CN102533925A - Method for in-vitro screening of PXR activation characteristics - Google Patents
Method for in-vitro screening of PXR activation characteristics Download PDFInfo
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Abstract
The invention relates to a method for in-vitro screening inducers, particularly to a method for in-vitro screening of PXR (pregnane X receptor) activation characteristics, which comprises the following steps: constructing a reporter gene vector; culturing cells, and screening G418 working concentration; carrying out transient transfection of cells; screening stably transfected cell clones; inducing and identifying stably transfected cell strain with a tested drug (ligand drug); and screening the PXR activation characteristics. When the PXR is activated by the ligand drug, the PXR can regulate the expression CYP3A gene. PXR gene-deficient mice lose drug inductivity of CYP3A. Contrarily, hPXR (human pregnane X receptor) receptor in activation state expressed in liver of a transgenic mouse can lead to continuous high expression of CYP3A enzyme. The method of the invention establishes critical technology for high-throughput screening based on the passway and carries out screening of PXR activation characteristics from mass compounds in a compound library at the early stage of drug development, and can reduce the risk of adverse drug interactions after the new drug comes into the market, and greatly reduce development cost.
Description
Technical field
The present invention relates to a kind of external evoked dose screening method, particularly external PXR activates the method method of characteristic examination.
Background technology
It must be that polar compound excretes with some exogenous material metabolic conversion that body is kept normal physiological function, and this removing and toxicide process mainly depend on liver system cells cytochrome p 450 (CYP) gene expression regulation and realize.The medicine of treating all kinds of diseases simultaneously is more and more, and drug combination has become common treatment means, and the untoward reaction that drug drug interaction causes also increases thereupon, and is wherein very most of relevant with drug interaction.It is relevant with drug metabolism that interactional mechanism takes place most medicines, especially with cytochrome P 450 enzymes (cytochrome P450, CYP450) relevant metabolism.The super large family that Cytochrome P450 is made up of many isozymes; That participates in drug metabolism mainly contains CYP3A4, CYP1A2, CYP2C9, CYP2C19, CYP2D6 etc., and wherein CYP3A4 is the gang of tool significance, and about 50% medicine is through its metabolism; Itself and drug metabolism are the most closely related; Therefore be necessary the drug safety evaluation is incorporated into the early stage of medicament research and development, find the drug interaction that possibly exist, the security that improves original new drug through the foundation of relevant triage techniques.Research show multiple medicine can pass through nuclear receptor PXR (pregnane X acceptor, Pregnane X Receptor, thereby PXR) induce the CYP3A expression of gene.434 amino acid of PXR albumen total length, its structure comprise the DNA land that aminoterminal is conservative and the ligand binding domain of carboxyl terminal.Medicine can directly combine with the interior PXR ligand binding domain of nuclear; Then with another nuclear receptor retinene1 X acceptor (Retinoid X receptor; RXR NR2B1) forms heterodimer, and this heterodimer combines with exogenous chemical response element on the target gene promoters; Promote the expression with zymoprotein of transcribing of CYP3A, strengthen its metabolic capacity simultaneously substrate.In view of PXR has brought into play important mediation in the generation of CYP3A enzyme, the activation of PXR acceptor can cause the synthetic acceleration of CYP3A enzyme, not only increases the metabolism of medicine itself, and the metabolism of the interior other drug of meeting acceleration bodies, induce drug-drug interaction.
The reporter gene method is predicted its inductive effect to CYP3A4 through detecting xenobiotics to the activation capability of PXR, is the interactional method of screening of medicaments commonly used at present.Compared reporter gene method and former human liver cell model of being commissioned to train foster dependency when prediction CYP3A4 inductor, the result shows that two kinds of methods have good dependency.Noracharttiyapot W etc. are the independent stable transfection HepG2 of reporter gene carrier cell, and screening has obtained Rifampin is had the functional monoclonal cell strain of clear and definite inductive effect, but its each lysing cell that all needs when detecting fluorescence.
