CN114107210A

CN114107210A - Cell screening model of label-free membrane receptor GPR84 and application thereof

Info

Publication number: CN114107210A
Application number: CN202010894186.5A
Authority: CN
Inventors: 梁鑫淼; 侯滔; 王纪霞; 赵耀鹏; 王志伟; 王俊; 陆金立
Original assignee: Taizhou Guokehuawu Biomedical Technologies Co ltd
Current assignee: Taizhou Guokehuawu Biomedical Technologies Co ltd
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2022-03-01

Abstract

The invention provides a cell screening model of a label-free membrane receptor GPR84 and application thereof. The invention is based on a label-free cell integration pharmacological technology, and establishes a method for screening GPR84 receptor agonist and antagonist by using a cell line stably expressing GPR 84. This method can also be used to study modulators that affect pathways downstream of the receptor. The GPR84 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 target pathway integration response, no damage to cells, reliable detection result, high sensitivity, high screening flux, short period, simplicity and convenience in operation and the like. It is used for searching agonists, antagonists and pathway modulators of GPR84 receptor and central nervous system diseases closely related to GPR84 receptor, such as multiple sclerosis, Alzheimer disease and the like, and drug screening of endotoxemia, obesity, type 2 diabetes, dyslipidemia, mixed lineage leukemia, inflammatory bowel disease and the like from natural product libraries, metabolite libraries and combinatorial chemistry libraries.

Description

Cell screening model of label-free membrane receptor GPR84 and application thereof

Technical Field

The invention relates to a construction method and application of a cell screening model of a label-free G protein-coupled receptor (GPCR), in particular to a cell screening model of a label-free membrane receptor GPR84 and application thereof.

Background

G protein-coupled receptors (GPCRs) are seven transmembrane receptors, the number of the G protein-coupled receptors is up to 800, the G protein-coupled receptors are the most important membrane receptors in cell signal transduction, and the G protein-coupled receptors play an important role in regulation and control in the occurrence and development of diseases, so the G protein-coupled receptors are the most concerned drug targets in the development of small molecule drugs. About 34% of the drug targets approved by FDA are GPCRs, and these drug targets only account for 20% of the total GPCRs, and a large number of GPCRs, especially orphan G-protein-coupled receptors (opgpcrs), remain unexplored and utilized. GPR84 is a member of the GPCRs family, and GPR84 is expressed primarily in the bone marrow and secondarily in peripheral leukocytes and lung, and in other tissues and organs, in humans in low amounts. GPR84 is normally expressed in a low amount, and its high expression is induced by the existence of some stimulus causing inflammatory response.

The current high-throughput screening methods for receptors mainly comprise a traditional radioligand receptor binding experiment method, a GTP gamma S binding experiment method, a cyclic adenosine monophosphate (cAMP) analysis method, a calcium flux detection method, a reporter gene detection method, a receptor endocytosis detection method, a beta-arrestin recruitment detection method and the like. However, the methods have certain limitations, the traditional radioligand receptor binding experiment method needs washing and filtering, the experiment period is long, the flux is low and the like, and the technology cannot distinguish agonists and antagonists of the receptor; the other GPCR detection methods mainly aim at the activation of a certain signal path, can not distinguish the activation of a plurality of paths, require fluorescent protein labeling or additional addition of an indicator, are complex to operate, and cause certain damage to cells due to the addition of the indicator, thereby influencing the reliability of a screening result.

