Method for evaluating the adhesion state of stratum corneum cells

Abstract

We propose a simple method for evaluating the adhesion state of stratum corneum cells without using special techniques. [Solution] The present invention is a method for evaluating the adhesion state of stratum corneum cells, comprising the following steps (A) to (C). (A) Steps to collect stratum corneum cells from a desired evaluation site by tape stripping. (B) A process of observing or analyzing the contours of individual stratum corneum cells using means that allow for identification of the contours of each cell. (C) A process for determining the amount of stratum corneum cells that can individually identify contours. [Effect] This invention makes it possible to easily evaluate the adhesion state of stratum corneum cells without using special techniques by minimally invasively collecting stratum corneum cells from the skin surface of a subject.

Description

Technical Field

[0001] The present invention relates to a technique for evaluating the adhesion state of stratum corneum cells by analyzing the stratum corneum cells.

Background Art

[0002] The skin consists of three layers: the epidermis, the dermis, and the subcutaneous tissue, in order from the surface layer where the human body contacts the outside air. The epidermis mainly consists of cells called keratinocytes (keratinized cells) and is classified into the basal layer, the spinous layer, the granular layer, and the stratum corneum (horny layer) from the deep part near the dermis.

[0003] The stratum corneum is formed by the division and differentiation of keratinocytes, and stratum corneum cells are mainly adhered to each other by corneodesmosomes, which are stratum corneum cell adhesion factors. Corneodesmosomes are composed of desmoglein 1, desmocollin 1, etc. When these proteins are decomposed by proteases such as kallikrein 5, stratum corneum cells peel off from the skin surface. When stratum corneum cells adhere to each other, they form a strong barrier to prevent external stimuli and water loss, while at the outermost layer of the skin, stratum corneum cells peel off as the stratum corneum cell adhesion factors are decomposed.

[0004] On the other hand, it has been reported that when the decomposition of stratum corneum cell adhesion factors is delayed due to a decrease in protease activity, etc., the stratum corneum accumulates excessively (Non-Patent Document 1). That is, the decomposition of the adhesion of stratum corneum cells and the proper progress of stratum corneum cell exfoliation at the outermost layer of the skin are important for maintaining normal epidermal turnover. Therefore, techniques for promoting the exfoliation of stratum corneum cells, such as stratum corneum exfoliating agents (Patent Document 1), have been developed. On the other hand, if the decomposition of stratum corneum cell adhesion is promoted more than necessary, it leads to a decrease in the skin barrier function, which is not preferable. From these facts, in order to determine whether a treatment for promoting stratum corneum cell exfoliation should be performed, a technique for evaluating the adhesion state of stratum corneum cells for each individual or for each site in a specific individual has been demanded.

[0005] Conventional methods for evaluating the adhesion state of stratum corneum cells have involved methods such as immunostaining with an anti-desmoglein 1 antibody on stratum corneum samples collected by tape stripping (Patent Document 2), which evaluate the amount of corneodesmosome constituent proteins. However, evaluating the amount of specific proteins requires specialized techniques and reagents, making it difficult to perform the procedure simply.

[0006] As a simple method for evaluating the adhesion state of stratum corneum cells, a method has been devised to assess the total amount of stratum corneum cells collected by tape stripping, determining that a smaller amount indicates stronger adhesion, making it difficult to remove the cells with tape. However, in reality, in skin with uneven adhesion, tape stripping can cause multiple layers of strongly adhered areas to peel off together (multilayer exfoliation), potentially increasing the amount of stratum corneum cells, making accurate evaluation of stratum corneum cell adhesion difficult. In short, a simple method for evaluating the adhesion state of stratum corneum cells is still unknown. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2024-075143 [Patent Document 2] Japanese Patent Publication No. 2019-214520 [Non-patent literature]

[0008] [Non-Patent Document 1] Rawlings AV. et al., Skin biology, xerosis, Barrier repair and measurement Drug Discovery Today. 5(2): e127-e136 (2008) [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The objective of this invention is to propose a simple method for evaluating the adhesion state of stratum corneum cells. [Means for solving the problem]

[0010] As a result of diligent research, the inventors of the present invention discovered that when stratum corneum collected by tape stripping is stained and observed at high magnification, the fewer the number of stratum corneum cells whose outlines can be individually identified, the stronger the adhesion of the stratum corneum cells. This led to the completion of the present invention.

