JP4805794B2 - Method for detecting stratum corneum protein, method for evaluating epidermal turnover and method for evaluating skin condition using the same - Google Patents

Description

  The present invention relates to a method for detecting a stratum corneum protein present in the stratum corneum, and more specifically, a method for peeling and collecting the stratum corneum by tape stripping, visualizing and detecting the stratum corneum by a simple method, and this The present invention relates to an evaluation method for skin turnover and a skin condition evaluation method using a skin.

  The epidermis is composed of various types of cells, and keratinocytes (keratinocytes) occupy the majority. As this keratinocyte grows and moves to the upper layer, the outermost stratum corneum is formed by keratinization. Epidermal cells play an important role in the normal keratinization and maintenance of the stratum corneum. In addition, it is said that abnormal cell adhesion of keratinocytes is a cause of skin diseases such as keratosis. Furthermore, it is known that skin troubles such as acne, dandruff and desquamation are caused by increased keratinization due to increased cell adhesion. In addition, there are many other stratum corneum proteins in the stratum corneum, and these are considered to be related to skin aging and the like.

  Examples of the stratum corneum protein include cell adhesion proteins, stratum corneum substances, and stratum corneum cell constituents. Of these, desmosomes are typical keratinocyte adhesion devices. Desmosomes are structures that are involved in the adhesion between epidermal cells and stratum corneum cells, and the adhesion between epidermal cells via desmosomes is linked to the cytoskeleton composed of keratin fibers, maintaining a strong structure in epithelial tissues. It is known that this is done (Non-Patent Document 1). The desmosome constituent proteins are known as desmogleins 1, 2, 3, 4, desmocollins 1, 2, 3, desmoplakin 1, 2, etc., and any structure or binding ability of these cell adhesion factor and cytoskeleton is known. Even if injured, the epidermal cytoskeleton network collapses and causes blistering (Non-patent Document 2).

  Among these constituent proteins of desmosome, especially desmoglein 1 has a close correlation between proteolysis of the stratum corneum desmosome component and cell detachment. For this reason, confirming the presence state of desmoglein in the stratum corneum is also an evaluation of the desmosome function, and is extremely important in evaluating the skin state. For this reason, various evaluation methods have been conventionally proposed. Specific examples include a method of evaluating the activity of chymotrypsin-like enzyme and trypsin-like enzyme from the observation of a structure with a microscope and the action of proteases present in the stratum corneum (see Patent Document 1).

However, in the conventional evaluation method, it is not clear what index can be used to accurately evaluate the adhesion function of horny layer cells or their peeling function, and the method described in Patent Document 1 It is not a simple method for measuring the amount of desmoglein remaining, and it has not been possible to show how desmoglein is present in the stratum corneum. Similarly, no method has been known for easily showing the existence of other stratum corneum proteins.
JP-A-8-68791 Standard Dermatology 7th Edition, Chapter 17, Medical School, 2004 Hiromi Aoyama, Yasuo Kitajima, Clinical Immunity 30 (suppl.18): 252,1998

  Accordingly, there has been a demand for the development of a skin evaluation method based on the discovery of a method for simply detecting the expression or presence of stratum corneum proteins such as desmoglein.

  In view of the above situation, as a result of intensive studies, the present inventors have found that the horny layer protein in the horny layer can be visualized and detected by peeling the horny layer from the epidermis by tape stripping and staining this with the fluorescent antibody method. I found it. Then, the stratum corneum at the same site is peeled off by tape stripping, and the presence of desmoglein in the stratum corneum can be detected by staining this with a fluorescent antibody method. The present invention was completed by discovering that the present invention can be used in an evaluation method for skin turnover and a skin condition evaluation method using the existing position of the stratum corneum protein as an index.

  That is, the present invention provides a method for detecting a stratum corneum protein comprising peeling and collecting the stratum corneum from the skin by tape stripping, and staining the collected stratum corneum sample by a fluorescent antibody method.

