CN115698315A - Methods for evaluating and/or selecting whole plasma secretion modulators - Google Patents

Abstract

The present invention provides techniques related to the regulation of whole plasma secretion of sebaceous gland cells. The invention provides a method for evaluating and/or selecting a whole plasma secretion regulator, which comprises the following steps: an observation step of observing the state of a sebaceous gland cell that fluoresces after a single or a plurality of sebaceous gland cells and a test substance are present; and a discrimination step of discriminating the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator on the basis of the state of the fluorescent sebaceous gland cells. The present invention also provides a three-dimensional structure of sebaceous gland cells whose surface layer is composed of mature sebaceous gland cells, and a method for producing a three-dimensional structure of sebaceous gland cells, wherein a plurality of seeded sebaceous gland cells are cultured on a cell adhesion-inhibiting treatment surface of a mortar-shaped recess to obtain a three-dimensional structure of sebaceous gland cells. The present invention also provides a whole plasma secretion regulator containing, as an active ingredient, a compound selected from the group consisting of an oligosaccharide and a macrolide compound.

Description

Methods for evaluating and/or selecting whole plasma secretion modulators

Technical Field

The present invention relates to a method for evaluating and/or selecting a holocrine (holocrine) regulator, a three-dimensional structure of sebaceous gland cells, a method for producing a three-dimensional structure of sebaceous gland cells, a holocrine secretion regulator, and the like.

Background

Sebaceous glands are present alongside parts having hair organs. Sebaceous glands secrete sebum from sebaceous gland openings that open onto the inner surface of pores. The sebaceous gland is of a bag-shaped structure, sebaceous gland cells are densely packed in the sebaceous gland, and the sebaceous gland cells are used for releasing sebum. Sebum is composed of wax esters, triglycerides, fatty acids, and the like, and this sebum is mixed with moisture such as sweat and emulsified into a skin surface film. The skin surface membrane, also known as sebum membrane, is used to protect the skin surface.

Examples of the site where sebaceous glands are present in large numbers include scalp, forehead including nose, underarm, chest, and vulva. Skin problems such as clogging of pores involving sebaceous glands tend to occur in the vicinity of these regions. Pore blockage is one of the skin problems that need to be prevented, ameliorated or treated.

Patent document 1 discloses a method for producing mature human sebaceous gland cells, which comprises a step of culturing immature human sebaceous gland cells under hypoxic conditions, for the purpose of providing cultured sebaceous gland cells capable of forming or accumulating oil droplets.

Non-patent document 1 discloses a laminated sheet obtained by laminating sebaceous gland cell layers (non-patent document 1).

Further, a method of embedding a plurality of sebaceous gland cells in matrigel and culturing the cells to form spheroids in which mature sebaceous gland cells are concentrated in the center has been disclosed (non-patent document 2).

Documents of the prior art

Patent literature

Patent document 1: japanese laid-open patent publication No. 4-35718

Non-patent literature

Non-patent document 1: christine Barrault, et al, experimental Dermatology.2012Apr,21 (4): 314-316.

Non-patent document 2: alona Feldman, et al, nature. 10:2348

Disclosure of Invention

Problems to be solved by the invention

It has long been considered that acne (hereinafter also referred to as "pore blockage") is formed due to thickening of the stratum corneum to block the pore outlet, and sebum accumulates and solidifies inside the pores. In addition, conventionally, the amount of sebum production has been focused on as one of the causes of pore clogging, and experimental systems for evaluating the amount of sebum production as described in patent documents 1 to 3 have been mainly studied and utilized.

However, such conventional findings cannot explain the formation of open comedones and micro comedones, which are a pathological condition in which the pores are clogged in a state in which the pore outlet is open. Thus, the detailed mechanism of pore blockage formation remains unexplained.

Accordingly, it is a primary object of the present invention to provide a technique relating to pore clogging formation.

Means for solving the problems

The present inventors have conducted intensive studies on the detailed mechanism of pore clogging formation, and as a result, found that pore clogging formation is associated with the full-plasma secretion of sebaceous gland cells. More specifically, the present inventors found that the whole plasma secretion state (normal or abnormal) of a sebaceous gland cell can be regulated by regulating autophagy conditions for the sebaceous gland cell, and that the whole plasma secretion state of the sebaceous gland cell is involved in pore blockage formation.

In the present specification, a sebaceous gland cell produces and stores sebum intracellularly, and finally the cell death is disrupted to secrete the sebum intracellularly into the outside, and this secretory form is referred to as a whole plasma secretion.

Further, the present inventors have further studied a technique relating to the regulation of the whole plasma secretion of sebaceous gland cells, and as a result, have found for the first time that, from the state of a living sebaceous gland cell that emits fluorescence, the fluorescence of the sebaceous gland cell disappears when the whole plasma secretion occurs, while the fluorescence of the sebaceous gland cell is maintained when the whole plasma secretion does not occur. The present inventors have found for the first time that the state of the whole plasma secretion of sebaceous gland cells and the condition for regulating the whole plasma secretion of sebaceous gland cells can be grasped over time by observing the state of the fluorescent sebaceous gland cells with fluorescence over time, and have found for the first time a method for evaluating and/or selecting a whole plasma secretion regulator using this technique. In addition, the inventor firstly discovers a novel three-dimensional structure of the sebaceous gland cell and a preparation method thereof, wherein the novel three-dimensional structure of the sebaceous gland cell can be used for better observing the whole-plasma secretion of the sebaceous gland cell.

Thus, the present invention provides a method for evaluating and/or selecting a panniculus adiposus cell-effective panniculus adiposus secretion regulator, a panniculus adiposus secretion regulator containing a substance selected by the method, a novel three-dimensional structure of a sebaceous gland cell, and a method for producing the same, as a technique related to pore formation.

Namely, the present invention is as follows.

The present invention provides a method for evaluating and/or selecting a whole plasma secretion regulator, comprising the steps of,

and (3) an observation procedure: fluorescence observation of the state of a fluorescent sebaceous gland cell after the presence of a single or a plurality of sebaceous gland cells and a test substance; and

a discrimination step: and discriminating the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator based on the state of the fluorescent sebaceous gland cells.

The whole plasma secretion regulator may be 1 or more than 2 selected from the group consisting of whole plasma secretion promoter, whole plasma secretion inhibitor, pore-clogging formation regulator, and autophagy regulator for sebaceous gland cells.

The sebaceous gland cell may be a three-dimensional structure composed of a plurality of sebaceous gland cells.

The observation may be made using time-lapse photography.

The observation may be performed using a fluorescence microscope.

In the observation step, the state of the fluorescent sebaceous gland cells can be observed fluorescently in the presence of a test substance and one or a plurality of sebaceous gland cells under the full-plasma secretion regulation conditions.

The whole plasma secretion regulatory condition is an autophagy regulatory condition, which may be an autophagy-inhibiting condition or an autophagy-inducing condition.

The state of the sebaceous gland cells that emit light can be determined based on the number of fluorescence-lost cells per unit volume.

The present invention also provides a method for evaluating and/or selecting a whole plasma secretion regulator, comprising the steps of,

and (3) an observation procedure: performing fluorescence observation on the state of the collected fluorescent angle plug after the single or multiple sebaceous gland cells exist with the tested substance; and

a discrimination step: and judging the tested substance to be a holoplasmic secretion regulator or a candidate of the holoplasmic secretion regulator according to the state of the fluorescent angle bolt.

The keratotic plugs in the observation step are a plurality of keratotic plugs which are picked up from the skin surface of the mammal by an adhesive sheet and attached to the adhesive sheet, and/or

The state of the plug in the determination step is a rate of change of the maximum plug diameter obtained by dividing an average value of the maximum plug diameter after use of the test substance by an average value of the maximum plug diameter before use.

The present invention provides a whole plasma secretion regulator comprising 1 or 2 compounds selected from oligosaccharide and macrolide compounds as active ingredients.

The present invention also provides a three-dimensional structure of sebaceous gland cells, wherein the surface layer of the three-dimensional structure is composed of mature sebaceous gland cells.

The present invention also provides a method for producing a three-dimensional structure of sebaceous gland cells, comprising culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting treated surface of a mortar-shaped recess to obtain a three-dimensional structure of sebaceous gland cells.

The three-dimensional structure may be spherical.

The three-dimensional structure can be used for fluorescence observation.

Can be used for the evaluation and/or selection method of the whole plasma secretion regulator.

The mortar-shaped recess may be shaped such that a plurality of sebaceous gland cells can aggregate in a bottom central region of the recess.

Effects of the invention

According to the present invention, a technique related to pore clogging formation can be provided. The effects described herein are not limited, and may be any effects described in the present specification.

Drawings

Fig. 1, a is a schematic cross-sectional view of a hair follicle organ. Fig. 1B shows the results of a confirmation test on the effect of the addition of an autophagy inhibitor on pore blockage in the vicinity of the human sebaceous gland ducts. In FIG. 1, B-1 is after incubation in the control (DMSO solution without autophagy inhibitor), B-2 is after incubation in the presence of autophagy inhibitor (DMSO solution containing baveromycin A1) in FIG. 1, and B-3 is after incubation in the presence of autophagy inhibitor (DMSO solution containing MHY 1485) in FIG. 1.

Fig. 2 is a result of a confirmation test concerning the effect of adding an autophagy inhibitor on pore clogging in the vicinity of a human-derived sebaceous gland duct. The hair-follicle-wall distance (. Mu.m) was measured before the culture, after the culture in the presence of a control (DMSO), after the culture in the presence of an autophagy inhibitor (baveromycin A1) and after the culture in the presence of an autophagy inhibitor (MHY-1485) in this order from the left. * MEAN ± SEM P <0.05, indicating a significant difference.

FIG. 3 shows a three-dimensional structure of sebaceous gland cells obtained by culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting treated surface of a mortar-shaped well subjected to a cell adhesion-inhibiting treatment. Fig. 3a is a schematic view of the bottom of the mortar-shaped concave portion in a V-shape, and fig. 3B is a schematic view of the bottom of the mortar-shaped concave portion in a U-shape. FIG. 3C is a photograph under an optical microscope showing the three-dimensional structure (spheroids) of sebaceous gland cells obtained by culturing on a cell adhesion-inhibiting treated surface (hydrophilized surface) of a mortar-shaped well.

Fig. 4 a is a photograph taken by confocal laser microscopy showing the overall appearance of the three-dimensional structure (spheroid) of the sebaceous gland cells when the mature sebaceous gland cells are fluorescently stained. At this time, as a maturation marker, a protein associated with fat differentiation (Adipophilin) was used for staining. Fig. 4B is a cross-sectional view of the stained spheroid shown in fig. 4 a in the depth direction from the surface toward the center, and is an image showing the location of mature sebaceous gland cells in the spheroid obtained in this embodiment.

FIG. 5A is an image of the spheroid obtained in the present embodiment at the start of observation (FIG. A-1) and at the end of observation (FIG. A-2) in culture under autophagy inducing conditions using starvation medium, and is a time-lapse image (particularly, an arrow portion) showing that sebaceous gland cells present in the surface layer of the spheroid are ruptured and the number of fluorescence-lost cells per unit volume is large. FIG. 5B is a graph of the spheroids obtained in the present embodiment, showing the start of observation (Panel B-1) and the end of observation (Panel B-2) during culture under autophagy-inhibiting conditions using a propagation medium, and a time-lapse graph (particularly, the arrow portion) showing that sebaceous gland cells present in the surface layer of the spheroids are not ruptured and the number of fluorescence-depleted cells per unit volume is small.

Fig. 6 is images a to C of spheroids obtained in the present embodiment when cultured under autophagy-inducing conditions using the spheroids and starvation medium. In FIG. 6, A to C show the numbers of cells in which fluorescence per unit volume disappears in the frame A (field 1: lower frame of field), B (field 2: upper left frame) in FIG. 6, and C (field 3: lower right frame) in FIG. 6. A quadrangular frame: 62.4. Mu. Mx 125. Mu. Mx50 μm. Cells with lost fluorescence were obtained from the fields of view 1 to 3 in the images a to C, and the average value of the cells was calculated as the number of cells with lost fluorescence per unit volume, and the results are shown in the lower part of the figure.

Fig. 7 is time-lapse images a to C of spheroids when cultured under autophagy-inducing conditions using the spheroids obtained in the present embodiment and a starvation medium. The process of fluorescence disappearance of 1 cell in the spheroid surface layer is shown in fig. 7 a (arrow portion at observation time 600 minutes), fig. 7B (arrow portion at observation time 610 minutes), and fig. 7C (arrow portion at observation time 620 minutes).

Fig. 8 is a view showing fluorescence observation of a state of a fluorescent corner plug by removing the corner plug by pasting with a tape, irradiating excitation light to a plurality of corner plugs on the tape to detect autofluorescence, reconstructing a three-dimensional structure of an image of the corner plugs, and observing the fluorescence.

Fig. 9 is a graph showing the rate of change in the maximum diameter of the keratotic plug as an evaluation of the effect of 4 weeks of continuous use of a cosmetic water with or without trehalose on the size of the keratotic plug. Rate of change of the maximum diameter of the corner key (sample =1 before use). The mean value of the trehalose-free group (N = 6) was 1.07500 (standard error 0.04200), and the mean value of the trehalose-containing group (N = 8) was 0.86644 (standard error 0.02755). For each subject sample, the maximum diameter of 11 to 24 corner pins was measured to obtain an average value. The rate of change of the maximum diameter of the kerato plug was calculated by dividing the average value of the test substance after use by the average value before use.

Detailed Description

The following describes embodiments for carrying out the present invention. The embodiments described below are merely examples of typical embodiments of the present invention, and do not limit the scope of the present invention. In the present specification, "%" represents mass% and "-" represents a numerical range unless otherwise specified, the range includes numerical values at both ends. The upper limit and the lower limit of the numerical values may be arbitrarily combined as needed.

<1. Method for evaluating and/or selecting whole milk secretion regulator according to the present embodiment >

In one aspect of the embodiment according to the present invention, it is also possible to provide a method for evaluating and/or selecting a modulator of the whole plasma secretion of sebaceous gland cells.

< method of the first embodiment of the present invention >

The method of the first embodiment of the present invention is a method for evaluating and/or selecting a whole plasma secretion regulator, and comprises the following steps. And (3) an observation procedure: allowing single or multiple sebaceous gland cells to exist with the tested substance, and carrying out fluorescence observation on the state of the sebaceous gland cells which emit fluorescence; and/or a discriminating step: and discriminating the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator based on the state of the fluorescent sebaceous gland cells. In this observation step, fluorescence observation is preferably performed under the whole plasma secretion-regulating condition.

As a conventional evaluation method of pore clogging, a method of evaluating whether or not pore clogging is small by microscopic observation using a magnifying glass; a method in which a region to be evaluated is photographed in a dark place by irradiating UVA light to the region using a facial imaging apparatus such as VISIA, and since a pore-clogging portion emits red or pale green light, it is possible to evaluate whether the number of light-emitting pores is reduced or not from a photographed image by photographing with this method; and a method of evaluating whether or not the rough feeling is reduced by palpation of a human, because the skin surface is rough when pores are clogged.

However, in the methods for evaluating a whole plasma secretion regulator or the methods for selecting a substance to be used as a whole plasma secretion regulator using these conventional evaluation methods, since a subject is targeted or sensory evaluation is performed, when the number of test substances is large, it is difficult to evaluate all the whole plasma secretion regulators quickly and to obtain reproducibility of the result. In addition, since pore clogging involves various mechanisms, these conventional evaluation methods are not suitable as a direct evaluation method of the secretion of the whole milk at the initial stage of pore clogging.

In contrast, in the method according to the first embodiment of the present invention, since an in vitro test can be performed using animal cells, and the test can be easily performed or reproduced, even if a plurality of test substances are present, the evaluation or selection can be easily performed, and the reproducibility of the result is high. In addition, in the present embodiment, since observation equipment (for example, fluorescence microscope-related equipment) and/or holoplasmic secretion regulation conditions can be used, the holoplasmic secretion regulation of sebaceous gland cells can be evaluated more objectively and with high accuracy, and these operations are not complicated.

Therefore, in the present embodiment, it is possible to easily evaluate whether or not a test substance is suitable as a whole plasma secretion regulator and/or to select a test substance as a whole plasma secretion regulator (specifically, a substance having a whole plasma secretion regulating effect). In addition, in the present embodiment, objectivity is high, and reproducibility of the result is also good.

The method according to the first embodiment of the present invention can be used to explain or analyze the skin problem with clogged pores, and can also provide a countermeasure against the skin problem with clogged pores.

For example, the problem of skin clogging with pores includes blackheads, open pores, rough pores, reddened pores, acne, seborrheic dermatitis, and the like.

In the present specification, the term "pore clogging" means that the stratum corneum is abnormally accumulated to clog the pore outlet, and sebum becomes hard in the pores to clog them.

In the present specification, the term "pore blackheads" means that substances clogging pores are oxidized by air to blacken.

In the present specification, the term "pore opening" means that sebum accumulates in the pores and hardens, and the pores on the surface are expanded.

In the present specification, rough pores refer to a feeling that sebum accumulates and hardens inside pores, and the pores slightly bulge.

In the present specification, it is considered that redness of pores occurs because sebum accumulates in pores and becomes hard, which changes the composition of sebum in hair follicles or changes the environment for bacterial growth present in skin (sebaceous glands, in hair follicles, on the surface layer, etc.).

In the present specification, it is considered that the formation of acne (acne) and seborrheic dermatitis is caused by the accumulation and hardening of sebum in pores, which causes the composition of sebum in hair follicles to change, or the environment for the growth of bacterial species present in skin (sebaceous glands, in-hair follicles, on the surface layer, etc.) to change.

The first embodiment of the present invention preferably includes the observation step and/or the discrimination step, and more preferably, the observation step is performed in the order of the discrimination step.

The first embodiment of the present invention more preferably includes a sebaceous gland cell culturing step, an observation step, and a discrimination step, and more preferably these steps are performed sequentially.

In the first embodiment of the present invention, it is preferable that the culture step and the observation step include a contact step of contacting the sebaceous gland cells with the test substance, more preferable that the culture step, the contact step, the observation step, and the discrimination step are included, and further preferable that these steps are performed in this order.

In the description of the whole plasma secretion regulator and the like in the first embodiment of the present invention, the following description of the second embodiment of the present invention and "2" to "3" may be applied to the first embodiment of the present invention and may be appropriately applied.

