CN103710385A - Construction method for cell model co-expressed with cytochrome P450 enzyme and application - Google Patents

Abstract

The invention provides a construction method of a cell model co-expressed with a cytochrome P450 enzyme (CYPA3A4). The construction method comprises the following steps: transfecting MDCK (Madin-Darby Canine Kidney)-mock and MDCK-hOCT1 (Organic Cation Transporter 1) cells by an expression plasmid pcDNA3.1-Hygro-CYP3A4 of CYPA3A4; obtaining monoclonal cells with stable resistance through hygromycin and G418 resistance selection; obtaining cells of stable single expression CYP3A4 and stable coexpression CYP3A4 and hOCT1 by means of detecting the protein expression of CYP3A4 through westernblot; and carrying out function verification through a classical substrate and an inhibitor. The cell model constructed according to the construction method provided by the invention can be used for researching the cell level specificity of CYP3A4 and the combined effect of hOCT1 and CYP3A4, and forecasting the treatment conditions of medicines on a liver and the medicine combined effect which may be caused by the CYP3A4 and hOCT1. The construction method has the advantages of reasonable design, specificity and good repeatability.

Description

Cell model construction and application with cytochrome P 450 enzymes coexpression

Technical field

The invention belongs to biological field, relate to the construction and application of a kind of coexpression human Organic Cation Transporter Gene 1 and cytochrome P 450 enzymes cell model.

Background technology

People's liver expression multi-medicament metabolic enzyme and transporter are the vitals of medicine disposition.Cytochrome P450 (CYP) is a topmost phase metabolic enzyme in people's liver, and the redox reaction of their mediation medicines, makes drug inactivation or activation.CYP3A4 expresses maximum CYP enzymes in liver, account for 30% of its total CYP enzyme, and in the medicine of clinical application at present, nearly 50% by CYP3A4 metabolism [1].Because CYP3A4 substrate spectrum is wide, therefore by the drug-drug interactions of its mediation also very general [2].Picked-up and the outer row of film transporter mediation high amount of drug and metabolite thereof be the deciding factor of a lot of medicine dispositions, so transporter also play an important role [3,4] in drug-drug interactions process.Having clinically nearly 40% oral pharmaceutical is alkaline compound, under physiological pH condition, partly or entirely with positively charged ion, there are [5], therefore their turnover cells often need to be mediated by protein called membrane transporters, Organic Cation Transporter Gene 1(organic cation transporter 1 is OCT1) wherein a kind of.People OCT1 is a kind of important protein called membrane transporters in liver, can mediate the picked-up [6] of many cation compounds.The vital role to disposition of drug in view of metabolic enzyme and transporter, the repercussion study of medicine and metabolic enzyme, transporter has become research contents indispensable in new drug development process, to it, becomes property of medicine evaluation to have great importance.Because transporter and metabolic enzyme exist substrate spectrum intercrossing widely, thereby in vitro study, need their common effects of comprehensive consideration.The conventional immortalized hepatocyte of in vitro study system, as HepG2, HepaRG, Huh7, and L-02 etc. [7,8], the disappearance of expressing due to metabolic enzyme and transporter or too low, is not suitable for carrying out drug transport and metabolism researchs; And the sources such as the organoid in people source, tissue are limited, cost is high, and be difficult to the acting in conjunction of the effect of the single-minded enzyme of research and/or itself and transporter.Therefore the transgenic cell model of stably express people transporter/CYP enzyme, is a kind of comparatively ideal in vitro study model.This research is on the basis of existing stably express hOCT1 cell model [9], the Martin who sets up stably express people CYP3A4 reaches than the mdck cell model of dog kidney epithelium (MDCK) cell model and coexpression hOCT1 and CYP3A4, for external single-minded research hOCT1, CYP3A4 and both actings in conjunction provide effective cell model.

Summary of the invention

The object of this invention is to provide cell model structure a kind of and cytochrome P 450 enzymes coexpression, it is cell model structure a kind of and cytochrome P 450 enzymes coexpression, also be a kind of stable high expression level CYP3A4 and coexpression hOCT1, the MDCK(Martin of CYP3A4 reaches than dog kidney epithelium) structure of cell model, by following steps, realize:

By design, contain Kpn I, (primer sequence is as SEQ ID No:1 for the primer of two restriction enzyme sites of Xho I, shown in 2), by before terminator codon with histidine-tagged people CYP3A4 cDNA(nucleotide sequence as shown in SEQ ID No:3) to be cloned in vector plasmid pcDNA3.1 (+)-Hygro upper, construction expression plasmid pcDNA3.1 (+)-Hygro-CYP3A4.Expression plasmid transfection MDCK-pcDNA3.1 (+) and MDCK-hOCT1 cell, after transfection, by aminoglycoside antibiotics G418 and hygromycin B, carry out resistance screening, select monoclonal cell, monoclonal cell carries out the protein expression of western blot checking CYP3A4, obtain MDCK-CYP3A4 and the MDCK-hOCT1-CYP3A4 cell of stably express CYP3A4, and select the consistent cell of CYP3A4 expressing quantity by Western blot, extract total RNA, by specific primer (primer sequence is as shown in SEQ ID No:4-9), carry out the mrna expression of CYP3A4 and hOCT1 in Real-time PCR checking cell, and compare with empty carrier cell and MDCK-hOCT1, and then it is active with P450-Glo CYP3A4 Assay test kit, to detect CYP3A4, and investigate the impact of classical inhibitor KETOKONAZOL on metabolic activity, thereby further confirm the function of transfectional cell CYP3A4.In addition application hOCT1 fluorogenic substrate 4-(4-(dimethylami-no) styryl)-N-methylpyridinium(ASP, ⁺), in conjunction with the function of hOCT1 inhibitor triethylammonium tetrakis (TEA) checking cell hOCT1.

Another object of the present invention is to provide the application of constructed model in the hepatotoxicity of research CYP3A4 and hOCT1 mediation.By following steps, realize: singly turn, two turn and mock cell gives the testing compound of different concns, hatch certain hour, adopt mtt assay to detect the survival rate of cell.By relatively singly expressing, coexpression hOCT1 is or/and the survival rate of the cell of CYP3A4 and mock cell illustrates hOCT1 and CYP3A4 role in the cytotoxicity of testing compound.

Take retrorsine as example, the cell of transfection CYP3A4 just can demonstrate toxicity, the metabolism that CYP3A4 is described is the toxigenous committed step of retrorsine, and MDCK-hOCT1-CYP3A4 cell demonstrate overt toxicity effect, illustrate that two albumen all play a role in toxicity process; Take nitidine chloride as example, MDCK-hOCT1 cell survival rate is minimum, the cellular uptake that hOCT1 mediation is described is the toxigenous committed step of compound, and two cell survival rate that turns hOCT1 and CYP3A4 is high compared with MDCK-hOCT1, illustrate that the metabolism that CYP3A4 mediates is attenuation process.Two examples all illustrate, two compounds are all common substrates of hOCT1 and CYP3A4.

The cell model that the present invention sets up can be for the effect of cell levels specificity research CYP3A4, can study the acting in conjunction of hOCT1 and CYP3A4 simultaneously, prediction medicine is in the disposal situation of liver, and prediction may be by CYP3A4, the drug interaction of hOCT1 mediation.The cell model that this research obtains, preliminary screening arrives the common substrate of several hOCT1 and CYP3A4, and is further applied to the toxicity research of chemical combination, has illustrated the hepatotoxicity mechanism of compound through hOCT1 and CYP3A4 mediation.The present invention is reasonable in design, specificity and reproducible.

Positively effect of the present invention is: (1) liver is the major organs of drug metabolism, and CYP3A4 is that in liver, expression amount is the highest, the CYP enzyme that substrate spectrum is the widest, but CYP enzyme is the interior stably express difficulty of mammalian cell in vitro, this research has successfully built the mdck cell model of stably express CYP3A4, can be for the effect at the single-minded research of cell levels CYP3A4.(2) CYP3A4 and hOCT1 are all at liver high expression level, the former is important metabolic enzyme, the latter is important transporter, both existence are substrate intercrossing widely, the cell model of stablizing coexpression hOCT1 and CYP3A4 is successfully set up in this research, can effectively study both actings in conjunction to drug metabolism transhipment, and the drug interaction of transporter, the common mediation of metabolic enzyme.(3) a lot of allogenic materials enter liver cell by transporter, may increase poison or attenuation by metabolism, the cell model that utilizes this research to set up, and intoxicating that can external single-minded this approach of research is machine-processed, seeks removing toxic substances way.

Accompanying drawing explanation

Fig. 1 is that plasmid pcDNA3.1 (+)-Hygro-CYP3A4 and corresponding enzyme thereof are cut the evaluation of product electrophoresis.

Fig. 2 is that Western blot detects the intracellular CYP3A4 protein expression of MDCK-CYP3A4-hOCT1.

Fig. 3 is that Western blot detects the intracellular CYP3A4 protein expression of MDCK-CYP3A4.

Fig. 4 is the protein expression of the more intracellular CYP3A4 of Western blot.

Fig. 5 is that Real-time PCR detects hOCT1 in different cells, the relative expression quantity of CYP3A4 mRNA.

Fig. 6 is the activity of Luciferin-IPA checking CYP3A4 when whether inhibitor exists.

Fig. 7 be different cells have or without hOCT1 inhibitor, exist under to ASP ⁺gather.

Fig. 8 is the toxicity of retrorsine to single expression, coexpression hOCT1 and CYP3A4 cell.

