JP2012016311A - Method for evaluating substance to be tested - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating a substance to be tested which evaluates a substance for suppressing an abnormality of a skin condition caused by sebum by a simple operation.SOLUTION: The method for evaluating a substance to be tested includes culturing a keratinocyte for expressing epimorphin or its differentiating cell structure under a differentiation condition while keeping contact with the substance to be tested or culturing a wild-type keratinocyte or its differentiating cell structure under a differentiation condition while keeping a contact with an unsaturated fatty acid and the substance to be tested, observing the morphology of the obtained cell structure and evaluating whether or not the substance to be tested is a substance for suppressing occurrence of an abnormality of a skin condition caused by sebum on the basis of the morphology of the cell structure.

Description

  The present invention relates to a test substance evaluation method. More specifically, the present invention relates to a method for evaluating a test substance useful for development of an external preparation, therapeutic agent or preventive agent for abnormal skin conditions caused by sebum.

  The human epidermis consists of a basal layer, a spiny layer, a granule layer, and a stratum corneum in order from the lower layer. Normal human skin usually turns over in a 28 day cycle. In normal skin turnover, keratinocytes are pushed up from the granular layer to the stratum corneum. At this time, enucleation occurs due to differentiation of keratinocytes, nucleated cells disappear, and a mature stratum corneum is formed.

  However, when cell growth in the epidermis is abnormally promoted by unsaturated fatty acids contained in sebum, keratinization in the skin is abnormally promoted, and enucleation may not occur in keratinocytes at the final differentiation stage. (Hereinafter referred to as “keratosis”). In this case, since the proportion of nucleated cells in the stratum corneum is increased, the barrier function of the stratum corneum of the skin is significantly reduced. Thus, the skin with a reduced barrier function tends to cause acne, rough skin, and the like. Accordingly, external preparations for abnormal skin conditions caused by sebum have been developed.

  Evaluation of external preparations for abnormal skin conditions such as keratinization caused by sebum, for example, causes keratinization by repeatedly applying oleic acid to the skin of animals such as mice. It is done by investigating the effects of external preparations on the skin, repeatedly applying and peeling adhesive tape to the skin of mice, etc. to cause thickening of the epidermis, and examining the effects of external preparations on the thickened epidermis (For example, refer to Patent Document 1). However, since the tendency of animal welfare has increased in recent years, there is a need for a method capable of evaluating external preparations and the like for abnormal skin conditions caused by sebum by simple operations without conducting animal tests. It has been.

  On the other hand, when the keratinocytes expressing exogenous epimorphin are cultured in three dimensions, the present inventors have characteristics similar to the pathological features of the skin accompanied by keratinization. Heading to show. However, the present inventors are currently in a state that exhibits characteristics resembling the pathological features of the skin with aberrant keratinization caused by epimorphin, and keratinization caused by unsaturated fatty acids such as oleic acid, etc. Abnormalities in skin conditions such as keratinization caused by sebum, and abnormalities in cells and cell structures caused by epimorphine or unsaturated fatty acids. We have not found any documents that specifically describe the relationship.

See JP2003-81834A

  The present invention has been made in view of the prior art, and an object thereof is to provide a test substance evaluation method capable of evaluating a substance that suppresses abnormal skin conditions caused by sebum by a simple operation. And

That is, the gist of the present invention is as follows.
(1) Culturing under differentiation conditions while contacting a keratinocyte expressing epimorphin or a differentiated cell construct thereof with a test substance, or differentiating while contacting a wild type keratinocyte or a differentiated cell construct thereof with an unsaturated fatty acid and a test substance Whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, based on the morphology of the cell structure after culturing under conditions and observing the morphology of the obtained cell structure A method for evaluating a test substance, characterized in that
(2) (A1) Cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in contact with a test substance in a support serving as a scaffold, or cell aggregates containing wild-type keratinocytes are used as scaffolds Culturing under differentiation conditions while contacting with an unsaturated fatty acid and a test substance in a support to obtain a cell structure,
(B1) Cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in a support body serving as a scaffold, or cell aggregates containing wild-type keratinocytes are contacted with unsaturated fatty acids in a support body serving as a scaffold. Culturing under differentiation conditions while obtaining a cell structure, and (C1) difference between the cell structure obtained in step (A1) and the cell structure obtained in step (B1) The method for evaluating a test substance according to (1), comprising the step of evaluating whether or not the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum,
(3) (A2) After culturing keratinocytes expressing epimorphin in a medium containing a test substance, the obtained keratinocytes are cultured under a differentiation condition in a medium containing calcium ionophore or unsaturated. Culturing wild-type keratinocytes in a medium containing a fatty acid and a test substance, and then culturing the obtained keratinocytes under a differentiation condition in a medium containing a calcium ionophore to obtain a cell structure;
(B2) After culturing keratinocytes expressing epimorphin in a medium containing calcium ionophore under differentiation conditions or culturing wild-type keratinocytes in a medium containing unsaturated fatty acids, the keratinocytes obtained are Culturing under differentiation conditions in a medium containing calcium ionophore to obtain a cell structure, and (C2) the cell structure obtained in step (A2) and the cell structure obtained in step (B2). The method for evaluating a test substance according to (1), comprising the step of evaluating whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, based on a difference from the body. And
(4) (A3) culturing the differentiated cell construct of keratinocytes expressing epimorphin in contact with the test substance under differentiation conditions, or contacting the differentiated cell construct of wild-type keratinocytes with the unsaturated fatty acid and the test substance Culturing under differentiation conditions to obtain a cell structure;
(B3) A differentiated cell construct of keratinocytes expressing epimorphin is cultured under differentiation conditions, or a differentiated cell construct of wild-type keratinocytes is cultured under differentiation conditions while contacting with an unsaturated fatty acid to obtain a cell structure And (C3) a skin condition in which the test substance is caused by sebum based on the difference between the cell structure obtained in step (A3) and the cell structure obtained in step (B3). It is related with the test substance evaluation method as described in said (1) including the step which evaluates whether it is a substance which suppresses generation | occurrence | production of abnormality of this.

  According to the test substance evaluation method of the present invention, there is an excellent effect that a substance that suppresses abnormal skin conditions caused by sebum can be evaluated by a simple operation.

In Experimental example 1, it is a drawing substitute photograph which shows the result of having investigated the relationship between the kind of culture medium, and the expression level of endogenous epimorphin in a HaCaT cell. In Experimental example 2, it is a drawing substitute photograph which shows the result of having investigated the relationship between the kind of culture medium, and the quantity of the secretion epimorphin in each of the culture supernatant of HaCaT-TE cell, and the culture supernatant of PT67-TE cell. In the manufacture example 4, it is a chart which shows the mass spectrum of a peptide. In manufacture example 4, it is a chart which shows the mass spectrum of the peptide after oxidation. (A) is a drawing-substituting photograph showing the result of observing the morphology of a cell cluster (cell structure) when using the medium of experiment number: 11 in Example 1, and (B) It is a drawing substitute photograph which shows the result of having observed the form of the cell cluster (cell structure) when the culture medium of experiment number: 10 was used. In Example 1, it is a graph which shows the result of having investigated the relationship between the kind of culture medium, and lumen formation rate. (A) is a drawing-substituting photograph showing the result of observing the morphology of a cell cluster (cell structure) when the medium of experiment number: 15 was used in Example 3, and (B) It is a drawing substitute photograph which shows the result of having observed the form of the cell cluster (cell structure) when the culture medium of experiment number: 14 was used. In Example 3, it is a graph which shows the result of having investigated the relationship between the kind of culture medium, and lumen formation rate. In Example 5, it is a graph which shows the result of having investigated the relationship between the kind of culture medium, and a confied envelope formation rate. In Example 7, it is a graph which shows the result of having investigated the relationship between the kind of culture medium or the kind of cell, and a confied envelope formation rate. (A) is a drawing substitute showing the result of observing the tissue morphology of the cell structure when the sample of Experiment No. 32 was applied without applying the 0.5 volume% oleic acid-containing ethanol solution in Example 9. The photograph (B) is a drawing-substituting photograph showing the result of observing the tissue morphology of the cell structure when the ethanol solution containing 0.5% by volume oleic acid and the sample of Experiment No. 33 were applied in Example 9. (C) is a photo, which substitutes for a drawing, showing the results of observing the tissue morphology of a cell structure when applying a 0.5 volume% oleic acid-containing ethanol solution and the sample of Experiment No. 34 in Example 9, (D ) Is a drawing-substituting photograph showing the results of observing the tissue morphology of a cell structure when a 0.5 volume% oleic acid-containing ethanol solution and a sample of Experiment No. 35 were applied in Example 9. FIG.

  The method for evaluating a test substance of the present invention comprises culturing keratinocytes expressing epimorphin or differentiated cell constructs thereof under differentiation conditions while contacting the test substances, or culturing wild-type keratinocytes or differentiated cell constructs thereof with unsaturated fatty acids and Culturing under differentiation conditions while in contact with the test substance, observing the morphology of the obtained cell structure, and based on the morphology of the cell structure, the test substance is subject to the occurrence of abnormal skin conditions caused by sebum. It is characterized by evaluating whether it is a substance to suppress.

The inventors have
(A) When oleic acid, which is one of unsaturated fatty acids contained in sebum, is contacted with keratinocytes expressing epimorphin, the amount of epimorphin secreted outside the keratinocytes increases,
(B) Cell structures obtained by culturing epikerphin-expressing keratinocytes or differentiated cell constructs thereof or wild-type keratinocytes or differentiated cell constructs thereof under differentiation conditions while in contact with oleic acid, exhibit keratinization. Abnormal tissue morphology similar to the pathological features of the skin is seen, whereas epimorphin-expressing keratinocytes or differentiated cell constructs thereof or wild-type keratinocytes or differentiated cell constructs thereof are treated with oleic acid and epimorphin. In a cell structure obtained by culturing under differentiation conditions while contacting with a cyclic peptide compound (SEQ ID NO: 1) that suppresses function, no abnormality in the tissue morphology is observed, and (c) by epimorphin Tissue morphology resembling the pathological features of the skin with induced keratinization and skin An abnormal skin condition, such as parakeratosis caused by found that are related to each other. The present invention is based on these findings.

  The evaluation method of the present invention comprises culturing keratinocytes expressing epimorphin or differentiated cell constructs thereof under differentiation conditions while contacting them with a test substance, or cultivating wild-type keratinocytes or differentiated cell constructs thereof with an unsaturated fatty acid and a test substance. One major feature is that the cells are cultured under differentiation conditions while being contacted. By performing a simple operation of culturing under the above differentiation conditions, the process of human skin differentiation in the presence of epimorphin expression or in the presence of unsaturated fatty acids is reproduced and caused by epimorphin or unsaturated fatty acids. The process of occurrence of abnormal skin conditions can be easily reproduced. This abnormal skin condition caused by epimorphine or unsaturated fatty acids is associated with abnormal skin conditions such as keratinization caused by sebum in human skin. Therefore, according to the evaluation method of the present invention, it is possible to evaluate whether or not the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum by a simple operation.

  Examples of abnormal skin conditions caused by sebum include acne, rough skin, and pimples.

  In the present specification, “wild-type keratinocytes” refers to keratinocytes that do not express epimorphin.

  Examples of wild-type keratinocytes include human wild-type keratinocytes and mouse wild-type keratinocytes. Among these, human wild-type keratinocytes are preferable from the viewpoint of sufficiently reproducing abnormal skin conditions caused by sebum in humans. Although it does not specifically limit as a human wild type keratinocyte, For example, a HaCaT cell, normal human skin epidermis keratinocyte, etc. are mentioned. Among human wild-type keratinocytes, HaCaT cells are preferred from the viewpoint of sufficiently reproducing abnormal skin conditions caused by sebum in humans.

  A keratinocyte expressing epimorphin is a cell obtained by introducing a nucleic acid encoding epimorphin into a wild type keratinocyte.

  The nucleic acid encoding epimorphin is not particularly limited, and examples thereof include a nucleic acid encoding mouse epimorphin and a nucleic acid encoding human epimorphin. Among them, a nucleic acid encoding mouse epimorphin is preferable from the viewpoint of abundant amount of information regarding expression and action mechanism of epimorphin and easy understanding of behavior in keratinocytes.

