JP7393780B2 - Screening method for gap junction function regulators - Google Patents

Description

The present invention relates to a method for evaluating or selecting a material that can control the fragmentation of gap junction plaques and the function of gap junctions.

In multicellular organisms including humans, intercellular communication is one of the most basic biological functions. Gap junctions are channels that directly communicate between adjacent cells, and membrane protein complexes called connexons (hemichannels) donated from adjacent cells bind together in the extracellular region to form macromolecular complexes (gap junction plaques). ) is formed. A connexon consists of four transmembrane proteins called connexins surrounding a central pore to form a hexamer. Gap junctions are known to pass small molecules with a molecular weight of less than 1000, such as ions, second messengers, and metabolites, and are essential for maintaining intercellular homeostasis and signal transmission during cell differentiation. .

Many connexin gene mutations have been reported to cause abnormalities in development, heartbeat, and nerve transmission, as well as diseases such as cataracts and hearing loss. For example, in recent years, the present inventors have shown that fragmentation of gap junction plaques is seen in the early molecular pathogenesis of mutations in the GJB2 gene, which encodes connexin 26, which is the most common cause of inherited hearing loss, and that fragmentation of gap junction plaques is observed in the presence of normal connexin 26. have shown that gap junctions containing other connexins accumulate and stabilize to form giant plaques, but when connexin 26 is mutated or deleted, gap junction plaques become fragmented (Non-patent Document 1). ).
On the other hand, it has been reported that connexin 26 is closely related to cancer metastasis, and that cancer metastasis is suppressed by biochemically suppressing the function of connexin 26 (Patent Document 1).

Therefore, it is thought that drugs that can control the fragmentation of gap junction plaques can serve as therapeutic agents for diseases associated with dysfunctional intercellular communication via gap junctions and cancer metastasis inhibitors.

Japanese Patent Application Publication No. 2001-17184

Kamiya, J. Clin. Invest. 124, 1598-1607 (2014)

The present invention relates to providing a method for simply and efficiently selecting a material that can control the fragmentation of gap junction plaques or the function of gap junctions.

The present inventor cultured cells expressing fragmented gap junction plaques on multiwell plates, imaged the formed gap junction plaques, and determined the maximum diameter and maximum diameter of gap junction plaques in contact with the cell area of one cell. We have found that by evaluating the area or minimum circularity, it is possible to efficiently screen for materials that can control the fragmentation of the gap junction plaque or the function of the gap junction.

That is, the present invention provides a method for evaluating or selecting a gap junction plaque fragmentation controlling agent or a gap junction function controlling agent, which includes the following steps (a) to (f).
(a) A step of culturing cells that can form fragmented gap junction plaques on a multiwell plate (b) A step of bringing a test substance into contact with the cultured cells cultured in the above step (c) A gap junction plaque, a cell membrane and (d) obtaining an image of the region containing the gap junction plaque; (e) measuring the diameter, area, or roundness of the gap junction plaque in contact with the cell region of one cell from the obtained image. , a step of selecting and recording the maximum diameter, maximum area, or minimum circularity from among them (f) comparing the maximum diameter, maximum area, or minimum circularity with a reference value, and comparing the maximum diameter, maximum area, or minimum circularity with a reference value, and determining the gap junction plaque fragmentation control agent or Step of evaluating or selecting a gap junction function controlling agent

According to the method of the present invention, gap junction shape data of 1,000 to 100,000 cells in one well of a multiwell plate can be obtained continuously and accurately at high speed, and gap junction plaque fragmentation control agent Alternatively, it is possible to efficiently select agents for controlling gap junction function on a large scale.

Cell images taken with a fully automatic imaging cytometer. Green: boundary of one cell (cell membrane), light blue: cell nucleus, red: gap junction plaque. The right image is an enlarged view (arrow: gap junction plaque). Schematic diagram showing plaque diameter and circularity in gap junction plaques. Measurement results of maximum diameter, maximum area, and minimum roundness of gap junction plaque of one cell after addition of test substance. The measurement results of the maximum diameter, maximum area, or minimum roundness of the gap junction plaque of one cell after addition of the gap junction plaque fragmentation inhibitor and the average value of the diameter, area, or roundness were recorded as data for one cell. Comparison with the measurement results in case.

