Method for evaluating and / or selecting total secretion regulator

Abstract

To provide a technique relating to total secretion regulation of sebaceous gland cells.SOLUTION: The present invention provides a total secretion regulator comprising a compound selected from the group consisting of oligosaccharides and macrolide compounds as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating and / or selecting a pan-secretion regulator, a three-dimensional structure of sebaceous gland cells, a method for producing a three-dimensional structure of sebaceous gland cells, a pan-secretion regulator, and the like. [Background technology]

[0002] Sebaceous glands are located in areas where hair organs are present. Sebaceous glands secrete sebum from the sebaceous gland openings that open onto the inner surface of the pores. Sebaceous glands have a sac-like structure with densely packed sebaceous gland cells inside, and these sebaceous gland cells are responsible for releasing sebum. Sebum is composed of wax esters, triglycerides, fatty acids, etc., and when this sebum mixes with moisture such as sweat, it is emulsified and forms a surface film of the skin. This surface film of the skin is also called the sebum film, and it plays a role in protecting the surface of the skin.

[0003] Areas with many sebaceous glands include the scalp, forehead, armpits, chest, and vulva. Skin problems involving clogged pores, which are caused by sebaceous glands, are likely to occur in these areas. Clogged pores are one of the skin problems that we want to prevent, improve, or treat.

[0004] Patent Document 1 discloses a method for producing mature human sebocytes, which comprises culturing immature human sebocytes under hypoxic conditions, with the aim of providing cultured sebocytes capable of forming or accumulating oil droplets.

[0005] Non-Patent Document 1 discloses a laminated sheet obtained by laminating layers of sebaceous gland cells (Non-Patent Document 1).

[0006] It has also been disclosed that multiple sebaceous gland cells are embedded in a matrix gel and cultured to create spheroids in which mature sebaceous gland cells gather in the center (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 4-35718 [Non-patent literature]

[0008] [Non-Patent Document 1] Christine Barrault, et al., Experimental Dermatology. 2012 Apr, 21(4):314-316. [Non-patent document 2] Alona Feldman, et al.,Nature.Communications.2019;10:2348 Summary of the Invention [Problem to be solved by the invention]

[0009] It has long been believed that the formation of comedones (hereinafter also referred to as "clogged pores") is caused by the blockage of pore outlets by a thickened stratum corneum, followed by the accumulation and solidification of sebum inside the pores. In the past, attention was focused on the amount of sebum produced as one of the causes of clogged pores, and experimental systems for evaluating the amount of sebum produced, such as those described in Patent Documents 1 to 3, have been investigated and utilized.

[0010] However, this conventional thinking has not always been able to explain the formation of open comedones and microcomedones, which are pathological conditions in which pores become clogged while the pore outlet remains open. Thus, the detailed mechanism of clogged pore formation has remained unknown until now.

[0011] Therefore, a main object of the present invention is to provide a technique relating to the formation of clogged pores. [Means for solving the problem]

[0012] As a result of extensive research into the detailed mechanism of clogged pore formation, the present inventors have newly discovered a relationship between clogged pore formation and holocytosis of sebaceous gland cells. More specifically, the present inventors have newly discovered that the holocytosis state (normal or abnormal) of sebaceous gland cells is regulated by adjusting the autophagy conditions for sebaceous gland cells, and that this holocytosis state of sebaceous gland cells is involved in the formation of clogged pores. In this specification, sebaceous gland cells produce and accumulate sebum within the cells, and eventually die and disintegrate, resulting in the secretion of the sebum from within the cells; this secretion pattern is referred to as holocrine secretion.

[0013] Furthermore, the present inventors further investigated techniques for regulating holocytosis of sebaceous gland cells, and as a result, they discovered that, from the state of fluorescent living sebaceous gland cells, the fluorescence of sebaceous gland cells is lost when holocytosis occurs, while the fluorescence of sebaceous gland cells is maintained when holocytosis does not occur.The present inventors then discovered that by fluorescently observing the state of fluorescent sebaceous gland cells over time, the state of holocytosis of sebaceous gland cells can be grasped over time, and new conditions for regulating holocytosis of sebaceous gland cells have been discovered, and a new method for evaluating and / or selecting holocytosis regulators using this technology.Furthermore, the present inventors have discovered a novel three-dimensional structure of sebaceous gland cells that allows for better observation of holocytosis of sebaceous gland cells, and a method for producing the same.

[0014] Thus, the present invention can provide, as technologies relating to the formation of clogged pores, a method for evaluating and / or selecting a holocrine regulator that is effective against sebaceous gland cells, a holocrine regulator containing a substance selected by said method, a new three-dimensional structure of sebaceous gland cells, and a method for producing the same. That is, the present invention is as follows.

[0015] The present invention includes an observation step of making one or more sebaceous gland cells and a test substance present therein, and then fluorescently observing the state of the fluorescent sebaceous gland cells; a discrimination step of discriminating the test substance as a pan-secretion regulator or a candidate pan-secretion regulator based on the state of the fluorescently-activated sebaceous gland cells; The present invention provides a method for evaluating and / or selecting a total secretion regulator, comprising: The holocrine regulator may be one or more selected from a holocrine promoter, a holocrine inhibitor, a regulator of clogged pore formation, and a regulator of sebocyte autophagy. The sebaceous gland cell may be a three-dimensional structure composed of a plurality of sebaceous gland cells. The observation may be an observation using time-lapse imaging. The observation may be performed using a fluorescence microscope. In the observation step, one or more sebaceous gland cells and a test substance may be present under conditions for holocytosis regulation, and the state of the fluorescent sebaceous gland cells may be observed by fluorescence. The holocytosis-regulating conditions may be autophagy-regulating conditions, and the autophagy-regulating conditions may be autophagy-inhibiting conditions or autophagy-inducing conditions. The state of the luminescent sebaceous gland cells may be determined based on the number of cells that have lost fluorescence per unit volume.

[0016] The present invention also provides a method for detecting keratinocytes, comprising: an observation step of fluorescently observing the state of keratinocytes that have been collected after allowing one or more sebaceous gland cells and a test substance to be present; a discrimination step of discriminating the test substance as a total secretion regulator or a candidate for a total secretion regulator based on the state of the fluorescent keratin plug; The present invention provides a method for evaluating and / or selecting a total secretion regulator, comprising: The keratinized plugs in the observation step are a plurality of keratinized plugs collected from the skin surface of a mammal with an adhesive sheet and attached to the adhesive sheet, and / or The state of the keratinized plug in the discrimination step may be a rate of change in the maximum diameter of the keratinized plug obtained by dividing the average value of the maximum diameter of the keratinized plug after use of the test substance by the average value of the maximum diameter of the keratinized plug before use.

[0017] The present invention provides a total secretion regulator containing, as active ingredients, one or two compounds selected from the group consisting of oligosaccharides and macrolide compounds.

[0018] The present invention also provides a three-dimensional structure of sebocytes, the surface layer of which is composed of mature sebocytes. The present invention also provides a method for producing a three-dimensional structure of sebaceous gland cells, in which a plurality of seeded sebaceous gland cells are cultured on a surface of a mortar-shaped depression treated to inhibit cell adhesion, thereby obtaining a three-dimensional structure of sebaceous gland cells. The three-dimensional structure may be spherical. The three-dimensional structure may be used for fluorescence observation. It may also be used in methods for evaluating and / or selecting global secretion regulators. The cone-shaped depression may have a shape that allows a plurality of sebaceous gland cells to gather in the central region of the bottom of the depression. [Effects of the Invention]

[0019] According to the present invention, a technique for preventing clogged pores can be provided. Note that the effects described herein are not necessarily limited to those described herein, and may be any of the effects described in this specification. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1A is a schematic diagram of a cross section of a hair follicle organ. Figure 1B shows the results of a confirmatory study on the effect of adding an autophagy inhibitor on pore clogging near the human sebaceous duct. Figure 1B-1 shows the results after culturing in a control (DMSO solution without an autophagy inhibitor); Figure 1B-2 shows the results after culturing in the presence of an autophagy inhibitor (DMSO solution containing Bafilomycin A1); and Figure 1B-3 shows the results after culturing in the presence of an autophagy inhibitor (DMSO solution containing MHY1485). [Figure 2]Figure 2 shows the results of a confirmation test on the effect of adding an autophagy inhibitor on clogged pores near human sebaceous gland ducts. From left to right, the distance between the hair and hair follicle wall (μm) is shown before culture; after culture in the presence of a control (DMSO); after culture in the presence of an autophagy inhibitor (Bafilomycin A1); and after culture in the presence of an autophagy inhibitor (MHY-1485). * indicates a significant difference (MEAN ± SEM P<0.05). [Figure 3] Figure 3 shows a three-dimensional structure of sebaceous gland cells obtained by culturing multiple seeded sebaceous gland cells on the cell adhesion-inhibiting surface of a cone-shaped depression that has been treated to inhibit cell adhesion. Figure 3A is a schematic diagram of the cone-shaped depression with a V-shaped bottom, and Figure 3B is a schematic diagram of the cone-shaped depression with a U-shaped bottom. Figure 3C is an optical microscope photograph showing a three-dimensional structure (spheroid) of sebaceous gland cells obtained by culturing on the cell adhesion-inhibiting surface (hydrophilized surface) of a cone-shaped depression. [Figure 4] Figure 4A is a confocal laser microscope photograph showing an overall image of a three-dimensional structure (spheroid) of sebaceous glands when mature sebaceous glands are fluorescently stained. Adipophilin was used as a maturation marker for staining. Figure 4B is a cross-sectional view of the stained spheroid in Figure 4A taken in the depth direction from the surface to the center, showing the location of mature sebaceous glands in the spheroid obtained in this embodiment. [Figure 5] Figure 5A shows time-lapse images taken at the start (Figure A-1) and end (Figure A-2) of observation of spheroids obtained in this embodiment cultured under autophagy-inducing conditions using a starvation medium, demonstrating that sebaceous gland cells present in the surface layer of the spheroids were disrupted and that the number of cells that lost fluorescence per volume unit was high (particularly the arrowed portion). Figure 5B shows time-lapse images taken at the start (Figure B-1) and end (Figure B-2) of observation of spheroids obtained in this embodiment cultured under autophagy-inhibiting conditions using a growth medium, demonstrating that sebaceous gland cells present in the surface layer of the spheroids were not disrupted and that the number of cells that lost fluorescence per volume unit was low (particularly the arrowed portion). [Figure 6] Figure 6 shows images A to C of spheroids obtained in this embodiment and spheroids cultured under autophagy-inducing conditions using starvation medium. Figures 6A to 6C show the number of cells that have lost fluorescence per volume unit within the frame in Figure 6A (field 1: field frame at the bottom), Figure 6B (field 2: field frame at the upper left), and Figure 6C (field 3: field frame at the lower right). Square frame: 62.4 μm x 125 μm x 50 μm. Cells that have lost fluorescence were obtained from fields 1 to 3 in these images A to C, respectively, and the average value was calculated as the number of cells that have lost fluorescence per volume unit, and the results are shown in the lower part of the figure. [Figure 7] Figure 7 shows time-lapse images A to C of the spheroids obtained in this embodiment and the spheroids cultured under autophagy-inducing conditions using starvation medium. Figure 7A (arrow at observation time 600 minutes), Figure 7B (arrow at observation time 610 minutes), and Figure 7C (arrow at observation time 620 minutes) show the process of one cell in the surface layer of the spheroid losing its fluorescence. [Figure 8] Figure 8 shows how keratinized plugs are removed by adhering them to adhesive tape, and then excitation light is irradiated onto multiple keratinized plugs on the tape to detect autofluorescence. The three-dimensional structure of these keratinized plug images is then reconstructed, and the state of the fluorescent keratinized plugs is observed by fluorescence. [Figure 9] Figure 9 shows the percentage change in maximum diameter of keratin plugs as an evaluation of the effect of four weeks of continuous use of lotions containing or not containing trehalose on the size of nasal keratin plugs. Percent change in maximum diameter of keratin plugs (value before use of sample = 1). The group without trehalose (N = 6) had an average value of 1.07500 (standard error 0.04200), and the group with trehalose (N = 8) had an average value of 0.86644 (standard error 0.02755). The maximum diameters of 11 to 24 keratin plugs were measured for each subject's sample and averaged. The percentage change in maximum diameter of keratin plugs was calculated by dividing the average value after use of the test substance by the average value before use. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, embodiments for carrying out the present invention will be described. Note that the embodiment described below shows one example of a typical embodiment of the present invention, and the scope of the present invention is not to be construed as being limited thereby. Note that in this specification, "%" indicates mass % unless otherwise specified, and when a numerical range is expressed using "to", the range is intended to include both end values. Furthermore, the upper and lower limit values ​​of the numerical values ​​can be arbitrarily combined as desired.

[0022] <1. Method for evaluating and / or selecting a total secretion regulator according to this embodiment> One aspect of the embodiment of the present invention may be to provide a method for evaluating and / or selecting a pan-secretion regulator for sebaceous gland cells.

[0023] <Method of the First Embodiment> The method of the first embodiment can provide a method for evaluating and / or selecting a holocytosis regulator, which includes an observation step of fluorescently observing the state of fluorescent sebaceous gland cells in the presence of one or more sebaceous gland cells and a test substance, and / or a discrimination step of discriminating the test substance as a holocytosis regulator or a candidate holocytosis regulator based on the state of the fluorescent sebaceous gland cells. In the observation step, fluorescent observation is preferably performed under holocytosis-regulating conditions.

[0024] Conventional methods for evaluating clogged pores include a method in which a person uses a magnifying glass to observe under a microscope to assess whether the clogged pores have become smaller; for example, a facial imaging device called VISIA is used to shine UVA light on the area to be evaluated and take an image in a dark place, where the clogged pores glow red or light green, and the image is then used to assess whether the number of glowing pores has decreased; and a method in which a person palpates the skin to assess whether the roughness has decreased, as the surface of the skin becomes rough when pores are clogged. However, in the method of evaluating the total secretion regulator or the method of selecting the substance to be used as the total secretion regulator using these conventional evaluation methods, because subjects are used or sensory evaluation is used, when there are a large number of test substances, it is difficult to quickly evaluate all of these total secretion regulators, and it is also difficult to obtain reproducible results.In addition, because it is thought that multiple mechanisms are involved in the clogging of pores, these conventional evaluation methods are inappropriate as a method of directly evaluating total secretion, which is the initial stage of clogging of pores.

[0025] In contrast, the method of the first embodiment allows for in vitro testing using animal cells, and the test is easy to perform or reproduce, making it easy to evaluate or select test substances even when there are a large number of them, and the results are highly reproducible. Furthermore, this embodiment allows for the use of observation equipment (e.g., fluorescence microscope-related) and / or holocytosis regulation conditions, making it possible to evaluate holocytosis regulation of sebaceous gland cells more objectively and accurately, and these tasks are not complicated. Therefore, in this embodiment, it is possible to easily evaluate whether a test substance is suitable as a holocytosis regulator and / or select a test substance as a holocytosis regulator (specifically, a substance having a holocytosis regulator effect). Furthermore, this embodiment is highly objective and the results are easily reproducible.

[0026] Furthermore, the method of the first embodiment can be used to clarify or analyze skin problems caused by clogged pores, and can also suggest countermeasures for skin problems caused by clogged pores. For example, skin problems caused by clogged pores include blackheads, enlarged pores, rough pores, red pores, acne, and seborrheic dermatitis. In this specification, clogged pores refers to clogging caused by abnormal accumulation of stratum corneum blocking the pore outlet and hardening of sebum inside the pore. In this specification, the term "blackening of pores" refers to the blackening of substances that have clogged pores due to oxidation by air. In this specification, enlarged pores refers to the phenomenon in which sebum accumulates and hardens inside pores, causing the pores to appear enlarged on the surface. In this specification, roughness of pores refers to the sensation felt when sebum accumulates and hardens inside the pores, causing the pores to swell slightly. In this specification, it is believed that redness of pores occurs when sebum accumulates and hardens inside the pores, causing changes in the composition of sebum in the hair follicles and changes in the growth environment of bacteria present on the skin (sebaceous glands, inside the hair follicles, on the surface, etc.), resulting in inflammation. In this specification, it is believed that acne and seborrheic dermatitis develop when sebum accumulates and hardens inside pores, causing a change in the composition of sebum in hair follicles or a change in the growth environment of bacteria present on the skin (sebaceous glands, inside hair follicles, on the surface, etc.).

[0027] The first embodiment preferably includes an observation step and / or a discrimination step, and more preferably, the observation step is performed first, followed by the discrimination step. More preferably, the first embodiment includes a step of culturing, observing, and distinguishing sebaceous gland cells, and it is more preferable to perform these steps sequentially. Furthermore, this first embodiment preferably includes a contact step of contacting the sebaceous gland cells with the test substance between the culture step and the observation step, and more preferably includes a culture step, a contact step, an observation step, and a discrimination step, and it is even more preferable to perform these steps sequentially. Furthermore, in the explanation of the total secretion regulator etc. in this first embodiment, the explanations of the second embodiment described below, "2." to "3.", can also be applied to this first embodiment and can be adopted as appropriate.