The adopted novel label-free cell integration pharmacological technology is based on a label-free Resonance Waveguide Grating (RWG) biosensor to convert the dynamic mass redistribution process of intracellular components caused by a medicament into an integral and dynamic wavelength shift response signal, namely a Dynamic Mass Reset (DMR) signal, and has the characteristics of no damage, high space-time resolution, high sensitivity, high flux, capability of target point-path integration research and simple operation, short experimental period and the like, no label or additional indicator is required in the detection process, and the effect of the medicament on the integral level of living cells is more truly responded. Therefore, the construction of a GPR84 receptor unmarked high-throughput cell screening model by adopting an unmarked cell integration pharmacological technology can greatly improve the discovery efficiency of endogenous ligands, agonists, antagonists and pathway regulators of GPR84, has great significance for describing the pharmacological and physiological functions of GPR84, and simultaneously provides guidance for drug screening of diseases such as central nervous system diseases, endotoxemia, obesity, type 2 diabetes, dyslipidemia, mixed lineage leukemia, inflammatory bowel disease and the like closely related to GPR84 receptor.

Disclosure of Invention

The invention aims to provide a cell screening model of a label-free membrane receptor GPR84 and application thereof, so as to solve the problems in the background technology.

In order to achieve the aim, by means of a novel label-free cell integration pharmacological technology, a cell screening model of a label-free membrane receptor GPR84 is provided, and the cell screening model is used for high-throughput screening of endogenous ligands, agonists, antagonists and pathway modulators of a GPR84 receptor and drug screening of diseases, such as central nervous system diseases, endotoxemia, obesity, type 2 diabetes, dyslipidemia, mixed lineage leukemia, inflammatory bowel disease and the like, closely related to a GPR84 receptor.

The technical scheme of the invention is as follows:

based on the marker-free cell integration pharmacological technology, a cell screening model of a GPR84 receptor is established by using a cell line HEK293T-Gi3-GPR84 stably expressing GPR84 and by means of known agonists and antagonists. And judging the agonistic activity and antagonistic activity of the sample to be tested or the regulation influence on a downstream passage according to the similarity and specificity of the DMR signal spectrum of the sample to be tested and the DMR characteristic signal spectrum of the known agonist and antagonist.

The label-free cell integration pharmacology technology is characterized in that a Resonance Waveguide Grating (RWG) biosensor is used for converting a dynamic redistribution phenomenon of intracellular components caused by a medicament into an integral and dynamic wavelength shift response signal, the signal is a response value (pm) of wavelength change, and the signal is realized through an Epic optical biosensor 384 micro-porous plate.

Wherein the cell screening model of the GPR84 receptor is obtained by inoculating HEK293T-Gi3-GPR84 cells in a cell-compatible 384-microplate with an optical biosensing function, and the density of the inoculated cells is 1.0-4.5 multiplied by 10⁴The number of the cells per well is 40 muL per well, and the cell culture time after inoculation is 18-24 h.

The application of a cell screening model of a label-free membrane receptor GPR84 is characterized in that a sample to be tested has the following screening steps of sensitivity, saturation and specificity:

(1) adding GPR84 receptor agonist GTPL5846 dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of GPR84 receptor agonist GTPL5846 is 0.1-1000 nM, and detecting the DMR characteristic signal spectrum;

(2) adding a GPR84 receptor antagonist GPR84 antagonist 8 dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of the GPR84 receptor antagonist GPR84 antagonist 8 is 1-100000 nM, and detecting the DMR characteristic signal spectrum;

(3) adding GTPL5846 with the lowest concentration corresponding to the highest response intensity into 384 micro-well plates of HEK293T-Gi3-GPR84 cells respectively added with GPR84 receptor agonist GTPL5846 and antagonist GPR84 antagonist 8 in the steps (1) and (2), and detecting the characteristic DMR signal spectrum;

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

The application of a cell screening model of a label-free membrane receptor GPR84 is that a sample to be tested has the following screening steps of agonist activity:

(2) adding a sample to be detected with the concentration of 0.01 nM-100 mu M into a micropore plate inoculated with HEK293T-Gi3-GPR84 cells, and detecting the DMR signal spectrum;

(3) performing correlation analysis on the DMR signal spectrums in the step (1) and the step (2), if the DMR signal spectrum in the step (2) has contour similarity with the DMR characteristic spectrum in the step (1);

(4) adding a GPR84 receptor antagonist GPR84 antagonist 8 (the concentration is 1-100000 nM) into a microplate inoculated with HEK293T-Gi3-GPR84 cells, pretreating for 5-90 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 GPR84 receptor if the intensity of the DMR signal is lower than that in the step (2).