Fig. 9 is that hOCT1 and CYP3A4 are on the Cytotoxic impact of nitidine chloride.

Embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.

embodiment 1

1. the structure of CYP3A4 expression plasmid and evaluation

Design upstream and downstream respectively with Kpn I, the people CYP3A4 specificity primer of Xho I restriction enzyme site, wherein restriction enzyme site underscore marks, the gene order for six Histidines of coding that marks, primer sequence is as follows:

SEQ ID No: 1 Forward: 5’- ATT GGTACCATGGCTCTCATCCCAGACTTGG-3’

SEQ ID No: 2 Reverse: 5’- ATT CTCGAGTCAATGATGATGATGATGATGGGCTCCACTTACGGTGCC-3’

Utilize above-mentioned primer to angle from people's gene storehouse, laboratory to get CYP3A4 cDNA(and before the terminator codon of this gene open reading frame, inserted the gene order of 6 Histidines of encoding) [10], its nucleotide sequence is as shown in SEQ ID No:3.PcDNA3.1 (+)-Hygro plasmid (purchased from Invitrogen company) of take is carrier, CYP3A4 cDNA is passed through to Kpn I, two restriction enzyme sites of Xho I are cloned on this carrier, and called after pcDNA3.1 (+)-Hygro-CYP3A4, as expression plasmid.With E.coli DH5 α bacterial strain amplification plasmid, bacterial classification is placed on the LB solid medium containing ammonia benzyl to 37 ℃ of incubator overnight incubation.Until next day, on substratum, grow after bacterial plaque, picking list bacterium colony is in increasing containing in the LB liquid nutrient medium of ammonia benzyl, use Axygen test kit to extract plasmid purification, use Kpn I, two restriction enzymes of Xho I carry out double digestion evaluation, and enzyme is cut system (10 μ L): pcDNA3.1 (+)-Hygro-CYP3A4 plasmid 5 μ L, 10 * M buffer, 1 μ L, Kpn I 1 μ L, Xho I 1 μ L, aqua sterilisa 2 μ L.37℃，2h。Agarose gel electrophoresis through 1% is identified (see figure 1), and enzyme is cut and obtained the CYP3A4 goal gene fragment of about 1.5kb and pcDNA3.1 (+)-Hygro carrier segments of about 5.6kb, identifies that correct plasmid is for cell transfecting.In Fig. 1,1,2,3,4 swimming lanes are four complete plasmid samples; 5,6,7,8 swimming lanes are that corresponding enzyme is cut sample, obtain two bands, the CYP3A4 goal gene of about 1.5kb, the carrier segments of about 5.6kb.Plasmid through order-checking show, successfully connect the CYP3A4 cDNA with Histidine (His) label, full length gene is without base mutation.

.MDCK the stable transfection of serial cell

MDCK-pcDNA3.1 (+) cell and MDCK-hOCT1 cell are with 2 * 10 ⁵/ hole kind, in 6 orifice plates, starts transfection when cell covers 70-80%.2.5 μ g pcDNA3.1 (+)-Hygro-CYP3A4 plasmids are diluted in to 100 μ l serum-free DMEM, add again 2.5 μ lPLUS Reagents, incubated at room 5min, mixes incubated at room 30min with the serum-free DMEM 100 μ l systems that contain 5 μ lLipofectamine LTX.Liposome plasmid complex compound is dripped in containing the treating in transfectional cell of 1000 μ l serum-free DMEM, front and back shake up gently, after 6h, be replaced by containing 10% serum DMEM substratum, continue to cultivate after 48h, 10% blood serum medium that contains being changed to containing 700 μ g/mL G418 and 400 μ g/mL hygromycin B carries out cell screening.Resistance screening 10-14 days, treats that cellular control unit is all dead, and transfection group cell raised growth, by cell dissociation, adopts limiting dilution assay kind to enter in 96 orifice plates.Under microscope, select monoclonal cell, with containing 700 μ g/mL G418, the substratum of 400 μ g/mL hygromycin B continues to cultivate, and changes every other day liquid.Continue to cultivate after approximately 10 days, monoclonal cell grows up to larger one, and digestion proceeds to 24 orifice plates and cultivates.MDCK-pcDNA3.1 (+) and the equal transfection pcDNA3.1 of MDCK-hOCT1 cell (+)-Hygro empty carrier plasmid are done with method resistance screening, in contrast cell simultaneously.So far, obtain altogether four kinds of cell MDCK-mock (MDCK cotransfection pcDNA3.1 (+) and pcDNA3.1 (+)-Hygro empty carrier plasmid) with stable resistance, MDCK-hOCT1 (MDCK cotransfection pcDNA3.1 (+)-hOCT1 plasmid and pcDNA3.1 (+)-Hygro empty carrier plasmid), MDCK-CYP3A4 (MDCK cotransfection pcDNA3.1 (+) empty carrier plasmid and pcDNA3.1 (+)-Hygro-CYP3A4 plasmid), MDCK-hOCT1-CYP3A4 (MDCK cotransfection pcDNA3.1 (+)-hOCT1 and pcDNA3.1 (+)-Hygro-CYP3A4 plasmid).