[0028] <1-1. Sebaceous gland cell culture process> In this first embodiment, a step of culturing sebaceous gland cells may be performed before the contacting step, or this culturing step may be omitted. This culturing step can be performed using the in vitro culture conditions for normal sebaceous gland cells (preferably derived from humans). In this culturing step, immature sebaceous gland cells are preferably seeded and cultured until they are capable of full secretion. This culturing step allows for the production of sebaceous gland cells in a full secretion state.

[0029] As used herein, the term "mature sebocytes (or mature sebaceous cells)" refers to differentiated sebaceous cells that contain sebum within the cells and are capable of full secretion. As used herein, the term "immature sebaceous gland cells (or immature sebaceous gland cells)" is a term used to distinguish them from the above-mentioned "mature sebaceous gland cells," and refers to undifferentiated sebaceous gland cells that are not the above-mentioned "mature sebaceous gland cells" or sebaceous gland cells that do not contain sebum within the cells.

[0030] In the sebaceous gland cell culture step of the first embodiment, it is preferable to seed and culture a plurality of sebaceous gland cells in a culture medium in a culture vessel (see FIG. 3). The culture medium is a growth medium for animal cells generally used for growth, such as proliferation, maintenance, and differentiation, and a growth medium normally used for culturing sebaceous gland cells, which will be described later in "Culture medium for inhibiting total secretion," can be appropriately adopted.

[0031] The sebaceous gland cells to be seeded are not particularly limited, but are preferably derived from mammals. Examples of such mammals include humans, hamsters, pigs, mice, and rats, with humans and / or hamsters being preferred from the standpoint of availability and culture maintenance. Commercially available sebaceous gland cells may also be used. In the case of human origin, examples include, but are not limited to, Caucasian, Asian, and African sebaceous gland cells. In this embodiment, any of these sebaceous gland cells may be used, but Caucasian sebaceous cells are preferred. The seeding concentration of sebocytes in the medium is preferably 1 x 10 2 ~1×10 5 cells / mL, preferably 5×10 2 ~2×10 4 cells / mL, more preferably 1 x 10 3 ~1×10 4 cells / mL. The culture vessel can be a culture vessel (e.g., made of plastic, glass, etc.) used under normal in vitro culture conditions for sebaceous gland cells, and examples include cell culture plates such as petri dishes and microplates (e.g., 6-well, 24-well, etc.).

[0032] The sebaceous gland cell culture process can be carried out under the same in vitro culture conditions as those used for normal sebaceous gland cells (preferably human-derived), and is preferably carried out under culture conditions using a growth medium (hereinafter also referred to as "growth medium culture conditions"). For example, the culture conditions are preferably a medium pH of about 7 to 8, a culture CO2 concentration of about 4 to 10% (preferably 4 to 6%) in the atmosphere, and a culture temperature of about 30 to 40°C, more preferably 33 to 38°C, and even more preferably about 36 to 37°C. The culture period is not particularly limited, but is preferably 5 to 15 days, more preferably 6 to 13 days, and even more preferably 7 to 12 days after seeding. The culture can be carried out under static, shaking, or rotation conditions, preferably static conditions, and the medium may be changed an average number of times per day, preferably once every 1 to 3 days, and more preferably once every 1 to 2 days.

[0033] Furthermore, it is more preferable that the sebaceous gland cell culture step be performed by applying the method for producing a three-dimensional sebaceous gland cell structure described below in "2." In this culture step, it is preferable to use a container having a cone-shaped depression for seeding and culturing multiple immature sebaceous gland cells. It is even more preferable that in this culture step, the seeded multiple sebaceous gland cells are cultured on a cell adhesion-inhibiting treated surface (more preferably a hydrophilized surface) of the inner surface of the cone-shaped depression. Furthermore, the sebaceous gland cells after the culturing step are preferably transferred to a container for a fluorescence microscope when used in the contacting step or the observing step, and the contacting step may be carried out at the time of transfer.

[0034] In the first embodiment, an animal cell culture device configured to perform the sebaceous gland cell culture process may be used, and the culture device may be used to maintain the observation target in the observation process. The animal cell culture device may be equipped with, for example, an incubator, humidity, temperature, and gas control units, etc.

[0035] <1-2. Contact process> In the first embodiment, it is preferable to provide a contacting step before the observing step. In the contacting step in the first embodiment, it is preferable to contact the sebaceous gland cells with the test substance under the growth medium culture conditions described above (preferably under the spheroid formation conditions described below). By including this contacting step, the holocytosis-regulating effect of the test substance on the sebaceous gland cells can be better observed by fluorescence in the next observation step, thereby making it easier to determine the holocytosis-regulating effect of the test substance. The contact period in the contact step is not particularly limited and can be adjusted appropriately taking into consideration the rate at which the test substance exerts its holocytosis-regulating effect on sebaceous gland cells. Possible causes of the rate at which the test substance exerts its holocytosis-regulating effect are, for example, but not limited to, the speed of the test substance's effect (fast acting or slow acting) and the solubility of the test substance (easily soluble or poorly soluble).

[0036] It is preferable to provide the contact period before adding the fluorescent reagent, from the viewpoint of making it easier to obtain accurate fluorescence observation results. The contact period in the contacting step is preferably at least 2 hours after contact with the test substance, with the preferred lower limit being more preferably 4 hours or more, even more preferably 6 hours or more, more preferably 8 hours or more, and more preferably 12 hours or more, and the preferred upper limit being not particularly limited, but examples include 48 hours or less, 36 hours or less, and 24 hours or less, etc. The preferred contact period is preferably 6 to 24 hours.

[0037] It is preferable to add a fluorescent reagent after the contact step and perform fluorescent observation of the sebaceous gland cells. However, it is also preferable to observe the sebaceous gland cells in a state where they are in contact with the test substance during this fluorescent observation period, from the viewpoint of being able to better observe the state of the total secretion regulating effect of the test substance, and in this case, fluorescent observation under total secretion regulating conditions is more preferable.

[0038] Furthermore, this first embodiment (preferably from the contacting step onward) is preferably performed in a container for a fluorescence microscope, and the container is preferably made of, for example, glass, plastic, or a combination thereof, more specifically, but not limited to, a glass-bottom dish. When sebaceous gland cells are contained in the container, it is preferable to appropriately employ the above-mentioned <Sebaceous Gland Cell Culturing Step> to set the inside of the container to conditions (e.g., CO2 concentration, temperature) that allow the sebaceous gland cells to survive.

[0039] In the first embodiment, a pretreatment step such as washing with the growth medium described above may be carried out before the contact step.

[0040] <1-3. Observation process> The observation step in the first embodiment will be described below. In the observation step of this first embodiment, it is preferable to place one or more sebaceous gland cells and a test substance in the presence of the test substance, and then observe the state of the fluorescent sebaceous gland cells. This allows fluorescent observation of the state of the holocytosis-regulating effect of the test substance on the sebaceous gland cells, and the state of the sebaceous gland cells at this time can also be obtained as an observation result (preferably an observation image). For example, the test substance can be evaluated and / or selected as a holocytosis-regulating agent or a candidate holocytosis-regulating agent, taking into account the difference from a control. The culture conditions for the observation step are not particularly limited, and can be appropriately selected, for example, under the culture conditions described in <1-1. Sebaceous gland cell culture step> above. The medium used here is not particularly limited, and any known or commercially available medium may be used, and the medium can be appropriately selected taking into consideration the state of the luminescent sebaceous gland cells during fluorescence observation. A more preferred embodiment of the observation step is to carry out the observation step under conditions for holocytosis regulation, and even more preferred is to place one or more sebaceous gland cells and a test substance in the presence of the test substance under conditions for holocytosis regulation, and then fluorescently observe the state of the fluorescing sebaceous gland cells.

[0041] <1-3-1. Conditions for regulating total secretion> In the first embodiment, the conditions for regulating holocytosis are preferably conditions that can regulate the induction of holocytosis and the inhibition of holocytosis in sebaceous gland cells (preferably mature sebaceous gland cells). Conditions for holocytosis regulation include, but are not limited to, one or a combination of two or more selected from the following: conditions for adding drugs such as physiologically active agents (stimulators or inhibitors); culture medium conditions (e.g., culture medium composition, temperature, etc.); and the state of sebaceous gland cells (e.g., a three-dimensional structure having mature sebaceous gland cells in the surface layer). Among these, using culture medium conditions is preferable because it allows for objectively accurate and highly reproducible observation results to be obtained. Furthermore, using the state of sebaceous gland cells is preferable because it facilitates fluorescent observation of the holocrine secretion regulatory activity. Even more preferable is a combination of culture medium conditions and the state of sebaceous gland cells.

[0042] In this first embodiment, the holocrine secretion regulating conditions are preferably holocrine secretion inducing conditions and / or holocrine secretion inhibiting conditions. In this first embodiment, by applying holocrine secretion inducing conditions, mature sebaceous gland cells die, and at this time, the cells themselves collapse and secretions containing sebum, which are the contents of the sebaceous gland cells, are released outside the sebaceous gland cells. In this first embodiment, by applying holocrine secretion inhibiting conditions, the sebaceous gland cells are maintained without collapse, and the contents of the sebaceous gland cells, such as sebum, are not released outside the sebaceous gland cells.

[0043] <1-3-1-1. Conditions for Regulating Autophagy> More specifically, autophagy-regulating conditions are preferred as holocrine secretion-regulating conditions in this first embodiment. In this embodiment, the autophagy-regulating conditions are preferably autophagy-inducing conditions and / or autophagy-suppressing conditions. By controlling the induction and / or suppression of autophagy, it becomes possible to easily regulate the total secretion of sebaceous gland cells, which is advantageous in that it is easy to work with and, further, its use allows for high accuracy and reproducibility in the evaluation and / or selection of total secretion regulators.

[0044] Autophagy is generally understood to be a survival system in which, when cells are in a state of starvation, they break down their own intracellular organelles and proteins to extract and reuse nutrients. However, the present inventors have newly discovered that when autophagy is induced in mature sebaceous gland cells, they are able to produce normal, full-body secretion.

[0045] More specifically, the present inventors have newly discovered that inducing autophagy in mature sebaceous cells causes the cell membrane of the mature sebaceous cells to break down and the internal components of the sebaceous cells to be secreted extracellularly (see Figures 5, 6, and 7). Thus, inducing autophagy in mature sebaceous cells can induce normal total secretion in sebaceous cells, thereby enabling normal total secretion in sebaceous cells.

[0046] Furthermore, the inventors newly discovered that inhibiting autophagy in mature sebaceous gland cells prevents the cell membranes of mature sebaceous gland cells from collapsing (see Figure 5), and that the resulting clumps become clogged in the ducts of pores, causing clogged pores (see Figures 1 and 2). Thus, inhibition of autophagy in mature sebocytes can suppress holocrine secretion, thereby rendering sebocytes holocrine dystrophic.

[0047] <1-3-1-2. Culture medium conditions for total secretion control> In the first embodiment, it is preferable to apply culture medium conditions to sebaceous gland cells as the holocytosis regulation conditions. Under these culture medium conditions, the state of holocytosis regulation of sebaceous gland cells can be observed.

[0048] The present inventors have newly discovered that the total secretion of sebaceous cells can be easily regulated by adjusting the medium composition conditions for sebaceous cells. By adopting this technique of adjusting the medium conditions for sebaceous cells, even non-experts can easily regulate the total secretion of sebaceous cells. This technique has the advantages of being highly operable and of being highly accurate and reproducible in the evaluation and / or selection of total secretion regulators.

[0049] The medium conditions are not particularly limited, but include, for example, medium composition, medium pH, CO2 concentration in the container, temperature, and period, and one or more selected from these can be used. Among the above culture conditions, it is preferable to adjust the culture medium composition, which allows for easy regulation of total secretion from sebaceous gland cells, and has the advantage of being highly workable and providing high accuracy and reproducibility in the evaluation and / or selection of total secretion regulators. The medium composition used under the medium conditions of this embodiment may be a medium composition for growth (specifically, proliferation, maintenance, differentiation, etc.) or starvation, and may contain one or more additives selected from additives for proliferation, antibiotics, serum, growth factors, etc., as appropriate, within the range that does not impair the effects of the present invention.

[0050] In this first embodiment, the medium conditions other than the adjustment of the medium composition can be those typically used for fluorescent observation of cultured animal cells. The observation step is preferably performed under conditions that allow observation while keeping sebaceous gland cells alive. For example, typical in vitro fluorescent observation conditions for human sebaceous gland cells can be employed, and the culture conditions described in <1-1. Culturing step> above may also be applied. For example, the pH of the medium is preferably about a culture pH (7-8), the CO2 concentration in the container is preferably a normal atmospheric concentration (0.04%) or a culture concentration (4-10% in air), and the temperature is preferably about room temperature (10-30°C) or a culture temperature of 30-40°C. Examples of conditions that allow sebaceous gland cells to survive include an animal cell culture device (preferably an incubator), a container for a fluorescence microscope, or a chamber.

[0051] The medium composition is preferably a medium for inhibiting holocrine secretion and / or a medium for inducing holocrine secretion. This first embodiment uses a medium for inhibiting total secretion, thereby suppressing or inhibiting total secretion in mature sebaceous gland cells, and can therefore be used as a model for inducing clogged pore formation. By adding a test substance to this model for inducing clogged pore formation, evaluation or selection of the test substance as a total secretion promoter can be performed easily and accurately. Then, under total secretion-suppressing conditions using the medium for inhibiting total secretion, the total secretion-promoting effect of the test substance on sebaceous gland cells can be observed by fluorescence. This observation allows for the determination of whether the test substance is suitable for preventing or improving clogged pores. This first embodiment uses a medium for inducing holocrine secretion, thereby inducing or promoting holocrine secretion in mature sebaceous gland cells, and can therefore be used as a model for inhibiting clogged pore formation. By adding a test substance to this model for inhibiting clogged pore formation, the test substance can be easily and accurately evaluated or selected as a holocrine secretion inhibitor. Then, under holocrine secretion-inducing conditions using this holocrine secretion-inducing medium, the holocrine secretion inhibitory effect of the test substance on sebaceous gland cells can be observed. This observation allows for the determination of whether the test substance is involved in promoting clogged pore formation, etc.

[0052] <Culture medium for total secretion inhibition> As the medium for inhibiting total secretion used under the conditions for inhibiting total secretion, it is preferable to use a growth medium for animal cell culture generally used for growth, maintenance, differentiation, etc. Examples of such growth medium for animal cell culture include, but are not limited to, a basal medium, a serum-reduced medium, a serum-free medium, etc. By using a general growth medium, total secretion of mature sebaceous gland cells can be inhibited. Generally, basal media are made by adding serum to the basal media components, including amino acids, vitamins, inorganic salts (e.g., calcium), and a carbon source such as glucose. Serum-free media are made by adding the basal media components, but replacing the serum with appropriate nutrients and hormones.

[0053] The medium for inhibiting total secretion used in this first embodiment is preferably a basal medium supplemented with serum or a serum-like component, or may be an autophagy-inhibiting medium. The medium for inhibiting total secretion is preferably not an amino acid-free medium that would cause nutrient starvation, and more preferably a growth medium whose basal medium composition contains amino acids to prevent nutrient starvation. The use of such a general growth medium can inhibit total secretion from mature sebaceous gland cells.

[0054] As the total secretion-inhibiting medium to be used in this first embodiment, a growth medium normally used for culturing sebaceous gland cells is more preferable, and more specifically, examples of basal media include William's E Medium, D-MEM, RPMI1640, Ham's F-12, Modified D-MEM / Ham's F-12 (1:1) (e.g., Sebomed™ basal medium), EpiLife medium series (Thermo Fisher Scientific), HuMedia medium series (Kurabo), and CnT-Prime medium series (CELLnTEC). Furthermore, it is preferable to use a basal medium to which one or more of serum, EGF, hydrocortisone, insulin, etc. have been added. In the basal medium, for example, serum is preferably 0.1% to 20%, more preferably 10%, EGF (Epidermal Growth Factor) is preferably 1 to 10 ng / mL, more preferably 3 to 6 ng / mL, hydrocortisone is preferably 0.1 to 20 μg / mL, more preferably 5 to 15 μg / mL, and insulin is preferably 5 to 50 μg / mL, more preferably 8 to 20 μg / mL.

[0055] Furthermore, the culture medium for inhibiting total secretion used in this first embodiment is preferably a differentiation medium capable of differentiating immature sebaceous gland cells, and more specifically, a differentiation medium that does not contain or has reduced amounts of differentiation inhibitors (e.g., EGF, BPE, etc.) is preferred. Examples of such differentiation media include, but are not limited to, William's E Medium, D-MEM, RPMI-1640, Ham's F-12, Modified D-MEM / Ham's F-12 (1:1) (e.g., Sebomed™ basal medium), EpiLife medium series (Thermo Fisher Scientific), HuMedia medium series (Kurabo Industries, Ltd.), and CnT-Prime medium series (CELLnTEC).

[0056] <Total secretion induction medium> The medium for inducing total secretion used in this first embodiment is preferably a medium obtained by removing some or all of the amino acids from the basal medium components of the above-mentioned "growth medium for animal cell culture," and more preferably, from the viewpoint of inducing total secretion from mature sebaceous cells, an amino acid-free medium, and even more preferably, a serum-free, serum-free, amino acid-free medium to which no serum has been added. Furthermore, a starvation medium that puts animal cells into a nutrient-starved state is more preferable, and examples thereof include a medium for inducing autophagy. By using a nutrient-starvation medium, total secretion from mature sebaceous cells can be induced. Among the mediums for animal cell culture, those for sebaceous cell culture are more preferable from the viewpoint of inducing total secretion from mature sebaceous cells.