Identified at present multiple can with PXR/RXR dimer bonded direct repeat sequence DR-3, DR-4, DR-5 and DR-8 etc. and response elements such as inverted repeats ER-6 and ER-8.Above-mentioned sequence is distributed in the multiple metabolic enzyme gene promotor relevant with pharmacokinetics, relevant with cyp3a4 mainly is be positioned at the proximal promoter zone ER-6, be positioned at the far-end xenobiotics increased response submodule body (XREM) at 7.2～7.8kb place, the transcription initiation site upper reaches and be positioned at the DR-3 etc. at 8.8kb place, the transcription initiation site upper reaches.Fu-Jun LIU etc. discovers that the DR-3 element in the far-end xenobiotics increased response submodule body (XREM) at 7.2～7.8kb place, the transcription initiation site upper reaches is induced most important to the Rifampin of PXR mediation, and the inducing action of its disappearance back Rifampin disappears basically.
Summary of the invention
The object of the invention is to provide a kind of external PXR to activate the method method of characteristic examination.
The present invention seeks to realize that the method that the external PXR of the present invention activates the characteristic examination comprises the steps: through following technical scheme
1, the structure of reporter gene carrier:
The design primer; With the two luciferase reporter gene plasmids of pGL3-3A4 is template; To dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer does
Table 1 carrier primer makes up table
2, cell cultures: routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole that contains G418, the G418 final concentration is 900 μ g/ml, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3, cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO (DMSO 99.8MIN.) and 10 μ M Rifampins (RIF) processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4, stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector (the hPXR expression vector is provided by Zhejiang University) cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5, the seized medicine of stable transfected cells strain (aglucon medicine) is induced evaluation: preceding 24 hours of dosing, and by 10 * 10
4In amount inoculation stable transfected cells strain to 96 well culture plate of cells/well, every hole adds seized medicine (aglucon medicine), and making seized medicine (aglucon medicine) final concentration is 10 μ M; Adding seized medicine (aglucon medicine) back, carry out fluorometric assay in 24h, 48h absorption cell culture fluid supernatant;
6, SAS 9.2 softwares are used in the statistical study of data, PXR is activated characteristic screen.
Seized medicine of the present invention can be any compound medicine.
Accompanying drawing:
Fig. 1 pGLuc-NEB-3A4-1 reporter gene vector construction
Fig. 2 pGLuc-dou-dis-3A4-9 reporter gene vector construction
The multiple contrast experiment is induced in No. 1, Fig. 3 reporter gene and No. 9 transfections
Fig. 4 a microscopically monoclonal cell; The monoclonal cell of growing in Fig. 4 b culturing bottle
Fig. 5 Rifampin is induced and is identified the functional monoclonal cell strain of stable transfection (each cell strain is compared with DMSO, and * represents P＜0.05)
The various drug-induced experiments of Fig. 6 (various medicines are compared with DMSO, and * represents P＜0.05)
Fig. 7 KETOKONAZOL suppresses experiment (compare with DMSO, * represents P＜0.05) to PXR
The present invention utilizes secretor type Gaussia luciferase reporter gene pGLuc-Basic system; With reporter gene carrier and people PXR expression vector cotransfection HepG2 cell; Obtain the stable transfected cells strain through the screening of G418 pressure; Thereby set up drug-induced dose external rapid screening method, predict whether certain medicine has the tendency of induce drug-drug interaction, the medicament research and development starting stage promptly carries out the examination of PXR activation characteristic and can select more superior candidate compound; Improve the security after new drug goes on the market, and then instructed drug development and clinical application thereof.
Experimental example 1
1 materials and methods
1.1 material
The HepG2 cell strain is available from the consonance cell centre; MEM substratum, non-essential amino acid (NEAA), foetal calf serum are available from Hyclone company; Rifampin is available from Merck company; T4 ligase enzyme, pGLuc-Basic reporter gene plasmid, restriction enzyme, uciferase activity assay kit be available from NEB company, transfection reagent Lipofectamine
TMLTX, G418 are available from invitrogen company; Carry in the plasmid, little extraction reagent kit and gel reclaim test kit available from Promega company; Probest Taq enzyme system is available from TaKaRa company, and the two luciferase reporter gene plasmids of the pGL3-3A4 that builds, hPXR expression vector are provided by Zhejiang University.
1.2 method
1.2.1 reporter gene vector construction: design primer (sequence is seen table 1); With the two luciferase reporter gene plasmids of pGL3-3A4 is template; With primer to dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11); Far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively, two double digestion products are connected, with primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then with the T4 ligase enzyme; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier; Transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-NEB-3A4-1 (hereinafter to be referred as No. 1).With dis-F-Bgl II/dis-R-EcoR I primer to the amplification distal fragment; With its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-dou-dis-3A4-9 (hereinafter to be referred as No. 9), said carrier primer is with table 1.