  The present invention also provides the presence of a stratum corneum protein in the stratum corneum characterized in that the stratum corneum at the same site on the skin is peeled off and collected a plurality of times by tape stripping, and the collected stratum corneum samples are stained by a fluorescent antibody method. A position detection method is provided.

  Furthermore, the present invention provides an epidermis turnover evaluation method and a skin condition evaluation method characterized by using the position of the stratum corneum protein in the stratum corneum as an index.

  According to the present invention, it becomes possible to visualize and detect horny layer proteins in the horny layer by a simple method. In addition, skin turnover can be easily evaluated. Furthermore, it is possible to easily evaluate skin conditions such as rough skin.

  In the present invention, the stratum corneum protein to be measured includes cell adhesion proteins, stratum corneum substances, stratum corneum cell components, and the like.

  Among these, as cell adhesion protein, desmoglein 1, 2, 3, 4, desmocollin 1, 2, 3, corneodesmosine, desmoplakin 1, 2, plamoglobin, placofilin and other desmosome components, claudin, occludin, ZO-1, 2, 3, MUPP-1, Tricellulin, E cadherin, N cadherin, P cadherin, α-catenin, β-catenin, p120, and the like.

  In addition, as the stratum corneum substance, lipids such as glucosylceramide, ceramide 1-9, cholesterol, free fatty acid, cholesterol sulfate, kallikrein 5, 6, 7, 8, 10, 11, 13, 14, plasmin, cathepsin D, E, G, L, transglutaminase 1, 2, 3, 5, 6, peptidyl arginine deiminase, steroid sulfatase, lipase, caspases and other enzymes, interleukin (IL) 1ra, 1α, 1β, 2, 3, 4, 5, 6, 8, 10, transforming growth factor (TGF) α, β, interferon (INF) α, β, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (Granulocyte macrophage) Cytokines such as colony stimulating factor (GM-CSF), filaggrin, pro Iragurin various phosphorylation-dephosphorylation enzymes, mention may be made of various amino acids and the like.

  Furthermore, examples of the stratum corneum cell component include loricrin, involucrin, and small proline-rich protein.

  Among the methods of the present invention, the stratum corneum is peeled off and collected from the skin by tape stripping, and the stratum corneum protein detection method for staining the collected stratum corneum sample by the fluorescent antibody method (hereinafter referred to as “first aspect invention”) is: For example, it can be carried out as follows.

  In order to implement the first aspect of the invention, it is first necessary to peel and collect the stratum corneum from the skin surface of the skin by tape stripping to obtain a stratum corneum sample. This tape stripping is a transparent tape that is sticky to the skin, for example, the adhesive layer is made of natural rubber or acrylic adhesive, and the transparent film is made of PET, cellophane, etc. Then, the stratum corneum is sampled by peeling the tape from the skin.

  In this tape stripping, the stratum corneum cells that are peeled off due to the adhesiveness of the tape in one operation are usually one layer. Therefore, the depth of the stratum corneum cells that can be peeled off and obtained is determined by the number of times of tape stripping. That is, by repeating this operation a plurality of times, stratum corneum cells can be sampled step by step from the upper layer to the inner (depth) direction.

  Next, stratum corneum proteins present in stratum corneum cells copied on the tape are stained and detected by a well-known fluorescent antibody method. This fluorescent antibody method is performed by, for example, washing a transparent tape with a stratum corneum obtained by tape stripping with phosphate buffered saline (PBS) or the like, and then subjecting this to fluorescently labeled target stratum corneum protein. The reaction is carried out by reacting the antibody for a predetermined time at an appropriate temperature such as room temperature, or by reacting the antibody against the target stratum corneum protein after washing the transparent tape and then reacting the antibody against the labeled antibody. As a preferable example of staining by the fluorescent antibody method, a mouse IgG antibody against a target stratum corneum protein (for example, desmoglein) is used as a primary antibody, and a fluorescent-labeled anti-mouse IgG antibody is used as a secondary antibody. The method of dyeing is mentioned.