  Examples of the nucleic acid encoding mouse epimorphin include a nucleic acid consisting of positions 153 to 1022 (SEQ ID NO: 2) of the base sequence represented by GenBank accession number: D10475. Moreover, as a nucleic acid which codes human epimorphin, the nucleic acid (sequence number: 4) etc. which consist of the 96th-995th positions of the base sequence shown by GenBank accession number: D14582, etc. are mentioned.

The nucleic acid encoding epimorphin may be a nucleic acid variant consisting of the base sequence shown in SEQ ID NO: 2 or 4 as long as the encoded polypeptide expresses the function as epimorphin. . As the variant, for example,
(A) a nucleic acid comprising a sequence different from the base sequence shown in SEQ ID NO: 2 or 4 through degeneracy,
(B) the amino acid sequence shown in SEQ ID NO: 3 or 5, comprising at least one, for example, a sequence having an insertion, substitution or deletion of one or several amino acid residues, and as epimorphin in keratinocytes A nucleic acid encoding a polypeptide that expresses the function of
(C) A sequence calculated by aligning the base sequence shown in SEQ ID NO: 2 or 4 under the conditions of Cost to open gap 11, Cost to extended gap 1, expect value 10, and wordsize 11 by the BLAST algorithm. From the viewpoint of sufficiently expressing the function as epimorphin, the homology value is preferably 60% or more, more preferably 80% or more, and even more preferably 90% or more. A nucleic acid which is a polypeptide in which the peptide expresses a function as epimorphin in keratinocytes,
(D) Hybridizes under stringent conditions with a nucleic acid consisting of a base sequence completely complementary to the base sequence shown in SEQ ID NO: 2 or 4, and the encoded polypeptide is converted into epimorphin in keratinocytes. Examples thereof include nucleic acids that are polypeptides that express functions.

  In the present specification, the stringent conditions refer to, for example, a nucleic acid having a base sequence represented by SEQ ID NO: 2 or 4 and a nucleic acid to be hybridized corresponding to the nucleic acid as a hybridization solution [ Composition: 6 × SSC (composition: 0.9 M sodium chloride, 0.09 M sodium citrate, adjusted to pH 7.0), 0.5 mass% sodium dodecyl sulfate, 5 × Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, Incubate for 10 hours at a temperature of room temperature or higher, a temperature of 42 ° C. or more as a more stringent condition, and a temperature of 60 ° C. or more as a more stringent condition, and then, for example, 2 × SSC More stringent conditions under ionic strength conditions of 0.1 × SSC, and It means conditions for washing at a temperature of room temperature or higher, a temperature of 42 ° C. or higher as a more stringent condition, and a temperature of 60 ° C. or higher as a more stringent condition.

  From the viewpoint of sufficiently reproducing the abnormal skin condition caused by sebum in humans, a signal peptide may be further added to the nucleic acid.

  The introduction of the nucleic acid into the wild type keratinocyte is not particularly limited. For example, a method using a viral vector such as a retrovirus vector or an adenovirus vector [for example, Molecular Cloning: A Laboratory Manual ), Sambrook et al., Cold Spring Harbor Press (published in 1989, etc.)].

  Observation of the morphology of the cell structure can be performed with the naked eye, a phase contrast microscope, or the like.

In one aspect, the evaluation method of the present invention is as follows.
(A1) Cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in contact with a test substance in a support used as a scaffold, or cell aggregates containing wild-type keratinocytes are used as a scaffold. Culturing under differentiation conditions while contacting with the test substance and unsaturated fatty acid in step to obtain a cell structure,
(B1) Cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in a support body serving as a scaffold, or cell aggregates containing wild-type keratinocytes are contacted with unsaturated fatty acids in a support body serving as a scaffold. Culturing under differentiation conditions while obtaining a cell structure, and (C1) difference between the cell structure obtained in step (A1) and the cell structure obtained in step (B1) The method includes a step of evaluating whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum (hereinafter referred to as “evaluation method 1”). Since the evaluation method 1 can easily follow the apoptosis, which is the final differentiation stage of epidermal keratinocytes, it is intended to evaluate the effectiveness of components premised on the use as a skin percutaneous preparation with high transdermal absorbability. It is suitable for the case.

  In step (A1), cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in contact with a test substance in a support serving as a scaffold, or cell aggregates containing wild-type keratinocytes are used as scaffolds. A cell structure is obtained by culturing under differentiation conditions while contacting with the test substance and unsaturated fatty acid in the support.

  Cell aggregates can be obtained by, for example, culturing keratinocytes expressing epimorphin or wild type keratinocytes in a culture vessel having a surface with extremely low adhesion to cells.

The swirling speed in the swirling culture is preferably 10 min ⁻¹ or more, more preferably 50 min ⁻¹ or more from the viewpoint of favorably forming cell aggregates, and preferably 180 min from the viewpoint of maintaining a good cell state. ^-1 or less, more preferably 150 min ^-1 or less.

  Since the carbon dioxide concentration in the culture atmosphere used for the swirl culture varies depending on the type of keratinocytes and the like, and cannot be determined unconditionally, it is preferably set as appropriate according to the type of keratinocytes. For example, when the keratinocytes are human keratinocytes or mouse keratinocytes, the carbon dioxide concentration is usually about 5% by volume.

  Since the culture temperature in the swirl culture varies depending on the type of keratinocytes and the like, it cannot be determined unconditionally. Therefore, it is preferable to set appropriately according to the type of keratinocytes. For example, when the keratinocytes are human keratinocytes or mouse keratinocytes, the culture temperature is usually preferably 36 to 38 ° C., and preferably 36.5 to 37.5 ° C., from the viewpoint of favorably growing the keratinocytes. Is more preferable.

  The medium used for swirl culture may be a medium suitable for the growth of keratinocytes. Examples of the medium include heat-inactivated serum-containing DMEM / HamF12 medium, heat-inactivated serum-containing DMEM, and the like. Among these, a heat-inactivated serum-containing DMEM / HamF12 medium is preferable from the viewpoint of favorably growing keratinocytes. Examples of heat-inactivated serum used in such a medium include heat-inactivated FCS. The medium may appropriately contain a cell growth promoting factor such as insulin.

  From the viewpoint of forming cell aggregates, the culture vessel used for swirling culture preferably has low cell adhesion, and more preferably non-cell adhesion. Note that “low cell adhesion” means adhesion that does not allow cells to adhere to the surface after seeding, preferably until the formation of cell aggregates is completed (10 hours or more). That means.

  Since the culture time in the swirl culture varies depending on the type of keratinocytes and the like, it cannot be determined unconditionally. Therefore, it is preferable to set appropriately according to the type of keratinocytes. For example, when the keratinocytes are human keratinocytes or mouse keratinocytes, the culture time is usually about 10 hours.

  Examples of the support to be used as a scaffold include collagen gel, gel composed of laminin and entactin, synthetic polymer compound containing collagen, laminin and the like. Examples of the synthetic polymer compound include methyl cellulose and polyvinyl alcohol. Among these, collagen gel is preferable from the viewpoint of efficiently differentiating keratinocytes contained in cell aggregates. Among collagen gels, a gel made of type I collagen is preferable from the viewpoint of efficiently differentiating keratinocytes contained in cell aggregates.

  In step (A1), when the support is a collagen gel, the cell aggregate can be embedded in the collagen gel and cultured under differentiation conditions. Embedding of the cell aggregates in the collagen gel can be performed by placing the cell aggregates in a mixture containing collagen and a medium, and allowing the resulting mixture to gel. The medium used for embedding cell aggregates in the collagen gel is the same as that in swirl culture.

  Culturing under differentiation conditions is performed by immersing a support containing cell aggregates in a medium and incubating.

  The differentiation condition in Evaluation Method 1 is the presence of an agent that embeds cell aggregates in a support and induces proliferation and / or differentiation of cell aggregates in a medium suitable for the growth of keratinocytes as necessary. It means culturing below.

  The carbon dioxide concentration and culture temperature in culture under differentiation conditions are the same as the carbon dioxide concentration and culture temperature in swirl culture.

  The medium used for culture under differentiation conditions is not particularly limited, and examples thereof include a medium in which fetal calf serum is added to a serum-free medium for keratinocytes so that the concentration thereof is about 5 to 20% by mass. The serum-free medium for keratinocytes is not particularly limited, and examples thereof include DMEM / HamF12 medium, α-MEM, DMEM and the like.

  The culture time in the culture under the differentiation condition is the time when keratinocytes in the cell aggregate are differentiated to form a cell cluster and a space is formed inside the cell cluster when the test substance is not used. Good. The culture time is preferably 3 days or more, more preferably 5 days or more from the viewpoint of securing the time, and suppresses cell death caused by factors other than differentiation, and improves the accuracy of test substance evaluation. From the viewpoint, it is preferably 10 days or less.

  In the step (A1), when the cell aggregate containing keratinocytes expressing epimorphin is used, the contact between the cell aggregate and the test substance is a medium containing the test substance as a medium used for culture under differentiation conditions. Can be performed by immersing a support containing cell aggregates in a medium containing such a test substance. In this case, since the concentration of the test substance in the medium varies depending on the type of the test substance and the like, it cannot be determined unconditionally. Therefore, it is preferable to set it appropriately according to the type of the test substance.

  On the other hand, in the step (A1), when a cell aggregate containing wild-type keratinocytes is used, the contact between the cell aggregate, the test substance and the unsaturated fatty acid is performed as a test substance as a medium used for culture under differentiation conditions. Can be carried out by immersing a support containing cell aggregates in a medium containing such test substance and unsaturated fatty acid. In this case, since the concentration of the test substance in the medium varies depending on the type of the test substance and the like, it cannot be determined unconditionally. Therefore, it is preferable to set it appropriately according to the type of the test substance. In addition, since the concentration of unsaturated fatty acid in the medium varies depending on the type of unsaturated fatty acid and the like, it cannot be determined unconditionally, so it is preferable to set it appropriately according to the type of unsaturated fatty acid. The concentration of the unsaturated fatty acid in the medium is usually preferably 0.0005% by mass or more, more preferably 0.001% by mass or more from the viewpoint of sufficiently expressing the effect of the unsaturated fatty acid on keratinocytes. From the viewpoint of suppressing cell death caused by these factors and improving the accuracy of evaluation of the test substance, it is preferably 0.1% by mass or less, more preferably 0.05% by mass or less.

  Next, in step (B1), cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in a support body serving as a scaffold, or cell aggregates containing wild-type keratinocytes are used as a scaffold body. And culturing under differentiation conditions while contacting with an unsaturated fatty acid to obtain a cell structure.

  When keratinocytes that express epimorphin are used in step (A1), keratinocytes that express epimorphin are used in step (B1). Further, when wild-type keratinocytes are used in step (A1), wild-type keratinocytes are used in step (B1).

  In step (B1), the culture under differentiation conditions is performed by the same operation as the culture under differentiation conditions in step (A1).

  Moreover, the cell aggregate used in step (B1), the support, the medium used for culture under differentiation conditions, the carbon dioxide concentration under differentiation conditions, the culture temperature and the culture time are used in step (A1). Cell aggregate, support, medium used for culture under differentiation conditions, carbon dioxide concentration under differentiation conditions, culture temperature and culture time are the same.

  In step (B1), when a cell aggregate containing wild-type keratinocytes is used, the contact between the cell aggregate and the unsaturated fatty acid is performed by using an unsaturated fatty acid as a medium used for culture under differentiation conditions. It can be performed by immersing a support containing cell aggregates in a medium containing such an unsaturated fatty acid using a medium containing the same. In this case, the concentration of the unsaturated fatty acid in the medium is the same as the concentration of the unsaturated fatty acid in the medium in step (A1).

  Next, in step (C1), the cell structure obtained in step (A1) [cell structure (A1)] and the cell structure obtained in step (B1) [cell structure (B1)]. Whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  When cell aggregates containing wild-type keratinocytes are cultured in a support body under differentiation conditions, a cell cluster containing wild-type keratinocytes and differentiated cells of wild-type keratinocytes is formed from the cell aggregates as the culture time elapses. The In this cell cluster, in the order from the outermost part of the cell cluster toward the inside, in the same manner as normal skin, a basal layer containing wild type keratinocytes and a differentiated cell layer containing differentiated cells of wild type keratinocytes A spiny layer, a granule layer, and a stratum corneum are formed, and a space is formed in the innermost part.

  In contrast, in the cell structure (B1), a space is not formed in the innermost part, and a form reflecting a state of keratinization caused by epimorphin or unsaturated fatty acid appears.

  However, when the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, the test substance suppresses keratinization caused by epimorphin or unsaturated fatty acid, so that the cell structure The body (A1) shows the same form as the cell cluster.

  Therefore, in step (C1), by examining the difference between the cell structure (A1) and the cell structure (B1), based on the difference, occurrence of abnormal skin condition caused by the sebum on the test substance It is possible to evaluate whether the substance is a substance that suppresses the above.

In another aspect of the evaluation method of the present invention,
(A2) After culturing keratinocytes expressing epimorphin in a medium containing a test substance, the obtained keratinocytes are cultured under differentiation conditions in a medium containing calcium ionophore, or tested with unsaturated fatty acids. Culturing wild-type keratinocytes in a medium containing a substance and then culturing the obtained keratinocytes under differentiation conditions in a medium containing a calcium ionophore to obtain a cell structure,
(B2) After culturing keratinocytes expressing epimorphin in a medium containing calcium ionophore under differentiation conditions or culturing wild-type keratinocytes in a medium containing unsaturated fatty acids, the keratinocytes obtained are Culturing under differentiation conditions in a medium containing calcium ionophore to obtain a cell structure, and (C2) the cell structure obtained in step (A2) and the cell structure obtained in step (B2). A method (hereinafter referred to as “evaluation method 2”) including a step of evaluating whether or not the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum based on the difference between the body and the body. is there. Evaluation method 2 is suitable for the purpose of evaluating a component for preventing aberrant keratinization that acts in the early stage of skin differentiation because differentiation from the basal layer to the intermediate layer of epidermal keratinocytes can be easily followed. is there.

  In step (A2), after culturing keratinocytes expressing epimorphin in a medium containing a test substance, the obtained keratinocytes are cultured under differentiation conditions in a medium containing calcium ionophore or unsaturated. After culturing wild-type keratinocytes in a medium containing a fatty acid and a test substance, the obtained keratinocytes are cultured under differentiation conditions in a medium containing a calcium ionophore to obtain a cell structure.

  When keratinocytes expressing epimorphin are used in step (A2), first, keratinocytes expressing epimorphin are cultured in a medium containing a test substance, and then the obtained keratinocytes contain a calcium ionophore. Incubate in culture medium under differentiation conditions. In this case, since the concentration of the test substance in the medium varies depending on the type of the test substance and the like, it cannot be determined unconditionally. Therefore, it is preferable to set it appropriately according to the type of the test substance. Moreover, it is preferable to adjust the density | concentration of the calcium vionophore in a culture medium so that it may become the quantity by which a cone envelope is formed when a test substance is not used. From the viewpoint of ensuring the concentration of the calcium vionophore in the medium so that the concentration of the calcium vionophore in the medium is usually an amount that forms a confined envelope when the test substance is not used, it is preferably 0.0002 mass. % Or more, more preferably 0.0005% by mass or more, from the viewpoint of suppressing cell death caused by factors other than differentiation and improving the accuracy of test substance evaluation, preferably 0.005% by mass or less Preferably it is 0.002 mass% or less.

  On the other hand, when using wild-type keratinocytes in step (A2), first, wild-type keratinocytes are cultured under differentiation conditions in a medium containing the test substance and unsaturated fatty acid, and then the obtained keratinocytes are Culture under differentiation conditions in a medium containing calcium ionophore. In this case, since the concentration of the test substance in the medium varies depending on the type of the test substance and the like, it cannot be determined unconditionally, so it is preferable to set it appropriately according to the test substance. Moreover, since the concentration of the unsaturated fatty acid in the medium varies depending on the type of the unsaturated fatty acid and the like, it cannot be determined unconditionally, and therefore it is preferably set as appropriate according to the unsaturated fatty acid. The concentration of the unsaturated fatty acid in the medium is usually preferably 0.0005% by mass or more, more preferably 0.001% by mass or more from the viewpoint of sufficiently expressing the effect of the unsaturated fatty acid on keratinocytes. From the viewpoint of suppressing cell death caused by these factors and improving the accuracy of evaluation of the test substance, it is preferably 0.1% by mass or less, more preferably 0.05% by mass or less. It is preferable to adjust the concentration of the calcium vionophore in the medium so that the amount of the coneified envelope is formed when the test substance is not used. The concentration of the calcium ionophore in the medium is usually preferably 0.0002% by mass from the viewpoint of ensuring the concentration of the calcium ionophore in the medium so that it becomes an amount that forms a cornified envelope when the test substance is not used. Above, more preferably 0.0005% by mass or more, from the viewpoint of suppressing cell death caused by factors other than differentiation and improving the accuracy of evaluation of the test substance, preferably 0.005% by mass or less, more preferably Is 0.002 mass% or less.

  The medium containing the test substance can be obtained by adding the test substance to the basic medium. A medium containing the test substance and unsaturated fatty acid can be obtained by adding the test substance and unsaturated fatty acid to the basic medium. A medium containing calcium ionophore can be obtained by adding calcium ionophore to a serum-free medium for keratinocytes. The basic medium is the same as the medium used in the evaluation method 1 for culturing under differentiation conditions.

  The differentiation condition in Evaluation Method 2 refers to culturing keratinocytes in a medium obtained by adding calcium ionophore to a serum-free medium for keratinocytes.

  The carbon dioxide concentration and culture temperature in the culture under differentiation conditions are the same as the carbon dioxide concentration and culture temperature in step (A1) of the evaluation method 1.

  It is preferable to adjust the culture time in the culture under the differentiation condition so as to be a time for forming a confined envelope when the test substance is not used. In general, the culture time is preferably 3 hours or more, more preferably 5 hours or more, from the viewpoint of securing the time for the formation of the conified envelope when the test substance is not used. In addition, since the upper limit of culture | cultivation time changes with the uses of an evaluation method, since it cannot determine unconditionally, it is preferable to set suitably according to the use of an evaluation method.

  Next, in step (B2), keratinocytes expressing epimorphin are cultured under differentiation conditions in a medium containing calcium ionophore, or wild type keratinocytes are cultured in a medium containing unsaturated fatty acids. Thereafter, the obtained keratinocytes are cultured under differentiation conditions in a medium containing calcium ionophore to obtain a cell structure.

  When keratinocytes that express epimorphin are used in step (A2), keratinocytes that express epimorphin are used in step (B2). Further, when wild type keratinocytes are used in step (A2), wild type keratinocytes are used in step (B2).

  In step (B2), when keratinocytes expressing epimorphin are used, keratinocytes expressing epimorphin are cultured in a basic medium, and then the obtained keratinocytes are subjected to differentiation conditions in a medium containing calcium ionophore. Incubate at In this case, the concentration of the calcium wionophore in the medium is the same as the concentration of the calcium ionophore in the medium in step (A2).

  When using wild-type keratinocytes in step (B2), first, wild-type keratinocytes are cultured in a medium containing unsaturated fatty acids, and then the obtained keratinocytes are differentiated in a medium containing calcium ionophore. Incubate under. In this case, the concentration of the unsaturated fatty acid in the medium is the same as the concentration of the unsaturated fatty acid in the medium in step (A2). In addition, the concentration of the calcium ionophore in the medium is the same as the concentration of the calcium ionophore in the medium in step (A2).

  A medium containing unsaturated fatty acids can be obtained by adding unsaturated fatty acids to the basic medium. The basic medium is the same as the medium used in the evaluation method 1 for culturing under differentiation conditions. A medium containing calcium ionophore can be obtained by adding calcium ionophore to a serum-free medium for keratinocytes.

  The carbon dioxide concentration, the culture temperature, and the culture time in the culture under differentiation conditions are the same as the carbon dioxide concentration, the culture temperature, and the culture time in step (A1) of the evaluation method 1.

  Next, in step (C2), the cell structure obtained in step (A2) [cell structure (A2)] and the cell structure obtained in step (B2) [cell structure (B2)]. Whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  When wild-type keratinocytes are cultured under differentiation conditions in a medium containing calcium ionophore, the wild-type keratinocytes differentiate with the passage of the culture time and form a cornified envelope.

  On the other hand, in the cell structure (B2), the state of keratinization caused by epimorphin or unsaturated fatty acid is reflected, and a confined envelope is not formed.

  However, when the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, the test substance suppresses keratinization caused by epimorphin or unsaturated fatty acid, so that the cell structure In (A2), a confined envelope is formed.

  Therefore, in step (C2), by examining the difference between the cell structure (A2) and the cell structure (B2), based on the difference, occurrence of an abnormal skin condition caused by sebum on the test substance It is possible to evaluate whether the substance is a substance that suppresses the above.

In another aspect of the evaluation method of the present invention,
(A3) Cultured under differentiation conditions while contacting the differentiated cell construct of keratinocytes expressing epimorphin with the test substance, or under differentiation conditions while contacting the differentiated cell construct of wild-type keratinocytes with the unsaturated fatty acid and the test substance Culturing in to obtain a cell structure,
(B3) A differentiated cell construct of keratinocytes expressing epimorphin is cultured under differentiation conditions, or a differentiated cell construct of wild-type keratinocytes is cultured under differentiation conditions while contacting with an unsaturated fatty acid to obtain a cell structure And (C3) a skin condition in which the test substance is caused by sebum based on the difference between the cell structure obtained in step (A3) and the cell structure obtained in step (B3). A method including a step of evaluating whether or not the substance suppresses occurrence of abnormalities (hereinafter referred to as “evaluation method 3”). Evaluation method 3 can track the overall differentiation of the skin and can evaluate many components in a short time. Therefore, the purpose is to screen active ingredients not only for external use but also for internal use. It is suitable for.

  In step (A3), the differentiated cell construct of keratinocytes expressing epimorphin is cultured under differentiation conditions while contacting with the test substance, or the differentiated cell construct of wild-type keratinocytes is contacted with the unsaturated fatty acid and the test substance. Culture under differentiation conditions to obtain cell structures.

  A differentiated cell construct is obtained by three-dimensionally culturing keratinocytes or wild-type keratinocytes expressing epimorphin on a substrate obtained by embedding fibroblasts in a porous polymer.

  Fibroblasts are preferably human fibroblasts from the viewpoint of sufficiently reproducing abnormal skin conditions caused by sebum in humans. Examples of the human fibroblasts include MRC-5 cells (ATCC CCL171) and primary human skin fibroblasts. Among these, MRC-5 cells are preferable because they can be cultured with the same composition as the keratinocyte medium.

  Examples of the porous polymer include synthetic polymer compounds such as collagen gel and polyvinyl alcohol, and chitosan. The porous polymer may further contain a substance that differentiates keratinocytes, a component similar to a basement membrane component, and the like.

  Examples of the base material include fibroblast-embedded collagen gel. Among these supports, fibroblast-embedded collagen gel is preferable from the viewpoint of sufficiently reproducing abnormal skin conditions caused by sebum. Among the collagen gels, a gel made of type I collagen is preferable from the viewpoint of reproducing human skin well. The fibroblast-embedded collagen gel can be prepared, for example, by seeding human fibroblasts in a solution containing type I collagen and gelling the resulting mixture. When the human fibroblasts are embedded, the collagen gel contracts by the action of the human fibroblasts and functions as a dermis equivalent.

  When the substrate is a human fibroblast-embedded collagen gel, from the viewpoint of securing a scaffold for the keratinocytes by adhering keratinocytes on the human fibroblast-embedded collagen gel, fibronectin is formed on the surface of the collagen gel. It is desirable to have cell adhesion factors such as

  The medium used for the three-dimensional culture of keratinocytes or wild-type keratinocytes expressing epimorphin is the same as the medium used for the swirl culture in Evaluation Method 1. Of these, a heat-inactivated FCS-containing DMEM / HamF12 medium is preferable from the viewpoint of being suitable for long-term high-density culture and suitable for cell colony growth.