The method of evaluating or selecting a gap junction plaque fragmentation controlling agent or a gap junction function controlling agent of the present invention includes the following steps (a) to (f).
(a) A step of culturing cells that can form fragmented gap junction plaques on a multiwell plate (b) A step of bringing a test substance into contact with the cultured cells cultured in the above step (c) A gap junction plaque, a cell membrane and (d) obtaining an image of the region containing the gap junction plaque; (e) measuring the diameter, area, or roundness of the gap junction plaque in contact with the cell region of one cell from the obtained image. , a step of selecting and recording the maximum diameter, maximum area, or minimum circularity from among them (f) comparing the maximum diameter, maximum area, or minimum circularity with a reference value, and comparing the maximum diameter, maximum area, or minimum circularity with a reference value, and determining the gap junction plaque fragmentation control agent or Step of evaluating or selecting a gap junction function controlling agent

In the present invention, "gap junction" refers to an intercellular junction that connects adjacent epithelial cells and has intercellular communication functions such as transport of small molecules and ions between adjacent cells and propagation of action potentials.
“Gap junctional plaque” refers to a complex consisting of several thousand gap junctional channels in which connexons on adjacent cell membranes join together. Here, connexon means a homohexamer of connexin (Cx).

More than 20 types of connexin protein families have been confirmed, and their distribution shows tissue specificity. For example, in the cochlea, connexins with a molecular weight of 26,000 and connexins with a molecular weight of 30,000 predominate, and in the heart, connexins with a molecular weight of 43,000 predominate. They are expressed as Cx26, Cx30, and Cx43, respectively, and are called connexin 26, connexin 30, and connexin 43.
Connexins expressed in each tissue are shown below. In some cases, multiple connexins coexist within the same tissue and work together to form gap junctions.
- Blood vessels (endothelium: Cx40, Cx37, smooth muscle: Cx43)
・Liver (Cx32, Cx26)
・Pancreas (β cell: Cx36, exocrine gland: Cx32)
・Lens (epithelium: Cx43, fibers: Cx46, Cx50)
・Inner ear (cochlea: Cx26, Cx30)
・Heart (sino-atrial node: Cx45, atrial muscle: Cx43, Cx45, Cx40, atrioventricular node: Cx45, bundle of His: Cx40, Purkinje fibers: Cx40, Cx43, ventricular muscle: Cx43, Cx45)
・Nervous system (astrocytes: Cx43, Cx30, Cx26, oligodendrocytes: Cx32, Cx29, Cx47, myelin sheath: Cx32, ependymal cells: Cx43, neurons: Cx36)

In step (a), "cells capable of forming fragmented gap junction plaques" refers to cells in which normal connexin expression is regulated and capable of forming fragmented gap junction plaques, such as Cx26 Inner ear cells derived from knockout mice, inner ear cells induced to differentiate from stem cells (iPS cells) derived from Cx26 knockout mice (Reference: Fukunaga, Stem Cell Reports, 2016, 7(6), 1023-1036), and fragmented inner ear cells Includes recombinant cells that have been genetically engineered to form gap junction plaques.
Examples of recombinant cells that have been genetically engineered to form fragmented gap junction plaques include HeLa cells that forcibly express only Cx30 (Non-Patent Document 1) and cells that have lost normal gap junction formation function. recombinant cells that have been genetically engineered to express connexin mutants.
Connexin mutants include the deletion of at least one amino acid residue, substitution with another amino acid residue, addition or insertion of another amino acid residue in the amino acids that constitute connexin, and normal gap junction formation function. Examples include connexin molecules in which the amino acid residue has been lost, preferably a mutant in which the amino acid residue is substituted with another amino acid residue. Mutations in amino acid residues may be introduced into one region in the amino acid sequence, or may be introduced into multiple different regions. The number of mutations may be at least one, preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5.
For example, a Cx26 mutant in which the 75th arginine residue of the Cx26 amino acid sequence (SEQ ID NO: 1) is replaced with a tryptophan residue (R75W), and a Cx26 mutant in which the 75th arginine residue in the Cx26 amino acid sequence (SEQ ID NO: 1) is replaced with a glutamine residue (R75Q). Cx26 mutant with the 143rd arginine residue replaced with a glutamine residue (R143Q), Cx26 mutant with the 179th aspartic acid residue replaced with an asparagine residue (D179N), and the 184th arginine residue replaced with an asparagine residue (D179N). Cx26 mutant with glutamine residue substituted (R184Q), Cx26 mutant with cysteine residue at position 235 deleted (235delC), glycine residue at position 45 replaced with glutamic acid residue and tyrosine residue at position 136 Examples include Cx26 mutants in which the group is replaced with other amino acid residues (G45E/Y136X).

