Method for evaluating test sample
阅读说明：本技术 被测试样的评价方法 (Method for evaluating test sample ) 是由 斋藤香织 藤田郁尚 冈田文裕 石井健 于 2019-03-14 设计创作，主要内容包括：本发明提供一种被测试样的评价方法,其是能够容易地评价被测试样是否具有抑制皮肤细胞中的细胞因子产生的作用的被测试样的评价方法,该评价方法基于由被测试样通过TRPM4表达细胞的TRPM4所引起的生理学现象,对被测试样所具有的细胞因子产生抑制作用进行评价。(The present invention provides a method for evaluating a test sample, which is capable of easily evaluating whether the test sample has an effect of inhibiting cytokine production in skin cells, and which evaluates the cytokine production-inhibiting effect of the test sample on the basis of a physiological phenomenon caused by TRPM4 in cells in which the test sample is expressed by TRPM 4.)
1. A method for evaluating a test sample, which evaluates the effect of the test sample on the inhibition of cytokine production in skin cells, characterized by bringing a TRPM 4-expressing cell into contact with the test sample, measuring a physiological phenomenon caused by the test sample passing through TRPM4, and evaluating the effect of the test sample on the inhibition of cytokine production based on the physiological phenomenon.
2. The method for evaluating a test sample according to claim 1, wherein the physiological phenomenon is cytokine production, wherein a TRPM 4-expressing cell having cytokine expression ability is used as the TRPM 4-expressing cell, wherein the physiological phenomenon is measured, and wherein the method for evaluating the inhibition effect of cytokine production in the test sample based on the physiological phenomenon comprises the steps of:
(A1) a step of contacting a TRPM 4-expressing cell having a cytokine-expressing ability with a test sample and a cytokine production-promoting substance, and measuring an expression level of the cytokine and/or a gene thereof in the TRPM 4-expressing cell;
(A2) a step of contacting a TRPM 4-expressing cell having a cytokine-expressing ability with a cytokine production-promoting substance and measuring the expression level of the cytokine and/or its gene in the TRPM 4-expressing cell;
(A3) a step of contacting a TRPM 4-deficient cell deficient in the function of TRPM4 in the TRPM 4-expressing cell with a test sample and a cytokine production promoting substance, and measuring the expression level of a cytokine and/or a gene thereof in the TRPM 4-deficient cell;
(A4) a step of contacting a TRPM 4-deficient cell deficient in the function of TRPM4 in the TRPM 4-expressing cell with a cytokine production promoting substance, and measuring the expression level of a cytokine and/or a gene thereof in the TRPM 4-deficient cell; and
(A5) and (c) evaluating the cytokine production inhibitory effect of the test sample based on the expression levels measured in the steps (a1) to (a 4).
3. The method for evaluating a test sample according to claim 1, wherein the operation of measuring the physiological phenomenon and evaluating the cytokine production inhibitory effect of the test sample based on the physiological phenomenon comprises:
(B1) a step of contacting the TRPM 4-expressing cell with a test sample, and measuring a change in TRPM4 activity in the TRPM 4-expressing cell before and after the contact with the test sample; and
(B2) a step of evaluating the cytokine production inhibitory effect possessed by the test sample based on the change in TRPM4 activity measured in step (B1).
4. A cytokine production inhibitor for use in an application of activating TRPM4 to inhibit cytokine production in skin cells, characterized by containing an aluminum compound as an active ingredient for activating TRPM 4.
Technical Field
The present invention relates to a method for evaluating a test sample. More specifically, the present invention relates to a method for evaluating a test sample for evaluating whether or not a test substance has an inhibitory effect on cytokine production, and to a cytokine production inhibitor.
Background
Inflammation of the skin causes acne scars, spots, wrinkles, and the like. Therefore, cosmetics and the like containing an anti-inflammatory agent for suppressing inflammation caused by the skin have been proposed (for example, see patent document 1). However, since the above-mentioned anti-inflammatory agents are agents for calming inflammation that has already occurred in the skin, there is a demand for development of cosmetics for preventing inflammation, agents for preventing inflammation, and the like, which can suppress the onset of inflammation by suppressing the production of cytokines, which are one of inflammation-causing factors.
Disclosure of Invention
Problems to be solved by the invention
The present invention has been made in view of the above-described conventional techniques, and an object thereof is to provide a method for evaluating a test sample that can easily evaluate whether or not the test sample has an effect of inhibiting cytokine production in skin cells, and a cytokine production inhibitor that can effectively inhibit cytokine production in skin cells.
Means for solving the problems
The present invention relates to:
(1) a method for evaluating a test sample, which evaluates the cytokine production inhibitory effect in skin cells possessed by the test sample, characterized by bringing a TRPM 4-expressing cell into contact with the test sample, measuring a physiological phenomenon caused by TRPM4 in the test sample-expressing cell that expresses TRPM4, and evaluating the cytokine production inhibitory effect possessed by the test sample on the basis of the physiological phenomenon.
(2) The method for evaluating a test sample according to (1) above, wherein the physiological phenomenon is cytokine production, wherein a TRPM 4-expressing cell having cytokine expression ability is used as the TRPM 4-expressing cell, wherein the physiological phenomenon is measured, and wherein the method for evaluating a test sample having an inhibitory effect on cytokine production based on the physiological phenomenon comprises the steps of:
(A1) contacting a test sample with a cell capable of expressing a cytokine-expressing TRPM4, and a cytokine production-promoting substance, and measuring the expression level of the cytokine and/or its gene in the TRPM 4-expressing cell;
(A2) contacting a cell capable of expressing a cytokine with a cell capable of expressing a TRPM4 with a cytokine production-promoting substance, and measuring the expression level of the cytokine and/or a gene thereof in the cell capable of expressing the
(A3) a step of contacting a test sample and a cytokine production promoting substance with a TRPM 4-deficient cell deficient in the function of TRPM4 in the TRPM 4-expressing cell, and measuring the expression level of the cytokine and/or its gene in the TRPM 4-deficient cell;
(A4) contacting a TRPM 4-deficient cell deficient in the function of TRPM4 in the TRPM 4-expressing cell with a cytokine production promoting substance, and measuring the expression level of the cytokine and/or its gene in the TRPM 4-deficient cell; and
(A5) and (c) evaluating the cytokine production inhibitory effect of the test sample based on the expression levels measured in the steps (a1) to (a 4).
