大鼠原代肝细胞药物相互作用模型的建立

Establishment of rat primary hepatocytes model for drug-drug interaction

目的建立并考察适用于药物相互作用研究的体外大鼠原代肝细胞模型。方法用改良Seglen两步灌流法分离肝细胞,以高糖DMEM为基础培养基培养细胞。通过检测细胞上清液中白蛋白(ALB)和尿素氮(BUN)水平、用底物探针法检测大鼠原代肝细胞中5种细胞色素P450(rCYP450)和葡萄糖醛酸转移酶(rUGT)代谢活性,考察培养时长对肝细胞功能和代谢酶活性水平的影响。原代肝细胞与利福平(RIF)、奥美拉唑(OMP)、β-萘黄酮(BNF)、苯巴比妥钠(PB)和地塞米松(DMS)共培养60 h,通过底物探针法检测代谢物的变化,用实时荧光聚合酶链反应法检测各组细胞代谢酶mRNA的表达水平,筛选多种代谢酶的阳性诱导剂。结果改良后的分离方法可得到活率大于90%,能正常分泌ALB和BUN的原代肝细胞,培养至第9天仍可维持基本功能和正常形态。rCYP1A2、rCYP2B1和rCYP3A1随培养时长活性下降迅速,48和72 h下降至20.54%~23.15%和0~7.80%。rCYP2D2和rUGT酶活性下降较缓,48和72 h可维持在57.41%~76.77%和25.25%~35.24%。诱导剂筛选结果显示:人代谢酶hCYP1A2的诱导剂OMP不能诱导rCYP1A2;hCYP2C和hCYP3A4诱导剂RIF不能诱导rCYP2C11和rCYP3A1。不同代谢酶可被一种或多种诱导剂诱导,DMS显著诱导除rCYP2B1外的其他6种代谢酶。结论本实验建立的大鼠原代肝细胞模型适用于体外药物相互作用研究。人和大鼠对代谢酶诱导剂的反应存在种属差异,DMS可作为大鼠肝细胞多种代谢酶的通用阳性诱导剂用于体外药物代谢和相互作用研究。

Objective To establish and investigate a rat primary hepatocyte model for the study of drug-drug interaction.Methods Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method,and then cultured with high glucose DMEM as basal medium.The effects of culture duration on hepatocytes function and metabolic activity were investigated by detecting secretion level of urea nitrogen(BUN)and albumin(ALB)and monitoring the metabolic activities of 5 rat cytochrome P450s(rCYP450)and UDP-glucuronosyltransferases(rUGT)using probes of respective substrate.Furthermore,the positive inducers of multiple rCYP450s and rUGT were screened according to the changes of metabolic activity using probes and of expression levels of mRNA detected by reverse transcription-polymerase chain reaction after rat primary hepatocytes were co-cultured with rifampicin,omeprazole,β-naphthoflavone,phenobarbital sodium and dexamethasone for 60 h.Results The hepatocytes keeping viability above 90%could be obtained using established isolation and culture method and the normal morphology and secretion function could be maintained after 9 days culture.The activity of rCYP1A2,rCYP2B1 and rCYP3A1 decreased rapidly with the extension of culture time.Their activities on 48 h and 72 h decreased to 20.54%-23.15%and 0-7.80%of those on 6 h respectively.But the activities of rCYP2D2 and rUGT showed slower decrease rate of remaining 54.41%-76.77%and 25.25%-35.24%on 48 h and 72 h respectively.The screening Results showed that human hCYP1A2 inducer of OMP could not induce rCYP1A2;hCYP2C and hCYP3A4 inducer of RIF could not induce rCYP2C11 and rCYP3A1.Each metabolic enzyme could be induced by one or several inducers.DMS appeared significant ability of induction for all metabolic enzymes except rCYP2B1.Conclusion A rat primary hepatocyte model has been established for the iv vitro study of drug-drug interaction.DMS can be used as a universal positive inducer for multiple metabolic enzymes of rat primary hepatocyte in respect of drug metabolism and drug-drug interaction.