A recombinant cell genetically engineered to express a connexin variant is a cell in which a nucleic acid encoding a connexin variant has been introduced into a host cell capable of expressing it, preferably a cell in which the gene encoding the connexin variant is constitutively present. A cell into which a nucleic acid encoding a connexin variant has been introduced in such a way that the nucleic acid can be replicated as an extrachromosomal element, or a cell in which the nucleic acid is Examples include cells into which the nucleic acid has been introduced so as to be replicable through chromosomal integration.

Here, the nucleic acid may be any of DNA, RNA, mRNA, cDNA, and cRNA. The cell into which the nucleic acid encoding the connexin variant has been introduced into a host cell capable of expressing the connexin variant contains the nucleic acid encoding the connexin variant and a vector (E. coli plasmid) suitable for expressing the connexin variant in the host cell used. , a yeast plasmid, or an animal virus vector such as a retrovirus vector), and the resulting DNA fragment can be obtained by introducing the resulting DNA fragment into a host cell. Nucleic acids encoding connexin variants can be introduced into host cells using, for example, known methods such as transformation and transfection, electroporation, calcium phosphate method, lipofection method, DEAE dextran method, particle gun method, and viruses. It can be carried out according to the method etc.

Nucleic acids encoding connexin variants include mutagenesis of one or more nucleotides on the connexin-encoding nucleic acid sequence, or substitution or insertion of another nucleotide sequence into the nucleotide sequence, or a part of the sequence of the gene. Alternatively, examples include nucleic acids that have been completely deleted. Specific methods for the above-mentioned mutation introduction and nucleotide sequence substitution or insertion include ultraviolet irradiation, site-specific mutagenesis, SOE-PCR method, homologous recombination method, and the like.

Further, the host cell may be any cell that can efficiently express the nucleic acid encoding the connexin variant polypeptide and is easy to culture, such as HeLa cells, Xenopus oocytes, Chinese hamster ovary cells, etc. (CHO), human embryonic kidney (HEK) cells, Sf-9 insect cells, and the like.

Cells capable of forming fragmented gap junction plaques are cultured on multiwell plates such as 96 wells, 384 wells, and 1536 wells. Here, the culture medium used includes ingredients suitable for the growth of the cells, such as glucose, amino acids, peptone, vitamins, cell growth promoting factors (e.g., cell growth factors, hormones, binding proteins, cell adhesion). Any medium may be used as long as it contains components such as factors, lipids), serum (eg, FBS, FCS, etc.), calcium chloride, magnesium chloride, etc. The medium may be a commercially available medium, such as D-MEM medium, Ham's F-12 medium, MEM medium, and the like.

In step (b), the cells capable of forming fragmented gap junction plaques are brought into contact with the test substance, for example, by adding the test substance to the culture medium in advance to a predetermined concentration, and then fragmenting the cells. Placing cells capable of forming fragmented gap junction plaques in a medium, or adding a test substance to a predetermined concentration to a medium containing cells capable of forming fragmented gap junction plaques. This can be done by
Here, the cell concentration at the time of seeding of cells capable of forming fragmented gap junction plaques is preferably 5,000 to 20,000 cells/well when a 96-well plate is used, for example. Further, the concentration of the test substance added is preferably 10 to 1000 nM.

In the present invention, the test substance is not particularly limited, and may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, or a compound. It may be a composition or a mixture.

For contact between cells capable of forming fragmented gap junction plaques and the test substance, it is preferable to culture them at room temperature (25° C. to 37° C.) for usually about 24 to 72 hours.

Then, in step (c), gap junction plaques, cell membranes and cell nuclei are visualized. Visualization is performed by fixing, permeabilizing, and blocking cells as necessary, and then staining them with a staining reagent such as a fluorescent dye and/or an antibody conjugate.
Staining of cells with these reagents is performed, for example, by adding the corresponding cell staining reagent to cells and incubating at room temperature to 37° C. for a predetermined period of time.
Visualization of gap junction plaques can be performed by using an immunofluorescent staining method using an anti-connexin antibody and a fluorescently labeled secondary antibody.
Cell membranes can be labeled with a fluorescently labeled cholera toxin B subunit (CtxB) that specifically recognizes the glycosphingolipid GM1, or by labeling cadherin, a transmembrane protein, with an anti-cadherin antibody. It is possible to visualize.
Visualization of cell nuclei can be performed using known nuclear staining reagents such as DAPI, propidium iodide (PI), Hoechst 33342, and the like.

