Application of calcium indicator GCaMP in calcium signal detection in human myocardial cell line

Abstract

The application provides application of a calcium ion indicator GCaMP in detection of a calcium signal of a human myocardial cell line, successfully applies the calcium ion indicator GCaMP to detection of a calcium signal of a myocardial cell, and increases the use of the calcium ion indicator GCaMP in detection of a calcium signal of a myocardial cell. Secondly, the application provides a method for detecting a calcium signal of a myocardial cell based on the calcium ion indicator GCaMP, and detecting agonism and / or inhibition of a myocardial cell PIEZO1 channel by a small molecule compound, and lays a foundation for research on a myocardial cell related calcium channel.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of a calcium ion indicator GCaMP in calcium signal detection of a human myocardial cell line. BACKGROUND

[0002] Calcium is the core element of the excitation-contraction coupling of myocardial cells. Under external stimulation, calcium ions enter the cell from the outside through calcium channels, increasing the local calcium ion concentration, activating the sarcoplasmic reticulum Ryanodine receptor (Ryanodine Receptor, Ryr), and ultimately causing myofilament sliding to promote myocardial cell contraction through calcium-induced calcium release. During the diastolic period of the myocardium, part of the calcium ions are reabsorbed into the sarcoplasmic reticulum for the next contraction. As a second messenger, calcium is widely involved in various signal transductions of myocardial cells responding to environmental changes. Myocardial cells can respond to environmental changes by using intracellular calcium ions as mediators to initiate various physiological or pathological reactions to cope with environmental changes. For many years, calcium signaling and its related research on the development and physiological and pathological processes of myocardial cells have been the focus of research in the field.

[0003] PIEZO1 channel is a new type of cardiac mechanical stress receptor. Under normal circumstances, PIEZO1 channel is not open, but under the stimulation of mechanical stretch, PIEZO1 channel agonist Yoda1, etc., PIEZO1 channel is open, increasing the influx of calcium ions, disrupting the intracellular calcium ion homeostasis, and then affecting the downstream signaling pathways to promote the occurrence and development of diseases. Therefore, in-depth research on the calcium signal in myocardial cells can help promote the research progress of PIEZO1 channel and a series of calcium-related channels.

[0004] Calcium imaging refers to a method for monitoring calcium ion concentration in tissues by using calcium ion indicators. Among calcium ion indicators, genetically encoded fluorescent calcium ion indicators can be divided into two categories: single-fluorophore genetically encoded calcium indicators and FRET-based / two-fluorescent protein genetically encoded calcium indicators. Among them, the GCaMP family of single-fluorophore genetically encoded calcium indicators is most widely used. GCaMP is composed of a circularly permuted enhanced green fluorescent protein (cpEGFP), a calmodulin (CaM) and a polypeptide sequence (M13) of myosin light chain kinase. In the absence of calcium ions, cpEGFP cannot function; when CaM-M13 specifically binds to calcium ions, cpEGFP undergoes a conformational change and is excited to release green fluorescence.

[0005] At present, the detection of myocardial cell calcium signal is still mainly based on chemical fluorescent dyes, but due to the problems of inaccurate subcellular localization, long-term incubation of chemical dyes which can cause cell toxicity, short residence of dyes in cells which limits the observation time, etc., the research progress of cardiac myocardial cells is limited. Therefore, it is necessary to develop a method for better detecting myocardial cell calcium signal for the research of myocardial cell calcium signal. SUMMARY

[0006] The purpose of the present application is to provide an application of calcium ion indicator GCaMP in human myocardial cell line calcium signal detection.

[0007] The present application adopts the following technical solutions:

[0008] An application of calcium ion indicator GCaMP in human myocardial cell line calcium signal detection.

[0009] An application of a lentivirus packaged with calcium ion indicator GCaMP in human myocardial cell line calcium signal detection.

[0010] A method for detecting human myocardial cell line calcium signal by using a lentivirus packaged with calcium ion indicator GCaMP.

[0011] Further, the method for detecting human myocardial cell line calcium signal by using a lentivirus packaged with calcium ion indicator GCaMP comprises the following steps:

[0012] (1) Resuscitation, culture and digestion subculture of human myocardial cell line;

[0013] (2) Lentivirus infection of human myocardial cell line;

[0014] (3) After 48h of infection, fluorescence microscope observation and quantitative analysis of fluorescence intensity were performed, and data were statistically analyzed.

