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Cell screening model for label-free histamine receptor H2

A histamine receptor, label-free technology, applied in the field of cell screening, can solve the problems of cumbersome operation, low throughput, long experimental period, etc., and achieve the effects of pathway integration research and simple operation, high temporal and spatial resolution, and short experimental period.

Pending Publication Date: 2020-06-30
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, such as the traditional radioligand-receptor binding assay requires washing and filtration, long experimental cycle and low throughput, and this technique cannot distinguish receptor agonists and antagonists; the rest The current GPCR detection method is mainly aimed at the activation of a certain signaling pathway, and often does not consider the activation of multiple pathways. It often requires fluorescent protein labeling or additional indicators, which makes the operation cumbersome, and the addition of these indicators will also cause cells. Certain damage, affecting the reliability of screening results

Method used

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  • Cell screening model for label-free histamine receptor H2
  • Cell screening model for label-free histamine receptor H2
  • Cell screening model for label-free histamine receptor H2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: DMR characteristic signal spectrum of agonist histamine on HEK-293-H2 cells

[0032] Human embryonic kidney cells HEK-293-H2 cells were derived from the cell bank independently constructed by the laboratory, the inverted microscope was purchased from OLYMPUS, histamine and cimetidine were purchased from Bailingwei and AMQUAR respectively. The cell culture plate is an Epic optical biosensing 384 microwell plate, purchased from Corning, and the detection platform is the third generation of Corning Imager, the detected signal is the wavelength shift caused by cell dynamic mass reset (DMR).

[0033] HEK-293-H2 cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cel...

Embodiment 2

[0034] Example 2: DMR characteristic signal spectrum of antagonist cimetidine on HEK-293-H2 cells

[0035] HEK-293-H2 cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches about 95%, and conduct an activity test. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 20mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1 hour; re-scan the baseline for 2 minutes, add different concentrations of cimetidine into the microwell plate, the volume of each well is 10 μL, the concentration is 100000nM, 33333.33nM, 11111.11nM, 3703.7nM, 1234.57nM, 411.52 nM, 137.17nM...

Embodiment 3

[0036] Example 3: Desensitized DMR characteristic signal spectrum of HEK-293-H2 cells

[0037] HEK-293-H2 cells in the logarithmic growth phase were seeded in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4 Firstly, place the inoculated cell plate in a cell incubator and culture it for 20-22 hours until the cell confluency reaches about 95%, and conduct an activity test. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 20mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1h; add different concentrations of histamine into the microwell plate to pretreat HEK-293-H2 cells for 1h, the volume of each well is 10μL, and the concentration is 100000nM, 33333.33nM, 11111.11nM, 3703.7nM, 1234.57nM, 411.52nM, 137.17nM, 45.72nM, 15.2...

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Abstract

The invention provides a cell screening model of a label-free histamine receptor H2. A method for screening an agonist and an antagonist of the H2 receptor is established based on a label-free cell integration pharmacology technology and using H2 stable expression cell lines. The method can further be used to study a modulator that affects a downstream pathway of the H2 receptor. The constructed H2 cell screening model does not require fluorescent labeling, no additional indicator is added during the detection process, and the cell screening model has the characteristics of target-pathway integration response, no damage to cells, reliable detection results, high sensitivity, high screening throughput and easy operation. The cell screening model is used for finding the agonist, the antagonist and the pathway modulator of the H2 receptor from natural product libraries, metabolite libraries and combinatorial chemical libraries, as well as H2-receptor-involved drug screening of acidity gastropathy such as hyperacidity and gastric and duodenal ulcers and neuropathic pain.

Description

technical field [0001] The invention relates to the field of cell screening, in particular to a cell screening model of unmarked histamine receptor H2. Background technique [0002] G protein-coupled receptor (GPCR) is the most important class of membrane receptors in cell signal transduction, and it is also one of the most concerned drug targets in the development of small molecule drugs, with about 34% of Modern drugs directly target this family of receptors. Histamine is a biogenic amine that widely exists in human tissues and exerts a wide range of physiological effects by binding to its receptors. So far, four subtypes of histamine receptors have been discovered: H1, H2, H3, and H4 receptors. Histamine receptors are members of the G protein-coupled receptor family, which conduct signal transduction by coupling and activating specific G proteins. Among them, the research on H2 receptors and their receptor antagonists began in the 1960s. H2 receptors are mainly related...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02
CPCC07K14/705G01N33/502G01N33/5041C12N2510/00C12N2503/02G01N2500/10C12Q1/02C12N5/10C12Q1/00
Inventor 梁鑫淼王志伟王纪霞单彩龙薛珍珍于广璞
Owner TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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