<1-1. Procedure for culturing sebaceous gland cells >

In the first embodiment of the present invention, the sebaceous gland cell culture step may be provided before the contact step, or the culture step may be omitted. The culture step can be performed under in vitro culture conditions for normal sebaceous gland cells (preferably of human origin). In this culture step, it is preferable to seed immature sebaceous gland cells and culture them in a state enabling full-plasma secretion. Through this culturing step, sebaceous gland cells in a state of being capable of full-plasma secretion can be obtained.

In the present specification, "mature sebaceous gland cells (or mature sebaceous gland cells)" refer to differentiated sebaceous gland cells that have sebum in them and are in a state of full-plasma secretion.

In the present specification, the term "immature sebaceous gland cells (or immature sebaceous gland cells)" is used separately from the term "mature sebaceous gland cells" described above, and refers to undifferentiated sebaceous gland cells other than the term "mature sebaceous gland cells" described above or to sebaceous gland cells that do not have sebum in their cells.

In the step of culturing sebaceous gland cells according to the first embodiment of the present invention, it is preferable that a plurality of sebaceous gland cells be seeded into the culture medium in the culture container and cultured (see fig. 3). The medium is a propagation medium for animal cells which is generally used for propagation, maintenance, differentiation, etc., and a propagation medium generally used for the culture of sebaceous gland cells described in the following < medium for suppressing total plasma secretion > can be suitably used.

The sebaceous gland cells to be seeded are not particularly limited, and are preferably derived from mammals. Examples of the mammal include humans, hamsters, pigs, mice, rats, and the like, and from the viewpoint of availability and culture maintenance, humans and/or hamsters are preferable. Commercially available products can be used for the sebaceous gland cells. In addition, human origin includes caucasian species, asian species, african species, and the like, but is not limited thereto. In the present embodiment, any species of the species may be used, and the caucasian species is preferable.

The seeding concentration of the sebaceous gland cells in the culture medium is preferably 1X 10 ² ～1×10 ⁵ cells/mL, more preferably 5X10 ² ～2×10 ⁴ cells/mL, more preferably 1X 10 ³ ～1×10 ⁴ cells/mL.

The culture vessel may be a culture vessel (for example, made of plastic or glass) used under ordinary conditions for in vitro culture of sebaceous gland cells, and examples thereof include a culture dish, a cell culture plate such as a microplate (for example, 6 well type, 24 well type, etc.), and the like.

The sebaceous gland cell culture step can be performed under normal in vitro culture conditions for sebaceous gland cells (preferably of human origin), preferably under culture conditions using a propagation medium (hereinafter also referred to as "propagation medium culture conditions"). As the culture conditions, for example, the pH of the medium is preferably about 7 to 8, and CO is cultured ₂ Preferably about 4 to 10% (preferably 4 to 6%) in the atmosphere, and the culture temperature is preferably about 30 to 40 ℃, more preferably about 33 to 38 ℃, and still more preferably about 36 to 37 ℃.

The culture period is not particularly limited, and is preferably 5 to 15 days, more preferably 6 to 13 days, and still more preferably 7 to 12 days after seeding.

The culture can be performed under the conditions of standing, shaking or rotation, preferably in a standing state, and the medium replacement may be performed in the number of replacement times of an average number of days, preferably 1 time for 1 to 3 days, and preferably 1 time for 1 to 2 days.

In the procedure for culturing sebaceous gland cells, the method for producing a three-dimensional structure of sebaceous gland cells described in "2" below is more preferably used. In this culture step, a container having a mortar-shaped well for seeding and culturing a plurality of immature sebaceous gland cells is preferably used. In this culture step, it is more preferable that the plurality of seeded sebaceous gland cells be cultured on a cell adhesion-inhibiting treated surface (more preferably, a hydrophilized surface) on the inner surface of the mortar-like concavity.

In addition, when the sebaceous gland cells after the culture step are used in the contact step or the observation step, the cells are preferably transferred to a container for a fluorescence microscope, and the contact step may be performed at the time of transfer.

In the first embodiment of the present invention, a culture apparatus for animal cells configured to perform a culture process of sebaceous gland cells may be used, and the culture apparatus may be used to maintain an observation target in an observation process. The animal cell culture apparatus may include, for example, an incubator, a humidity, temperature, and gas control unit, and the like.

<1-2. Contact Process >

In the first embodiment of the present invention, the contact step is preferably provided before the observation step.

In the contacting step in the first embodiment of the present invention, it is preferable that the sebaceous gland cells are contacted with the test substance under the culture conditions of the propagation medium (suitably, under the conditions for spheroid production described below). By including this contact step, the whole plasma secretion regulatory action of the test substance on the sebaceous gland cells can be more favorably observed by fluorescence in the subsequent observation step, and thus the whole plasma secretion regulatory action of the test substance can be more easily discriminated.

The contact period in the contact step is not particularly limited, and may be appropriately adjusted in consideration of the rate of the whole plasma secretion regulatory action of the test substance on the sebaceous gland cells. The factors for the rapid and slow whole plasma secretion regulating action include, for example, the onset rate of the test substance (immediate effect, delayed effect), the solubility of the test substance (high solubility, low solubility), and the like, but are not limited thereto.

The contact period is preferably set before the addition of the fluorescent reagent, from the viewpoint of easily obtaining the fluorescence observation result with high accuracy.

The contact period in the contact step is preferably at least 2 hours after the contact with the test substance, and an appropriate lower limit value is more preferably 4 hours or more, further preferably 6 hours or more, further preferably 8 hours or more, and more preferably 12 hours or more, and an appropriate upper limit value is not particularly limited, and examples thereof include 48 hours or less, 36 hours or less, and 24 hours or less. The suitable contact period is preferably 6 to 24 hours.

In addition, it is preferable to perform fluorescence observation of the sebaceous gland cells by adding a fluorescent reagent after the contact step, but from the viewpoint of enabling the full-plasma secretion regulating action of the test substance to be observed more favorably, it is preferable to perform observation in a state where the sebaceous gland cells are in contact with the test substance also during the fluorescence observation, and in this case, it is more preferable to perform fluorescence observation under the full-plasma secretion regulating condition.

In the first embodiment of the present invention (preferably after the contact step), the reaction is preferably carried out in a container for a fluorescence microscope, and the material of the container is preferably, for example, glass, plastic, or a combination thereof, and more specifically, a glass-bottom petri dish or the like is mentioned, but the present invention is not limited thereto. When the vessel contains sebaceous gland cells, the above-mentioned method is preferably adopted in the vessel<Procedure for culturing sebaceous gland cells>Conditions for the production of sebaceous gland cells (e.g. CO) ₂ Concentration, temperature).

In the first embodiment of the present invention, a pretreatment step, such as washing with the propagation medium, may be performed before the contact step.

<1-3. Observation Process >

The following describes an observation step in the first embodiment of the present invention.

In the observation step in the first embodiment of the present invention, it is preferable that the state of the fluorescent sebaceous gland cells is observed by fluorescence in the presence of a test substance in one or a plurality of sebaceous gland cells. This makes it possible to perform fluorescence observation of the effect of the test substance on the regulation of the whole-plasma secretion of the sebaceous gland cells, and to obtain the state of the sebaceous gland cells at that time as an observation result (preferably, an observation image). For example, the test substance may also be evaluated and/or selected as a whole plasma secretion modulator or a candidate for a whole plasma secretion modulator, taking into account the difference from the comparison control.

In the observation step, the culture conditions are not particularly limited, and for example, the culture conditions described in <1-1. Sebaceous gland cell culture step > above can be appropriately used. The medium used in this case is not particularly limited, and may be any known or commercially available medium, and may be appropriately selected in consideration of the state of the sebaceous gland cells that emit light during fluorescence observation.

In a more preferred embodiment of the observation step, the observation step is performed under whole-plasma secretion regulatory conditions, and more preferably, under whole-plasma secretion regulatory conditions, the state of a sebaceous gland cell that fluoresces is observed fluorescently by allowing a single or a plurality of sebaceous gland cells to exist together with the test substance.

<1-3-1. Total plasmatic secretion Regulation Condition >

The condition for regulating the whole plasma secretion in the first embodiment of the present invention is preferably a condition capable of regulating the induction of whole plasma secretion and the inhibition of whole plasma secretion in a sebaceous gland cell (preferably, a mature sebaceous gland cell).

The conditions for regulating the whole plasma secretion include, for example, 1 or 2 or more kinds of combinations selected from the group consisting of conditions for adding a drug such as a physiologically active agent (an accelerator or an inhibitor), medium conditions (for example, medium composition, temperature, and the like), the state of sebaceous gland cells (for example, a three-dimensional structure having mature sebaceous gland cells in the surface layer), and the like, but are not limited thereto.

Among these, the use of the medium conditions is preferred because observation results with high accuracy and high reproducibility can be objectively obtained. In addition, the use of the state of sebaceous gland cells is preferred because the whole plasma secretion-regulating action can be easily observed by fluorescence. More preferably, the conditions are a combination of the culture medium conditions and the state of the sebaceous gland cells.

The whole plasma secretion regulatory condition in the first embodiment of the present invention is preferably a whole plasma secretion inducing condition and/or a whole plasma secretion inhibiting condition. In the first embodiment of the present invention, by using the induction conditions of the whole plasma secretion, the mature sebaceous gland cells die, at which time the cells themselves are ruptured and simultaneously the secretions including sebum as the contents of the sebaceous gland cells are released outside the sebaceous gland cells. In the first embodiment of the present invention, the use of the full-plasma secretion inhibiting conditions allows the cells of the sebaceous gland cells to be maintained without rupture, and sebum and the like as the contents of the sebaceous gland cells are not released outside the sebaceous gland cells.

<1-3-1-1. Conditions for autophagy modulation >

More specifically, autophagy-regulating conditions are preferred as the whole plasma secretion-regulating conditions in the first embodiment of the present invention.

The autophagy-regulating condition in the present embodiment is more preferably an autophagy-inducing condition and/or an autophagy-inhibiting condition. By regulating the induction and/or inhibition of autophagy, it is possible to easily regulate the whole plasma secretion of sebaceous gland cells, and therefore the operability is excellent, and by adopting such conditions, there is an advantage that the accuracy and reproducibility of evaluation and/or selection of the whole plasma secretion regulator are high.

Autophagy is generally considered to be a living system in which, when a cell is starved, intracellular small organs or proteins of the cell are decomposed to obtain nutrients and the nutrients are reused. However, the present inventors have found for the first time that mature sebaceous gland cells are capable of normally performing whole plasma secretion when autophagy is induced on the cells.

More specifically, the present inventors have found for the first time that autophagy is induced in mature sebaceous gland cells, whereby the cell membrane of the mature sebaceous gland cells is disrupted, and the internal components of the sebaceous gland cells are secreted outside the cells (see fig. 5, 6, and 7). By inducing autophagy in mature sebaceous gland cells in this manner, it is possible to normally induce full-plasma secretion in the sebaceous gland cells, and thus it is possible to cause normal full-plasma secretion in the sebaceous gland cells.

Furthermore, the present inventors have found for the first time that autophagy is inhibited in mature sebaceous gland cells, and that the cell membranes of mature sebaceous gland cells are not ruptured (see fig. 5), and that the resulting clumps block the pores by blocking the ducts of the pores, etc. (see fig. 1 and 2).

By inhibiting autophagy in mature sebaceous gland cells in this way, it is possible to inhibit the whole-plasma secretion of the sebaceous gland cells, and thus it is possible to cause abnormal whole-plasma secretion of the sebaceous gland cells.

<1-3-1-2. Culture Medium Condition for Total plasma secretion Regulation >

As the whole plasma secretion regulatory condition in the first embodiment of the present invention, it is preferable to adopt a medium condition for sebaceous gland cells. Under these conditions, the state of regulation of the whole plasma secretion of the sebaceous gland cells can be observed.

The present inventors have found for the first time that the whole plasma secretion of sebaceous gland cells can be easily regulated by adjusting the medium composition conditions for the sebaceous gland cells. Thus, by using a technique for adjusting the medium conditions for the sebaceous gland cells, it is possible to easily adjust the whole plasma secretion of the sebaceous gland cells even by an unskilled person. This technique has the advantage of high operability and excellent accuracy and reproducibility in evaluation and/or selection of the whole plasma secretion regulatory agent.

The medium conditions are not particularly limited, and include, for example, the medium composition, the medium pH, and CO in a vessel ₂ The concentration, temperature, period and the like may be 1 or 2 or more selected from them.

Among the above medium conditions, the medium composition is preferably adjusted, whereby the whole plasma secretion of sebaceous gland cells can be easily adjusted, and therefore, there are advantages of high operability and excellent accuracy and reproducibility in the evaluation and/or selection of a whole plasma secretion regulator.

To the composition of the medium used under the medium conditions of the present embodiment, 1 or 2 or more kinds selected from additives for proliferation and the like, antibiotics, serum, growth factors and the like may be appropriately added to the composition of the medium for proliferation (specifically, proliferation, maintenance, differentiation and the like) or for starvation and the like within a range that does not impair the effects of the present invention.

In the first embodiment of the present invention, the medium conditions other than the adjustment of the composition of the medium may be the usual conditions for observing fluorescence of cultured animal cells. The observation step is preferably carried out under conditions that allow observation while allowing the sebaceous gland cells to survive, and for example, normal conditions for in vitro fluorescence observation of human sebaceous gland cells may be used, or the above-mentioned conditions may be used<1-1 cultivation step>The culture conditions of (1). For example, the pH of the medium is excellentSelecting CO in a container with the culture pH value of about 7-8 ₂ The concentration is preferably a normal atmospheric concentration (0.04%) or an incubation concentration (4 to 10% in the atmosphere), and the temperature is preferably about room temperature (10 to 30 ℃) or an incubation temperature of 30 to 40 ℃. Examples of the conditions for survival of sebaceous gland cells include the inside of a culture apparatus (preferably a culture chamber) for animal cells, the inside of a container for a fluorescence microscope, and the inside of a chamber.

The medium composition is preferably a medium for inhibiting whole plasmatic secretion and/or a medium for inducing whole plasmatic secretion.

The first embodiment of the present invention can inhibit or inhibit the whole plasma secretion of mature sebaceous gland cells by using a medium for inhibiting whole plasma secretion, and therefore can be used as an induction model for pore formation. In the induction model for pore clogging formation, the presence of the test substance enables the test substance to be evaluated or selected as a whole plasma secretion promoter in a simple manner and with high accuracy. Furthermore, the effect of the test substance on promoting the secretion of the whole plasma of the sebaceous gland cells can be observed under the condition of inhibiting the secretion of the whole plasma using the medium for inhibiting the secretion of the whole plasma. This observation makes it possible to determine whether or not the test substance is suitable for preventing or ameliorating pore clogging or the like.

The first embodiment of the present invention can induce or promote the full-plasma secretion of mature sebaceous gland cells by using a medium for inducing full-plasma secretion, and thus can be used as a model for suppressing pore formation. In the inhibition model for pore clogging formation, the test substance is present, and thus the test substance can be evaluated or selected as a whole plasma secretion inhibitor easily and accurately. Furthermore, the inhibitory effect of the test substance on the whole plasma secretion of sebaceous gland cells can be observed under the whole plasma secretion-inducing conditions using the medium for whole plasma secretion-inducing. This observation makes it possible to determine whether or not the test substance is involved in promotion of pore formation.

< culture Medium for inhibiting Total plasma secretion >

As the culture medium for suppressing total plasma secretion used under the condition of total plasma secretion suppression, a propagation medium for animal cell culture generally used for propagation such as propagation, maintenance, differentiation and the like is preferably used. Examples of the propagation medium for culturing animal cells include, but are not limited to, basal medium, serum-reduced medium, and serum-free medium. By using a normal propagation medium, the whole plasma secretion of mature sebaceous gland cells can be suppressed.

In general, a basal medium is prepared by adding serum to a basal medium component containing carbon sources such as amino acids, vitamins, inorganic salts (e.g., calcium) and glucose. Serum-free media are media in which serum is replaced with appropriate nutrients and hormones in the basal media components.

The whole plasma secretion-inhibiting medium used in the first embodiment of the present invention is preferably a medium in which serum or the same component as serum is added to the basic medium components, and which may be an autophagy-inhibiting medium. The medium for suppressing whole plasma secretion is preferably a medium not containing an amino acid and not in a nutrient-starved state, and more preferably a propagation medium containing an amino acid in a basal medium composition so as not to be in a nutrient-starved state. By using this ordinary propagation medium, the whole plasma secretion of mature sebaceous gland cells can be suppressed.

As the whole plasma secretion-inhibiting Medium used in the first embodiment of the present invention, a propagation Medium generally used for the culture of sebaceous gland cells is more preferable, and more specifically, examples thereof include William's E Medium, D-MEM, RPMI1640, ham's F-12, modified D-MEM/Ham's F-12 (1) (e.g., sebomedT M basal Medium), epiLife Medium series (Thermo Fisher Scientific Co., ltd.), huMedia Medium series (KURABO Co., ltd.); cnT-Prime Medium series (CELLnTEC Co.). It is preferable that 1 or 2 or more selected from the group consisting of serum, EGF, hydrocortisone, insulin and the like are added to the basal medium, and for example, the serum is preferably 0.1 to 20%, more preferably 10%, the EGF (Epidermal Growth Factor) is preferably 1 to 10ng/mL, more preferably 3 to 6ng/mL, the hydrocortisone is preferably 0.1 to 20. Mu.g/mL, more preferably 5 to 15. Mu.g/mL, and the insulin is preferably 5 to 50. Mu.g/mL, more preferably 8 to 20. Mu.g/mL.

In addition, as the medium for suppressing the secretion of the whole plasma used in the first embodiment of the present invention, a differentiation medium capable of differentiating immature sebaceous gland cells is preferably used, and more specifically, a differentiation medium containing no differentiation inhibitory factor (for example, EGF, BPE, etc.) or reduced amounts thereof is preferably used. Examples of the differentiation Medium include William's E Medium, D-MEM, RPMI-1640, ham's F-12, modified D-MEM/Ham's F-12 (1) (e.g., sebomedTM basal Medium), epiLife Medium series (Thermo Fisher Scientific Co., ltd.), huMedia Medium series (KURABO Co., ltd.); cnt-Prime Medium series (CELLnTEC), but the present invention is not limited thereto.

< culture Medium for inducing Whole plasma secretion >

The culture medium for inducing total plasma secretion used in the first embodiment of the present invention is preferably a culture medium in which a part or all of the amino acids are removed from the basic medium components of the aforementioned "propagation medium for animal cell culture", and is more preferably a culture medium containing no amino acids from the viewpoint of inducing total plasma secretion of mature sebaceous gland cells, and is even more preferably a serum-free amino acid-free culture medium to which no serum is added. More preferably, the animal cells are starved for nutrients, and examples thereof include a culture medium for inducing autophagy. By using a nutrient starvation medium, it is possible to induce full plasma secretion of mature sebaceous gland cells. The medium for culturing animal cells is more preferably a medium for culturing sebaceous gland cells from the viewpoint of inducing the full-plasma secretion of mature sebaceous gland cells.

