CN112575051A

CN112575051A - Cell screening model of beta 1 adrenergic receptor

Info

Publication number: CN112575051A
Application number: CN201910922559.2A
Authority: CN
Inventors: 梁鑫淼; 张鹏宇; 王纪霞; 王志伟; 单彩龙; 于广璞; 薛珍珍
Original assignee: Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Current assignee: Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2021-03-30

Abstract

The invention provides a cell screening model of a beta 1 adrenergic receptor. The invention is based on the non-marker cell integration pharmacology technology, and utilizes a cell line stably expressed by a beta 1 adrenergic receptor to establish a method for screening agonists and antagonists of the beta 1 adrenergic receptor. The method can also be used to study modulators that affect the downstream pathway of the β 1 adrenergic receptor. The beta 1 adrenergic receptor cell screening model constructed by the invention does not need fluorescent labeling, does not need an additional indicator in the detection process, and has the characteristics of no labeling, no harm, high flux, high sensitivity and the like. It is used for searching agonist, antagonist and pathway active molecules or active compounds of beta 1 adrenergic receptors from natural product libraries, metabolite libraries and combinatorial chemistry libraries, and drug screening of arrhythmia, angina pectoris, myocardial infarction, heart failure and hypertension related diseases in which the beta 1 adrenergic receptors participate.

Description

Cell screening model of beta 1 adrenergic receptor

Technical Field

The invention relates to the field of cell screening, in particular to a cell screening model of a beta 1 adrenergic receptor.

Background

Adrenergic receptors are a class of tissue receptors that mediate the action of catecholamines, and are G-protein coupled. G-protein-coupled receptors (GPCRs) are the most important class of membrane receptors in cell signaling. They are classified into adrenergic alpha receptors and beta receptors according to their response to norepinephrine. Norepinephrine is relatively more sensitive to the action of alpha receptors than epinephrine, which is more sensitive to the action of beta receptors. The beta receptors are distributed in human body organs and blood vessels, and the adrenergic receptors are divided into 3 subtypes of beta 1, beta 2 and beta 3 according to different amino acid residues of the adrenergic beta receptors. Among them, β 1 receptors are mainly distributed in organs such as heart, kidney, gastrointestinal tract, etc., and β 2 receptors are mainly distributed in liver, arteriovenous, uterus, bronchus, skeletal muscle, etc.

Beta receptor antagonists are widely used in clinical treatment of cardiac arrhythmias, angina pectoris, myocardial infarction, heart failure, hypertension, such as quinidine, propranolol, carteolol, carvedilol, and the like. The main reason for the action of β receptor antagonists is to selectively antagonize β 1 receptors, whereas antagonizing β 2 receptors causes adverse effects such as bronchospasm and peripheral vascular resistance. Therefore, a selective beta 1 receptor screening model is constructed to find endogenous ligands, highly active agonists, antagonists and pathway modulators of the beta 1 receptor, to find highly selective beta 1 drugs.

The current high-throughput screening methods for receptors mainly comprise the traditional radioligand binding technology, FLIPR, Western Blotting and the like. These methods have certain limitations, for example, the traditional radioligand binding technology has to select the correct ligand, but has the disadvantages of few ligands for selection, long experimental period, low flux and the like, and the technology can not distinguish the receptor agonist from the receptor antagonist; western Blotting requires addition of a marker, is complicated in operation and long in experimental period, and can only detect a signal of a protein level, so that the reliability of a screening result is influenced.

Disclosure of Invention

The object of the present invention is to provide a cell screening model for β 1 adrenergic receptors, which solves the problems set forth in the background art described above.

In order to achieve the above objects, a cell screening model of β 1 adrenergic receptor is provided by means of a novel label-free cell integration pharmacological technology to screen β 1 receptor agonists, antagonists and pathway modulators, and drug screening applications of β 1 receptor-involved arrhythmia, angina pectoris, myocardial infarction, heart failure, hypertension-related diseases at high throughput.

The technical scheme of the invention is as follows:

based on a marker-free cell integration pharmacological technology, a cell screening model of a beta 1 receptor is established by using a cell line HEK-293-beta 1 which stably expresses the beta 1 and by means of known agonists and antagonists. And judging the agonistic activity, the antagonistic activity or the regulation influence of a downstream passage of the sample to be detected according to the similarity of the DMR signal spectrum of the sample to be detected and the DMR characteristic signal spectrum of the known agonist and antagonist.