The application of a cell screening model of a label-free membrane receptor GPR84 is characterized in that a sample to be tested has the screening steps of antagonistic activity:

(1) respectively adding a sample to be detected and GTPL5846 into a micropore plate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of GTPL5846 is 0.1-1000 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 GTPL5846 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; and (3) if the DMR signal is weaker than the signal of the GTPL5846 in the step (1), and the DMR signal spectrum of the DMR signal spectrum is similar to the DMR spectrum profile of the GTPL5846 in the step (1), judging that the sample to be tested is the antagonist of the GPR84 receptor.

The application of a cell screening model of a label-free membrane receptor GPR84 is that a sample to be tested has the activity of regulating a GPR84 pathway:

(2) continuously adding GTPL5846 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 1-90 min; if the DMR signal is different from the signal of GTPL5846 in step (1) at one of the ascending phase, plateau phase and lag phase, if the sample to be tested in step (1) does not have a DMR response signal, it can be determined that the sample to be tested is a GPR84 receptor downstream signal pathway inhibition modulator;

(3) continuously adding GTPL5846 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 1-90 min; if the DMR signal is different from the GTPL5846 signal in step (1) in one of the ramp-up, plateau and lag phases; and (2) if the sample to be detected in the step (1) has a DMR response signal, adding a GPR84 receptor antagonist GPR84 antagonist 8 into a micropore plate inoculated with HEK293T-Gi3-GPR84 cells, pretreating for 5-90 min until the concentration of the GPR84 receptor antagonist GPR84 antagonist 8 is 1-10000 nM, adding the sample to be detected with the same concentration as that in the step (1), detecting a DMR signal, and if the DMR signal spectrum is consistent with that of the sample in the step (1), judging that the sample to be detected is a GPR84 receptor downstream signal pathway activation regulator.

Wherein the rise period is 1-30 min, the plateau period is 30-60 min and the lag period is 60-90 min.

The invention utilizes the established unmarked GPR84 cell model to carry out high-throughput screening on a commercial small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier to obtain the agonist, antagonist and pathway regulator of the GPR84 receptor. In addition, according to the relevance of the target point and the disease, the GPR84 receptor is found to play an important role in diseases such as central nervous system diseases, endotoxemia, obesity, type 2 diabetes, dyslipidemia, mixed lineage leukemia, inflammatory bowel disease and the like, and can also be used for carrying out drug screening on relevant diseases.

Drawings

FIG. 1: (A) DMR signature profiles of GTPL5846 at different concentrations on HEK293T-Gi3-GPR84 cells;

(B) concentration-response dependence curves of various concentrations of GTPL5846 on HEK293T-Gi3-GPR84 cells.

FIG. 2: (A) after HEK293T-Gi3-GPR84 cells are pretreated for 90 min by GTPL5846 with different concentrations, a DMR signal spectrum of the GTPL5846 with fixed concentration is obtained;

(B) after HEK293T-Gi3-GPR84 cells are pretreated for 90 min by GTPL5846 with different concentrations, a concentration-response dependence curve corresponding to a DMR signal spectrum of GTPL5846 with fixed concentration is obtained.

FIG. 3: (A) DMR signature profiles of different concentrations of GPR84 antagonist 8 on HEK293T-Gi3-GPR84 cells;

FIG. 4: (A) DMR signal profiles of fixed concentration GTPL5846 after 90 min pretreatment of HEK293T-Gi3-GPR84 cells with different concentrations of GPR84 antagonist 8;

(B) after different concentrations of GPR84 antagonist 8 pre-treating HEK293T-Gi3-GPR84 cells for 90 min, the DMR signal profile of the fixed concentration GTPL5846 corresponds to a concentration-response dependent curve.