[0015] In step (2), the method comprises:

[0016] a) The human myocardial cells treated in step (1) were plated into the well plate;

[0017] b) The lentivirus sample amount per well was calculated according to the optimal multiplicity of infection (MOI), and the infection solution was prepared by mixing the lentivirus with DMEM high-sugar medium containing 5 μg / mL Polybrene;

[0018] c) After the old culture medium in the well plate was discarded, the infection solution was added; after 16h of infection, the infection solution was discarded, the well plate was washed twice with PBS, fresh DMEM high-sugar medium was added, and the culture was continued until 48h of infection.

[0019] Preferably, the optimal MOI in step (2) is 20.

[0020] Further, the lentivirus is pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE.

[0021] Use of a lentivirus packaged with a calcium ion indicator GCaMP in detecting agonism and / or inhibition of human myocardial cell line PIEZO1 channel by a compound.

[0022] A method for detecting agonism and / or inhibition of human myocardial cell line PIEZO1 channel by a compound, comprising the following steps:

[0023] (1) Resuscitation, culture and digestion subculture of human myocardial cell line AC16;

[0024] (2) Lentivirus infection of human myocardial cell line AC16 treated in step (1);

[0025] I) The human myocardial cells AC16 treated in step (1) were plated into the well plate;

[0026] II) The well plate was divided into two groups for lentivirus infection:

[0027] (i) Control group A: calculate the amount of lentivirus per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar medium containing 5 μg / mL Polybrene; add the infection solution after discarding the old culture medium in the well plate; after 16 h of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-sugar medium, and culture until 48 h after infection; continue to culture for another 24 h;

[0028] (ii) Experimental group A: calculate the amount of lentivirus per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar medium containing 5 μg / mL Polybrene; add the infection solution after discarding the old culture medium in the well plate; after 16 h of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-sugar medium, and culture until 48 h after infection; continue to culture for another 24 h after adding 20 μM of the test small molecule compound;

[0029] (3) Observe under a fluorescence microscope, quantitatively analyze the fluorescence intensity, and count the data; obtain the relative fluorescence intensity of experimental group A relative to control group A, and the test small molecule compound with an increase of more than 20% compared with the control group is an agonist of the PIEZO1 channel;

[0030] (4) Select the test small molecule compound with no significant difference in the relative fluorescence intensity of experimental group A relative to control group A;

[0031] (5) Lentivirus infection of human myocardial cell line AC16 treated by step (1);

[0032] I) Spread the well-treated human myocardial cell AC16 into the well plate;

[0033] II) Divide the well plate into two groups for lentivirus infection:

[0034] (i) Control group B: calculate the amount of lentivirus per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar medium containing 5 μg / mL Polybrene; add the infection solution after discarding the old culture medium in the well plate; after 16 h of infection, discard the infection solution, wash twice with PBS, add fresh DMEM high-sugar medium, and culture until 48 h after infection; continue to culture for another 24 h after adding 20 μM Yoda1;

[0035] (ii) Experimental group B: calculate the amount of lentivirus per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar culture medium containing 5 μg / mL Polybrene; after the old culture medium in the well plate is discarded, the infection solution is added; after 16 h of infection, the infection solution is discarded, washed twice with PBS, and fresh DMEM high-sugar culture medium is added for culture until 48 h after infection; 20 μM Yoda1 and 20 μM of the to-be-tested small-molecule compound screened in step (4) are added, and then the culture is continued for 24 h;

[0036] (6) Under the fluorescence microscope, the fluorescence intensity is quantitatively analyzed, and the data are counted; the relative fluorescence intensity of experimental group B relative to control group B is obtained, and the to-be-tested small-molecule compound with a decrease of more than 20% compared with the control group is an inhibitor of the PIEZO1 channel.

[0037] Further, in step (4), the to-be-tested small-molecule compound with a relative fluorescence intensity of experimental group A relative to control group A of 80% to 120% is selected.

[0038] The application has the beneficial effects that the application provides the application of the calcium ion indicator GCaMP in the detection of the calcium signal of the human myocardial cell line, successfully applies the calcium ion indicator GCaMP to the detection of the calcium signal of the myocardial cell, and increases the use of the calcium ion indicator GCaMP in the detection of the calcium signal of the myocardial cell.

