JP5406582B2 - Screening method for materials for improving multifaceted skin barrier function - Google Patents

Description

  The present invention relates to a screening method for a skin barrier function improving material useful for screening an active ingredient of a skin external preparation such as cosmetics.

  The barrier function in the skin is one of the important functions in terms of preventing foreign bodies from entering the living body. Such insufficiency of the barrier function causes rough skin, inflammation, and the like, and its conservation measures are a major issue in the cosmetics field. Such skin barrier function factors include the stratum corneum barrier function due to the shape and bonding state of the stratum corneum cells, skin moisture retention such as the water content of the epidermis and dermis, and is not simple, It is already known that many factors are intertwined. Of particular interest in these factors in recent years is the tight junction protein present in the epidermal granule layer, and the theory that the skin barrier function is reduced due to the failure of cell junction via the tight junction protein ( For example, see Patent Document 1 and Patent Document 2.) A screening method for a cosmetic material paying attention to the function of the tight junction is also disclosed in the aforementioned patent document. This method can evaluate numerically the components that improve the decrease in skin barrier function due to the failure of tight junction function, but it is not possible to visually grasp how screening materials are working against tight junction failure. It is difficult, and there has been a demand for such a differentiation method that can visually grasp the improvement of the skin barrier function.

In addition to tight junctions, it is also known that for the skin barrier function, the amount, distribution state and type of ceramide are important factors for the skin barrier function (for example, Patent Document 3 and Patent Document 4). , See Patent Document 5). In addition, labeled ceramides for examining the biodistribution of ceramides are already known (see, for example, Patent Document 6), and there are commercially available products.
However, there is no example in which the distribution of ceramide exerted by the specimen and the effect of improving recovery from tight junction injury were examined simultaneously under comparable conditions. For this reason, regarding the decrease and improvement of the skin barrier function, it is often said that only one factor is spoken while recognizing that the factors are multifaceted.

JP 2008-26092 A JP 2006-250786 A JP 2002-102177 A JP 2007-108060 A Special table 2006-511462 gazette Japanese Patent Laid-Open No. 09-2978

  The present invention has been made under such circumstances, and provides a screening method for a skin barrier function-enhancing material capable of distinguishing the improvement effect from various viewpoints in the reduction of skin barrier function with many factors. This is the issue.

In view of such a situation, the present inventors have eagerly studied for a screening method for a skin barrier function improving material that can distinguish the improvement effect from various viewpoints in the reduction of skin barrier function with many factors. As a result, the skin or cultured skin three-dimensional model is cultured with the labeled ceramide, and then the presence of the labeled ceramide in the skin or cultured skin three-dimensional model is observed, thereby affecting the ceramide distribution. Whether the skin or cultured skin three-dimensional model, which is the same experimental subject, has a recovery effect from injury of the tight junction against the tight junction injury skin model obtained by treating the tight junction with injury. By identifying whether or not, the effect on tight junctions can also be identified, and these can be combined. The found that the effect of the analyte to multilateral skin barrier function can distinguish, and completed the invention. That is, the present invention is as follows.
(1) Intraceramic tissue characterized by culturing a three-dimensional model of skin or cultured skin with labeled ceramide and then observing the presence of labeled ceramide in the three-dimensional model of skin or cultured skin Transportation discrimination method.
(2) In the method for distinguishing intracerebral transport of ceramide as described in (1), the effect of the specimen on intratracheal transport of labeled ceramide is identified, and the intracerebral transport of ceramide is compared with the condition in which no specimen exists. The method for screening a material for improving skin barrier function, characterized in that, when promoted, the specimen is identified as having an action of improving skin barrier function due to ceramide deficiency.
(3) Furthermore, it is discriminated whether or not the specimen has an effect of recovering from a tight junction injury skin model with respect to a tight junction injury skin model obtained by performing injury treatment on a skin or cultured skin three-dimensional model tight junction. The screening method for the skin barrier function improving material according to (2), wherein the screening method is characterized in that it is identified as a more preferable material for improving skin barrier function when it has an effect of recovering from tight junction injury .

  ADVANTAGE OF THE INVENTION According to this invention, in the fall of the skin barrier function with many factors, the screening method of the skin barrier function improvement material which can distinguish the improvement effect from a multilateral viewpoint can be provided.

<1> differentiation method of tissue transport of ceramide of differentiation method invention tissue transport of ceramide of the present invention, the skin or cultured skin three-dimensional model incubated with labeled ceramide, thereafter, skin or It is characterized by observing the presence of the label in the cultured skin three-dimensional model. The skin is preferably a skin fragment collected from a living body by removing body hair as desired, and excluding the stratum corneum, and composed of an epidermal granule layer, an epidermal basal layer, and a dermis portion, such as a mouse, a rat, Guinea pig, pig and rabbit skin fragments are preferred. In addition, as the cultured skin three-dimensional model, the skin cells such as normal (non-cancerous) keratinocytes and fibroblasts collected from human or non- human animal skin are cultured, and a three-dimensional structure is constructed. preferably those obtained by pseudo the structure of the skin, can also be used to purchase a commercially available product of such a form. Preferable examples of commercially available products include “EFT-400” (normally cultured human three-dimensional skin model) sold by Kurashiki Boseki Co., Ltd. In particular, “EPI-200” composed only of an epidermis component, “EpiDerm” sold by USA MaTek, and the like can be more suitably exemplified. Such a three-dimensional model of skin or cultured skin is cultured in a medium containing labeled ceramide for 1 to 10 hours, preferably 3 to 5 hours, and the labeled ceramide is incorporated into the cells. After washing with PBS (phosphate buffered saline) or liquid medium, replace with fresh medium and continue culturing. After 6 to 24 hours, preferably after 12 to 18 hours, the medium is collected and the fluorescence intensity is measured, or the tissue is processed into sections and observed under a fluorescence microscope to capture the amount of ceramide incorporated as fluorescence. Quantify and identify the location. The amount of ceramide in the collected medium is large, or in the cultured skin three-dimensional model, when the horny layer side between the epidermal cells is the upper part, ceramide formation is promoted more when ceramide is transported to the upper part. Has an effect of improving the barrier function. For further differentiation of the sections, the observation image of the fluorescence microscope is taken into a computer or the like, and image processing software such as “Photoshop” manufactured by Adobe is used to perform binarization or the like, and the fluorescence sites are identified. The existence position can be grasped as a two-dimensional distribution by separating the non-fluorescent site. In addition, the fluorescence intensity can be discriminated from the area ratio of the white spot and the black spot. Such intracerebral transport status of ceramide can be used as an index for screening methods for skin barrier function improving materials described later.

<2> Screening method for skin barrier function-improving material of the present invention The screening method for a material for improving skin barrier function of the present invention is the above-described screening method for intracerebral transport of ceramide. When the intracerebral transport of ceramide is promoted as compared with the condition in which the specimen does not exist, the specimen is identified as having an action of improving the decrease in skin barrier function due to ceramide deficiency. It is characterized by that. As described above, the intracerebral transport of ceramide can be identified by the increase in the area of the fluorescent portion and the spread of the fluorescent portion.

Using a three-dimensional model of skin or cultured skin, treatment with a tight-junction injury agent such as capric acid makes it possible to differentiate the recovery action from tight-junction injury under almost the same conditions as in the above-mentioned method for distinguishing intracerebral transport of ceramide Therefore, it is possible to perform screening for a skin barrier function improving material in consideration of such recovery promoting action, and it is preferable to perform such screening. Screening for recovery from such tight junction injury can be performed as follows.