Keratinocytes that express epimorphin, wild-type keratinocytes, or culture of these differentiated cells in three dimensions, for example,
(1) seeding keratinocytes expressing epimorphin, wild-type keratinocytes or differentiated cells thereof on the substrate;
(2) A step of culturing keratinocytes, wild-type keratinocytes, or differentiated cells thereof expressing the epimorphin on the substrate by immersing the seeded substrate obtained in step (1) in a medium, and ( 3) The surface of the keratinocyte, wild type keratinocyte or differentiated cell expressing epimorphin in the culture obtained in the step (2) is lifted to the contact surface between the air and the medium, and the epimorphin is placed. Culturing keratinocytes expressing, wild-type keratinocytes or differentiated cells thereof,
(Air-lift method) or the like.

  The differentiation conditions in Evaluation Method 3 are keratinocytes, wild-type keratinocytes that express epimorphin, or differentiated cells thereof are cultured to a confluent state on a substrate, and keratinocytes, wild-type keratinocytes, or these that express epimorphin It means that the surface of the differentiated cell is lifted up to the contact surface between the air and the medium, exposed to the gas phase, and cultured for 14 days or more while changing the medium once every two days.

  The medium, carbon dioxide concentration, and culture temperature used for culture under differentiation conditions are the same as the medium, carbon dioxide concentration, and culture temperature used for culture under differentiation conditions in Evaluation Method 1.

  The culture time in the culture under the differentiation condition is preferably 14 days or more, more preferably 20 days or more from the viewpoint of satisfactorily reproducing the turnover in the skin, preferably from the viewpoint of maintaining the tissue in a good state. Is 28 days or less, more preferably 25 days or less.

  In the step (A3), when a differentiated cell construct of keratinocytes that express epimorphin is used, the contact between the differentiated cell construct and the test substance is performed on the keratinocytes expressing epimorphin or the upper surface of these differentiated cells in the differentiated cell construct. In addition to lifting and arranging the contact surface between the air and the medium, a solution containing a test substance is applied to the upper surface of the differentiated cell construct, and the differentiated cell construct is cultured. In this case, since the concentration of the test substance in the solution varies depending on the type of the test substance and the like, it is preferable to set appropriately according to the test substance.

  On the other hand, in the case where a differentiated cell construct of wild type keratinocytes is used in step (A3), the contact between the differentiated cell construct and the test substance and the unsaturated fatty acid causes the keratinocytes expressing epimorphin in the differentiated cell construct or the differentiated cells of Place the upper surface up to the contact surface between the air and the medium, apply the solution containing the test substance and the solution containing unsaturated fatty acid to the upper surface of the differentiated cell construct, and culture the differentiated cell construct Is done by. In this case, since the concentration of the test substance in the solution varies depending on the type of the test substance and the like, it cannot be determined unconditionally. Moreover, since the concentration of the unsaturated fatty acid in the solution varies depending on the type of the unsaturated fatty acid and the like and cannot be determined unconditionally, it is preferably set as appropriate according to the unsaturated fatty acid. The concentration of the unsaturated fatty acid in the solution is preferably 0.05% by mass or more, more preferably, from the viewpoint of ensuring a sufficient amount to express the effect of the unsaturated fatty acid when the test substance is not used. From the viewpoint of ensuring ease of handling, it is preferably 2% by mass or less, and more preferably 1% by mass or less.

  Next, in step (B3), the differentiated cell construct of keratinocytes expressing epimorphin is cultured under differentiation conditions, or the differentiated cell construct of wild-type keratinocytes is cultured under differentiation conditions while contacting with an unsaturated fatty acid. To obtain a cell structure.

  The differentiated cell construct and medium used in step (B3) and the carbon dioxide concentration, culture temperature and culture time in the culture under differentiation conditions are the same as the differentiated cell construct and medium used in step (A3) and the differentiation conditions. It is the same as the carbon dioxide concentration, culture temperature and culture time in the culture.

  When a differentiated cell construct of keratinocytes that express epimorphin is used in step (A3), a differentiated cell construct of keratinocytes that express epimorphin is used in step (B3). In addition, when a differentiated cell construct of wild type keratinocytes is used in step (A3), a differentiated cell construct of wild type keratinocytes is used in step (B3).

  In the step (B3), when the differentiated cell construct of wild type keratinocytes is used, the contact between the differentiated cell construct and the unsaturated fatty acid can be performed by applying a solution containing the unsaturated fatty acid on the surface of the differentiated cell construct. it can. In this case, the concentration of the unsaturated fatty acid in the solution containing the unsaturated fatty acid is the same as the concentration of the unsaturated fatty acid in the solution containing the unsaturated fatty acid in step (A3).

  Next, in step (C3), the test substance is caused by sebum based on the difference between the cell structure obtained in step (A3) and the cell structure obtained in step (B3). Evaluate whether the substance suppresses the occurrence of abnormal skin conditions.

  When a differentiated cell construct of wild-type keratinocytes is cultured under differentiation conditions, mature cells having no nucleus appear on the outermost surface of the obtained cell structure as the culture time elapses.

  On the other hand, in the cell structure (B3), nucleated cells appear on the outermost surface, and a form reflecting the state of keratinization caused by epimorphin or unsaturated fatty acid appears.

  However, when the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, the cell structure (A3) is suppressed because aberrant keratinization caused by epimorphin or unsaturated fatty acid is suppressed. Then, mature cells that do not have a nucleus on the outermost surface appear.

  Therefore, in the step (C3), by examining the difference between the cell structure (A3) and the cell structure (B3), based on the difference, occurrence of abnormal skin condition caused by sebum in the test substance It is possible to evaluate whether the substance is a substance that suppresses the above.

  In the present invention, in each of the step (C1) of the evaluation method 1, the step (C2) of the evaluation method 2, and the step (C3) of the evaluation method 3, as a positive control, for example, the formula (I):

[Wherein, Xaa ¹ and Xaa ⁵ are each independently a seryl group which may have a substituent, a threonyl group which may have a substituent, or a tyrosinyl group which may have a substituent, Xaa ² Is an isoleucyl group which may have a substituent, a valyl group which may have a substituent or a leucyl group which may have a substituent, Xaa ³ and Xaa ⁴ each independently have a substituent An optionally substituted asparaginyl group, an optionally substituted glutaminyl group, an optionally substituted asparatyl group or an optionally substituted glutamyl group, Cys is a cysteinyl group, R ¹ is a compound of the formula (II) ):

(In the formula, n represents an integer of 1 to 10)
Or a group represented by formula (III):

(In the formula, n represents an integer of 1 to 10)
Represents a group represented by
Or a pharmacologically acceptable salt thereof may be used. Such a cyclic peptide compound or a pharmacologically acceptable salt thereof is a substance selected as a substance that suppresses the occurrence of abnormal skin conditions caused by sebum by the evaluation method of the present invention. The formula (I) corresponds to SEQ ID NO: 1.

Xaa ¹ is a seryl group which may have a substituent, a threonyl group which may have a substituent, or a tyrosinyl group which may have a substituent. The said substituent should just be a functional group which does not inhibit the effect of this invention. Examples of the substituent include a thioglycosyl group derived from a monosaccharide or a polysaccharide, an O-glycosyl group derived from a monosaccharide or a polysaccharide, an N-glycosyl group derived from a monosaccharide or a polysaccharide, a phosphate group, and the like. Is mentioned. Of the Xaa ^1, a seryl group is preferred from the viewpoint of sufficiently expressing the inhibitory action.

Xaa ² is an isoleucil group that may have a substituent, a valyl group that may have a substituent, or a leucyl group that may have a substituent. The said substituent should just be a functional group which does not inhibit the effect of this invention. Among the Xaa ^2, an isoleucyl group is preferable from the viewpoint of sufficiently expressing the inhibitory action.

Xaa ³ is an asparaginyl group which may have a substituent, a glutaminyl group which may have a substituent, an asparatyl group which may have a substituent, or a glutamyl group which may have a substituent. The substituent may be a functional group that does not hinder the object of the present invention. When Xaa ³ is an optionally substituted asparaginyl group, examples of the substituent include a thioglycosyl group derived from a monosaccharide or a polysaccharide, an O-glycosyl group derived from a monosaccharide or a polysaccharide, a monosaccharide Alternatively, an N-glycosyl group derived from a polysaccharide can be mentioned. When Xaa ³ is a glutaminyl group which may have a substituent, examples of the substituent include an amino group. When Xaa ³ is an aspartyl group which may have a substituent, examples of the substituent include a succinimide group and a phosphate group. When Xaa ³ is a glutamyl group which may have a substituent, examples of the substituent include a carboxyl group. Of the Xaa ^3, a glutamyl group is preferred from the viewpoint of sufficiently expressing the inhibitory action.

Xaa ⁴ is an asparaginyl group which may have a substituent, a glutaminyl group which may have a substituent, an asparatyl group which may have a substituent, or a glutamyl group which may have a substituent. The said substituent should just be a functional group which does not inhibit the effect of this invention. Examples of the substituent include the same substituents as in Xaa ³ . Of the Xaa ^4, a glutaminyl group is preferable from the viewpoint of sufficiently expressing the inhibitory action.

Xaa ⁵ is a seryl group which may have a substituent, a threonyl group which may have a substituent, or a tyrosinyl group which may have a substituent. The said substituent should just be a functional group which does not inhibit the effect of this invention. Examples of the substituent include the same substituents as in Xaa ¹ . Among the Xaa ^5, a seryl group is preferable from the viewpoint of sufficiently expressing the inhibitory action.

  In formula (I), n is an integer of 1-10. The n is 10 or less, preferably 8 or less, more preferably 5 or less, still more preferably 3 or less, and particularly preferably 1 from the viewpoint of sufficiently expressing the inhibitory action.

Xaa ^{1 to} Xaa ⁵ , R ¹ and Cys may each be an L-functional group or a D-functional group. From the viewpoint of adaptability to human skin, Xaa ¹ to Xaa ⁵ , R ¹ and Cys are preferably L-functional groups.

  Pharmacologically acceptable salts include acid addition salts and base addition salts. Examples of acid addition salts include inorganic acid salts and organic acid salts. Examples of the inorganic acid salt include hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, and the like. Organic salts include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate Salt, paratoluenesulfonate, and the like. Examples of the base addition salt include inorganic base salts and organic base salts. Examples of inorganic base salts include sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, and the like. Examples of the organic base salt include organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt.

In the cyclic peptide compound represented by the formula (I) or a pharmacologically acceptable salt thereof, from the viewpoint of sufficiently expressing the inhibitory action, in the formula (I), Xaa ¹ is a seryl group and Xaa ² is A compound in which n is 1 is preferably an isoleucyl group, Xaa ³ is a glutamyl group, Xaa ⁴ is a glutaminyl group, Xaa ⁵ is a seryl group, R ¹ is a group represented by the formula (III).

  As described above, according to the evaluation method of the present invention, human skin differentiation under the expression of epimorphin or in the presence of unsaturated fatty acids can be performed by performing a simple operation of culturing under differentiation conditions. The process of the occurrence of abnormal skin conditions caused by epimorphine or unsaturated fatty acids can be easily reproduced. The abnormal skin condition caused by epimorphine or unsaturated fatty acid is associated with abnormal skin conditions such as keratinization caused by sebum in human skin. Therefore, it is possible to evaluate whether or not the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum by a simple operation. Therefore, the evaluation method of the present invention is useful for development, screening, validation, and the like of substances that suppress abnormal skin conditions caused by sebum.

  Next, the present invention will be described in more detail based on examples. However, the present invention is not limited to such examples. In the following, Cys represents a cysteinyl group, Ser represents a seryl group, Gln represents a glutaminyl group, Glu represents a glutamyl group, Ile represents an isoleucyl group, and Gly represents a glycyl group.

(Production Example 1)
Heat-inactivated fetal calf serum (FCS) was added to DMEM / HamF12 (manufactured by Sigma-Aldrich) to a concentration of 10% by mass, and heat-inactivated FCS-containing DMEM / HamF12 medium (hereinafter referred to as “DH10”). Medium)).

  HaCaT cells, which are normal human epidermal keratinocyte cell lines, were cultured at 37 ° C. in a 5% by volume carbon dioxide atmosphere in the DH10 medium.

  DNA encoding T7-tag (SEQ ID NO: 6) is added to the site corresponding to the N-terminal side of epimorphin in cDNA encoding mouse epimorphin (GenBank accession number: E06629, SEQ ID NO: 2); CDNA encoding T7-tag epimorphin was prepared. Next, cDNA encoding the T7-tag epimorphin was inserted into the EcoRI recognition site of the retrovirus expression vector pQCXIN (Clontech) to prepare an expression plasmid for T7-tag epimorphin.