[0011] In other words, the present invention is a method for evaluating the adhesion state of stratum corneum cells, comprising the following steps (A) to (C). (A) Steps to collect stratum corneum cells from a desired evaluation site by tape stripping. (B) A process of observing or analyzing the contours of individual stratum corneum cells using means that allow for identification of the contours of each cell. (C) A process for determining the amount of stratum corneum cells that can individually identify contours. [Effects of the Invention]

[0012] This invention makes it possible to easily evaluate the adhesion state of stratum corneum cells without using special techniques by minimally invasively collecting stratum corneum cells from the skin surface of a subject. [Brief explanation of the drawing]

[0013] [Figure 1] Examples of stained images of tape-stripped stratum corneum cells and images with masked stratum corneum cells whose individual contours could be visually identified. [Figure 2] Graph showing the correlation between desmoglein 1 level and the number of identifiable stratum corneum cells. [Figure 3] Graph showing the correlation between desmoglein 1 level and identifiable stratum corneum cell area. [Figure 4] A graph showing a comparison of the number of identifiable stratum corneum cells in areas with and without blemishes. [Modes for carrying out the invention]

[0014] In the present invention, the adhesion state of stratum corneum cells refers to the state regarding the strength of adhesion between stratum corneum cells when they are adjacent to other stratum corneum cells. In addition to the strength of adhesion of individual stratum corneum cells, it also includes the uniformity of adhesion strength when multiple cells are grasped as a group, the degree of change in the adhesion strength in the depth direction, and the like.

[0015] In the present invention, it is necessary to identify the contour of individual stratum corneum cells. Individual stratum corneum cells refer to each stratum corneum cell maintaining its cell state. For stratum corneum cells with defects or deformations, it is not necessary to regard them as individual stratum corneum cells. However, in relative evaluation, if the comparison targets are analyzed under the same conditions, there is no problem in regarding stratum corneum cells with defects or deformations as individual stratum corneum cells. When two-dimensionally grasping the cell morphology of stratum corneum cells collected by tape stripping from the tape surface side, if the outermost periphery of the cells can be generally identified, it can be considered that the contour can be identified. Even if the stratum corneum cells overlap each other, the contour can be identified. Regarding the judgment of whether the contour can be identified or has been identified, there is not necessarily such a condition, and a criterion for making a judgment that the contour can be identified for each observation method or judge can be set. For example, when 80% or more of the contour can be grasped, it may be considered that the contour can be identified. Even when the position of the contour is not clear, if it can be grasped within an error of 2 μm, it may be considered that the contour can be identified.

[0016] In the step of "collecting stratum corneum cells from an arbitrary evaluation site by tape stripping", it is possible to collect stratum corneum cells from an arbitrary site where the adhesion state is to be evaluated. Regarding the tape used in tape stripping, at least one side is a sticky substance, and there is no problem as long as it has an adhesive force sufficient to collect the stratum corneum. The detailed technique of collecting stratum corneum cells is not questioned. However, when comparing multiple samples, collection under the same conditions is preferred, which enables more accurate evaluation. For example, before sticking the tape on the skin, the evaluation site is washed to remove sebum and dirt on the skin surface that inhibit the adhesion of stratum corneum cells, and when sticking the tape on the skin, pressing with the same strength makes the evaluation result more accurate.

[0017] In the step of "observing or analyzing the contours of individual horny layer cells by a distinguishable means", since each horny layer cell is as small as around 30 μm in diameter, for example, a method of observing and analyzing at a high magnification can be mentioned. For example, by using a microscope to observe the horny layer cells collected by tape stripping at a high magnification, it is possible to observe each small horny layer cell that cannot be easily visually recognized. In addition, since the horny layer cells have high transparency and it is difficult to distinguish the contours as they are, it is possible to make the evaluation more accurate by making the contours easier to distinguish by methods such as staining, image processing, and adjusting the light irradiation method in microscopic observation. These processes for making the contours easier to distinguish may be carried out at the necessary timing. For example, in the case of staining, it may be before or after tape stripping. Also, it is not necessarily required to go through the step of observation. For example, by means of machine learning, from the overall image of the horny layer cells collected by tape stripping, etc., by analyzing or predicting the contours of the horny layer cells, it is possible to obtain the information necessary in the step of "determining the amount of horny layer cells whose contours can be individually distinguished".