.Western blot detects the protein expression of monoclonal cell CYP3A4

The preparation of 3.1 protein samples

The monoclonal cell of the single transfection CYP3A4 obtaining and cotransfection hOCT1 and CYP3A4 is continued to cultivate, after covering with cell bottle, cleans three times with ice-cold PBS in cell, with cell scraper, scrape, in 4 ℃, the centrifugal 5min of 2000g, collecting cell, add and contain PMSF, the RIPA lysate 150 μ l of the proteinase inhibitor such as Aprotimin and Leupeptin, piping and druming mixes, cracking 30min on ice, during this time repeatedly piping and druming several times, after cracking completes 4 ℃, the centrifugal 5min of 13000g, get supernatant liquor and be total protein sample, be sub-packed in-80 ℃ of preservations stand-by.Protein sample adopts BCA method to measure protein concentration, calculates 50 μ g albumen desirable proteins sample volumes, and adds 4 μ l 5 * SDS sample-loading buffers, and adding that water supplies is 20 μ l loading systems, and sample boils 10min and gets final product loading in boiling water.

3.2 Western blot method is selected the monoclonal cell that CYP3A4 expresses

SDS-PAGE gel electrophoresis adopts 5% concentrated glue, 7.5% separation gel, and 20 μ g albumen loadings, 90V electrophoresis 30min, 120V electrophoresis 60min, pvdf membrane is at 200mA, transferring film 2h under condition of ice bath.With TBST damping fluid, prepare 7.5% skim-milk, transferring film finishes to take out transfer film and in skimmed milk, seals 2h, object band (CYP3A4) primary antibodie is the anti-His monoclonal antibody of mouse 1:1200 dilution, internal reference (GAPDH) primary antibodie is the anti-GAPDH monoclonal antibody of mouse 1:20000 dilution, 4 ℃ of overnight incubation, TBST washes film 5min * 6 time, the anti-1:5000 dilution of horseradish peroxidase mark goat anti-mouse two, incubated at room 2h, washes film with preceding method.EZ-ECL Chemiluminescence Detection Kit(ECL) chemical illuminating reagent A liquid, B liquid are appropriate, 1:1 mixes and drips on film, and reaction 3min, faces up membranin to place in exposure box, with the film for medical X-ray radiography compressing tablet several seconds, automatic processor is developed a film.Select result referring to Fig. 2, Fig. 3.In Fig. 2, D1-D91 is the dual-transfected hOCT1 that expresses of the obvious CYP3A4 of having of picking out and the monoclonal cell of CYP3A4; Mock is the mdck cell of transfection empty carrier, and M/O is the cell of single hOCT1 of expression, as two negative controls; Sf9 is for to confirm to express the insect cell expression system of CYP3A4, as positive control.S3 in Fig. 3, S4, S56, S57 is the monoclonal cell of single transfection CYP3A4 of expressing of the obvious CYP3A4 of having of picking out; Mock, the negative contrast of MDCK-hOCT1; The positive contrast of Sf9.

Through western blot, repeatedly compare, from above cell, pick out single transfection and dual-transfected cell that CYP3A4 expression amount is suitable, so that the relatively effect of hOCT1 in the situation that enzymic activity is suitable in follow-up study.Referring to Fig. 4, be numbered single transfectional cell of S4 and be numbered in the dual-transfected cell of D60 CYP3A4 expressing quantity basically identical, definite designation is MDCK-CYP3A4 and MDCK-hOCT1-CYP3A4.In Fig. 4, S4 is the monoclonal cell of single transfection CYP3A4 of expressing of the obvious CYP3A4 of having of picking out, D60 be with S4 in the suitable coexpression CYP3A4 of CYP3A4 expression amount and the monoclonal cell of hOCT1, mock is empty carrier control cells; M/O is MDCK-hOCT1 cell.

detect monoclonal cell hOCT1, the expression amount of CYP3A4 mRNA

Use RNAsimple Total RNA Kit total RNA extraction reagent box (RNAsimple Total RNA Kit, day root), extract MDCK-mock, MDCK-CYP3A4, MDCK-hOCT1, total RNA of MDCK-hOCT1-CYP3A4 cell, carries out total rna concentration mensuration after having extracted.Use PrimeScript ^tMrT reagent Kit (Perfect Real Time, Takara) is cDNA by total RNA reverse transcription, and reaction system is pressed table 1 preparation, and RNA is in-80 ℃ of preservations for residue.Reverse transcription condition is: 37 ℃, and 15 min(reverse transcription reactions); 85 ℃, the inactivation reaction of 5 sec(ThermoScript II); 4 ℃.