[0057] Suitable media for inducing complete secretion include amino acid-free D-MEM media obtained by removing one or more or all of the amino acids contained in standard D-MEM media, and serum-free D-MEM media that is completely free of amino acids is more suitable from the viewpoint of achieving a more effective state of nutrient starvation. Standard D-MEM media contains the amino acids L-arginine hydrochloride, L-cystine, L-glutamine, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0058] Examples of autophagy induction media, which are examples of holocrine secretion induction media, include, but are not limited to, serum-free D-MEM (Dulbecco's modified Eagle's medium), serum-free amino acid-free D-MEM (high glucose (4500 mg / L)) (containing sodium pyruvate (110 mg / L) and amino acid-free) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and Hank's Balanced Salt Solution (serum-free). Of these, serum-free amino acid-free D-MEM is preferred.

[0059] <1-3-1-3. Conditions for adding drugs to regulate total secretion> In the observation step in the first embodiment, an additive agent may be appropriately used to cause one or more sebaceous gland cells and a test substance to be present, and the state of the sebaceous gland cells that have been made fluorescent may be observed by fluorescence. The conditions for adding a drug to regulate holocrine secretion used in this first embodiment include, for example, conditions for adding a holocrine secretion promoter and / or a holocrine secretion inhibitor. Furthermore, by combining the medium conditions using the above-mentioned "growth medium for animal cell culture" with conditions for adding a holocrine promoter, holocrine secretion-inducing conditions can be achieved. Examples of the additive used in the holocrine secretion-promoting conditions include an autophagy promoter. Furthermore, by combining the medium conditions using the above-mentioned "growth medium for animal cell culture" with conditions for adding a holocytosis inhibitor, holocytosis-suppressing conditions can be achieved. Examples of the additive drug used in the holocytosis-inhibiting conditions include autophagy inhibitors. Among animal cell cultures, those for sebaceous gland cell culture are more suitable. The amount of the added drug can be determined by referring to literature on conventional autophagy inhibition or promotion experiments.

[0060] Generally, autophagy begins with (1) a small vesicle called an autophagosome encapsulating an item to be degraded within the cell, followed by (2) fusing with a vesicle called a lysosome, which is filled with degradative enzymes, and (3) the item to be degraded is broken down inside the autolysosome, which is formed by the fusion of the two vesicles mentioned above.

[0061] As a drug that inhibits the process before (1), for example, MHY-1485 (C 17 H 21 These include, but are not limited to, N7O4 (CAS No: 326914-06-1), amino acids, etc. MHY-1485 and amino acids inhibit the step that turns on autophagy itself (before step (1) above), and this inhibition is caused by activation of a protein called mTORC1, which negatively regulates autophagy. Drugs that inhibit the initial stage of (1) above include, but are not limited to, 3-methyladenine (3-MA). Drugs that inhibit the above (2) (inhibition of fusion between autophagosomes and lysosomes) include, but are not limited to, bafilomycins such as bafilomycin A1, chloroquine, and the like. Examples of inhibitors of degradative enzymes in (3) above include, but are not limited to, pepstatin A. Among these, an mTORC1 activator (preferably MHY-1485) and / or an autophagosome-lysosome fusion inhibitor (preferably bafilomycins) are preferred. One or more autophagy inhibitors selected from the above-mentioned autophagy inhibitors can be used.

[0062] By adopting general culture conditions using a growth medium and conditions for adding drugs for holocytosis regulation, holocytosis of sebaceous gland cells can be easily regulated and desired holocytosis regulation conditions can be obtained. Under these holocytosis regulation conditions, the holocytosis regulation effect of a test substance on sebaceous gland cells can be observed by fluorescence. By using these holocytosis regulation conditions, observation results can be obtained that are objectively accurate and highly reproducible.

[0063] <Sebaceous gland cells> The sebaceous gland cells used in this first embodiment may be sebaceous gland cells prepared in advance in the above-mentioned "sebaceous gland cell culture process" and stored frozen and used as needed, or the process of this embodiment may include a sebaceous gland cell culture process. The sebaceous gland cells used in this first embodiment are preferably mature sebaceous gland cells in a state capable of full secretion. The sebaceous gland cells used in this first embodiment are preferably a three-dimensional structure obtained by three-dimensional culture rather than a plurality of cells obtained by two-dimensional culture. The three-dimensional structure is preferably a three-dimensional structure formed by the aggregation of a plurality of cells, and examples thereof include, but are not limited to, a cell group, a cell mass, and a three-dimensional structure (more preferably, a spheroid).

[0064] Examples of the three-dimensional structure of sebaceous gland cells include, but are not limited to, laminated sheets, tubular, rod-like, and spheroidal structures, and one or more of these may be selected. Three-dimensional structures of sebaceous gland cells can be formed by the aggregation and coagulation of multiple sebaceous gland cells. Generally, a spheroid is a spherical cell aggregate formed by the aggregation and coagulation of cells.

[0065] Furthermore, the sebaceous gland cells used in this first embodiment are even more preferably a three-dimensional structure of sebaceous gland cells in which the surface layer is composed of mature sebaceous gland cells (see Figures 3 and 4). From the perspective of using this three-dimensional structure to observe holocrine secretion, it is more preferable that the three-dimensional structure has a configuration in which the mature sebaceous gland cells present in the surface layer become the surface layer after they disappear due to holocrine secretion. It is even more preferable to use the three-dimensional structure of sebaceous gland cells related to this embodiment, which will be described in "2." below.

[0066] <Test substance> The test substance used in the first embodiment and the like is not particularly limited and may be either naturally or artificially derived, and may be any of a single substance, a mixture, or a composition containing a single substance or a mixture. The composition may be, for example, a preparation, a cosmetic, a topical skin preparation, a pharmaceutical composition, or a food or beverage composition. The concentration of the test substance may be adjusted as appropriate. For example, aqueous solutions containing different concentrations of the test substance may be prepared and used to observe concentration-dependent effects.

[0067] The test substance is preferably one or more selected from compounds, microorganisms or their cultures, extracts, mixtures thereof, compositions, etc. The compound may be either an inorganic compound or an organic compound.

[0068] Examples of inorganic compounds include, but are not limited to, compounds of metal elements (main group elements, transition elements) or non-metal elements (boron, silicon, etc.).

[0069] Organic compounds are compounds containing carbon, and include, but are not limited to, lipid compounds, sugar compounds, peptide compounds, nucleic acid compounds, alkaloid compounds, steroid compounds (terpene compounds), enzymes, agonists, antagonists, hormones, polymer compounds, and antibiotics.

[0070] Examples of microorganisms include, but are not limited to, bacteria (e.g., eubacteria, archaea, etc.), fungi (e.g., molds, mushrooms, yeasts, etc.), microalgae, viruses, etc., and decomposition products thereof. Examples of cultures include, but are not limited to, cultures of microorganisms, and may be fermented products of beneficial bacteria such as lactic acid bacteria, bifidobacteria, and yeast. Extracts include, but are not limited to, those extracted from natural sources (for example, animals, plants, microorganisms, etc.), organic compounds, inorganic compounds, cultures, fermentation products, minerals, and the like.

[0071] <Observation> In this first embodiment, it is preferable to perform fluorescent observation of the state of fluorescing sebaceous gland cells in the presence of a test substance after the contact step. This makes it possible to easily perform fluorescent observation of the total secretion state of sebaceous gland cells in response to the test substance. More preferably, fluorescent observation is performed under total secretion-regulating conditions after the contact step. As the state of the fluorescent sebaceous gland cells, normal or abnormal total secretion can be detected from the change in fluorescence emission of the sebaceous gland cells over time. The state of the fluorescent sebaceous gland cells is preferably determined based on the number of deflorescence-loss cells per unit volume, where one deflorescence-loss cell per unit volume is counted when the fluorescence of one sebaceous gland cell disappears within a specified region and a specified observation period.

[0072] When holocytosis of sebaceous glands occurs normally, the number of cells that lose fluorescence over a certain period of observation increases. Normally, when a single living sebaceous gland cell disintegrates, the fluorescence stored inside the cell is released outside the cell, causing the fluorescence to disappear. In particular, when holocytosis occurs normally in sebaceous gland cells present in the surface layer of the three-dimensional structure, the fluorescence of these sebaceous gland cells disappears in the surface layer within a short period of time. After that, the fluorescent sebaceous gland cells present underneath move to the surface layer, so the surface layer of the three-dimensional structure appears to be constantly fluorescent. In this way, the overall secretion-stimulating effect of the test substance can be determined with high accuracy based on the change in fluorescence emission of each sebaceous gland cell in the observed image.

[0073] When the total secretion of sebaceous glands is abnormal, the number of cells that lose their fluorescence during a certain period of observation decreases. In particular, when the total secretion of sebaceous glands in the surface layer of the three-dimensional structure is abnormal, the fluorescence of these sebaceous glands remains undiminished in the surface layer. In this way, based on the change in the fluorescence emission of each sebaceous gland cell in the observed image, the total secretion inhibitory effect of the test substance can be determined with high accuracy.

[0074] <Fluorescent reagent addition process> In the first embodiment, it is preferable to add a fluorescent reagent to the sebaceous gland cells and the test substance after the contacting step. In this case, it is preferable to make the living cells fluoresce, since this makes it easier to determine the holocrine state (normal or abnormal) of the sebaceous gland cells. When adding the fluorescent reagent to the sebaceous gland cells and the test substance, it is more preferable to do so under holocrine secretion-regulating conditions after the contacting step.

[0075] The fluorescent reagent is a fluorescent reagent containing a fluorescent dye that causes living cells to fluoresce and loses its fluorescence when the cells die. For the fluorescence observation in this embodiment, a fluorescent reagent containing a fluorescent dye that can at least cause living sebaceous gland cells to fluoresce is more suitable. By using such a fluorescent reagent, the number of cells that lose fluorescence per unit volume can be determined over time, and further, changes in the number of sebaceous gland cells over time can be detected based on the number of cells that lose fluorescence per unit volume. As the fluorescent reagent, for example, a commercially available Live / Dead™ kit can be used, and examples of such kits include, but are not limited to, the Live / Dead™ Cell Imaging Kit (488 / 570) (Thermo Fisher Scientific). The state of fluorescent live sebaceous gland cells can be observed by following the procedures described in the instruction manual attached to such a commercially available kit. For example, when observing live sebaceous gland cells using green fluorescence, the wavelength of the excitation light is preferably 480 to 490 nm, and the wavelength of the detection light at this time is preferably 490 to 580 nm.

[0076] <Fluorescence microscope observation process> The observation in the first embodiment is preferably performed using a fluorescence microscope. The fluorescence microscope can use, as a light source, one or more light sources selected from, for example, a laser, an ultra-high pressure mercury lamp, a xenon lamp, and an LED (e.g., ultraviolet LED). Examples of the fluorescence microscope include simple fluorescence microscopes that do not use a laser as a light source, laser scanning microscopes, confocal laser microscopes such as confocal laser scanning microscopes, multiphoton excitation microscopes such as two-photon laser scanning excitation microscopes, and light sheet fluorescence microscopes. One or more of these can be used. Generally, simple fluorescence microscopes primarily use one or more light sources selected from, for example, an ultra-high pressure mercury lamp, a xenon lamp, and an LED capable of irradiating ultraviolet light, and are capable of observing a sample as a two-dimensional structure. Among these, laser scanning microscopes, confocal laser microscopes, multiphoton excitation microscopes, and light sheet fluorescence microscopes are preferred because they allow the sample to be observed as a three-dimensional structure, with confocal laser microscopes being more preferred. Furthermore, in the fluorescence observation of this first embodiment, it is more preferable to perform image analysis of the observed sebaceous gland cells using image analysis software (more preferably high-resolution 3D / 4D image analysis software). The observation step of this embodiment can use one or more combinations of known or commercially available devices equipped with an observation unit configured for fluorescence microscopy, an imaging unit configured for imaging the object under observation, an image analysis unit configured for analyzing the captured observation image, and the like. Examples of known or commercially available devices include, but are not limited to, a confocal laser scanning microscope (e.g., Carl Zeiss LSM800) and an image analysis device (e.g., a computer) that stores and executes image analysis software (preferably, high-resolution 3D / 4D image analysis software (e.g., Imaris)). Generally, 3D / 4D image analysis software enables multidimensional visualization of image data (e.g., three-dimensional image construction) and analysis of 3D / 4D images (e.g., quantitative analysis).

[0077] In the observation of the first embodiment, it is preferable to capture an image (moving image and / or still image) of the observation target irradiated with irradiation light. By irradiating the irradiating light (excitation light) onto the sebaceous gland cells of the observation target, it is possible to detect fluorescence emitted from the sebaceous gland cells, and it is preferable that the luminescent sebaceous gland cells are living cells. It is preferable that the light be irradiated from the irradiating unit only when imaging and not irradiated at other times in order to reduce damage to the object of observation (sebaceous gland cells).It is preferable that the observed image be stored in an internal or external storage unit.

[0078] Furthermore, it is preferable to set a "certain region" within the entire observation target as a "field of view," and observe the number of cells that have lost fluorescence within that field of view for a predetermined period of time. The field of view can be set at any location, and the number of fields of view may be single or multiple (e.g., 1 to 5), with three being preferable. The range of one field of view is preferably set to 50 to 70 μm × 100 to 140 μm × 25 to 70 μm. The predetermined period is preferably the observation time from the addition of the fluorescent reagent (starting point), and is not particularly limited, but is preferably 8 to 20 hours, more preferably 10 to 15 hours, and even more preferably 11 to 13 hours. It is preferable to start imaging of the sebaceous gland cells when observation of the sebaceous gland cells begins. It is preferable to calculate the number of fluorescence-quenching cells per unit volume in one visual field, and it is more preferable to calculate the average number of fluorescence-quenching cells per unit volume in multiple visual fields.

[0079] When the object to be observed is a three-dimensional structure, it is preferable to capture images by changing the focal plane from the surface of the three-dimensional structure toward the center in increments of 0.5 to 2 μm (preferably 1 μm) within a range of 100 μm from the surface in the depth direction (toward the center). By three-dimensionally reconstructing the images captured at each focal plane, the three-dimensional structure can be represented and evaluated in three dimensions.

[0080] The observation in this first embodiment is preferably performed using time-lapse imaging, which allows a large number of observation images (preferably still images) to be acquired at predetermined intervals in chronological order to obtain time-lapse images.The state of sebaceous gland cells can be observed based on the time-lapse images, and the effect of the test substance on sebaceous gland cells in regulating total secretion can be determined.Based on the time-lapse images, the number of cells that have lost fluorescence within a predetermined period and a predetermined field of view can be preferably counted. Generally, "time lapse images," also known as slow-speed or time-lapse imaging, are an imaging method that creates the appearance of frame-by-frame images by connecting and playing back images captured one frame at a time over several seconds (or several minutes). It is also possible to capture a video in advance and then capture a large number of still images from this video at predetermined intervals to create image data similar to time lapse images.

[0081] In the first embodiment, the conditions for capturing time-lapse images can be set as appropriate. For example, time-lapse images can be acquired by capturing and storing observation images in chronological order, each of which includes fluorescence emitted from the observation target in response to the irradiated light. It is preferable to irradiate the observation target with irradiated light (excitation light) from a light source when capturing the time-lapse images or immediately before capturing the images. This reduces damage to the observation target and enables fluorescence observation over a long period of time.

[0082] Specifically, if the imaging interval is short, the amount of damage to the sebaceous gland cells by the irradiated light will be large, and there is a risk that the fluorescence of the sebaceous gland cells will be lost, while if the imaging interval is long, there is a risk that important images of the total secretion-regulating effect of the test substance on the sebaceous gland cells will be missed. Therefore, the imaging interval (in minutes) for each frame under imaging conditions is preferably one frame every 3 to 20 minutes, more preferably one frame every 5 to 15 minutes, even more preferably one frame every 8 to 12 minutes, and even more preferably one frame every 9 to 11 minutes (and even more preferably one frame every 10 minutes). This reduces damage to sebaceous gland cells and enables accurate long-term observation of the total secretion state of sebaceous gland cells.

[0083] The imaging time under the imaging conditions is preferably 8 to 20 hours, more preferably 10 to 15 hours, and even more preferably 11 to 13 hours. The starting point of the imaging time is not particularly limited, but is preferably after the addition of the fluorescent reagent. Furthermore, more preferred imaging conditions are an imaging interval of preferably one frame every 9 to 11 minutes (more preferably every 10 minutes), and an imaging time of preferably 11 to 13 hours (more preferably 60 to 90 frames, even more preferably 66 to 78 frames).