The unmarked cell integration pharmacology technology records Dynamic Mass Redistribution (DMR) generated by the stimulation of a cell by a medicament as a visual spectral line through a resonant waveguide grating biosensor, so that the action target and the path of the medicament are reflected. The label-free cell integration pharmacology technology confirms the activity result mainly through three experiments of excitation, desensitization and antagonism.

Wherein, the establishment process of the cell screening model of the beta 1 adrenergic receptor comprises the following steps:

1) HEK-293-beta 1 cells are inoculated in 384 micro-porous plates with the function of resonant waveguide grating biosensor, and the density of the inoculated cells is 2.0-2.5 multiplied by 10⁴One/hole, thinThe volume of the cell culture solution is 40 muL/hole, and the cell culture time after inoculation is 18-24 h.

2) Adding non-selective beta agonist adrenalin with the concentration of 0.6-5000 nM into a 384 micro-well plate inoculated with HEK-293-beta 1 cells, and detecting the DMR characteristic signal spectrum;

3) adding a selective beta 1 agonist ICI118,587 into a 384 micro-perforated plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, and detecting the DMR characteristic signal spectrum;

4) adding non-selective beta 1 and beta 2 adrenoceptor antagonist propranolol into a 384 micro-well plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, and detecting the DMR characteristic signal spectrum;

5) all the obtained DMR characteristic signal spectrums have concentration-response dependence and have sensitivity, saturation and specificity.

Further, the screening step of the sample to be tested for having the agonistic activity is as follows:

1) adding adrenalin or ICI118,587 into a 384 micro-perforated plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, and detecting the DMR characteristic signal spectrum;

2) adding a sample to be detected into a micropore plate inoculated with HEK-293-beta 1 cells by 0.01 nM-100 mu M, and detecting the DMR signal spectrum;

3) correlating and analyzing the DMR signal spectra in the step 1) and the step 2), wherein if the DMR signal spectrum in the step 2) has no similarity with the DMR characteristic spectrum in the step 1), the sample has no agonist activity; if the contour similarity exists, the next step is carried out;

4) adding the nonselective beta 1 and beta 2 adrenoceptor antagonist propranolol into a micropore plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 2), detecting a DMR signal of the sample, and judging the sample to be an agonist of the beta 1 receptor if the DMR signal intensity is lower than that in the step 2).

Further, the screening step of the sample to be tested for having antagonistic activity is as follows:

1) respectively adding a sample to be detected and adrenalin or ICI118 and 587 into a micropore plate inoculated with HEK-293-beta 1 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of adrenalin or ICI118 and the concentration of 587 are 0.6-5000 nM, and detecting a DMR signal spectrum;

2) if the sample to be detected in the step 1) does not cause the DMR signal spectrum, continuously adding the adrenaline or ICI118,587 with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting the DMR signal spectrum; if the DMR signal is weaker than the signal of the epinephrine or ICI118,587 in step 1), it can be determined that the sample is an antagonist of the β 1 receptor.

Further, the step of the test sample having the activity of regulating the β 1 pathway is as follows:

2) continuously adding epinephrine or ICI118,587 with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting the DMR signal spectrum for 1-60 min; if the DMR signal is different from the adrenaline or ICI118,587 signal in step 1) at one stage of the ramp, plateau and lag periods;

3) adding the nonselective beta 1 and beta 2 adrenoceptor antagonist propranolol into a micropore plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 1), detecting a DMR signal of the sample, and judging that the sample to be detected is a regulator of a beta 1 receptor downstream signal path if the DMR signal spectrum is consistent with that of the sample in the step 1).

The technical method used in the invention is a label-free cell integration pharmacology technology, and Dynamic Mass Redistribution (DMR) generated by the stimulation of the cell by the drug is recorded as a visual spectral line by a resonant waveguide grating biosensor, so that the action target and the channel of the drug are reflected, and the label-free cell integration pharmacology technology has the characteristics of no label, no harm, high flux, high sensitivity and the like. Therefore, the method for constructing the beta 1 label-free high-throughput screening model by adopting the label-free cell integration pharmacological technology can greatly improve the discovery efficiency of agonists, antagonists and pathway regulators of the beta 1, has great significance for explaining the pharmacological and physiological functions of the beta 1, and simultaneously provides guidance for the beta 1 receptor to participate in the drug screening of related diseases such as arrhythmia, angina pectoris, myocardial infarction, heart failure and hypertension.

Drawings

FIG. 1 (A) DMR signature spectra on HEK-293- β 1 cells at various concentrations of epinephrine; (B) concentration-response dependence curves of various concentrations of epinephrine on HEK-293- β 1 cells.