The medium for inducing whole plasma secretion is preferably an amino acid-free D-MEM medium obtained by removing 1, 2 or more or all of the amino acids from a normal amino acid-containing D-MEM medium, and more preferably a serum-free D-MEM medium containing no amino acids at all, from the viewpoint of enabling a more favorable nutritional starvation. In addition, the usual D-MEM medium contains 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 as amino acids.

Examples of the culture medium for inducing autophagy for inducing whole plasma secretion include, but are not limited to, serum-free D-MEM (Dulbecco's modified Eagle's medium), serum-free D-MEM (high sugar (4500 mg/L)) (containing pyruvic acid Na (110 mg/L) and no amino acid) (Fuji film, wako pure chemical industries, ltd.), and Hank's Balanced Salt Solution (serum-free). Among them, serum-free and amino acid-free D-MEM is preferable.

<1-3-1-3. Conditions for adding drugs for regulating secretion of Total pulp >

In the observation step of the first embodiment of the present invention, the state of the fluorescent sebaceous gland cells can be observed fluorescently by using an appropriate additive agent and allowing single or multiple sebaceous gland cells and the test substance to exist.

The conditions for adding a drug for controlling total plasma secretion used in the first embodiment of the present invention include, for example, the conditions for adding a total plasma secretion promoter and/or a total plasma secretion inhibitor.

In addition, the whole plasma secretion inducing conditions can be formed by combining the medium conditions using the above-mentioned "propagation medium for animal cell culture" with the whole plasma secretion promoter addition conditions. Examples of the additive agent used in the conditions for adding the whole plasma secretion promoter include autophagy promoters and the like.

In addition, the condition for inhibiting the secretion of the whole plasma can be formed by combining the medium condition using the above-mentioned "propagation medium for culturing animal cells" with the condition for adding the whole plasma secretion inhibitor. Examples of the additive agent used under the conditions for adding the whole plasma secretion inhibitor include autophagy inhibitors.

For culturing animal cells, sebaceous gland cells are more preferred.

The amount of the additive agent to be added may be determined by reference to the literature relating to the existing autophagy inhibition experiment or promotion experiment.

Autophagy is typically subjected to the following procedures: the target of degradation in the cell is encapsulated by vesicles called autophagosomes, (1) then, (2) a degrading enzyme called lysosomes is fused with a large number of stacked vesicles, and (3) the target of degradation is degraded by autolysosomes formed by fusion of the 2 vesicles.

Examples of the agent before the inhibition of the above-mentioned (1) include MHY-1485 (C) ₁₇ H ₂₁ N ₇ O ₄ : CAS No: 326914-06-1), amino acids, etc., but is not limited thereto. MHY-1485, the stage where amino acid inhibition turns on autophagy itself (before said (1)), this inhibition is caused by the activation of a protein called mTORC1, which negatively regulates autophagy.

Examples of the agent for inhibiting the initial stage of the above-mentioned (1) include, but are not limited to, 3-Methyladenine (3-MA).

Examples of the agent for inhibiting (2) (inhibiting the fusion of autophagosome and lysosome) include, but are not limited to, barflumycins such as barflunomycin A1, chloroquine, and the like.

Examples of the inhibitor of the degrading enzyme in the above-mentioned (3) include pepstatin A, etc., but the inhibitor is not limited thereto.

Among them, mTORC1 activator (suitably MHY-1485) and/or fusion inhibitor of autophagosome and lysosome (suitably bavlomycin).

1 or 2 or more selected from the autophagy inhibiting agents can be used.

By adopting the usual medium conditions using the propagation medium and the drug-added conditions for the regulation of the whole plasma secretion, the whole plasma secretion of the sebaceous gland cells can be easily regulated, and desired conditions for the regulation of the whole plasma secretion can be obtained. Under the condition of the whole-plasma secretion regulation, the whole-plasma secretion regulation effect of the test substance on the sebaceous gland cells can be subjected to fluorescence observation. By using the whole plasma secretion regulation condition, a highly accurate and highly reproducible observation result can be objectively obtained.

< sebaceous gland cells >

The sebaceous gland cells used in the first embodiment of the present invention may be stored in a frozen state and used as needed, which is prepared in advance in the "culture step of sebaceous gland cells", and the step of the present embodiment may include a culture step of sebaceous gland cells.

The sebaceous gland cells used in the first embodiment of the present invention are preferably mature sebaceous gland cells in a state of being secretionable in whole plasma.

The sebaceous gland cells used in the first embodiment of the present invention are more preferably three-dimensional structures obtained by three-dimensional culture than a plurality of cells obtained by two-dimensional culture. The three-dimensional structure is preferably a structure obtained by three-dimensionally aggregating a plurality of cells, and examples thereof include, but are not limited to, a cell group, a cell mass, a three-dimensional structure (more preferably, a spheroid), and the like.

Examples of the three-dimensional structure of the sebaceous gland cells include a laminated sheet, a tube, a rod, and a spheroid, but not limited thereto, 1 or 2 or more kinds of them may be selected.

Three-dimensional structures of sebaceous gland cells can be formed by aggregation and aggregation of multiple sebaceous gland cells. Generally, spheroids are spherical cell aggregates in which cells are aggregated and aggregated.

The sebaceous gland cells used in the first embodiment of the present invention are more preferably three-dimensional structures of sebaceous gland cells whose surface layer is composed of mature sebaceous gland cells (see fig. 3 and 4). From the viewpoint of use in observing the whole plasma secretion, the three-dimensional structure preferably has a structure in which mature sebaceous gland cells existing in the superficial layer are completely secreted and disappear, and then the sebaceous gland cells therebelow become the superficial layer. Further, the three-dimensional structure of the sebaceous gland cell according to the present embodiment described in "2" below is preferably used.

< test substance >

The test substance used in the first embodiment of the present invention and the like is not particularly limited, and may be naturally derived or artificially prepared, or may be a monomer or a mixture or a composition containing a monomer or a mixture. The composition may be, for example, a pharmaceutical preparation, a cosmetic, an external preparation for skin, a pharmaceutical composition, a food or drink composition, or the like.

The concentration of the test substance can be appropriately adjusted, and for example, aqueous solutions containing different concentrations of the test substance can be prepared for observing the concentration-dependent effect.

The test substance is preferably 1 or 2 or more selected from the group consisting of a compound, a microorganism or a culture thereof, an extract thereof, and a mixture or a composition thereof. The compound may be either an inorganic compound or an organic compound.

Examples of the inorganic compound include, but are not limited to, compounds of metal elements (main group elements, transition elements) or nonmetal elements (boron, silicon, and the like).

Examples of the organic compound include carbon-containing compounds, such as lipid compounds, sugar compounds, peptide compounds, nucleic acid compounds, alkaloid compounds, steroid compounds (terpene compounds), enzymes, agonists, antagonists, hormones, polymer compounds, and antibiotics, but are not limited thereto.

Examples of the microorganism include, but are not limited to, bacteria (e.g., eubacteria, archaea, etc.), fungi (e.g., molds, mushrooms, yeasts, etc.), microalgae, viruses, and their lysates.

The culture may be, for example, a culture obtained by culturing microorganisms, but is not limited to this, and may be, for example, a fermentation product of beneficial bacteria such as lactic acid bacteria, bifidobacteria, and yeast.

Examples of the extract include, but are not limited to, extracts obtained from natural products (for example, animals, plants, microorganisms, etc.), organic compounds, inorganic compounds, cultures, fermented products, minerals, and the like.

< Observation >

In the first embodiment of the present invention, it is preferable that the test substance is present after the contacting step, and the state of the fluorescent sebaceous gland cells is observed by fluorescence. This makes it possible to easily observe the state of the plasma-complete secretion of the test substance by the sebaceous gland cells. More preferably, the fluorescence observation is performed under the whole plasma secretion-regulating condition after the contacting step.

The state of the fluorescent sebaceous gland cells can be detected as the normality or abnormality of the whole plasma secretion from the change in fluorescence of the sebaceous gland cells with time.

The state of the fluorescent sebaceous gland cells is preferably based on the number of fluorescent-disappeared cells per unit volume. The number of fluorescence-disappeared cells per unit volume was counted as 1 when the fluorescence of 1 sebaceous gland cell disappeared within a predetermined region and a predetermined observation period.

When the whole plasma secretion of sebaceous gland cells normally occurs, the number of cells in which fluorescence disappears increases in observation for a certain period of time. Normally, when 1 sebaceous gland cell that survives is lysed, fluorescence accumulated inside the cell is released outside the cell, and the fluorescence disappears. In particular, when the whole plasma secretion normally occurs in the sebaceous gland cells existing in the surface layer of the three-dimensional structure, the fluorescence of the sebaceous gland cells disappears in the surface layer for a short period of time. Thereafter, the fluorescent sebaceous gland cells present therebelow migrate to the surface layer side, and thus the surface layer of the three-dimensional structure appears to be constantly fluorescent.

Thus, the plasma secretion promoting effect of the test substance can be determined with high accuracy from the change in fluorescence of each sebaceous gland cell in the observation image.

When the secretion of the sebaceous gland cells is abnormal in the whole plasma, the number of cells in which fluorescence disappears decreases in observation for a certain period of time. In particular, when the plasma secretion is abnormal without occurring normally in the sebaceous gland cells present in the surface layer of the three-dimensional structure, the fluorescence of the sebaceous gland cells is maintained without disappearing in the surface layer.

Thus, the pancreatin inhibitory action of the test substance can be determined with high accuracy from the change in fluorescence of each sebaceous gland cell in the observation image.

< fluorescent reagent addition step >

In the first embodiment of the present invention, it is preferable to add a fluorescent reagent to the sebaceous gland cells and the test substance after the contact step. In this case, since the state of the whole plasma secretion (normal or abnormal) of the sebaceous gland cell can be easily discriminated, it is preferable to cause the living cell to emit fluorescence. When a fluorescent reagent is added to the sebaceous gland cells and the test substance, the treatment is more preferably performed under the full-plasma secretion regulation conditions after the contact step.

The fluorescent reagent is a fluorescent reagent containing a fluorescent dye having a characteristic that a living cell emits fluorescence and that fluorescence disappears after the cell dies. In the fluorescence observation of the present embodiment, a fluorescent reagent containing a fluorescent dye that allows at least living sebaceous gland cells to emit fluorescence is more preferable. By using such a fluorescent reagent, the number of fluorescence-lost cells per unit volume can be determined over time, and the change in the number of sebaceous gland cells over time can be detected from the number of fluorescence-lost cells per unit volume.

As the fluorescent reagent, for example, a commercially available Live/Dead (TM) Kit can be used, and as the Kit, for example, live/Dead (TM) Cell Imaging Kit (488/570) (Thermo Fisher Scientific Co., ltd.) and the like can be mentioned, but the Kit is not limited thereto. The state of cells that fluoresce or sebaceous glands can be observed according to the procedures described in the instructions attached to the commercial kit.

For example, when living cells of sebaceous gland cells are observed by green fluorescence, the wavelength of excitation light is preferably 480 to 490nm, and the wavelength of detection light in this case is preferably 490 to 580nm.

< fluorescent microscope Observation step >

The observation according to the first embodiment of the present invention is preferably performed using a fluorescence microscope. The fluorescence microscope may use, for example, 1 or more selected from a laser, an ultra-high pressure mercury lamp, a xenon lamp, an LED (ultraviolet LED, etc.) and the like as a light source. Examples of the fluorescence microscope include a simple fluorescence microscope not using a laser beam as a light source, a laser scanning microscope, a confocal laser microscope such as a confocal laser scanning microscope, a multiphoton excitation microscope such as a two-photon laser scanning excitation microscope, and a light sheet fluorescence microscope, and 1 or 2 or more kinds selected from them can be used. In general, the simple fluorescence microscope mainly uses 1 or more light sources selected from, for example, an ultra-high pressure mercury lamp, a xenon lamp, and an LED that can irradiate ultraviolet rays, and can observe a sample as a two-dimensional structure. Among these, from the viewpoint of enabling observation of a sample as a three-dimensional structure, a laser scanning microscope, a confocal laser microscope, a multiphoton excitation microscope, and a light sheet fluorescence microscope are preferable, and a confocal laser microscope is more preferable.

In the fluorescence observation according to the first embodiment of the present invention, it is preferable to perform image analysis on the sebaceous gland cells under observation using image analysis software (more preferably, high-definition 3D/4D image analysis software). In the observation step of the present embodiment, 1 or 2 or more kinds of known or commercially available devices provided with the above-described observation unit configured to perform observation with a fluorescence microscope, the imaging unit configured to image an observation target, the image analysis unit configured to analyze an image of an observed image obtained by imaging, and the like can be used in combination. Examples of the known or commercially available device include a confocal laser microscope device (e.g., carat zeiss LSM 800) and an image analysis device (e.g., a computer or the like) that is installed with image analysis software (preferably, 3D/4D image analysis software (e.g., high definition 3D/4D image analysis software)) and executes the image analysis software, but the present invention is not limited thereto, and the 3D/4D image analysis software can generally realize multidimensional visualization (e.g., stereoscopic image construction) of image data and analysis (e.g., quantitative analysis) of 3D/4D images.

In the observation according to the first embodiment of the present invention, it is preferable that the observation target irradiated with the irradiation light is photographed as an observation image (a moving image and/or a still image). When irradiation light (excitation light) is applied to a sebaceous gland cell to be observed, the sebaceous gland cell can be caused to emit fluorescence and detected, and the sebaceous gland cell that emits light is a living cell.

From the viewpoint of reducing damage to the observation target (sebaceous gland cells), the light to be irradiated is preferably irradiated from the irradiation section during imaging and is not irradiated except during imaging. The observation image is preferably stored in an internal or external storage unit.

It is preferable that the "certain region" in the entire observation target is set as a "field of view" and the number of fluorescence-disappeared cells in the field of view is observed for a predetermined period. The setting position of the field of view can be arbitrarily set, and the number of fields of view may be single or plural (for example, 1 to 5), and the number of fields of view is preferably 3. The range of 1 visual field is preferably 50 to 70 μm × 100 to 140 μm × 25 to 70 μm. The predetermined period is preferably, but not particularly limited to, an observation time after the addition of the self-fluorescent reagent (starting point), and is preferably 8 to 20 hours, more preferably 10 to 15 hours, and further preferably 11 to 13 hours. It is preferable to start imaging the sebaceous gland cells at the start of observation of the sebaceous gland cells.

The number of fluorescence-lost cells per unit volume in 1 field is preferably calculated, and more preferably, the average value of the number of fluorescence-lost cells per unit volume in a plurality of fields is calculated.

When the observation target is a three-dimensional structure, it is preferable to take an image by gradually changing the focal plane from the surface of the three-dimensional structure to the central portion of the three-dimensional structure in the depth direction (central portion direction) by 0.5 to 2 μm (preferably 1 μm) within a range of 100 μm. By performing stereoscopic reconstruction of the images of the respective focal planes thus captured, a three-dimensional structure can be three-dimensionally represented and evaluated.

The observation according to the first embodiment of the present invention is preferably performed using time-lapse photography, whereby a plurality of observation images (preferably still images) can be obtained at predetermined intervals in time series to obtain time-lapse images. The state of the sebaceous gland cells is observed based on the time-delay image, and the full-plasma secretion regulation effect of the tested substance on the sebaceous gland cells can be judged. Preferably, the number of fluorescence-disappeared cells in a predetermined period and a predetermined field of view is counted from the delayed image.

In general, "time-lapse image" is called low-speed photography or micro-speed photography, and is a photographing method in which several seconds (or fractions) are photographed frame by frame and the resultant images are linked and played back, thereby presenting frame by frame images. Further, it is also possible to obtain a video image in advance, obtain a plurality of image data as still images from the video image at predetermined intervals, and form image data such as a time-lapse image.

In the first embodiment of the present invention, the shooting conditions for the time-lapse image can be set appropriately. The time-lapse image can be obtained, for example, by capturing and storing observation images containing fluorescence emitted from the observation target under the action of irradiation light in time series. Preferably, irradiation light (excitation light) is irradiated from a light source to the observation target at the time of or before the time-lapse image is captured, whereby damage to the observation target can be reduced and fluorescence observation can be performed for a long time.

Specifically, when the imaging interval is short, the damage amount of the irradiated light to the sebaceous gland cells is large, and therefore, the fluorescence of the sebaceous gland cells may disappear, whereas when the imaging interval is long, an important image in the full-plasma secretion regulation action of the test substance on the sebaceous gland cells may be missed.

Therefore, the shooting interval (in units of minutes) per frame in the shooting conditions is preferably 3 to 20 minutes per frame, more preferably 5 to 15 minutes per frame, further preferably 8 to 12 minutes per frame, and more preferably 9 to 11 minutes per frame (more preferably 10 minutes). This enables the whole plasma secretion state of the sebaceous gland cells to be observed with high accuracy over a long period of time while reducing damage to the sebaceous gland cells.

The imaging time under the imaging conditions is preferably 8 to 20 hours, more preferably 10 to 15 hours, and still more preferably 11 to 13 hours. The starting point of the imaging time is not particularly limited, and is preferably after the fluorescent reagent is added.

Further, preferable imaging conditions are that the imaging interval is preferably 9 to 11 minutes (more preferably 10 minutes) per frame, and the imaging time is preferably 11 to 13 hours (more preferably 60 to 90 frames, and further preferably 66 to 78 frames).

In the first embodiment of the present invention, it is more preferable to use a fluorescence detection device configured to be capable of irradiating an observation target with irradiation light (excitation light), detecting fluorescence emitted from the observation target, and observing the observation target with fluorescence. The fluorescence detection device more preferably includes a microscope, and further preferably includes a light source (e.g., a laser, an LED, or the like) configured to irradiate irradiation light (excitation light) to an observation target (preferably, a sebaceous gland cell that emits fluorescence), a detection unit (e.g., an image sensor or the like) configured to receive and detect fluorescence from the observation target, and an imaging unit configured to image the observation target. The excitation light and the detection light may be appropriately set according to the fluorescent reagent used.