[0084] In this first embodiment, it is more preferable to use a fluorescence detection device configured to irradiate an observation object with irradiation light (excitation light), detect fluorescence emitted from the observation object, and perform fluorescence observation of the observation object. The fluorescence detection device preferably includes a microscope, and further includes a light source (e.g., a laser, an LED, etc.) configured to irradiate the observation object (preferably, fluorescent sebaceous gland cells) with irradiation light (excitation light), a detection unit (e.g., an image sensor, etc.) configured to receive and detect fluorescence from the observation object, and an imaging unit configured to capture an image of the observation object. The excitation light and detection light can be set appropriately depending on the fluorescent reagent used.

[0085] Examples of fluorescence detection devices include fluorescence microscopes, confocal laser microscopes, multiphoton excitation microscopes, and light sheet microscopes, but are not limited to these as long as they are capable of fluorescent observation of an object. Among these, one or more selected from confocal laser microscopes, multiphoton excitation microscopes, and light sheet microscopes are preferred because they allow three-dimensional observation of the object, with confocal laser microscopes being even more preferred. For example, a confocal laser microscope irradiates a laser from a light source, focuses the laser on the object (sebaceous gland cells) with an objective lens, and acquires the fluorescence emitted from the object as detection light. Furthermore, for example, a light sheet microscope irradiates a sheet of excitation light from a light source only onto the focal plane of the object, and acquires the fluorescence emitted from the object as detection light, thereby enabling the acquisition of an optical section image.

[0086] A fluorescence detection device can capture images of an object to be observed using an image sensor such as a CCD or CMOS, and monitor the state of the object over time. The fluorescence detection device can perform imaging processing such as analog-to-digital (AD) conversion of analog image signals or imaging signal processing. The images output by the fluorescence detection device for observation can be either color images or black-and-white images, although color images are preferred. Color images can be represented, for example, by RGB (red, green, blue), and black-and-white images can be represented, for example, by brightness.

[0087] Preferably, the fluorescence detection device further includes a time-lapse imaging device configured to perform time-lapse imaging, or an external time-lapse imaging device is provided so as to be capable of transmitting and receiving data to and from the outside. The time-lapse imaging device is preferably configured to be capable of performing imaging in accordance with the above-described time-lapse image capturing conditions. The fluorescence detection device may also be provided, externally or internally, with an image analysis unit or device configured to perform image analysis of the captured observation image.

[0088] <1-4. Discrimination process> The determination step in the first embodiment will be described below. In the discrimination step of the first embodiment, it is preferable to discriminate the test substance as a pan-secretion regulator or a candidate pan-secretion regulator based on the state of the fluorescently-activated sebaceous gland cells.

[0089] In the first embodiment, the state of fluorescent sebaceous gland cells can be determined based on the number of cells that have lost their fluorescence per unit volume. By using the number of cells that have lost their fluorescence per unit volume as an index, the overall secretion-regulating effect of a test substance on sebaceous gland cells can be objectively and simply determined. More specifically, if the number of cells that have lost fluorescence is large, the test substance can be determined to have a stronger holocrine secretion-promoting effect, and conversely, if the number of cells that have lost fluorescence is small, the test substance can be determined to have a stronger holocrine secretion-inhibiting effect. In a more preferred embodiment, the discrimination is based on the number of fluorescence-quenched cells under holocytosis-regulating conditions. Even more preferably, if the number of fluorescence-quenched cells is large under holocytosis-inhibiting conditions, the test substance can be determined to have a stronger holocytosis-promoting effect, whereas if the number of fluorescence-quenched cells is small under holocytosis-inducing conditions, the test substance can be determined to have a stronger holocytosis-inhibiting effect.

[0090] In this first embodiment, methods for evaluating the regulatory effect of a test substance on total secretion include, but are not limited to, the following methods (1) to (3).

[0091] For example, (1) a positive control (indicator substance) for holocytosis regulation can be used as an indicator. More specifically, when the number of fluorescence-quenching cells per unit volume of the test substance is comparable to that of the positive control, the test substance is evaluated as a holocytosis regulator and / or selected as a substance with holocytosis regulation activity. The standard for "equivalent" is, for example, 100%±30%, preferably 100%±20%, and more preferably 100%±10%, of the number of fluorescence-quenching cells per unit volume of the positive control, where the number of fluorescence-quenching cells per unit volume of the test substance is taken as 100%. For example, if the number of fluorescent cells quenched in culture using starvation medium as the positive control is 100%±30%, the test substance can be identified as a holocytosis promoter or candidate holocytosis promoter and selected as a substance with holocytosis regulation activity. Furthermore, for example, if the number of fluorescent cells lost in culture using growth medium as a positive control is 100% ± 30%, the test substance can be identified as a global secretion inhibitor or a candidate global secretion inhibitor and selected as a substance with global secretion inhibitory activity. It is preferable that the observation period for both comparisons be the same.

[0092] More specifically, when a test substance exhibits a cell count of fluorescence loss equivalent to or greater than that of a positive control that promotes holocytosis, the test substance can be evaluated or selected as a holocytosis promoter. For example, the "cell count of fluorescence loss per unit volume in the positive control that promotes holocytosis" can be set to the "cell count of fluorescence loss per unit volume in a starvation medium without the addition of the test substance." It is preferable to use the same time-lapse imaging period for both comparisons. When evaluating or selecting a test substance as a holocytosis promoter, it is preferable to perform the imaging under holocytosis-inhibiting conditions. For example, if the number of fluorescence-quenched cells of a test substance is comparable to or less than that of a positive control that inhibits pan-secretion, the test substance can be evaluated or selected as a pan-secretion inhibitor. For example, the "number of fluorescence-quenched cells per unit volume of the positive control that inhibits pan-secretion" can be set to the "number of fluorescence-quenched cells per unit volume using growth medium without the addition of the test substance." It is preferable to use the same time-lapse imaging period for both comparisons. When evaluating or selecting a test substance as a pan-secretion inhibitor, it is preferable to perform the imaging under pan-secretion-inducing conditions.

[0093] For example, (2) the test substance exhibits a concentration-dependent increase in the holocytosis-regulating activity. More specifically, the above observation step is performed using a plurality of different concentrations of the test substance, the number of cells that have lost fluorescence per unit volume for each concentration is obtained, and the correlation coefficient between these numbers of cells that have lost fluorescence and the concentration is calculated to determine the degree of correlation. In this case, if the correlation coefficient is preferably 0.4 or higher, the test substance is determined to have a concentration-dependent increase in effect, and the test substance is evaluated as a holocytosis-regulating agent and / or selected as a substance having a holocytosis-regulating activity. If the correlation coefficient is 0.7 or higher, it is determined to have a stronger correlation, and it can be determined that the test substance is more likely to have a stronger holocytosis-regulating activity. It is preferable that the observation periods for these experiments be the same.

[0094] More specifically, for example, if the correlation coefficient between the concentration of the test substance and the number of cells that have lost fluorescence is 0.4 or higher and shows a positive slope indicating a holocytosis-promoting effect, the test substance is determined to have a holocytosis-promoting effect, and the test substance can be evaluated or selected as a holocytosis-promoting agent. When evaluating or selecting a test substance as a holocytosis-promoting agent, it is preferable to perform the evaluation or selection under holocytosis-inhibiting conditions. Furthermore, for example, if the correlation between the concentration of the test substance and the number of cells that have lost fluorescence is 0.4 or higher and shows a negative slope indicating a pan-secretion inhibitory effect, the test substance is determined to have a pan-secretion inhibitory effect, and the test substance can be evaluated or selected as a pan-secretion inhibitor. When evaluating or selecting a test substance as a pan-secretion inhibitor, it is preferable to carry out the evaluation or selection under pan-secretion-inducing conditions.

[0095] For example, (3) determining the ranking of multiple test substances. More specifically, based on the observation process for multiple test substances, the number of cells that have lost fluorescence per unit volume for each test substance can be obtained, and based on this number of cells that have lost fluorescence, the ranking of the strength of the overall secretion regulating effect of the test substances can be determined. Furthermore, the ranking may be determined in order of the value closest to the positive control in (1) above, or, preferably, in order of the highest correlation coefficient in (2) above. It is preferable that these observation periods are the same.

[0096] More specifically, as an example, a plurality of test substances are used, and the observation step is performed for each test substance. Based on the number of cells that have lost fluorescence, the test substances are ranked in descending order of the number of cells that have lost fluorescence. Test substances with a stronger holocrine secretion-promoting effect can be evaluated or selected as holocrine secretion promoters or candidates thereof. When a test substance is evaluated or selected as a holocrine secretion promoter, it is preferable to perform the evaluation or selection under holocrine secretion-inhibiting conditions. As an example, multiple test substances are used, and the observation step is performed for each test substance. Based on the number of cells that have lost fluorescence, the test substances are ranked in descending order of the number of cells that have lost fluorescence. Test substances with the strongest holocrine inhibitory effect can be evaluated or selected as holocrine inhibitors or candidates for holocrine inhibitors. When evaluating or selecting a test substance as a holocrine inhibitor, it is preferable to perform the evaluation or selection under holocrine-inducing conditions. Based on the results of this evaluation or selection, the accuracy of the results may be improved by combining the above methods (1) and (2).Furthermore, based on the results of this evaluation or selection, the compounds may be subjected to in vivo testing, animal evaluation testing on humans, etc., in descending order of their ranking.

[0097] <Evaluation and / or selection of total secretion regulators> This first embodiment makes it possible to easily evaluate whether a test substance is suitable as a holocrine regulator and / or to select a test substance as a holocrine regulator (specifically, a substance having a holocrine regulator effect). The holocrine secretion regulator is not particularly limited, but examples thereof include holocrine secretion regulators of sebaceous gland cells (more preferably holocrine secretion promoters and holocrine secretion inhibitors), clogged pore formation regulators (more preferably clogged pore formation promoters and clogged pore inhibitors), and autophagy regulators of sebaceous gland cells (more preferably autophagy promoters and autophagy inhibitors), and one or more of these can be selected.

[0098] The test substance evaluated and / or selected as a pan-secretion regulator according to this first embodiment can be selected as a substance having a pan-secretion regulator action, and the selected substance may then be subjected to further in vitro tests, in vivo tests, and animal evaluation tests such as human tests to further confirm the pan-secretion regulator action. The selected substance obtained by the method of the first embodiment can be incorporated into a total secretion regulator, as will be described later, to exert a total secretion regulating effect. Furthermore, in the description of the total secretion regulator etc. in this first embodiment, the description in "3." below can also be applied to <Evaluation and / or Selection of Total Secretion Regulator> and can be adopted as appropriate.

[0099] <Method of the Second Embodiment> In explaining the method of this second embodiment, explanations of each component such as holocrine secretion, test substance, holocrine secretion regulator, fluorescence microscope, image analysis software, etc., which overlap with those of the above-mentioned <method of the first embodiment> will be omitted as appropriate, but the explanations of the <method of the first embodiment> and the explanations of "2." and "3." described below also apply to this second embodiment, and such explanations can be adopted as appropriate.

[0100] The present inventor has explained in <Test Example 1> of the Examples below that there is a relationship between the formation of clogged pores and the total secretion of sebaceous gland cells, and further that when the duct part of the pore of the cultured skin piece is prone to insufficient total secretion, the rupture of sebaceous gland cells, poor digestion, etc. cause the accumulation of dead sebaceous gland cells, sebum, stratum corneum, etc., and the keratin plug, which is the deposit, swells laterally, making it impossible to expel these dead bodies and other substances to the outside.The present inventor has also found that, instead of measuring the diameter of the pore outlet, it is possible to estimate whether the total secretion of sebaceous gland cells has occurred appropriately by collecting the keratin plug clogged inside the pore and measuring the maximum diameter at which the keratin plug causing the clogging swells the most.

[0101] Therefore, the method of the second embodiment can provide a method for evaluating and / or selecting a holocytosis regulator, which includes an observation step of fluorescently observing the state of the collected keratinocytes after allowing one or more sebaceous gland cells and a test substance to be present, and / or a discrimination step of discriminating the test substance as a holocytosis regulator or a candidate holocytosis regulator based on the state of the keratinocytes that have been made fluorescent.Furthermore, since the state of the keratinocytes that have been made fluorescent does not require sensory evaluation, there is also the advantage that the method for evaluating and / or selecting a holocytosis regulator, which includes a discrimination step of discriminating as a holocytosis regulator or a candidate holocytosis regulator, can be performed with greater accuracy.

[0102] As a result, the method of the second embodiment can achieve the main objective of providing a technology related to the formation of clogged pores. More specifically, the method of the second embodiment can also solve the main objective of providing a method for more easily estimating the state of holocrine secretion, or for evaluating and / or selecting a holocrine secretion regulator, rather than evaluating changes in pore diameter on the skin surface (i.e., the apparent prominence of pores). Furthermore, the method of the second embodiment can be used to clarify or analyze skin problems caused by clogged pores, and can also suggest countermeasures for skin problems caused by clogged pores.

[0103] The method of the second embodiment can be performed to determine or evaluate whether a test substance has a holocytosis regulator in a human evaluation test, an animal evaluation test using a mammal other than a human (e.g., a mouse, a rat, or a pig), or a test using a skin fragment from a mammal (e.g., a human, a pig) containing a pilosebaceous gland. This allows for the evaluation of the test substance as a holocytosis regulator and / or the selection of a substance that can be used as an active ingredient of a holocytosis regulator from the test substance.

[0104] The holocrine regulator is preferably one or more selected from holocrine promoters, holocrine inhibitors, pore clogging regulators, and sebaceous gland cell autophagy regulators.

[0105] The second embodiment preferably includes an observation step and / or a discrimination step, and more preferably, the observation step is performed first, followed by the discrimination step. The second embodiment preferably includes a contacting step, an observing step, and a determining step, and it is even more preferable that these steps are performed in sequence.

[0106] <Contact process> In the contacting step in the second embodiment, it is preferable to bring one or more sebaceous gland cells and a test substance into contact with each other.

[0107] Furthermore, in the second embodiment, it is more preferable that the contacting step in the animal evaluation test be to contact the test substance or a sample containing the test substance with the skin surface of a mammalian subject animal. By contacting the test substance with the skin, the test substance also comes into contact with one or more sebaceous gland cells present in the pilosebaceous region, etc. The mammal is not particularly limited, and is preferably one or more species selected from humans, pigs, mice, rats, etc. Furthermore, taking into consideration differences in sebum secretion due to gender and age, men (males), women (females), children, adults (young people (15 to 34 years old), middle-aged people (35 to 64 years old), elderly people (65 years old or older), etc.) may be appropriately selected. The contacting part of mammal is not particularly limited, but is preferably the part where sebum secretion occurs a lot, for example, the part where there are many sebaceous glands (for example, scalp, nose, forehead etc. in human), and more preferably the nose in human.In addition, the contacting part is preferably the part where keratin plug is once removed and then keratin plug is formed again in order to adjust the formation period of keratin plug between test animals.

[0108] In the contact step, it is preferable to administer the test substance so that one or more sebaceous gland cells and the test substance are present. The administration method is not particularly limited, and examples include, but are not limited to, injection, application, spraying, penetration, oral administration, etc. Among these, non-invasive administration, such as application, spraying, penetration, etc., is preferred. When administering, the test substance may be prepared into a sample containing the test substance, and examples of such samples include cosmetics or topical skin preparations containing the test substance. Examples of such cosmetics or topical skin preparations include, but are not limited to, lotions, emulsions, impregnated packs, skin patch sheets, creams, etc.

[0109] The contact conditions (e.g., period, etc.) are not particularly limited and may be set in consideration of normal animal evaluation tests such as human evaluation tests, and the test substance may be used in the normal living environment of the test animal. The number of administrations may be once a day or may be divided into multiple doses per day. The administration interval may be either continuous or intermittent. The administration period or contact period is, for example, 1 week to 2 months, preferably 2 weeks to 6 weeks, and more preferably 3 weeks to 5 weeks.

[0110] Furthermore, in the second embodiment, when a test is performed using the skin piece, from the viewpoint of holocytosis regulation, the skin piece is preferably a mammalian skin piece containing one or more pilosebaceous glands. Examples of the skin piece include, but are not limited to, a skin piece collected from a mammal, a cultured skin piece obtained by culturing such a skin piece, and the like. The skin piece may be a biological sample, or may be a skin piece collected during livestock slaughter.

[0111] In the second embodiment, a culture step of culturing the collected skin piece or the like may be included before or during the contact step in the test using the skin piece. For example, as in <Test Example 1> described later, the culture step of the skin piece may be performed using a general skin piece culture medium under conditions of 37°C and 5% CO. Furthermore, in the skin slice culture process, the medium conditions for holocytosis regulation described above in <1-3-1. Conditions for holocytosis regulation> may be applied as appropriate, thereby regulating the induction of holocytosis and the inhibition of holocytosis in sebaceous gland cells present in the skin slice, in order to facilitate evaluation or selection of the holocytosis regulation of the test substance.

[0112] In the second embodiment, in the contact step in the test using a skin slice, the test substance or a sample containing the test substance may be applied by coating, spraying, or pasting onto the skin slice so as to contact one or more sebaceous gland cells contained in the pilosebaceous portion of the skin slice, or the test substance may be added to a culture medium. The sample may be prepared as a general cosmetic or topical skin preparation, as in animal evaluation tests. The conditions for the contact step can be set taking into account the general culture conditions for skin slices. For example, the contact period is not particularly limited, but may be, for example, 1 day to 4 weeks.