Obtain after the cDNA of cell, design hOCT1, CYP3A4, the exclusive primer of dog GAPDH carries out Real-time PCR, detects hOCT1 in cell, the mrna expression situation of CYP3A4, take dog GAPDH as reference gene simultaneously, hOCT1 to different cells, the mRNA of CYP3A4 carries out relative quantification, and the primer sequence is referring to table 2.Take the cDNA that obtains as template (dilute 10 times of uses afterwards), use SYBR premix Ex Taq ^tM(TliRNaseH Plus) carries out RealTime PCR on StepOne plus Real-Time PCR system, and reaction system is prepared in Table 3.

Real-time PCR reaction conditions is: 95 ℃ of reaction of degeneration 1min, and amplification cycles 40 times, loop parameter: 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 72 ℃ are extended 10s.Primer is according to above-mentioned conditioned response, and melt curve analysis is special unimodal figure.Experimental result adopts

relative quantification method, wherein △ CT=CTtarget gene-Avg.CT GAPDH.The demonstration of Real-time PCR result, hOCT1 and/or CYP3A4 mRNA be high expression level (Fig. 5 A, 5B) in its corresponding transfectional cell all, and in the cell of untransfected, all almost can't detect its corresponding mrna expression.At MDCK-hOCT1, in MDCK-hOCT1-CYP3A4 cell, hOCT1 mrna expression amount consistent (Fig. 5 A).Similar with hOCT1, the expression amount of CYP3A4mRNA in MDCK-CYP3A4 and MDCK-hOCT1-CYP3A4 cell is also at same level (Fig. 5 B).Fig. 5 be take people GAPDH as internal reference, and the cell of transfection hOCT1 is with respect to the equal high expression level hOCT1 of blank cell, and the cell of transfection CYP3A4 is with respect to the equal high expression level CYP3A4 of blank cell, and data represent with mean ± SD, n=3.

. exclusive substrate checking cell hOCT1, the function of CYP3A4

the checking of 5.1 cell CYP3A4 metabolic activities

Adopt P450-Glo CYP3A4 Assay (Luciferin-IPA) test kit to identify the function of CYP3A4, the omnidistance lucifuge operation of identification experiment.Cell is with 5 * 10 ⁴/ hole kind 96 orifice plates, carry out functional verification next day.Substratum is abandoned in suction, with fresh culture, Luciferin-IPA is diluted to 3 μ M, the 50 every hole of μ L application of samples, and 37 ℃ of cell culture incubators are hatched 1h; Inhibitor control group is set simultaneously, and inhibitor group is first by the substratum that contains 1 μ M KETOKONAZOL 50 μ L preincubate 20min, then the substratum 50 μ L that contain 1 μ M KETOKONAZOL and 3 μ MLuciferin-IPA are hatched with method; Alternative is got blank well and is operated with method, as background, contrasts; Hatch after end, get 25 μ L Incubating Solutions, add equivalent Luciferin Detection Reagent to start luminous reaction, after room temperature reaction 20min, with Chemiluminescence Apparatus, detect luminous intensity.The cell of hatching is washed three times with PBS, adds 0.1%SDS cracking, and collecting cell lysate is measured protein concentration with BCA method, to proofread and correct luminous intensity.CYP3A4 metabolic activity represents with the net luminescence intensity of sample unit's protein concentration, after sample luminous intensity deduction background luminescence intensity divided by sample total protein content (Luminescence/ μ g protein).Result shows, the cell of transfection CYP3A4 is significantly higher than mock cell to Luciferin-IPA metabolic activity, and the cell metabolic activity of single transfection CYP3A4 and cotransfection CYP3A4 and hOCT1 is suitable, add after CYP3A4 inhibitor KETOKONAZOL, metabolic activity is subject to obvious inhibition, illustrate and obtain the cell of stablizing high expression level CYP3A4, referring to Fig. 6, in Fig. 6, Luciferin-IPA is high-sensitive CYP3A4 substrate, the cell of transfection CYP3A4 all demonstrates the metabolic activity that more blank cell is high, in figure, data represent with mean ± SD, n=3.