(3) The method for evaluating a test sample according to item (1), wherein the physiological phenomenon is measured, and the cytokine production inhibitory action of the test sample is evaluated based on the physiological phenomenon, the procedure comprising:
(B1) a step of contacting the TRPM 4-expressing cell with a test sample, and measuring a change in TRPM4 activity in the TRPM 4-expressing cell before and after the contact with the test sample; and
(B2) and (c) evaluating the cytokine production inhibitory effect of the test sample on the basis of the change in TRPM4 activity measured in step (B1). And
(4) a cytokine production inhibitor for use in an application of activating TRPM4 to inhibit cytokine production in skin cells, characterized by containing an aluminum compound as an active ingredient for activating
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, there are provided a method for evaluating a test sample that can easily evaluate whether the test sample has an effect of inhibiting cytokine production in skin cells, and a cytokine production inhibitor that can effectively inhibit cytokine production in skin cells.
Drawings
Fig. 1 is a drawing substitute photograph showing the result of western blot analysis in reference example 1.
FIG. 2 shows the results of examining the expression level of IL-1. alpha. gene in each of the cells obtained in examples 1 to 2 and comparative examples 1 to 2 in test example 1.
FIG. 3 shows the results of examining the expression level of IL-8 gene in each of the cells obtained in examples 1 to 2 and comparative examples 1 to 2 in test example 1.
FIG. 4 shows the results of examining the expression levels of TNF genes in the cells obtained in examples 1 to 2 and comparative examples 1 to 2 in test example 1.
FIG. 5 shows the results of examining the expression level of IL-8 gene in each of the cells obtained in comparative examples 3 to 6 in test example 2.
Fig. 6 is a graph showing the results of examining the relationship between the presence or absence of TRPM4 agonist and the relative fluorescence intensity in reference example 2.
FIG. 7 is a graph showing the results of examining the relationship between the type of sample to be tested and the relative fluorescence intensity in example 3.
Fig. 8 is a graph showing the results of examining the expression level of IL-1 α in each of the epidermal keratinocytes obtained in example 4 and comparative example 7 in test example 3.
FIG. 9 is a graph showing the results of examining the expression level of IL-6 in each of epidermal keratinocytes obtained in example 4 and comparative example 7 in test example 3.
Detailed Description
1. Method for evaluating test sample
The method for evaluating a test sample of the present invention is a method for evaluating a test sample for evaluating an inhibitory effect on cytokine production in skin cells of the test sample, and is characterized by bringing a TRPM 4-expressing cell into contact with the test sample, measuring a physiological phenomenon caused by TRPM4 in the test sample-expressing cell through TRPM4, and evaluating the inhibitory effect on cytokine production in the test sample based on the physiological phenomenon.
According to the method for evaluating a test sample of the present invention, since the operation of measuring a physiological phenomenon caused by TRPM4 in a test sample cell expressed by TRPM4 and evaluating the cytokine production inhibitory effect of the test sample based on the physiological phenomenon is employed, it is possible to accurately evaluate whether or not the test sample inhibits the cytokine production caused by the activation of TRPM4 in a skin cell. Therefore, according to the method for evaluating a test sample of the present invention, it can be easily evaluated whether or not the test sample has an effect of inhibiting cytokine production in skin cells. In addition, according to the method for evaluating a test sample of the present invention, since TRPM 4-expressing cells are used, the cytokine production inhibitory effect of the test sample can be evaluated with good reproducibility, and the evaluation can be performed simultaneously a plurality of times under the same condition.
The test sample is a sample to be evaluated for the presence or absence of an inhibitory effect on cytokine production. Examples of the sample to be measured include inorganic compounds, organic compounds, plant extracts, cell cultures, cell extracts, and the like, but the present invention is not limited to the above examples. When the sample to be tested is a liquid, it may be used as it is or may be diluted with a solvent as necessary. In addition, when the sample to be tested is a solid, it can be dissolved in a solvent and used.
The solvent is not determined in a general manner because it differs depending on the type of the sample to be tested, the type of the physiological phenomenon of the measurement target, and the like, and therefore, it is preferably determined depending on the type of the sample to be tested, the type of the physiological phenomenon of the measurement target, and the like. Examples of the solvent include ethanol, physiological saline, phosphate buffered physiological saline, purified water, a culture medium, and a calcium-containing solution [ composition: 140mM sodium chloride, 5mM potassium chloride, 2mM magnesium chloride, 2mM calcium chloride, 10mM glucose and 10mM 2- [4- (2-hydroxyethyl) piperazin-1-yl ] ethanesulfonic acid (HEPES) hydrochloric acid buffer (ph7.4) ], a calcium-free solution [ composition: 140mM sodium chloride, 5mM potassium chloride, 2mM magnesium chloride, 5mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, 10mM glucose, and 10mM HEPES buffer (pH7.4), etc., but the present invention is not limited to the above examples. The culture medium contains media components suitable for growth of TRPM 4-expressing cells. Examples of the medium components include glucose, amino acids, peptone, vitamins, cell growth promoting factors, serum, calcium chloride, magnesium chloride, and the like, but the present invention is not limited to the above examples. The medium may be a medium produced by supplementing a minimal medium with medium components, or may be a commercially available medium. The minimal medium is not particularly limited, and examples thereof include Eagle's minimal medium (hereinafter referred to as "MEM"), Dulbecco's modified Eagle medium (hereinafter referred to as "DMEM"), RPMI-1640 medium, and the like, but the present invention is not limited to the above examples. Since the medium cannot be determined in a lump because it differs depending on the type of TRPM 4-expressing cells and the like, it is preferably determined depending on the type of TRPM 4-expressing cells and the like.
The TRPM 4-expressing cells are cells that exhibit physiological functions equivalent to those of cells expressing wild-
Examples of the endogenous TRPM 4-expressing cells include normal human epidermal keratinocytes, HaCaT cells, and keratinocytes such as NCTC 2544; t cells such as human cardiac muscle cells, human T cells, Jurkat cells, MOLT-4 cells, U-937 cells and the like; mast cells such as human mast cells and HMC-1; human monocytes, monocytes such as THP-1, etc., but the present invention is not limited to the above examples.