[0113] <Observation process> In the observation step in the second embodiment, it is preferable to perform fluorescence observation of the state of the collected keratin plug after allowing one or more sebaceous gland cells and a test substance to be present.

[0114] The keratin plugs collected are preferably those collected from the skin surface that has been in contact with a test substance, and more preferably those collected from the skin surface that has been in contact with the test substance after a predetermined period of time has passed since the keratin plugs were removed. The predetermined period of time after removal is not particularly limited and can be set appropriately, and in the case of humans, it can be set to, for example, 3 to 5 weeks. The keratin plugs used in the control test are preferably those collected from the skin surface without contact with the test substance, and more preferably those collected from the skin surface after the keratin plugs have been removed in advance and a predetermined period of time has passed without contact with the test substance.

[0115] As a more preferred embodiment, the same test animal is used to carry out a control test, followed by a test using the test substance after a predetermined period of time, and the state of the keratin plugs collected in each test is observed by fluorescence, so that the overall secretion regulation of the test substance can be determined more accurately and efficiently.Also, the test animal can be divided into a use group and a control group, and the tests can be carried out at the same time. Furthermore, before starting the test for the presence or absence of contact with the test substance, as a preliminary step, keratinized plugs may be removed in advance to standardize the time for keratinized plug formation among the test animals and reduce variation among the test animals.

[0116] Alternatively, different skin surface areas (e.g., the left and right parts of a human nose, the left and right parts of a pig's back, etc.) may be set up on the same skin surface of a test animal but in similar regions, and the test may be performed simultaneously. Then, by comparing the state of keratin plugs collected from the skin surface of the area not contacted with the test substance with the skin surface of the area contacted with the test substance, the total secretion of the test substance can be determined simultaneously. Furthermore, for the area not contacted with the test substance, a sample without the test substance may be used, or a sample with a control drug added (so-called positive or negative) may be used.

[0117] The state of the keratin plug includes, but is not limited to, for example, the shape of the keratin plug (three-dimensional shape or size, cross-sectional shape or size, etc.), the state of fluorescence of the keratin plug (intensity, fluorescent color, autofluorescence, etc.).Among these, the shape of the keratin plug is preferred, and the size of the three-dimensional shape (preferably the maximum diameter of the three-dimensional shape of the keratin plug) is more preferred.The fluorescence of the keratin plug may be obtained using a fluorescent reagent as described in the first embodiment above, but autofluorescence without using a fluorescent reagent is preferred.Furthermore, it is more preferred to observe the size of the three-dimensional shape of the keratin plug using autofluorescence. In this specification, the cross-sectional shape refers to the shape of the plane perpendicular to the height direction of the three-dimensional shape of the keratin plug (more preferably, a shape like a vertically elongated ellipsoid).In addition, the three-dimensional image of the three-dimensional shape of the keratin plug is composed of many cross-sectional shapes stacked in the height direction, and the maximum diameter of the keratin plug in this specification refers to the diameter of the cross section of the most bulging part of the three-dimensional shape of this keratin plug (see Figure 8).

[0118] The keratinized plugs to be observed with fluorescence are preferably a plurality of keratinized plugs that have been collected from the skin surface of a mammalian subject using an adhesive sheet and adhered to the adhesive sheet. The adhesive sheet is not particularly limited, and a commercially available product for removing keratin plugs from the skin may be used. The skin surface is preferably any of the above-mentioned contact sites, more preferably the nose.

[0119] In the second embodiment, the fluorescence observation is preferably performed by irradiating the collected keratinocytes with excitation light and observing the fluorescence from the keratinocytes. When observing the fluorescence from the keratinocytes, a known fluorescent reagent may be used to cause the keratinocytes to fluoresce, and the fluorescent keratinocytes may then be observed. More preferably, from the standpoints of work efficiency and simplicity, fluorescence observation of the autofluorescence of the collected keratinocytes is preferred. Generally, the fluorescence wavelength used in cell biology observations is typically in the range of 300 to 800 nm. Therefore, when observing the autofluorescence of keratinocytes, the excitation wavelength and fluorescence wavelength may be set taking this range into consideration. When observing the autofluorescence of keratinocytes, the fluorescence wavelength may be set so that all of the autofluorescence emitted from the keratinocytes can be detected by excitation light (e.g., near 550 nm). In a more specific preferred embodiment, the excitation light (excitation wavelength) irradiated onto the keratinocytes is near 540 to 570 nm, and the fluorescence wavelength for detecting the autofluorescence of the keratinocytes is 545 nm or longer (more preferably 545 to 575 nm or longer).

[0120] In the fluorescence step in this second embodiment, it is preferable to fluorescently observe one or more of the three-dimensional shapes of one keratin plug (the entire image of the keratin plug).It is also preferable to photograph the cross-sectional shape of one keratin plug so that the layers can be stacked, and it is further preferable to stack this layer data and finally reconstruct one keratin plug into a three-dimensional image. In the fluorescence observation in the second embodiment, it is preferable to measure the maximum diameter of the single keratin plug. Furthermore, it is preferable to measure the maximum diameter of the entire image of the reconstructed keratin plug.

[0121] A more preferred mode of the fluorescence process in the second embodiment is to fluorescently observe one or more of the three-dimensional shape of one keratin plug (the whole image of keratin plug), and measure the maximum diameter of the diameter of this one keratin plug.When fluorescently observing a plurality of keratin plugs, it is preferred to calculate the average value of the maximum diameter of keratin plugs from the sum of the maximum diameters of these keratin plugs / the total number of these keratin plugs.

[0122] The fluorescence detection device used for fluorescent observation of the state of keratinocyte plugs may be any of the configurations of the fluorescence detection device and fluorescence microscope described in the first embodiment above, and is preferably a confocal laser microscope or a confocal laser microscope device. A suitable fluorescence detection device for use in the second embodiment includes an observation unit configured for fluorescence microscopy, an imaging unit configured for capturing an image of an object to be observed, and an image analysis unit configured for analyzing the captured observation image. Furthermore, the fluorescence detection device used in this second embodiment preferably includes an observation unit configured to enable fluorescent observation of one or more three-dimensional shapes of a single keratinocyte (the entire image of the keratinocyte), and more preferably includes an imaging unit configured to capture images of the layers of the cross-sectional shape of a single keratinocyte so that they can be stacked. This allows the layered data to be stacked using image analysis software, ultimately reconstructing a single keratinocyte into a three-dimensional image. The fluorescence detection device may also include an image analysis unit configured to determine the maximum diameter of the single keratinocyte.

[0123] Furthermore, the fluorescence detection device is preferably equipped with an image analysis unit that is capable of photographing an entire image of a single keratinocyte, and is further configured to reconstruct the photographed keratinocyte image into a three-dimensional image using image analysis software (preferably 3D / 4D image analysis software capable of constructing a three-dimensional image, such as ZEN Black Edition or IMARIS), and the data of the reconstructed keratinocyte image may be stored in a memory unit so that it can be read out. Furthermore, it is more preferable to measure the maximum diameter of the lateral bulge of the keratinocyte plug from the overall image of the reconstructed keratinocyte plug using image analysis software, and the image analysis software may be stored and executed in the fluorescence detection device.

[0124] <Discrimination process> In the discrimination step in the second embodiment, it is preferable to discriminate the test substance as a holocytosis regulator or a candidate holocytosis regulator based on the state of the fluorescent keratinocyte plugs, thereby enabling more accurate and simple discrimination of the holocytosis regulation of the test substance.

[0125] In a more preferred embodiment, the state of the keratin plug is the rate of change in the maximum diameter of the keratin plug, calculated by dividing the average value of the maximum diameter of the keratin plug after application of the test substance by the average value of the maximum diameter of the keratin plug before application, and the rate of change in the maximum diameter of the keratin plug before application of the test substance can be defined as 1. In this case, the number of keratin plugs used to calculate this rate of change is preferably 5 to 30, more preferably 11 to 24. The average value in this case is the average value obtained by averaging the total value of the maximum diameter of the keratin plug in the three-dimensional image of the keratin plug reconstructed by image analysis. This allows the state of total secretion to be easily and accurately determined.

[0126] More preferably, in the more preferred embodiment, the test group that does not use test substance in contacting step (that is, control test group) is compared with the change rate of the maximum diameter of keratin plug in the group that uses test substance in contacting step, so as to determine the total secretion regulation of test substance.Furthermore, it is more preferable to calculate the average value of the change rate of the maximum diameter of keratin plug in both groups, and compare these two values, so as to determine the total secretion regulation of test substance.

[0127] In a more preferred embodiment, the state of the keratin plugs in the discrimination step is determined by comparing the rate of change in the maximum diameter of the keratin plugs in the group using the test substance with the rate of change in the maximum diameter of the keratin plugs in the group not using the test substance, and if the rate of change in the group using the test substance is smaller than that in the group not using the test substance, it is determined that the test substance has a holocrine secretion promoting effect, and / or if the rate of change in the group using the test substance is larger than that in the group not using the test substance, it is determined that the test substance has a holocrine secretion inhibiting effect.In this case, it is preferable that the sex and contact conditions (duration, etc.) of the test animals are the same.

[0128] For example, as shown in Figure 9, the rate of change in the maximum diameter of keratinocytes in subjects (specifically, men) who used the test substance (specifically, trehalose) was smaller than the rate of change in the maximum diameter of keratinocytes in the control test. This indicates that the test substance effectively promotes total secretion, and therefore it can be evaluated or determined that the test substance has a total secretion-promoting effect.

[0129] <Method of the Third Embodiment> The method of the third embodiment may be a combination of the method of the first embodiment and the method of the second embodiment, for example, the method of the first embodiment may be performed followed by the method of the second embodiment, or vice versa, the method of the second embodiment may be performed followed by the method of the first embodiment, thereby enabling a more accurate determination of whether or not a test substance has a global secretion regulating effect.

[0130] <1-5. Device or system for evaluating and / or selecting a total secretion regulator> As another aspect of this embodiment, an apparatus or system for evaluating and / or selecting a total secretion regulator can be provided.

[0131] The device for evaluating and / or selecting a holocytosis regulator according to this embodiment is preferably configured to be able to execute the holocytosis regulator evaluation and / or selection method described above (e.g., the method of the first embodiment, the method of the second embodiment, or a combination thereof), and more preferably includes a control unit configured to execute the holocytosis regulator evaluation and / or selection method described above. The control unit or the device including the control unit may further include a communication unit that enables wireless and / or wired transmission and reception between the control unit and another unit (unit or part) or another device.

[0132] The device or system of this embodiment preferably includes a management device including the control unit, and / or a fluorescence detection device including a fluorescence microscope. Furthermore, when culturing sebaceous gland cells, skin slices, etc., the device or system of this embodiment preferably includes a management device including the control unit, an animal cell culture device, and a fluorescence detection device including a fluorescence microscope. The fluorescence detection device including the fluorescence microscope preferably includes an observation unit configured for fluorescence microscope observation, an imaging unit configured for imaging the observation target, and an image analysis unit configured for image analysis of the captured observation image. The fluorescence detection device or fluorescence detection system including the fluorescence microscope may be a fluorescence microscope observation device or a fluorescence microscope observation system.

[0133] The method according to this embodiment can also be realized by a device (e.g., a computer, PLC, server, cloud service, etc.) including a CPU for managing the evaluation and / or selection of a total secretion regulator, or a control unit including a CPU provided in the management device. The method according to this embodiment can also be stored as a program in hardware resources including a recording medium or a storage unit (non-volatile memory (e.g., USB memory), HDD, CD, DVD, Blu-ray, server, cloud service, etc.) and realized by a control unit. In this way, it is also possible to provide a control unit capable of executing the method according to this embodiment or a device provided with the control unit. The management device may also include an input unit such as a keyboard or touch panel, a communication unit such as a network, a display unit such as a display or touch panel, etc.

[0134] <2. Three-dimensional structure of sebaceous gland cells and method for producing the three-dimensional structure of sebaceous gland cells> In the description of the three-dimensional structure of sebaceous gland cells and the method for manufacturing the three-dimensional structure of sebaceous gland cells in this embodiment, the description of each component, each processing method, each device, etc. of the total secretion regulator, the evaluation, the selection, etc., which overlaps with the above-mentioned "1." will be omitted as appropriate, but the description of "1." also applies to this embodiment and can be adopted as appropriate. Furthermore, in the description of the total secretion regulator, etc. in this embodiment, the description of "3." described below can also apply to this embodiment and can be adopted as appropriate.

[0135] Another aspect of the present embodiment is to provide a three-dimensional structure of sebaceous gland cells and / or a method for producing the three-dimensional structure of sebaceous gland cells. This makes it possible to provide a three-dimensional structure of sebaceous gland cells and / or a method for producing the same, and in particular, to provide a novel three-dimensional structure and a method for producing the same.

[0136] <2-1. Three-dimensional structure of sebaceous gland cells according to this embodiment> This embodiment can provide a three-dimensional structure of sebaceous gland cells in which the surface layer is composed of mature sebaceous gland cells (see FIG. 3). This three-dimensional structure is suitable for use in fluorescence observation. Furthermore, from the perspective of use in observing holocrine secretion, a three-dimensional structure configured so that, after the mature sebaceous gland cells present in the surface layer have undergone holocrine secretion and collapsed, the sebaceous gland cells present below them become the surface layer is even more suitable. This embodiment has the advantage that, because mature sebaceous gland cells are present in the surface layer, observation of the state or status of holocrine secretion regulation (preferably confocal laser observation) can be easily performed.

[0137] The three-dimensional structure of sebaceous gland cells of this embodiment can be used in techniques (experimental systems, models, etc.) related to holocrine secretion or dermatitis involving sebaceous gland cells. For example, it can be used in a method for evaluating or selecting holocrine secretion regulators, a cell experimental system for pore clogging formation, a cell experimental system for elucidating the mechanism of holocrine secretion of sebaceous glands, a cell experimental system for elucidating the relationship between holocrine secretion and autophagy, a cell experimental system for elucidating the mechanisms of onset of seborrheic dermatitis, acne, etc., or models of these.

[0138] In conventional experiments using sebaceous gland cells, only the amount of sebum production has been evaluated. Therefore, in two-dimensional cultures in which sebaceous gland cells are cultured in two dimensions, the area of ​​sebum stained with a fluorescent dye is observed in a planar manner under a microscope to measure the area of ​​sebum, or sebum is extracted from the sebaceous gland cells of the two-dimensional culture, and the extracted sebum is quantified to evaluate the amount of sebum production. Moreover, since the amount of sebum production is taken into consideration in two-dimensional cultures, the present inventor believes that the sebaceous gland cells have not matured to the stage of full secretion. There is also a technique for evaluating the amount of sebum production by obtaining a laminated sheet by laminating layers of skin cells and observing the distribution of sebum production on the cross section of this sheet (Non-Patent Document 1). Also, by embedding multiple sebaceous gland cells in a matrix gel and culturing them, spheroids in which mature cells gather in the center are obtained, and the amount of sebum production present inside these spheroids is evaluated (Non-Patent Document 2). Thus, in the prior art, only sebum production has been evaluated in both two-dimensional and three-dimensional cultures.

[0139] In response to this, the present inventors have newly elucidated the mechanism of clogged pore formation, in which induction of autophagy in sebaceous gland cells results in normal holocrine secretion, while impaired autophagy results in abnormal holocrine secretion, resulting in sebaceous gland cells failing to properly disintegrate and clogging pores. They have found that the state of holocrine secretion (normal or abnormal) of sebaceous gland cells, rather than the amount of sebum produced by sebaceous gland cells, is deeply involved in the formation of clogged pores (see Figures 1 and 2). This has led to the new concept and technology of holocrine secretion of sebaceous gland cells. Furthermore, based on the concept and technology of holocrine secretion of sebaceous gland cells, the present inventors have investigated a method for culturing sebaceous gland cells that allows for better observation, and have been able to obtain a novel three-dimensional structure of sebaceous gland cells in which the surface layer is composed of mature sebaceous gland cells.

[0140] The shape of the three-dimensional structure of sebaceous gland cells in this embodiment is not particularly limited, and examples include tubular, spherical, and sheet shapes, with a roughly spherical shape being preferred, and a spheroid being more preferred (see Figures 3 and 4). The size of the three-dimensional sebaceous gland cell structure of this embodiment is preferably 200 to 600 μm in minimum length, more preferably 250 to 550 μm, and even more preferably 300 to 500 μm, and preferably 300 to 700 μm in maximum length, more preferably 350 to 650 μm, and even more preferably 400 to 600 μm. When the cross section is elliptical (approximately spheroidal), the minimum length can be replaced with the minor axis, and the maximum length can be replaced with the major axis. When the three-dimensional structure of sebaceous gland cells of this embodiment is approximately spherical, the diameter is more preferably 250 to 650 μm, even more preferably 300 to 600 μm, even more preferably 350 to 550 μm, and more preferably 400 to 500 μm.