  The antibody used in the above may be a monoclonal antibody, a polyclonal antibody, or a combination thereof. In addition, antibodies against these target stratum corneum proteins are either known or can be easily obtained by a well-known method described in the literature. In addition, fluorescent labeling for antibodies can be easily carried out by a known method.

  Then, by observing the fluorescently stained sample with a fluorescence microscope, the location of the stratum corneum protein (eg, desmoglein) can be visualized and detected simultaneously with the shape of the stratum corneum. In addition, it is also possible to perform image processing on the obtained microscopic image and to evaluate the fluorescence distribution of the stratum corneum protein in numerical values.

  Also, in the method of the present invention, the stratum corneum at the same site in the skin is peeled and collected multiple times by tape stripping, and the location of the stratum corneum protein in the stratum corneum is detected by staining the collected plurality of stratum corneum samples by the fluorescent antibody method. The method (hereinafter referred to as “second aspect invention”) can be carried out by repeating the first aspect invention.

  That is, as described above, in tape stripping, the stratum corneum cells that are peeled off due to the adhesiveness of the tape in a single operation are usually one layer. For example, tape stripping is first performed on the same part of the skin. Corn layer sample (surface sample), stratum corneum sample obtained by tape stripping about 10-15 times (intermediate layer shallow sample), stratum corneum sample obtained by tape stripping about 20-30 times (intermediate layer deep sample) ) If four samples of stratum corneum samples (deep layer samples) obtained by tape stripping about 35 to 40 times are obtained, and the target stratum corneum protein is detected for each of these samples, the location and distribution of the stratum corneum It becomes clear (for example, in which part it is often present and in what form it exists). Needless to say, this method is not limited to using four samples as described above, and may be performed simply with three samples or with four or more samples when the distribution is accurately obtained.

  Epidermal turnover evaluation method (hereinafter referred to as “third aspect invention”) using the present position of stratum corneum protein in the stratum corneum as an index, and skin condition using the present position of stratum corneum protein in the stratum corneum as an index The evaluation method (hereinafter referred to as “fourth aspect invention”) can be easily implemented by utilizing the second aspect invention.

  That is, after investigating the relationship between the location of specific horny layer proteins and epidermal turnover for multiple panels, the location of the subject's horny layer proteins to be evaluated is examined, and the epidermal turnover can be evaluated accordingly. Good. For example, a method for evaluating epidermis turnover by selecting desmoglein as a stratum corneum protein and using this location as an index is as follows.

  Skin turnover can be determined by examining the stratum corneum in the depth direction to determine whether it is in a normal state. Therefore, first, according to the second aspect of the invention, it is possible to examine in which position of the stratum corneum the desmoglein is present, and from this, it is possible to evaluate in which state the keratinization and thus the epidermis turnover is present. And, for example, desmoglein is present on the entire cell surface mainly in the stratum corneum deep layer sample, but in the stratum corneum surface sample, if it is present on the cell edge, the keratinization is in a good state, It can be evaluated that the skin has been turned over properly. On the other hand, when desmoglein is present on the entire cell surface even in the stratum corneum surface sample, it can be evaluated that the keratinization is not good and the skin is not turned over properly. Moreover, the state of the epidermis turnover by the part of the skin can be compared by changing the part where the tape stripping is performed, for example, by comparing the face and the upper arm.

  In addition, the skin condition can be evaluated in the state of the outermost layer of the stratum corneum, that is, the fourth aspect invention detects the stratum corneum protein according to the first aspect invention, detects its abundance, and determines the skin condition from this abundance. It is something to evaluate. For example, transepidermal water transpiration (TEWL), stratum corneum water content, and oil content are well known as parameters representing the stratum corneum state or skin state. A correlation was found between the expression state of the layer's desmoglein. In particular, it has been found that there is a high correlation between TEWL and water content. Therefore, according to the first aspect of the invention, for example, when desmoglein is stained and is present mainly in the peripheral part of the horny layer cells, it can be evaluated that the skin condition is good, and conversely, desmoglein is When it exists mainly on the entire surface of the stratum corneum, it can be evaluated that the skin condition is not good.