[0039] Secondly, the application provides a sample processing method for detecting the calcium signal of the myocardial cell based on the calcium ion indicator GCaMP, detects the agonizing or inhibiting effect of the small-molecule compound on the PIEZO1 channel of the myocardial cell based on GCaMP, and lays a foundation for the research on the related calcium channel of the myocardial cell. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is used for verifying the infection efficiency in example 1.

[0041] Figure 2 It is the structural formula of the compound MXY003-005.

[0042] Figure 3 It is the accuracy verification result of the method of the application.

[0043] Figure 4 It is the agonizing effect diagram of the first group of compounds in example 2.

[0044] Figure 5 It is the agonizing effect diagram of the second group of compounds in example 2.

[0045] Figure 6 It is the agonizing effect diagram of the third group of compounds in example 2.

[0046] Figure 7 Inhibition effect diagram of the first group of compounds in Example 3.

[0047] Figure 8 Inhibition effect diagram of the second group of compounds in Example 3.

[0048] Figure 9 Inhibition effect diagram of the third group of compounds in Example 3. DETAILED DESCRIPTION

[0049] The present application will be described in detail below with reference to examples and drawings.

[0050] Detection of calcium signal of human myocardial cell line AC16 cells in Example 1

[0051] (1) Resuscitation, culture and digestion passage of human myocardial cell line AC16

[0052] The human myocardial cell line AC16 cells were taken out from the -80°C refrigerator, thawed in a 37°C water bath, and when the cells were almost completely thawed, 1 mL pipette was used to suck the cells in a super-clean bench and add them to 3 mL of DMEM (high sugar) culture medium containing 10% FBS, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and 4 mL of DMEM (high sugar) culture medium containing 10% FBS was used to resuspend and spread in a 10 cm culture dish. After the cells were shaken, they were placed in a 37°C, 5% CO2, saturated humidity cell incubator for culture.

[0053] The composition of DMEM (high sugar) culture medium is: containing 4.5 g / L D-glucose, containing 580 mg / L L-glutamine, containing 110 mg / L sodium pyruvate (Gicbo, C11995500BT).

[0054] The growth state of the cells was observed every day, and the cells were changed every 1-2 days. Cells with good growth state and a confluence rate of 90% were taken for digestion passage. The culture medium was discarded, and the cells were washed twice with phosphate buffered saline (PBS), 1 mL of 37°C preheated 0.25% trypsin + 0.02% EDTA solution was added to digest for 1-2 min, and the cells were placed under a microscope for observation. When more than half of the cells were shrunk and moved like sand, 3 mL of 10% FBS DMEM (high sugar) culture medium was added to terminate the digestion. It was placed in a centrifuge at 1000 rpm for 5 min. The supernatant was discarded, and DMEM (high sugar) culture medium (90% DMEM (high sugar) culture medium + 10% FBS + 1% PS) was added to resuspend and plate.

[0055] PS contains the following components: Penicillin: 10000 U / mL, Streptomycin: 10000 μg / mL (VivaCell, C3420-0100). Trypsin contains trypsin, pancreatic amylase and pancreatic lipase (Sunway, CR25200).

[0056] (2) Determining the optimal multiplicity of infection (MOI) for lentivirus infection

[0057] a) The AC16 cells treated in step (1) were plated into 96-well plates at a cell count of 1 x 10 4 The cells were divided into different MOI (without Polybrene) experimental groups, different MOI (with Polybrene) experimental groups and blank cell groups. The MOI was set to 0, 10, 20 and 40. DMEM (high sugar) medium was used for culture.

[0058] b) The next day, the cell state was observed, and when the cell growth state was good and the confluence reached 60-70%, the culture medium was discarded, and lentivirus containing the corresponding volume was added, and DMEM (high sugar) medium or DMEM (high sugar) medium containing Polybrene was used for infection according to the experimental groups.

[0059] The concentration of Polybrene in the culture medium was 5 μg / mL. The amount of virus added per well = MOI x cell count at the time of infection / dose (TU / mL) x 10 3 The lentivirus was pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE (and Yuan Biology).

[0060] c) After 16 hours of infection, the cell state was observed under a microscope, the culture medium was discarded, and the cells were washed twice with PBS, and then new DMEM (high sugar) medium was added for culture.