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 spectra of GPR84 receptor agonist GTPL5846 on HEK293T-Gi3-GPR84 cells

Human embryonic kidney cells HEK293T-Gi3-GPR84 cells were obtained from the institute of chemical and physical, university of Chinese academy of sciences, and purchased from OLYMPUS under an inverted microscope. GTPL5846 (cat # HY-1276 (4)) and GPR84 antagonist 8 (cat # HY-11256 (2)) from MedChemexpress Inc. culture Medium DMEM (cat # 01-052-1 ACS) was purchased from Biolocal Industries, Inc. Balanced salt solutions HBSS (cat # 14065-. The cell culture plate is an Epic optical biosensing 384 micro-porous plate, which is purchased from corning company. The detection platform is the kang ning third generation Epic^®The imager, available from corning, detects a wavelength shift due to cell Dynamic Mass Resetting (DMR).

Inoculating HEK293T-Gi3-GPR84 cells in logarithmic growth phase into a 384-compatible micro-perforated plate, wherein the used culture medium is DMEM, the inoculation volume of each hole is 40 mu L, and the inoculation density is 2.5 multiplied by 10⁴And (4) performing activity detection on the inoculated cell plate after culturing for 20-24 hours in a cell culture box until the cell fusion degree reaches about 95%. The cell culture solution in the plate was changed to Hank's Balanced salt solution (HBSS, 20 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 90 min on the imager; the baseline was rescanned for 2min and GTPL5846 was added to the plates in a volume of 10. mu.L per well at concentrations of 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.2 nM, 15.6 nM, 7.8 nM, 3.9 nM, 2.0 nM, 1.0 nM, 0.5 nM, 0.2 nM and 0.1 nM 4 times in parallel, and the DMR signal was monitored in real time on an Epic instrument for 90 min, and the EC for GTPL5846 was calculated based on the maximum DMR response of the cells over 90 min of GTPL5846 exposure₅₀The values, results are shown in FIG. 1. The experimental result shows that GTPL5846 activates GPR84 receptor to generate DMR signal response and presents dose dependence, the dose response curve presents a single-phase S-shaped and reaches a saturation response, the highest DMR response value reaches about 600 pm, and the EC of the DMR response value reaches₅₀The value was 59.8. + -. 5.6 nM.

Example 2: desensitization DMR signature profile of HEK293T-Gi3-GPR84 cells

Inoculating HEK293T-Gi3-GPR84 cells in logarithmic growth phase into a 384-compatible micro-perforated plate, wherein the used culture medium is DMEM, the inoculation volume of each hole is 40 mu L, and the inoculation density is 2.5 multiplied by 10⁴And (4) performing activity detection on the inoculated cell plate after culturing for 20-24 hours in a cell culture box until the cell fusion degree reaches about 95%. The cell culture solution in the microplate is replaced by Hank's balanced salt solution(HBSS, 20 mM HEPES), 30. mu.L volume per well, after addition, was placed in Epic^®Balancing for 90 min on the imager; different concentrations of GTPL5846 were added to the microplate to pre-treat HEK293T-Gi3-GPR84 cells for 90 min, with a volume of 10. mu.L per well, at concentrations of 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.2 nM, 15.6 nM, 7.8 nM, 3.9 nM, 2.0 nM, 1.0 nM, 0.5 nM, 0.2 nM and 0.1 nM, in parallel 4 times; rescanning the baseline for 2min, adding a fixed concentration of 125 nM GTPL5846 to the microplate, adding a volume of 10. mu.L per well, paralleling 4 times, placing on an Epic apparatus to monitor the DMR signal in real time for 90 min, and calculating the IC based on the maximum DMR response value within 90 min of the cells being subjected to the GTPL5846₅₀The values, results are shown in FIG. 2. The results of the experiment show that GTPL5846 desensitizes GPR84 receptor in a dose-dependent manner, the dose response curve is monophasic in an "S" form and all reach a saturation response, and the IC thereof₅₀The value was 43.05. + -. 7.5 nM.