[0141] In the three-dimensional structure of sebaceous glands of this embodiment, it is preferable that mature sebaceous glands are present in the surface layer on the observation side. It is more preferable that the three-dimensional structure of sebaceous glands has many mature sebaceous glands in the surface layer, and it is even more preferable that the three-dimensional structure of sebaceous glands has many immature sebaceous glands in the interior near the center (see FIG. 4). In the three-dimensional structure of sebaceous glands, immature sebaceous glands mature from the interior to the exterior, and mature sebaceous glands are present in the surface layer. The thickness (depth direction) of the surface layer where the mature sebaceous gland cells are present is preferably within 50 μm from the surface side, more preferably within 40 μm, and even more preferably within 30 μm. From the viewpoint of observation, it is preferable that the surface layer is densely populated with mature sebaceous gland cells. The proportion of mature sebaceous gland cell regions present on the surface of the three-dimensional structure is preferably, for example, about 60% or more, about 70% or more, about 80% or more, or about 90% or more of the surface. The percentage of the mature cell region can be calculated based on the total area of ​​fluorescent emission of mature sebaceous cells in the two-dimensional surface image by fluorescently staining the mature sebaceous cells present in the three-dimensional structure, observing the three-dimensional structure with fluorescence, and obtaining a surface image (or surface photograph) of the three-dimensional structure. The percentage can be calculated based on the total area of ​​fluorescent emission of mature sebaceous cells in the two-dimensional surface image by multiplying the total area of ​​fluorescent emission of mature cells by the surface image area of ​​the three-dimensional structure of sebaceous cells by 100 (%).

[0142] Since mature sebaceous gland cells are present in the surface layer of the three-dimensional structure of this embodiment, it is easy to stain, fluorescently stain, or fluorescently observe the mature sebaceous gland cells, and therefore the three-dimensional structure of this embodiment is suitable for use in staining observation or fluorescent observation. Furthermore, the three-dimensional structure of this embodiment is suitable for use in confocal laser microscope observation, since it is easy to fluorescently stain and observe live mature sebaceous gland cells present in the surface layer. In this way, staining or fluorescent staining can be easily performed on mature sebaceous gland cells in the surface layer. Furthermore, since this surface layer can be easily observed fluorescently, the evaluation and / or selection method for a total secretion regulator can also be easily performed. For this reason, the three-dimensional structure of this embodiment is suitable for use in the evaluation and / or method of the embodiment.

[0143] <2-2. Method for producing a three-dimensional structure of sebaceous gland cells according to this embodiment> This embodiment can provide a method for producing a three-dimensional structure of sebaceous gland cells, in which a plurality of seeded sebaceous gland cells are cultured on a cell adhesion-inhibiting surface (more preferably a hydrophilized surface) of a cone-shaped depression to obtain a three-dimensional structure of sebaceous gland cells. This allows for the production of a three-dimensional structure of sebaceous gland cells of this embodiment, more preferably a three-dimensional structure of sebaceous gland cells whose surface layer is composed of mature sebaceous gland cells.

[0144] The bottom of the cone-shaped recess is preferably shaped so that multiple sebaceous gland cells can gather in the central region of the bottom of the recess. More preferably, the bottom of the recess is V-shaped or U-shaped, which makes it easier for seeded sebaceous gland cells to gather in the V-shaped or U-shaped depression, resulting in a better spherical cell cluster shape and making it easier for mature sebaceous gland cells to exist in the surface layer.

[0145] The shape of the container for seeding the sebaceous gland cells includes, but is not limited to, a petri dish, a flask, a plate with multiple holes, etc. Disposable products are preferred. The material of the container includes, but is not limited to, glass, plastic resin (preferably polystyrene resin), etc. The wells into which the sebaceous gland cells are seeded are preferably subjected to a cell adhesion-inhibiting treatment; this surface treatment can inhibit adhesion of the cells to the surface of the wells, making it easier to peel off the three-dimensional structure after culture. This surface treatment allows the seeded sebaceous gland cells to gather in the central region, facilitating adhesion between the cells, resulting in a better spherical cell cluster shape, and making it easier for mature sebaceous gland cells to reside in the surface layer. The cell adhesion-inhibiting treatment is not particularly limited, but examples include hydrophilic surface treatment. The recesses are preferably subjected to a hydrophilic surface treatment, and examples of the hydrophilic treatment include, but are not limited to, plasma treatment, corona discharge treatment, oxidizing agent treatment, and coating with a hydrophilic substance (preferably, a cell adhesion inhibitor such as polyethylene glycol). Examples of the hydrophilic substance coating include a high molecular weight polymer coating.

[0146] The production method of this embodiment can be carried out under the same in vitro culture conditions as for normal human sebaceous gland cells, except for the cone-shaped depression. Furthermore, the production method of this embodiment can be applied to the culture conditions described in "1." above (particularly the autophagy-inhibiting conditions), and these culture conditions may be used as spheroid formation conditions. In this case, it is preferable to use a growth medium for animal cell culture generally used for growth, maintenance, differentiation, etc., and among these, a differentiation medium is preferred. More specifically, a differentiation medium that does not contain or has reduced amounts of differentiation inhibitors (e.g., EGF, BPE, etc.) is preferred. Examples of growth media for animal cell culture include, but are not limited to, basal medium, serum-reduced medium, and serum-free medium. The culture conditions in this embodiment are preferably those used for ordinary cultured animal cells, such as a pH of about 7 to 8, a CO2 concentration of about 4 to 10% in the atmosphere, and a culture temperature of about 36 to 37° C. The culture period is not particularly limited, but is preferably 5 to 15 days, more preferably 6 to 13 days, and even more preferably 7 to 12 days after seeding. The culture can be carried out under static or rotating conditions, preferably under static conditions. The seeding concentration of sebocytes in the medium was 1 x 10 2 ~1×10 5 cells / mL, preferably 5×10 2 ~2×10 4 cells / mL, more preferably 1 x 10 3 ~1×10 4 cells / mL.

[0147] <3. Total secretion regulator> In the explanation of the total secretion regulator in this embodiment, the explanations of the total secretion regulator, its evaluation, its selection, etc., each configuration, each processing method, each device, etc. that overlap with the above-mentioned "1." to "2." will be omitted as appropriate, but the explanations of "1." to "2." also apply to this embodiment, and these explanations can be adopted as appropriate.

[0148] Another aspect of this embodiment is to provide a substance having a holocytosis-regulating effect, or an active ingredient of a holocytosis-regulating agent, and / or a holocytosis-regulating agent. This can provide a technology related to holocytosis regulation of sebaceous gland cells, and specifically, a substance having a holocytosis-regulating effect, or an active ingredient of a holocytosis-regulating agent, and / or a holocytosis-regulating agent. This can provide a drug for skin problems such as clogged pores.

[0149] By using the method for evaluating and / or selecting a holocytosis regulator in this embodiment (e.g., the methods of the first to third embodiments), a selected substance (hereinafter also referred to as a "selected substance") can be provided as a substance having a holocytosis regulator effect or as an active ingredient of a holocytosis regulator. Furthermore, the method for evaluating and / or selecting a total secretion regulator may be the device or system for evaluating and / or selecting a total secretion regulator of this embodiment.

[0150] Examples of holocytosis regulating effects include, but are not limited to, regulating holocytosis of sebaceous glands, regulating the formation of clogged pores, and regulating autophagy of sebaceous glands. Regulating effects include promoting or inducing effects, inhibiting or suppressing effects, and maintaining effects. More specifically, examples include promoting holocytosis of sebaceous glands, inhibiting holocytosis of sebaceous glands, promoting the formation of clogged pores, inhibiting the formation of clogged pores, promoting autophagy of sebaceous glands, and inhibiting autophagy of sebaceous glands. One or more of these effects can be selected. Therefore, the selected substance obtained by the method for evaluating and / or selecting a holocytosis regulator of this embodiment has a holocytosis regulator action, and can therefore be effective in regulating clogged pores.

[0151] The selected substance is not particularly limited and may be either naturally or artificially derived, and may be either a single substance or a mixture. The selected substance is preferably one or more substances selected from compounds, microorganisms or their cultures, extracts, mixtures thereof, and compositions. The compound may be either an inorganic compound or an organic compound.

[0152] The selected substance may preferably be, for example, a retinoid such as tretinoin; benzoyl peroxide; oleic acid; curcuminoid such as curcumin; oligosaccharide such as trehalose; vitamin D; macrolide compounds such as rapamycin and bafilomycin; phosphatidylinositol 3-kinase inhibitors such as wortmannin; chloroquine; or the like, and one or more substances selected from the group consisting of these may be used. Among these, one or more compounds selected from the group consisting of macrolide compounds, curcuminoids, retinoids, oligosaccharides, and phosphatidylinositol 3-kinase inhibitors are preferred, and oligosaccharides and / or macrolide compounds are more preferred. Among these, retinoids such as tretinoin, benzoyl peroxide, and oleic acid are suitable for use as pore-clogging regulators, while curcumin, trehalose, vitamin D, rapamycin, wortmannin, and chloroquine are suitable for use as autophagy regulators.

[0153] Examples of oligosaccharides used in this embodiment include disaccharides such as trehalose (α,α-trehalose), neotrehalose (α,β-trehalose), isotrehalose (β,β-trehalose), and sucrose; and disaccharides to decasaccharides (preferably di- to tetrasaccharides) such as trisaccharides such as raffinose. Of these, non-reducing oligosaccharides such as trehalose are more preferred.

[0154] The macrolide compound used in this embodiment is preferably a macrocyclic lactone compound having 12 or more ring members. Examples of macrolide compounds include rapamycin and bafilomycin. Of these, rapamycin and bafilomycin are more preferred.

[0155] Examples of curcuminoids used in this embodiment include curcumin and curcumin analogues such as desmethoxycurcumin, bisdesmethoxycurcumin, and tetrahydrocurcumin. Of these, curcumin is more preferred.

[0156] The retinoid used in this embodiment may be either natural or synthetic, and examples thereof include retinol, retinol derivatives (retinol acetate, retinol palmitate, retinol propionate, etc.), tretinoin, isotretinoin, motretinide, etretinate, acitretin, all-trans retinoic acid or all-trans zinc retinoate, synthetic retinoids (adapalene, etc.), etc. Of these, retinol and tretinoin are more preferred.

[0157] Suitable compounds that have an autophagy-promoting effect include non-reducing disaccharides such as trehalose, macrolide compounds such as rapamycin, curcuminoids such as curcumin, and retinoids such as retinol or tretinoin, and these compounds can be used as active ingredients of autophagy promoters or holocrine promoters. In addition, compounds with autophagy inhibitory activity are preferably macrolide compounds such as bafilomycin and phosphatidylinositol 3-kinase inhibitors such as wortmannin, and these compounds can be used as active ingredients of autophagy inhibitors or pansecretion suppressors.

[0158] In this embodiment, one or more selected substances from these can be used.

[0159] The selected substance can be contained as an active ingredient in a holocrine regulator (preferably a holocrine promoter or a holocrine inhibitor), a clogged pore formation regulator (preferably a clogged pore formation promoter or a clogged pore formation inhibitor), an autophagy regulator (preferably an autophagy promoter or an autophagy inhibitor), or an agent for preventing, improving, or treating clogged pores (hereinafter also referred to as a "holocrine regulator, etc."), or can be used in the holocrine regulator, etc. The agent may also be a composition. The selected substance can also be used to produce a total secretion regulator, etc. The present embodiment can also provide the selected substance or use thereof for regulating holocrine secretion, or for regulating holocrine secretion, etc. The holocrine secretion regulation, etc. may be one or more substances selected from holocrine secretion regulation (preferably promoting holocrine secretion or inhibiting holocrine secretion), regulation of clogged pore formation (preferably promoting clogged pore formation or inhibiting clogged pore formation), regulation of autophagy (preferably promoting autophagy or inhibiting autophagy), and prevention, improvement, or treatment of clogged pores. The present embodiment can also provide a method for regulating holocytosis, a method for regulating autophagy, and a method for preventing, improving, and treating clogged pores using the selected substance, or a method for regulating holocytosis, a method for regulating autophagy, and a method for preventing, improving, and treating clogged pores using an agent containing the selected substance.

[0160] <Diseases and symptoms> The selected substance exhibits the physiologically active action and can therefore be used in methods for preventing, improving or treating diseases caused by clogged pores or symptoms of clogged pores. Diseases caused by clogged pores or symptoms of clogged pores include, but are not limited to, blackening of pores, enlarged pores, roughness of pores, redness of pores, acne, seborrheic dermatitis, etc., and one or more of these may be selected from the group consisting of these. Areas where clogged pores are likely to occur include areas with many sebaceous glands, more specifically, the scalp, facial skin (nose, forehead, etc.), and chest. Furthermore, examples of uses of the present embodiment include, but are not limited to, prevention, improvement, or treatment of clogged pores.

[0161] In this embodiment, "prevention" refers to preventing or delaying the onset of symptoms or diseases in a subject, or reducing the risk of developing symptoms or diseases in a subject. In this technology, "improvement" refers to improving or maintaining the disease, symptoms, or condition in a subject; preventing or delaying deterioration; or reversing, preventing, or delaying progression.

[0162] In this embodiment, the selected substance can be used in, for example, cosmetics, topical skin preparations, quasi-drugs, foods and beverages, feeds, etc., but is not particularly limited to these. The "total secretion regulator, etc." may be used, for example, as cosmetics, topical skin preparations, quasi-drugs, foods and beverages, feeds, etc., but is not particularly limited thereto. Among these, cosmetics, topical skin preparations, pharmaceuticals, quasi-drugs, etc. are preferred. Furthermore, the "total secretion regulator, etc." may be used as a compounding agent or additive to a composition, and may be, for example, a "total secretion regulator, etc." used to be compounded or added to compositions such as cosmetics, topical skin preparations, quasi-drugs, foods and beverages, and feeds.

[0163] In the case where this embodiment is, for example, a cosmetic or topical skin preparation, the selected substance or total secretion regulator, etc., can be blended into various forms of cosmetic or topical skin preparation, such as, but not limited to, emulsion, cream, lotion, pack, cleanser, makeup cosmetic, dispersion, ointment, liquid, aerosol, patch, cataplasm, liniment, etc.

[0164] When this embodiment is, for example, a pharmaceutical product or a quasi-drug, the selected substance or the total secretion regulator etc. can be incorporated into, for example, oral preparations such as tablets, capsules, granules, powders, liquids, and suspensions; external preparations such as dermatological preparations, patches, eye drops, nasal drops, oral preparations, and suppositories; and parenteral preparations such as infusions and injections, but are not limited to these.

[0165] The selected substance or the total secretion regulator can be produced by a known production method. The selected substance may be a commercially available product.

[0166] The content of the selected substance is not particularly limited, and the selected substance can be contained in an amount of preferably 0.001 to 99% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.01 to 90% by mass of the total amount of the formulation. The "formulation" in "of the total amount of the formulation" may be the "total secretion regulator, etc."

[0167] The selected substance or the total secretion regulator can be applied to humans and non-human animals (for example, pets, livestock, etc.), etc. Among these, humans and pets are preferred, and humans are more preferred.

[0168] Methods for using the selected substance or total secretion regulator include, but are not limited to, administration such as transdermal administration, oral administration, and administration by injection; oral ingestion; and application to the skin. The amount of the selected substance used or administered is not particularly limited as long as it is an amount that can obtain the effects of the present invention, and may be adjusted appropriately depending on the dosage form of the preparation, the application site, age, sex, etc.

[0169] The total secretion regulator etc. can be produced using known production methods. In addition to the selected substance, the total secretion regulator etc. can also contain optional components such as various additives, if necessary.

[0170] As the optional ingredient, ingredients acceptable for cosmetics, topical skin preparations, pharmaceuticals, foods and beverages, feeds, etc. can be appropriately blended. For example, one or more selected from excipients, colorants, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, moisturizers, pH adjusters, etc. may be appropriately used, thereby allowing the desired dosage form to be obtained.

[0171] The present technology can also employ the following configuration. [1] an observation step of contacting one or more sebaceous gland cells with a test substance and then observing the state of the fluorescent sebaceous gland cells; and a discrimination step of discriminating the test substance as a pan-secretion regulator or a candidate pan-secretion regulator based on the state of the fluorescently-activated sebaceous gland cells; A method for evaluating and / or selecting a total secretion regulator, comprising: In a preferred embodiment, in the observation step, a single or multiple sebaceous gland cells and a test substance are present under conditions for holocytosis regulation, and the state of the fluorescent sebaceous gland cells is observed by fluorescence. In a more preferred embodiment, in the observation step, one or more sebaceous gland cells are contacted with the test substance under growth medium culture conditions (preferably under spheroid formation conditions), and then the state of the sebaceous gland cells that have been made fluorescent under holocrine secretion control conditions is observed. [2] The method for evaluating and / or selecting a holocrine regulator according to [1], wherein the holocrine regulator is one or more selected from a holocrine promoter, a holocrine inhibitor, a regulator of clogged pore formation, and a regulator of autophagy of sebaceous gland cells. [3] The method for evaluating and / or selecting a total secretion regulator according to [1] or [2], wherein the sebaceous gland cells are three-dimensional structures composed of multiple sebaceous gland cells. [4] The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [3], wherein the observation is performed using time-lapse imaging. [5] The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [4], wherein the observation is performed using a fluorescence microscope. The observation is preferably performed using a fluorescence detection device or a fluorescence microscope observation device comprising an observation unit configured for fluorescence microscope observation, an imaging unit configured for imaging the observation target, and an image analysis unit configured for image analysis of the captured observation image, and may further comprise an animal cell culture unit for culturing the observation target in order to maintain it. [6] In the observation step, after contacting one or more sebaceous gland cells with a test substance, the state of the fluorescent sebaceous gland cells is observed; the holocytosis-regulating conditions are autophagy-regulating conditions, The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [5] above, wherein the autophagy-regulating conditions are autophagy-suppressing conditions or autophagy-inducing conditions. [7] The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [6], wherein the state of the fluorescent sebaceous gland cells is determined based on the number of cells that have lost fluorescence per unit volume. [8] The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [7], wherein the fluorescently labeled sebaceous gland cells are viable cells that are fluorescently labeled and made to emit light. [9] The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [8], wherein the sebaceous gland cells are a three-dimensional structure of sebaceous gland cells, the surface layer of which is composed of mature sebaceous gland cells.

