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Cell screening model of [beta]1 adrenergic receptor

A technology of adrenaline and cells, applied in the field of cell screening, can solve the problems affecting the reliability of screening results, inability to distinguish receptor agonists and antagonists, and few ligands

Pending Publication Date: 2021-03-30
TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have certain limitations. For example, the traditional radioligand binding technology must select the correct ligand, but there are few ligands to choose from, long experimental cycle and low throughput. This technology cannot distinguish receptors. agonists and antagonists; Western Blotting needs to add markers, complicated operation, long experiment period, and can only detect the signal at the protein level, which affects the reliability of the screening results

Method used

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  • Cell screening model of [beta]1 adrenergic receptor
  • Cell screening model of [beta]1 adrenergic receptor
  • Cell screening model of [beta]1 adrenergic receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: DMR characteristic spectrum without selective beta agonist adrenaline in HEK-293-β1 cells

[0040] Human embryonic renal cell HEK-293-β1 cells are from laboratory autonomous build cell libraries, inverted microscope to buy Olympus, adrenaline, ICI 118, 587 and Popranolol purchase to Tocris. Cell culture plates are EPIC optical biotomizers 384 microplate, purchased in Corning, the test platform for Corning third generation EPIC® imager, the detected signal is the wavelength displacement caused by cell dynamic mass reset (DMR).

[0041] The HEK-293-β1 cells will be in a long period of time, inoculated in the 384 microplate, the medium used for DMEM (# sh3002.01b, thermo), the inoculation volume of 40 μL per well, the cells inoculated each well 2.0 × 10 4 For the cultivation of the cellular plates in the cell incubator to cultivate 20 ~ 22 h, to about 95%, and the active experiment is performed.

[0042] The cell culture fluid in the microplate is changed to a Hank'...

Embodiment 2

[0044] Example 2: Selective β1 agonist ICI 118, 587 DMR feature spectrum on HEK-293-β1 cells

[0045] The HEK-293-β1 cells will be in a long period of time, inoculated in the cell-compatible 384 microplate, the medium used is DMEM (# SH3002.01B, thermo), and the inoculation volume of each well is 40 μL, each well. The number of cells inoculated was 2.0 × 10 4 For the cultivation of the cellular plates in the cell incubator to cultivate 20 ~ 22 h, to about 95%, and the active experiment is performed.

[0046] The cell culture fluid in the microplate is changed to a Hank's balance salt solution (containing 10 mm HEPES), and the volume of 30 μL per hole is added, after adding, placed in EPIC ® The imager is equilibrated with 1 h; the baseline of 2 min will be added to the microplate, and the volume is 10 μL per well, the concentration is 5000 nm, 2500 nm, 1250 nm, 625 nm, 312.5 nm, 156.3 nm, 78.1. Nm, 39.1 nm, 19.5 nm, 9.8 nm, 4.9 nm, 2.4 nm, 1.2 nm, and 0.6 nm, parallel 3 times, pla...

Embodiment 3

[0048] Example 3: DMR characteristics spectrum of antagonist proportellol in HEK-293-β1 cells

[0049] The HEK-293-β1 cells will be in a long period of time, inoculated in the 384 microplate, the medium used for DMEM (# sh3002.01b, thermo), the inoculation volume of 40 μL per well, the cells inoculated each well 2.0 × 10 4 For the cultivation of the cellular plates in the cell incubator to cultivate 20 ~ 22 h, to about 95%, and the active experiment is performed.

[0050] The cell culture fluid in the microplate is changed to a Hank's balance salt solution (containing 10 mm HEPES), and the volume of 30 μL per hole is added, after adding, placed in EPIC ® The imager is equilibrated with 1 h; the baseline of 2 min is re-scanned, and the fragrant of different concentrations of propranolol is added to the microplate, and the volume is 10 μl per well, the concentration is 5000 nm, 2500 nm, 1250 nm, 625 nm, 312.5 nm, 156.3 nm, 78.1 nm, 39.1 nm, 19.5 nm, 9.8 nm, 4.9 nm, 2.4 nm, 1.2 nm an...

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Abstract

The invention provides a cell screening model of a [beta]1 adrenergic receptor. On the basis of an unmarked cell integration pharmacology technology, a method for screening an agonist and an antagonist of a [beta]1 adrenergic receptor is established by utilizing a cell line stably expressed by the [beta]1 adrenergic receptor. The method can also be used to study modulators that affect the downstream pathway of the [beta]1 adrenergic receptor. A [beta]1 adrenergic receptor cell screening model constructed by the invention does not need fluorescence labeling, does not need additional indicatorsin the detection process, and has the characteristics of no label, no harm, high flux, high sensitivity and the like. The method is used for searching agonists, antagonists and pathway active molecules or active compounds of [beta]1 adrenergic receptors from a natural product library, a metabolite library and a combinatorial chemistry library, and screening drugs for arrhythmia, angina pectoris, myocardial infarction, heart failure and hypertension related diseases participated by the [beta]1 adrenergic receptor.

Description

Technical field [0001] The present invention relates to the field of cell screening, and more particularly to a cell screening model of β1 adrenaline receptors. Background technique [0002] Adrenergic receptors are a type of tissue receptor that mediates cantamine effects for G-protein coupling. G-protein-coupled receptor (GPCR) is the most important of cell signaling is the most important film receptor. According to the different reaction of the norepinephrine, it is divided into an adrenergic alpha receptor and a beta receptor. Relatively speaking, norepinephrine is more sensitive to α receptors than adrenaline, while the action of adrenaline to beta receptors is more sensitive. The beta receptor is distributed in the human body organ and the vascular system. The adrenaline receptor is divided into β1, β2 and β3 a total of 3 subtypes depending on the amino acid residue of the adrenergic beta receptor. Among them, β1 receptors are mainly distributed in organs such as heart, kid...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N21/84
CPCG01N33/502G01N21/84
Inventor 梁鑫淼张鹏宇王纪霞王志伟单彩龙于广璞薛珍珍
Owner TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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