Examples of the fluorescence detection device include a fluorescence microscope device, a confocal laser microscope device, a multiphoton excitation microscope device, and a light sheet microscope device, and the fluorescence detection device is not limited to this as long as it can perform fluorescence observation of an observation target. Among them, 1 or 2 or more selected from a confocal laser microscope device, a multiphoton excitation microscope device, and a light sheet microscope device are preferable, and a confocal laser microscope device is more preferable, since three-dimensional observation of an observation object can be performed. For example, a confocal laser microscope device can irradiate a laser beam from a light source, focus the laser beam on an observation target (sebaceous gland cells) with an objective lens, and obtain fluorescence emitted from the observation target as detection light. In addition, for example, the light sheet microscope device may obtain an optical section image by irradiating only a focal plane of an observation target with excitation light in a sheet form from a light source, and obtaining fluorescence emitted from the observation target as detection light.

The fluorescence detection device can photograph an object to be observed with an image sensor such as a CCD or a CMOS, and monitor the state of the object to be observed with time. The fluorescence detection device can perform imaging processing such as AD (Analog to Digital) conversion of an Analog image signal or imaging signal processing, for example. In the fluorescence detection device, the image to be output for observation may be either a color image or a black-and-white image, but a color image is preferable. A color image can be represented by RGB (red, green, blue), for example, and a black-and-white image can be represented by luminance, for example.

Preferably, the fluorescence detection device further includes a time-lapse imaging device configured to be capable of time-lapse imaging, or is provided externally with a time-lapse imaging device in a state capable of transmitting and receiving information to and from the outside. The time-lapse imaging device is preferably configured to be implemented according to the imaging conditions of the time-lapse image. The fluorescence detection device may be provided with an image analysis unit or device configured to analyze an image of the captured observation image, either externally or internally.

<1-4. Determination step >

The discrimination step in the first embodiment of the present invention will be explained below.

In the discrimination step according to the first embodiment of the present invention, it is preferable that the test substance is discriminated as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator based on the state of the fluorescent sebaceous gland cells.

In the first embodiment of the present invention, the state of the fluorescent sebaceous gland cells can be discriminated from the number of fluorescent-disappeared cells per unit volume. By using the number of fluorescence-lost cells per unit volume as an index, the whole plasma secretion regulatory effect of the test substance on the sebaceous gland cells can be objectively and easily determined.

More specifically, when the number of fluorescence-disappeared cells is large, it can be judged that the test substance has a stronger action of promoting the whole plasma secretion, while when the number of fluorescence-disappeared cells is small, it can be judged that the test substance has a stronger action of inhibiting the whole plasma secretion.

More preferably, the number of fluorescence-depleted cells under the condition of whole plasma secretion regulation is used for the discrimination. More preferably, when the number of fluorescence-lost cells is large under the condition of full-plasma secretion inhibition, it can be judged that the test substance has a stronger full-plasma secretion promoting effect, and when the number of fluorescence-lost cells is small under the condition of full-plasma secretion induction, it can be judged that the test substance has a stronger full-plasma secretion inhibiting effect.

In the first embodiment of the present invention, examples of the method for evaluating the whole plasma secretion modulating action of a test substance include the following (1) to (3), but the present invention is not limited thereto.

For example, (1) a positive control (indicator substance) for whole plasma secretion regulation is used as an indicator. More specifically, when the number of fluorescence-lost cells per unit volume of the test substance is about the same as the number of fluorescence-lost cells per unit volume of the positive control in comparison with the positive control, the test substance is evaluated as a whole plasma secretion regulator and/or the test substance is selected as a substance having a whole plasma secretion regulating effect. On the same basis, when the number of fluorescence-lost cells per unit volume of the positive control is defined as 100%, the number of fluorescence-lost cells per unit volume of the test substance is, for example, 100% ± 30%, preferably 100% ± 20%, and more preferably 100% ± 10%. For example, when the number of fluorescent cells lost in culture using a starvation medium as a positive control is 100% ± 30%, the test substance can be identified as a whole plasma secretion promoter or a candidate for a whole plasma secretion promoter, and can be selected as a substance having a whole plasma secretion promoting effect. For example, when the number of fluorescent cells lost in culture using a propagation medium as a positive control is 100% ± 30%, the test substance can be identified as a candidate for a holoplasmic secretion inhibitor or a holoplasmic secretion inhibitor, and can be selected as a substance having a holoplasmic secretion inhibitory action. The observation periods of the two compared are preferably the same.

More specifically, as an example, in the case where the test substance is the number of fluorescence-lost cells equal to or more than that of the positive control in comparison with the positive control that promotes the whole plasma secretion, the test substance may be evaluated or selected as the whole plasma secretion promoter. For example, the "number of fluorescence-lost cells per unit volume of the positive control that promotes total plasma secretion" may be set to "the number of fluorescence-lost cells per unit volume using a starvation medium without addition of a test substance", and it is preferable that the time-lapse imaging periods of the two to be compared be the same. When the test substance is evaluated or selected as a whole-plasma secretion promoter, it is preferably performed under whole-plasma secretion-inhibiting conditions.

In addition, as an example, in the case where the test substance is the number of fluorescence-disappeared cells equal to or less than that of the positive control in comparison with the positive control for inhibiting the whole plasma secretion, the test substance may be evaluated or selected as the whole plasma secretion inhibitor. For example, the "number of fluorescence-disappeared cells per unit volume of the positive control inhibiting the whole plasma secretion" may be set to "the number of fluorescence-disappeared cells per unit volume using the propagation medium without adding the test substance", and it is preferable that the time-lapse imaging period of the two to be compared is the same. When the test substance is evaluated or selected as a whole plasma secretion inhibitor, it is preferably performed under whole plasma secretion inducing conditions.

For example, (2) the concentration-dependent increase in the whole secretion regulation of the test substance. More specifically, the above observation step is performed using a plurality of test substances of different concentrations to obtain the number of fluorescence-lost cells per unit volume at each concentration, and the correlation coefficient between the number of fluorescence-lost cells and the concentration is obtained to determine the degree of the correlation. In this case, it is preferable that the effect of the test substance is determined to have concentration dependency when the correlation coefficient is 0.4 or more, and the test substance is evaluated as the whole plasma secretion regulator and/or selected as a substance having a whole plasma secretion regulating action. When the correlation coefficient is 0.7 or more, the correlation is judged to be stronger, and it can be judged that the possibility of having a stronger whole plasma secretion regulating action is high. These observation periods are preferably the same.

More specifically, as an example, when the correlation coefficient between the concentration of the test substance and the number of fluorescence-lost cells is 0.4 or more and the test substance tends to have a full-plasma secretion promoting effect (positive), the test substance is judged to have a full-plasma secretion promoting effect, and the test substance can be evaluated or selected as a full-plasma secretion promoting agent. When the test substance is evaluated or selected as a whole plasma secretion promoter, it is preferably performed under whole plasma secretion-inhibiting conditions.

In addition, as an example, when the correlation coefficient between the concentration of the test substance and the number of fluorescence-lost cells is 0.4 or more and the test substance tends to exhibit a full-plasma secretion inhibitory effect (negative), it can be determined that the test substance has a full-plasma secretion inhibitory effect, and the test substance can be evaluated or selected as a full-plasma secretion inhibitor. When the test substance is evaluated or selected as a whole plasma secretion inhibitor, it is preferably performed under whole plasma secretion inducing conditions.

For example, (3) a plurality of test substances are sorted. More specifically, the number of fluorescence-lost cells per unit volume of each test substance is obtained from the observation step of a plurality of test substances, and the order of the level of the holoplasmic secretion regulatory action of the test substances can be determined from the number of fluorescence-lost cells. The order may be based on a value close to the positive control in (1) above, and preferably, the order is based on the correlation coefficient in (2) above. These observation periods are preferably the same.

Specifically, for example, the observation step is performed using each of a plurality of test substances, and the test substances are sorted in descending order of the number of fluorescence-lost cells based on the number of fluorescence-lost cells. From the higher order of the cistron, the test substance can be evaluated or selected as a whole plasma secretion promoter or a candidate thereof as a cistron having a strong whole plasma secretion promoting effect. When the test substance is evaluated or selected as a whole plasma secretion promoter, it is preferably performed under whole plasma secretion-inhibiting conditions.

In addition, as an example, the observation step is performed using each of a plurality of test substances, and the test substances are sorted in ascending order according to the number of fluorescence-lost cells based on the number of fluorescence-lost cells. From the higher order of the cisposition, the test substance can be evaluated or selected as a whole plasma secretion inhibitor or a candidate thereof as a cisposition having a strong whole plasma secretion inhibitory action. When the test substance is evaluated or selected as a whole plasma secretion inhibitor, it is preferably performed under whole plasma secretion inducing conditions.

Based on the evaluation or selection result, the accuracy of the result can be further improved by using the method (1) or (2). Further, according to the evaluation or selection result, an in vivo test, an animal evaluation test such as a human test, or the like can be performed on the host.

< evaluation and/or selection of Whole plasma secretion regulator >

According to the first embodiment of the present invention, it is possible to evaluate whether or not a test substance is suitable as a whole plasma secretion regulator and/or to easily select a test substance as a whole plasma secretion regulator (specifically, a substance having a whole plasma secretion regulating action).

The whole plasma secretion regulator is not particularly limited, and examples thereof include a whole plasma secretion regulator of sebaceous gland cells (more preferably a whole plasma secretion promoter and a whole plasma secretion inhibitor), a pore-clogging formation regulator (more preferably a pore-clogging formation promoter and a pore-clogging inhibitor), and an autophagy regulator of sebaceous gland cells (more preferably an autophagy promoter and an autophagy inhibitor), and 1 or 2 or more kinds thereof can be selected.

With the first embodiment of the present invention, as the whole plasma secretion regulator, the test substance to be evaluated and/or selected may be selected as a substance having a whole plasma secretion regulating action. Thus, the selected substance can be further subjected to in vitro tests, in vivo tests, animal evaluation tests such as human tests, and the whole plasma secretion regulating effect can be further confirmed.

The selected substance obtained by the method of the first embodiment of the present invention may be contained in a whole plasma secretion regulator to exert a whole plasma secretion regulating effect, as described later.

In the description of the whole plasma secretion regulator and the like according to the first embodiment of the present invention, the following description of "3" may be applied to the < evaluation and/or selection of the whole plasma secretion regulator >, and may be appropriately applied.

< method of the second embodiment of the present invention >

In the description of the method according to the second embodiment of the present invention, the description of the whole plasma secretion, the test substance, the whole plasma secretion regulator, the fluorescence microscope, and the like, the respective configurations of the image analysis software, and the like, which are repeated as in the above-described < method of the first embodiment > will be appropriately omitted, and the description of the < method of the first embodiment > and the following "2." and "3." will also be applied to the second embodiment of the present invention, and this description can be appropriately applied.

The present inventors have explained the following by < experimental example 1> and the like of [ example ] below: pore blockage formation is associated with the full plasma secretion of sebaceous gland cells; when the pore canal portion of the skin sheet after the culture tends to be deficient in the secretion of the whole plasma, debris of the sebaceous gland cells, sebum, stratum corneum and the like are accumulated due to rupture of the sebaceous gland cells, poor digestion and the like, and the keratotic plug as the accumulation is laterally swollen, and the debris and the like cannot be discharged to the outside. The present inventors have also found that it is possible to estimate whether or not the whole plasma secretion of sebaceous gland cells occurs normally by collecting a keratoplug that has clogged inside the pore without measuring the diameter of the pore outlet, and measuring the maximum diameter at which the keratoplug that causes clogging swells to the maximum.

Thus, the method of the second embodiment of the present invention may provide a method for the evaluation and/or selection of a whole plasma secretion modulator comprising: an observation step of performing fluorescence observation of the collected keratotic plug state after the presence of the test substance in one or a plurality of sebaceous gland cells; and/or a discrimination step of discriminating the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator on the basis of the state of the fluorescent plug. Further, since the state of the fluorescent keratotic plug does not need to be evaluated by a sensory evaluation, there is an advantage that a method for evaluating and/or selecting a whole plasma secretion regulatory agent including a determination step of determining the keratotic plug as a whole plasma secretion regulatory agent or a candidate for the whole plasma secretion regulatory agent with higher accuracy can be performed.

Thus, with the method of the second embodiment of the present invention, when it is intended to provide a technique relating to pore clogging formation as a main object, the object can be achieved. More specifically, the method according to the second embodiment of the present invention can solve the above-mentioned problems, when it is intended to provide a method for evaluating and/or selecting a whole plasma secretion controlling agent, etc., by estimating the whole plasma secretion state more easily without evaluating the change in pore diameter on the skin surface (i.e., the apparent pore state).

The method according to the second embodiment of the present invention can be used for the elucidation or analysis of the skin problem of pore clogging, and a countermeasure for solving the skin problem of pore clogging can be proposed.

The method of the second embodiment of the present invention can be used for determining or evaluating whether or not a test substance has a whole plasma secretion regulatory action in a human evaluation test, an animal evaluation test using a mammal other than human (mouse, rat, pig, etc.), or a test using a skin patch of a mammal (human, pig, etc.) containing a pilosebaceous portion. This provides a method for selecting a substance that can be used as an active ingredient of a whole plasma secretion regulator from among test substances and/or from among test substances.

The whole plasma secretion regulator is preferably 1 or more than 2 selected from the group consisting of a whole plasma secretion promoter, a whole plasma secretion inhibitor, a pore-clogging formation regulator, and an autophagy regulator for sebaceous gland cells.

The second embodiment of the present invention preferably includes the observation step and/or the discrimination step, and more preferably is performed in the order from the observation step to the discrimination step.

The second embodiment of the present invention more preferably includes a contacting step, an observing step, and a discriminating step, and further preferably sequentially performs these steps.

< contact step >

The contacting step in the second embodiment of the present invention is preferably carried out such that a single sebaceous gland cell or a plurality of sebaceous gland cells are present with the test substance.

In the second embodiment of the present invention, as the contacting step in the animal evaluation test, it is more preferable that the skin surface of a mammal as a test subject is contacted with the test substance or a sample containing the test substance. By bringing the skin into contact with the test substance, the test substance also comes into contact with a single or a plurality of sebaceous gland cells present in the pilosebaceous section or the like.

The mammal is not particularly limited, but preferably 1 or 2 or more species selected from the group consisting of human, pig, mouse, rat, and the like. In addition, in consideration of differences in sebum secretion due to sex and age, men (males), women (females), children, adults (young (15 to 34 years), middle (35 to 64 years), elderly (65 years or older), and the like) and the like can be appropriately set.

The contact site of the mammal is not particularly limited, but a site with high sebum secretion, for example, a site with high sebaceous glands (scalp, nose, forehead, etc. in the case of a human being), is preferable, and in the case of a human being, the nose is more preferable. In order to adjust the formation period of the corner plugs between the test animals, the contact portion is preferably a portion where the corner plugs are once removed and then formed again.

In the contacting step, the test substance is preferably administered in such a manner that a single or a plurality of sebaceous gland cells can exist together with the test substance. The administration is not particularly limited, and examples thereof include injection, coating, spraying, osmosis, oral administration, and the like, but are not limited thereto. Among them, non-invasive administration such as coating, spraying, penetration and the like is preferable. When a test substance is administered, a sample containing the test substance can be prepared, and examples of the sample include a cosmetic or an external preparation for skin containing the test substance. Examples of the cosmetic or skin preparation for external use include, but are not limited to, lotions, milky lotions, dipping bags, sheets for skin application, creams, and the like.

The contact conditions (e.g., period) are not particularly limited, and the test substance may be used in a normal living environment of the test animal, taking into consideration the setting of a normal animal evaluation test such as a human. The number of administrations may be 1 administration on 1 day or 1 divided administration on 1 day. The dosing interval may be continuous or intermittent. The administration period or the contact period is, for example, 1 week to 2 months, preferably 2 weeks to 6 weeks, and more preferably 3 weeks to 5 weeks.

In the second embodiment of the present invention, in the case of the test using the skin patch, the skin patch is more preferably a mammalian skin patch containing one or more pilosebaceous portions from the viewpoint of the regulation of the secretion of the whole body fluid. Examples of the skin sheet include, but are not limited to, a skin sheet collected from a mammal, and a cultured skin sheet obtained by culturing the skin sheet. The skin patch may be a biological sample, or may be a skin patch collected when meat treatment is performed on livestock.

In the second embodiment of the present invention, the culture step of culturing the collected skin piece or the like may be included before or in the contact step in the test using the skin piece, for example, as follows<Test example 1>Described in (1), can be measured at 37 ℃ and 5% CO ₂ The skin patch culturing step is carried out under the conditions using a normal culture medium for skin patch culture.

In addition, in the skin sheet culturing step, the whole plasma secretion regulation of the test substance can be evaluated or screened easily by appropriately using the culture medium conditions for whole plasma secretion regulation of <1-3-1. Whole plasma secretion regulation condition > or the like, and therefore, the induction of whole plasma secretion and the inhibition of whole plasma secretion in sebaceous gland cells present in the skin sheet can be regulated.

In the second embodiment of the present invention, in the contact step in the test using the skin patch, the test substance may be added to the culture medium by applying, spraying, sticking, or the like to the skin patch so that the test substance or the sample containing the test substance can contact one or more sebaceous gland cells contained in the pilosebaceous portion of the skin patch. The sample can be prepared into a general cosmetic or skin preparation for external use, as in the case of animal evaluation tests. The conditions of the contact step may be set in consideration of the culture conditions of a normal skin sheet, and for example, the contact period is not particularly limited, and may be, for example, 1 day to 4 weeks.

< Observation step >

In the observation step in the second embodiment, it is preferable to perform fluorescence observation of the state of the collected keratotic plug after the presence of a single or a plurality of sebaceous gland cells and the test substance.

The collected keratotic plug is preferably one collected from the skin surface exposed to the test substance, and more preferably one collected from the skin surface exposed to the test substance after a predetermined period of time has elapsed since the removal of the keratotic plug. The predetermined period after the removal is not particularly limited, and may be set as appropriate, and in the case of a human, for example, it may be set to 3 to 5 weeks.

The keratoplug used in the control test is preferably one collected from the skin surface not contacted with the test substance, and more preferably one collected from the skin surface not contacted with the test substance after a predetermined period of time has passed since the keratoplug was removed in advance.

More preferably, the whole plasma secretion control of the test substance can be determined with high accuracy and high efficiency by performing the test using the same test animal in the order of performing the control test first, then performing the test using the test substance after a predetermined period of time, and observing the state of each collected keratotic plug by fluorescence. In addition, the test animals may be divided into the use group and the control group at the same time.

Before starting the test with or without contact of the test substance, as a preliminary stage, the keratotic plug may be removed in advance in order to equalize the time for forming the keratotic plug between the test animals and reduce the variation between the test animals.