[0018] In the step of "determining the amount of horny layer cells whose contours can be individually distinguished", there is no particular limitation on the method for determining the amount of horny layer cells whose contours can be individually distinguished. For example, the number of horny layer cells whose contours can be individually distinguished may be measured, or it may be obtained from the total value of the areas of the horny layer cells whose contours can be individually distinguished. When obtaining the area of the horny layer cells, for example, it is possible to calculate it from the number of pixels in the region occupied by the horny layer cells whose contours can be individually distinguished in the acquired image, or an index that enables relative comparison of areas, such as using the number of pixels itself as an index of area, may also be used. Note that the area of the horny layer cells refers to the area of the horny layer cells when the horny layer cells are peeled off by the tape stripping method and then the cell morphology is two-dimensionally grasped from the tape surface side.

[0019] In implementing the present invention, the relatively smaller the amount of stratum corneum cells that can individually identify contours, the stronger the intercellular adhesion is evaluated. Thus, the present invention compares the relative strength of stratum corneum cell adhesion. Basically, it determines and compares the amount of identifiable stratum corneum cells among multiple measurement targets, and evaluates the target with a smaller amount of identifiable stratum corneum cells as having stronger intercellular adhesion. Furthermore, for example, by using stratum corneum cells collected by tape stripping from multiple subjects with good skin condition, calculating the amount of identifiable stratum corneum cells and setting it as an ideal value, the strength of adhesion of the stratum corneum cells of the evaluation target can be evaluated relative to the ideal value by comparing the amount of identifiable stratum corneum cells of the evaluation target with the ideal value.

[0020] Furthermore, since a relatively smaller number of stratum corneum cells capable of individually identifying contours indicates stronger cell adhesion, it becomes possible to analyze the uniformity of cell adhesion strength in the evaluation area by, for example, subdividing a region of the same stratum corneum cell sample obtained by tape stripping and analyzing the variation in the amount of stratum corneum cells capable of individually identifying contours in each subdivided region. Additionally, it becomes possible to evaluate changes in cell adhesion strength in the depth direction by, for example, performing tape stripping on the same area multiple times and comparing the results.

[0021] In the steps of "observing or analyzing the contours of individual stratum corneum cells using means that allow for the identification of their contours" and "determining the amount of stratum corneum cells whose contours can be individually identified," when comparing multiple samples, it is desirable to unify the conditions for observation, analysis, and determination. For example, if observation or analysis is performed using different methods, or if determinations are made by different evaluators, the amount of stratum corneum cells whose contours can be individually identified may vary depending on the method or evaluator. [Examples]

[0022] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0023] [Analysis of the relationship between the number of stratum corneum cells identifiable at the single-cell level and the amount of adhesion proteins] <Method> Four different points on one subject were designated as test sites. After washing each site, stratum corneum samples were collected from each site using a skin checker (promo tool) via tape stripping. The skin checker measured approximately 25 mm. 2 The area to be stained was marked with a water-repellent pen, and the following procedures were performed sequentially within that area. 90 μL of 3% BSA / PBS(-) solution was added, incubated at room temperature for 30 minutes, and then washed in PBS(-) for 5 minutes. Anti-Dsg1 antibody solution (anti-Desmoglein 1 mouse monoclonal, Dsg1-p124, supernatant (PROGEN)) was diluted 10-fold with 1% BSA / PBS(-) solution, 90 μL was added, incubated at room temperature for 1 hour, and then washed in PBS(-) for 5 minutes. Goat Anti-Mouse IgG H&L (Alexa Fluor 568) preadsorbed (ab175701, Abcam) was diluted 200-fold with 1% BSA / PBS(-) solution, 90 μL was added, incubated in the dark at room temperature for 1 hour, and then washed in PBS(-) for 5 minutes.

[0024] After air drying, the skin checkers were observed using a fluorescence microscope (BZ-X700, KEYENCE) with a 40x objective lens, and five fluorescence images and five visible light images were acquired for each skin checker. By analyzing the acquired visible light images with ImageJ, the total area of ​​regions containing stratum corneum cells in the images was determined and defined as the total stratum corneum cell area. Since desmoglein 1 is present in the fluorescence detection sites in the acquired fluorescence images, the fluorescence area can be considered to represent the amount of desmoglein 1 in the image area. By correcting the fluorescence area by dividing it by the total stratum corneum cell area, the fluorescence area ratio (amount of desmoglein 1) was obtained. The fluorescence area ratio is the amount of desmoglein 1 in the field of view corrected for the amount of stratum corneum cells.