1.2.2 the screening of cell cultures and G418 working concentration routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃; Substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole (the G418 final concentration is respectively 500,600,700,800,900,1000 μ g/ml) that contains G418; Keep the G418 concentration stabilize constant when changing liquid, continue to cultivate for 3 weeks.
1.2.3 cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA.24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates.During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9; Be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h, cultivate 12h after; Be replaced by the drug-treated substratum that contains DMSO and 10 μ M Rifampins, continue to cultivate and detecting uciferase activity on 24, the 48 little time points after the dosing.Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole.
1.2.4 stable transfected cells clone's screening screening adopts G418 pressure to select and the limiting dilution assay cloning.With No. 9 and PXR expression vector cotransfection HepG2 cell; Wink change cell and use trysinization, do the cell counting, by 200 cell transfer to 96 orifice plates in every hole; Add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml; After cultivating for 3 weeks, obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively.The cell strain of stable transfection is kept cultivation with the full substratum that contains 500 μ g/ml.
1.2.5 inducing, identifies and preceding 24 hours of various drug-induced experiment dosing stable transfected cells strain Rifampin, by 10 * 10
4The amount of cells/well is seeded in 96 well culture plates, and every hole adds medicine, and making the medicine final concentration is 10 μ M.Different time points is drawn the cell culture fluid supernatant after dosing, carries out fluorometric assay.
1.2.6 SAS 9.2 softwares are used in the statistical study of statistical analysis data.
2 results
2.11 the structure of number reporter gene plasmid is a template with the two luciferase reporter gene plasmids of pGL3-3A4; With primer to dis-F/dis-R and pro-F/pro-R increase respectively the CYP3A4 distal promoter regulating and controlling sequence (Dis fragment) that obtains about 600bp and the proximal promoter sequence (Pro fragment) of about 400bp; With the T4 ligase enzyme two double digestion products are connected behind the double digestion separately; Amplification obtains the far-end regulating and controlling sequence of about 1kb and the fragment of near-end regulating and controlling sequence linear series to dis-F/pro-R with primer then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier; Transformed into escherichia coli is cut and sequence verification through enzyme, is built into No. 1 reporter gene plasmid (Fig. 1).
2.29 the structure of number reporter gene plasmid with dis-F-Bgl II/dis-R-EcoR I primer to about 600bp distal fragment that increases; It is connected into the Bgl II/EcoR I site of No. 1 carrier; Transformed into escherichia coli is after the PCR checking has positive colony at the 1.7kb place; Cut and sequence verification through enzyme, be built into No. 9 reporter gene plasmids (Fig. 2).
2.3 change in wink the plain enzyme activity assay of cell fluorescence with No. 1 with No. 9 reporter gene plasmids respectively with hPXR carrier for expression of eukaryon corotation HepG2 cell, behind the transfection 12h, dosing continues processing cell 48h, uses BioLux
TMGaussia Luciferase Assay Kit detects.The result shows that 10 μ M Rifampins can obviously be induced No. 1 and No. 9 luciferase reporter genes are expressed; 3.98 times and 5.72 times of the highest raisings of the relative negative control group difference of its fluorescent value; And on 12h, 24h, 36h, four time points of 48h, No. 9 the multiple of inducing all will be higher than No. 1, sees Fig. 3.
On 12h, 24h, 36h, four time points of 48h, No. 9 RIF induces and contrasts fluorescent value intensity and all will see table 2 apparently higher than No. 1.
Table 2 reporter gene changes RIF wink No. 1 and No. 9 and induces and contrast fluorescent value intensity
2.4G418 definite 1000,900,800 μ g/ml holes of working concentration were in the 3rd day, 700 and 600 μ g/ml holes were in the 4th day, and 500 μ g/ml holes began to occur cell growth-inhibiting phenomenon in the 10th day, and cell begins dissolving.After 20 days, each porocyte stable growth of G418 concentration 800 μ g/ml, and two porocytes of 1000,900 μ g/ml are all dead in succession.Therefore, can think that the best effort concentration of G418 is 900 μ g/ml under this experiment condition.
