Cell model for screening in-vitro activity of URAT1 inhibitor as well as construction method and screening method of cell model

A cell model and construction method technology, applied in the field of pharmacology and genetic engineering, can solve the problems of harmful environment, pollution, high cost of use, etc., and achieve the effect of high specificity, high sensitivity and low requirements

Pending Publication Date: 2021-06-01
SOUTH CHINA UNIV OF TECH
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this radioisotope method has the advantage of high sensitivity, its use is expensive and harmful to the environment, and the operator should take special safety precautions when using it to avoid contamination and dispose of waste

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Cell model for screening in-vitro activity of URAT1 inhibitor as well as construction method and screening method of cell model
  • Cell model for screening in-vitro activity of URAT1 inhibitor as well as construction method and screening method of cell model
  • Cell model for screening in-vitro activity of URAT1 inhibitor as well as construction method and screening method of cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Constructing a cell line that stably and highly expresses the hURAT1 gene

[0066] 1. Preparation of related solutions and reagents:

[0067] (1) Ampicillin liquid storage solution: Accurately weigh 0.5 g of ampicillin powder with an analytical balance, place it in a 15 mL centrifuge tube, add 3 mL of distilled water to dissolve it, and then dilute to 5 mL to obtain ampicillin with a final concentration of 100 mg / mL penicillin stock solution. Filter and sterilize with a 0.22 μm nitrocellulose membrane in an ultra-clean bench, aliquot and store in a -20°C refrigerator. When in use, dilute the ampicillin stock solution to a working concentration of 100 μg / mL according to the volume of the medium.

[0068] (2) Complete medium for HEK293T cells: Add 10wt% fetal bovine serum, 1wt% penicillin / streptomycin and 1wt% L-glutamine to DMEM high-glucose basal medium, mix well and filter with 0.22 μm nitrocellulose Sterilize by membrane filtration and store in a 4°C refr...

Embodiment 2

[0118] Example 2 Establishment of the in vitro activity screening model of URAT1 inhibitors based on fluorescence method

[0119] 1. Solution preparation

[0120] (1) HBSS (Cl-free - ) solution preparation: take by weighing solid sodium gluconate (125mmol / L), potassium gluconate (4.8mmol / L), calcium gluconate (1.3mmol / L), magnesium sulfate (1.2mmol / L), dihydrogen phosphate Dissolve potassium (1.2mmol / L) in a beaker with distilled water, add HEPES (25mmol / L), D-glucose (5.6mmol / L) to 1000mL, then add NaHCO 3 The pH of the solution was brought to 7.3-7.4, sterilized by filtration through a 0.22 μm nitrocellulose filter, and placed in a 4°C refrigerator for later use.

[0121] (2) Preparation of 0.5% MTT solution: Weigh 0.5g of MTT in a 100mL volumetric flask, and use prepared HBSS (Cl-free - ) solution was dissolved and fixed to volume, filtered and sterilized through a 0.22 μm nitrocellulose filter membrane, aliquoted into 15 mL centrifuge tubes and wrapped in tinfoil, seale...

Embodiment 4

[0165] Embodiment 4 is based on the application of the in vitro screening model of fluorescence method (can refer to Figure 6 flow chart)

[0166] 1. Solution preparation

[0167] (1) Preparation of common solutions: For the preparation of HBSS (Cl-free) solution, 0.5% MTT solution, HEK293T complete medium containing 2% fetal bovine serum, fluorescent substance solution and URAT1 inhibitor solution, refer to Example 2.

[0168] (2) Preparation of test compound storage solution: Take 10 compounds, accurately weigh each test compound in a 1.5mL centrifuge tube, add 1ml DMSO to dissolve, and prepare a 200mM storage solution.

[0169] There are 10 compounds to be tested, which are marked as Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, and Compound 10. Wherein, the structural formula of compound 1 is: The structural formula of compound 2 is: The structural formula of compound 3 is: The structural formula of com...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a cell model for screening the in-vitro activity of a URAT1 inhibitor as well as a construction method and a screening method of the cell model. The construction method comprises the following steps: connecting an hURAT1 gene to a plasmid, transfecting the plasmid into a cell, and carrying out drug screening, DNA identification and protein identification to obtain the cell model for URAT1 inhibitor in-vitro activity screening. The screening method comprises the following steps: adding a drug to be detected and fluorescein into a culture medium, inoculating the cell model, performing co-culturing, removing the culture medium to obtain cultured cells, performing cracking, testing a fluorescence spectrophotometric value, and judging whether the drug to be detected is the URAT1 inhibitor or not according to the fluorescence spectrophotometric value. Compared with the existing method for marking 14C-uric acid through radioactivity, the method disclosed by the invention has the advantages of high sensitivity, quantification, high specificity, low requirements on equipment and test environment, capability of avoiding radioactive contamination and the like, and can be used for high-throughput screening of URAT1 inhibitors.

Description

technical field [0001] The invention relates to the fields of pharmacology and genetic engineering, in particular to a cell model for screening in vitro activity of URAT1 inhibitors, a construction method and a screening method thereof. Background technique [0002] Uric acid is the product of purine metabolism in the human body. In nature, only humans, birds and some primates use uric acid as the final product of purine metabolism. Due to the lack of enzymes that can further decompose uric acid into soluble allantoin, humans are prone to Hyperuricemia occurs. Human uric acid is formed in the liver, about 1 / 3 is excreted through the intestines, and about 2 / 3 is excreted through the kidneys. Studies have found that the classic mode of renal urate transport is: glomerular filtration, renal tubular reabsorption, renal tubular secretion, and post-secretion reabsorption. More than 98% of the uric acid filtered by the glomerulus is reabsorbed by the proximal renal tubule and the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N5/10C12N15/12C12N15/65C12N15/85C12Q1/02G01N21/64C12R1/91
CPCC12N5/0686C07K14/47C12N15/85C12N15/65G01N33/5044G01N21/6428C12N2510/00C12N2503/02G01N2500/10
Inventor 李晶周海燕彭雯张雷
Owner SOUTH CHINA UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap