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Construction and application of co-expressed uptake transporters and drug-metabolizing enzyme models

A technology of uptake transporters and co-expression, applied in the biological field, can solve the problems of lack of comprehensive understanding of the synergy between drug transporters and metabolic enzymes, and few in vitro models

Active Publication Date: 2016-12-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is still a lack of comprehensive understanding of the synergy between drug transporters and metabolic enzymes, and there are few in vitro models available for research in this direction

Method used

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  • Construction and application of co-expressed uptake transporters and drug-metabolizing enzyme models
  • Construction and application of co-expressed uptake transporters and drug-metabolizing enzyme models
  • Construction and application of co-expressed uptake transporters and drug-metabolizing enzyme models

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of recombinant plasmids pcDNA3.1(+) / OAT1 and pcDNA3.1 / Hygro(+) / CYP1A2

[0030] 1.1 Construction of recombinant plasmid pcDNA3.1(+) / OAT1

[0031] (1) Design two PCR primers according to the DNA sequence of human OAT1. The upstream and downstream primers respectively design a restriction site, EcoR I and Nhe I (underlined)

[0032] Upstream primer: 5’-CTA GCTAGC GACATGGCCTTTAATGACCTCCTGCAG-3';

[0033] Downstream primer: 5’-CAG GAATTC TCAGAGTCCATTCTTCTCTTGTGCTGA-3'.

[0034] (2) PCR was carried out using the pCMV6-OAT1 plasmid as a template, and the reaction system and reaction conditions are shown in Table 1.

[0035]

[0036]

[0037] The PCR product was subjected to 1% agarose gel electrophoresis, and a 1.7kb electrophoresis band was obtained, which was consistent with the gene in the database OAT1 Same size.

[0038] (3) The target band was recovered by gel, and the pcDNA3.1 (+) plasmid and OAT1 gene fragment were respectively used E...

Embodiment 2

[0047] Example 2 MDCK cell transfection and OAT1 functional identification

[0048] 2.1 Transfection of pcDNA3.1(+) / hOAT1 plasmid into MDCK cells

[0049] MDCK cells preserved in our laboratory were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. Cells were placed at 37°C, 5% CO 2 cultured under conditions, passaged after trypsinization. The day before transfection, 2 × 10 per well 5 / mL for seeding and transfection when the cells reached 80% confluence. The pcDNA3.1(+) and pcDNA3.1(+) / hOAT1 plasmids used for transfection were extracted by a plasmid extraction kit and their concentrations were determined. Follow transfection reagent Lipofectamine TM 2000 MDCK cells were transfected according to the instructions. Add 100 μL of serum-free and antibiotic-free DMEM medium to two 1.5 mL Ependorff tubes. Add 6 μL of transfection reagent to one tube, and add 2-3 μg of plasmid to the other tube. The above two solutions were mixed and allowed to stand f...

Embodiment 3

[0065] Application of Example 3 in hOAT1 Inhibitor and Substrate Screening

[0066] Investigate glycyrrhizic acid, glycyrrhetinic acid, ligustrazine, ferulic acid, tanshinone, salvianolic acid A, salvianolic acid sodium, tanshinone I, salvianolic acid B, cryptotanshinone I, dihydrotanshinone I, aristolochic acid I, etc. Inhibitory effects of traditional Chinese medicine monomers on the uptake of fluorescent substrate 6-CFL by MDCK-OAT1 cells. The MDCK-OAT1 monoclonal cells were planted in 48-well plate, 1×105cell / well, and the accumulation experiment was carried out after 48h. The cells were divided into the control group and the test group, washed twice with HBSS solution, the cells in the control group were added with 200 μL HBSS solution containing 4 μmol / L 6-CFL, and the cells in the test group were added with 4 μmol / L 6-CFL and 100 μmol / L test drug (Probenecid was used as the inhibitor positive control with a concentration of 1 mmol / L) in 200 μL of HBSS solution, incubat...

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Abstract

The invention provides construction of a model of co-expression uptake of a carrier and a drug-metabolizing enzyme. The construction comprises the following steps: amplifying a human gene OAT1cDNA used as a template to obtain a human OAT1 gene segment, connecting the obtained human OAT1 gene segment to a carrier pcDNA3.1(+) to obtain a plasmid pcDNA3.1(+) / OAT1, transfecting MDCK (Madin Darby Canine Kidney) cells, and screening to obtain MDCK-hOAT1 cells in which the OAT1 is highly expressed; connecting a human CYP1A2 gene segment to a carrier pcDNA3.1(+) / Hygro(+) to obtain a plasmid pcDNA3.1 / Hygro(+) / CYP1A2, transfecting the MDCK-hOAT1 cells, screening by using a hygromycin B to obtain cells of co-expression of OAT1 and CYP1A2, then carrying out mRNA verification on the cells and also carrying out functional verification by virtue of a classic substrate and an inhibitor. The cell model can be applied to cellular-level screening based on the medical toxic effects of OAT1 and CYP1A2, and therefore, prediction of the synergistic effect of the two proteins in a medicine disposition process is realized and a basis can be provided for clinical rational medications. The cell model is rational in design and high in repeatability.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the construction and application of a cell model co-expressing an uptake transporter OAT1 and a drug metabolizing enzyme CYP1A2. technical background [0002] After drugs enter the body, they often go through processes such as absorption, distribution, metabolism, and excretion. The drug enters the blood circulation through the shielding membrane composed of cells from the outside or the site of administration, and enters the liver through the portal vein. Under the influence of the drug metabolizing enzymes in the liver, the drug prototype or metabolites are rapidly distributed in various tissues through the blood circulation. As an important excretory organ of the human body, the kidney plays an important role in the disposal of drugs. The excretion process of drugs through the kidney includes three processes of glomerular filtration, renal tubular secretion and reabsorption...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/12C12N15/53C12Q1/02
Inventor 曾苏赵垒胡海红余露山蒋惠娣周惠徐思云
Owner ZHEJIANG UNIV
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