d-Penicillamine2,5-enkephalin作为底物考察三明治大鼠原代肝细胞的转运体功能研究  

Application of d-Penicillamine2,5-enkephalin to characterization of sandwich-cultured hepatocytes transport activities

作　　者：郭岑[1] 赫蕾[1] 姚丹[2] 潘国宇[2] 王广基[1] 

机构地区：[1]中国药科大学药物代谢动力学重点实验室 [2]中国科学院上海药物研究所药物安全评价研究中心,上海201203

出　　处：《中国临床药理学与治疗学》2013年第5期506-512,共7页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：国家自然科学基金项目(81173116);江苏省药代动力学重点实验室提升项目(BM20120102)

摘　　要：目的:通过液相色谱-串联质谱(LC/MS/MS)法测定d-Penicillamine2,5-enkephalin(DP-DPE)在肝细胞和Hank's缓冲液中的浓度,研究DPDPE作为底物用于"三明治"培养大鼠原代肝细胞(SCRH)的转运体功能。方法:将SCRH与溶于Hank's缓冲液的DPDPE共温孵,采用LC/MS/MS法测定细胞内蓄积的和外排到缓冲液中的DPDPE浓度,用于评价模型药物α-萘异硫氰酸酯(ANIT)对参与DPDPE摄取的Oatp和负责DPDPE外排的Mrp2的转运功能的影响。结果:建立的样品处理和测定方法能够满足体外转运实验需要的灵敏度和线性范围,在Hank's缓冲液中线性范围为0.5～50ng/mL(r2=0.9995),在细胞基质中线性范围为0.1～5ng/well(r2=0.9997),样品处理操作简便。日内日间精密度(RSD)均小于15%;提取回收率大于65%。ANIT可抑制DPDPE在SCRH胆管侧的外排及基底侧的摄取,对基底侧外排无显著影响。结论:该分析方法选择性好,灵敏度高,操作简便,并成功地用于将DPDPE作为探针底物研究SCRH模型上转运体的功能。AIM： To develop a sensitive and rapid method for determining the concentration of d-Penicillamine2,5-enkephalin（DPDPE） in hepatocytes and Hank＇s buffer by LC/MS/MS.And to evaluate the function of transporters in sandwich-cultured rat hepatocytes（SCRH） using DPDPE as the probe substrate.METHODS： Co-incubate DPDPE with SCRH treated by alpha-naphthylisothiocyanate,the model compound,and measure the accumulation and the efflux of DPDPE by SCRH.The concentration of DPDPE in hepatocytes or Hank＇s buffer was determined by LC/MS/MS.RESULTS：DPDPE had a good linearity from 0.5 to 50 ng/mL（r2=0.9995） in Hank＇s buffer and from 0.1 to 5 ng/well（r2=0.9997） in hepatocytes in the LC/MS/MS method established in this study.This method was sensitive enough to quantify DPDPE used as probe substrate in SCRH transport studies.The inter-and intra-day precision（RSD） were less than 15% and the recoveries were above 65%.The basolateral uptake and canalicular efflux of DPDPE by SCRH was inhibited by ANIT without change in the basolateral efflux.CONCLUSION： The analytical method established here is selective,sensitive and feasible to determine DPDPE used as probe substrate in the evaluation of membrane transporters in SCRH.

关 键 词：转运体 “三明治”培养大鼠原代肝细胞 LC MS MS DPDPE 

分 类 号：R969.1[医药卫生—药理学]