  In other words, skin troubles such as acne, dandruff, and tanning are caused by stratification of the stratum corneum due to increased cell adhesion, so it is an indicator of where in the stratum corneum desmoglein is present. By doing so, it is possible to evaluate the roughness of the skin, the desquamation state, and the like. Moreover, it can confirm that the skin state by a skin site | part differs by changing the site | part which carries out a tape stripping, for example by comparing with a face and the upper arm inside.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not restrict | limited at all by these Examples.

Example 1
Detection of desmoglein using the human inner upper arm:
(Sample preparation)
A transparent double-sided tape (manufactured by Nichiban Co., Ltd.) is cut into a size of 1.8 cm × 2.0 cm, and this is attached to a slide glass. Then, the adhesive surface of this slide glass was lightly pressed against the inner side of the human upper arm so that the entire surface of the tape adhered to the skin, and then peeled off from the skin to obtain an outermost layer sample. Moreover, it applied to the same site | part using the new slide glass similarly, tape stripping was repeated 2 to 39 times, and the sample of the middle layer, the deep part, and the deepest part was prepared.

(Primary antibody reaction)
An appropriate amount of an anti-desmoglein 1 mouse IgG monoclonal antibody (PROGEN) solution is applied to the tape surface of the slide glass sample and allowed to react at room temperature for 1 hour. Then, it is immersed in phosphate buffered saline (PBS) to wash away unreacted antibodies.

(Secondary antibody reaction)
An appropriate amount of a 200-fold PBS diluted solution of fluorescently labeled anti-mouse IgG antibody (manufactured by Invitrogen) is applied to the tape surface of the slide glass sample subjected to the primary antibody reaction, and reacted at room temperature for 1 hour. Then, it is immersed in PBS to wash away unreacted antibodies.

(Enclosed)
Mount Quick Aqueous (SPI SUPPLIES) was dropped on the surface of the slide glass sample reacted with the secondary antibody, and the sample was covered with a cover glass to obtain an observation sample.

(Observation)
The embedded slide glass sample was observed using an inverted fluorescent microscope manufactured by Olympus. This observation screen is shown in FIGS.

(result)
As shown in FIG. 1, a stained image on a dot centered around the stratum corneum cells was obtained, and desmoglein could be visualized and detected. Moreover, it was confirmed from FIGS. 1-4 that the expression state of the desmoglein in the deep part and surface of a stratum corneum has changed. From this, desmoglein disappears toward the upper layer of the epidermis, indicating that cell adhesion is weakened and the stratum corneum is finally peeled off. From this, it was shown that the skin turnover can be evaluated by the method of the present invention.

Example 2
Detection of desmoglein using human face, lips and outer upper arm:
In the same manner as in Example 1, the sampling site was changed to the human face, lips, and outer upper arm, and the epidermis of the first layer was peeled off. The facial image thus obtained is shown in FIG. 5, the lip image in FIG. 6, and the outer upper arm in FIG.

(result)
1-4 and FIGS. 5-7, desmoglein was detected in the entire stratum corneum cells like a deep part inside the upper arm on the face. This is considered to be because the face that is susceptible to external environmental stimulation is more keratinized than the inner side of the upper arm, which is difficult to receive stimulation, and the skin is rough. In the lips with a very thin stratum corneum, desmoglein was detected from the entire stratum corneum cells, as in the deep part inside the upper arm. Conversely, the stratum corneum outside the upper arm was close to the inside.

