稳定表达hMATE1及共表达hMATE1与hOCT1或hOCT2细胞模型的构建
|
摘要
为构建稳定表达人多药及毒素外排转运体1(h MATE1)的转基因细胞模型,提取人肾总m RNA,经逆转录PCR获得h MATE1 c DNA,借助HindⅢ、KpnⅠ两个酶切位点与pc DNA3.1(+)重组获得重组质粒。将pc DNA3.1(+)-h MATE1重组质粒转染至MDCK、MDCK-h OCT1和MDCK-h OCT2细胞中,经潮霉素B抗性筛选后,以4',6-二脒基-2-苯基吲哚(DAPI)和N-甲基-4-苯基吡啶(MPP+)的积聚实验筛选获得具有良好h MATE1功能的单克隆。测定筛选获得的细胞中转运体m RNA的表达量,并表征其对二甲双胍的积聚或对西咪替丁的转运能力。结果表明,本研究构建的MDCK-h MATE1、MDCK-h OCT1/h MATE1、MDCK-h OCT2/h MATE1细胞模型均高表达h MATE1 m RNA,MDCK-h MATE1细胞对二甲双胍的积聚为转染空载体细胞的17.6倍;MDCK-h OCT1/h MATE1和MDCK-h OCT2/h MATE1细胞对西咪替丁的净外排率分别为17.5和3.65。因此,本研究成功构建了稳定表达h MATE1及共表达h MATE1与h OCT1或h OCT2的细胞模型,可用于h MATE1及其与h OCT1或h OCT2共同参与的药物转运或药物-药物相互作用的体外研究。
To establish single- and double-transfected transgenic cells stably expressing h MATE1, h MATE1 c DNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pc DNA3.1(+) plasmid by virtue of both HindⅢ and KpnⅠ restriction enzyme sites. Subsequently, the recombined pc DNA3.1(+)-h MATE1 plasmid was transfected into MDCK, MDCK-h OCT1 or MDCK-h OCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 μg·m L-1, all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The m RNA content of h MATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed h MATE1 m RNA. The cellular accumulation of metformin in MDCK-h MATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 μmol·L-1 cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-h OCT1/h MATE1 and MDCK-h OCT2/h MATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good h MATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving h MATE1 alone or together with h OCT1/2 in vitro.
To establish single- and double-transfected transgenic cells stably expressing h MATE1, h MATE1 c DNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pc DNA3.1(+) plasmid by virtue of both HindⅢ and KpnⅠ restriction enzyme sites. Subsequently, the recombined pc DNA3.1(+)-h MATE1 plasmid was transfected into MDCK, MDCK-h OCT1 or MDCK-h OCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 μg·m L-1, all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The m RNA content of h MATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed h MATE1 m RNA. The cellular accumulation of metformin in MDCK-h MATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 μmol·L-1 cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-h OCT1/h MATE1 and MDCK-h OCT2/h MATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good h MATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving h MATE1 alone or together with h OCT1/2 in vitro.
引文
[1]Tanaka Y,Hipolito CJ,Maturana AD,et al.Structural basis for the drug extrusion mechanism by a MATE multidrug transporter[J].Nature,2013,496:247-297.
[2]Otsuka M,Matsumoto T,Morimoto R,et al.A human transporter protein that mediates the final excretion step for toxic organic cations[J].Proc Natl Acad Sci USA,2005,102:17923-17930.
[3]Motohashi H,Inui K.Organic cation transporter OCTs (SLC22)and MATEs(SLC47)in the human kidney[J].AAPS J,2013,15:581-588.
[4]Ciarimboli G.Role of organic cation transporters in drug-induced toxicity[J].Expert Opin Drug Metab Toxicol,2011,7:159-232.
[5]Motohashi H,Nakao Y,Masuda S,et al.Precise comparison of protein localization among OCT,OAT,and MATE in human kidney[J].J Pharm Sci,2013,102:3302-3309.
[6]Tu MJ,Li LP,Lei HM,et al.Involvement of organic cation transporter 1 and CYP3A4 in retrorsine-induced toxicity[J].Toxicology,2014,322:34-42.
[7]Hu HH,Su C,Jiang Y,et al.Construction and application of double-transfected cells expressing the human transporter P-glycoprotein and cytochrome P450 3A4[J].Pharmazie,2013,68:816-835.
[8]Fahrmayr C,Konig J,Auge D,et al.Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2(MRP2)using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells[J].Br J Pharmacol,2012,165:1836-1847.
[9]Lwai M,Minematsu T,Li Q,et al.Utility of P-glycoprotein and organic cation transporter 1 double-transfected LLC-PK1cells for studying the interaction of YM155 monobromide,novel small-molecule survivin suppressant,with P-glycoprotein[J].Drug Metab Dispos,2011,39:2314-2333.
[10]Rius M,Keller D,Brom M,et al.Vectorial transport of nucleoside analogs from the apical to the basolateral membrane in double-transfected cells expreesing the human concentrative nucleoside transporter h CNT3 and the export pump ABCC4[J].Drug Metab Dispos,2010,38:1054-1116.
[11]Boron WF,De Weer P.Active proton transport stimulated by CO2/HCO3-,blocked by cyanide[J].Nature,1976,259:240-241.
[12]Yasujima T,Ohta KY,Inoue K,et al.Evaluation of 4',6-diamidino-2-phenylindole as a fluorescent probe substrate for rapid assays of the functionality of human multidrug and toxin extrusion proteins[J].Drug Metab Dispos,2010,38:715-735.