the functional verification of cell hOCT1

The cell that screening is obtained carries out hOCT1 functional verification, to confirm in screening process that in cell, whether hOCT1 function is stable.Cell is with 5 * 10 ⁴/ hole kind, in 96 orifice plates, is tested after 24h.Select the fluorogenic substrate ASP of hOCT1 ⁺gather experiment, inhibitor control group is set simultaneously, select the classical inhibitor TEA of hOCT1 to suppress the picked-up of substrate.Substratum is abandoned in suction, and with the PBS cleaning cell twice of 37 ℃ of preheatings, substrate group contains 1 μ M ASP ⁺hBSS solution, inhibitor group contains 1 μ M ASP ⁺and the HBSS solution of 3mM TEA, dosage is the 50 every holes of μ L, and 37 ℃ of cell culture incubator lucifuges are hatched 15min, choose the hole of the full cell of same kind, only give equivalent HBSS and hatch as background contrast, microplate reader is measured fluorescence intensity, excitation wavelength 475nm, emission wavelength 605nm.After cells accumulation finishes, with PBS, clean cell three times, add 0.1%SDS lysing cell, collect lysate and measure protein concentration, with correction of fluorescence intensity.Sample accumulated amount represents with the relative value of itself and mock cell, after fluorescent intensity deduction background than upper protein concentration again with the ratio of mock cell.Result shows, ASP in the cell of transfection hOCT1 ⁺gather and be all significantly higher than mock cell, and MDCK-hOCT1 and MDCK-hOCT1-CYP3A4 are to ASP ⁺centralize ability suitable, hOCT1 inhibitor TEA significantly reduces ASP in transfectional cell ⁺gather that (Fig. 7, in figure, data represent with mean ± SD, n=3.), illustrate that cell hOCT1 function is good.

In conjunction with mRNA, protein expression, and functional verification, showing MDCK-hOCT1, the hOCT1 in MDCK-hOCT1-CYP3A4 cell is compared with mock cytotostatic high expression level, and function approaches; MDCK-CYP3A4, CYP3A4 in MDCK-hOCT1-CYP3A4 cell is compared with mock cytotostatic high expression level, and active in same level, the above results shows: list is expressed hOCT1 respectively, CYP3A4 and coexpression hOCT1, the cell model of CYP3A4 successfully constructs, can be for research hOCT1, the synergy of CYP3A4.

application example

6.1 cell models are for retrorsine Study of cytotoxicity

Retrorsine (RTS) is the stronger alkaloid of a kind of hepatotoxicity in Pyrrolizidine alkaloid.Research shows, in human body, it mainly produces electrophilicity active metabolite by CYP3A4 metabolism, and with DNA, thereby the macromole such as albumen are in conjunction with producing toxicity.Utilize the cell model of this research and establishment, we investigate the toxicity process of hOCT1, CYP3A4 mediation.MDCK-mock, MDCK-hOCT1, MDCK-CYP3A4, MDCK-hOCT1-CYP3A4 cell is 2000/ hole kind 96 orifice plates respectively.Cultivate after 24h, the substratum 200 μ L that contain 200 μ M RTS are hatched 72h, and the Incubating Solution that contains equivalent DMSO of usining is hatched as blank group with method.Add the PBS solution 20 μ L that contain 5mg/mL MTT and continue to hatch 4h, inhale and abandon Incubating Solution, add 150 μ L DMSO, concussion 10min fully dissolves crystallization.Select 570nm, 630nm wavelength is measured absorbancy in microplate reader, and the percentage ratio of the white control group of administration group absorbancy duty of take calculates survival rate (blank group is 100%).Result as shown in Figure 8, the cell survival rate of transfection CYP3A4 obviously reduce ( p<0.001), illustrate that CYP3A4 produces in cytotoxic process and plays keying action at RTS; And dual-transfected cell survival rate compared with other cell all low ( p<0.001), illustrate that hOCT1 also plays an important role in the toxicity process of RTS.Fig. 8 is that cell is hatched after 72h in containing the substratum of 200 μ MRTS, carries out MTT experiment.Survival rate is organized with the percentage ratio of blank group (DMSO group) and is represented with RTS, and in figure, data represent with mean ± SD, n=3.With the comparison of mock cell, * * * p< 0.001; With MDCK-CYP3A4 comparison, ^### p<0.001.

cell model is for toxicity and the attenuation mechanism research of nitidine chloride

Nitidine chloride belongs to protoberberine alkaloid, the cell model that utilizes this research to set up, and we have verified hOCT1 and the vital role of CYP3A4 in this alkaloid cytotoxicity.MDCK-mock, MDCK-hOCT1, MDCK-hOCT1-CYP3A4 cell is all with 1 * 10 ⁴/ hole kind, in 96 orifice plates, is cultivated after 24h, and the substratum 200 μ L that contain different concns nitidine chloride continue to hatch 24h, and the substratum that contains equivalent DMSO of usining is hatched as blank with method.Hatch after end, by " 6.1 " described method, carry out MTT experiment, the cytotoxicity of checking nitidine chloride.Experimental result as shown in Figure 9, the cell survival rate of the cell of transfection hOCT1 and cotransfection hOCT1 and CYP3A4 be starkly lower than mock cell ( p<0.001), and the cell survival rate of cotransfection hOCT1 and CYP3A4 more singly turn the obvious rising of having of hOCT1 ( p<0.001).Illustrate that hOCT1 plays keying action in the cytotoxic process of nitidine chloride, and CYP3A4 plays attenuation by the metabolism of mediation nitidine chloride.Fig. 9 is that cell and different concns nitidine chloride are hatched after 24h, carries out MTT experiment.The cell survival rate of drug treating group is with the percentage ratio of the white control group of its duty (DMSO group), and in figure, data represent with mean ± SD, n=3.* * p<0.001, compares with mock cell; ^### p<0.001, compares with MDCK-hOCT1 cell.