First, a tight junction injury skin model is prepared according to the following procedure. The tight junction injury skin model is for screening for a skin barrier function-improving component, and is characterized by injury treatment of a tight junction of a skin or a cultured skin three-dimensional model. The skin is preferably a skin fragment collected from a living body by removing body hair as desired, and excluding the stratum corneum, and composed of an epidermal granule layer, an epidermal basal layer, and a dermis portion, such as a mouse, a rat, Guinea pig, pig and rabbit skin fragments are preferred. In addition, as the cultured skin three-dimensional model, the skin cells such as normal (non-cancerous) keratinocytes and fibroblasts collected from human or non- human animal skin are cultured, and a three-dimensional structure is constructed. preferably those obtained by pseudo the structure of the skin, can also be used to purchase a commercially available product of such a form. Preferable examples of commercially available products include “EPI-200” (normally cultured human three-dimensional skin model) sold by Kurashiki Boseki Co., Ltd. Such a three-dimensional model of skin or cultured skin damages a tight junction portion by taking an injury means from the granule layer side. For example, ultraviolet rays and chemical substances can be preferably exemplified as the injury means, and in the case of ultraviolet rays, ultraviolet rays having a wavelength of 280 to 320 nm are per unit of 7.5 to 200 mJ / cm ² , more preferably 50 to 160 mJ / cm ² . rather I be irradiated with the energy, if chemical treatments, 0 aliphatic saturated straight chain fatty acid or a monovalent metal salt of capric acid, chain length in such caprylic acid (8-12 carbon atoms) .1～10MM, more preferably a solution from the dermis side or epidermal basal layer side of 0.5 to 2 mm, 5 to 24 hours, more preferably not good if caused to act for 10-15 hours. A neutralizing antibody that recognizes the extracellular domain of occludin or claudin can also be used. Among these treatments for injury, particularly preferred is treatment for chemical injury, and treatment with a capric acid solution is particularly preferred. This is because homogeneous damage can occur to tight junctions. Such homogeneous injury is a very important factor in accurately identifying the mechanism in screening for a recovery accelerator for injury tight junction described later. The post-treatment injury means is immediately removed from the skin or cultured skin three-dimensional model. Detachment can be accomplished by terminating the irradiation if ultraviolet irradiation is performed, and can be accomplished by washing with a medium or PBS (phosphate buffered saline) or the like if chemical injury is involved. The tight junction injury skin model obtained in this way is used in a method for identifying a tight junction function recovery agent described later, and is suitably used for screening a recovery accelerator for an injured tight junction.

Next, prepare a tight junction function tray circuit. Tight junction function tracer kit includes a biotin but it may also be labeled, a tight junction function tracer kit consisting of avidin but it may also be labeled, either of the biotin or avidin that is fluorescently labeled It is characterized by. Examples of the label include fluorescent labels such as fluorescein, rhodamine, fluotetramethylrhodamine, dansyl, luminescent labels such as luciferin, radioactive labels such as gallium, enzyme labels such as horseradish peroxidase, and the like. In order to visually distinguish the tight junction function, it is preferable to use a fluorescent label for either biotin or avidin. To distinguish the function of the tight junctions, in the kit of the present invention, the biotin but it may also be labeled, by acting from the dermis side or epidermal basal layer side, to act against the epidermal granular layer. In this case, biotin function of tight junctions but it may also be labeled if it is normal is halt in the epidermal granular layer side, it does not move in the corner layer side. If the function of the tight junction dysfunction, biotin but it may also be labeled to move to the corner layer side. Movement of the labeled even though good biotin in such skin tissue, by the action of avidin but it may also be labeled, can be visualized. Any avidin may be used as long as it has an irreversible binding property to biotin. For example, streptavidin can be preferably exemplified from its availability. Such avidin is preferably fluorescently labeled. The kits of the present invention, be labeled as a substance to act from the dermal side or epidermal basal layer side select avidin but it may also, be labeled as a visible means of avidin but it may also be labeled in the tissue Although it is also possible to use a not good biotin, the precise differentiation of tight junction function, with biotin but it may also be labeled as substances which act from the dermal side, it is labeled as a visible means of the biotin it is more preferable to use a not good avidin.