2.5 stable transfection HepG2 monoclonal cell strain screening is with No. 9 and PXR expression vector cotransfection HepG2 cell; After cultivating for 3 weeks with the G418 screening of medium of 900 μ g/ml; Obtain the monoclonal cell (Fig. 4) of stable growth; It is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively, obtains 8 strain of strain monoclonal cell, called after 9+PXR-1～9+PXR-8 successively altogether.
2.6 stable transfected cells strain Rifampin induces evaluation and various drug-induced experiment dosing to handle preceding 24 hours, and 8 strain monoclonal cells are pressed 10 * 10
4The amount of cells/well is seeded in 96 well culture plates; 10 μ M Rifampins were handled after 24 hours, and the 9+PXR-3 cell strain shows the best effect of inducing, with solvent control DMSO group ratio; Inducing multiple is 4.05 (Fig. 5), selects this cell strain to carry out further various drug-induced experiments.
Under 10 μ M concentration, clotrimazole, DEXAMETHASONE BP98, nifedipine, omeprazole, troglitazone are respectively 1.9 times and 1.9 times, 1.8 times and 2.2 times, 2.8 times and 3.4 times, 2.5 times and 3.1 times, 3.8 times and 3.6 times (the P value is all less than 0.05) at the multiple of inducing of 24h and 48h; Phenytoin Sodium Salt is respectively 1.3 times and 1.3 times at the multiple of inducing of 24h and 48h, and comparing with solvent control DMSO group does not have significant difference (Fig. 6).Known PXR suppressor factor KETOKONAZOL, under 10 μ M, two kinds of concentration of 25 μ M, that has all suppressed Rifampin significantly induces effect (Fig. 7).
3, conclusion
When PXR was activated by the aglucon medicine, people's PXR/RXR dimer can be incorporated on the response element in the target CYP gene promoter (Promoter), thus regulation and control CYP3A genetic expression.PXR genetically deficient mouse loses the drug-induced property of CYP3A.The persistence high expression level that can cause on the contrary, the CYP3A enzyme at the hPXR acceptor of transgenic mice liver expression state of activation.Because the sub theory of crucial transcriptional control that PXR induces CYP3A to express as xenobiontics is widely accepted; Therefore the present invention has set up based on the gordian technique of this path high flux screening and in medicament research and development and promptly the magnanimity compound in the compound library has been carried out the examination of PXR activation characteristic in early days; Can reflect indirectly whether it has the inducing properties of CYP3A; The compound that can activate the PXR acceptor possibly cause drug interaction or the toxigenous meta-bolites do not expected; The research and development starting stage is promptly eliminated the risk that these compounds can reduce the bad drug interaction of new drug listing back generation, and the research and development expense is reduced greatly.
Among the present invention; The contriver has designed No. 9, two far-end enhanser reporter gene carriers, sees from inducing experimental result, and Rifampin is to No. 9 No. 1 carrier (Fig. 3) of inducing multiple to be higher than to have only a far-end enhanser; And No. 9 fluorescent value also will be higher than No. 1 (table 2) far away; The existence of two far-end enhansers is described, has been improved the usefulness of reporter gene carrier, such carrier is not seen bibliographical information before this as yet.
Chemical compound lot comprise prescription drugs Rifampin, St.John ' s Wort and some agrochemicals can with the PXR receptors bind.Medicine is the major cause that causes the clinical medicine interphase interaction to the inductive effect of metabolic enzyme CYP450.Therefore in drug research and performance history; Set up the method for drug-induced dose of examination CYP450; Eliminate the lead compound of inducing CYP450 to express; And the new drug of multiple exhibition number of C YP450 transparent (CYP450-transparent does not promptly have CYP450 and induces or suppress active), for reducing the new drug development cost and avoiding drug interaction clinically significant.