The test can be performed simultaneously by setting different skin surface portions (for example, the left and right portions of the nose of a human, the left and right portions of the back of a pig, and the like) in the same skin surface portion and similar region of the test animal. Then, the state of the keratotic plugs collected from the skin surface of the portion not in contact with the test substance and the state of the keratotic plugs collected from the skin surface of the portion in contact with the test substance are compared, whereby the whole plasma secretion of the test substance can be simultaneously determined. In addition, in the portion not contacted with the test substance, a sample to which the test substance is not added or a sample to which a control agent is added (so-called negative or positive) may be used.

Examples of the state of the corner key include, but are not limited to, the shape of the corner key (three-dimensional shape or size, cross-sectional shape or size, etc.), the fluorescence state of the corner key (intensity, fluorescence color, autofluorescence, etc.), and the like. Among them, the shape of the corner pin is preferable, and the size of the three-dimensional shape (the maximum diameter of the three-dimensional shape of the corner pin is preferable) is more preferable. The fluorescence of the corner pins may be the fluorescent reagent described in the first embodiment, but autofluorescence of the fluorescent reagent is preferably not used. Further, it is more preferable to perform fluorescence observation with the size of the three-dimensional shape of the diagonal plug by autofluorescence.

The cross-sectional shape in the present specification means a shape of a plane perpendicular to the height direction of the three-dimensional shape of the corner key (more preferably, a shape such as a vertically long ellipsoid). In addition, the three-dimensional image of the three-dimensional shape of the corner key has a shape in which a plurality of cross sections are stacked in the height direction, and the maximum diameter of the corner key in the present specification means the diameter of the cross section of the most protruding portion in the three-dimensional shape of the corner key (see fig. 8).

The keratotic plugs to be observed by fluorescence are preferably a plurality of keratotic plugs which are collected from the skin surface of a mammal as a test animal by an adhesive sheet and attached to the adhesive sheet.

The pressure-sensitive adhesive sheet is not particularly limited, and any commercially available product may be used as long as it is commercially available as a sheet for removing keratotic plugs. The skin surface is preferably any of the above-mentioned contact sites, more preferably the nose.

In the fluorescence observation according to the second embodiment of the present invention, it is preferable that the collected corner plugs are irradiated with excitation light, and fluorescence observation is performed on fluorescence from the corner plugs. When observing the fluorescence from the angle plug, the angle plug may be caused to fluoresce using a known fluorescent reagent, and the angle plug that fluoresces may be observed. From the viewpoint of the operation efficiency and the simplicity, it is more preferable to perform fluorescence observation of the autofluorescence of the collected keratotic plugs. Since the fluorescence wavelength generally used for observation in cell biology is in the range of 300 to 800nm, when fluorescence observation is performed on autofluorescence of the keratotic plug, the excitation wavelength and the fluorescence wavelength can be set in consideration of the range of the fluorescence wavelength. When observing the fluorescence of the kerato plug, the fluorescence wavelength can be set so as to be able to detect all the autofluorescence emitted from the kerato plug by the excitation light (for example, about 550 nm). More specifically, the excitation light (excitation wavelength) for irradiation to the kerato plug is about 540 to 570nm, and the fluorescence wavelength for detecting the autofluorescence of the kerato plug is 545nm or more (more preferably 545 to 575nm or more).

In the fluorescence step in the second embodiment of the present invention, it is preferable to perform fluorescence observation on one or more three-dimensional shapes (overall appearance of the corner key) of 1 corner key. It is also preferable to perform imaging so that layers of the cross-sectional shapes of 1 corner key can be stacked, and it is further preferable to stack the layer data and finally reconstruct 1 corner key into a three-dimensional image.

In the fluorescence observation according to the second embodiment of the present invention, it is preferable to measure the maximum diameter of the 1 corner pin. Furthermore, the maximum diameter of the overall aspect of the reconstructed corner bolt is preferably measured.

As a more preferable mode of the fluorescence process in the second embodiment of the present invention, a single or a plurality of three-dimensional shapes (overall appearance of the corner key) of 1 corner key are subjected to fluorescence observation and the maximum diameter of the 1 corner key is measured as the maximum diameter. When a plurality of corner pins are observed by fluorescence, it is preferable to obtain an average value of the maximum diameters of the corner pins from the total value of the maximum diameters of the corner pins/the total number of the corner pins.

The fluorescence detection device used for observing the state of the diagonal pin by fluorescence may be configured as appropriate from the fluorescence detection device and the fluorescence microscope described in the first embodiment, and is preferably a confocal laser microscope or a confocal laser microscope device.

A preferred fluorescence detection device used in the second embodiment of the present invention includes an observation unit configured to perform fluorescence microscope observation, an imaging unit configured to image an observation target, and an image analysis unit configured to perform image analysis on an observed image that is imaged.

The fluorescence detection device used in the second embodiment of the present invention preferably includes an observation unit configured to enable fluorescence observation of one or more three-dimensional shapes (the overall shape of the corner key) of the 1 corner key, and more preferably includes an imaging unit configured to image in a manner such that layers of the cross-sectional shapes of the 1 corner key can be stacked. Thus, the image analysis software can stack the layered data and finally reconstruct 1 corner key into a three-dimensional image. The fluorescence detection device may further include an image analysis unit configured to be able to determine a maximum diameter of the 1 corner pin.

The fluorescence detection device preferably has an image analysis unit configured to reconstruct the captured image of the corner plugs into a three-dimensional image using image analysis software (preferably 3D/4D image analysis software capable of constructing a stereoscopic image, such as ZEN Black Edition or IMARIS), and the fluorescence detection device preferably has a storage unit configured to store the data of the reconstructed corner plug image in a readable manner.

Furthermore, it is more preferable to use image analysis software that can be stored in the fluorescence detection device for execution, and that measures the maximum diameter of the corner bolt in the lateral direction from the overall appearance of the reconstructed corner bolt.

< determination step >

In the determination step in the second embodiment of the present invention, it is preferable that the test substance is determined as the whole plasma secretion regulator or a candidate for the whole plasma secretion regulator based on the state of the fluorescent plug. This makes it possible to easily determine the whole plasma secretion control of a test substance with higher accuracy.

In a more preferable aspect, the state of the plug is preferably a rate of change in the maximum diameter of the plug obtained by dividing an average value of the maximum diameter of the plug after the use of the test substance by an average value of the maximum diameter of the plug before the use of the test substance, and the rate of change in the maximum diameter of the plug before the use of the test substance may be 1. In this case, the number of angular pins for obtaining the rate of change is preferably 5 to 30, and more preferably 11 to 24. The average value in this case is an average value obtained by averaging the total value of the maximum diameters of the corner pins in the three-dimensional image of the corner pins reconstructed by image analysis. This makes it possible to easily determine the state of whole plasma secretion with high accuracy.

In a more preferred embodiment, the whole pulp secretion regulation of the test substance is determined by comparing the rate of change in the maximum diameter of the keratoplug in the test group in which the test substance is not used in the contact step (i.e., the control test group) with the rate of change in the maximum diameter of the keratoplug in the group in which the test substance is used in the contact step. Further, it is preferable to determine the whole plasma secretion regulation of the test substance by averaging the change rates of the maximum diameters of the two groups of the corner pins and comparing the two groups.

In a more preferred aspect, the state of the keratoplug in the determination step is determined to have an all-serous secretion promoting effect when the rate of change of the keratoplug maximum diameter of the group using the test substance is smaller than that of the group not using the test substance, and/or determined to have an all-serous secretion inhibiting effect when the rate of change of the group using the test substance is larger than that of the group not using the test substance, by comparing the rate of change of the keratoplug maximum diameter of the group using the test substance with the rate of change of the keratoplug maximum diameter of the group not using the test substance. In this case, the sex and the contact conditions (period, etc.) of the test animals are preferably the same.

For example, as shown in fig. 9, the test substance (specifically, trehalose) used in a subject (specifically, a male) has a smaller rate of change in the maximum diameter of keratoplug than that in the control test, and therefore the test substance favorably promotes the whole plasma secretion, and it can be evaluated or determined that the test substance has a whole plasma secretion-promoting effect.

< method of the third embodiment of the present invention >

The method according to the third embodiment of the present invention may be performed by appropriately combining the method according to the first embodiment and the method according to the second embodiment, and may be performed, for example, in the order of the method according to the first embodiment and the method according to the second embodiment, or may be performed in the order of the method according to the second embodiment and the method according to the first embodiment. Thus, it is possible to determine with high accuracy whether or not the test substance has the full-plasma secretion regulating action.

<1-5. Apparatus or System for evaluation and/or selection of Whole pulp secretion Modulator >

In addition, as another aspect of the present embodiment, a device or system for evaluating and/or selecting a whole plasma secretion regulator can be provided.

The apparatus for evaluating and/or selecting a whole plasma secretion regulatory agent according to the present embodiment is preferably configured to be able to execute the method for evaluating and/or selecting a whole plasma secretion regulatory agent described above (for example, the method according to the first embodiment, the method according to the second embodiment, and a method combining these methods), and more preferably includes a control unit configured to be able to execute the method for evaluating and/or selecting a whole plasma secretion regulatory agent. The control section or the device provided with the control section and other sections (units or portions) or other devices may be further provided with a communication section that can transmit and receive information by wireless and/or wired.

The device or system of the present embodiment preferably includes a management device including the control unit and/or a fluorescence detection device including a fluorescence microscope. Further, when culturing sebaceous gland cells, skin pieces, or the like, the device or system of the present embodiment more preferably includes a management device provided with the control unit, a culture device for animal cells, and a fluorescence detection device including a fluorescence microscope. The fluorescence detection device including a fluorescence microscope preferably includes an observation unit configured to perform observation by the fluorescence microscope, an imaging unit configured to image an observation target, and an image analysis unit configured to analyze an image of the imaged observation image. The fluorescence detection device or the fluorescence detection system including the fluorescence microscope may be a fluorescence microscope observation device or a fluorescence microscope observation system.

The method according to the present embodiment may be implemented by a device (for example, a computer, a PLC, a server, a cloud service, or the like) including a CPU or the like for managing evaluation, selection, or the like of the whole secretion regulator, or a control unit including a CPU or the like provided in the management device. The method according to the present embodiment may be implemented by the control unit as a program stored in a hardware resource including a recording medium, a storage unit (a nonvolatile memory (such as a USB memory), an HDD, a CD, a DVD, a blu-ray, a server, a cloud service, and the like). In this way, a control unit that can execute the method according to the present embodiment or a device including the control unit can be provided. The management device may include an input unit such as a keyboard and a touch panel, a communication unit such as a network, a display unit such as a display and a touch panel, and the like.

<2 > preparation method of three-dimensional Structure of sebaceous gland cell and three-dimensional Structure of sebaceous gland cell >

In the explanation of the three-dimensional structure of sebaceous gland cells and the method for producing the three-dimensional structure of sebaceous gland cells in the present embodiment, explanations of each configuration, each processing method, each apparatus, and the like, such as the whole plasma secretion regulator, evaluation, and selection, which are sufficient to the above-mentioned "1", may be appropriately omitted, but the explanation of "1" is also applicable to the present embodiment, and the explanation thereof may be appropriately adopted. In the description of the whole plasma secretion regulator and the like in the present embodiment, the following description of "3" can be applied to the present embodiment and can be appropriately applied.

In another aspect of the present embodiment, it is also possible to provide a three-dimensional structure of sebaceous gland cells and/or a method for producing a three-dimensional structure of sebaceous gland cells. Thus, a three-dimensional structure of sebaceous gland cells and/or a method for producing the same, particularly a novel three-dimensional structure and a method for producing the same, can be provided.

<2-1 > three-dimensional Structure of sebaceous gland cells according to the present embodiment >

This embodiment can provide a three-dimensional structure of sebaceous gland cells whose surface layer is composed of mature sebaceous gland cells (see fig. 3). The three-dimensional structure is preferably used for fluorescence observation. From the viewpoint of observation for the use in the whole plasma secretion, a three-dimensional structure having a structure in which mature sebaceous gland cells present in the surface layer undergo whole plasma secretion and sebaceous gland cells present therebelow after lysis become the surface layer is more preferable. In the present embodiment, since the surface layer has mature sebaceous gland cells, there is an advantage that observation (preferably confocal laser observation) relating to the state or condition of the regulation of the whole plasma secretion can be easily performed.

The three-dimensional structure of the sebaceous gland cell of the present embodiment can be used in the technology (experimental system, model, or the like) related to the panniculus secretion in which the sebaceous gland cell participates or dermatitis. For example, the present invention can be used for a method for evaluating or selecting a whole plasma secretion regulator, a cell experiment system for pore clogging formation, a cell experiment system for elucidating a whole plasma secretion mechanism of sebaceous glands, a cell experiment system for elucidating a relation between whole plasma secretion and autophagy, a cell experiment system for elucidating pathogenesis such as seborrheic dermatitis and acne, or models thereof.

In the conventional experiments using sebaceous gland cells, only the amount of sebum production was evaluated. Therefore, in a two-dimensional culture in which sebaceous gland cells are two-dimensionally cultured, a sebum region stained with a fluorescent dye is observed with a plane microscope, the sebum region is measured, sebum is extracted from the sebaceous gland cells of the two-dimensional culture, and the amount of sebum produced is evaluated by quantifying the extracted sebum. Furthermore, in the two-dimensional culture, considering the amount of sebum production, the present inventors considered that the sebaceous gland cells were not mature until the stage of the whole plasma secretion.

In addition, conventionally, a laminate sheet is obtained by laminating layers of skin cells, and the distribution of sebum production in the cross section is observed to evaluate the amount of sebum production (non-patent document 1). Further, a plurality of sebaceous gland cells were embedded in matrigel and cultured to obtain spheroids in which mature cells are aggregated in the central part, and the amount of sebum production present inside was evaluated (non-patent document 2).

Thus, in the prior art, both two-dimensional culture and three-dimensional culture have been evaluated only for sebum production.

In contrast, the present inventors have for the first time elucidated a pore-clogging mechanism in which autophagy induction of sebaceous gland cells normally causes full-plasma secretion, autophagy failure causes abnormal full-plasma secretion, sebaceous gland cells cannot normally lyse to clog pores, and regardless of the amount of sebum production found in the sebaceous gland cells, the full-plasma secretion state (normal or abnormal state) of the sebaceous gland cells closely participates in pore-clogging formation (see fig. 1 and 2). Thus, the present inventors have obtained a novel concept and technique of full-plasma secretion of sebaceous gland cells. Further, the present inventors have studied a culture method of a sebaceous gland cell which can be observed more favorably based on the concept and technique of the whole plasma secretion of a sebaceous gland cell, and as a result, have obtained a three-dimensional structure of a sebaceous gland cell whose surface layer is composed of mature sebaceous gland cells for the first time.

The shape of the three-dimensional structure of the sebaceous gland cell of the present embodiment is not particularly limited, and examples thereof include a tubular shape, a spherical shape, a sheet shape, and the like, but a substantially spherical shape is preferable, and a spheroid is more preferable (see fig. 3 and 4).

The size of the three-dimensional structure of the sebaceous gland cell of the present embodiment is preferably 200 to 600 μm, more preferably 250 to 550 μm, and even more preferably 300 to 500 μm in minimum length, and is preferably 300 to 700 μm, more preferably 350 to 650 μm, and even more preferably 400 to 600 μm in maximum length. When the cross section is an ellipse (approximately a rotational ellipsoid), the minimum length may be replaced by a short diameter and the maximum length may be replaced by a long diameter.

When the three-dimensional structure of the sebaceous gland cell of the present embodiment is substantially spherical, the diameter is more preferably 250 to 650 μm, still more preferably 300 to 600 μm, yet more preferably 350 to 550 μm, and yet more preferably 400 to 500 μm.

In the three-dimensional structure of the sebaceous gland cell of the present embodiment, it is preferable that mature sebaceous gland cells are present in the surface layer on the observation side. The three-dimensional structure of the sebaceous gland cells preferably has a large number of mature sebaceous gland cells in the surface layer, and more preferably has a large number of immature sebaceous gland cells in the interior near the center (see fig. 4). The three-dimensional structure of the sebaceous gland cells is from the inside to the outside, and after the immature sebaceous gland cells are matured, mature sebaceous gland cells exist and exist on the surface layer.

The thickness (depth direction) of the layer in the surface layer where the mature sebaceous gland cells are present is preferably within 50 μm, more preferably within 40 μm, and still more preferably within 30 μm from the surface side.

From the viewpoint of observation, it is preferable that mature sebaceous gland cells are densely present in the surface layer.

The proportion of the mature sebaceous gland cell region 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 proportion of the mature cell region can be calculated as follows: after the mature sebaceous gland cells existing in the three-dimensional structure are subjected to fluorescent staining, the three-dimensional structure is subjected to fluorescent observation, the three-dimensional structure is subjected to two-dimensional photographing to obtain a surface image (or a surface photograph), and the surface image (or the surface photograph) is calculated by [ the total area of the fluorescent luminescence of the mature sebaceous gland cells/the area of the surface image of the three-dimensional structure of sebaceous gland cells ] × 100 (%) ] based on the total area of the fluorescent luminescence of the mature sebaceous gland cells in the two-dimensional surface image.

Since mature sebaceous gland cells are present in the surface layer of the three-dimensional structure of the present embodiment, staining, fluorescence observation, and the like of mature sebaceous gland cells are easily performed, and therefore the three-dimensional structure of the present embodiment is suitably used for staining observation or fluorescence observation.

The three-dimensional structure of the present embodiment is suitable for confocal laser microscope observation because living cells of mature sebaceous gland cells present in the superficial layer can be easily subjected to fluorescent staining and fluorescence observation. Thus, mature sebaceous gland cells of the surface layer can be easily stained or fluorescently stained. Furthermore, since the surface layer can be easily observed by fluorescence, the method for evaluating and/or selecting the whole plasma secretion regulatory agent can also be easily performed. Therefore, the three-dimensional structure of the present embodiment is suitably used for the evaluation and/or method of the embodiment.

<2-2 > method for producing a three-dimensional structure of sebaceous gland cells according to the present embodiment >

The present embodiment can provide a method for producing a three-dimensional structure of sebaceous gland cells, which comprises culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting treated surface (more preferably a hydrophilized surface) of a mortar-like well to obtain a three-dimensional structure of sebaceous gland cells. This makes it possible to obtain the three-dimensional structure of the sebaceous gland cell of the present embodiment, more preferably the three-dimensional structure of the sebaceous gland cell whose surface layer is composed of mature sebaceous gland cells.