The method of distinguishing the tight junction function recovery agent is to treat the tight junction injury skin model with the specimen and the untreated one with the tight junction function tray circuit to determine the distribution status of the tracer in the skin tissue. If the probability that the tracer movement from the basal layer to the granule layer is hindered by treatment with the specimen is significantly higher than that of the untreated, the administration of the specimen is It has the effect of recovering the junction, and it is characterized in that the recovery improves the skin barrier function. The probability can be easily distinguished by observing a tight junction injury skin model under a fluorescence microscope. The significance of the difference in probability is that the observation image of the fluorescence microscope is taken into a computer, etc., and image processing software such as “Photoshop” made by Adobe is used for image processing such as binarization, and the fluorescence site is not detected. It can be clarified by dividing the fluorescent part and obtaining the total area ratio of the fluorescent part and statistically processing it. It can be discriminated that the larger the difference between the values of the untreated sample and the treated sample, the better the tight junction injury recovery action of the specimen, in other words, the better barrier function recovery action. It is also preferable to set the concentration of the specimen so that it can be extrapolated when actually administered to the skin, and observe the dose effect by shaking the dose. In the discrimination method of the present invention, recovery from tight junction injury can be discriminated as an image, so that the mechanism of the recovery process of tight junction can be known by following the bias of the fluorescent part, time-series changes, etc. I can do it. The above work is divided into processes and expressed as follows.
(Step 1) Step of creating a tight junction injury skin model by injuring tight junction of skin or cultured skin three-dimensional model with injury means (Step 2) Step of processing tight junction injury skin model with specimen (Step 3) The process of treating the tight junction injury skin model treated with the specimen 2 and the tight junction injury skin model not treated with the specimen with one of the tight junction functional tray circuits, preferably labeled biotin (Step 4) and the tracer treatment of Step 3 Step of preparing a specimen for microscope from the skin model (Step 5) The specimen of Step 4 is treated with the other of the tray circuit, preferably with fluorescently labeled avidin (Step 6). The specimen of Step 4 is preferably under the microscope. Is a step of observing under a fluorescence microscope (preferably step 7 The microscopic image of step 6 is taken into a computer as an image image, image-processed, divided into a fluorescent portion and a non-fluorescent portion, and a quantitative ratio of the fluorescent portion (preferably step 8). , The process of determining the degree of correlation between specimen processing and identifying the presence or absence of the recovery action of tight junction function

  Thus, the intracerebral transport of the identified ceramide and the recovery promoting action from the injury of the tight junction are integrated, and it is distinguished whether or not it is useful as a material for improving the skin barrier function.

  In the above integration, the results of the screening evaluation are as follows: 1) a material that does not promote recovery from tight junction injury or ceramide production, and 2) that promotes recovery from injury of tight junction, Material that does not promote ceramide production 3) Does not promote recovery from injury of tight junctions, but has material that promotes production of ceramide 4) Promotes recovery from injury of tight junctions It is preferable to classify into four materials that also have a production promoting effect, and to determine the rank for each category. In this classification, materials that are classified into 4 and that exhibit both effects at the actual use concentration are particularly preferable as active ingredients of the external preparation for skin. Such ingredients are formulated into an external preparation for skin such as cosmetics together with optional ingredients.

  Hereinafter, the present invention will be described in more detail with reference to examples.

(Step 1) “EPI-200” was cultured in a medium containing 1 mM sodium caprate for 12 hours.
(Steps 2 and 3) The culture was continued by exchanging with a medium containing no mixture of 1 mM sodium caprate containing a mixture of 500 μg / ml ε, γ-glutamyllysine and 0.1% palmaria extract as a treated specimen. One hour before collecting “EPI-200”, “EZ-Link ^™ -sulfo-NHS-LC-biotin” (Pierce) was added as a tracer to a final concentration of 0.1 mg / ml. On the other hand, 500 μg / ml ε, γ-glutamyllysine and a 70% ethanol aqueous solution not containing 0.1% palmaria extract were added as untreated samples, and the culture was continued in the same manner.
(Step 4) “EPI-200” treated in Steps 1 to 3 was collected, immersed in “OCT Compound” (manufactured by Sakura Finetech), and frozen in liquid nitrogen. Subsequently, a frozen section specimen was prepared with a frozen section preparation apparatus.
(Step 5) The specimen is reacted with a rabbit anti-occludin antibody (manufactured by Invitrogen), and then reacted with a FITC-labeled donkey anti-rabbit antibody (ICN-pharmaceutical) and streptavidin-Texas red (manufactured by Oncogene). It was.
(Step 6) The specimen obtained in Step 5 was observed under a fluorescence microscope at two wavelengths of 488 and 543 nm.

