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Label-free arginine vasopressin receptor cell model construction and ligand screening method

A technology of arginine vasopressin and receptor cells, applied in the field of pharmacology research and drug screening research

Pending Publication Date: 2021-05-28
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Representative drugs targeting V1a receptors include Conivaptan (YM078) and Relcovaptan (Relcovaptan), which are currently used to treat isolytic hyponatremia, but in the long-term drug safety evaluation, There are certain disadvantages

Method used

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  • Label-free arginine vasopressin receptor cell model construction and ligand screening method
  • Label-free arginine vasopressin receptor cell model construction and ligand screening method
  • Label-free arginine vasopressin receptor cell model construction and ligand screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Model Construction of V1a Receptor on HEK293 Cell Line

[0033] Seed HEK293 cells in Epic optical biosensor 384 microwell plate, about 1.5×10 per well 4 After culturing for 24 hours, discard the medium, add 30 μL of HBSS buffer after incubation, let stand for 1 hour until the cell state is stable, and place it on the signal detection position of the Epic instrument. After establishing a baseline of 2 minutes on the Epic instrument system, add a gradient V1a receptor agonist (such as arginine vasopressin, starting from the final concentration of 80nM, and gradually decreasing the concentration by 1 / 2 times to 0.15625nM, a total of 10 Concentration point) or antagonist (such as SR49059, wherein SR49059 starts from the final concentration of 200nM, and the concentration is gradually decreased to 0.390625nM by 1 / 2 times, a total of 10 concentration points), 10μL per well, continue to monitor the cell response signal for 1h, record In step S1, the DMR signal curve of the pr...

Embodiment 2

[0036] Model Construction of V1a Receptor on CHO-V1a Cell Line

[0037] Seed CHO-V1a cells in Epic optical biosensor 384 microwell plate, about 1.5 × 10 per well 4 After culturing for 24 hours, discard the medium, add 30 μL of HBSS buffer after incubation, let stand for 1 hour until the cell state is stable, and place it on the signal detection position of the Epic instrument. After establishing a 2min baseline on the Epic instrument system, add a gradient V1a receptor agonist (such as arginine vasopressin, starting from a final concentration of 200nM, and gradually decreasing the concentration by 1 / 2 times to 0.024nM, a total of 14 Concentration point) or antagonist (such as SR49059, starting from the final concentration of 2000nM, the concentration is gradually decreased by 1 / 2 times to 0.98pM, a total of 12 concentration points), 10μL per well, continue to monitor the cell response signal for 1h, recorded as step S1, the DMR signal curve of the probe molecule is obtained. ...

Embodiment 3

[0039] Example 3 Ligand screening of V1a receptor

[0040] Seed CHO-V1a cells in Epic optical biosensor 384 microwell plate, about 1.5 × 10 per well 4 After culturing for 24 hours, discard the medium, add 30 μL of HBSS buffer after incubation, let stand for 1 hour until the cell state is stable, and place it on the signal detection position of the Epic instrument. After establishing a baseline of 2 minutes on the Epic instrument system, add the distillate fraction with a final concentration of 50 μg / mL, 10 μL per well, and treat it for 60 min, record it as S1, and obtain the DMR signal curve of the distillate fraction. Re-establish the baseline on the Epic instrument system, add arginine vasopressin (10 μL) at a final concentration of 50 nM to each well, monitor the cell response signal for 1 h, and obtain the signal effect of the test compound on the agonist arginine vasopressin, Denoted as S2, the desensitized DMR signal curve of the Jindenglan medicinal fraction was obtain...

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Abstract

The invention relates to a label-free arginine vasopressin receptor cell model construction and ligand screening method, in particular to a method for building the models of a subtype (V1a receptor) of an arginine vasopressin receptor on different cell lines and for carrying ligand screening by using a label-free cell dynamic mass reset technology. The V1a receptor model disclosed by the invention is constructed on an HEK293 cell line for endogenous expression of the V1a receptor and a transfected CHO-V1a cell line. The method has the characteristics of being close to an in-vivo real environment, free of labels, capable of monitoring in real time, high in flux and simple to operate, and ligands with V1a receptor activity, including agonists and antagonists, can be screened.

Description

technical field [0001] The present invention belongs to the field of pharmacological research and drug screening research, in particular to subtypes of arginine vasopressin receptors, namely arginine receptor subtype 1a (V1a) receptors on endogenous cells and transfected cells The model establishment of the present invention also relates to the ligand screening method of V1a receptor. Background technique [0002] Arginine vasopressin (AVP) is a 9-peptide cyclic hormone produced by the supraoptic nucleus and paraventricular nucleus cells of the hypothalamus, stored in the neurohypophysis, and acting on the arginine vasopressin receptor (AVPR) , mediates a series of physiological functions such as regulating circulation and water balance. Arginine vasopressin receptors belong to the rhodopsin A family of G protein-coupled receptors. According to different transmission mechanisms, arginine vasopressin receptors have three subtypes in the human body: V1a, V1b and V2. V1a rec...

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N5/071
CPCG01N33/56966C12N5/0686C12N5/0682
Inventor 梁鑫淼曲腊腊侯滔刘艳芳王纪霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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