Example 3: GPR84 receptor antagonist GPR84 antagonist 8 DMR characteristic signal profile on HEK293T-Gi3-GPR84 cells

Inoculating HEK293T-Gi3-GPR84 cells in logarithmic growth phase into a 384-compatible micro-perforated plate, wherein the used culture medium is DMEM, the inoculation volume of each hole is 40 mu L, and the inoculation density is 2.5 multiplied by 10⁴And (4) performing activity detection on the inoculated cell plate after culturing for 20-24 hours in a cell culture box until the cell fusion degree reaches about 95%. The cell culture solution in the plate was changed to Hank's Balanced salt solution (HBSS, 20 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 90 min on the imager; different concentrations of GPR84 antagonist 8 were added to the plate at a volume of 10. mu.L per well at 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.2 nM, 78.1 nM, 39.0 nM, 19.5 nM, 9.8 nM, 4.9 nM, 2.4 nM and 1.2 nM by rescanning the baseline for 2min, and 4.9 nM, 2.4 nM and 1.2 nM were plated 4 times in parallel and placed on an Epic instrument to monitor the DMR signal for 90 min in real time, the results are shown in FIG. 3. The experimental results show that the DMR response signal for different concentrations of GPR84 antagonist 8 was close to zero.

Example 4: antagonistic DMR signature profiles of HEK293T-Gi3-GPR84 cells

Inoculating HEK293T-Gi3-GPR84 cells in logarithmic growth phase into a 384-compatible micro-perforated plate, wherein the used culture medium is DMEM, the inoculation volume of each hole is 40 mu L, and the inoculation density is 2.5 multiplied by 10⁴And (4) performing activity detection on the inoculated cell plate after culturing for 20-24 hours in a cell culture box until the cell fusion degree reaches about 95%. The cell culture solution in the plate was changed to Hank's Balanced salt solution (HBSS, 20 mM HEPES), and 30. mu.L of the solution was added to each well, and after the addition, the plate was placed in Epic^®Equilibrate on imager for 90 min. Different concentrations of the GPR84 receptor antagonist GPR84 antagonist 8 were added to the microplate to pre-treat the cells for 90 min at a volume of 10 μ L per well at concentrations of 10000 nM, 5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.2 nM, 78.1 nM, 39.0 nM, 19.5 nM, 9.8 nM, 4.9 nM, 2.4 nM and 1.2 nM, in parallel 4 times; rescanning the baseline for 2min, adding a fixed concentration of 125 nM GTPL5846 to the microplate, adding a volume of 10. mu.L per well, paralleling 4 times, placing on an Epic apparatus to monitor the DMR signal in real time for 90 min, and calculating the IC based on the maximum DMR response value within 90 min of the cells being subjected to the GTPL5846₅₀The values, results are shown in FIG. 4. The experimental results show that the GPR84 antagonist 8 antagonizes GPR84 receptor in a dose-dependent manner, the dose response curve is in a monophasic S type and all reach saturation response, and the IC thereof₅₀The value was 192.75. + -. 36.32 nM.

The invention establishes a GPR84 unmarked screening model based on the unmarked cell integration pharmacological technology, the model has the advantages of no need of fluorescent labeling and no need of adding an indicator in the detection process, and efficiently and reliably screens a commercial small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier to obtain the medicaments for treating central nervous system diseases, endotoxemia, obesity, type 2 diabetes, dyslipidemia, mixed lineage leukemia, inflammatory bowel disease and other diseases closely related to GPR84 receptor agonists, antagonists and pathway regulators and GPR84 receptor.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A cell screening model for the unlabeled membrane receptor GPR84 characterized in that: based on the marker-free cell integration pharmacological technology, a cell screening model of a GPR84 receptor is established by using a cell line HEK293T-Gi3-GPR84 stably expressing GPR84 and by means of known GPR84 receptor agonists and antagonists.

