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Cell model for screening [kappa] opioid receptor agonist, and screening method thereof

A technology for agonizing opioid receptors and opioid receptors, which is applied in the fields of cell biology and pharmacy, and can solve the problems of expensive use, large funds and manpower, and long experimental cycles

Active Publication Date: 2016-04-06
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] According to literature reports, the current methods for screening OPRK agonists are mostly based on radioligand receptor binding experiments of cell membrane receptors, cAMP detection kits, dual luciferase reporter gene analysis, etc., all of which have the following shortcomings, including the need for The amount of cells is large, the process is cumbersome, the experimental cycle is long, and some require radioactive measurement, the use of expensive substrates or cofactors, and the investment of funds and manpower is large.
[0014] So far, there have been no reports of cell models based on high-content analysis and high-throughput screening of OPRK agonists

Method used

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  • Cell model for screening [kappa] opioid receptor agonist, and screening method thereof
  • Cell model for screening [kappa] opioid receptor agonist, and screening method thereof
  • Cell model for screening [kappa] opioid receptor agonist, and screening method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1: Establishment of cell lines stably expressing human OPRK and PKAcat-EGFP

[0097] 1. Vector Construction

[0098] The pcDNA3.1 / Hygro(+)-oprk eukaryotic expression vector containing the full-length cDNA of human OPRK was constructed.

[0099] The purchased plasmid pcDNA3.1(+)-hKOR (purchased from Missouri University of Science & Technology) and pcDNA3.1 / Hygro(+) plasmid were digested with HindIII and XbaI restriction enzymes, and the digested products were separated by 1% agarose gel electrophoresis. The DNA fragment band (hKOR fragment, SEQ ID NO: 4) was excised under an ultraviolet transilluminator. Agarose Gel DNA Recovery Kit to recover DNA fragments. The recovered target fragment and carrier fragment were ligated at a molar ratio (1:1). The ligation reaction product was transformed into DH5α fresh competent cells, and screened on LB plates (containing ampicillin). Several single clones were inoculated and expanded in 5 mL liquid LB medium (containin...

Embodiment 2

[0112] Example 2: Identification of Chinese Hamster Ovary Cells (CHO-PKAcat-EGFP / OPRK)

[0113] The cell line prepared in Example 1 was treated with the agonist U50488 to activate OPRK and work together with forskolin to induce the redistribution of PKAcat-EGFP. The human κ opioid receptor in the established cell line binds to its specific ligand U50488, and the receptor is activated, which regulates the activity of AC, and then affects the concentration of cAMP. PKAcat-EGFP redistribution occurs after cooperating with forskolin.

[0114]

[0115] In order to quantitatively detect the degree of redistribution of PKAcat-EGFP induced by U50488, this example uses InCellAnalyzer1000 or InCellAnalyzer2000 to obtain the redistribution cell image of PKAcat-EGFP, and uses the multi-target analysis module (MultiTargetAnalysis) to analyze the obtained cell image, and the particle formation index Indicates the degree of redistribution of PKAcat-EGFP. The particle formation index is...

Embodiment 3

[0127] Example 3: U50488 induces the half effective concentration of PKAcat-EGFP redistribution determination

[0128] exist[ 35 S] EC of U50488 in GTPγS binding assay 50 =3.1nM (ZhuJ, LuoLY, LiJG. Activationoftheclonedhumankappaopioidreceptorbyagonistssenhances[ 35 S] GTPgammaSbindingtomembranes: determinationofpotenciesandefficaciesofligands[J].JPharmacolExpTher, 1997,282(2):676-84).

[0129] Proceed as follows:

[0130] 1) The cells prepared in Example 1 were made into 1×10 5 cells / mL of cell suspension, inoculated in a black transparent-bottom 96-well culture plate, 100 μL / well.

[0131] 2) At 37°C, 5% CO 2 , 95% humidity incubator for 24 hours.

[0132] 3) Wash the cells once with the cell analysis solution, 100 μL / well; discard the solution, and then add 100 μL / well of the cell analysis solution.

[0133] 4) Add U50488 diluted in cell analysis solution to make the final concentration 5.1×10 -11 , 1.5 / 10 -10 , 4.6×10 -10 , 1.4×10 -9 , 4.1×10 -9 , 1.2×10 -...

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Abstract

The present invention belongs to the field of cell biology and pharmacy, and relates to a cell model for screening an OPRK opioid receptor agonist, and a screening method thereof. Particularly the present invention relates to a cell, which expresses an opioid receptor and PKA. The present invention further relates to a method for screening the opioid receptor agonist, a method for evaluating excitation activity or cytotoxicity of a compound or composition on the opioid receptor, and uses of the cell in opioid receptor agonist screening. According to the present invention, the established cell model is sensitive, efficient and reliable, and can be used for high-throughput screening and / or high content screening of the opioid receptor, especially the OPRK opioid agonist.

Description

technical field [0001] The invention belongs to the fields of cell biology and pharmacy, and relates to a cell model and a screening method for screening κ opioid receptor (κ opioid receptor, OPRK) agonists. Background technique [0002] Drug screening models are an essential research platform for drug development. High content analysis or high content screening (High Content Screening, HCS) is a cell as the detection object, relying on high-resolution cell imaging system, fully integrated sample preparation technology, automation equipment, data management system, detection reagents and bioinformatics With the advantages of resources such as these, we can realize the diversified, rapid and large-scale screening of drug candidates at the cellular or molecular level. HCS technology can simultaneously detect the effects of candidate drugs on cell morphology, growth, differentiation, migration, apoptosis, metabolic pathways and signal transduction on the premise of maintaining...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12Q1/02G01N21/64C12R1/91
Inventor 宫泽辉李玉蕾王莉莉龙隆李微苏瑞斌颜慧周培兰
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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