The exogenous TRPM 4-expressing cell may be any cell that overexpresses
Examples of the nucleic acid encoding TRPM4 include, for example, GenBank accession No.: NM-017636, and cDNA derived from the mRNA, but the present invention is not limited to the above examples. Specific examples of the nucleic acid encoding TRPM4 include, for example, a nucleic acid encoding TRPM4 represented by seq id no: 1, or a polypeptide consisting of the amino acid sequence shown in the specification; the code is composed of the following components: 1 and a mutant polypeptide nucleic acid which has an amino acid sequence having a sequence identity of 80% or more and exhibits a TRPM4 activity, but the present invention is not limited to the above-mentioned examples. "sequence identity" refers to the relationship between the amino acid sequence (query sequence) to be evaluated and the sequence number: 1 (reference sequence) and the amino acid sequences shown in (1) are aligned. From the viewpoint of exhibiting TRPM4 activity, the sequence identity is 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more, and the upper limit thereof is 100%.
Examples of TRPM4 activity include regulatory activity of potassium ion flux and sodium ion flux in cells, regulatory activity of membrane potential in cells, and the like, but the present invention is not limited to the above examples. These TRPM4 activities are exhibited, for example, by TRPM4 being activated by TRPM4 agonists.
The host cell may be a cell expressing endogenous TRPM4 or a cell not expressing
The TRPM 4-expressing cell is preferably a TRPM 4-expressing cell having cytokine expression ability, from the viewpoint of evaluating with high accuracy whether or not the test sample has a cytokine production inhibitory effect. Examples of the TRPM 4-expressing cells having cytokine-expressing ability include HaCaT cells, but the present invention is not limited to the above examples.
Examples of physiological phenomena caused by TRPM4 include a decrease in the expression level of a cytokine gene due to activation of TRPM4, a decrease in the expression level of a cytokine due to activation of TRPM4, and expression of the activity of TRPM4 itself, but the present invention is not limited to the above examples.
When the decrease in the expression level of a cytokine gene caused by the activation of TRPM4 is used as a physiological phenomenon caused by TRPM4, the following
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A method in which a physiological phenomenon is cytokine production, a TRPM 4-expressing cell having a cytokine-expressing ability is used as a TRPM 4-expressing cell, the physiological phenomenon is measured, and the cytokine production inhibitory action of the test sample is evaluated on the basis of the physiological phenomenon, wherein the method comprises the steps of:
(A1) a step of contacting a TRPM 4-expressing cell having a cytokine-expressing ability with a test sample and a cytokine production-promoting substance, and measuring the expression level of the cytokine and/or its gene in the TRPM 4-expressing cell;
(A2) a step of contacting a cell capable of expressing a cytokine with a cell capable of expressing a TRPM4 with a cytokine production-promoting substance and measuring the expression level of the cytokine and/or a gene thereof in the cell capable of expressing the
(A3) a step of contacting a test sample and a cytokine production promoting substance with a TRPM 4-deficient cell deficient in the function of TRPM4 in the TRPM 4-expressing cell, and measuring the expression level of the cytokine and/or its gene in the TRPM 4-deficient cell;
(A4) contacting a TRPM 4-deficient cell deficient in the function of TRPM4 in the TRPM 4-expressing cell with a cytokine production promoting substance, and measuring the expression level of the cytokine and/or its gene in the TRPM 4-deficient cell; and
(A5) and (D) evaluating the cytokine production inhibitory effect of the test sample based on the expression levels measured in the steps (A1) to (A4).
In the
In the step (a1), a TRPM 4-expressing cell having cytokine-expressing ability is contacted with a test sample and a cytokine production-promoting substance, and the expression level of a cytokine and/or its gene in the TRPM 4-expressing cell is measured.
Examples of the cytokine include interleukin-1 α (hereinafter referred to as "IL-1 α"), interleukin-1 β (hereinafter referred to as "IL-1 β"), interleukin-2 (hereinafter referred to as "IL-2"), interleukin-6 (hereinafter referred to as "IL-6"), interleukin-8 (hereinafter referred to as "IL-8"), and tumor necrosis factor (hereinafter referred to as "TNF"), but the present invention is not limited to the above examples. Of these cytokines, IL-1. alpha. and IL-8 are preferred in terms of accurately evaluating the effect of inhibiting the onset of inflammation in the skin. Examples of the cytokine gene include IL-1. alpha. gene, IL-1. beta. gene, IL-2 gene, IL-6 gene, IL-8 gene, TNF gene, etc., but the present invention is not limited to these examples. Among these cytokine genes, the IL-1. alpha. gene and the IL-8 gene are preferable from the viewpoint of accurately evaluating the effect of suppressing the onset of inflammation in the skin.
Examples of the cytokine production-promoting substance include tumor necrosis factor α (hereinafter referred to as "TNF α"), lipopolysaccharide, phorbol 12-myristate 13-acetate (hereinafter referred to as "PMA"), interferon γ (hereinafter referred to as "IFN γ"), IL-17, IL-22, IL-33, histamine and the like, but the present invention is not limited to the above examples.
Examples of the order of contacting a TRPM 4-expressing cell with a test sample and a cytokine production-promoting substance include: (a1) a sequence of contacting a TRPM 4-expressing cell with a cytokine production promoting substance after contacting the TRPM 4-expressing cell with a test sample; (a2) a sequence of simultaneously contacting the test sample and the cytokine production promoting substance with the TRPM 4-expressing cell; (a3) a sequence of contacting a TRPM 4-expressing cell with a test sample after contacting the TRPM 4-expressing cell with a cytokine production-promoting substance; however, the present invention is not limited to the above examples.
In the case where the procedure of (a1) is adopted in step (a1), examples of a method for contacting a TRPM 4-expressing cell with a test sample include a method of incubating a TRPM 4-expressing cell in a test sample, but the present invention is not limited to the above-described examples. In addition, as a method for contacting a TRPM 4-expressing cell with a cytokine production-promoting substance, a method in which a cytokine production-promoting substance is added to a mixture of a test sample and a TRPM 4-expressing cell after incubation and the resulting mixture is incubated may be mentioned, but the present invention is not limited to the above-mentioned examples. Reagents used for measurement of physiological phenomena of a measurement object can be used for contact between a TRPM 4-expressing cell and a test sample and contact between a TRPM 4-expressing cell and a cytokine production promoting substance.
The contact temperature between the TRPM 4-expressing cell and the test sample and the contact temperature between the TRPM 4-expressing cell and the cytokine production promoting substance are not generally determined depending on the type of TRPM 4-expressing cell and the like, and therefore, it is preferable to determine the contact temperature depending on the type of TRPM 4-expressing cell and the like. The contact temperature of the TRPM 4-expressing cell with the test sample and the contact temperature of the TRPM 4-expressing cell with the cytokine production promoting substance are generally preferably 35 to 40 ℃ from the viewpoint of ensuring temperature conditions suitable for expression of TRPM4 activity in the TRPM 4-expressing cell. The contact time between the TRPM 4-expressing cell and the test sample is not generally determined depending on the type of TRPM 4-expressing cell, the type of test sample, and the like, and therefore is preferably determined depending on the type of TRPM 4-expressing cell, the type of test sample, and the like. From the viewpoint of accurately evaluating the effect of a test sample on TRPM4 activity, the contact time of a TRPM 4-expressing cell with a test sample is preferably 1 minute to 24 hours in general. The contact time between the TRPM 4-expressing cell and the cytokine production promoting substance differs depending on the type of TRPM 4-expressing cell, the type of cytokine production promoting substance, the type of substance to be measured for the expression level, and the like, and therefore cannot be determined in a lump, and therefore is preferably determined depending on the type of TRPM 4-expressing cell, the type of cytokine production promoting substance, the type of substance to be measured for the expression level, and the like. The contact time between the TRPM 4-expressing cell and the cytokine production-promoting substance is preferably 6 to 72 hours in the case of evaluating the expression level of the cytokine and preferably 1 to 12 hours in the case of evaluating the expression level of the cytokine gene, from the viewpoint of accurately evaluating the influence of the test sample on the TRPM4 activity.
In the case of contacting a TRPM 4-expressing cell with a test sample, the number of TRPM 4-expressing cells per 1mL of the test sample is preferably determined in accordance with the type of TRPM 4-expressing cell, the type of the test sample, and the like, because the number cannot be determined uniformly depending on the type of TRPM 4-expressing cell, the type of the test sample, and the like, and the number of TRPM 4-expressing cells per 1mL of the test sample is preferably 1 × 10 from the viewpoint of improving the accuracy of measurement of physiological phenomena caused by TRPM43More than one, more preferably 1 × 104More than one, 1 × 10 is preferable from the viewpoint of accurately producing physiological phenomena caused by TRPM4 in TRPM4 cells9Less than, more preferably 1 × 108One below.
In the case of contacting a test sample with a TRPM 4-expressing cell, the amount of the cytokine production promoting substance per 1mL of the test sample cannot be determined in a lump because it differs depending on the type of the cytokine production promoting substance, the type of the TRPM 4-expressing cell, and the like, and therefore, it is preferably determined depending on the type of the cytokine production promoting substance, the type of the TRPM 4-expressing cell, and the like. From the viewpoint of accurately evaluating the effect of a test sample on TRPM4 activity, the amount of the cytokine production promoting substance per 1mL of the test sample is preferably 5 to 500ng in general.
In step (a1), in the case of adopting the order of (a2) or (a3), the number of TRPM 4-expressing cells per 1mL of test sample and the amount of the cytokine production promoting substance per 1mL of test sample are the same as the number of TRPM 4-expressing cells per 1mL of test sample and the amount of the cytokine production promoting substance per 1mL of test sample when adopting the order of (a 1).
The contact temperature between the TRPM 4-expressing cell and the test sample and the cytokine production promoting substance differs depending on the type of TRPM 4-expressing cell and the like, and therefore cannot be determined in a lump, and therefore, it is preferably determined depending on the type of TRPM 4-expressing cell and the like. The contact temperature of the TRPM 4-expressing cell with the test sample and the cytokine production-promoting substance is generally preferably 35 to 40 ℃ from the viewpoint of ensuring temperature conditions suitable for expression of TRPM4 activity in the TRPM 4-expressing cell.
The contact time between the TRPM 4-expressing cell and the test sample and the cytokine production-promoting substance differs depending on the type of TRPM 4-expressing cell, the type of test sample, the type of cytokine production-promoting substance, the type of substance to be measured for the expression level, and the like, and therefore cannot be determined in general, and therefore, it is preferable to determine the contact time depending on the type of TRPM 4-expressing cell, the type of test sample, the type of cytokine production-promoting substance, the type of substance to be measured for the expression level, and the like. Regarding the contact time of the TRPM 4-expressing cell with the test sample and the cytokine production-promoting substance, from the viewpoint of accurately evaluating the influence of the test sample on the TRPM4 activity, it is generally preferable to evaluate the expression level of the cytokine for 6 to 72 hours, and to evaluate the expression level of the cytokine gene for 1 to 12 hours.
Examples of the method for measuring the expression level of the cytokine include western blotting, enzyme-linked immunosorbent assay (ELISA), and the like, but the present invention is not limited to the above-mentioned examples. Examples of the method for measuring the expression level of the cytokine gene include northern blotting, quantitative RT-PCR, and the like, but the present invention is not limited to the above-mentioned examples.
Examples of the antibody used in western blotting and ELISA include an anti-cytokine antibody and an antibody fragment thereof, but the present invention is not limited to the above examples. The anti-cytokine antibody may be a monoclonal antibody or a polyclonal antibody. From the viewpoint of improving the quantitativity of the expression amount of the cytokine, a monoclonal antibody is preferable. Monoclonal antibodies are obtained, for example, as follows: the antibody can be obtained by culturing a hybridoma that produces a monoclonal antibody reactive with a cytokine or a part thereof to obtain a culture supernatant, and purifying the obtained culture supernatant by ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like as necessary. Hybridomas are obtained, for example, as follows: the antibody-producing cells are obtained by administering a cytokine or a part thereof to an animal intravenously, subcutaneously or intraperitoneally to immunize the animal, fusing the antibody-producing cells with myeloma cells, and culturing the resulting fused cells in HAT medium. In addition, polyclonal antibodies were obtained as follows: an animal is immunized by administering a cytokine or a part thereof intravenously, subcutaneously, or intraperitoneally to the animal to obtain antiserum, and the obtained antiserum is purified by ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like as necessary, to obtain the antibody. Antibody fragments are obtained, for example, as follows: the anti-cytokine antibody is digested with a digestive enzyme, and the obtained degradation product is purified as necessary, for example. In addition, as the anti-cytokine antibody and the antibody fragment thereof, commercially available anti-cytokine antibodies and antibody fragments thereof can be used.
Examples of the probe used in northern blotting include a nucleic acid comprising all or a part of a nucleic acid encoding a cytokine gene, a nucleic acid comprising all or a part of an antisense strand of a nucleic acid encoding a cytokine gene, and the like, but the present invention is not limited to the above examples. In the case of a nucleic acid comprising a part of a nucleic acid encoding a cytokine gene or a nucleic acid comprising a part of the antisense strand of the nucleic acid, the length of the probe cannot be determined in a single row depending on the type of the cytokine gene and the like, and therefore, it is preferably determined depending on the type of the cytokine gene and the like. From the viewpoint of improving the accuracy of measurement of the expression level of the cytokine gene, the length of the probe is preferably 20 to 500 nucleotides.
Examples of the primer set used in the quantitative RT-PCR method include, for example, a primer composed of a part of the nucleotide sequence of a nucleic acid encoding a cytokine gene and a primer composed of a part of the nucleotide sequence of the antisense strand of the nucleic acid, but the present invention is not limited to the above examples. The length, GC content and Tm value of each primer constituting the primer pair and the distance between primers on the nucleic acid encoding the cytokine gene are different depending on the type of the cytokine gene and the like, and therefore cannot be determined in general, and therefore, it is preferably determined depending on the type of the cytokine gene and the like. From the viewpoint of improving the accuracy of measurement of the expression level of the cytokine gene, the length of the primer is preferably 12 to 30 nucleotides. The GC content of the primer is preferably 40 to 60% in general, from the viewpoint of improving the accuracy of measurement of the expression level of the cytokine gene. The Tm value of the primer is preferably 55 to 80 ℃ from the viewpoint of improving the accuracy of measurement of the expression level of the cytokine gene. In order to rapidly measure the expression level of the cytokine gene, the distance between primers on the nucleic acid encoding the cytokine gene is preferably 100 to 800 nucleotides. In addition, from the viewpoint of improving the accuracy of measurement of the expression level of the cytokine gene, each primer constituting the primer pair preferably has a sequence that does not include both a sequence in which thymine residues or cytosine residues are continuous (polypyrimidine sequence) and a sequence in which adenine residues or guanine residues are continuous (polypurine sequence). From the viewpoint of improving the accuracy of measurement of the expression level of the cytokine gene, each primer constituting the primer set is preferably constituted of a part of the nucleotide sequence of the nucleic acid encoding the cytokine gene or the antisense strand thereof and a sequence not included in the sequences of other genes and the like. The sequence used for the Primer can be obtained by using a Primer design program such as Primer BLAST. The primer pair may also be a commercially available primer pair.
In the step (a2), the TRPM 4-expressing cell having the cytokine-expressing ability is contacted with the cytokine production-promoting substance, and the expression level of the cytokine and/or its gene in the TRPM 4-expressing cell is measured.
The method for contacting the TRPM 4-expressing cell and the cytokine production promoting substance used in step (a2), the method for measuring the contact temperature of the TRPM 4-expressing cell and the cytokine production promoting substance, the contact time of the TRPM 4-expressing cell and the cytokine production promoting substance, and the respective expression amounts of the cytokine gene and the cytokine are the same as the method for contacting the TRPM 4-expressing cell and the cytokine production promoting substance used in step (a1), the contact temperature of the TRPM 4-expressing cell and the cytokine production promoting substance, the contact time of the TRPM 4-expressing cell and the cytokine production promoting substance, and the respective expression amounts of the cytokine and its gene. The amount of the cytokine production promoting substance contacted with the TRPM 4-expressing cell is the same as the amount of the cytokine production promoting substance used in the contacting of the TRPM 4-expressing cell with the test substance and the cytokine production promoting substance in step (a 1).
In the step (a3), a TRPM 4-deficient cell deficient in the function of TRPM4 in a TRPM 4-expressing cell is contacted with a test sample and a cytokine production promoting substance, and the expression level of a cytokine and/or its gene in the TRPM 4-deficient cell is measured.
TRPM 4-deficient cells the same TRPM 4-expressing cells used in step (a1) and step (a2) except that TRPM4 is functionally deficient. TRPM 4-deficient cells can be obtained by disrupting the TRPM4 gene in TRPM 4-expressing cells using gene knock-out; causing a functional deficiency of TRPM4 in a TRPM4 expressing cell; and the like. Examples of the gene deletion method include a method of disrupting the TRPM4 gene by homologous recombination, a method of disrupting the TRPM4 gene by genome editing technology, and the like, but the present invention is not limited to the above examples. Further, as a method for making a function of TRPM4 deficient, a method of suppressing the expression of TRPM4 gene by RNA silencing method using siRNA or shRNA, a method of suppressing the function of normal TRPM4 by expressing dominant negative mutant in TRPM 4-expressing cells, and the like can be cited, but the present invention is not limited to the above examples.
The same procedure as in step (a1) was carried out except that in step (A3), TRPM 4-deficient cells were used instead of TRPM 4-expressing cells having cytokine-expressing ability used in step (a1), whereby the expression level of cytokines and/or their genes was determined.
In the step (a4), a TRPM 4-deficient cell deficient in the function of TRPM4 in a TRPM 4-expressing cell is contacted with a cytokine production promoting substance, and the expression level of a cytokine and/or a gene thereof in the TRPM 4-deficient cell is measured.
The same procedure as in step (a2) was carried out except that in step (a4), TRPM 4-deficient cells were used instead of TRPM 4-expressing cells having cytokine-expressing ability used in step (a2), whereby the expression level of cytokines and/or their genes was determined.
In the method for evaluating a test sample according to the present invention, it is preferable to correct the expression level when a test sample is used, using a control sample, in order to improve the evaluation accuracy. The control sample may be any liquid that does not contain the sample to be tested, and examples thereof include a solvent used for diluting the sample to be tested, a solvent used for dissolving the sample to be tested, and the like. When a control sample is used, the expression level of the cytokine and/or its gene is measured in the same manner as in steps (a1) to (a4) except that the control sample is used instead of the test sample in steps (a1) to (a 4).
In step (a5), the cytokine production inhibitory action of the test sample is evaluated based on the expression levels measured in steps (a1) to (a 4).
When the expression level measured in step (a1) is less than the expression level measured in step (a2) and the expression level measured in step (A3) is equivalent to the expression level measured in step (a4), the test sample can be evaluated as a substance having an action of activating TRPM4 and inhibiting cytokine production in skin cells.
When the expression of TRPM4 itself is used as a physiological phenomenon caused by TRPM4, the following
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A method for measuring the physiological phenomenon and evaluating the cytokine production inhibitory effect of the test sample on the basis of the physiological phenomenon, the method comprising the steps of:
(B1) a step of contacting the TRPM 4-expressing cell with a test sample, and measuring a change in TRPM4 activity in the TRPM 4-expressing cell before and after the contact with the test sample; and
(B2) and (c) evaluating the cytokine production inhibitory effect of the test sample on the basis of the change in TRPM4 activity measured in step (B1).
The TRPM 4-expressing cell used in
Examples of the change in TRPM4 activity include a change in the amount of sodium ions introduced into a cell by TRPM4 in a TRPM 4-expressing cell before and after the test sample contact, an increase in current due to activation of TRPM4 in a TRPM 4-expressing cell before and after the test sample contact, and the like, but the present invention is not limited to the above examples.
When the change in the amount of sodium ion introduced into the cell by TRPM4 in the TRPM 4-expressing cell before and after the test sample is contacted is used as the change in TRPM4 activity, examples of a method for measuring the amount of sodium ion introduced into the cell by TRPM4 include a method for measuring the amount of sodium ion introduced into the cell by using the intracellular calcium ion concentration of the TRPM 4-expressing cell as an index of the amount of sodium ion introduced into the cell, but the present invention is not limited to the above-described examples.
When the intracellular calcium ion concentration is used as an index for measuring the amount of sodium ions introduced into the cell by TRPM4, the intracellular calcium ion concentration can be measured, for example, by the following method or the like: a calcium indicator specifically binding to calcium ions is introduced into a TRPM 4-expressing cell, the calcium indicator is allowed to bind to calcium ions in the TRPM 4-expressing cell, and the amount of the calcium indicator bound to calcium ions in the TRPM 4-expressing cell is measured.
In TRPM 4-expressing cells in which TRPM4 was activated, the amount of TRPM 4-mediated influx of sodium ions from the outside of TRPM 4-expressing cells to the inside of TRPM 4-expressing cells was increased and the intracellular sodium ion concentration of TRPM 4-expressing cells was increased, as compared to TRPM 4-expressing cells in which TRPM4 was not activated. As the intracellular sodium ion concentration of the TRPM 4-expressing cell increases, the intracellular calcium ion concentration of the TRPM 4-expressing cell decreases. In addition, as shown in the examples described later, activation of TRPM4 is associated with inhibition of cytokine production in skin cells. Therefore, in the case where the intracellular calcium concentration of the TRPM 4-expressing cell contacted with the test sample is lower than the intracellular calcium concentration of the TRPM 4-expressing cell not contacted with the test sample, it can be evaluated that the test sample is a substance having an action of activating TRPM4 and inhibiting cytokine production in skin cells. On the other hand, when the intracellular calcium concentration of the TRPM 4-expressing cell contacted with the test sample is higher than or equal to the intracellular calcium concentration of the TRPM 4-expressing cell not contacted with the test sample, the test sample can be evaluated as a substance having no effect of inhibiting cytokine production in skin cells.
When intracellular calcium ion concentration is used as an index for measuring the amount of sodium ion introduced into a cell by TRPM4, TRPM4 activation can be evaluated by a method including the following steps, for example:
(I) a step of determining intracellular calcium ion concentration a of the TRPM 4-expressing cell using a calcium indicator;
(II) a step of contacting a TRPM 4-expressing cell with a test sample, and determining the intracellular calcium ion concentration B of the TRPM 4-expressing cell using a calcium indicator; and
(III) comparing the intracellular calcium ion concentration A obtained in the step (I) with the intracellular calcium ion concentration B obtained in the step (II), and evaluating the TRPM4 activating effect of the test sample.
In step (I), the intracellular calcium ion concentration a of the TRPM 4-expressing cells was determined using calcium indicators. As the TRPM 4-expressing cell, HEK293 cells expressing exogenous TRPM4 and the like are preferably used.
The calcium indicator is preferably a reagent capable of detecting a change before and after binding to calcium ions by using a change in optical characteristics or the like, because the amount of calcium indicator bound to calcium ions can be measured by a simple operation. Examples of the change in optical characteristics include a change in fluorescence intensity and a change in absorbance, but the present invention is not limited to these examples. Examples of the calcium indicator include a fluorescent calcium indicator in which the fluorescence intensity changes before and after binding to calcium ions, but the present invention is not limited to the above examples. Specific examples of the calcium indicator include 1- [ 6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy ] -2- (2-amino-5-methylphenoxy) ethane-N, N '-tetraacetic acid pentaacetoxymethyl ester (Fura 2-AM), 1- [ 2-amino-5- (2, 7-dichloro-6-hydroxy-3-oxo-9-xanthenyl) phenoxy ] -2- (2-amino-5-methylphenoxy) ethane-N, N' -tetraacetic acid tetraacetoxy methyl ester (Fluo 3-AM), 1- [ 2-amino-5- (2, and fluorescent calcium indicators such as 7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl) phenoxy ] -2- (2-amino-5-methylphenoxy) ethane-tetraacetoxymethyl tetraacetate (Fluo 4-AM), but the present invention is not limited to the above examples. Among calcium indicators, fluorescent calcium indicators that change in fluorescence intensity before and after binding to calcium ions are preferred in terms of ease of measurement and ease of dynamic discrimination from inclusion substances present in TRPM 4-expressing cells.
The fluorescent calcium indicator may have one excitation wavelength or two or more excitation wavelengths. When the calcium indicator is a fluorescent calcium indicator, the fluorescent calcium indicator preferably has two excitation wavelengths because the fluorescence intensity is easily measured and the detection intensity is high. In the case of using a fluorescent calcium indicator having one excitation wavelength when determining the change in intracellular calcium concentration, the change in intracellular calcium concentration can be determined based on the fluorescence intensity at the excitation wavelength. In the case of using a fluorescent calcium indicator having two or more excitation wavelengths when measuring the change in the intracellular calcium concentration, two excitation wavelengths (the 1 st excitation wavelength and the 2 nd excitation wavelength) selected from the two or more excitation wavelengths may be selected from the viewpoint of improving the evaluation accuracy of the cytokine production inhibitory effect of the test sample, and the change in the intracellular calcium concentration may be measured based on the fluorescence intensity ratio calculated from the fluorescence intensity at each of the 1 st excitation wavelength and the 2 nd excitation wavelength. In the case of using, for example, FURA2-AM as a fluorescent calcium indicator having two excitation wavelengths when determining the intracellular calcium concentration, it is possible to use the fluorescence intensity at an excitation wavelength of 340nm as the fluorescence intensity at the 1 st excitation wavelength (hereinafter also referred to as "1 st fluorescence intensity") and the fluorescence intensity at an excitation wavelength of 380nm as the fluorescence intensity at the 2 nd excitation wavelength (hereinafter also referred to as "2 nd fluorescence intensity"). The fluorescence intensity ratio can be determined, for example, based on the formula (I).
[ fluorescence intensity ratio ] ([ 1 st fluorescence intensity ]/[ 2 nd fluorescence intensity ] (I)
Examples of the method for measuring the intracellular calcium ion concentration a using a fluorescent calcium indicator include the following
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A method, comprising the steps of:
introducing a fluorescent calcium indicator into a TRPM 4-expressing cell to obtain an indicator-introduced cell;
contacting the indicator-introduced cells with calcium ions to bind the calcium ions to the fluorescent calcium indicator introduced into the cells; and
and a step of measuring the fluorescence intensity of the fluorescent calcium indicator bound to the calcium ion introduced into the cell as the indicator.
Examples of a method for introducing a fluorescent calcium indicator into a TRPM 4-expressing cell include a method in which a buffer containing a fluorescent calcium indicator is circulated in a reflux chamber containing a TRPM 4-expressing cell, but the present invention is not limited to the above examples. Examples of the buffer include HEPES buffer, but the present invention is not limited to the above examples.
As a method for bringing the TRPM 4-expressing cell into contact with calcium ions, for example, a method of circulating a buffer containing calcium ions in a reflux chamber containing the TRPM 4-expressing cell into which a fluorescent calcium indicator is introduced can be cited, but the present invention is not limited to the above-mentioned examples. Examples of the buffer include HEPES buffer, but the present invention is not limited to the above examples.
The contact temperature between the TRPM 4-expressing cell into which the fluorescent calcium indicator is introduced and the buffer containing calcium ions differs depending on the type of TRPM 4-expressing cell and the like, and therefore cannot be determined in a lump, and therefore is preferably determined depending on the type of TRPM 4-expressing cell and the like. It is to be noted that, in order to exclude the influence of temperature on TRPM4 activity, the TRPM 4-expressing cells are preferably contacted with a buffer containing calcium ions under a constant temperature environment.
The number of TRPM 4-expressing cells used in step (I) is not determined in a lump because it differs depending on the type of the means for measuring the calcium ion concentration a, and is therefore preferably determined depending on the type of the means for measuring the calcium ion concentration a. For example, when the calcium ion concentration a is measured by observing cells at a magnification of 100 times using an inverted microscope, the number of TRPM 4-expressing cells per 1 field (data analysis range) is preferably 10 or more, more preferably 100 or more, from the viewpoint of improving the reliability of the measurement result, and is preferably 300 or less, more preferably 200 or less, from the viewpoint of ensuring the distance between cells so that the cells are not too dense.
In step (II), a TRPM 4-expressing cell is contacted with a test sample, and the intracellular calcium ion concentration B of the TRPM 4-expressing cell is determined using a calcium indicator.
Examples of the method for measuring the intracellular calcium ion concentration B using a fluorescent calcium indicator include the following
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A method, comprising the steps of:
introducing a fluorescent calcium indicator into a TRPM 4-expressing cell to obtain an indicator-introduced cell;
a step of bringing an indicator into contact with a test sample;
contacting the cells with the test sample with calcium ions to bind the calcium ions to the fluorescent calcium indicator in the cells; and
a step of measuring the fluorescence intensity of the fluorescent calcium indicator bound to calcium ions in the cell.
As a method for bringing the TRPM 4-expressing cells into contact with the test sample, a method of circulating a buffer containing a fluorescent calcium indicator in a refluxing chamber containing the TRPM 4-expressing cells into which the fluorescent calcium indicator is introduced, and the like can be cited, but the present invention is not limited to the above-mentioned examples. Examples of the method of contacting the cells after the test sample with calcium ions include a method of circulating a buffer solution containing calcium ions but not containing the test sample, but the present invention is not limited to the above-mentioned examples. In the present invention, the buffer containing the sample to be tested may be circulated in the refluxing room containing the TRPM 4-expressing cells into which the fluorescent calcium indicator has been introduced, and then the buffer containing the sample to be tested and calcium ions may be circulated.
The method for measuring intracellular calcium ion concentration B, the contact temperature and the number of TRPM 4-expressing cells used in step (II) are the same as those for measuring intracellular calcium ion concentration A, the contact temperature and the number of TRPM 4-expressing cells used in step (I).