[10] The method for evaluating and / or selecting a total secretion regulator according to any one of [1] to [9], wherein the sebaceous gland cells are a three-dimensional structure of sebaceous gland cells obtained by culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting surface (more preferably a hydrophilized surface) in a mortar-shaped depression.

[0172]

[11] A three-dimensional structure of sebaceous glands, the surface layer of which is composed of mature sebocytes.

[12] The three-dimensional structure of sebaceous gland cells according to

[11] , wherein the three-dimensional structure is spherical.

[13] The three-dimensional structure of sebaceous gland cells according to

[11] or

[12] , which is used for fluorescence observation, preferably for fluorescence microscopy, more preferably for one or more observations selected from confocal laser scanning microscopy, multiphoton excitation microscopy, and light sheet microscopy, and even more preferably for confocal laser scanning microscopy.

[14] A three-dimensional structure of sebaceous gland cells according to any one of

[11] to

[13] , which is used in a method for evaluating and / or selecting a total secretion regulator.

[0173]

[15] A method for producing a three-dimensional structure of sebaceous gland cells, in which a plurality of seeded sebaceous gland cells are cultured on a cell adhesion-inhibiting surface (preferably a hydrophilized surface) of a mortar-shaped depression to obtain a three-dimensional structure of sebaceous gland cells. The cell adhesion-inhibiting treated surface or the hydrophilized surface is preferably treated by one or more methods selected from plasma treatment, corona discharge treatment, oxidizing agent treatment, hydrophilic substance coating treatment, etc., and among the hydrophilic substance coatings, treatment with a cell adhesion inhibitor such as a photocrosslinkable hydrophilic polymer is preferred.

[16] The method for producing a three-dimensional structure of sebaceous gland cells according to

[15] , wherein the mortar-shaped depression has a shape that allows a plurality of sebaceous gland cells to gather in the central region of the bottom of the depression. The bottom of the cone-shaped recess is preferably V-shaped or U-shaped.

[0174]

[17] An observation step of fluorescently observing the state of the collected keratin plug after allowing one or more sebaceous gland cells and a test substance to be present; and a discrimination step of discriminating the test substance as a total secretion regulator or a candidate for a total secretion regulator based on the state of the fluorescent keratin plug; A method for evaluating and / or selecting a total secretion regulator, comprising:

[18] The method for evaluating and / or selecting a holocrine regulator according to

[17] , wherein the holocrine regulator is one or more selected from a holocrine promoter, a holocrine inhibitor, a regulator of clogged pore formation, and a regulator of autophagy of sebaceous gland cells.

[19] The method for evaluating and / or selecting a total secretion regulator according to

[17] to

[18] , wherein the observation is performed using a fluorescence microscope. The observation is preferably performed using a fluorescence detection device or a fluorescence microscope observation device including an observation unit configured to perform fluorescence microscope observation, an imaging unit configured to image the observation target, and an image analysis unit configured to analyze the captured observation image. 〔20〕 The keratinized plugs in the observation step are a plurality of keratinized plugs collected from the skin surface of a mammal with an adhesive sheet and attached to the adhesive sheet, and / or The method for evaluating and / or selecting a total secretion regulator according to any one of

[17] to

[19] , wherein the state of the keratinocyte plug in the discrimination step is the rate of change in the maximum diameter of the keratinocyte plug obtained by dividing the average value of the maximum diameter of the keratinocyte plug after use of the test substance by the average value of the maximum diameter of the keratinocyte plug before use.

[0175]

[21] A selected substance evaluated and / or selected by the method for evaluating and / or selecting a total secretion regulator according to any one of [1] to

[10] and

[17] to

[20] , or a total secretion regulator containing the selected substance as an active ingredient.

[22] A total secretion regulator containing a selective substance, etc. The selection substance is preferably one or more selected from compounds, microorganisms, fermented products, extracts, mixtures thereof, compositions, etc. The selection substance may be either natural or synthetic.

[23] The selective substance according to

[21] or a total secretion regulator containing the selective substance as an active ingredient, or the total secretion regulator according to

[22] , wherein the selective substance is one or more selected from retinoid, benzoyl peroxide, oleic acid, curcumin, trehalose, vitamin D, rapamycin, wortmannin, and chloroquine, or a mixture thereof.

[24] The selective substance according to

[21] or a total secretion regulator containing the selective substance as an active ingredient, or the total secretion regulator according to

[22] , wherein the selective substance is one or more compounds (more preferably oligosaccharides and / or macrolide compounds) selected from the group consisting of oligosaccharides, retinoids, curcuminoids, macrolide compounds, and phosphatidylinositol 3-kinase inhibitors, or a mixture thereof.

[25] A cosmetic, topical skin preparation, quasi-drug, pharmaceutical, or food product containing the selected substance according to any one of

[21] to

[24] above, or a total secretion regulator containing the selected substance.

[26] The selected substance according to any one of

[21] to

[24] above or its use for producing a total secretion regulator or the like.

[27] The selective substance according to any one of

[21] to

[24] above, or its use for regulating total secretion or the like.

[28] A method for regulating total secretion, a method for regulating clogged pore formation, a method for regulating autophagy, or a method for preventing, improving, or treating clogged pores, which uses the selected substance according to any one of

[21] to

[24] above in a mammal. [Example]

[0176] The following examples and comparative examples will be used to explain the embodiments of the present invention, but the scope of the present invention is not limited to these examples.

[0177] Ideal skin appearance is generally characterized by both uniformity and smoothness, both of which are known to be adversely affected by morphological changes in pores (e.g., enlarged pores, acne). Clogged pores are generally considered one of the main causes of morphological changes in pores. It has long been believed that the formation of clogged pores is caused by the blockage of the pore outlet by a thickened stratum corneum, followed by the accumulation and solidification of sebum inside the pores. However, this conventional view cannot explain the formation of open comedones and microcomedones, which are conditions in which the pore outlet remains open and the pore becomes clogged, and the detailed mechanism of clogged pore formation remains unknown.

[0178] <Test Example 1: Confirmation test of clogged pores using human excised skin and autophagy inhibitors> Sample: Skin (without clogged pores) piece (1cm 2 ): Abdominal skin of a Caucasian woman in her 30s. The skin contains pilosebaceous glands. <Sebum staining method for hair organs> The skin pieces were cultured for 6 days in William's E Medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Then, the pilosebaceous glands of all pores were excised from the skin pieces and fixed overnight at 4°C using 4% paraformaldehyde solution. They were then stained with 1 μg / mL Nile red solution (Sigma) and DAPI stain (Sigma) for 30 minutes at room temperature to stain the sebum present in the pores and sebaceous ducts. The stained sebum was then observed under a fluorescence microscope.

[0179] <Fluorescence observation of cultured skin under autophagy-regulating conditions> As shown in the following Test Examples 1a and 1b, skin samples were cultured while inhibiting autophagy by adding an autophagy inhibitor, and then fluorescently stained and observed. In Test Example 1a, bafilomycin A1 was used as the autophagy inhibitor, and in Test Example 1b, MHY-1485 was used as the autophagy inhibitor. Test Example 1c was used as a negative control and was performed in the same manner as Test Example 1a, except that DMSO solvent containing no autophagy inhibitor was used. For Test Examples 1a, 1b, and 1c, skin sections (without clogged pores) were cultured for 6 days in William's E Medium supplemented with 10% fetal bovine serum at 37°C and 5% CO. In Test Examples 1a and 1b, DMSO containing an autophagy inhibitor was added to the medium, and the skin sections were cultured for 6 days in contact with the autophagy inhibitor.

[0180] In Test Examples 1a, 1b, and 1c, after culturing a piece of skin (without clogged pores) for 6 days, the entire pilosebaceous region was excised from the skin piece and fixed overnight at 4°C using a 4% paraformaldehyde solution. Then, the sebaceous region was stained with 1 μg / mL Nile red solution (Sigma) and DAPI stain (Sigma) for 30 minutes at room temperature to stain the sebum present in the pores and sebaceous ducts. The stained sebum was observed under a fluorescence microscope, focusing on the hair contained in the pilosebaceous region, the opposing hair follicle wall, and the sebaceous duct leading to the pores. At this time, the distance between the hair follicle side and the hair follicle wall facing the hair was measured, and the longest distance in this area where the fluorescent dye was densely filled was taken as the hair-to-hair follicle wall distance (between the tips of the opposing triangles in the figure).

[0181] Human skin was cultured for six days with bafilomycin A1 or MHY-1485, drugs that inhibit autophagy through different mechanisms. After this, pores and sebaceous glands were observed three-dimensionally using a confocal laser scanning microscope (Carl Zeiss LSM800) and the image analysis software Imaris (high-definition 3D / 4D image analysis software). Figure 1B-1 shows Test Example 1c, where the cells were cultured in the control (DMSO solution without added autophagy inhibitor); Figure 1B-2 shows Test Example 1a, where the cells were cultured in the presence of an autophagy inhibitor (DMSO solution containing Bafilomycin A1); and Figure 1B-3 shows Test Example 1b, where the cells were cultured in the presence of an autophagy inhibitor (DMSO solution containing MHY1485). The distance between the two triangular arrows indicates the point between the hair (left side) and the hair follicle wall (right side) that is filled with dye-stained sebum oil and is the longest distance (hair-follicle wall distance: μm). As shown in Figures 1 and 2, the hair-to-follicle wall distance (μm) when the autophagy inhibitor (Bafilomycin A1) was added after culturing; and when the autophagy inhibitor (MHY-1485) was added after culturing was 1.8 times greater for Bafilomycin A1 and 1.6 times greater for MHY-1485 compared to the control (DMSO), with significant differences in both cases (p<0.05).

[0182] The above results confirmed that the inhibition of autophagy causes clogged pores. These results demonstrate that impaired autophagy in sebaceous gland cells leads to poor secretion of sebaceous gland secretions, resulting in clogged sebaceous ducts and hair follicles. Based on the above results, the inventors were able to elucidate for the first time the mechanism of clogged pore formation. The inventors found that in clogged skin, impaired autophagy in sebocytes leads to total sebocyte secretion failure and cell accumulation in the sebaceous ducts and hair follicles. Thus, impaired autophagy in sebocytes is thought to lead to clogged pores. The inventors' discovery provides important insight into the molecular mechanisms that maintain healthy pores. Furthermore, this study is expected to lead to the development of new skin care products that prevent enlarged pores and acne by maintaining appropriate autophagy in sebaceous gland cells, resulting in beautiful, even, and smooth skin.

[0183] <Test Example 2: Method for creating and observing a three-dimensional structure of sebaceous gland cells> <How to create spheroids> The sebaceous gland cells used were human-derived sebaceous gland cells (2 × 10 6 The cells are derived from Caucasian donors and are iPS cell-derived sebocytes (PCi-SEB), which are immature sebocytes. Phenocell also offers donors of Asian and African descent. Frozen human sebaceous gland cells (1 mL) were thawed in a warm bath at approximately 30°C. The thawed sebaceous gland cells were suspended in 4 mL of medium, and the suspension was centrifuged. The supernatant was removed and the sebaceous gland cells were collected. CnT-Prime differentiation medium (CELLnTEC) was added to the sebaceous gland cells, and 5 x 10 4 A cell suspension of 100 cells / mL was prepared.

[0184] In a 96-well plate having wells with a V-shaped or U-shaped vertical cross section of the bottom of the well, where multiple sebaceous gland cells can gather in the central region of the bottom of the cone-shaped depression, 100 μL of cell suspension was added per well, and 5 × 10 cells were collected per well. 3 Sebaceous gland cells were seeded onto the wells to form spheroids (see Figure 3). After seeding, the sebaceous gland cell suspension was cultured statically in an incubator (37°C, 5% CO2) for 11 days. During this period, the spheroid-forming medium was changed once a day. During this period, multiple sebaceous gland cells were cultured while gathering in the central region of the well, resulting in the formation of spheroids of sebaceous gland cells, and thus a three-dimensional structure of sebaceous gland cells was obtained.

[0185] The obtained three-dimensional structure of sebaceous gland cells was approximately spherical, with a minimum major axis of 400 μm and a maximum major axis of 500 μm, and this diameter was generally within the range of 400 to 500 μm (see FIGS. 3 and 4). The obtained three-dimensional structure of sebaceous glands was observed by fluorescently staining mature sebaceous glands with adipophilin as a maturation marker. Figure 4A is a photograph showing an overall image of the three-dimensional structure (spheroid) of sebaceous glands when the mature sebaceous glands were fluorescently stained. Figure 4B is a cross-sectional view of the stained spheroid of Figure 4A in the depth direction from the surface layer, showing the location of mature sebaceous glands in the spheroid obtained in this embodiment. As shown in Figure 4, adipophilin fluorescent staining was observed in most areas near the surface layer of the three-dimensional structure.

[0186] As a result, the surface layer of the obtained three-dimensional structure of sebaceous glands was densely populated with mature sebaceous glands, while the closer to the center, the fewer mature sebaceous glands were observed.The thickness of the surface layer containing the mature sebaceous glands was approximately 30 μm. The proportion of mature sebaceous gland cells present on the surface of the three-dimensional structure was approximately 80-100% of the surface. The proportion of this area was calculated based on the total area of ​​fluorescent emission of mature sebaceous gland cells in the surface image, obtained by fluorescently staining the mature sebaceous gland cells present in the three-dimensional structure, observing the three-dimensional structure under fluorescent observation, and multiplying the total area of ​​fluorescent emission of mature sebaceous gland cells by 100%.

[0187] <Total secretion regulation conditions> On day 11 after cell seeding, spheroids of sebaceous glands were transferred to glass-bottom dishes at room temperature (20°C) in the atmosphere. After extensive research, the inventors discovered that by changing the type of commercially available medium, mature sebaceous gland cells present in the surface layer of the three-dimensional structure could be either fully secreted or not, and thus discovered the conditions for regulating the full secretion of the three-dimensional structure of sebaceous glands.

[0188] <Starvation medium (medium for inducing autophagy)> Amino acid-free basal medium (serum-free): D-MEM (Dulbecco's Modified Eagle's Medium / high glucose; containing sodium pyruvate; amino acid-free) medium (Fujifilm Wako Pure Chemical Industries, Ltd.: 048-33575).

[0189] <Growth medium (medium for inhibiting autophagy)> D-MEM (Dulbecco's Modified Eagle's / L-glutamic acid; phenol red-free) medium (Fujifilm Wako Pure Chemical Industries, Ltd.: 040-30095) supplemented with 10% fetal bovine serum.

[0190] <Cell / Dead Cell Imaging Kit> Live / Dead Cell Imaging Kit TM Cell Imaging Kit (488 / 570): When detecting the fluorescence of live cells, live cells emit green fluorescence at an excitation wavelength of 488 nm and a detection wavelength of 515 nm. <Confocal laser microscope> Using a confocal laser microscope (Carl Zeiss LSM800) and image analysis software (Imaris (high-definition 3D / 4D image analysis software)): At room temperature (10-30°C), fluorescent observation was performed using time-lapse imaging of the three-dimensional structure of sebaceous gland cells in a glass-bottom dish (containing starvation medium or growth medium, test substance, CO2 concentration 5% CO2, atmospheric pressure).

[0191] <A state in which mature sebaceous gland cells undergo full secretion (under conditions using starvation medium)> A small amount of starvation medium was added to a glass-bottom dish containing spheroids of mature sebaceous gland cells, and the starvation medium was removed to wash the spheroids with starvation medium. The spheroids were then allowed to adapt to the starvation medium, and the spheroids and starvation medium were then allowed to come into contact with each other in a container in an incubator (37°C, 5% CO2). Then, in the incubator, 300 μL of spheroid and starvation medium and a live / dead cell imaging kit (LIVE / DEAD TMA mixture (cell suspension) containing spheroids, starvation medium, and fluorescent material was prepared by mixing 300 μL of the Cell Imaging Kit (488 / 570) with the spheroids and the starvation medium (D-MEM, high glucose, sodium pyruvate-containing, amino acid-free) in a glass-bottom dish (5% CO).

[0192] As described above, under conditions conducive to holocytosis, a fluorescent substance for detecting live cells was added to a glass-bottom dish containing spheroids and starvation medium, and mixed. Live sebaceous cells were fluorescently stained, and the three-dimensional structure of fluorescent sebaceous cells contained within the glass-bottom dish was observed using a confocal laser microscope. The glass-bottom dish was maintained at 37°C and 5% CO2. The time-dependent changes in the live cells were observed using time-lapse imaging, using the number of cells per unit volume that lost fluorescence over time as an indicator (see Figures 5-7).