When visualizing the cells, if necessary, apoptotic markers such as fragmented PARP and anti-active caspase 3 can be labeled and detected in order to count cell death due to side effects of the test substance.

Then, in step (d) an image of the region containing the gap junction plaque is acquired.
That is, visualized images of gap junctions, cell membranes, cell nuclei, and even apoptosis markers visualized in the previous step are obtained. Image acquisition can be performed using a fully automatic imaging cytometer (GE, IN Cell Analyzer 2200, etc.).
For example, regarding gap junctions, the focused part of green fluorescence labeled with connexin is automatically recognized, and a composite image (2.5D) with a thickness of about 4 μm in the Z-axis direction is obtained with the focused focus as the center. This provides an overall image of the entire gap junction plaque from the superficial side to the basal side. Images of red fluorescence, which identifies cell boundaries (cell membranes), and DAPI fluorescence, which identifies nuclei, are similarly acquired (see FIG. 1). Images are preferably acquired from 10 to 50 fields per well.

Next, in step (e), the diameter, area, or circularity of the gap junction plaque in contact with the cell area of one cell is measured from the acquired image, and the maximum diameter, maximum area, or minimum circularity is selected from among them. recorded.
Image analysis is performed using image analysis software (GE, IN Cell Investigator, etc.), and the shape data of the gap junction plaque is obtained by the following procedure.
First, the cell region of each single cell is recognized from the cell membrane information image. Specifically, the entire range indicated by the red fluorescence is recognized as the Cell-Pre, and a part of the range where the fluorescence intensity is stronger than the surrounding area is recognized as the Membrane. Cell nucleus information is recognized as an indicator of a single cell. Cells in which only one cell nucleus information exists within the range enclosed when Membrane is subtracted from Cell-Pre are analyzed as one cell unit.

Next, gap junction plaque information is recognized as a target. Next, the diameter, area, and circularity of the gap junction plaque that contains or has a contact point with the recognized cell and the Target are measured, and the measured values of the maximum diameter, maximum area, or minimum circularity are determined as 1. Recorded as cell data.
Here, the diameter of the gap junction plaque is the maximum diameter of the object labeled with connexin immunostaining, the area is the area of the same object, and the roundness is the closer the object is to a perfect circle. The value is closer to 1, and conversely, the closer the shape is to an ellipse, flattened, or linear shape (see FIG. 2), the closer the value is to 0 (see FIG. 2).
With this measurement, it is possible to obtain gap junction size data for a large number of cells at once. For example, 100 cells per field of view are measured in 10 fields of view in one well of a 96-well plate. This makes it possible to obtain numerical data for 1,000 cells or more per well.

Next, in step (f), the maximum diameter, maximum area, or minimum roundness of the plaque is compared with a reference value, and a gap junction plaque fragmentation controlling agent or a gap junction function controlling agent is evaluated or selected.
As shown in the Examples below, the addition of a gap junction plaque fragmentation control agent increases the maximum diameter and maximum area values and decreases the minimum circularity value of gap junction plaques.
Therefore, the maximum diameter, maximum area, or minimum roundness level is determined by determining whether the maximum diameter or maximum area value of the gap junction plaque in cells to which the test substance has been added increases compared to a predetermined reference value. If the minimum roundness is decreased compared to a predetermined reference value, the test substance can be evaluated or selected as a gap junction plaque fragmentation inhibitor and furthermore, as a gap junction function improver. If the maximum diameter and maximum area values of the gap junction in the added cells are decreased compared to the predetermined reference value, and the minimum roundness is increased compared to the predetermined reference value, then The test substance can be evaluated or selected as a gap junction plaque fragmentation promoting agent and thus a gap junction function inhibitor.

The predetermined standard values include, for example, the gap junction size value measured in the absence of the test substance, as well as the size value of fragmented gap junction plaques and/or normal gap junction plaques determined by pre-measurement. Can be mentioned.
For example, the size value of fragmented gap junction plaques (x(0)), the size value of normal gap junction plaques (x(1)) determined by pre-measurement, and the size value of gap junction plaques after contact with the test substance. The gap junction plaque fragmentation inhibitor or gap junction function improver can be evaluated by calculating the gap junction recovery rate from the size value (x) as shown in the following formula.