  The obtained expression plasmid for T7-tag epimorphin was used as a packaging cell [manufactured by Invitrogen, trade name: Lipofectamine and Invitrogen, trade name: Plus Reagent] to be used for packaging cells [Clontech, PT67. Cells (mouse fibroblast-derived cells)]. Next, among the obtained cells, the culture supernatant was collected from cells exhibiting resistance to 500 μg / mL geneticin (Gibco Laboratories, trade name: G418). Retrovirus was obtained from the collected culture supernatant. The obtained retrovirus was infected with the HaCaT cells and cultured for 8 days at 37 ° C. in a 5% by volume carbon dioxide atmosphere in the presence of 500 μg / mL geneticin (Gibco Laboratories, trade name: G418). Thereafter, keratinocytes (HaCaT-TE cells) producing T7-tag epimorphin were isolated by examining epimorphin expression in the cultured cells.

(Production Example 2)
PT67 cells were cultured at 37 ° C. in a 5% by volume carbon dioxide atmosphere in DH10 medium.

  The expression plasmid for T7-tag epimorphin of Production Example 1 was introduced into PT67 cells using a gene introduction reagent (trade name: Lipofectamine and Invitrogen, trade name: Plus reagent). Next, the obtained cells were cultured at 37 ° C. for 8 days in a 5% by volume carbon dioxide atmosphere in the presence of 500 μg / mL geneticin (Gibco Laboratories). Then, the fibroblast (PT67-TE cell) which produces T7-tag epimorphin was isolated by investigating the epimorphin expression in the cell after culture | cultivation.

(Production Example 3)
A nucleic acid (SEQ ID NO: 7) encoding IL-2 signal peptide is added to the site corresponding to the N-terminus of epimorphin in cDNA (SEQ ID NO: 2) encoding epimorphin, and IL-2 signal peptide binding epithelium is added. A nucleic acid encoding morphine was obtained. The obtained nucleic acid was inserted into an EcoRI recognition site of a retrovirus expression vector (Clontech, trade name: pQCXIN) to prepare an expression plasmid for cell surface epimorphin.

  The obtained expression plasmid for cell surface epimorphin was introduced into PT67 using a trade name: Lipofectamine (Invitrogen) and Plus reagent (Invitrogen). A retrovirus obtained in the same manner as in Production Example 1 was infected with HaCaT cells. Next, the obtained cells were cultured at 37 ° C. for 8 days in a 5% by volume carbon dioxide atmosphere in the presence of 500 μg / mL geneticin (manufactured by Gibco Laboratory, trade name: G418). Then, the cell (HaCaT-EPM cell) which produces an extracellular epimorphin was isolated by investigating the epimorphin expression in the cell after culture | cultivation.

(Experimental example 1)
5 volumes of HaCaT cells in DH10 medium (experiment number: 1), 0.01 vol% oleic acid-containing DH10 medium (experiment number: 2) or 0.025 vol% oleic acid-containing DH10 medium (experiment number: 3) The cells were cultured at 37 ° C. for 3 days in a% carbon dioxide atmosphere.

  The cultured cells are isolated, and the obtained cells are solubilized with a solubilizing reagent [composition: 2% by volume sodium dodecyl sulfate (SDS), 10% by volume glycerol, 5% by volume 2-mercaptoethanol, 0.008% by volume bromophenol blue. , 0.65 M Tris-HCl buffer (pH 6.8)] to obtain a cell extract.

  Western blotting was performed using the obtained cell extract and an anti-epimorphin antibody (R & D Systems) to examine the expression level of endogenous epimorphin in HaCaT cells. The expression level of β-actin in HaCaT cells was examined in the same manner as described above except that an anti-β-actin antibody was used instead of the anti-epimorphin antibody.

  The results of examining the relationship between the type of medium and the expression level of endogenous epimorphin in HaCaT cells in Experimental Example 1 are shown in FIG. In FIG. 1, lane 1 is a band corresponding to endogenous epimorphin when the culture medium of experiment number 1 is used, lane 2 is a band corresponding to endogenous epimorphin when the culture medium of experiment number 2 is used, Lane 3 shows a band corresponding to endogenous epimorphin when using the medium of experiment number: 3.

  From the results shown in FIG. 1, it can be seen that the expression level of endogenous epimorphin in HaCaT cells is comparable even when any of the culture media of Experiment Nos. 1 to 3 is used. In addition, it turns out that the expression level of (beta) -actin in a HaCaT cell is also comparable even if it uses any culture medium of experiment number: 1-3. Therefore, these results indicate that oleic acid has little effect on the expression level of endogenous epimorphin in HaCaT cells.

(Experimental example 2)
The HaCaT-TE cells obtained in Production Example 1 or the PT67-TE cells obtained in Production Example 2 were treated with DH10 medium (Experiment No .: 4), 0.01 vol% oleic acid-containing DH10 medium (Experiment No. 5) or The cells were cultured in a DH10 medium containing 0.025% by volume oleic acid (Experiment No. 6) at 37 ° C. for 3 days in a 5% by volume carbon dioxide atmosphere. In addition, the HaCaT-TE cells obtained in Production Example 1 or the PT67-TE cells obtained in Production Example 2 are irradiated with ultraviolet rays B (UVB) so that the irradiation dose is 10 mJ / cm ^2, and then In DH10 medium, the cells were cultured for 3 days at 37 ° C. in an atmosphere of 5% by volume of carbon dioxide (Experiment No. 7).

  The obtained culture was subjected to centrifugation [1000 × g, 30 minutes] to obtain a culture supernatant.

  Secreted epimorphin was recovered from the obtained culture supernatant using an anti-T7 tag antibody (manufactured by Novagen) and protein G sepharose beads (manufactured by GE Healthcare). Western blotting was performed using the collected secreted epimorphin and an HRP-labeled anti-T7 tag antibody (manufactured by Novagen), and the secreted epimorphin in each of the culture supernatant of HaCaT-TE cells and the culture supernatant of PT67-TE cells was The amount was examined.

  FIG. 2 shows the results of examining the relationship between the type of medium and the amount of secreted epimorphin in each of the culture supernatant of HaCaT-TE cells and the culture supernatant of PT67-TE cells in Experimental Example 2. In FIG. 2, lane 1 is a band corresponding to secreted epimorphin when the medium of experiment number: 4 is used, lane 2 is a band corresponding to secreted epimorphin when the medium of experiment number: 5 is used, lane 3 Indicates a band corresponding to secreted epimorphin when the medium of experiment number: 6 is used, and lane 4 indicates a band corresponding to secreted epimorphin when the medium of experiment number: 7 is used.

  From the results shown in FIG. 2, the amount of secreted epimorphin in the culture supernatant of HaCaT-TE cells, which are keratinocytes, is the highest when the medium of Experiment No. 6 with the highest concentration of oleic acid was used, and UVB It can be seen that the amount is equal to the amount of secreted epimorphin in the culture supernatant of the HaCaT-TE cells irradiated with. On the other hand, it can be seen that the amount of secreted epimorphin in the culture supernatant of HaCaT-TE cells is lowest when the medium of experiment number 4 containing no oleic acid is used. In contrast, secreted epimorphin is not detected in the culture supernatant of PT67-TE cells, which are fibroblasts, regardless of the concentration of oleic acid. These results indicate that oleic acid causes epimorphin secretion from keratinocytes. Thus, it is suggested that the cutaneous keratinization caused by oleic acid is associated with abnormal skin conditions caused by epimorphin.

(Production Example 4)
In the formula (I), a compound in which Xaa ¹ is a seryl group, Xaa ² is an isoleucyl group, Xaa ³ is a glutamyl group, Xaa ⁴ is a glutaminyl group, Xaa ⁵ is a seryl group, and n = 1 is as follows: And synthesized.

(1) Synthesis of peptide As a starting material, Fmoc-Cys (Trt) -Trt (2-Cl) resin [the amount of Fmoc-Cys (Trt) per 1 g of 2-chlorotrityl chloride resin is 0.70 mmol] equivalent to 0.25 mmol The amount was put into an automatic peptide synthesizer (Applied Biosystem, trade name: 430A).

  First, under the control of the program of the automatic peptide synthesizer, 2 mmol of Fmoc-Ser (OBu), which is an Fmoc-amino acid derivative, was added to a coupling agent [0.45M O- (benzotriazol-1-yl) -N, N, N The above reaction is activated with dimethylformamide containing ', N'-tetramethyluronium hexafluorophosphate (hereinafter referred to as “HBTu”) and 0.45M1-hydroxybenzotriazole (hereinafter referred to as “HOBt”). Put in the tank. Thereby, in the said reaction tank, the coupling reaction of the amino acid residue on resin and Fmoc-amino acid derivative was performed, and the Fmoc group protection peptide chain was produced | generated.

  Next, the Fmoc group in the Fmoc group-protected peptide chain on the resin was removed with 20% by volume piperidine-containing N-methylpyrrolidone solution (deprotection) and washed. Thereafter, Fmoc-Gln (Trt), Fmoc-Glu (OBu), Fmoc-Ile, Fmoc-Ser (OBu), Fmoc-Gly and Fmoc-Cys (Trt) are used in this order to perform the same operation as described above. Thus, according to the amino acid sequence shown in SEQ ID NO: 8, the corresponding Fmoc-amino acid derivative is sequentially introduced into the Fmoc group-protected peptide chain on the resin, and the Fmoc group-protected peptide having the amino acid sequence shown in SEQ ID NO: 8 is obtained. A resin containing was obtained. The success or failure of the coupling reaction was confirmed as appropriate by performing a Kaiser test.

  The obtained resin containing the Fmoc group-protected peptide is mixed with trifluoroacetic acid (hereinafter referred to as “TFA”), triisopropylsilane (hereinafter referred to as “TIS”), water and ethanedithiol (hereinafter referred to as “DT”). In a liquid [TFA / TIS / water / DT (volume ratio) 92.5 / 2.5 / 2.5 / 2.5], incubation at room temperature for 2 hours to remove the peptide chain from the resin. I did it. The 2-chlorotrityl chloride resin was filtered off from the mixed solution after incubation to obtain a filtrate. The obtained filtrate was concentrated under reduced pressure to remove TFA from the filtrate. Cold diethyl ether was added to the resulting residue to recover about 700 mg of a crude peptide precipitate.

  About 700 mg of the resulting crude product was subjected to a high-performance liquid chromatography fractionator [(stock) with a reverse-phase column (manufactured by Zorbax, octadecyl silica column, column inner diameter: 30 mm, column length 250 mm). ) Made by Shimadzu Corporation, trade name: Model LC8A]. Then, using 0.1% by volume trifluoroacetic acid aqueous solution and 0.1% by volume trifluoroacetic acid-containing acetonitrile solution, acetonitrile in the eluent has a concentration gradient of 1 to 60% by volume. Chromatography was performed for 25 minutes at a flow rate of 1.0 mL / min. The fraction containing the target peptide was collected, and acetonitrile was distilled off from the fraction. Next, the residue was freeze-dried to obtain 110 mg of the target peptide trifluoroacetate salt. Thereafter, the trifluoroacetate salt of the target peptide was desalted to obtain the target peptide (linear peptide). From the results of Kaiser test, it was confirmed that the peptide was a peptide consisting of the amino acid sequence shown in SEQ ID NO: 8.

(2) Cyclization of peptide 110 mg (0.135 mmol) of the peptide obtained in the above (1) was added to 130 mL of 50% by volume acetic acid aqueous solution. To the obtained mixture, 210 μL (0.8 equivalent) of 0.5 M aqueous iodine solution was added, and the mixture was stirred at room temperature for 3 hours. Thereby, the thiol group of two cysteinyl groups in the peptide was oxidized to form a disulfide bond. Thereafter, 70 mg of ascorbic acid was added to the resulting mixture.

  Next, the obtained mixture was mixed with a reverse-phase high-performance chromatographic fractionation apparatus [(stock), equipped with a reverse-phase column [manufactured by Zorbax, octadecyl silica column, column inner diameter: 30 mm, column length 250 mm]. ) Made by Shimadzu Corporation, trade name: Model LC8A]. Then, using 0.1% by volume trifluoroacetic acid aqueous solution and 0.1% by volume trifluoroacetic acid-containing acetonitrile solution, acetonitrile in the eluent has a concentration gradient of 1 to 60% by volume. Chromatography was performed for 25 minutes at a flow rate of 1.0 mL / min. This gave 20 mg of product.