1 to 3 are untreated controls, and FIGS. 4 to 6 are skin model tissue slice images when treated with 1 mM sodium caprate. Similarly, in FIGS. 7 to 24, a portion surrounded by a dotted line in each drawing indicates a stratum corneum. 1 and 4 are stained images of the tracer, FIGS. 2 and 5 are stained images of the anti-occludin antibody (arrows), and FIGS. 3 and 6 are images obtained by superimposing the stained images of the tracer and the anti-occludin antibody, respectively. 1-3, it can be seen that the tracer added from the epidermal basal layer side (bottom) passes through the cells and stops at the position of the tight junction (arrow, anti-occludin antibody staining site), that is, at the granular layer. It can be seen from ˜6 that the tracer leaks in part of the horny layer above the granular layer by damaging the tight junction with sodium caprate treatment (FIG. 6 *). FIGS. 7-9 are skin model tissue slice images after 3 hours, and FIGS. 10-12 are skin model tissue images after 6 hours. On the other hand, FIGS. 13 to 15 show 3 hours after culturing in a medium containing a mixture of 500 μg / ml ε, γ-glutamyl lysine and 0.1% palmaria extract after tight junction injury, and FIGS. 16 to 18 show 6 hours after that. FIG. 7, 10, 13, and 16 are tracer staining images, FIGS. 8, 11, 14, and 17 are staining images (arrows) of anti-occludin antibodies, and FIGS. 9, 12, 15, and 18 are staining of tracer and anti-occludin antibodies, respectively. It is an image obtained by superimposing images. 7-9 and 10-12, the tracer remained leaking into the stratum corneum even after the sodium caprate was released after the tight junction injury with sodium caprate (FIG. 9, 12 *), FIG. It can be seen that leakage to the stratum corneum of the tracer is prevented by treatment with a mixture of ε, γ-glutamyllysine and palmaria extract as shown in -15 and 16-18. In addition, only 500 microgram / ml (epsilon), (gamma) -glutamyl lysine is added to FIGS. 19-21 after a tight junction injury, and only the 0.1% palmaria extract is added to FIGS. 22-24 after a tight junction injury, and the result 6 hours after. Also shown. 19 and 22 are stained images of the tracer, FIGS. 20 and 23 are stained images of the anti-occludin antibody (arrows), and FIGS. 21 and 24 are images obtained by superimposing the stained images of the tracer and the anti-occludin antibody, respectively. It can be seen that ε, γ-glutamyllysine and palmaria extract alone prevent leakage to the stratum corneum of the tracer. That is, it can be seen that this discrimination method is suitable for screening for a skin barrier improving material involved in the tight junction function.

<Step 1> “EPI-200” is transferred to a culture plate, and maintenance medium (EPI-) containing 200 μl of 5 μM “BODIPY (registered trademark) FL-C5-Ceramide-BSA” (manufactured by Molecular Probes) on the epidermal basal layer side. 100, manufactured by Kurabo Industries) was added and incubated at 37 ° C. and 5% carbon dioxide for 4 hours, and then allowed to stand in a 4 ° C. refrigerator for 1 hour.
<Step 2> The medium was replaced with 500 μl of a fresh maintenance medium, and cultured at 37 ° C. and 5% CO ₂ for 20 hours.
<Step 3> The medium was replaced with a maintenance medium containing 300 μl of 5% defatted bovine serum albumin and allowed to stand at 4 ° C. for 30 minutes.
<Step 4> Step 3 was repeated twice.
<Step 5> A fresh maintenance medium and a maintenance medium containing 1 mM sodium caprate were respectively replaced, and cultured at 37 ° C. and 5% carbon dioxide.
<Step 6> After 12 hours of culture, the culture medium was replaced with a fresh maintenance medium not containing 1 mM sodium caprate.
<Step 7> After culturing for a certain period of time, “EPI-200” was detached from the support, embedded in “OCT compound” (manufactured by Sakura Finetech), and frozen with liquid nitrogen.
<Step 8> Frozen sections were prepared and observed under a fluorescence microscope at an excitation wavelength of 470 nm / fluorescence wavelength of 525 nm.