Furthermore, in the method of the present invention, toxicity evaluation of a test substance can be performed simultaneously with the selection or evaluation of a gap junction function controlling agent or a gap junction plaque fragmentation controlling agent.
In this case, a labeled apoptotic marker (eg, active caspase 3) is determined as a dead cell by colocalization with a nuclear label. The ratio of dead cells to the total number of nuclei (apoptotic marker positive ratio) is calculated as the dead cell ratio. For example, the value obtained by subtracting the obtained dead cell rate from the gap junction recovery rate is used as the Gap junction Restoration and Toxicity Index (GRT value), and a gap junction plaque fragmentation inhibitor that takes side effects into account or It becomes possible to determine gap junction function improving agents. That is, if the dead cell rate without the addition of the test substance is y(0) and the dead cell rate after the addition of the test substance is y, the test substance-induced dead cell rate is calculated as shown in the formula below,

The GRT value can be determined by subtracting the test substance-induced cell death rate from the gap junction recovery rate.
GRT value = (gap junction recovery rate) - (test substance-induced cell death rate)

Here, the GRT value is considered to be obtained in the range of 0-1, but the higher the value, the higher the efficacy and safety can be evaluated, and it becomes an index for drug screening for clinical application.
For example, even if the gap junction recovery rate is 0.95 (95% recovery), if the test substance-induced cell death rate is 0.5 (50% cell death due to the test substance), the GRT value = 0. 95-0.5=0.45. On the other hand, if the gap junction recovery rate was 0.78 (78% recovery) and the test substance-induced cell death rate was 0.08 (8% cell death due to the test substance), the GRT value = 0.78-0. 08=0.7. In other words, the latter test substance or administration concentration is considered to have higher efficacy and safety or administration conditions.
Using this index, it becomes possible to screen safe and effective drugs on a large scale and select gap junction plaque fragmentation inhibitors or gap junction function improving agents.

The test substance evaluated or selected as a gap junction plaque fragmentation inhibitor or a gap junction function enhancer by the above method can be used as a therapeutic agent for diseases associated with abnormalities in intercellular communication via gap junctions, such as skin diseases ( It can be used as a therapeutic agent for diseases such as congenital ichthyosis), cardiovascular diseases, airway epitheliitis, alveolar tissue disorders, hearing disorders (e.g., hereditary hearing loss), endothelial lesions, visual disorders (e.g., cataracts), and gap junction plaque fragmentation. A test substance evaluated or selected as a promoter or gap junction function inhibitor can be a cancer metastasis inhibitor.

(1) Cell culture and drug addition Hela cells stably expressing Cx30, Cx26, or Cx26 mutants were seeded on a 96-well plate for cell imaging, Lumox multi-well 96-well plate (Sarstedt).
A gene encoding Cx30, Cx26, or Cx26 mutant (R75W), or a plasmid carrying a drug resistance gene such as a neomycin resistance gene is introduced into HeLa cells by lipofection, and then a drug such as neomycin is added to induce the gene introduction. Cells that did not contain the gene were killed, and only the cells that had been introduced with the gene were cultured to survive. Specifically, 24 hours after gene introduction was carried out in a 6 cm dish, the seeds were reseeded in a 10 cm dish at a rate of 1/10 to 1/40 depending on the growth rate. After another 24 hours, the medium was exchanged with a medium containing a drug matching the selection marker contained in the expression vector, and the medium was exchanged twice a week until colonies grew. Approximately 14 to 16 days after adding the drug, the appearance of colonies was confirmed, and the colonies were isolated, and Vaseline was pressed against the bottom of the cap to allow the colonies to enter. A medium was placed in the cap, peeled off by pipetting, and transferred to a 24-hole dish. Whether the gene had been introduced was confirmed by PCR method or the like.
After seeding each cell at 9600 cells/well, the cells were cultured for 3 days at 37° C. and 5% CO 2 in D-MEM medium containing 10% fetal bovine serum. After confirming a uniform cell density of approximately 60-80% of confluence (saturation), test drugs were added at concentrations of 50, 100, 500, and 1000 nM. 24 to 48 hours after addition, the cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes, and then the solution was exchanged with PBS and stored at 4°C.