2. The cell screening model of the unlabeled membrane receptor GPR84 according to claim 1, characterized in that: HEK293T-Gi3-GPR84 cells are inoculated in a 384 micro-well plate which is compatible with cells and has an optical biosensing function, and the density of the inoculated cells is 1.0-4.5 multiplied by 10⁴The number of the cells per well is 40 muL per well, and the cell culture time after inoculation is 18-24 h.

3. Use of a cell screening model for the unlabeled membrane receptor GPR84, characterized in that: the screening steps of the sample to be detected with sensitivity, saturation and specificity are as follows:

(1) adding GPR84 receptor agonist GTPL5846 dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of GTPL5846 is 0.1-1000 nM, and detecting the DMR characteristic signal spectrum;

(3) adding GTPL5846 with the lowest concentration corresponding to the highest response intensity into the cell plate added with GPR84 receptor agonist GTPL5846 and antagonist GPR84 antagonist 8 in the step (1) and the step (2), and detecting a desensitized DMR characteristic signal spectrum and an antagonistic DMR characteristic signal spectrum;

(4) all the obtained DMR characteristic signal spectrums have the characteristics of concentration-response dependency relationship, sensitivity, saturation and specificity.

4. Use of a cell screening model for the unlabeled membrane receptor GPR84, characterized in that: the screening steps of the sample to be tested for having the agonistic activity are as follows:

(4) adding GPR84 receptor antagonist GPR84 antagonist 8 into a microplate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of GPR84 receptor antagonist GPR84 antagonist 8 is 1-100000 nM, pretreating for 5-90 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 GPR84 receptor if the DMR signal intensity is lower than that in the step (2).

5. Use of a cell screening model for the unlabeled membrane receptor GPR84, characterized in that: the screening steps of the samples to be tested for antagonistic activity are as follows:

(1) respectively adding a sample to be detected and a GPR84 receptor agonist GTPL5846 into a micropore plate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the GTPL5846 is 0.1-1000 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 GTPL5846 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 GTPL5846 in step (1), the test sample is determined to be an antagonist of the GPR84 receptor.

6. Use of a cell screening model for the unlabeled membrane receptor GPR84, characterized in that: the screening procedure for the test sample to be a modulator of activation of the signalling pathway downstream of the GPR84 receptor is as follows:

(1) respectively adding a sample to be detected and a GPR84 receptor agonist GTPL5846 into a micropore plate inoculated with HEK293T-Gi3-GPR84 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the GTPL5846 is 2.4-20000 nM, and detecting a DMR signal spectrum;

(2) continuously adding GTPL5846 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 1-90 min; if the DMR signal is different from the signal of GTPL5846 in step (1) at one stage of the ascending phase, the plateau phase and the lag phase, if the sample to be tested does not have a DMR response signal, it can be determined that the sample to be tested is an inhibitory modulator of the GPR84 receptor downstream signal pathway;

(3) continuously adding GTPL5846 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 1-90 min; if the DMR signal is different from the signal of GTPL5846 in the step (1) in one stage of ascending phase, plateau phase and lag phase, if the sample to be tested has a DMR response signal, GPR84 receptor antagonist GPR84 antagonist 8 is added into a micropore plate inoculated with HEK293T-Gi3-GPR84 cells, the concentration of GPR84 receptor antagonist GPR84 antagonist 8 is 1-10000 nM, pretreatment is carried out for 5-90 min, the sample to be tested with the same concentration as that in the step (1) is added, the DMR signal is detected, and if the DMR signal spectrum is consistent with that of the sample in the step (1), the sample to be tested can be judged to be a GPR84 receptor downstream signal pathway activation regulator.

7. The model for cell screening of the unlabeled membrane receptor GPR84 according to claim 6, characterized in that: the ascending period is 1-30 min, the plateau period is 30-60 min and the delay period is 60-90 min.