The present invention utilizes secretor type Gaussia luciferase reporter gene pGLuc-Basic system; Made up the reporter gene carrier that comprises CYP3A4 gene promoter regulating and controlling sequence; Through with the external cotransfection HepG2 of people PXR expression vector cell, obtained the strain of stable transfection monoclonal cell down in G418 pressure screening, owing to the luciferase of reporter gene is secreted into outside the born of the same parents; Therefore directly detect cell conditioned medium and get final product, avoided lysing cell.Rifampicin medicine is induced experiment, identifies the 9+PXR-3 cell strain and has the clear and definite effect (Fig. 5) of inducing.The contriver utilizes this cell strain further to detect the activation capability of 7 kinds of existing medicines to PXR; Wherein, nifedipine is known PXR agonist, and KETOKONAZOL is known PXR antagonist; Contriver's result sees (Fig. 6,7), shows that further the inventive method reporter gene method accurately, reliably.The foundation of this screening system is for the generation of prediction CYP3A enzyme induction and drug interaction, for clinical drug application directs and new drug development provide useful information significant.This research meets sets up the new model that early stage, sensitive, quick, reliable drug safety is estimated; Improve the accuracy that drug safety is estimated; Belong to external drug safety and estimate alternative model, can become fast the safety evaluation screening implement and become drug safety and estimate the integral part in the gordian technique.
Embodiment 1: external PXR activates the method for characteristic examination
1), the structure of reporter gene carrier:
The design primer; With the two luciferase reporter gene plasmids of pGL3-3A4 is template; To dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/HindIII site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer is seen table 1
2), cell cultures: routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole that contains G418, the G418 final concentration is 900, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3), cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO and 10 μ M RIF processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4), stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5), stable transfected cells strain clotrimazole induces evaluation: preceding 24 hours of dosing, by 10 * 10
4In amount inoculation stable transfected cells strain to 96 well culture plate of cells/well, every hole adds clotrimazole, and making the clotrimazole final concentration is 10 μ M; After adding clotrimazole, draw the cell culture fluid supernatant, carry out fluorometric assay in 24h, 48h;
6), the statistical study of data uses SAS 9.2 softwares, PXR activated characteristic screen, clotrimazole is respectively 1.9 times and 3.4 times (the P value is all less than 0.05) at the multiple of inducing of 24h and 48h.
Embodiment 2: external PXR activates the method for characteristic examination, and this method comprises the steps:
1), the structure of reporter gene carrier:
The design primer; With the two luciferase reporter gene plasmids of pGL3-3A4 is template; To dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer is seen table 1
2), cell cultures: routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole that contains G418, the G418 final concentration is 900, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3), cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO and 10 μ M RIF processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4), stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5), stable transfected cells strain induced by dexamethasone identifies: preceding 24 hours of dosing, by 10 * 10
4In amount inoculation stable transfected cells strain to 96 well culture plate of cells/well, every hole adds DEXAMETHASONE BP98, and making dexamethasone concentration is 10 μ M; After adding DEXAMETHASONE BP98, draw the cell culture fluid supernatant, carry out fluorometric assay in 24h, 48h;
6), the statistical study of data uses SAS 9.2 softwares, PXR activated characteristic screen, DEXAMETHASONE BP98 is respectively 1.9 times and 2.5 times (the P value is all less than 0.05) at the multiple of inducing of 24h and 48h.
Embodiment 3: external PXR activates the method for characteristic examination
1) structure of reporter gene carrier:
The design primer; With the two luciferase reporter gene plasmids of pGL3-3A4 is template; To dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer is seen table 1
2), cell cultures: routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole that contains G418, the G418 final concentration is 900, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3), cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO and 10 μ M RIF processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4), stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5), stable transfected cells strain nifedipine induces evaluation: preceding 24 hours of dosing, by 10 * 10
4In amount inoculation stable transfected cells strain to 96 well culture plate of cells/well, every hole adds nifedipine, and making nifedipine concentration is 10 μ M; After adding nifedipine, draw the cell culture fluid supernatant, carry out fluorometric assay in 24h, 48h;
6), the statistical study of data uses SAS 9.2 softwares, PXR activated characteristic screen, nifedipine is respectively 1.8 times and 3.1 times (the P value is all less than 0.05) at the multiple of inducing of 24h and 48h.