(2) Visualization of gap junctions and single cells by immunofluorescence staining 2-1. Visualization of gap junctions, cell membranes, and apoptosis markers 1) Permeabilization of fixed cells: 0.1% TritonX-100 (10% solution 10 μl/ml PBS) (50 ul/well) for 5 min.
2) Washing: PBS 5 minutes twice (approximately 150 μl each wash).
3) Blocking: 2% BSA 30 minutes (50ul/well).
4) Primary antibody reaction: The following antibodies are reacted as primary antibodies. (The diluent is a 1% BSA solution)
・Anti-Cx26 antibody (Rb Invitrogen) 1:600
・Anti-Cx30 antibody (Rb Invitrogen) 1:600
・Anti-active caspase 3 antibody (Rb Promega) 1:400
5) Cover with parafilm and let react overnight in a shaker in the refrigerator.
6) Washing: PBS co-wash once + PBS 10 minutes twice.
7) Secondary antibody reaction and cell membrane staining: React the following antibodies with 1% BSA. 1 hour at room temperature. At the same time, cell membrane visualization treatment using CxtB was performed.
・Secondary antibody: Alexa488-Rb 1:1000
・Visualization of cell membrane: CxtB-Alexa555 1:400
8) Washing: PBS 10 minutes twice.

2-2. Visualization of cell nuclei 1) Staining of cell nuclei: DAPI (Dojindo) 1:3000 in PBS for about 3 minutes.
2) Mounting: Remove the liquid, wash once with PBS, and mount with FluoSave.
3) Storage: Cover with a plate sticker and store in the refrigerator, protected from light.

(3) Automatically capture fluorescence images using an imaging cytometer Capture images of labeled fluorescence of gap junctions, cell membranes, and apoptosis markers using a fully automatic imaging cytometer (GE, IN Cell Analyzer 2200, etc.) on the imaging plate stained above. did. Regarding gap junctions, the focused portion of green fluorescence labeled with connexin 30 or connexin 26 is automatically recognized, and a composite image (2.5D) with a thickness of approximately 4 μm in the Z-axis direction centered on the focal point is obtained. As a result, an image is obtained in which the entire gap junction plaque can be visualized from the superficial side to the basal side.
Images of red fluorescence, which identifies cell boundaries, and DAPI fluorescence, which recognizes nuclei, are similarly acquired. Similarly, images of 10 fields of view are acquired per well.
In addition, when staining with an anti-active caspase 3 antibody, the entire dead cells are stained green, which is used to count cell death.

(4) Analysis of gap junction size using image analysis software The formation and recovery of gap junction plaques were analyzed using image analysis software (GE, IN Cell Investigator, etc.) using image analysis software using the following analysis method.
1) Cell Recognition First, an analysis program is made to automatically recognize the cell region of each single cell from an image of red fluorescence indicating cell membrane information such as Alexa555-labeled CtxB. The entire range indicated by the red fluorescence is recognized by Cell-Pre, and the part where the fluorescence intensity is stronger than the surrounding area is recognized as the membrane. The fluorescence of nuclear staining DAPI is recognized as an indicator of single cells. Cells in which only one DAPI label exists within the range enclosed when Membrane is subtracted from Cell-Pre are analyzed as one cell unit.

2) Recognition of gap junction plaque Next, the gap junction plaque region is recognized as a target from the green fluorescence information.
The diameter, area, and circularity of a gap junction plaque containing or having a contact point with one cell recognized in the above analysis and the above Target were measured. Among them, the measured values of the maximum plaque diameter, maximum plaque area, and minimum roundness were recorded as data for one cell. Through this measurement, gap junction size data for multiple cells (for example, 100 cells) was obtained at once.

3) Calculation of gap junction recovery rate The number of fragmented Cx30 gap junctions (approximately 1-2 μm) to which no drug has been added is x (0), and the average value of accumulated gap junctions such as Cx26 gap junctions (3- 5 μm) is x (1), and the average of the values after drug addition (maximum diameter, maximum area, and minimum roundness of gap junction plaques) is x, then the gap junction recovery rate can be calculated from the following formula. .

4) Calculation of drug-induced dead cell rate Green fluorescence labeled using activated caspase 3 antibody etc. is measured as dead cells by colocalization with nuclear labeling by DAPI. The ratio of dead cells to the total number of nuclei (active caspase 3 positive ratio) is calculated as the dead cell ratio. By subtracting the resulting dead cell rate from the gap junction recovery rate, it becomes possible to determine the optimal concentration in consideration of side effects. When the dead cell rate without the addition of the drug is y(0) and the dead cell rate after the addition of the drug is y, the drug-induced dead cell rate can be calculated from the following formula.