FIG. 2 (A) DMR signature spectra on HEK-293- β 1 cells of ICI118,587 at various concentrations; (B) concentration-response dependence curves of different concentrations of ICI118,587 on HEK-293- β 1 cells.

FIG. 3 DMR signature spectra of propranolol on HEK-293-beta 1 cells.

FIG. 4 (A) DMR signal spectra of fixed concentrations of epinephrine after 1h pretreatment of HEK-293- β 1 cells with various concentrations of epinephrine; (B) after HEK-293-beta 1 cells are pretreated for 1h by adrenaline with different concentrations, the DMR signal spectrum of adrenaline with fixed concentration corresponds to a concentration-response dependence curve.

FIG. 5 (A) DMR signal spectra of fixed concentration ICI118,587 after 1h of different concentrations of epinephrine pretreatment of HEK-293- β 1 cells; (B) after HEK-293-beta 1 cells were pretreated with different concentrations of adrenalin for 1h, the DMR signal spectrum of ICI118,587 at a fixed concentration corresponded to a concentration-response dependence curve.

FIG. 6 (A) DMR signal spectra of fixed concentration ICI118,587 after 1h pretreatment of HEK-293- β 1 cells with different concentrations of ICI118,587; (B) after HEK-293-beta 1 cells are pretreated for 1h by ICI118,587 with different concentrations, a concentration-response dependence curve corresponding to a DMR signal spectrum of ICI118,587 with fixed concentration is obtained.

FIG. 7 (A) DMR signal spectra of fixed concentrations of epinephrine after 1h of pretreatment of HEK-293- β 1 cells with different concentrations of ICI118,587; (B) after HEK-293-beta 1 cells were pretreated for 1h with different concentrations of ICI118,587, the DMR signal spectra of fixed concentrations of epinephrine corresponded to concentration-response dependence curves.

FIG. 8 (A) DMR signal spectra of fixed concentrations of epinephrine after 1h of pretreatment of HEK-293- β 1 cells with varying concentrations of propranolol; (B) after propranolol with different concentrations pretreats HEK-293-beta 1 cells for 1h, the corresponding concentration-response dependence curve of the DMR signal spectrum of epinephrine with fixed concentration is obtained.

FIG. 9 (A) DMR signal spectra of fixed concentration ICI118,587 after 1h of propranolol pretreatment of HEK-293- β 1 cells at different concentrations; (B) after the propranolol with different concentrations is used for pre-treating HEK-293-beta 1 cells for 1h, a concentration-response dependence curve corresponding to a DMR signal spectrum of ICI118,587 with fixed concentration is obtained.

Detailed Description

The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.

Example 1: DMR signature profiles on HEK-293-beta 1 cells without selective beta agonist epinephrine

Human embryonic kidney cells HEK-293-. beta.1 cells were obtained from a laboratory self-constructed cell bank, and were purchased from OLYMPUS, epinephrine, ICI118,587 and propranolol from Tocris. The cell culture plate is an Epic optical biosensing 384 micro porous plate purchased from Corning company, the detection platform is a Corning third generation Epic imager, and the detected signal is wavelength shift caused by cell Dynamic Mass Resetting (DMR).

HEK-293-beta 1 cells in logarithmic growth phase were seeded in 384-well plates using DMEM (# SH30022.01B, Thermo) in a seeding volume of 40 μ L per well and a number of cells seeded per well of 2.0X 10⁴And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment.

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; rescanning the baseline for 2 min, adding into the microplate, adding 10 μ L per well, and keeping the concentration at 5000nM, 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, 3 replicates, and was placed on an Epic instrument to monitor the DMR signal for 1h in real time and calculate the EC for epinephrine at the DMR maximum response value within 40 min of epinephrine on cells₅₀The values, results are shown in FIG. 1.

The study shows that adrenalin is in dosage-dependent beta 1 receptor agonism, the dosage response curve is in a single-phase S shape and reaches the saturation response, the highest DMR response value reaches 550 pm, and the corresponding EC₅₀The value was 289.3. + -. 22.7 nM.

Example 2: DMR signature profiling of selective β 1 agonist ICI118,587 on HEK-293- β 1 cells

HEK-293-beta 1 cells in logarithmic growth phase were seeded in cell-compatible 384-well plates using DMEM (# SH30022.01B, Thermo) in a seeding volume of 40 μ L per well and a number of cells seeded per well of 2.0X 10⁴And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment.