Embodiment 4: external PXR activates the method for characteristic examination
1), the structure of reporter gene carrier:
The design primer; With the two luciferase reporter gene plasmids of pGL3-3A4 is template; To dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer di s-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer is seen table 1
2), cell cultures: routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole that contains G418, the G418 final concentration is 900, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3), cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO and 10 μ M RIF processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4), stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5), stable transfected cells strain omeprazole induces evaluation: preceding 24 hours of dosing, by 10 * 10
4In amount inoculation stable transfected cells strain to 96 well culture plate of cells/well, every hole adds omeprazole, and making the omeprazole final concentration is 10 μ M; After adding seized medicine, draw the cell culture fluid supernatant, carry out fluorometric assay in 24h, 48h;
6), the statistical study of data uses SAS 9.2 softwares, PXR activated characteristic screen, omeprazole is respectively 2.2 times and 3.8 times (the P value is all less than 0.05) at the multiple of inducing of 24h and 48h
Embodiment 5: external PXR activates the method for characteristic examination
1), the structure of reporter gene carrier:
The design primer; With the two luciferase reporter gene plasmids of pGL3-3A4 is template; To dis-F/dis-R and pro-F/pro-R increase respectively CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer is seen table 1
2), cell cultures: the conventional HepG2 cell of cultivating is in 6 orifice plates; 5%CO2,37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density; Add the nutrient solution 1ml/ hole that contains G418; The G418 final concentration is 900, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3), cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO and 10 μ M RIF processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4), stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5), stable transfected cells strain troglitazone induces evaluation: preceding 24 hours of dosing, by 10 * 10
4In amount inoculation stable transfected cells strain to 96 well culture plate of cells/well, every hole adds troglitazone, and making the troglitazone final concentration is 10 μ M; After adding seized medicine, draw the cell culture fluid supernatant, carry out fluorometric assay in 24h, 48h;
6), the statistical study of data uses SAS 9.2 softwares, PXR activated characteristic screen, troglitazone is induced multiple 2.8 times and 3.6 times (the P value is all less than 0.05) respectively.
Claims (3)
1. an external PXR activates the method for characteristic examination, and this method comprises the steps:
1), the structure of reporter gene carrier:
The design primer; To dis-F/dis-R and pro-F/pro-R increase respectively people CYP3A4 distal promoter regulating and controlling sequence (7833～-7208) and proximal promoter sequence (361～+ 11), far-end regulating and controlling sequence and near-end regulating and controlling sequence are carried out EcoR I-EcoR V and EcoR V-Hind III double digestion respectively with primer, two double digestion products are connected with the T4 ligase enzyme; With primer dis-F/pro-R is carried out far-end regulating and controlling sequence and the segmental clone of near-end regulating and controlling sequence linear series then; Be connected into the EcoR I/Hind III site of pGLuc-Basic carrier, transformed into escherichia coli is cut and sequence verification through enzyme; Carrier called after pGLuc-NEB-3A4-1, promptly No. 1; To the amplification distal fragment, with its Bgl II/EcoR I site that is connected into No. 1 carrier, transformed into escherichia coli is cut and sequence verification through enzyme, carrier called after pGLuc-dou-dis-3A4-9, promptly No. 9 with dis-F-Bgl II/dis-R-EcoR I primer; Said carrier primer does
The carrier primer makes up table
2), the screening of cell cultures and G418 working concentration: routine is cultivated the HepG2 cell in 6 orifice plates, 5%CO
2, 37 ℃, substratum is MEM+10%FBS+1%NEAA, reaches 50% when being covered with to cell density, adds the nutrient solution 1ml/ hole that contains G418, the G418 final concentration is 900 μ g/ml, keeps the G418 concentration stabilize constant when changing liquid, continues to cultivate for 3 weeks;
3), cell transient transfection: the HepG2 cell is cultivated with MEM+10%FBS+1%NEAA; 24h before the transfection is by 20 * 10
4The amount of cells/well is seeded in 24 well culture plates; During transfection with 500ng hPXR expression vector; 300ng1 number or No. 9, be added to 1 culture hole after hatching with 2.5 μ L LipofectamineTM LTX transfection reagents, be replaced by the MEM+10%FBS substratum behind the transfection 7h; After cultivating 12h; Be replaced by and contain 1 μ L DMSO and 10 μ M RIF processing substratum, continue to cultivate, after adding RIF, detecting uciferase activity on 24, the 48 little time points; Multiple 3 times of transfection and detection gross weight, each transfection all is provided with multiple hole;
4), stable transfected cells clone: screening adopts G418 pressure to select and the limiting dilution assay cloning, with No. 9 and PXR expression vector cotransfection HepG2 cell, wink changes cell and uses trysinization; Do the cell counting; By 200 cell transfer to 96 orifice plates in every hole, add the full substratum of MEM that contains G418 by final concentration 900 μ g/ml, cultivated for 3 weeks after; Obtain the cell clone of stable growth, it is transferred to 24 orifice plates, 6 orifice plates and culturing bottle enlarged culturing successively; The cell strain of stable transfection is kept cultivation with the full substratum of MEM that contains 500 μ g/ml G418;
5), the seized medicine of stable transfected cells strain (aglucon medicine) induces evaluation: preceding 24 hours of dosing, in amount inoculation stable transfected cells strain to 96 well culture plate by 10 * 10 cells/well, every hole adds seized medicine, making seized medicine final concentration is 10 μ M; After adding seized medicine, draw the cell culture fluid supernatant, carry out fluorometric assay in 24h, 48h;
6), the statistical study of data uses SAS 9.2 softwares, PXR is activated characteristic screens.