The bottom of the mortar-shaped recess is preferably in a shape such that a central region of the bottom of the recess can collect a plurality of sebaceous gland cells. The bottom of the concave portion is more preferably V-shaped or U-shaped, and the seeded sebaceous gland cells easily gather in the V-shaped or U-shaped depression, and thus can form a more favorable globular cell mass shape, and mature sebaceous gland cells easily exist on the surface layer.

The shape of the vessel to be seeded with sebaceous gland cells may be exemplified by a culture dish, a flask, a plate having a plurality of wells, and the like, but is not limited thereto, and disposable products are preferred. Examples of the material include glass, plastic resin (preferably polystyrene resin), and the like, but the material is not limited thereto.

Preferably, the dents into which sebaceous gland cells are seeded are subjected to a cell adhesion-inhibiting treatment, and this surface treatment can inhibit the adhesion of cells to the surface of the dents, and the three-dimensional structure can be easily peeled off after culture. By this surface treatment, the seeded sebaceous gland cells are apt to aggregate in the central region, and the cells are apt to adhere to each other, whereby a more favorable spherical cell mass shape can be formed, and mature sebaceous gland cells are apt to exist in the surface layer thereof. The cell adhesion-inhibiting treatment is not particularly limited, and examples thereof include a hydrophilization surface treatment and the like.

The recesses are more preferably subjected to a hydrophilization surface treatment, and examples of the hydrophilization include a plasma treatment, a corona discharge treatment, an oxidizing agent treatment, a coating treatment with a hydrophilic substance (preferably a cell adhesion inhibitor such as polyethylene glycol), and the like, but the hydrophilization is not limited thereto. Examples of the hydrophilic substance coating include a polymer coating.

In the production method of the present embodiment, the outside of the mortar-like well can be performed under normal conditions for culturing human sebaceous gland cells in vitro. In the production method of the present embodiment, the culture conditions (particularly, autophagy-inhibiting conditions) described in "1" above may be applied, and the culture conditions may be used as conditions for producing spheroids. In this case, it is preferable to use a propagation medium for animal cell culture which is generally used for propagation, maintenance, differentiation and the like, among which a differentiation medium is preferable, and more specifically, a differentiation medium which does not contain a differentiation inhibitory factor (e.g., EGF, BPE and the like) or which is reduced in amount is preferable. Examples of the propagation medium for culturing animal cells include, but are not limited to, basal medium, serum-reduced medium, and serum-free medium.

The culture conditions in the present embodiment are preferably normal conditions for culturing animal cells, for example, pH 7 to 8, and CO is preferably cultured ₂ About 4-10% in the atmosphere, and the culture temperature is about 36-37 ℃. The culture period is not particularly limited, but is preferably 5 to 15 days, more preferably 6 to 13 days, and still more preferably 7 to 12 days after seeding.

The culture may be performed under static or gyratory conditions, preferably in a static state.

The seeding concentration of the sebaceous gland cells in the culture medium is preferably 1X 10 ² ～1×10 ⁵ cells/mL, more preferably 5X10 ² ～2×10 ⁴ cell/mL, more preferably 1X 10 ³ ～1×10 ⁴ cells/mL.

<3. Holoplasmic secretion regulator >

In the description of the whole plasma secretion regulatory agent in the present embodiment, the description of each configuration, each processing method, each apparatus, and the like, such as the whole plasma secretion regulatory agent, evaluation, and selection, which are repeated in the above-described "1." "to" 2. "is appropriately omitted, but the description of" 1. "" to "2." is also applicable to the present embodiment, and the description thereof can be appropriately adopted.

In another aspect of the present embodiment, it is possible to provide a substance having a function of regulating the secretion of whole plasma, an active ingredient of a whole plasma secretion regulator, and/or a whole plasma secretion regulator. Thus, it is possible to provide a technique relating to the regulation of the whole plasma secretion of sebaceous gland cells, specifically, a substance having a function of regulating the whole plasma secretion, or an active ingredient of a whole plasma secretion regulator, and/or a whole plasma secretion regulator. This enables provision of a drug that addresses the skin problem of pore clogging.

The method for evaluating and/or selecting a whole plasma secretion regulator according to the present embodiment (for example, the methods according to the first to third embodiments of the present invention) can provide a selected substance (hereinafter also referred to as "selected substance") as a substance having a whole plasma secretion regulating action or as an active ingredient of a whole plasma secretion regulator.

The method for evaluating and/or selecting a whole plasma secretion regulator may be a device or system for evaluating and/or selecting a whole plasma secretion regulator according to the present embodiment.

Examples of the function of regulating the secretion of the whole plasma include, but are not limited to, a function of regulating the secretion of the whole plasma of sebaceous gland cells, a function of regulating pore formation, and a function of regulating autophagy of sebaceous gland cells. Examples of the regulatory action include a promoting or inducing action, an inhibiting or inhibiting action, and a maintaining action. More specifically, examples thereof include an action of promoting the whole plasma secretion of sebaceous gland cells, an action of inhibiting the whole plasma secretion of sebaceous gland cells, an action of promoting pore formation, an action of inhibiting pore formation, an action of promoting the autophagy of sebaceous gland cells, and an action of inhibiting the autophagy of sebaceous gland cells. From these, 1 or more than 2 species can be selected.

Therefore, the selected substance obtained by the method for evaluating and/or selecting a whole plasma secretion regulator according to the present embodiment has a whole plasma secretion regulating effect, and can exert an effect of regulating pore clogging.

The selected substance is not particularly limited, and may be a natural source or artificially prepared, and may be a monomer or a mixture. The selected substance is preferably 1 or 2 or more selected from the group consisting of compounds, microorganisms, cultures thereof, extracts thereof, mixtures thereof, and combinations thereof. The compound may be either an inorganic compound or an organic compound.

Preferred examples of the selected substance include retinoids (retinoids) such as retinoic acid; benzoyl peroxide; oleic acid; curcumin such as curcumin; oligosaccharides such as trehalose; vitamin D; macrolide compounds such as rapamycin and bavlomycin; phosphatidylinositol 3-kinase inhibitors such as wortmannin; chloroquine and the like, and 1 or 2 or more selected from them can be used.

Among them, 1 or 2 or more compounds selected from the group consisting of macrolide compounds, curcuminoids, retinoids, oligosaccharides and phosphatidylinositol 3-kinase inhibitors are preferable, and oligosaccharides and/or macrolide compounds are more preferable.

Among them, retinoids such as tretinoin, benzoyl peroxide and oleic acid are preferably used as pore-clogging regulators. Among them, curcumin, trehalose, vitamin D, rapamycin, wortmannin, chloroquine are preferably used as autophagy regulators.

Examples of the oligosaccharide used in the present embodiment include disaccharides such as trehalose (α, α -trehalose), neotrehalose (α, β -trehalose), isocystulose (β, β -trehalose), and sucrose; and 2 to 10 saccharides (preferably di-to tetrasaccharides) such as trisaccharides (e.g., raffinose), and among them, non-reducing oligosaccharides such as trehalose are preferable, and trehalose is more preferable.

The macrolide compound used in the present embodiment is a macrolide, preferably a compound of ring member 12 or more. Examples of the macrolide compound include rapamycin and bafilomycin. Among them, rapamycin and bafilomycin are more preferable.

Examples of the curcumin used in the present embodiment include curcumin; curcumin analogs such as demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin, and the like. Among them, curcumin is more preferable.

The retinoid used in the present embodiment may be a natural retinoid or a synthetic retinoid, and examples thereof include retinol, retinol derivatives (retinol acetate, retinol palmitate, retinol propionate, and the like), tretinoin, isotretinoin, motretinide, avermectin, acitretin, all-trans-retinoic acid or zinc all-trans-retinoic acid, and synthetic retinoids (adapalene and the like). Among them, retinol and tretinoin are more preferable.

Further, as the compound having an autophagy promoting action, a macrolide compound such as trehalose and non-reducing disaccharide like, rapamycin, a curcumin such as curcumin, a retinoid such as retinol or retinoic acid, and the like are preferable, and these compounds are useful as active ingredients of autophagy promoters or whole plasma secretion promoters.

The compound having an autophagy-inhibiting action is preferably a macrolide compound such as bafilomycin or a phosphatidylinositol 3-kinase inhibitor such as wortmannin, and these compounds are useful as active ingredients of autophagy inhibitors or whole plasma secretion inhibitors.

In this embodiment, 1 or 2 or more selected substances selected from them can be used.

The selected substance may be contained as an active ingredient of a whole-plasma secretion regulator (preferably, a whole-plasma secretion promoter or a whole-plasma secretion inhibitor), a pore-clogging formation regulator (preferably, a pore-clogging formation promoter or a pore-clogging formation inhibitor), an autophagy regulator (preferably, an autophagy promoter or an autophagy inhibitor), a pore-clogging prevention/improvement/treatment agent (hereinafter, also referred to as a "whole-plasma secretion regulator or the like"), or may be used as a whole-plasma secretion regulator or the like. The agent may be a composition.

In addition, the selected substances can be used for preparing a whole plasma secretion regulator and the like.

In addition, the present embodiment may also provide the selected substances or their use for regulating or for the regulation of whole plasma secretion or the like. The regulation of the secretion of whole plasma and the like may be 1 or 2 or more selected from the group consisting of regulation of the secretion of whole plasma (preferably promotion of the secretion of whole plasma and inhibition of the secretion of whole plasma), regulation of pore formation (preferably promotion of pore formation and inhibition of pore formation), regulation of autophagy (preferably promotion of autophagy and inhibition of autophagy), and prevention, improvement and treatment of pore formation.

The present embodiment can also provide a method for regulating whole plasma secretion, a method for regulating autophagy, a method for preventing, improving, and treating pore clogging, or a method for regulating whole plasma secretion, a method for regulating autophagy, and a method for preventing, improving, and treating pore clogging, using a drug containing the selected substance.

< diseases, symptoms >

The selected substance exerts such a physiological activity and is therefore useful in a method for preventing, ameliorating or treating a disease caused by pore clogging or a symptom of pore clogging.

The disease caused by pore clogging or the symptom of pore clogging is not particularly limited, and examples thereof include blackheads of pores, pore dehiscence, pore roughness, pore redness, acne (acne), seborrheic dermatitis, and the like, and 1 or 2 or more kinds thereof can be selected.

Further, the site where pores are likely to be clogged may be a site with sebaceous glands, and more specifically, scalp, facial skin (nose, forehead, etc.), chest, and the like.

Examples of the application of the present embodiment include, but are not limited to, prevention, amelioration, or treatment of pore clogging.

In the present embodiment, "prevention" refers to prevention or delay of onset of symptoms or diseases of the subject to be applied, reduction of risk of onset of symptoms or diseases of the subject to be applied, or the like. In the present technology, "amelioration" refers to improvement or maintenance of a disease, symptom, or state of a subject to which it is applied; prevention or delay of deterioration; reversal, prevention, or delay of progression.

In the present embodiment, the selected substance can be used in, for example, cosmetics, skin preparations for external use, quasi drugs, foods, drinks, feeds, and the like, but is not limited thereto.

The "whole plasma secretion regulator and the like" can be used, for example, as a cosmetic, an external preparation for skin, a quasi-drug, a food, a drink, a feed, and the like, but is not limited thereto. Among them, cosmetics, external preparations for skin, medicines, quasi drugs, and the like are preferable.

The "whole plasma secretion regulator and the like" may be used as a compounding agent or an additive in a composition, and may be used as a "whole plasma secretion regulator and the like" for compounding or adding to a composition such as a cosmetic, an external preparation for skin, a quasi drug, a food, a drink, a feed, and the like.

In the case of cosmetics and external preparations for skin, for example, the selected substance or whole milk secretion regulator may be blended with various forms of cosmetics or external preparations for skin, such as, for example, milky lotion, cream, lotion, pack, face toilet, color cosmetic, dispersion, ointment, liquid, aerosol, patch, plaster, liniment, etc., but the present invention is not limited thereto.

In the case of a pharmaceutical or quasi-drug according to the present embodiment, the selected substance or the whole plasma secretion regulator may be incorporated into an oral preparation such as a tablet, a capsule, a granule, a powder, a liquid, or a suspension; external preparations such as skin preparation, patch, eye drop, nose drop, oral preparation, and suppository; non-oral preparations such as drops and injections, but are not limited thereto.

The selected substance or whole plasma secretion regulator and the like can be prepared by a known preparation method. Commercially available materials can be used as the selected material.

The content of the selected substance is not particularly limited, and the selected substance may be contained in the total amount of the preparation preferably in a range of 0.001 to 99% by mass, more preferably in a range of 0.005 to 90% by mass, and even more preferably in a range of 0.01 to 90% by mass. The "preparation" of "in the total amount of the preparation may be the" whole plasma secretion regulator and the like ".

The object of application of the selected substance or the whole plasma secretion regulator and the like may be human and non-human animals (e.g., pets, livestock, and the like) and the like. Of these, humans and pets are preferred, and humans are more preferred.

Examples of the method of using the selected substance, the whole plasma secretion regulator, and the like include transdermal administration, oral administration, injection administration, and the like; oral ingestion; skin coating, etc., but is not limited thereto.

The amount of the selected substance to be used or administered is not particularly limited as long as the effect of the present invention can be obtained, and can be appropriately adjusted depending on the form, site of application, age, sex, and the like of the preparation.

The whole milk secretion regulator and the like can be prepared by a known preparation method. In addition to the above-mentioned selected substances, the whole milk secretion regulator and the like may be used in combination with any of various additives, if necessary.

As the optional component, a component acceptable for cosmetics, external skin preparations, medicines, foods, drinks, feeds, or the like may be appropriately blended, and for example, 1 or 2 or more selected from excipients, colorants, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, humectants, pH regulators, and the like may be appropriately used, whereby a desired dosage form can be obtained.

In addition, the present technology can adopt the following configurations.

[ 1 ] A method for evaluating and/or selecting a whole milk secretion regulator, which comprises

And (3) an observation procedure: observing the state of fluorescent sebaceous gland cells after contacting the single or plurality of sebaceous gland cells with a test substance; and

a discrimination step: and discriminating the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator based on the state of the fluorescent sebaceous gland cells.

Preferably, in the observation step, the state of the fluorescent sebaceous gland cell is observed fluorescently in the presence of a test substance and one or more sebaceous gland cells under the condition of whole plasma secretion regulation.

More preferably, in the observation step, the state of the sebaceous gland cells that fluoresce is observed under the whole plasma secretion-regulating conditions after the sebaceous gland cell or cells are brought into contact with the test substance under the growth medium culture conditions (preferably under the spheroid production conditions).

[ 2 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to [ 1 ] above, wherein the whole plasma secretion regulator is 1 or 2 or more selected from the group consisting of a whole plasma secretion promoter, a whole plasma secretion inhibitor, a pore-clogging formation regulator and an autophagy regulator for sebaceous gland cells.

[ 3 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to the above [ 1 ] or [ 2 ], wherein the sebaceous gland cell is a three-dimensional structure composed of a plurality of sebaceous gland cells.

[ 4 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of the above [ 1 ] to [ 3 ], wherein the observation is an observation using time-lapse photography.

[ 5 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of [ 1 ] to [ 4 ] above, wherein the observation is an observation using a fluorescence microscope. The observation is preferably performed using a fluorescence detection device or a fluorescence microscope observation device, and these devices include an observation unit configured to perform fluorescence microscope observation, an imaging unit configured to image an observation target, an image analysis unit configured to analyze an imaged observation image, and a culture unit for animal cells cultured to maintain the observation target.

[ 6 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of [ 1 ] to [ 5 ], wherein the observation step comprises observing the state of a fluorescent sebaceous gland cell after contacting a single or a plurality of sebaceous gland cells with a test substance,

the whole plasma secretion regulatory condition is an autophagy regulatory condition.

The autophagy-modulating condition is an autophagy-inhibiting condition or an autophagy-inducing condition.

[ 7 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of [ 1 ] to [ 6 ] above, wherein the state of the fluorescent sebaceous gland cells is discriminated on the basis of the number of fluorescent disappeared cells per unit volume.

[ 8 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of [ 1 ] to [ 7 ] above, wherein the living cells are fluorescently labeled and caused to emit light by the fluorescent sebaceous gland cells.

[ 9 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of [ 1 ] to [ 8 ] above, wherein the sebaceous gland cell is a three-dimensional structure of a sebaceous gland cell whose surface layer is composed of mature sebaceous gland cells.

[ 10 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to any one of [ 1 ] to [ 9 ] above, wherein the sebaceous gland cells are three-dimensional structures of sebaceous gland cells obtained by culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting treated surface (more preferably a hydrophilized surface) of a mortar-like well.

11A three-dimensional structure of sebaceous gland cells, the surface layer of which is composed of mature sebaceous gland cells.

[ 12 ] the three-dimensional structure of a sebaceous gland cell according to [ 11 ] above, wherein said three-dimensional structure is spherical.

[ 13 ] the three-dimensional structure of sebaceous gland cells according to [ 11 ] or [ 12 ] above, wherein the fluorescence observation is preferably carried out using a fluorescence microscope. More preferably used for 1 or 2 or more selected from confocal laser microscope observation, multiphoton excitation microscope observation and light sheet microscope observation, and further preferably used for confocal laser microscope.

[ 14 ] the three-dimensional structure of sebaceous gland cells according to any one of [ 11 ] to [ 13 ] above for use in a method for the evaluation and/or selection of a whole plasma secretion modulator.

[ 15 ] A method for producing a three-dimensional structure of sebaceous gland cells, wherein a three-dimensional structure of sebaceous gland cells is obtained by culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting treated surface (preferably a hydrophilized surface) of a mortar-like well.

The cell adhesion-inhibiting treated surface or the hydrophilized surface is preferably 1 or 2 or more selected from plasma treatment, corona discharge treatment, oxidant treatment, hydrophilic substance coating treatment, and the like, and in hydrophilic substance coating, it is preferable to treat with a cell adhesion inhibitor such as a photo-crosslinking hydrophilic polymer.

[ 16 ] the method for producing a three-dimensional structure of sebaceous gland cells according to [ 15 ], wherein the mortar-like well has a shape such that a plurality of sebaceous gland cells can be collected in the bottom central region of the well.

The bottom of the mortar-shaped concave part is preferably V-shaped or U-shaped.

17A method for evaluating and/or selecting a whole plasma secretion regulator, which comprises

And (3) an observation procedure: performing fluorescence observation on the collected keratotic plug state after the single or multiple sebaceous gland cells exist with the tested substance; and

a discrimination step: and according to the state of the fluorescent angle bolt, judging the tested substance as a holoplasmic secretion regulator or a candidate of the holoplasmic secretion regulator.

