Application of FRT cell strain in preparation of preparation or kit for screening Chrm3 regulator

A cell line and regulator technology, applied in the field of biomedicine, can solve the problems of interference, long cycle, complicated operation and so on

Pending Publication Date: 2021-02-12
JINLIN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Calcium flow detection usually uses fluorescent dyes, such as Flou-3, Flou-4, etc., using fluorescent dyes and Ca 2+ After the combination, a strong fluorescent signal is generated, and the change of the...

Method used

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  • Application of FRT cell strain in preparation of preparation or kit for screening Chrm3 regulator
  • Application of FRT cell strain in preparation of preparation or kit for screening Chrm3 regulator
  • Application of FRT cell strain in preparation of preparation or kit for screening Chrm3 regulator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080]Example 1 Determination of endogenous expression of Chrm3 in FRT cells

[0081]1 RT-PCR detection of Chrm3 expression

[0082]First use TRIzol method to extract total RNA from FRT cells. The general method is as follows: Harvest FRT cells (1~5)×106, Add 1ml Trizol to lyse the cells and add 0.2ml chloroform to shake and mix, then centrifuge, centrifuge at 10000g for 15min. Take the upper colorless water phase, add 0.5ml isopropanol, and precipitate RNA with alcohol. After standing at room temperature for 10 minutes, centrifuge at 10000g for 10 minutes. The RNA was washed with 75% ethanol in DEPC water and centrifuged at 7500g for 5 min. After drying at room temperature, add 20 μl DEPC water to obtain an RNA solution. Use Nanodrop 2000 to detect RNA concentration, perform agarose gel electrophoresis on RNA solution to detect RNA integrity, and then use reverse transcription kit to reverse transcription to obtain cDNA solution. This study used 2×EasyTaq PCR SuperMix, and designed and s...

Embodiment 2

[0100]Example 2 Construction of cell model co-expressing ANO1 and YFP-H148Q / I152L

[0101]1 Construction of a cell line co-expressing ANO1 and YFP-H148Q / I152L

[0102]The FRT cell line expressing ANO1 has been constructed in our laboratory in the early stage, and the YFP-H148Q / I152 plasmid was transfected into FRT cells stably expressing ANO1 according to the Lipofectamine 3000 instructions, and the transfected cells were screened with the optimal concentration of G418 antibiotic Two weeks later, a suitable single-cell clone group was selected for limiting dilution in a 96-well plate, and the positive cloned cell line was expanded and cultured to obtain an FRT cell line co-expressing ANO1-YFP-H148Q / I152L.

[0103]2 Fluorescence quenching kinetics experiment to identify the validity of the cell model

[0104]The FRT cells stably co-transfected with ANO1-YFP-H148Q / I152L were seeded in a 96-well plate with a black wall and a transparent bottom, and cultured for 18 hours. The cells were d...

Embodiment 3

[0105]Example 3 Verification of the effectiveness of cell model screening of Chrm3 modulators

[0106]1 Detection of Ca by Fura-2 fluorescent probe method2+The change

[0107]The FRT cells stably co-expressing ANO1-YFP-H148Q / I152L were digested and centrifuged to make a cell suspension, added Fura-2 / AM (final concentration 5μM), incubated at 37°C for 30 min, and gently shaken. The cells were washed once with calcium and magnesium-free PBS buffer to remove Fura-2 / AM remaining outside the cells. After centrifugation, calcium and magnesium-free PBS buffer was added to make a cell suspension. In the Fluostar multi-function microplate reader, the fluorescence intensity was recorded at 510nm using 340 and 380nm dual excitation sources. During the measurement, the fluorescence ratio of 340nm / 380nm was recorded at rest and after adding different concentrations of CCh (Chrm3 activator). TritonX-100 and EGTA were added to determine the maximum and minimum fluorescence. Calculate Ca based on...

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Abstract

The invention relates to the field of biological medicine, and in particular, relates to an application of an FRT cell strain in preparation of a preparation or a kit for screening a Chrm3 regulator.FRT cells endogenously express Chrm3 in mRNA and protein levels; an FRT cell model capable of stably co-expressing ANO1 and YFP-H148Q/I152L is successfully constructed; the cell model can sensitivelydetect the intracellular calcium concentration, the slope value of fluorescence change and the concentration of the Chrm3 regulator form a dose-dependent relationship, and the cell model can be used for screening the Chrm3 regulator; the Z' factor value of the model is 0.678, and the requirement of high-throughput screening is met. The cell model realizes high-throughput screening of the Chrm3 regulator by sensitively detecting a calcium signal, and can also be applied to screening of other GPCR targets related to the calcium ion signal.

Description

Technical field[0001]The present invention relates to the field of biomedicine, in particular to the application of FRT cell strains in preparing preparations or kits for screening Chrm3 modulators.Background technique[0002]Muscarinic acetylcholine receptor (mAChR) refers to a choline receptor that can specifically bind to muscarine and is activated. mAChR is a member of the G-protein-coupled receptor (GPCR) family and is divided into five different subtypes Chrm1~Chrm5. Muscarinic acetylcholine receptor Ⅲ subtype (Chrm3) is mainly distributed in exocrine glands, smooth muscle, vascular endothelium, brain and autonomic ganglia, etc., mediating many important physiological functions, such as: vascular smooth muscle relaxation, gastrointestinal and Contraction of smooth muscle of bladder, relaxation of sphincter, reduction of pupils, increased secretion of glands, etc. Chrm3 activator and antagonist have different effects and can treat many diseases in clinic. Among them, the activato...

Claims

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Application Information

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IPC IPC(8): C12Q1/6897C12Q1/02
CPCC12Q1/6897G01N33/5008G01N33/5038C12N2503/02G01N2500/10
Inventor 郑锴郝峰李欣王坤胡城解宇浩张嘉琪
Owner JINLIN MEDICAL COLLEGE
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