Above-mentioned two application examples show, the cell model that this research is set up can be used for studying OCT1 and CYP3A4 independent and common effect in the disposal process of medicine.

The reference the present invention relates to:

[1] Perera MA. The missing linkage: what pharmacogenetic associations are left to find in CYP3A Expert Opin Drug Metab Toxicol 2010;6:17-28。

[2] Thummel K, Wilkinson G. In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol 1998;38:389-430。

[3] Huang S-M, Woodcock J. Transporters in drug development: advancing on the Critical Path. Nature Reviews Drug Discovery 2010;9:175-6。

[4] Müller F, Fromm MF. Transporter-mediated drug–drug interactions. Pharmacogenomics 2011;12:1017-37。

[5] Neuhoff S, Ungell A-L, Zamora I, Artursson P. pH-dependent bidirectional transport of weakly basic drugs across Caco-2 monolayers: implications for drug–drug interactions. Pharm Res 2003;20:1141-8。

[6] Hagenbuch B. Drug uptake systems in liver and kidney: a historic perspective. Clin Pharmacol Ther 2009;87:39-47。

[7] Ji L, Zhao X, Chen L, Zhang M, Wang Z. Pyrrolizidine alkaloid clivorine inhibits human normal liver L-02 cells growth and activates p38 mitogen-activated protein kinase in L-02 cells. Toxicon 2002;40:1685-90。

[8] Gomez-Lechon M, Lahoz A, Gombau L, Castell J, Donato M. In vitro evaluation of potential hepatotoxicity induced by drugs. Curr Pharm Des 2010;16:1963-77。

[9] Tu M, Sun S, Wang K, Peng X, Wang R, Li L, et al. Organic cation transporter 1 mediates the uptake of monocrotaline and plays an important role in its hepatotoxicity. Toxicology 2013;311:225-30。

[10] Wang Xiaowen, once Soviet Union. human-cytochrome P450 3A4 mutant CYP3A4. 3, CYP3A4. 4, and the heterogenous expression of CYP3A4. 5 and CYP3A4. 18 recombinases is with active. Chinese J Pharmacol Toxicol 2009; 23:456-63.

<110> Zhejiang University

The cell model construction and application of <120> and cytochrome P 450 enzymes coexpression

<160> 9

<210> 1

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> is with the people CYP3A4 specificity upstream primer of restriction enzyme site

<440> 1

att ggt acc atg gct ctc atc cca gac ttg g

<210> 2

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> is with restriction enzyme site and histidine-tagged CYP3A4 specificity downstream primer

<440> 2

att ctc cag tca atg atg atg atg atg atg ggc tcc act tac ggt gcc

<210> 3

<211> 1530

<212> DNA

<213> artificial sequence

<220>

<223> termination codon is before with six histidine-tagged people CYP3A4 cDNA sequences