(3) Confirmation of Cyclic Peptide Compound Each of the peptide obtained in (1) and the product (oxidized peptide) obtained in (2) is mass spectrometer [trade name: manufactured by Shimadzu Corporation LC-MS-2010], the mass spectrum of each of the peptide obtained in (1) and the oxidized peptide obtained in (2) was examined. In Production Example 4, the mass spectrum of the peptide is shown in FIG. 3, and in Production Example 4, the mass spectrum of the oxidized peptide is shown in FIG.

  From the results shown in FIG. 3, in the mass spectrum of the peptide obtained in the above (1), a peak is seen at 826.2 m / z near the theoretical value of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 8. I understand that. From this result, it was confirmed that the peptide obtained in the above (1) was a peptide consisting of the amino acid sequence shown in SEQ ID NO: 8.

From the results shown in FIGS. 3 and 4, the mass spectrum of the peptide obtained in (1) above shows a peak at 826.2 m / z (see FIG. 3), whereas (2 In the mass spectrum of the oxidized peptide obtained in (1), it can be seen that a peak is observed at 824.2 m / z, which is two hydrogen atoms less. From these results, the peptide obtained in the above (1) is cyclized in the oxidized peptide obtained in the above (2) by forming a disulfide bond between two cysteinyl groups. Was supported. Therefore, the oxidized peptide obtained in the above (2) has the formula (I), wherein Xaa ¹ is a seryl group, Xaa ² is an isoleucyl group, Xaa ³ is a glutamyl group, Xaa ⁴ is a glutaminyl group, and Xaa ⁵ is ceryl. This is a cyclic peptide compound in which R ¹ is a group represented by formula (III) and n is 1. When the purity of the cyclic peptide compound was examined by high performance liquid chromatography (hereinafter referred to as “HPLC”), it was confirmed to be 98.2%.

(Production Example 5)
(1) Preparation of linear peptide In Production Example 4, instead of Fmoc-Cys (Trt) -Trt (2-Cl) resin, Fmoc-Asp (OBu) -Alko-resin [Fmoc-Asp (OBu per 1 g of resin) ) Is used in an amount equivalent to 0.25 mmol, and as Fmoc-amino acid derivatives, Fmoc-Ser (OBu), Fmoc-Gln (Trt), Fmoc-Glu (OBu), Fmoc-Ile, Instead of using Fmoc-Ser (OBu), Fmoc-Gly and Fmoc-Cys (Trt) in this order, Fmoc-Gln (Trt), Fmoc-Glu (OBu), Fmoc-Ile and Fmoc-Ser (OBu) The same operation as in Production Example 4 was performed except that the sequences were used in order, and shown in SEQ ID NO: 9. A linear peptide consisting of the amino acid sequence was obtained.

Example 1
(1) Preparation of test sample The test peptide 1 was obtained by dissolving the cyclic peptide compound obtained in Production Example 4 as a test substance in purified water so that its concentration was 10 μg / mL. In addition, the linear peptide obtained in Production Example 5 as a test substance was dissolved in purified water so as to have a concentration of 10 μg / mL, and Test Sample 2 was obtained.

(2) Medium preparation In the following experiment, DH10 medium was used as the medium of Experiment No. 8. Moreover, 1 volume% oleic acid containing ethanol solution was added to DH10 culture medium so that the concentration of oleic acid might be 0.05 volume%, and the culture medium was obtained (experiment number: 9). Further, 1% by volume oleic acid-containing ethanol solution and test sample 2 were added to DH10 medium so that the concentration of oleic acid was 0.05% by volume and the concentration of linear peptide was 0.0001% by mass. Thus, a medium was obtained (experiment number: 10). Also, 1% by volume oleic acid-containing ethanol solution and test sample 1 are added to the DH10 medium so that the concentration of oleic acid is 0.05% by volume and the concentration of the cyclic peptide compound is 0.0001% by mass. A medium was obtained (Experiment No. 11).

(3) Calculation of cell cluster formation and lumen formation rate HaCaT cells (wild type keratinocytes) were cultured at 37 ° C. in a DH10 medium in a 5% by volume carbon dioxide atmosphere.

Next, the obtained HaCaT cells were suspended in 350 μL of a DH10 medium containing 1000 U / mL DNase I (Sigma-Aldrich). The obtained suspension was subjected to swirl culture at 37 ° C. for 24 hours in a 5-well dish (manufactured by Corning, ultra-low adhesion surface) at 100 min ⁻¹ in a 5% by volume carbon dioxide atmosphere. Smooth and round cell aggregates were formed.

  The formed cell aggregate was embedded in a high-density collagen gel prepared with a 0.5 mass% type IA collagen solution [manufactured by Koken Co., Ltd.]. The collagen gel is a support that serves as a scaffold for cells.

  Next, the cell aggregates after embedding were placed in a 5 vol% carbon dioxide atmosphere in the medium of experiment number 8: the medium of experiment number: 9, the medium of experiment number: 10 or the medium of experiment number: 11. By culturing at 37 ° C. for 4 days, cell clusters (cell structures) were formed. The morphology of the obtained cell cluster was observed under a phase contrast microscope.

  In the cell cluster, the outermost cell is in an undifferentiated state because it is in contact with collagen. However, the cells located inside the undifferentiated cells existing in the outermost layer usually start to differentiate rapidly and reach anoikis. Therefore, in the cell cluster, a lumen that can be easily discriminated is formed when 3 to 4 days have elapsed since the start of culture in a normal state. Thus, 100 randomly selected cell clusters were observed, and the ratio of cell clusters in which clear lumen formation was observed in all 100 cell clusters was examined to calculate the lumen formation rate.

  In Example 1, the result of observing the morphology of the cell cluster (cell structure) when using the culture medium of Experiment No. 11 is shown in FIG. 5 (A). In Example 1, the culture medium of Experiment No. 10 is used. FIG. 5 (B) shows the result of observing the morphology of the cell cluster (cell structure) when it was present. In the figure, the scale bar indicates a length of 100 μm.

  Moreover, in Example 1, the result of having investigated the relationship between the kind of culture medium and lumen formation rate is shown in FIG. In the figure, lane 1 is the lumen formation rate in the cell cluster when the medium of experiment number: 8 is used, lane 2 is the lumen formation rate in the cell cluster when the medium of experiment number: 9 is used, and lane 3 is Experiment No: The lumen formation rate in the cell cluster when the medium of 10 is used, and Lane 4 shows the lumen formation rate in the cell cluster when the medium of Experiment No. 11 is used.

  From the results shown in FIG. 5 (A), FIG. 5 (B) and FIG. 6, the cell cluster when using the medium of experiment number: 11 has a high lumen formation rate, whereas the experiment number: It can be seen that the cell cluster when the medium of 10 is used has a low lumen formation rate. From these results, the culture medium of Experiment No. 11 containing the test sample 1 suppressed the occurrence of lumen formation abnormality in the cell cluster caused by oleic acid, but the experiment No. 10 containing the test sample 2 was suppressed. It can be seen that the medium does not inhibit the occurrence of lumen formation abnormalities in cell clusters caused by oleic acid.

  Since the lumen formation in the cell cluster reflects the differentiation and state in the skin, abnormalities in the lumen formation in the cell cluster caused by oleic acid are caused by abnormalities such as oleic acid contained in the sebum. It is thought to reproduce the keratinization, which is one of the abnormal skin conditions caused by saturated fatty acids. Therefore, it is suggested that the cyclic peptide compound contained in the test sample 1 is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  From the above results, a cell structure obtained by culturing cell aggregates containing wild-type keratinocytes under differentiation conditions in a test body and a support body serving as a scaffold while contacting with an unsaturated fatty acid such as oleic acid, The test substance is caused by sebum on the basis of the difference between the cellular structure containing wild-type keratinocytes and the cell structure obtained by culturing under the differentiation conditions in the support as a scaffold in contact with the unsaturated fatty acid. It is suggested that it can be evaluated whether it is a substance that suppresses the occurrence of abnormal skin conditions.

(Example 2)
As a test substance, among the cyclic peptide compounds represented by the formula (I), a compound other than the cyclic peptide compound obtained in Production Example 4 was used and the same operation as in Example 1 was performed. It is evaluated whether it is a substance that suppresses the occurrence of abnormal skin conditions caused by.

  As a result, it can be seen that this test substance shows the same tendency as the cyclic peptide compound obtained in Production Example 4.

(Example 3)
(1) Preparation of culture medium In the following experiment, DH10 culture medium was used as a culture medium of experiment number: 12. 1 μL of purified water was added to 1 mL of DH10 medium to obtain a medium (Experiment No. 13). Furthermore, the test sample 2 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the linear peptide was 0.0001% by mass to obtain a medium (Experiment No. 14). Moreover, the test sample 1 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the cyclic peptide compound was 0.0001% by mass to obtain a medium (Experiment No. 15).

(2) Formation of cell cluster (cell structure) and calculation of lumen formation rate In Example 1, instead of the HaCaT cell, the HaCaT-EPM cell obtained in Production Example 3 was used. Experiment No. 8 medium, Experiment No. 9 medium, Experiment No. 10 medium or Experiment No. 11 medium instead of Experiment No. 12 medium, Experiment No. 13 medium, Experiment No. 14 Except that the medium or the medium of experiment number: 15 was used, the same operation as in Example 1 was performed, and the lumen formation rate was calculated.

  In Example 3, the result of observing the morphology of the cell cluster (cell structure) when using the medium of Experiment No. 15 is shown in FIG. 7A. In Example 3, the medium of Experiment No. 14 is used. FIG. 7 (B) shows the result of observing the morphology of the cell cluster (cell structure) when it was present. In the figure, the scale bar indicates a length of 100 μm.

  Moreover, in Example 3, the result of having investigated the relationship between the kind of culture medium and a lumen formation rate is shown in FIG. In the figure, lane 1 is the lumen formation rate in the cell cluster when the medium of experiment number: 12 is used, lane 2 is the lumen formation rate in the cell cluster when the medium of experiment number: 13 is used, and lane 3 is Experiment No .: lumen formation rate in the cell cluster when the medium of 14 is used, lane 4 shows the lumen formation rate in the cell cluster when the medium of experiment number: 15 is used. In the figure, the data is based on three counts and is shown as mean ± standard error. In the figure, * is P <0.05.

  From the results shown in FIG. 7 (A), FIG. 7 (B), and FIG. 8, the cell cluster when using the medium of experiment number: 15 has a high lumen formation rate, whereas the experiment number: It can be seen that the cell cluster when 14 mediums are used has a low lumen formation rate. From these results, the culture medium of Experiment No. 14 containing the test sample 1 suppressed the occurrence of lumen formation abnormality in the cell cluster caused by epimorphin, but the experiment No. 15 containing the test sample 2 was suppressed. It can be seen that the medium does not inhibit the occurrence of lumen formation abnormalities in cell clusters caused by epimorphin.

  Lumen formation in the cell cluster reflects differentiation and state in the skin. In addition, the results shown in Experimental Example 2 suggest that skin keratinization caused by oleic acid is associated with abnormal skin condition caused by epimorphin. Therefore, it is suggested that the cyclic peptide compound contained in the test sample 1 is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  Based on the above results, cell structures obtained by culturing cell aggregates containing epikerphin-expressing keratinocytes under a differentiation condition in a support body as a scaffold while in contact with a test sample, and expressing epimorphin Based on the difference between the cellular structure containing keratinocytes and the cell structure obtained by culturing under the differentiation condition in the support body as the scaffold, the occurrence of abnormal skin condition caused by sebum in the test substance It is suggested that it is possible to evaluate whether it is a suppressive substance.

Example 4
As a test substance, among the cyclic peptide compounds represented by the formula (I), a compound other than the cyclic peptide compound obtained in Production Example 4 was used, and the same operation as in Example 3 was performed. It is evaluated whether it is a substance that suppresses the occurrence of abnormal skin conditions caused by.

  As a result, it can be seen that this test substance shows the same tendency as the cyclic peptide compound obtained in Production Example 4.