[0025] The skin checker was immersed in a staining solution (0.1% amide black, 9% acetic acid, 0.9% sodium acetate, 90% water) for 24 hours, and then washed with tap water. Using a fluorescence microscope (BZ-X700, KEYENCE) with a 40x objective lens, 10 visible light images were acquired for each skin checker, each taken from a different position. Corneocytes whose individual contours could be identified by visual inspection in the images were identified, and the number of identifiable corneocytes and the total area of ​​corneocytes were measured to determine the identifiable cell quantity. The relationship between the amount of desmoglein 1 and the identifiable cell quantity was plotted. Regarding corneocytes whose individual contours could be identified by visual inspection in the images, Figure 1 shows the stained image of tape-stripped corneocytes used in this study, and an example of corneocytes whose individual contours could be identified by visual inspection.

[0026] <Result> As shown in Figures 2 and 3, the amount of desmoglein 1 showed a strong negative correlation with the amount of identifiable cells, which was determined from the number of identifiable stratum corneum cells and the total area of ​​stratum corneum cells. Therefore, it was shown that skin with a lower amount of identifiable cells in the stratum corneum collected by tape stripping, i.e., the amount of stratum corneum cells capable of individually identifying contours, could be evaluated as having a higher amount of desmoglein 1. Desmoglein 1 is a component of corneodesmosomes and is known to play a major role in stratum corneum cell adhesion. Therefore, a lower amount of identifiable cells can be considered to indicate a higher amount of corneodesmosomes and stronger stratum corneum cell adhesion. Although the results of measuring the amount of identifiable cells varied depending on the evaluator, a similarly strong negative correlation was observed between the amount of desmoglein 1 and the amount of identifiable cells even when measured by different evaluators. This is because, although the criteria for determining the amount of identifiable cells differ among evaluators, they remain constant within the test, so the relative evaluation results between samples do not change.

[0027] [Comparison of stratum corneum cell adhesion strength between areas with and without blemishes] <Method> The skin of one individual, including the area with a blemish and the surrounding area, was cleansed. Stratum corneum cells were collected using tape stripping with a skin checker, ensuring that the sample included both the blemish area and the surrounding non-blemish area. The skin checker was immersed in a staining solution (0.1% amide black, 9% acetic acid, 0.9% sodium acetate, 90% water) for 24 hours, and then rinsed with tap water. Using a fluorescence microscope (BZ-X700, KEYENCE) with a 40x objective lens, approximately 300 visible light images were acquired from different positions for both the non-blemish and blemish areas. The number of keratinocytes whose individual contours could be visually identified in the images was measured and defined as the identifiable cell quantity. The relationship between desmoglein 1 level and identifiable cell quantity was plotted.

[0028] <Result> As shown in Figure 4, the amount of identifiable cells was lower in the blemished areas compared to the non-blemished areas. From this, it can be evaluated that cell adhesion is stronger in the blemished areas than in the non-blemished areas. In fact, it has been reported that stratum corneum cell adhesion is stronger in blemished areas than in non-blemished areas, and this result confirms that the amount of identifiable cells can indeed be used as an indicator to evaluate the state of stratum corneum cell adhesion. When implementing the present invention, the amount of identifiable cells between multiple measurement targets can be determined and compared in this way, and it can be evaluated and estimated that the cell adhesion is stronger in targets with a smaller amount of identifiable cells.

Claims

1. A method for evaluating the state of stratum corneum cell adhesion, comprising the following steps (A) to (C). (A) A process of collecting stratum corneum cells from an arbitrary evaluation site by tape stripping. (B) A process of observing or analyzing the contours of individual stratum corneum cells using means that allow for identification of the contours of each cell. (C) A step to determine the amount of stratum corneum cells that can individually identify the contour.

2. A method for evaluating the adhesion state of stratum corneum cells according to claim 1, characterized by staining stratum corneum cells.

3. A method for evaluating the adhesion state of stratum corneum cells according to claim 1 or claim 2, characterized in that the relatively smaller the amount of stratum corneum cells that can individually identify contours, the stronger the adhesion between cells is evaluated to be.

4. A method for evaluating the adhesion state of stratum corneum cells according to claim 1 or claim 2, characterized in that the amount of corneodesmosomes is evaluated as being greater when the amount of stratum corneum cells capable of individually identifying contours is relatively small.

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