[0061] d) After 48 hours of infection, the cell state was observed under a microscope, and 3 fields of view were randomly selected for photography to verify the infection efficiency. The optimal infection condition was determined to be MOI = 20 (with Polybrene), and the results are shown in Figure 1 .

[0062] The FITC filter was selected for the inverted fluorescence microscope, the excitation wavelength was 490 nm, and the emission wavelength was 520 nm.

[0063] (3) Lentivirus infection of human myocardial cell line AC16

[0064] a) The human myocardial cell line AC16 cells were plated into 96-well plates at a cell count of 1 x 10 4The medium uses DMEM (high sugar) medium.

[0065] b) The next day, observe the cell state, when the cell growth state is good, the confluence reaches 60-70%, discard the culture medium, calculate the virus addition amount per well according to the optimal MOI = 20 (add Polybrene) determined in step (2), and add DMEM (high sugar) medium containing the corresponding volume of lentivirus and Polybrene for infection. Among them, the concentration of Polybrene in the culture medium is 5 μg / mL; the virus amount added per well = MOI x cell number at the time of infection / dose (TU / mL) x 10 3 .

[0066] c) After 16h of infection, observe the cell state under a microscope, discard the culture medium, wash with PBS for 2 times, and add new DMEM (high sugar) medium for culture.

[0067] (4) Calcium ion fluorescence signal detection

[0068] After 48h of infection, the cells were observed under an inverted fluorescence microscope. Among them, the FITC filter was selected for the inverted fluorescence microscope, the excitation wavelength was 490nm, and the emission wavelength was 520nm. Randomly select 3 fields of view for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the image, and the data was statistically analyzed. According to the statistical results, the calcium level of cardiomyocytes was quantitatively characterized (Zheng Q, et al. Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites. Cell. 2022 Oct 27; 185(22): 4082-4098. e22.).

[0069] (5) Accuracy verification

[0070] To judge the accuracy of this method, a small molecule compound HBST271 (structure as shown in Figure 2 ) was introduced before testing. It is known that this compound has no agonistic effect on PIEZO1.

[0071] AC16 cells were divided into 3 groups. Control group (Con): simple lentivirus infection. Agonist group (Yoda1): lentivirus infection, then add PIEZO1 agonist Yoda1 (20 μM) for stimulation for 24h. Small molecule compound group (HBST271): lentivirus infection, then add small molecule compound (20 μM) for stimulation for 24h.

[0072] Cells were placed under an inverted fluorescence microscope for observation. Randomly selected 3 fields were photographed. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images, and the data was statistically analyzed. The results are shown in Table 1 and Figure 3

[0073] Table 1 Relative fluorescence intensity of negative control

[0074]

[0075] The results show that the relative fluorescence intensity of PIEZO1 channel agonist Yoda1 is significantly higher than that of the blank control group, and the relative fluorescence intensity of the negative control group has no significant difference with the blank control group. It can be seen that the activation of PIEZO1 channel can enhance the calcium signal level in cardiomyocytes, indicating that this method not only can detect the calcium signal of cardiomyocytes, but also can judge the agonistic or inhibitory effect of compounds on PIEZO1.

[0076] Example 2 Detection of the agonistic effect of small molecule compounds on AC16 PIEZO1 channel

[0077] The small molecule compound is a substituted benzyl sulfide oxadiazole compound, which may have a regulatory effect on PIEZO1 ion channel, can specifically agonize or inhibit the function of PIEZO1 ion channel, has low potential cardiotoxicity, and has small toxic and side effects.

[0078] The structure of the above compound is shown in Table 2.

[0079] Table 2 Preferred compound number and corresponding structural formula

[0080]

[0081]

[0082] To determine whether the above small molecule compound is a PIEZO1 channel agonist, the method described in Example 1 was used to divide AC16 cells into the following 3 groups for experiment.

[0083] (i) Control group: simple lentivirus infection, same as Example 1.

[0084] (ii) Agonist group: lentivirus infection, 16h after infection, discard the culture medium, wash with PBS for 2 times, add new DMEM (high sugar) culture medium for culture, until 48h after infection, add PIEZO1 agonist Yoda1 (20μM), stimulate for 24h, and then observe under fluorescence microscope.