[ 18 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to [ 17 ] above, wherein the whole plasma secretion regulator is 1 or 2 or more selected from the group consisting of a whole plasma secretion promoter, a whole plasma secretion inhibitor, a pore-clogging formation regulator and an autophagy regulator for sebaceous gland cells.

[ 19 ] the method for evaluating and/or selecting a whole plasma secretion regulator according to the above [ 17 ] or [ 18 ], wherein the observation is an observation using a fluorescence microscope. The observation is preferably performed using a fluorescence detection device or a fluorescence microscope observation device, and these devices include an observation unit configured to perform fluorescence microscope observation, an imaging unit configured to image an observation target, and an image analysis unit configured to analyze an image of the captured observation image.

·〔20〕

The method for evaluating and/or selecting a whole plasma secretion regulator according to any one of the above [ 17 ] to [ 19 ], wherein the keratotic plug in the observation step is a plurality of the keratotic plugs attached to an adhesive sheet collected from the skin surface of a mammal using the adhesive sheet; and/or

The state of the plug in the determination step is a rate of change in the maximum plug diameter obtained by dividing the average value of the maximum plug diameters after the test substance is used by the average value of the maximum plug diameters before the test substance is used.

[ 21 ] a whole milk secretion regulator or the like which is a selected substance evaluated and/or selected by the evaluation and/or selection method of the whole milk secretion regulator described in any one of [ 1 ] to [ 10 ] and [ 17 ] to [ 20 ]; or contains the selected substance as an active ingredient.

22A whole plasma secretion regulator and the like, which contains a selective substance.

The selection substance is preferably 1 or 2 or more selected from compounds, microorganisms, fermentation products, extracts, and mixtures thereof, and compositions. The selected substance may be natural or synthetic.

[ 23 ] the selective substance according to [ 21 ] above, or the whole plasma secretion regulator comprising the selective substance as an active ingredient, or the whole plasma secretion regulator according to [ 22 ] above, wherein the selective substance is 1 or 2 or more selected from the group consisting of retinoids, benzoyl peroxide, oleic acid, curcumin, trehalose, vitamin D, rapamycin, wortmannin and chloroquine, or a mixture thereof.

[ 24 ] the selective substance according to [ 21 ] above, or the whole plasma secretion regulator comprising the selective substance as an active ingredient, or the whole plasma secretion regulator according to [ 22 ] above, wherein the selective substance is 1 or 2 or more compounds (more preferably oligosaccharide and/or macrolide compounds) selected from the group consisting of oligosaccharide, retinoid, curcumin, macrolide compounds, and phosphatidylinositol 3-kinase inhibitor, or a mixture thereof.

[ 25 ] A cosmetic, a skin external preparation, a quasi-drug, a medicine or a food, which contains the selective substance according to any one of [ 21 ] to [ 24 ] above, or a whole-plasma secretion regulator containing the selective substance.

[ 26 ] the selected substance according to any one of [ 21 ] to [ 24 ] above for use in the preparation of a whole milk secretion regulator or the like, or a use thereof.

The selective substance according to any one of [ 21 ] to [ 24 ] above for use in, for example, regulation of whole plasma secretion or the like, or use thereof.

(28) use of the selected substance according to any one of [ 21 ] to [ 24 ] in a method for regulating secretion of whole plasma, a method for regulating pore formation, a method for regulating autophagy, or a method for preventing, improving or treating pore clogging in a mammal.

[ examples ] A method for producing a compound

Embodiments of the present invention will be described below with reference to examples and comparative examples. The scope of the present invention is not limited to the examples.

Generally, an ideal skin appearance has both uniformity and smoothness characteristics, both of which are adversely affected by pore morphology changes (pore enlargement, acne, etc.). Pore clogging is generally considered to be one of the main causes of pore morphology change. The formation of pore clogging has long been considered to be related to the occlusion of pore outlets by a thickened stratum corneum and the subsequent accumulation and solidification of sebum within the pores. However, this conventional concept cannot explain the morbid state of pore clogging when pore outlets are opened, that is, the formation of open acne (open comedo) and micro acne (fine acne), and also fails to explain the detailed mechanism of pore clogging formation.

< test example 1: confirmation test of pore clogging by removal of skin and autophagy inhibitor Using human >

Sample preparation: skin (without pore clogging) sheet (1 cm) ² ): the abdominal skin of a 30's white female. The skin contains the pilosebaceous part.

< method for sebum staining of Hair organ >

Adding skin piece at 37 deg.C and 5% CO ₂ After 6 days of culture using William's E Medium supplemented with 10% fetal bovine serum under the conditions, the pilo-sebaceous gland part of the whole pore was removed from the skin piece, fixed overnight at 4 ℃ using 4% paraformaldehyde solution, and thereafter stained with 1 μ g/mL nile red solution (Sigma) and DAPI stain (Sigma) at room temperature for 30 minutes, the sebum present in the pore and sebaceous gland duct was stained, and the stained sebum was observed by a fluorescence microscope.

< fluorescence observation of cultured skin under autophagy-regulating conditions >

As described in test examples 1a to 1b below, an autophagy inhibitor was added, and the skin cultured while inhibiting autophagy was collected, subjected to fluorescent staining, and then subjected to fluorescent observation. In test example 1a, bafilomycin A1 was used as an autophagy inhibitor, and in test example 1b, MHY-1485 was used as an autophagy inhibitor. Test example 1c was performed in the same manner as in test example 1a, except that a DMSO solvent not containing an autophagy inhibitor was used as a negative control.

As the culture conditions of test examples 1a, 1b and 1c, CO was 5% at 37 ℃% ₂ Skin (no pore clogging) sheets were cultured for 6 days under the conditions using William's E Medium supplemented with 10% fetal bovine serum. In test examples 1a and 1b, the culture medium was supplemented with a DMSO solvent containing an autophagy inhibitor, and cultured for 6 days in a state where the skin section was brought into contact with the autophagy inhibitor.

In test examples 1a, 1b and 1c, after the skin (without pore clogging) sheet was cultured for 6 days, the whole hair follicle sebaceous gland portion was removed from the skin sheet, fixed with 4% paraformaldehyde solution at 4 ℃ overnight, and then stained with 1. Mu.g/mL nile red solution (Sigma) and DAPI stain (Sigma) at room temperature for 30 minutes to stain the sebum present in the pores and sebaceous gland ducts. The stained sebum is observed by a fluorescence microscope with the hair contained in the pilosebaceous portion, the hair follicle wall facing the hair follicle, and the sebaceous gland duct connected to the pore as the center.

At this time, the distance between the hair follicle side of the hair and the portion sandwiched between the hair follicle walls facing the hair is measured, and the portion is densely filled with fluorescent dye, and the longest distance is defined as the hair-follicle wall distance (between the tips of the opposing triangles in the figure).

After 6 days of human skin culture with the agents bafilomycin A1 or MHY-1485 that inhibit autophagy by different mechanisms, the pores and sebaceous glands were observed three-dimensionally using a confocal laser microscope (carl zeiss LSM 800) and image analysis software Imaris (high precision 3D/4D image analysis software)).

FIG. 1B-1 is the incubation with the control (DMSO solution without autophagy inhibitor added) in test example 1c, FIG. 1B-2 is the incubation in the "presence" of autophagy inhibitor (DMSO solution containing Barflomycin A1) in test example 1a, and FIG. 1B-3 is the incubation in the "presence" of autophagy inhibitor (DMSO solution containing MHY 1485) in test example 1B.

The distance between the triangular arrow and the triangular arrow indicates the position between the hair (left side) and the hair follicle wall (right side) filled with pigmented sebum oil and the longest distance (hair-follicle wall distance: μm).

As shown in fig. 1 and 2, when an autophagy inhibitor (baverromycin A1) is added after the culture; the hair-follicle wall distance (. Mu.m) after incubation with autophagy inhibitor (MHY-1485) was 1.8 fold for baverromycin A1 and 1.6 fold for MHY-1485, respectively, with a significant difference (p < 0.05).

The results confirmed pore blockage due to autophagy inhibition. These results indicate that autophagy failure of sebaceous gland cells prevents smooth progress of the whole-plasma secretion of sebaceous gland cells, and that sebaceous gland ducts and hair follicles are clogged.

The present inventors have clarified the mechanism of pore clogging formation for the first time from the above results. The present inventors found that autophagy failure of sebaceous gland cells in skin with pore blockage caused hypoplasmatic secretion of sebaceous gland cells and cell accumulation at sebaceous gland ducts and hair follicles. It is thus believed that the autophagy disorder of sebaceous gland cells causes pore blockage.

The above findings of the present inventors provide important insights related to the molecular mechanisms for maintaining pore health. Furthermore, the present study is expected to provide a novel skin care that can maintain autophagy of sebaceous gland cells appropriately, prevent pore enlargement and acne, and create a beautiful, uniform, smooth skin.

< test example 2: method for producing three-dimensional structure of sebaceous gland cell and method for observing the same

< method for producing spherical body >

The sebaceous gland cells used are human-derived sebaceous gland cells (2X 10) ⁶ (cell/visual) Phenocell, white human species as the donor, and sebaceous gland cells (PCi-SEB) derived from iPS cells, which are immature sebaceous gland cells as the stage. Incidentally, it is also available from Phenocell corporation in Asian race or African race as the source donor.

Frozen human sebaceous gland cells (1 mL) were lysed in a warm bath at about 30 ℃. The solubilized sebaceous gland cells were suspended in 4mL of a culture medium, and the suspension was centrifuged to remove the supernatant and recover the sebaceous gland cells. To the sebaceous gland cells, a differentiation medium (CELLnTEC) containing CnT-Prime was added, and 5X10 cells were prepared in the medium ⁴ cells/mL of cell suspension.

In a 96-well plate having wells each having a V-shaped or U-shaped vertical cross section at the bottom and a shape allowing accumulation of a plurality of sebaceous gland cells in the bottom center region of a mortar-shaped well, 100. Mu.L of a cell suspension was added per well at a rate of 5X10 per well ³ Sebaceous gland cells were seeded as cells (see FIG. 3). After seeding the sebaceous gland cells, the cell suspension of the sebaceous gland cells was subjected to the incubation at 37 ℃ with 5% CO ₂ Conditions of (d) was cultured in a static state for 11 days. During this period, the spheroid preparation medium was changed once a day. Meanwhile, a plurality of sebaceous gland cells are accumulated in the central region of the well and cultured, and as a result, spheroids of the sebaceous gland cells can be formed, whereby a three-dimensional structure of the sebaceous gland cells can be obtained.

The three-dimensional structure of the obtained sebaceous gland cells was substantially spherical, had a minimum diameter of 400 μm and a maximum diameter of 500 μm, and the diameters were approximately in the range of 400 to 500 μm (see FIGS. 3 and 4).

And (3) observing the three-dimensional structure of the obtained sebaceous gland cells by using the fat differentiation related protein as a maturation marker and carrying out fluorescent staining on the mature sebaceous gland cells. Fig. 4 a is a photograph showing the overall appearance of the three-dimensional structure (spheroid) of the sebaceous gland cells when the mature sebaceous gland cells are fluorescently stained. Fig. 4B is a sectional view in the depth direction from the surface layer of the spheroid stained in fig. 4 a, and is an image showing the position where mature sebaceous gland cells exist in the spheroid obtained in the present embodiment. As shown in fig. 4, fluorescent staining of the adipose differentiation-related protein was confirmed in most of the range near the surface layer of the three-dimensional structure.

Thus, mature sebaceous gland cells are densely present in the surface layer of the three-dimensional structure of the obtained sebaceous gland cells, but the mature sebaceous gland cells are less observed as the distance from the center part is closer. It should be noted that the thickness of the layer in the surface layer where the mature sebaceous gland cells exist is about 30 μm.

The proportion of mature sebaceous gland cell areas present on the surface of the three-dimensional structure is about 80 to 100% of the surface. The proportion of this region is calculated as follows: after the mature sebaceous gland cells present in the three-dimensional structure are subjected to fluorescent staining, a surface image of the three-dimensional structure is photographed by fluorescent observation of the three-dimensional structure, and the surface image is calculated by multiplying [ the total area of the fluorescent emission of the mature sebaceous gland cells/the surface image area of the three-dimensional structure of the sebaceous gland cells ]. Times.100% on the basis of the total area of the fluorescent emission of the mature sebaceous gland cells in the surface image.

< Condition for regulating Total plasma secretion >

Spheroids of sebaceous gland cells on day 11 after cell seeding were transferred to glass-bottom culture dishes at room temperature (20 ℃) under atmospheric air. As a result of intensive studies, the present inventors have found for the first time that mature sebaceous gland cells present in the surface layer of a three-dimensional structure are in a "whole plasma secretion state or incomplete plasma secretion" state by changing the type of a commercially available culture medium, and thus have found conditions for regulating whole plasma secretion of a three-dimensional structure of sebaceous gland cells.

< starvation Medium (autophagy-inducing Medium) >

Basal medium without amino acids (serum not supplemented): D-MEM (Dulbecco's modified Eagle/high sugar; sodium pyruvate; without amino acids) medium (Fuji film & Wako pure chemical industries: 048-33575).

< propagation Medium (autophagy-inhibiting Medium) >

D-MEM (Dulbecco's modified-Eagle/L-glutamic acid; phenol red-free) medium supplemented with 10% fetal bovine serum (Fuji film and Wako pure chemical industries: 040-30095).

< kit for cell/dead cell imaging >

cell/DEAD cell imaging kit (LIVE/DEAD) ^TM Cell Imaging Kit (488/570): when the living cells are subjected to fluorescence detection, the living cells emit green fluorescence under the excitation wavelength of 488 nm/the detection wavelength of 515 nm.

< confocal laser microscope >

Confocal laser microscope (carl zeiss LSM 800) and image analysis software (Imaris (high precision 3D/4D image analysis software)): at normal temperature (10-30 deg.C), adding hunger culture medium or reproduction culture medium, test substance and CO ₂ Concentration of 5% CO ₂ Atmospheric pressure) were observed by fluorescence using time-lapse photography.

< State in which mature sebaceous gland cells produce whole plasma secretion (in the case of using starvation medium conditions) >

Adding a small amount of starvation medium to a glass-bottomed culture dish containing mature sebaceous gland cell spheroids, removing the starvation medium, washing the spheroids with the starvation medium to adapt the spheroids to the starvation medium, and then adding CO in an incubator (37 ℃, 5% ₂ Below) contacting the spheroids and starved medium within the container.

Thereafter, 300. Mu.L of spheroid and starvation medium were mixed with LIVE/DEAD cell imaging kit (LIVE/DEAD) in the incubator ^TM Cell Imaging Kit (488/570)) 300. Mu.L was mixed to prepare a mixture (Cell suspension) containing spheroids, starving medium and fluorescent substance, and the mixture was added to a glass-bottom dish (Cell suspension was added)5％CO ₂ ). As the starvation medium, the above-mentioned D-MEM (high sugar; sodium pyruvate-containing; amino acid-free) medium was used.

As described above, the fluorescent substance for detecting living cells was added to and mixed with the glass-bottomed culture dish containing spheroids and starvation medium under the condition that the whole plasma secretion occurred, living cells of the sebaceous gland cells were subjected to fluorescent staining, and the three-dimensional structure of the fluorescent sebaceous gland cells contained in the glass-bottomed culture dish was observed using a confocal laser microscope. The content of CO in the glass-bottomed culture dish was adjusted to 37 ℃ and 5% ₂ . The change of the living cells with time was observed by time-lapse photography using the number of cells with time eliminated per unit volume as an index (see fig. 5 to 7).

When a substance having a full-plasma secretion inhibitory effect is evaluated and/or selected, a test substance is added to a spheroid-producing medium containing spheroids before a fluorescent substance is added for at least 2 hours. For a test substance having a slow action, the test substance was added to the spheroid preparation medium containing spheroids 24 hours before the fluorescent substance was added.

In this way, after culturing the mixture containing the spheroids, the test substance, and the spheroid preparation medium for a predetermined time, the spheroids are transferred to the test substance and the starvation medium, and the fluorescent substance is further added to the mixture. The spheroids in the mixed solution containing these fluorescent substances were subjected to fluorescence observation over time under conditions that cause panchromatic secretion. In the time-lapse fluorescence observation, the number of time-lapse fluorescence-lost cells per unit volume is used as an index to determine whether or not the test substance has a full-plasma secretion inhibitory effect, and the evaluation and/or selection of the full-plasma secretion inhibitor can be performed.

< State in which mature sebaceous gland cells do not produce plasma secretion (in the case of using the conditions of the propagation medium) >

Adding a small amount of propagation medium to a glass-bottomed culture dish containing spheroids of mature sebaceous gland cells, removing the propagation medium, washing the spheroids with the propagation medium to adapt the spheroids to the propagation medium, and then adding CO in an incubator (37 ℃, 5% ₂ Below) spheroids and breedingAnd (5) contacting the culture medium.

Thereafter, 300. Mu.L of spheroids and propagation medium were mixed with LIVE/DEAD cell imaging kit (LIVE/DEAD) in the incubator ^TM Cell Imaging Kit (488/570)) 300 μ L, mixing, and making CO content at 37 deg.C and 5% ₂ The spheroids are then contacted with a propagation medium. Preparing a mixture (cell suspension) containing spheroids, a propagation medium and a fluorescent substance, and adding the mixture to a glass-bottomed dish (5% by addition of CO) ₂ ). As the propagation medium, the above-mentioned medium (Fuji film and Wako pure chemical industries: 040-30095) supplemented with 10% fetal bovine serum D-MEM (Dulbecco's modified-Eagle/L-glutamic acid; phenol red-free) was used.

As described above, under the condition that the whole plasma secretion does not occur, the fluorescent substance for detecting living cells was added to and mixed with the glass-bottomed culture dish containing spheroids and the propagation medium, living cells of sebaceous gland cells were subjected to fluorescent staining, and the three-dimensional structure of fluorescent sebaceous gland cells contained in the glass-bottomed culture dish was observed using a confocal laser microscope. The content of CO in the glass-bottomed culture dish was adjusted to 37 ℃ and 5% ₂ . The time-lapse change of the living cells was observed by time-lapse photography using the number of fluorescence-disappeared cells per unit volume as an index (see fig. 5).

In the evaluation and/or selection of the drug having the full-plasma secretion promoting effect, the test substance is added to the spheroid production medium containing spheroids at least 2 hours before the fluorescent substance is added. For a test substance having no significant effect, it is preferable to add the test substance to the spheroid-producing medium containing spheroids 24 hours before adding the fluorescent substance.