[13]Wang K,Sun S,Li L,et al.Involvement of organic cation transporter 2 inhibition in potential mechanism of antidepressant action[J].Prog Neuropsychopharmacol Biol Psychiatry,2014,53:90-97.
[14]Artursson P.Epithelial transport of drugs in cell culture.I:A model for studying the passive diffusion if drugs over intestinal absorptive(Caco-2)cells[J].J Pharm Sci,1990,79:476-482.
[15]Sun X,Tian Y,Zhang Z,et al.A single LC-tandem mass spectrometry method for the simultaneous determination of four H2antagonists in human plasma[J].J Chromatogr B Analyt Techenol Biomed Life Sci,2009,877:3953-3961.
[16]Alexander RT,Dimke H,Cordat E.Proximal tubular NHEs:sodium,protons and calcium?[J].Am J Physiol-Renal Physiol,2013,305:F229-F236.
[17]Semiz S,Dujic T,Causevic A.Pharmacogenetics and personalized treatment of type 2 diabetes[J].Biochem Med(Zagreb),2013,23:154-224.
[18]Somogyi A,Stockley C,Keal J,et al.Reduction of metformin renal tubular secretion by cimetidine in man[J].Br J Clin Pharmacol,1987,23:545-595.
[19]Meyer zu Schwabedissen HE,Verstuyft C,Kroemer HK,et al.Human multidrug and toxin extrusion 1(MATE1/SLC47A1)transporter:functional characterization,interaction with OCT2(SLC22A2),and single nucleotide polymorphisms[J].Am J Physiol-Renal Physiol,2010,298:F997-F1005.
[2]Otsuka M,Matsumoto T,Morimoto R,et al.A human transporter protein that mediates the final excretion step for toxic organic cations[J].Proc Natl Acad Sci USA,2005,102:17923-17930.
[3]Motohashi H,Inui K.Organic cation transporter OCTs (SLC22)and MATEs(SLC47)in the human kidney[J].AAPS J,2013,15:581-588.
[4]Ciarimboli G.Role of organic cation transporters in drug-induced toxicity[J].Expert Opin Drug Metab Toxicol,2011,7:159-232.
[5]Motohashi H,Nakao Y,Masuda S,et al.Precise comparison of protein localization among OCT,OAT,and MATE in human kidney[J].J Pharm Sci,2013,102:3302-3309.
[6]Tu MJ,Li LP,Lei HM,et al.Involvement of organic cation transporter 1 and CYP3A4 in retrorsine-induced toxicity[J].Toxicology,2014,322:34-42.
[7]Hu HH,Su C,Jiang Y,et al.Construction and application of double-transfected cells expressing the human transporter P-glycoprotein and cytochrome P450 3A4[J].Pharmazie,2013,68:816-835.
[8]Fahrmayr C,Konig J,Auge D,et al.Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2(MRP2)using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells[J].Br J Pharmacol,2012,165:1836-1847.
[9]Lwai M,Minematsu T,Li Q,et al.Utility of P-glycoprotein and organic cation transporter 1 double-transfected LLC-PK1cells for studying the interaction of YM155 monobromide,novel small-molecule survivin suppressant,with P-glycoprotein[J].Drug Metab Dispos,2011,39:2314-2333.
[10]Rius M,Keller D,Brom M,et al.Vectorial transport of nucleoside analogs from the apical to the basolateral membrane in double-transfected cells expreesing the human concentrative nucleoside transporter h CNT3 and the export pump ABCC4[J].Drug Metab Dispos,2010,38:1054-1116.
[11]Boron WF,De Weer P.Active proton transport stimulated by CO2/HCO3-,blocked by cyanide[J].Nature,1976,259:240-241.
[12]Yasujima T,Ohta KY,Inoue K,et al.Evaluation of 4',6-diamidino-2-phenylindole as a fluorescent probe substrate for rapid assays of the functionality of human multidrug and toxin extrusion proteins[J].Drug Metab Dispos,2010,38:715-735.
[13]Wang K,Sun S,Li L,et al.Involvement of organic cation transporter 2 inhibition in potential mechanism of antidepressant action[J].Prog Neuropsychopharmacol Biol Psychiatry,2014,53:90-97.
[14]Artursson P.Epithelial transport of drugs in cell culture.I:A model for studying the passive diffusion if drugs over intestinal absorptive(Caco-2)cells[J].J Pharm Sci,1990,79:476-482.
[15]Sun X,Tian Y,Zhang Z,et al.A single LC-tandem mass spectrometry method for the simultaneous determination of four H2antagonists in human plasma[J].J Chromatogr B Analyt Techenol Biomed Life Sci,2009,877:3953-3961.
[16]Alexander RT,Dimke H,Cordat E.Proximal tubular NHEs:sodium,protons and calcium?[J].Am J Physiol-Renal Physiol,2013,305:F229-F236.
[17]Semiz S,Dujic T,Causevic A.Pharmacogenetics and personalized treatment of type 2 diabetes[J].Biochem Med(Zagreb),2013,23:154-224.
[18]Somogyi A,Stockley C,Keal J,et al.Reduction of metformin renal tubular secretion by cimetidine in man[J].Br J Clin Pharmacol,1987,23:545-595.
[19]Meyer zu Schwabedissen HE,Verstuyft C,Kroemer HK,et al.Human multidrug and toxin extrusion 1(MATE1/SLC47A1)transporter:functional characterization,interaction with OCT2(SLC22A2),and single nucleotide polymorphisms[J].Am J Physiol-Renal Physiol,2010,298:F997-F1005.