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; rescanning the baseline for 2 min, adding to the microplate at a volume of 10. mu.L per well at concentrations of 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, 3 times in parallel, monitoring the DMR signal in real time on an Epic instrument for 1h, calculating the EC of ICI118,587 based on the maximum DMR response within 40 min of ICI118,587 on the cells₅₀The values, results are shown in FIG. 2.

The study shows that ICI118,587 is a dose-dependent beta 1 receptor agonist, the dose response curve is a monophasic S type and all achieve saturation response, the highest DMR response value reaches 100 pm, and the corresponding EC is₅₀The value was 1.2. + -. 0.6 nM.

Example 3: DMR characteristic signal profile of antagonist propranolol on HEK-293-beta 1 cells

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; different concentrations of propranolol were added to the plates in a volume of 10. mu.L per well at 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 by rescanning the baseline for 2 min, 3 replicates, and the DMR signal was monitored in real time on an Epic instrument for 1h, with the results shown in FIG. 3.

Studies have shown that the DMR response signal is close to zero for different concentrations of propranolol.

Example 4: adrenaline-to-adrenaline desensitization DMR signature spectra

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; adding epinephrine of different concentrations to the pretreated HEK-293-beta 1 cells in microwell plates for 1h, adding epinephrine of fixed concentration to the microwell plates in a volume of 10 μ L per well, at concentrations of 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, parallelizing 3 times, rescanning the baseline for 2 min, adding epinephrine of fixed concentration to the microwell platesThe wells were plated at 10. mu.L/well at 400 nM in parallel 3 times, and the DMR signals were monitored in real time on an Epic instrument for 1h, and IC was calculated based on the maximum DMR response within 40 min of adrenergic action on the cells₅₀The values, results are shown in FIG. 4.

Studies have shown that adrenalin presents a dose-dependent desensitized beta 1 receptor, the dose response curve presents a monophasic "S" shape and all reach a saturation response, the corresponding IC₅₀The value was 176.5. + -. 16.1 nM.

Example 5: DMR signature spectra for epinephrine-to-ICI 118,587 desensitization

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; adding epinephrine of different concentrations into a microplate to pretreat HEK-293-beta 1 cells for 1h, with a volume of 10 μ L per well, 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 in parallel for 3 times, rescanning the baseline for 2 min, adding ICI118,587 of fixed concentration into the microplate with a volume of 10 μ L per well, a concentration of 100 nM in parallel for 3 times, monitoring the DMR signal for 1h in real time on an Epic instrument, calculating the IC based on the maximum response value of the DMR within 40 min of ICI118,587 acting on the cells₅₀The values, results are shown in FIG. 5.

Studies have shown that ICI118,587 presents dose-dependent desensitized β 1 receptors, dose-response curves are monophasic "S" and all reach saturation response (negative signal), corresponding IC₅₀The value was 188.6. + -. 20.7 nM.

Example 6: DMR characteristic signal spectrum for ICI118,587 desensitization to ICI118,587

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; pretreating HEK-293-beta 1 cells for 1h by adding ICI118,587 with different concentrations into a microplate, adding the ICI118,587 with the volume of 10 muL in each well and the concentrations of 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 in each well, paralleling for 3 times, rescanning the base line for 2 min, adding ICI118,587 with the fixed concentration into the microplate, adding the ICI118,587 with the volume of 10 muL in each well and the concentration of 100 nM in each well, paralleling for 3 times, monitoring the DMR signal for 1h in real time on an Epic instrument, calculating the IC based on the maximum response value of the DMR within 40 min of the ICI118,587 effect on the cells₅₀The values, results are shown in FIG. 6.

The study showed that ICI118,587 is a dose-dependent desensitized beta 1 receptor, the dose response curve is monophasic "S" type and all reach saturation response, corresponding IC₅₀The value was 1.9. + -. 0.6 nM.

Example 7: DMR signature spectra for ICI118,587 desensitization to epinephrine

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; will be differentICI118,587 was added to the plates for 1h to pre-treat HEK-293- β 1 cells at a concentration of 10 μ L per well, 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 in parallel 3 times, the base line was rescanned for 2 min, epinephrine was added to the plates at a fixed concentration of 10 μ L per well, 400 nM in parallel 3 times, DMR signal was monitored in real time on an Epic instrument for 1h, and IC was calculated based on the maximum DMR response value of epinephrine over 40 min on the cells₅₀The values, results are shown in FIG. 7.