In this way, after culturing the mixed solution containing the spheroids, the test substance, and the spheroid preparation medium for a predetermined period, the spheroids are transferred to the test substance and the propagation medium, and the fluorescent substance is added to the mixed solution. The spheroids in the mixed solution containing these fluorescent substances were subjected to fluorescence observation over time under conditions in which global secretion did not occur. In the time-lapse fluorescence observation, whether or not the test substance has a full-plasma secretion promoting effect is determined using the number of time-lapse fluorescence-lost cells per unit volume as an index, and the full-plasma secretion promoter can be evaluated and/or selected.

< confocal laser microscope Observation >

Using confocal laser microscope, at 37 ℃ and 5% CO ₂ Time-lapse imaging is performed on spheroids in a mixed solution containing the test substance under the condition (1).

The objective lens x20 of the confocal laser microscope was used to take images of living cells by changing focal planes gradually from 1 μm to 100 μm from the surface layer of the spheroid using a laser excitation wavelength of 488nm and a detection wavelength of 490 to 570 nm. Further, 1 set of laser irradiation and fluorescence imaging was performed on the three-dimensional structure of the sebaceous gland cells every 10 minutes, and the change with time of the living cells of the sebaceous gland cells was observed for 12 hours. The current imaging condition was that 1 frame (still image) was taken every 10 minutes for 12 hours (72 frames).

The captured images obtained as described above were captured at intervals of 1 μm in the depth direction, and the two-dimensional captured images were constructed as three-dimensional images using analysis software Imaris. In this case, the depth was arbitrarily cut out to a depth of 50 μm in a range of 62.4 μm in the vertical direction (or horizontal direction) and 125 μm in the horizontal direction (or vertical direction), and 3 spots were cut out this time. The obtained still image was reproduced as a video, and the number of cells in which green fluorescence disappeared within 12 hours from the start of imaging (when the fluorescent reagent was added) was counted.

The total number of cells in which the green fluorescence disappeared in these 3 spots was counted, and the average value was determined as the number of cells in which the fluorescence disappeared per unit volume (average value). This makes it possible to determine the state of the plasma secretion of mature cells present in the surface layer of the three-dimensional structure (see FIG. 6).

Under the conditions where the whole plasma secretion occurs, the average number of fluorescence-disappeared cells per unit volume was about 10 in the observation 12 hours after the addition of the fluorescent substance (see a in fig. 5 and 6). On the other hand, under the condition that full-plasma secretion does not occur, the average number of fluorescence-disappeared cells per unit volume was about 1 in the observation after 12 hours from the addition of the fluorescent substance (see B in fig. 5).

FIG. 5A is a graph showing the start time (Panel A-1) and the end time (Panel A-2) of observation of spheroids obtained in the present embodiment in culture under autophagy inducing conditions using starved medium, and a time-lapse graph (particularly, an arrow portion) showing the decrease in the number of cells that fluoresce per unit volume due to the lysis of sebaceous gland cells present in the surface layer of spheroids.

FIG. 5B is a graph showing the images at the start of observation (Panel B-1) and at the end of observation (Panel B-2) of the spheroids obtained in the present embodiment using the autophagy-inhibiting conditions in the propagation medium, and a time-lapse image (particularly, the arrow portion) showing that sebaceous gland cells present in the surface layer of the spheroids are not lysed and the number of cells that fluoresce per unit volume is not reduced.

In the culture under the autophagy induction condition, the sebaceous gland cells with disappeared green fluorescence are the sebaceous gland cells with full plasma secretion, and in the delayed image, the disappearance of the fluorescence of 1 sebaceous gland cell in a short period of time can be observed. In the disappeared area, the sebaceous gland cells that fluoresce right below the cells are exposed on the outermost surface (see fig. 5 a and 6). Thus in the water-in-pulp secretion process, sebaceous gland cells lyse cell membranes and release internal sebum out of the cells, and the released sebum can be well released to the skin surface through sebaceous gland ducts and hair follicles without clogging ducts and pores. The mature sebaceous gland cells normally generate full plasma secretion, thereby preventing and improving pore blockage.

On the other hand, in the culture under the autophagy inhibition condition, the sebaceous gland cells with the green fluorescence not disappeared were the sebaceous gland cells without the occurrence of the whole plasma secretion, and in the delayed image, the sebaceous gland cells existing on the surface of the three-dimensional structure were observed after 12 hours of non-disappearance. Since such dead cells are not secreted in the whole plasma, the dead cells are accumulated in the sebaceous gland duct and the hair follicle, and the pores are clogged. The full plasma secretion of mature cells does not occur normally, and pore blockage occurs, which is aggravated.

< test example 3: method for evaluating and/or selecting whole plasma secretion regulator >

As test example 3A, under the global secretion regulatory conditions described in the above-mentioned < state in which no global secretion occurs in mature sebaceous gland cells (in the case of using a propagation medium) > evaluation and/or selection of a global secretion regulator for a test substance was performed by observing with a fluorescent reagent, spheroids, and a confocal laser microscope using trehalose as the test substance.

Under spheroid preparation conditions using a CnT-Prime differentiation medium (CELLnTEC), 1 spheroid of the present embodiment was contacted with trehalose as a test substance for 6 hours to 24 hours.

In an incubator (37 ℃ C., 5% CO) ₂ Next), after the spheroids and trehalose were contacted, the microspheres were washed with 10% fetal bovine serum added D-MEM (Dulbecco modified-Eagle/L-glutamic acid; phenol red-free) medium in the presence of trehalose, and a cell/DEAD cell imaging kit (LIVE/DEAD) in the presence of trehalose ^TM The fluorescent reagent of Cell Imaging Kit (488/570) was added to a glass-bottomed culture dish in the incubator, and mixed to perform fluorescent staining on live cells of sebaceous gland cells. At 37 ℃ C, 5% CO ₂ Under the conditions of (1) fluorescence observation was performed on the state of a fluorescent spheroid in a glass-bottomed culture dish in the presence of a propagation medium, a spheroid and trehalose using a confocal laser microscope (carl zeiss LSM 800) and image analysis software (Imaris (high-precision 3D/4D image analysis software) using time-lapse photography (1 frame per 10 minutes) after 720 minutes of adding a fluorescent reagent, and laser irradiation and fluorescence detection were performed 1 time per 10 minutes in cooperation with the time-lapse photography.

The number of fluorescence-lost cells per unit volume in 720-minute imaging was calculated as the state of the sebaceous gland cells that fluoresce, and when the number of fluorescence-lost cells in culture using a starvation medium as a positive control was 100% ± 30%, the trehalose was identified as a candidate for a whole plasma secretion promoter or a whole plasma secretion promoter, and a substance that does not have a whole plasma secretion promoting effect was selected.

By using trehalose as a selected substance, a full-plasma secretion promoter containing trehalose as an active ingredient can be provided in the present embodiment; a whole plasma secretion inducer; pore clogging preventive, ameliorating or therapeutic agents, and the like.

In addition, instead of trehalose, a retinoid, benzoyl peroxide, oleic acid, curcumin, vitamin D, rapamycin, wortmannin, and chloroquine were used as test substances, and the retinoid, benzoyl peroxide, curcumin, vitamin D, and rapamycin were used as whole plasma secretion promoters, and oleic acid, wortmannin, and chloroquine were used as whole plasma secretion inhibitors, in the same manner as in test example 3A.

In addition, as test example 3B, a method of evaluating and/or selecting a whole plasma secretion inhibitor of a test substance was performed under autophagy-inducing conditions. In this case, after 1 spheroid of the present embodiment is brought into contact with trehalose as a test substance for 6 hours or more and 24 hours or less under spheroid preparation conditions, the spheroid is brought into the presence of trehalose under autophagy induction conditions using an autophagy induction medium, and a fluorescent reagent is added to perform fluorescent staining and fluorescent observation. The concentrations of the test substance in the medium used in the contacting step and the observation step are the same. Fluorescence staining and fluorescence observation were performed in the same manner as in test example 3A.

The number of fluorescence-lost cells per unit volume in 720-minute imaging was calculated as the state of the sebaceous gland cells that fluoresce, and when the number of fluorescence-lost cells in culture using a propagation medium as a positive control was 100% ± 30%, the test substance was identified as a candidate for a pancreatic secretion inhibitor or a pancreatic secretion inhibitor, and was selected as a substance having a pancreatic secretion inhibitory effect.

Thereby enabling the evaluation or selection of a whole plasma secretion inhibitor from a plurality of test substances.

The test substances shown in Table 1 were evaluated for the presence or absence of the full-plasma secretion promoting effect or the full-plasma secretion inhibiting effect using spheroids according to test example 3A described above, and compounds having these effects were selected. The spheroids used were prepared according to the < spheroid preparation method > of test example 2 described above.

Trehalose and rapamycin are carried out in a propagation medium. As the propagation medium, the "D-MEM (Dulbecco's modified-Eagle/L-glutamic acid; phenol red-free) medium supplemented with 10% fetal bovine serum" described in test example 3A above was used. As a starvation medium (autophagy-inducing medium), "serum-free amino acid-free D-MEM (high sugar (4500 mg/L)) (pyruvic acid-containing Na (110 mg/L)) containing no amino acid) (Fuji film and Wako pure chemical industries, ltd.".

The "total number of cells of sebaceous glands with disappeared fluorescence (3 spots)" in Table 1 is the number of cells after disappearance of fluorescence (corresponding to the "number of disappeared fluorescence cells" in test example 3) per unit volume (62.4. Mu. M.times.125. Mu. M.times.50 μm; as shown in FIG. 6). The 3 numbers "13,8, 10" in this column are the measurements at 3 different points within the same spheroid.

In table 1, the denominator of "fluorescence-depleted cell ratio (/ starvation medium/no drug added) (%)" in the selection of all plasma secretion promoter candidates was "starvation medium" and the "average" of drug added (—: no) "was 9.7.

The number of fluorescence-lost cells per unit volume in the 720-minute image was calculated as the state of fluorescent sebaceous gland cells, and starvation medium was used as a positive control

When the number of fluorescent cells lost during culture is 100% +/-30%, the test substance is determined as a whole plasma secretion promoter or a candidate for a whole plasma secretion promoter, and is selected as a substance having a whole plasma secretion promoting effect.

When the number of fluorescence-lost cells per unit volume in 720-minute imaging is calculated as the state of fluorescent sebaceous gland cells, and the number of fluorescence-lost cells in culture using a propagation medium as a positive control is 100% ± 30%, the test substance can be determined as a candidate for a whole plasma secretion inhibitor or a whole plasma secretion inhibitor, and can be selected as a substance having a whole plasma secretion inhibitory effect.

This result confirmed that trehalose and rapamycin have a better plasma secretion promoting effect or autophagy promoting effect and are useful as active ingredients of a plasma secretion regulator.

[ TABLE 1 ]

< test example 4: evaluation or selection of holo-plasmic secretion modulating action of test substance based on evaluation test >

< analysis of the Effect of continuous 4-week trehalose lotion on nasal Angle suppository size >

Application of trehalose toning lotion and collection of keratome

The subject: male with severe nasal keratotic plug between 20-40 years old

Number of subjects: cosmetic water containing trehalose (3%) 8 groups

The cosmetic lotion containing no trehalose is used in 6 groups

< test details >

1. The nose plugs were removed by sticking and pulling them before 4 weeks from the start of the use of the test toilet water (this operation was performed for collecting the plugs at the start of the use of the toilet water after 4 weeks, and the plugs were formed uniformly among the subjects, and therefore the pulled plugs were discarded).

2. After 4 weeks, the nasal plugs were removed from the nasal plugs and the plugs were frozen in the state of adhering to the nasal strips for observation under a confocal microscope. The lotion was applied from that day.

3. After 4 weeks, the nasal plugs were removed from the nasal plugs and the plugs were frozen in the state of adhering to the nasal strips for observation under a confocal microscope.

As described above, for each subject, 2 corner plugs samples before and after use of the lotion were collected, whereby the corner plugs before and after use were collected from each subject, respectively.

< Angle bolt Observation and diameter measurement Using confocal laser microscope >

1. The nasal patch with the keratome attached thereto was thawed and then flattened, and the keratome attached surface was fixed to the observation stage of a confocal microscope (ZEISS, upright confocal laser microscope LSM 700) with the keratome attached surface as the observation surface. The excitation wavelength of the confocal microscope was set to 555nm, and the fluorescence wavelength for detection was set to 559nm or more. The fluorescence wavelength generally used for observation in cell biology is generally in the range of 300 to 800 nm.

2. The kerato plug was irradiated with a laser beam of 555nm (excitation light), and the autofluorescence emitted from the kerato plug was detected at a fluorescence wavelength of 559nm or more. The method is adopted to shoot continuous optical tomography images of 1 corner bolt as a whole, and the shot continuous images are used for reconstructing three-dimensional images by using ZEN black edition of analytic software of ZEISS company. After the three-dimensional image of the corner bolt was constructed, the maximum diameter was measured using the software.

Continuous tomograms (continuous tomograms) of the corner pins are taken, and finally a three-dimensional image can be produced.

The reason for reconstructing the three-dimensional image of the kerato plug is not to be compared with the three-dimensional structure of the cell, but to determine the position where the diameter of the kerato plug is the largest. In this case, the keratin plug is a plug in which the keratin plug is composed of a mixture of human keratin cells and a mixture of human keratin cells.

2. The above procedure was performed for 11-24 corner plugs for 1 sample (1 nose patch with corner plug attached).

3. The average value of the maximum diameters of all the kerato plugs was calculated for 1 sample, and the rate of change in the maximum diameter of the kerato plug was obtained by dividing the average value after the use of the lotion by the average value before the use of the lotion.

In this analysis method, it is not intended to evaluate the change in pore diameter on the skin surface (i.e., pore appearance), but it is intended to estimate whether or not the whole plasma secretion of sebaceous gland cells (rupture and digestion of sebaceous gland cells) occurs normally. In this method, the diameter of the pore outlet is not measured, but is judged to be suitable for measuring the diameter of the keratoplug clogged in the pore.

< results and examination >

When the secretion of the whole sebaceous gland plasma proceeds smoothly, the remains of the sebaceous gland cells are accumulated in the pores. The result of the accumulation confirmed that the size of the corner pins, that is, the maximum diameter of the corner pins, became large. In other words, if the secretion of the whole sebaceous gland plasma is promoted, the accumulation of the sebaceous gland cell remains in pores can be reduced, and the maximum diameter of the keratotic plug can be reduced. Furthermore, from the present results, it is considered that the maximum diameter is reduced by using a cosmetic water to which trehalose is added as an autophagy promoter (considered as a whole plasma secretion promoter).

Claims (16)

1. A method for the evaluation and/or selection of whole plasma secretion modulators comprising:

an observation step of observing the state of a sebaceous gland cell that fluoresces after a single or a plurality of sebaceous gland cells and a test substance are present; and

and a determination step of determining the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator based on the state of the fluorescent sebaceous gland cells.

2. The method for evaluating and/or selecting a whole plasma secretion regulator according to claim 1, wherein,

the whole plasma secretion regulator is 1 or more than 2 selected from whole plasma secretion promoter, whole plasma secretion inhibitor, pore blocking regulator, and autophagy regulator of sebaceous gland cell.

3. The method for evaluating and/or selecting a whole plasma secretion regulator according to claim 1 or 2, wherein,

the sebaceous gland cell is a three-dimensional structure composed of a plurality of sebaceous gland cells.

4. The method for evaluating and/or selecting a whole plasma secretion modulator according to any one of claims 1 to 3, wherein,

the observation is an observation using time-lapse photography.

5. The method for evaluating and/or selecting a whole plasma secretion modulator according to any one of claims 1 to 4, wherein,

the observation was performed using a fluorescence microscope.

6. The method for evaluating and/or selecting a whole plasma secretion modulator according to any one of claims 1 to 5, wherein,

in the observation step, a test substance is allowed to exist in one or a plurality of sebaceous gland cells under the full-plasma secretion regulation condition, and the state of the sebaceous gland cells that emit fluorescence is observed under fluorescence.

7. The method for evaluating and/or selecting a whole plasma secretion modulator according to any one of claims 1 to 6, wherein,

in the observation step, a test substance is allowed to exist in one or a plurality of sebaceous gland cells under the condition of whole plasma secretion regulation, and the state of the sebaceous gland cells that emit fluorescence is observed by fluorescence,

the whole plasma secretion regulatory condition is an autophagy regulatory condition,

the autophagy-modulating condition is an autophagy-inhibiting condition or an autophagy-inducing condition.

8. The method for evaluating and/or selecting a whole plasma secretion modulator according to any one of claims 1 to 7, wherein,

and judging the state of the luminous sebaceous gland cells according to the number of fluorescence-lost cells per unit volume.

9. A method for the evaluation and/or selection of whole plasma secretion modulators comprising:

an observation step of performing fluorescence observation of the state of the collected fluorescent keratotic plug after the presence of the test substance in one or a plurality of sebaceous gland cells; and

and a determination step of determining the test substance as a whole plasma secretion regulator or a candidate for a whole plasma secretion regulator, based on the state of the fluorescent plug.

10. The method for evaluating and/or selecting a whole plasma secretion regulator according to claim 9, wherein,

the keratome in the observation step is a plurality of keratomes which are taken from the skin surface of a mammal by an adhesive sheet and attached to the adhesive sheet; and/or

The state of the plug in the determination step is a rate of change in the maximum plug diameter obtained by dividing the average value of the maximum plug diameter after use of the test substance by the average value of the maximum plug diameter before use.

11. A whole milk secretion regulator comprising 1 or 2 compounds selected from the group consisting of oligosaccharide and macrolide compounds as active ingredients.

12. A three-dimensional structure of sebaceous gland cells, the surface layer of which is composed of mature sebaceous gland cells.

13. The three-dimensional structure of sebaceous gland cells according to claim 12, wherein,

the three-dimensional structure is spherical.

14. The three-dimensional structure of sebaceous gland cells according to claim 12 or 13, wherein said three-dimensional structure is used for fluorescence observation and/or for methods of evaluation and/or selection of whole plasma secretion regulators.

15. A method for producing a three-dimensional structure of sebaceous gland cells, comprising culturing a plurality of seeded sebaceous gland cells on a cell adhesion-inhibiting treated surface of a mortar-shaped recess, thereby obtaining a three-dimensional structure of sebaceous gland cells.

16. The method for producing a three-dimensional structure of sebaceous gland cells according to claim 15, wherein,

the mortar-like recess is in the shape of a central region in the bottom of which a plurality of sebaceous gland cells can aggregate.

Images