(Example 5)
(1) Preparation of culture medium In the following experiment, DH10 culture medium was used as a culture medium of experiment number: 16. Further, a 1% by volume oleic acid-containing ethanol solution was added to the DH10 medium so that the concentration of oleic acid was 0.02% by volume to obtain a medium (Experiment No. 17). Further, the ethanol solution containing 1% by volume of oleic acid and the test sample 2 obtained in Example 1 (1) were prepared so that the concentration of oleic acid was 0.02% by volume and the concentration of linear peptide was 0.000001. It added to DH10 culture medium so that it might become mass%, and the culture medium was obtained (experiment number: 18). In addition, a 1% by volume oleic acid-containing ethanol solution and the test sample 1 obtained in Example 1 (1) were prepared using a oleic acid concentration of 0.02% by volume and a cyclic peptide compound concentration of 0.000001 mass. % Was added to DH10 medium to obtain a medium (Experiment No. 19). A 1% by volume oleic acid-containing ethanol solution and the test sample 2 obtained in Example 1 (1) were prepared using a oleic acid concentration of 0.02% by volume and a linear peptide concentration of 0.00001% by mass. Was added to DH10 medium to obtain a medium (experiment number: 20). A 1% by volume oleic acid-containing ethanol solution and the test sample 1 obtained in Example 1 (1) were prepared such that the concentration of oleic acid was 0.02% by volume and the concentration of the cyclic peptide compound was 0.00001% by mass. Thus, it added to DH10 culture medium and obtained the culture medium (experiment number: 21). In addition, a 1% by volume oleic acid-containing ethanol solution and the test sample 2 obtained in Example 1 (1) were prepared using a oleic acid concentration of 0.02% by volume and a linear peptide concentration of 0.0001. It added to DH10 culture medium so that it might become mass%, and the culture medium was obtained (experiment number: 22). A 1% by volume oleic acid-containing ethanol solution and the test sample 1 obtained in Example 1 (1) were prepared such that the concentration of oleic acid was 0.02% by volume and the concentration of the cyclic peptide compound was 0.0001% by mass. Thus, it added to the DH10 culture medium and obtained the culture medium (experiment number: 23).

(2) Calculation of Cornified Envelope Formation Rate HaCaT cells were cultured for 3 days at 37 ° C. in a 5 vol% carbon dioxide atmosphere in any medium of Experiment Nos. 16-23.

  Next, the obtained HaCaT cells were washed with phosphate buffered physiological saline, and then incubated at 37 ° C. for 3 minutes in 500 μL of a trypsin-EDTA solution (manufactured by SIGMA).

Thereafter, the obtained HaCaT cells were suspended in a serum-free DH medium (DMEM / HamF12, Sigma Aldrich) so as to be 1.0 × 10 ⁵ cells / mL. To the obtained suspension, calcium ionophore A23187 (manufactured by Sigma-Aldrich) that causes calcium influx was added to a concentration of 20 ng / mL, and 5% by volume of HaCaT cells contained in the obtained mixture was added. The cells were cultured at 37 ° C. for 5 hours in a carbon atmosphere.

  The resulting cells were washed with phosphate buffered saline. The cells after washing were incubated for 10 minutes in a lysate (composition: 2% by mass SDS, 20 mM dithiothreitol, remaining purified water). Thereafter, the number of residual insolubilized cells caused by the insoluble corfined envelope after calcium influx by calcium ionophore A23187 was counted under an optical microscope, and the number of total cells and the number of residual insolubilized cells was used to determine the rate of formation of the conified envelope. Calculated.

  FIG. 9 shows the results of examining the relationship between the type of medium and the rate of formation of the conformed envelope in Example 5. In the figure, 1 is the rate of formation of the confined envelope when the culture medium of experiment number: 16 is used, 2 is the rate of formation of the conformed envelope when the culture medium of experiment number: 17 is used, and 3 is the culture medium of experiment number: 18 Cornified envelope formation rate when used, 4 is the conified envelope formation rate when using the medium of the experiment number: 19, 5 is the conified envelope formation rate when using the medium of the experiment number: 20, 6 Experiment No .: Cornified envelope formation rate when using the culture medium of 21; 7 is the Cornified envelope formation rate when using the culture medium of Experiment No. 22; 8 is the Corni when using the culture medium of Experiment No. 23 The fido envelope formation rate is shown. In the figure, the data is based on three counts and is shown as mean ± standard error. In the figure, ** is P <0.01, and *** is P <0.001.

  From the results shown in FIG. 9, it was confirmed that the experiment No. 19 (4 in the figure), 21 (6 in the figure) and 23 (8 in the figure) including the test sample 1 were used. The envelope formation rate is the rate of formation of the conified envelope when each of the culture media of test numbers 18 (3 in the figure), 20 (5 in the figure) and 22 (7 in the figure) including the test sample 2 is used. It can be seen that it is higher than that.

  Since the formation of the cornified envelope reflects the differentiation and condition in the skin, the cornified envelope formation rate in the presence of oleic acid correlates with the cornified envelope formation rate in the case of skin keratinization. it is conceivable that. Therefore, it is suggested that the cyclic peptide compound contained in the test sample 1 is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  From the above results, the wild-type keratinocytes were obtained by culturing the wild-type keratinocytes under differentiation conditions in a medium containing a test substance, an unsaturated fatty acid such as oleic acid, and a calcium ionophore, and the wild-type keratinocytes, Suppresses the occurrence of abnormal skin conditions caused by sebum based on differences between cell structures obtained by culturing under differentiation conditions in a medium containing unsaturated fatty acids and calcium ionophores It is suggested that it can be evaluated whether it is a substance to do.

(Example 6)
As the test substance, among the cyclic peptide compounds represented by the formula (I), a compound other than the cyclic peptide compound obtained in Production Example 4 was used, and the same operation as in Example 5 was performed. It is evaluated whether it is a substance that suppresses the occurrence of abnormal skin conditions caused by.

  As a result, it can be seen that this test substance shows the same tendency as the cyclic peptide compound obtained in Production Example 4.

(Example 7)
(1) Preparation of medium In the following experiment, DH10 medium was used as the medium of experiment number 24 or 25. Test sample 2 obtained in Example 1 (1) was added to DH10 medium so that the concentration of the linear peptide was 0.000001% by mass to obtain a medium (Experiment No. 26). Moreover, the test sample 1 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the cyclic peptide compound was 0.000001% by mass to obtain a medium (Experiment No. 27). The test sample 2 obtained in Example 1 (1) was added to a DH10 medium so that the concentration of the linear peptide was 0.00001% by mass to obtain a medium (Experiment No. 28). The test sample 1 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the cyclic peptide compound was 0.00001% by mass to obtain a medium (Experiment No. 29). Further, the test sample 2 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the linear peptide was 0.0001% by mass to obtain a medium (experiment number: 30). . The test sample 1 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the cyclic peptide compound was 0.0001% by mass to obtain a medium (Experiment No. 31).

(2) Calculation of Cornified Envelope Formation Rate HaCaT cells were cultured in the medium of Experiment No. 24 at 37 ° C. for 3 days in a 5% by volume carbon dioxide atmosphere.

  In addition, when HaCaT-EPM cells were used, the HaCaT-EPM cells were cultured in a medium of any of experiment numbers: 25 to 31 at 37 ° C. in a 5% by volume carbon dioxide atmosphere for 3 days.

  Next, the obtained HaCaT cells or HaCaT-EPM cells were washed with phosphate buffered saline, and then incubated at 37 ° C. for 3 minutes in 500 μL of trypsin-EDTA solution.

Thereafter, the obtained HaCaT cells or HaCaT-EPM cells were suspended in a serum-free DH medium so as to be 1.0 × 10 ⁵ cells / mL. Calcium ionophore A23187 causing calcium influx was added to the obtained suspension so that the concentration was 20 ng / mL, and HaCaT cells contained in the obtained mixture were added at 37 ° C. in a 5% by volume carbon dioxide atmosphere. Cultured for 5 hours.

  The same operation as in Example 5 was performed to calculate the confied envelope formation rate.

  FIG. 10 shows the results of examining the relationship between the type of medium or the type of cells and the rate of formation of the conified envelope in Example 7. In the figure, 1 is the rate of formation of the confined envelope when the culture medium of experiment number 24 is used, 2 is the rate of formation of the confined envelope when the culture medium of experiment number 25 is used, and 3 is the culture medium of experiment number 26 Conformed envelope formation rate when used, 4 is the conformed envelope formation rate when using the medium of the experiment number: 27, 5 is the conified envelope formation rate when using the medium of the experiment number: 28, 6 is Experiment number: Cornified envelope formation rate when using the culture medium of 29, 7 is the Cornified envelope formation rate when using the culture medium of the experiment number: 30, and 8 is Corni when using the culture medium of the experiment number: 31 The fido envelope formation rate is shown. In the figure, the data is based on three counts and is shown as mean ± standard error. In the figure, * indicates P <0.05, and ** indicates P <0.01.

  From the results shown in FIG. 10, it was confirmed that the experiment No. 27 (4 in the figure), 29 (6 in the figure), and 31 (8 in the figure) including test sample 1 were used. Envelope formation rate is the rate of formation of the confined envelope when each of the media of test numbers 26 (3 in the figure), 28 (5 in the figure) and 30 (7 in the figure) including the test sample 2 is used. It can be seen that it is higher than that. Since the formation of the corrided envelope reflects the differentiation and state in the skin, the formation rate of the corrided envelope when epimorphin is expressed is the rate of formation of the conified envelope in the case of skin keratinization It is thought that this is correlated. Therefore, it is suggested that the cyclic peptide compound contained in the test sample 1 is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  From the above results, cell structures obtained by culturing keratinocytes expressing epimorphin under a differentiation condition in a medium containing a test substance and a calcium ionophore, and keratinocytes expressing epimorphin were converted into calcium ionophores. Whether or not the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, based on the difference between the cell structure obtained by culturing under differentiation conditions in a medium containing It is suggested that it can be evaluated.

(Example 8)
As a test substance, among the cyclic peptide compounds represented by the formula (I), a compound other than the cyclic peptide compound obtained in Production Example 4 was used, and the same operation as in Example 7 was performed. It is evaluated whether it is a substance that suppresses the occurrence of abnormal skin conditions caused by.

  As a result, it can be seen that this test substance shows the same tendency as the cyclic peptide compound obtained in Production Example 4.

Example 9
(1) Preparation of sample In the following experiment, DH10 culture medium was used as a sample of experiment number: 32. 1 μL of purified water was added to 1 mL of DH10 medium to obtain a sample (experiment number: 33). In addition, the test sample 1 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the cyclic peptide compound was 0.001% by mass to obtain a sample (Experiment No. 34). The test sample 2 obtained in Comparative Example 1 (1) was added to the DH10 medium so that the concentration of the linear peptide was 0.001% by mass to obtain a sample (Experiment No. 35).

(2) Observation of tissue morphology of cell structure obtained by three-dimensional culture α-MEM (Gibco Laboratory Co., Ltd.) containing 10% by mass heat-inactivated FCS from human fetal lung fibroblast MRC-5 cells (Product made) In 10 mL, it culture | cultivated at 37 degreeC by the 5 volume% carbon dioxide atmosphere for 72 hours.

A support [manufactured by BD Biosciences, trade name: cell culture insert] was placed in the well of a 24-well culture plate. In the support, the MRC-5 cells and a collagen gel mixed solution (collagen type I, manufactured by Nitta Gelatin Co., Ltd.) were mixed. The resulting mixture was gelled to obtain a cell-embedded gel (1.7 × 10 ⁵ / mL). 0.05 ml of a 1 mg / mL fibronectin aqueous solution (manufactured by BD Bioscience) was added to the upper surface of the obtained cell-embedded gel, and the cell-embedded gel was left at room temperature for 1 hour. Next, the MRC-5 cells in the cell-embedded gel were incubated overnight at 37 ° C. in a 5% by volume carbon dioxide atmosphere in DH10 medium.

Thereafter, 0.4 μg / mL hydrocortisone (manufactured by Sigma-Aldrich), 100 μg / mL gentamicin (manufactured by Gibco Laboratory), insulin (5 μg / ml), and 50 μg / mL ascorbic acid (manufactured by Sigma-Aldrich) are contained. HaCaT cells (7.0 × 10 ⁴ cells) suspended in 0.2 mL of DH10 medium were seeded on the cell-embedded gel.