[0193] When evaluating and / or selecting a substance with a global secretion inhibitory effect, add the test substance to the spheroid-forming medium containing the spheroids at least 2 hours before adding the fluorescent substance. For test substances that take time to exhibit efficacy, it is preferable to add the test substance to the spheroid-forming medium containing the spheroids at least 24 hours before adding the fluorescent substance. After culturing the mixture containing the spheroids, test substance, and spheroid-forming medium for a predetermined period of time, the spheroids are transferred to a test substance and starvation medium, and a fluorescent substance is added to the mixture. The spheroids in the mixture containing the fluorescent substance are subjected to fluorescence observation over time under conditions conducive to holocytosis. In the fluorescence observation over time, the number of cells per unit volume that lose fluorescence over time can be used as an indicator to determine whether the test substance has a holocytosis inhibitory effect, and this can be used to evaluate and / or select holocytosis inhibitors.

[0194] <A state in which mature sebaceous gland cells do not fully secrete (when using a growth medium)> A small amount of growth medium was added to a glass-bottom dish containing spheroids of mature sebaceous gland cells, and the growth medium was removed to wash the spheroids with the growth medium. The spheroids were then allowed to absorb the growth medium and then placed in an incubator (37°C, 5% CO2) to allow the spheroids to come into contact with the growth medium. Then, in the incubator, spheroids and 300 μL of growth medium were placed in a live / dead cell imaging kit (LIVE / DEAD TM The spheroids were mixed with 300 μL of the Cell Imaging Kit (488 / 570) and allowed to come into contact with the growth medium at 37°C under 5% CO2. A mixture (cell suspension) containing spheroids, growth medium, and fluorescent material was prepared, and this mixture was added to a glass-bottom dish (containing 5% CO2). The growth medium used was the Dulbecco's Modified Eagle's Medium (D-MEM) with L-glutamic acid (phenol red-free) supplemented with 10% fetal bovine serum (Fujifilm Wako Pure Chemical Industries, Ltd.: 040-30095).

[0195] As described above, under conditions that prevent secretion, a fluorescent substance for detecting live cells was added to a glass-bottom dish containing spheroids and growth medium, and mixed. Live sebaceous cells were fluorescently stained, and the three-dimensional structure of fluorescent sebaceous cells contained in the glass-bottom dish was observed using a confocal laser microscope. The glass-bottom dish was maintained at 37°C and 5% CO2. The time-dependent changes in the live cells were observed using time-lapse imaging, using the number of cells per unit volume that lost fluorescence over time as an indicator (see Figure 5).

[0196] When evaluating and / or selecting drugs with a pansecretory effect, add the test substance to the spheroid-forming medium containing the spheroids at least 2 hours before adding the fluorescent substance. For test substances that are unlikely to be effective, it is preferable to add the test substance to the spheroid-forming medium containing the spheroids at least 24 hours before adding the fluorescent substance. After culturing the mixture containing the spheroids, test substance, and spheroid-forming medium for a predetermined period of time, the spheroids are transferred to the test substance and growth medium, and a fluorescent substance is further added to the mixture. The spheroids in the mixture containing the fluorescent substance are subjected to fluorescence observation over time under conditions that do not cause holocrine secretion. In the fluorescence observation over time, the number of cells that have lost fluorescence over time per unit volume is used as an indicator to determine whether the test substance has a holocrine secretion-promoting effect, and this can be used to evaluate and / or select holocrine secretion-promoting agents.

[0197] <Confocal laser microscope observation> The spheroids in the mixture containing the test substance were imaged using a confocal laser microscope under time-lapse conditions of 37°C and 5% CO2. The confocal laser microscope had a 20x objective lens, and a laser excitation wavelength of 488 nm and detection wavelengths of 490-570 nm were used to observe live cells. Images were taken within a range of 100 μm from the spheroid surface, with the focal plane shifted in 1 μm increments. Furthermore, one set of laser irradiation and fluorescence imaging of the three-dimensional structure of sebaceous gland cells was performed every 10 minutes, and the time-dependent changes in live sebaceous gland cells were observed for 12 hours. The imaging conditions were one frame (still image) every 10 minutes, for a total of 12 hours (72 frames). The acquired images were converted into a three-dimensional image using the analysis software Imaris, based on depth-direction images captured at 1 μm intervals. A 62.4 μm (or horizontal) x 125 μm (or vertical) x 50 μm depth range could be arbitrarily cut out; three areas were cut out in this study. The acquired still images were played back as a video, and the number of cells whose green fluorescence had disappeared was counted from the start of imaging (when the fluorescent reagent was added) up to 12 hours.

[0198] The total number of cells whose green fluorescence had disappeared in these three locations was counted and averaged. This average value was used as the number of cells that had lost fluorescence per unit volume (mean value). This allowed us to determine the total secretion state of mature cells present in the surface layer of the three-dimensional structure (see Figure 6). Under conditions where total secretion occurs, the average number of cells that lost fluorescence per unit volume observed 12 hours after the addition of the fluorescent substance was approximately 10 (see Figures 5A and 6). On the other hand, under conditions where total secretion does not occur, the average number of cells that lost fluorescence per unit volume observed 12 hours after the addition of the fluorescent substance was approximately 1 (see Figure 5B).

[0199] Figure 5A shows time-lapse images taken at the start (Figure A-1) and end (Figure A-2) of observation when spheroids obtained in this embodiment were cultured under autophagy-inducing conditions using starvation medium, demonstrating that the sebaceous gland cells present in the surface layer of the spheroids had collapsed and the number of fluorescent cells per unit volume had decreased (particularly the area indicated by the arrow). Figure 5B shows images taken at the start (Figure B-1) and end (Figure B-2) of observation of spheroids obtained in this embodiment cultured under autophagy-suppressing conditions using a growth medium. These are time-lapse images showing that the sebaceous gland cells present in the surface layer of the spheroids did not collapse and the number of fluorescent cells per unit volume did not decrease (particularly the area indicated by the arrow).

[0200] In cultures under autophagy-inducing conditions, sebocytes that lost their green fluorescence were those undergoing holocytosis. Time-lapse images showed that the fluorescence of individual sebocytes disappeared over a short period of time. In the area where the fluorescence disappeared, the fluorescent sebocytes directly below them were exposed at the surface (see Figures 5A and 6). Thus, during the holocytosis process, sebocytes break down their cell membranes, releasing the sebum from within. The released sebum passes through the sebaceous gland ducts and hair follicles, allowing it to be efficiently excreted to the skin surface without clogging the ducts or pores. Normal holocytosis by mature sebocytes can prevent and improve clogged pores.

[0201] On the other hand, when cultured under autophagy-inhibiting conditions, sebaceous gland cells whose green fluorescence did not disappear were sebaceous gland cells that had not undergone holocytosis. In time-lapse images, sebaceous gland cells present on the surface of the three-dimensional structure could be observed even after 12 hours had passed. It is believed that the lack of holocytosis of these dead cells leads to accumulation in the sebaceous gland ducts and hair follicles, resulting in clogged pores. The inability of mature cells to undergo normal holocytosis can lead to clogged pores or worsen clogged pores.

[0202] <Test Example 3: Method for evaluating and / or selecting total secretion regulators> In Test Example 3A, under the total secretion regulation conditions described above under "Conditions in which total secretion does not occur in mature sebaceous gland cells (conditions using growth medium)", a fluorescent reagent, spheroids, and confocal laser microscope observations are used, and trehalose is used as the test substance, and a method for evaluating and / or selecting a test substance as a total secretion regulator is performed. Under spheroid formation conditions using CnT-Prime differentiation medium (CELLnTEC), one spheroid of this embodiment is brought into contact with trehalose, a test substance, for 6 hours to 24 hours. After contacting the spheroids with trehalose in an incubator (37°C, 5% CO2), the spheroids were grown under autophagy-inhibiting conditions using a growth medium of D-MEM (Dulbecco's Modified Eagle's / L-glutamic acid; phenol red-free) supplemented with 10% fetal bovine serum, and then visualized with a cell / dead cell imaging kit (LIVE / DEAD). TMThe fluorescent reagent from the Cell Imaging Kit (488 / 570) was added to the glass-bottom dish in the incubator and mixed to fluorescently stain live sebaceous cells. The state of the fluorescent spheroids in the glass-bottom dish containing growth medium, spheroids, and trehalose was observed at 37°C and 5% CO2 using a confocal laser microscope (Carl Zeiss LSM800) and image analysis software (Imaris, high-resolution 3D / 4D image analysis software) by time-lapse imaging (one frame every 10 minutes) for 720 minutes after the addition of the fluorescent reagent. Laser irradiation and fluorescence detection were performed every 10 minutes to synchronize with the time-lapse imaging. The state of the fluorescent sebaceous gland cells was determined by calculating the number of cells that lost fluorescence per unit volume during 720 minutes of imaging. If the number of lost fluorescent cells was 100% ± 30% of the number of cells cultured using starvation medium as a positive control, the trehalose was identified as a pansecretory agent or candidate for a pansecretory agent, and selected as a substance with pansecretory activity. By using trehalose as the selected substance in this manner, this embodiment can provide a holocrine secretion promoter, a holocrine secretion inducer, an agent for preventing, improving or treating clogged pores, and the like, all of which contain trehalose as an active ingredient.

[0203] Furthermore, by performing the same test as in Test Example 3A above using retinoids, benzoyl peroxide, oleic acid, curcumin, vitamin D, rapamycin, wortmannin, and chloroquine as test substances instead of trehalose, it will be possible to evaluate or select retinoids, benzoyl peroxide, curcumin, vitamin D, and rapamycin as total secretion promoters, and oleic acid, wortmannin, and chloroquine as total secretion inhibitors.

[0204] Furthermore, in Test Example 3B, a method for evaluating and / or selecting a test substance as a global secretion inhibitor under autophagy-inducing conditions was performed. In this case, one spheroid of this embodiment was contacted with the test substance trehalose for 6 to 24 hours under spheroid-forming conditions, and then the spheroid and trehalose were placed in the presence of an autophagy-inducing medium under autophagy-inducing conditions, and a fluorescent reagent was added to perform fluorescent staining and fluorescent observation. The test substance used in the contacting step and the observation step in the medium was the same concentration. Fluorescent staining and fluorescent observation were performed in the same manner as in Test Example 3A above. The state of the fluorescent sebaceous gland cells is determined by calculating the number of cells that have lost fluorescence per unit volume during 720 minutes of imaging. If the number of lost fluorescent cells is 100% ± 30% of the number of cells cultured using growth medium as a positive control, the test substance is identified as a total secretion inhibitor or candidate for a total secretion inhibitor, and is selected as a substance with total secretion inhibitory activity. This allows evaluation or selection of a total secretion inhibitor from a large number of test substances.

[0205] The test substances shown in Table 1 were evaluated for their holocrine secretion-promoting or holocrine secretion-inhibiting effects using spheroids according to the above-mentioned Test Example 3A, and compounds having these effects were selected. The spheroids used were produced according to the <Spheroid Production Method> in the above-mentioned Test Example 2.

[0206] Trehalose and rapamycin were added to the growth medium. The growth medium used was the "D-MEM (Dulbecco's Modified Eagle's / L-glutamic acid; phenol red-free) medium supplemented with 10% fetal bovine serum" described in Test Example 3A above. The starvation medium (autophagy induction medium) used was "serum-free, amino acid-free D-MEM (high glucose (4500 mg / L)) (containing sodium pyruvate (110 mg / L) and amino acids-free) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)."

[0207] The "total number of sebaceous gland cells that lost their fluorescence (three locations)" in Table 1 refers to the number of cells that lost their fluorescence per unit volume (62.4 μm × 125 μm × 50 μm; as shown in Figure 6) (corresponding to the "number of fluorescent cells that lost" in Test Example 3). The three numbers in this column, such as "13, 8, and 10," represent measurements taken at three different locations within the same spheroid.

[0208] The denominator of the "fluorescence loss cell ratio ( / starvation medium, no drug added) (%)" in the selection of all secretagogue candidates in Table 1 is 9.7, which is the "average" of "starvation medium with added drug (-: no drug)."

[0209] The state of the fluorescent sebaceous gland cells was determined by calculating the number of cells that lost fluorescence per unit volume during 720 minutes of imaging. If the number of lost fluorescent cells was 100% ± 30% of the number of cells cultured using starvation medium as a positive control, the test substance was determined to be a pansecretory agent or a candidate pansecretory agent, and was selected as a substance with pansecretory activity.

[0210] The state of the fluorescent sebaceous gland cells is determined by calculating the number of cells that have lost fluorescence per unit volume during 720 minutes of imaging. If the number of cells that have lost fluorescence is 100% ± 30% of the number of cells that have lost fluorescence in culture using growth medium as a positive control, the test substance can be identified as a total secretion inhibitor or a candidate for a total secretion inhibitor, and can be selected as a substance with total secretion inhibitory activity.

[0211] As a result, it was confirmed that trehalose and rapamycin have a more effective holocytosis-promoting effect or autophagy-promoting effect, and that they can be used as active ingredients of holocytosis regulators.

[0212] [Table 1]

[0213] <Test Example 4: Evaluation or selection of the overall secretion-regulating effect of a test substance by human evaluation test> <Analysis of the effect of trehalose lotion on the size of nasal blackheads after four weeks of continuous use> Use of trehalose lotion and collection of blackheads Subjects: Men aged 20-40 with noticeable blackheads on their noses Number of subjects: 8 people in the trehalose-containing (3%) lotion group Trehalose-free lotion group: 6 people

[0214] <Exam Contents> 1. Four weeks before starting to use the test lotion, blackheads were removed from the nose using a nasal blackhead removal pack (this was done to standardize the time it took for blackheads to form among the subjects, in preparation for the collection of blackheads when starting to use the lotion four weeks later. Therefore, the removed blackheads were discarded). 2. After the four weeks described above, blackheads were removed from the nose using a nasal blackhead removal pack, and the removed blackheads were stored frozen while still attached to the pack for confocal microscopy observation. From this day onwards, the subject began using the lotion. 3. After the four weeks described above, keratin plugs were removed from the nose using a nasal keratin plug removal pack, and the removed keratin plugs were stored frozen while still attached to the pack for confocal microscopic observation. As described above, two samples of keratin plugs were collected from each subject, one before and one after use of the lotion, thereby obtaining keratin plugs from each subject before and after use.

[0215] <Observation and diameter measurement of keratinocytes using a confocal laser microscope> 1. After thawing the nose pack sheet with keratin plugs attached, the sheet was flattened and placed on the observation stage of a confocal microscope (ZEISS, upright confocal laser microscope LSM700) with the keratin plug-attached surface as the observation surface. Furthermore, the excitation wavelength of the confocal microscope was set to 555 nm, and the fluorescence wavelength for detection was set to 559 nm or higher. The fluorescence wavelength used for cell biology observations is typically in the range of 300 to 800 nm. 2. The keratinocyte was irradiated with a 555 nm (excitation light) laser, and the autofluorescence emitted from the keratinocyte was detected at a fluorescence wavelength of 559 nm or higher. Using this method, successive optical tomographic images of the entire keratinocyte were taken, and the successive images were reconstructed into a 3D image using ZEISS's analysis software, ZEN Black Edition. After constructing a 3D image of the keratinocyte, the maximum diameter was measured using the same software. By taking successive cross-sectional photographs (sequential tomographic images) of the keratin plug, we were finally able to create a three-dimensional image. The reason for reconstructing a 3D image of this keratin plug is not to resemble the 3D structure of cells, but to determine the point at which the diameter of the keratin plug is greatest.The 3D cellular structure is an aggregate of living sebaceous gland cells, imitating the sebaceous gland of a living body, while the keratin plug in this case is a solidified mixture of dead sebaceous gland cells, sebum, and the stratum corneum.

[0216] 2. The above procedure was carried out on 11-24 keratin plugs per sample (one nose pack sheet with keratin plugs attached).

[0217] 3. For one sample, the maximum diameter of all keratin plugs was averaged, and the average value after using the lotion was divided by the value before using the lotion to determine the rate of change in the maximum diameter of keratin plugs. This analysis method is not intended to evaluate changes in pore diameter on the skin surface (i.e., the apparent prominence of pores), but rather to estimate whether or not holocytosis of sebaceous gland cells (rupture and digestion of sebaceous gland cells) has occurred appropriately. For this purpose, we adopted this method because we determined that it would be more appropriate to measure the diameter of the keratin plugs clogged inside the pores rather than the diameter of the pore outlet.

[0218] <Results and Discussion> When sebaceous glands are not secreting properly, dead sebaceous gland cells accumulate in the pores. As a result of this accumulation, it was confirmed that the size of the keratinized plug, i.e., the maximum diameter of the keratinized plug, increases. In other words, it can be assumed that promoting sebaceous gland secretion reduces the accumulation of dead sebaceous gland cells in the pores, thereby reducing the maximum diameter of the keratinized plug. Furthermore, these results suggest that this maximum diameter was reduced by using a lotion containing trehalose, an autophagy promoter (considered to be a holocytosis promoter).

Claims

[Claim 1] A total secretion regulator comprising one or two compounds selected from the group consisting of oligosaccharides and macrolide compounds as active ingredients.

Images