Studies have shown that adrenalin presents a dose-dependent desensitized beta 1 receptor, the dose response curve presents a monophasic "S" shape and all reach a saturation response, the corresponding IC₅₀The value was 70.9. + -. 8.0 nM.

Example 8: antagonistic DMR signature spectra of propranolol against ICI118,587

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; propranolol of different concentrations was added to the pretreated cells in microwell plates for 1h, in a volume of 10 μ L per well, at concentrations of 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, in 3 replicates; rescanning the baseline for 2 min, adding ICI118,587 with fixed concentration into the microplate, adding the volume of 10 muL and the concentration of 100 nM into each well, paralleling for 3 times, placing the microplate on an Epic instrument to monitor the DMR signal for 1h in real time, and calculating IC based on the maximum response value of the DMR within 40 min of the ICI118,587 acting on the cells₅₀The values, results are shown in FIG. 8.

The research shows that propranolol antagonizes beta 1 receptor in a dose-dependent manner, the dose response curve is in a single-phase S shape and all reach saturation response, and corresponding IC₅₀The value was 1.5. + -. 0.4. mu.M.

Example 9: antagonistic DMR signature profile of propranolol for epinephrine

The cell culture solution in the plate was changed to Hank's balanced salt solution (containing 10 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Balancing for 1h on the imager; propranolol of different concentrations was added to the pretreated cells in microwell plates for 1h, in a volume of 10 μ L per well, at concentrations of 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, in 3 replicates; rescanning baseline for 2 min, adding epinephrine with fixed concentration into the microplate, adding epinephrine with fixed concentration into each well with volume of 10 μ L and concentration of 100 nM, paralleling for 3 times, monitoring DMR signal for 1h in real time on Epic instrument, and calculating IC based on maximum response value of DMR within 40 min of epinephrine acting on cells₅₀The values, results are shown in FIG. 9.

The research shows that propranolol antagonizes beta 1 receptor in a dose-dependent manner, the dose response curve is in a single-phase S shape and all reach saturation response, and corresponding IC₅₀The value was 52.7. + -. 6.3 nM.

The invention establishes a beta 1 receptor unmarked screening model based on unmarked cell integration pharmacology technology, the model has the characteristics of unmarked, harmless, high flux, high sensitivity and the like, and can efficiently screen a commercialized small molecule library, an autonomously prepared natural product extract, a component or compound library and a chemical modifier so as to obtain agonists, antagonists and pathway modulators of the beta 1 receptor and medicines for treating beta 1 receptor-regulated arrhythmia, angina pectoris, myocardial infarction, heart failure and hypertension related diseases.

Claims

1. A cell screening model for the β 1 adrenergic receptor, characterized by: judging the agonistic activity, the antagonistic activity or the regulation influence of a downstream channel of a sample to be detected by utilizing a cell line HEK-293-beta 1 for stably expressing beta 1 and by means of known agonists and antagonists according to the similarity of a DMR signal spectrum of the sample to be detected and a DMR characteristic signal spectrum of the known agonists and antagonists;

the establishment process comprises the following steps:

1) HEK-293-beta 1 cells are inoculated in an Epic 384-hole biosensor microplate, and the density of the inoculated cells is 2.0-2.5 multiplied by 10⁴The number of the cells is one, the volume of a cell culture solution is 40 muL/hole, and the cell culture time after inoculation is 18-24 h;

2) adding non-selective adrenergic receptor agonist adrenalin into a 384 micro-perforated plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, and detecting the DMR characteristic signal spectrum;

3) adding a selective beta 1 adrenergic receptor agonist ICI118,587 into a 384 micro-perforated plate inoculated with HEK-293-beta 1 cells at the concentration of 0.6-5000 nM, and detecting the DMR characteristic signal spectrum;

5) all DMR signature spectra obtained have a concentration-response dependence.

2. The model for screening cells for β 1 adrenergic receptors according to claim 1, wherein: the screening steps of the sample to be tested for having the agonistic activity are as follows:

3. The model for screening cells for β 1 adrenergic receptors according to claim 1, wherein: the screening steps of the sample to be tested for having the antagonistic activity are as follows:

4. The model for screening cells for β 1 adrenergic receptors according to claim 1, wherein: the steps of the sample to be tested having the activity of regulating the beta 1 adrenergic receptor pathway are as follows:

5. The model for screening cells for β 1 adrenergic receptors according to claim 4, wherein: the ascending period is 1-40 min, the plateau period is 40-50 min and the delay period is 50-60 min.