5) Calculation of gap junction recovery/toxicity evaluation index By subtracting the drug-induced dead cell rate in 4) from the gap junction recovery rate in 3) above, ((gap junction recovery rate) - (drug-induced dead cell rate) ), the gap junction recovery/toxicity evaluation index (GRT value) can be calculated.

(5) Results 1) Repair of gap junction plaque fragmentation was confirmed in advance by microscopic observation using immunostaining of connexin molecules, and hyperfunction of gap junctions was confirmed by dye transfer assay using Neurobiotin tracer. Maximum diameter of gap junction plaques when two drugs (proteasome inhibitors) A and B and drug C, which was confirmed to repair gap junction plaque fragmentation and not to enhance gap junction function, were added. Figure 3 shows the measurement results of (Target Length), maximum area (Target Area), and minimum roundness (Form Factor). For comparison, Figure 4 shows a comparison of measurement results when drug A or B was added and the average values of diameter, area, and roundness of all gap junction plaques were recorded as data for one cell. Shown below.
As shown in FIG. 3, no effect was observed with drug C, but with drugs A and B, the maximum diameter and maximum area of gap junction plaques generally increased in a concentration-dependent manner, and the minimum circularity decreased. At high concentration (1000 nM), the effect of B was more pronounced than that of A. However, at low concentrations (50 nM), A was more effective than B. When side effects are taken into consideration, A, which is effective at low concentrations, is considered to be more appropriate as a drug.
On the other hand, when the maximum diameter, maximum area, or minimum circularity was not used as data for one cell, no concentration-dependent changes were reflected at all (Fig. 4, right).

2) When the gap junction recovery rate of drugs A (A50) and B (B50) at 50 nM is calculated from the formula shown in 3) of (4) above, it is calculated as follows.

In this calculated value, the maximum diameter of the gap junction plaque of CX26, which is considered to have normal gap junction function in the inner ear, was set as x(1) as a positive control value.
If the measured value is the same as the maximum plaque diameter x (0) of CX30 without the addition of drugs and no increase in the maximum plaque diameter due to the drug is observed, it will be around 0, and it will recover to the same level as the maximum plaque diameter of gap junctions of CX26. In this case, the value will be around +1. If the maximum diameter increases further than CX26, the value will exceed 1. In other words, at low concentrations, the recovery rate of drug A at 50 mM is 1.31, which is higher than the recovery rate of drug B, 0.43, indicating that A is more effective than B at low concentrations.

3) When the drug-induced cell death rate of drugs A (A50) and B (B50) at 50 nM is calculated from the formula shown in 4) of (4) above, it is calculated as follows. It can be seen that the number of dead cells due to side effects is higher in the

4) Then, the gap junction recovery/toxicity evaluation index (GRT value) can be calculated as follows.
GRT value (A50) = 1.31-0.0015 = 1.3085
GRT value (B50) = 0.43-0.0177 = 0.4123
As a result, it can be determined that among drug A and drug B, both of which were found to be effective, drug A has the highest efficacy and safety when used at a low concentration of 50 nM.

Claims (5)

A method for evaluating or selecting a gap junction plaque fragmentation controlling agent or a gap junction function controlling agent, comprising the following steps (a) to (f).
(a) A step of culturing cells that can form fragmented gap junction plaques on a multiwell plate (b) A step of bringing a test substance into contact with the cultured cells cultured in the above step (c) A gap junction plaque, a cell membrane and (d) obtaining an image of the region containing the gap junction plaque; (e) measuring the diameter, area, and roundness of the gap junction plaque in contact with the cell region of one cell from the obtained image. (f) Comparing the maximum diameter, maximum area and minimum circularity with a reference value, and determining the gap junction plaque fragmentation control agent or the gap junction. Process of evaluating or selecting a function control agent 2. The method according to claim 1, wherein in step (a), the cells capable of forming fragmented gap junction plaques are cells expressing Cx26 mutant or Cx30 wild type. 3. The method according to claim 2, wherein the Cx26 mutant is a mutant in which the 75th arginine residue in the amino acid sequence of Cx26 is replaced with a tryptophan residue or a glutamine residue. The method according to any one of claims 1 to 3, wherein in step (e), measurement of 100 cells per field is performed in 10 fields. The method according to any one of claims 1 to 4 , wherein the dead cell rate is further calculated using an apoptosis marker, and toxicity evaluation by the test substance is also performed.