  25 is untreated with sodium caprate, FIG. 26 is 12 hours after treatment with 1 mM sodium caprate, and FIG. 27 is replaced with fresh maintenance medium not containing sodium caprate after 12 hours after treatment with 1 mM sodium caprate, and 18 hours later. It is a fluorescence image of the frozen section of “EPI-200”. Fluorescence (ceramide) in the epidermal granule layer (arrow) directly under the stratum corneum is observed in the upper part between cells when sodium caprate is not treated (FIG. 25), but disappeared by treatment with sodium caprate (FIG. 26). Thereafter, after replacing with a fresh maintenance medium not containing sodium caprate, fluorescence (ceramide) is observed again in the upper part (arrow) between cells of the epidermal granule layer (FIG. 27). That is, it can be seen that sodium caprate has the action of reversibly inhibiting the action of epidermal cells that secrete ceramide in the stratum corneum direction. That is, it can be seen that this discrimination method is suitable for evaluating the degree of involvement of tight junctions in the intracerebral transport of ceramide.

  <Step 6> is performed in the same manner as in Example 2, but after <Step 6>, separate "EPI-200" is contained alone at 500 µg / mlε, γ-glutamyllysine and 0.1% palmaria extract, respectively. The culture medium was replaced with the maintenance medium to be cultured for 6 hours. Thereafter, the medium was collected, and the fluorescence intensity (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) of “BODIPY FL-C5-Ceramide-BSA” secreted outside the cells was measured using ARVO SX Multilabel Counter (manufactured by PerkinElmer). . Furthermore, it advanced to <process 8> after that.

  The results are shown in FIG. 28. As for ε, γ-glutamyllysine and palmaria extract, the amount of extracellularly secreted fluorescently labeled ceramide “BODIPY FL-C5-Ceramide-BSA” is significantly higher than that of the control without addition. Facilitate. FIG. 29 shows 12 hours after treatment with 1 mM sodium caprate and 12 hours after replacement with maintenance medium. FIG. 30 shows 12 hours after treatment with 1 mM sodium caprate and replacement with maintenance medium containing 500 μg / ml ε, γ-glutamyllysine. 12 hours later, FIG. 31 is a fluorescence image of a frozen section of “EPI-200” after 12 hours after treatment with 1 mM sodium caprate and after replacement with a maintenance medium containing 0.1% palmaria extract. is there. In FIG. 29, not much fluorescence (ceramide) in the epidermal granule layer immediately below the stratum corneum is observed in the upper part between cells, whereas in FIGS. 30 and 31, the upper part (arrow) between the cells in the epidermal granule layer. Many are recognized. That is, it turns out that (epsilon), (gamma) -glutamyl lysine and a palmaria extract each have the effect which accelerates | stimulates the function of the epidermal cell which secretes ceramide toward a stratum corneum. That is, it can be seen that this discrimination method is suitable for screening of a skin barrier improving material based on the degree of involvement of tight junctions in the intracerebral transport of ceramide in consideration of the results of Examples 1 and 2.

  The present invention can be applied to the evaluation of active ingredients of external preparations for skin such as cosmetics.