In step (III), the intracellular calcium ion concentration a obtained in step (I) and the intracellular calcium ion concentration B obtained in step (II) are compared, and the TRPM4 activation effect of the test sample is evaluated. In step (III), in the case where the calcium ion concentration B is decreased as compared with the calcium ion concentration a, it can be evaluated that the test sample has TRPM4 activation, and further, the larger the difference between the calcium ion concentration a and the calcium ion concentration B, the higher the TRPM4 activation of the test sample can be evaluated.
In the case where an increase in current due to activation of TRPM4 in a TRPM 4-expressing cell before and after the test sample contact is used as a change in TRPM4 activity, the activation of TRPM4 can be evaluated, for example, by a method comprising the following steps:
(i) a step of measuring an electric current A in a TRPM 4-expressing cell at a certain potential;
(ii) (ii) a step of contacting a TRPM 4-expressing cell with a test sample and measuring the current B in the TRPM 4-expressing cell at the same potential as that in step (i); and
(iii) (iii) comparing the current A obtained in the step (i) with the current B obtained in the step (ii), and evaluating the TRPM4 activating effect of the test sample.
In step (i), the current a caused by the activation of TRPM4 in TRPM 4-expressing cells was measured at a certain potential. Examples of the method for measuring the current a include a patch clamp method, but the present invention is not limited to the above examples. According to the patch clamp method, a current caused by activation of 1 or more TRPM4 present in the cell membrane of a TRPM 4-expressing cell can be measured. Examples of the patch clamp method include a whole cell method and a cell adhesion method, but the present invention is not limited to the above examples.
In step (ii), a TRPM 4-expressing cell is contacted with the test sample, and the current B in the TRPM 4-expressing cell is measured at the same potential as that in step (i). The method for measuring the current B used in step (ii) is the same as the method for measuring the current a used in step (i).
In step (iii), the current a obtained in step (i) and the current B obtained in step (ii) are compared, and the TRPM4 activation effect of the test sample is evaluated. In step (iii), in the case where the current a is smaller than the current B, it can be evaluated that the test sample has TRPM4 activation. In addition, the greater the difference between current a and current B, the higher TRPM4 activation that the test sample has can be assessed.
As described above, according to the method for evaluating a test sample of the present invention, since the operation of measuring a physiological phenomenon caused by TRPM4 in a cell in which the test sample is expressed by TRPM4 and evaluating the effect of inhibiting the production of a cytokine contained in the test sample based on the physiological phenomenon is employed, it is possible to easily and accurately evaluate whether or not the test sample has the effect of inhibiting the production of a cytokine in a skin cell. Therefore, the method for evaluating a test sample of the present invention is expected to be applicable to the development of cosmetics for preventing inflammation, agents for preventing inflammation, and the like.
2. Cytokine production inhibitor
The cytokine production inhibitor of the present invention is a cytokine production inhibitor used for an application of activating TRPM4 to inhibit cytokine production in skin cells, and is characterized by containing an aluminum compound as an active ingredient for activating
Since the cytokine production inhibitor of the present invention contains an aluminum compound as an active ingredient for activating TRPM4, cytokine production in skin cells can be effectively inhibited. Therefore, the cytokine production inhibitor of the present invention can be suitably used for inhibition of cytokine production in skin cells, and the like.
The content of the aluminum compound in the production inhibitor of the present invention is not generally determined depending on the type of the aluminum compound, the use of the cytokine production inhibitor of the present invention, and the like, and therefore is preferably set as appropriate depending on the type of the aluminum compound, the use of the cytokine production inhibitor of the present invention, and the like. The content of the aluminum compound in the cytokine production inhibitor of the present invention is preferably 0.0001% by mass or more, more preferably 0.005% by mass or more, still more preferably 0.008% by mass or more, and particularly preferably 0.01% by mass or more in view of sufficiently exhibiting the effect of activating TRPM4 to inhibit cytokine production in skin cells, and is preferably 100% by mass or less in view of inhibiting a load on the skin.
The cytokine production inhibitor of the present invention may contain other components such as water, a pH adjuster, and a stabilizer within a range not interfering with the object of the present invention. In the case where the cytokine production inhibitor of the present invention contains other components, the aluminum compound and the other components may form a complex in the cytokine production inhibitor of the present invention within a range not interfering with the object of the present invention.
As described above, the cytokine production inhibitor of the present invention can effectively inhibit cytokine production in skin cells, and thus can be suitably used for inhibiting the onset of inflammation in the skin in the past. Therefore, the cytokine production inhibitor of the present invention is expected to be used in applications such as cosmetics for preventing inflammation in the skin and agents for preventing inflammation in the skin.
When the cytokine production inhibitor of the present invention is used in a cosmetic for preventing inflammation in the skin or an agent for preventing inflammation in the skin, the content of the cytokine production inhibitor of the present invention in the cosmetic for preventing inflammation or the agent for preventing inflammation is not generally determined depending on the use of the cosmetic for preventing inflammation or the agent for preventing inflammation, the type of the aluminum compound contained in the cytokine production inhibitor of the present invention, and the like. The content of the cytokine production inhibitor of the present invention in the cosmetic preparation for inflammation prevention or the agent for inflammation prevention is usually preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, further preferably 0.0008% by mass or more, and particularly preferably 0.01% by mass or more, from the viewpoint of sufficiently exhibiting the action of activating TRPM4 to inhibit inflammation in the skin, and is desirably adjusted to preferably 20% by mass or less, more preferably 10% by mass, from the viewpoint of inhibiting the load on the skin. Other components such as a solvent, a surfactant, a humectant, a thickener, a preservative, an antioxidant, and a pH adjuster may be added to the inflammation preventing cosmetic or the inflammation preventing agent within a range that does not interfere with the action of activating TRPM4 to inhibit inflammation in the skin. The amount and the number of applications of the cosmetic preparation for preventing inflammation or the inflammation preventive agent are different depending on the type of inflammation, the type of the subject to be administered, the age of the subject to be administered, the weight of the subject to be administered, and the like, and therefore cannot be determined in a general manner.