2. external PXR as claimed in claim 1 activates the method for characteristic examination, and the seized medicine in this method is any compound.
3. external PXR as claimed in claim 2 activates the method for characteristic examination, and the seized medicine in this method is clotrimazole, DEXAMETHASONE BP98, nifedipine, omeprazole or troglitazone.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104974933A (en) * | 2014-04-04 | 2015-10-14 | 上海泰因生物技术有限公司 | Device and method of continuously and repeatedly suspending transient transfection expression recombination protein on large scale |
| CN108174823A (en) * | 2018-02-01 | 2018-06-19 | 中国检验检疫科学研究院 | A kind of Pregnane X Receptor transgene mouse model, its construction method and its application |
| CN108728417A (en) * | 2018-01-12 | 2018-11-02 | 湖南农业大学 | Method and products thereof for screening fodder antibiotics substitute |
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| CN101906469A (en) * | 2010-07-06 | 2010-12-08 | 中南大学 | A high-throughput drug screening method expressing PXR-MRP2 fluorescent reporter gene technology platform |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906469A (en) * | 2010-07-06 | 2010-12-08 | 中南大学 | A high-throughput drug screening method expressing PXR-MRP2 fluorescent reporter gene technology platform |
Non-Patent Citations (6)
| Title |
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| 《中国博士学位论文全文数据库》 20110715 陆倍倍 "液质联用及药物筛选技术在中药毒性研究中的应用探索" 正文第29-32页"报告基因载体的构建"部分，第33页"HepG2细胞常规培养"和"G418工作浓度筛选"部分，第34页"细胞瞬时转染"部分，第35页"稳定转染细胞克隆的筛选"部分、"阳性化学药物对鉴定稳定转染细胞株荧光诱导活性"部分、"PXR抑制剂酮康唑对鉴定稳定转染细胞株荧光诱导活性的影响"部分，第36页"统计学分析"部分，以及第43页第一段， 1-3 , * |
| 《药学学报》 20060112 王宇光等 "PXR受体调控的CYP3A诱导及其在药物代谢中的重要意义" 全文 1-2 第41卷, 第1期 * |
| 《药学学报》 20100812 张庆颢等 "基于体外CYP3A4抑制和诱导数据定量预测体内药物-药物相互作用" 全文 1-2 第45卷, 第8期 * |
| 张庆颢等: ""基于体外CYP3A4抑制和诱导数据定量预测体内药物-药物相互作用"", 《药学学报》, vol. 45, no. 8, 12 August 2010 (2010-08-12) * |
| 王宇光等: ""PXR受体调控的CYP3A诱导及其在药物代谢中的重要意义"", 《药学学报》, vol. 41, no. 1, 12 January 2006 (2006-01-12) * |
| 陆倍倍: ""液质联用及药物筛选技术在中药毒性研究中的应用探索"", 《中国博士学位论文全文数据库》, 15 July 2011 (2011-07-15) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104974933A (en) * | 2014-04-04 | 2015-10-14 | 上海泰因生物技术有限公司 | Device and method of continuously and repeatedly suspending transient transfection expression recombination protein on large scale |
| CN104974933B (en) * | 2014-04-04 | 2017-08-15 | 上海泰因生物技术有限公司 | A kind of extensive continuous several times, which suspend, turns the apparatus and method of expression recombinant protein wink |
| CN108728417A (en) * | 2018-01-12 | 2018-11-02 | 湖南农业大学 | Method and products thereof for screening fodder antibiotics substitute |
| CN108174823A (en) * | 2018-02-01 | 2018-06-19 | 中国检验检疫科学研究院 | A kind of Pregnane X Receptor transgene mouse model, its construction method and its application |
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