It is a figure which shows the dyeing | staining image of the tracer in untreated control. The part surrounded by the dotted line in the figure is the stratum corneum, and the part below it is the epidermis layer. The same applies below. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) FIG. 2 is a diagram illustrating an image obtained by superimposing stained images of a tracer and an anti-occludin antibody (arrow), similar to FIG. 1. (Drawing substitute photo) It is a figure which shows the dyeing | staining image of the tracer in 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) FIG. 5 is a view showing an image obtained by superimposing stained images of a tracer and an anti-occludin antibody (arrow), similar to FIG. * Indicates a leak of the tracer into the stratum corneum. (Drawing substitute photo) It is a figure which shows the dyeing | staining image of the tracer 3 hours after replacing | exchanging to the culture medium which does not contain sodium caprate after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) It is a figure which shows the image which overlap | superposed each stained image of a tracer and an anti- occludin antibody (arrow) similar to FIG. * Indicates a leak of the tracer into the stratum corneum. (Drawing substitute photo) It is a figure which shows the dyeing | staining image of the tracer 6 hours after replacing | exchanging to the culture medium which does not contain sodium caprate after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) It is a figure which shows the image which overlap | superposed each stained image of a tracer and an anti- occludin antibody (arrow) similar to FIG. * Indicates a leak of the tracer into the stratum corneum. (Drawing substitute photo) The figure which shows the dyeing | staining image of the tracer 3 hours after replacing | exchanging to the culture medium which does not contain sodium caprate, but contains 500 micrograms / ml epsilon, (gamma) -glutamyl lysine, and a 0.1% palmaria extract after 1 mM sodium caprate treatment. It is. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) It is a figure which shows the image which overlap | superposed each stained image of a tracer and an anti- occludin antibody (arrow) similar to FIG. * Indicates a leak of the tracer into the stratum corneum. (Drawing substitute photo) The figure which shows the dyeing | staining image of the tracer 6 hours after replacing | exchanging to the culture medium which does not contain sodium caprate, but contains 500 micrograms / ml epsilon, (gamma) -glutamyl lysine, and a 0.1% palmaria extract after 1 mM sodium caprate treatment. It is. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) FIG. 17 is a view showing an image obtained by superimposing stained images of a tracer and an anti-occludin antibody (arrow), similar to FIG. (Drawing substitute photo) It is a figure which shows the dyeing | staining image of the tracer 6 hours after replacing | exchanging to the culture medium which does not contain sodium caprate but contains 500 microgram / ml (epsilon), gamma-glutamyl lysine after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) FIG. 20 is a view showing an image obtained by superimposing stained images of a tracer and an anti-occludin antibody (arrow), similar to FIG. (Drawing substitute photo) It is a figure which shows the dyeing | staining image of the tracer 6 hours after replacing | exchanging to the culture medium which does not contain sodium caprate but contains 0.1% palmaria extract after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the dyeing | staining image (arrow) of an anti- occludin antibody similar to FIG. (Drawing substitute photo) FIG. 23 is a view showing an image obtained by superimposing stained images of a tracer and an anti-occludin antibody (arrow), similar to FIG. (Drawing substitute photo) It is a figure which shows the fluorescence image of the frozen section of "EPI-200" untreated with sodium caprate. (Drawing substitute photo) It is a figure which shows the fluorescence image of the frozen section of "EPI-200" 12 hours after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the fluorescence image of the frozen section of "EPI-200" 18 hours after replacing with the fresh maintenance medium which does not contain sodium caprate 12 hours after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the influence of (epsilon), (gamma) -glutamyl lysine, and a Palmaria extract with respect to the secretion amount to the extracellular side of a fluorescent labeling ceramide. (Drawing substitute photo) It is a figure which shows the fluorescence image of the frozen section of "EPI-200" 12 hours after replacing with a maintenance medium 12 hours after 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the fluorescence image of the frozen section of "EPI-200" 12 hours after substituting to the maintenance culture medium containing 500 micrograms / ml epsilon and (gamma) -glutamyl lysine 12 hours after a 1 mM sodium caprate treatment. (Drawing substitute photo) It is a figure which shows the fluorescence image of the frozen section of "EPI-200" 12 hours after substituting to the maintenance culture medium containing a 0.1% palmaria extract 12 hours after a 1 mM sodium caprate treatment. (Drawing substitute photo)

Claims (2)

(1) A measurement process in which measurement of the following sample-free control and measurement under the sample-added condition are performed,
  Due to the presence and location of the labeled ceramide in the skin cultured with the labeled ceramide and / or the action of epidermal cells secreting ceramide in the direction of the stratum corneum in the culture medium exchanged after the culture. Sample-free control measurement process for measuring the abundance of labeled ceramide secreted extracellularly,
  The amount and location of the labeled ceramide in the skin cultured with the labeled ceramide and further cultured with the specimen, and / or ceramide in the culture medium exchanged after the culture is secreted in the horny layer direction. A sample addition measurement step for measuring the abundance of labeled ceramide secreted out of the cell by the action of epidermal cells,
(2) The presence position and amount of labeled ceramide in the skin and / or the amount of labeled ceramide present in the culture medium obtained in the sample-free addition control measurement step and the sample addition measurement step A step of comparing,
  Compared with the above-mentioned sample-free addition control measurement step, the sample addition measurement step has more ceramide labeled on the horny layer side of epidermal cells and / or the amount of labeled ceramide present in the culture medium is larger. In this case, a screening method for a skin barrier function improving material, wherein the specimen is used as a skin barrier function improving material.   The method for screening a skin barrier function-improving material according to claim 1, wherein the skin has been subjected to injury treatment of a tight junction.

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