  Next, the cell-embedded gel seeded with the HaCaT cells was immersed in DH10 in the well of a 24-well culture plate. Thereafter, the cells contained in the cell-embedded gel were incubated for 5 days at 37 ° C. in a 5% by volume carbon dioxide atmosphere to obtain a cell construct. Next, the surface of the HaCaT cell in the obtained cell construct is lifted up to the contact surface between air and DH10 medium, and the cell construct is cultured at 37 ° C. for 15 days in a 5% by volume carbon dioxide atmosphere. A differentiated cell construct containing HaCaT cells was obtained.

  Thereafter, in Experiment No. 32, 5 μL of the sample of Experiment No. 36 was applied to the surface of the differentiated cell construct, and the differentiated cell construct was further cultured at 37 ° C. in a 5% by volume carbon dioxide atmosphere for 1 day. On the other hand, in Experiment Nos. 33 to 35, 5 μL of 0.5 volume% oleic acid-containing ethanol solution and 5 μL of any sample of Experiment Nos. 33 to 35 were applied to the surface of the differentiated cell construct, and differentiated cells were applied. The construct was further cultured for 1 day at 37 ° C. in a 5% by volume carbon dioxide atmosphere.

  A frozen section was prepared from the obtained cell structure. The frozen section was stained with hematoxylin-eosin to stain the cell nucleus blue-purple and most of the cytoplasmic cytoplasm was stained red. Using the frozen section after staining, the tissue morphology of the cell structure was observed under a phase contrast microscope.

  FIG. 11 (A) shows the result of observing the tissue morphology of the cell structure when the sample of Experiment No. 32 was applied without applying the 0.5 volume% oleic acid-containing ethanol solution in Example 9. In Example 9, the result of observing the tissue morphology of the cell structure when 0.5 volume% oleic acid-containing ethanol solution and the sample of Experiment No. 33 were applied is shown in FIG. The result of observing the tissue morphology of the cell structure when the acid-containing ethanol solution and the sample of Experiment No. 34 were applied is shown in FIG. 11 (C), and the ethanol solution containing 0.5% by volume oleic acid and the experiment No. 35 of FIG. FIG. 11D shows the result of observing the tissue morphology of the cell structure when the sample was applied. In the figure, the scale bar indicates a length of 50 μm.

  From the results shown in FIGS. 11 (A) and 11 (C), in the cell structure when 0.5 vol% oleic acid-containing ethanol solution and the sample of Experiment No. 34 including the test sample 1 were applied, It can be seen that cell nuclei are not observed as in the case of the cell structure when the sample of Experiment No. 32 was applied without applying the ethanol solution containing 5% by volume of oleic acid. Therefore, it turns out that the cyclic peptide compound contained in the test sample 1 can suppress the occurrence of keratinization caused by oleic acid.

  On the other hand, from the results shown in FIGS. 11 (B) and 11 (D), the cell structure and 0.5% by volume when 0.5 volume% oleic acid-containing ethanol solution and the sample of Experiment No. 33 were applied. It can be seen that cell nuclei are seen in the cell structure when the sample of Experiment No. 35 containing the oleic acid-containing ethanol solution and the test sample 2 is applied. Therefore, it turns out that the linear peptide contained in the test sample 2 cannot suppress the occurrence of keratinization caused by oleic acid.

  From these results, it is suggested that the cyclic peptide compound contained in the test sample 1 is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  From the above results, a cell structure obtained by culturing a differentiated cell construct of keratinocytes expressing epimorphin under differentiation conditions while contacting with a test sample, and a differentiated cell construct of keratinocytes expressing epimorphin, Whether or not the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum can be evaluated based on differences from cell structures obtained by culturing under differentiation conditions It is suggested.

(Example 10)
As a test substance, among the cyclic peptide compounds represented by the formula (I), a compound other than the cyclic peptide compound obtained in Production Example 4 was used, and the same operation as in Example 9 was performed. Evaluate whether it is a substance that suppresses the occurrence of abnormal skin conditions.

  As a result, it can be seen that this test substance shows the same tendency as the cyclic peptide compound obtained in Production Example 4.

(Example 11)
In Example 9, the same as in Example 9 except that the HaCaT-EPM cell obtained in Production Example 3 was used instead of the HaCaT cell and the ethanol solution containing 0.5% by volume oleic acid was not used. The operation is performed to evaluate whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

  As a result, as in Example 9, it can be evaluated that the cyclic peptide compound obtained in Production Example 4 is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum.

(Example 12)
As a test substance, among the cyclic peptide compounds represented by formula (I), a compound other than the cyclic peptide compound obtained in Production Example 4 was used, and the same operation as in Example 11 was performed. Evaluate whether it is a substance that suppresses the occurrence of abnormal skin conditions.

  As a result, it can be seen that this test substance shows the same tendency as the cyclic peptide compound obtained in Production Example 4.

  Based on the above results, the cell structure obtained by culturing the differentiated cell construct of keratinocytes expressing epimorphin in contact with the test sample under differentiation conditions and the differentiated cell construct of keratinocytes expressing epimorphin are differentiated. It is suggested that the test substance can evaluate whether it is a substance that suppresses the occurrence of abnormal skin condition caused by sebum, based on the difference between cell structures obtained by culturing under The

  Further, when evaluating whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, the compound represented by formula (I) is an occurrence of abnormal skin conditions caused by sebum. It is suggested that it can be used as a positive control for substances that suppress stagnation.

(Comparative Example 1)
The test sample 1 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the cyclic peptide compound was 0.001% by mass to obtain a medium (Experiment No. 36). Moreover, the test sample 2 obtained in Example 1 (1) was added to the DH10 medium so that the concentration of the linear peptide was 0.001% by mass to obtain a medium (Experiment No. 37). A culture medium was obtained in the same manner as described above except that the same amount of purified water (control) as the purified water contained in test sample 1 was used instead of test sample 1.

  In the obtained medium, the HaCaT-EPM cells obtained in Production Example 3 were cultured at 37 ° C. in a 5% by volume carbon dioxide atmosphere, and the cells were collected over time. To the collected cells, a reagent A [manufactured by Dojindo Laboratories, trade name: WST-1] attached to a cell counting kit (manufactured by Dojindo Laboratories, trade name: Cell Counting Kit) and 110 μL of a mixed solution with the solution B [volume ratio of the reagent A and the solution B (reagent A / solution B) = 10/1] was added and incubated for 3 hours. About the obtained mixture, the light absorbency in wavelength 450nm was measured using the spectrophotometer [The product name: ARVOtmSX 1420 MULTILABEL COUNTER made from a Wallac (WALLAC) company], and the influence on growth of HaCaT-EPM cell was investigated.

  Table 1 shows the results of examining the growth state of the HaCaT-EPM cells when the experiment No. 36 or 37 medium was used. The evaluation criteria in Table 1 are as follows.

+: The growth state of HaCaT-EPM cells is significantly better than when the control is used (risk rate of 0.01 or less).
-: The growth state of HaCaT-EPM cells is the same as the growth state of HaCaT-EPM cells when a control is used.

  From the results shown in Table 1, it can be seen that the growth state of the HaCaT-EPM cells cultured in the medium of Experiment No. 36 including the test sample 1 is good. On the other hand, it can be seen that the growth state of the HaCaT-EPM cells cultured in the medium of Experiment No. 37 including the test sample 2 is poor.

  However, it is unclear from these results whether or not the cyclic peptide compound contained in the test sample 1 has an effect on keratinization. Therefore, by examining the influence of the test substance on the growth state of HaCaT-EPM cells, it cannot be evaluated whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum. it is conceivable that.

SEQ ID NO: 1 is the sequence of a cyclic peptide compound. A disulfide bond is formed between the first cysteinyl group and the eighth cysteinyl group. The second Xaa is —CO— (CH ₂ ) _n —NH— (wherein n is an integer of 1 to 10). The third Xaa is Ser that may have a substituent, Thr that may have a substituent, or Tyr that may have a substituent. The fourth Xaa is Ile which may have a substituent, Val which may have a substituent, or Leu which may have a substituent. The fifth Xaa is Asn which may have a substituent, Gln which may have a substituent, Asp which may have a substituent, or Glu which may have a substituent. The sixth Xaa is Asn which may have a substituent, Gln which may have a substituent, Asp which may have a substituent, or Glu which may have a substituent. The seventh Xaa is Ser that may have a substituent, Thr that may have a substituent, or Tyr that may have a substituent.

  SEQ ID NO: 6 is a T7 tag sequence.

  SEQ ID NO: 7 is the sequence of the IL-2 signal peptide.

SEQ ID NO: 8 is a partial sequence of a cyclic peptide compound. The second Xaa is —CO— (CH ₂ ) _n —NH— (wherein n is an integer of 1 to 10). The third Xaa is Ser that may have a substituent, Thr that may have a substituent, or Tyr that may have a substituent. The fourth Xaa is Ile which may have a substituent, Val which may have a substituent, or Leu which may have a substituent. The fifth Xaa is Asn which may have a substituent, Gln which may have a substituent, Asp which may have a substituent, or Glu which may have a substituent. The sixth Xaa is Asn which may have a substituent, Gln which may have a substituent, Asp which may have a substituent, or Glu which may have a substituent. The seventh Xaa is Ser that may have a substituent, Thr that may have a substituent, or Tyr that may have a substituent.

  SEQ ID NO: 9 is a linear peptide sequence.

Claims (4)

  Culturing keratinocytes expressing epimorphin or their differentiated cell constructs under differentiation conditions in contact with the test substance, or contacting wild type keratinocytes or their differentiated cell constructs with unsaturated fatty acids and the test substance under differentiation conditions Culture, observe the morphology of the resulting cell structure, and evaluate whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum based on the morphology of the cell structure The test substance evaluation method characterized by the above-mentioned. (A1) Cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in contact with a test substance in a support used as a scaffold, or cell aggregates containing wild-type keratinocytes are used as a scaffold. Culturing under differentiation conditions while contacting with an unsaturated fatty acid and a test substance to obtain a cell structure,
(B1) Cell aggregates containing keratinocytes expressing epimorphin are cultured under differentiation conditions in a support body serving as a scaffold, or cell aggregates containing wild-type keratinocytes are contacted with unsaturated fatty acids in a support body serving as a scaffold. Culturing under differentiation conditions while obtaining a cell structure, and (C1) difference between the cell structure obtained in step (A1) and the cell structure obtained in step (B1) The test substance evaluation method according to claim 1, further comprising a step of evaluating whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum. (A2) After culturing keratinocytes expressing epimorphin in a medium containing a test substance, the obtained keratinocytes are cultured under differentiation conditions in a medium containing calcium ionophore, or tested with unsaturated fatty acids. Culturing wild-type keratinocytes in a medium containing a substance and then culturing the obtained keratinocytes under differentiation conditions in a medium containing a calcium ionophore to obtain a cell structure,
(B2) After culturing keratinocytes expressing epimorphin in a medium containing calcium ionophore under differentiation conditions or culturing wild-type keratinocytes in a medium containing unsaturated fatty acids, the keratinocytes obtained are Culturing under differentiation conditions in a medium containing calcium ionophore to obtain a cell structure, and (C2) the cell structure obtained in step (A2) and the cell structure obtained in step (B2). The test substance evaluation method according to claim 1, comprising a step of evaluating whether the test substance is a substance that suppresses the occurrence of abnormal skin conditions caused by sebum, based on a difference from the body. (A3) Cultured under differentiation conditions while contacting the differentiated cell construct of keratinocytes expressing epimorphin with the test substance, or under differentiation conditions while contacting the differentiated cell construct of wild-type keratinocytes with the unsaturated fatty acid and the test substance Culturing in to obtain a cell structure,
(B3) A differentiated cell construct of keratinocytes expressing epimorphin is cultured under differentiation conditions, or a differentiated cell construct of wild-type keratinocytes is cultured under differentiation conditions while contacting with an unsaturated fatty acid to obtain a cell structure And (C3) a skin condition in which the test substance is caused by sebum based on the difference between the cell structure obtained in step (A3) and the cell structure obtained in step (B3). The method for evaluating a test substance according to claim 1, comprising a step of evaluating whether the substance is a substance that suppresses the occurrence of abnormalities.