[0085] ​(iii) Small molecule compound group: The difference is that after 16 hours of infection, the culture medium was discarded, the cells were washed twice with PBS, and new DMEM (high glucose) culture medium was added for culture. After 48 hours of infection, the small molecule compound to be tested (20 μM) was added, and fluorescence microscopy observation was performed after 24 hours of stimulation.

[0086] Cells were observed under an inverted fluorescence microscope. Three randomly selected fields of view were photographed. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and generate statistical data.

[0087] Small molecules whose relative fluorescence intensity increased by 20% or more compared with the control group were classified as possible agonists of the PIEZO1 channel.

[0088] The compounds I-1 to I-18 were tested in three batches for their agonistic effects on PIEZO1 at a concentration of 20 μM. The results of the three groups are as follows.

[0089] (1) The first group of compounds

[0090] The compounds were tested for agonism, and the results are shown in Table 3 and Figure 4 shown.

[0091] Table 3 Relative fluorescence intensity of the first batch of compounds

[0092]

[0093] In the agonist effect test of the first group of compounds, the fluorescence intensity of I-3 increased by 61.68%, which was better than Yoda1 (33.95%). The fluorescence intensity of I-2 increased by 31.18%, which was comparable to Yoda1.

[0094] (2) The second group of compounds

[0095] The compounds were tested for agonism, and the results are shown in Table 4 and Figure 5 shown.

[0096] Table 4 Relative fluorescence intensity of the second group of compounds

[0097]

[0098] In the agonist effect test of the second group of compounds, no compound had a better agonist effect.

[0099] (3) The third group of compounds

[0100] The compounds were tested for agonism, and the results are shown in Table 5 and Figure 6 shown.

[0101] Table 5 Relative fluorescence intensity of the third group of compounds

[0102]

[0103]

[0104] In the third group of compounds, the fluorescence intensity increased more obviously: I-5 (75.72%), I-6 (39.91%), I-8 (70.54%), I-16 (26.87%) and I-17 (40.26%), but all were weaker than Yoda1 (139.79%).

[0105] In summary, based on the standard of 20% increase in fluorescence intensity compared with the control group, the agonistic effects of compounds I-2, I-3, I-5, I-6, I-8, I-16 and I-17 were better, and the agonistic effect of I-3 was better than Yoda1.

[0106] Example 3: Detection of the inhibitory effect of small molecule compounds on AC16 Peizo1 channel

[0107] According to the results of Example 2, the compounds with no obvious difference compared with the control group were selected for further inhibition detection.

[0108] To determine whether the small molecule compounds are PIEZO1 channel inhibitors, the AC16 cells were divided into two groups.

[0109] (i) Control group: lentivirus infection, after 16h of infection, the culture medium was discarded, washed twice with PBS, and new DMEM (high sugar) culture medium was added for culture, until 48h of infection, PIEZO1 agonist Yoda1 (20μM) was added, and fluorescence microscopy observation was performed after 24h of stimulation.

[0110] (ii) Small molecule compound group: lentivirus infection, after 16h of infection, the culture medium was discarded, washed twice with PBS, and new DMEM (high sugar) culture medium was added for culture, until 48h of infection, PIEZO1 agonist Yoda1 (20μM) and small molecule compound (20μM) were added at the same time, and fluorescence microscopy observation was performed after 24h of stimulation.

[0111] The cells were observed under an inverted fluorescence microscope. Three fields were randomly selected for photography. ImageJ software was used to quantitatively analyze the fluorescence intensity of the images and the data was statistically analyzed. The relative fluorescence intensity was compared with the control group, and the small molecules with a decrease of 20% or more were listed as possible PIEZO1 channel inhibitors.

[0112] (1) First group of compounds

[0113] The compounds in Table 3 with no obvious difference compared with the control group were selected for inhibition detection, and the results are shown in Table 6 and Figure 7 Table 7.

[0114] Table 6 Relative fluorescence intensity of the first group of compounds

[0115]

[0116] The fluorescence intensity of I-1 was reduced by 28.87% after the agonistic effect was selected and compared with the negative control.

[0117] (2) The second group of compounds

[0118] The compounds in Table 4 were selected for inhibition detection, and the results are shown in Table 7 and Figure 8 .

