性研究 建立复合因素（CCl₄+高脂低蛋白饲料+乙醇）大鼠肝纤维化模型。参照文献制备肝切片,以培养基中乳酸脱氢酶（LDH）漏出、肝组织中谷胱甘肽S-转移酶（GST）活性和肝细胞噻唑蓝（MTT）还原能力为指标,观察切片厚度、培养液pH值和培养时间对肝切片活力的影响。在确定的最佳制备与培养条件下,将切片与肝药酶诱导剂苯巴比妥钠(PB,0.5mmol.L^-1)、乙醇(50mmol.L^-1)或β-萘黄酮(β-NF,0.05mmol.L^-1)分别孵育0、2、4和6h后,制备肝切片细胞浆（S₉）,检测细胞色素P450（CYP450）1A（7-ethoxyresorufin O-deethylase,EROD）、CYP2E1（aniline hydroxylase,ANH）、CYP3A1/2（erythromycin N-demethylase,ERD）、尿苷二磷酸葡糖醛酸转移酶（uridine diphosphate-glucuronate transferase,UGT）和GST活性,并与对照组相比较。结果显示,大鼠经复合因素处理后,3周即可造成肝纤维化早期病理改变。在切片厚度300μm、培养液pH7.0的条件下,肝切片在6h内能够维持最低的LDH漏出量和最高的GST活性以及MTT还原量。培养6h内,对照组上述各酶活性无明显改变。而乙醇诱导组的CYP2E1活性、PB诱导组的CYP3A和UDPGT活性较对照组明显增高,且呈时间依赖性递增,在6h达高峰,分别为0时酶活性的2.3、4.4和1.6倍（P<0.05）。上述结果表明,肝纤维化状态下的精密肝切片技术,其切片厚度300μm、培养液pH7.0、培养时间6h为最佳培养条件。肝纤维化肝切片能稳定维持较高的Ⅰ、Ⅱ相药物代谢酶活性,其中的CYP2E1、CYP3A1/2和UGT在6h培养时间内,能被肝药酶诱导剂所诱导。 二、维拉帕米在正常和肝纤维化切片中的代谢对比研究 分别制备正常和肝纤维化肝切片。切片在含有5μg.mL^-1维拉帕米的培养基中分别孵育0、2、5、15、30、45、60、120、240、360min,培养完毕后,利用反相高效液更多还原

【Abstract】 Use of precision-cut liver slices （PCLS） as a valuable tool for studies of xenobiotic （drug and toxicant）metabolism and toxicity in vitro is increasing in recent years. However, the metabolizing characteristic of pathologic PCLS such as fibrotic PCLS has not been reported until now. This study was undertaken to grope the optimal culture condition, test the stability and inducibility of phase I and phase II enzymes of drug metabolism, and use this technique to study and compare the metabolism of a calcium antagonist-verapamil in both normal and fibrotic rat liver slices.I. Establishment of fibrotic rat precision-cut liver slice technique and inducibility in vitro of drug-metabolizing enzymesComplex factors （higher fat diet, alcohol drinking and CCl₄ injection） were administered in rats for 3 weeks to make liver fibrosis. Fibrotic PCLS were prepared and incubated. Lactate dehydrogenase （LDH） leakage, glutathione S-transferase （GST） activity and MTT reduction were chosen as indexes to assess the viability of fibrotic PCLS. Meanwhile, PCLS were treated with specific cytochrome P450 （CYP450） inducers, and the activities of CYP1A1, CYP2E1, CYP3A1/2, GST and UDP-glucuronosyltransferase （UDPGT） were monitored. Rats were in the earlier status of hepatic fibrosis after treatment of complex factors for 3 weeks. When the thickness of slices was 300 μm and pH of medium was 7.0, the LDH leakage, GST activity and MTT reduction of fibrotic slice could maintain on a steady level during 6 h of culture. Meanwhile, there were no significant changes in all the enzymatic activities after 6 h incubation; while the induction of CYP2E1 by ethanol （50 mM） and CYP3A1/2 as well as UDPGT by phenobarbital （0.5 mM） was demonstrated in the time-dependent manners, cresting at 6 h, which were 2.3, 4.4- and 1.6-folds, respectively, of their controls （P < 0.05）. A 300 urn of the thickness, 7.0 of the medium pH and 6 h of culture time are the optimal slicing and culturing conditions for fibrotic liver slice. Phase I and phase II enzymes of fibrotic PCLS could maintain the steady levels, and the activities of CYP2E1, CYP3A1/2 and UDPGT could be induced invitro by inducers within 6 h incubation.II. Metabolism of verapamil by normal and fibrotic rat precision-cut liver slicesNormal and fibrotic rat precision-cut liver slices were prepared and incubated individually in the presence of 10 uM verapamil. After 2, 5, 15, 30, 45, 60, 90, 120, 240 and 360 min of incubation, the incubation medium was removed for analysis. The concertration of verapamil from the medium was determined by HPLC. Subsequent to the incubation, each slice was removed and homogenized by hand and its protein content was determined. Intrinsic clearance （CLints） was calculated as the ratio of its initial concentration to the area under concentration-time curve （AUC）, and correlated to the average protein content. The AUC was model-independently calculated by employing WinNonlin Standard Edition Version 4.0.1 （Pharsight, USA）. Significant differences in the CLjnts （|il.min"1.nig¹） obtained with normal （9.7 ± 1.8） and fibrotic （5.6 ± 1.4） rat liver slices were observed （P < 0.01）. The result demonstrates that the rate of verapamil metabolism with rat liver slices was affected by status of fibrosis. These findings add further support to the use of fibrotic liver slices as a tool for studying drug metabolism and instructing clinical medication.更多还原