<440> 3

atg gct ctc atc cca gac ttg gcc atg gaa acc tgg ctt ctc ctg 45

gct gtc agc ctg gtg ctc ctc tat cta tat gga acc cat tca cat 90

gga ctt ttt aag aag ctt gga att cca ggg ccc aca cct ctg cct 135

ttt ttg gga aat att ttg tcc tac cat aag ggc ttt tgt atg ttt 180

gac atg gaa tgt cat aaa aag tat gga aaa gtg tgg ggc ttt tat 225

gat ggt caa cag cct gtg ctg gct atc aca gat cct gac atg atc 270

aaa aca gtg cta gtg aaa gaa tgt tat tct gtc ttc aca aac cgg 315

agg cct ttt ggt cca gtg gga ttt atg aaa agt gcc atc tct ata 360

gct gag gat gaa gaa tgg aag aga tta cga tca ttg ctg tct cca 405

acc ttc acc agt gga aaa ctc aag gag atg gtc cct atc att gcc 450

cag tat gga gat gtg ttg gtg aga aat ctg agg cgg gaa gca gag 495

aca ggc aag cct gtc acc ttg aaa gac gtc ttt ggg gcc tac agc 540

atg gat gtg atc act agc aca tca ttt gga gtg aac atc gac tct 585

ctc aac aat cca caa gac ccc ttt gtg gaa aac acc aag aag ctt 630

tta aga ttt gat ttt ttg gat cca ttc ttt ctc tca ata aca gtc 675

ttt cca ttc ctc atc cca att ctt gaa gta tta aat atc tgt gtg 720

ttt cca aga gaa gtt aca aat ttt tta aga aaa tct gta aaa agg 765

atg aaa gaa agt cgc ctc gaa gat aca caa aag cac cga gtg gat 810

ttc ctt cag ctg atg att gac tct cag aat tca aaa gaa act gag 855

tcc cac aaa gct ctg tcc gat ctg gag ctc gtg gcc caa tca att 900

atc ttt att ttt gct ggc tat gaa acc acg agc agt gtt ctc tcc 945

ttc att atg tat gaa ctg gcc act cac cct gat gtc cag cag aaa 990

ctg cag gag gaa att gat gca gtt tta ccc aat aag gca cca ccc 1035

acc tat gat act gtg cta cag atg gag tat ctt gac atg gtg gtg 1080

aat gaa acg ctc aga tta ttc cca att gct atg aga ctt gag agg 1125

gtc tgc aaa aaa gat gtt gag atc aat ggg atg ttc att ccc aaa 1170

ggg gtg gtg gtg atg att cca agc tat gct ctt cac cgt gac cca 1215

aag tac tgg aca gag cct gag aag ttc ctc cct gaa aga ttc agc 1260

aag aag aac aag gac aac ata gat cct tac ata tac aca ccc ttt 1305

gga agt gga ccc aga aac tgc att ggc atg agg ttt gct ctc atg 1350

aac atg aaa ctt gct cta atc aga gtc ctt cag aac ttc tcc ttc 1395

aaa cct tgt aaa gaa aca cag atc ccc ctg aaa tta agc tta gga 1440

gga ctt ctt caa cca gaa aaa ccc gtt gtt cta aag gtt gag tca 1485

agg gat ggc acc gta agt gga gcc cat cat cat cat cat cat tga 1530

<210> 4

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> is for the specificity upstream primer of the hOCT1 of quantitative PCR

<440> 4

ccc aca ttc gtc agg aac ctc gga

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> is for the specificity downstream primer of the hOCT1 of quantitative PCR

<440> 5

caa gca ggc cca aca ccg ca

<210> 6

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> is for the specificity upstream primer of the CYP3A4 of quantitative PCR

<440> 6

tcc att cct cat ccc aat tct tga

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> is for the specificity downstream trip primer of the CYP3A4 of quantitative PCR

<440> 7

tcc act cgg tgc ttt tgt gt

<210> 8

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> is for the specificity upstream primer of the dog GAPDH of quantitative PCR

<440> 8

agg agc gag atc ccg cca aca

<210> 9

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> is for the specificity upstream primer of the dog GAPDH of quantitative PCR

<440> 9

ccg cct ttc aag tga gcc cca

Claims (3)

1. one kind builds with the cell model of cytochrome P 450 enzymes coexpression, it is characterized in that, by following steps, realizes:

Design contains Kpn I, the primer of two restriction enzyme sites of Xho I, primer sequence is as SEQ ID No:1, shown in 2, by before terminator codon, with histidine-tagged people CYP3A4 cDNA, be cloned on vector plasmid pcDNA3.1 (+)-Hygro, construction expression plasmid pcDNA3.1 (+)-Hygro-CYP3A4, expression plasmid transfection MDCK-pcDNA3.1 (+) and MDCK-hOCT1 cell, after transfection, by aminoglycoside antibiotics G418 and hygromycin B, carry out resistance screening, select monoclonal cell, monoclonal cell carries out the protein expression of western blot checking CYP3A4, obtain MDCK-CYP3A4 and the MDCK-hOCT1-CYP3A4 cell of stably express CYP3A4, and select the consistent cell of CYP3A4 expressing quantity by Western blot, extract total RNA, specific primer by primer sequence as shown in SEQ ID No:4-9 carries out the mrna expression of CYP3A4 and hOCT1 in Real-time PCR checking cell, and compare with empty carrier cell and MDCK-hOCT1, and then it is active with P450-Glo CYP3A4 Assay test kit, to detect CYP3A4, and investigate the impact of KETOKONAZOL on metabolic activity, thereby confirm the function of transfectional cell CYP3A4, application hOCT1 fluorogenic substrate 4-(4-(dimethylami-no) styryl)-N-methylpyridinium, in conjunction with the function of hOCT1 inhibitor triethylammonium tetrakis checking cell hOCT1, wherein the nucleotide sequence of people CYP3A4 cDNA is as shown in SEQ ID No:3.

2. the application of the cell model of a kind of and cytochrome P 450 enzymes coexpression that method builds according to claim 1 in the hepatotoxicity of research CYP3A4 and hOCT1 mediation.

3. application according to claim 2, it is characterized in that, by following steps, realize: singly turn, two turn and mock cell gives the testing compound of different concns, hatch certain hour, adopt mtt assay to detect the survival rate of cell, by relatively more singly expressing, coexpression hOCT1 is or/and the survival rate of the cell of CYP3A4 and mock cell confirms that hOCT1 and CYP3A4 act in the cytotoxicity of testing compound.

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