[0119] Table 7 Relative fluorescence intensity of the second group of compounds

[0120]

[0121] The fluorescence intensity of I-1 was reduced by 28.87% after the agonistic effect was selected and compared with the negative control.

[0122] (3) The third group of compounds

[0123] The compounds in Table 5 were selected for inhibition detection, and the results are shown in Table 8 and Figure 9 .

[0124] Table 8 Relative fluorescence intensity of the third group of compounds

[0125]

[0126] The fluorescence intensity of I-1 was reduced by 28.87% after the agonistic effect was selected and compared with the negative control.

[0127] Based on the above results, the fluorescence intensity was reduced by 20% compared with the control group, and the compounds I-1, I-10, I-13 and I-14 had inhibition effect.

[0128] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, alternative combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are included in the scope of the present application.

Claims

1. Use of a lentivirus packaged with calcium ion indicator GCaMP, pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE, in detecting the agonistic and / or inhibitory effect of substituted benzyl sulfide oxadiazole compounds on human myocardial cell line PIEZO1 channel.

2. The application of a lentivirus pSLenti-CMV-jGCaMP7f-PGK-Puro-WPRE packaged with a calcium ion indicator GCaMP in detecting the agonism and / or inhibition of substituted benzyl sulfide oxadiazole compounds on human myocardial cell line PIEZO1 channel according to claim 1, characterized in that, It comprises the following steps: (1) Resuscitation, culture and digestion of human myocardial cell line AC16; (2) Lentivirus infection of human myocardial cell line AC16 treated in step (1); I) The human myocardial cells AC16 treated in step (1) are plated into the well plate; II) The well plate is divided into two groups for lentivirus infection: (i) Control group A: calculate the lentivirus sample amount per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar medium containing 5 μg / mL Polybrene; after discarding the old culture medium in the well plate, add the infection solution; after 16 h of infection, discard the infection solution, wash twice with PBS, and add fresh DMEM high-sugar medium for culture until 48 h after infection; continue to culture for another 24 h; (ii) Experimental group A: calculate the lentivirus sample amount per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar medium containing 5 μg / mL Polybrene; after discarding the old culture medium in the well plate, add the infection solution; after 16 h of infection, discard the infection solution, wash twice with PBS, and add fresh DMEM high-sugar medium for culture until 48 h after infection; add 20 μM test small molecule compound and continue to culture for another 24 h; (3) Observe under a fluorescence microscope, quantitatively analyze the fluorescence intensity, and count the data; obtain the relative fluorescence intensity of experimental group A relative to control group A, and the test small molecule compound with an increase of more than 20% compared with the control group is an agonist of PIEZO1 channel; (4) Select the test small molecule compound with no significant difference in relative fluorescence intensity of experimental group A relative to control group A; (5) Lentivirus infection of human myocardial cell line AC16 treated in step (1); I) The human myocardial cells AC16 treated in step (1) are plated into the well plate; II) The well plate is divided into two groups for lentivirus infection: (i) Control group B: calculate the lentivirus sample amount per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar medium containing 5 μg / mL Polybrene; after discarding the old culture medium in the well plate, add the infection solution; after 16 h of infection, discard the infection solution, wash twice with PBS, and add fresh DMEM high-sugar medium for culture until 48 h after infection; add 20 μM Yoda1 and continue to culture for another 24 h; (ii) Experimental group B: calculate the amount of lentivirus per well according to MOI = 20, and configure it into an infection solution with DMEM high-sugar culture medium containing 5 μg / mL Polybrene; after the old culture medium in the well plate is discarded, the infection solution is added; after 16 h of infection, the infection solution is discarded, washed twice with PBS, fresh DMEM high-sugar culture medium is added, and cultured until 48 h after infection; 20 μM Yoda1 and 20 μM of the small molecule compound to be tested screened in step (4) are added, and then cultured for another 24 h; (6) Under a fluorescence microscope, the fluorescence intensity is quantitatively analyzed, and the data are counted; The relative fluorescence intensity of experimental group B relative to control group B is obtained, and the small molecule compound with a decrease of more than 20% compared with the control group is an inhibitor of the PIEZO1 channel.

3. The method of claim 2, wherein, In step (4), the small molecule compound to be tested with a relative fluorescence intensity of experimental group A relative to control group A of 80% to 120% is selected.

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