Example 3
Examination of correlation between skin condition and expression state of stratum corneum desmoglein:
(1) Method for measuring TEWL / water content (skin measurement)
Using healthy volunteers who performed informed consent as subjects, transepidermal water transpiration (TEWL) and stratum corneum water were measured by the following methods. First, the test site of the subject was washed with a cleaning agent, and then rested for 15 minutes in a test room maintained at room temperature 22 ° C. and humidity 50%. Next, Delfin Technologies Vapometer
TEWL was measured 4 times with the SWL2 type, and the average value was used. The water content was measured four times with a skin surface angle layer water content measuring device SKICON-200EX manufactured by IBS, and the average value was used.

(2) Image analysis method From the measurement site | part of the test subject who measured TEWL etc. in said (1), tape stripping was performed once using the transparent double-sided tape (made by Nichiban), and the stratum corneum sample was extract | collected. This stratum corneum sample was stained with desmoglein by a fluorescent antibody method, and then photographed with a CCD camera attached to a fluorescence microscope. This photographed image was analyzed with image analysis software, WinR00F, manufactured by Mitani Corporation. Specifically, three square areas of 1 mm ² were randomly extracted from the image taken with a microscope, the number of fluorescent dots included in the range was measured with WinR00F, and the average value was used. And quantified.

(3) Correlation study The correlation between the expression state of desmoglein quantified in (2) above, the stratum corneum water content obtained in (1), and TEWL was examined. As shown in Fig. 2, a high correlation was found between the expression level of desmoglein and these two skin parameters.

  That is, desmoglein is found on the entire stratum corneum cells, and the greater the amount, the lower the water content and the higher the TEWL (so-called rough skin). Conversely, desmoglein was observed confined to the stratum corneum cell margin, and it was found that the smaller the amount, the higher the water content and the lower the TEWL (so-called ideal skin). This result suggests that desmoglein in the stratum corneum is involved in skin barrier formation. At the same time, it was shown that this method is effective for the evaluation of the stratum corneum and the barrier state.

Example 4
Detection of desmoglein 3 from the lips:
The lips of some volunteers who gave informed consent were stripped once using a transparent double-sided tape (manufactured by Nichiban Co., Ltd.), and a lip horny layer sample was obtained. For this lip horny layer sample, desmoglein 3 was detected in the same manner as in Example 1 using an anti-desmoglein 3 mouse monoclonal antibody (Progen) and a FITC-labeled anti-mouse IgG antibody.

  And, since the expression of desmoglein 3 is much in the rough lips, and the expression of desmoglein 1 is not greatly influenced by the state of the lips, by detecting desmogleins 1 and 3 and looking at their ratio, It has been found that it is an indicator of the rough state of the lips.

Example 5
Involucrin detection:
Tape stripping was performed from the stratum corneum in the same manner as in Example 1. The obtained tape was washed with PBS, and then reacted with an anti-involucrin antibody (abcam manufactured by Abcam) diluted 500 times with PBS for 1 hour. Thereafter, Alexa Fluor 546 anti-mouse IgG antibody diluted 200-fold with PBS was reacted for 1 hour and observed with a fluorescence microscope. As a result, it was revealed that the entire surface of the stratum corneum cells were stained and cells that were hardly stained were observed (FIG. 10). This result also suggests that it can be used as a method for evaluating the turnover of the stratum corneum or the skin condition, similar to the result of desmograin.

  The method for detecting desmoglein in the stratum corneum, the method for evaluating skin turnover and the method for evaluating skin condition according to the present invention are not only used for the development of pharmaceuticals and cosmetics but also objectively indicate the condition of the customer's skin. Therefore, it can also be used as an index for selecting a cosmetic suitable for the customer.

Fluorescence micrograph of stratum corneum (outermost layer: one tape strip) obtained from the inner side of the upper arm Fluorescence micrograph of stratum corneum (middle layer: 9 tape strips) obtained from the inner side of the upper arm Fluorescence micrograph of stratum corneum (depth: tape strip 29 times) obtained from the inner side of the upper arm Fluorescence micrograph of stratum corneum (deepest part: 39 times of tape strip) obtained from the inner side of the upper arm Fluorescence micrograph of stratum corneum obtained from the face Fluorescence micrograph of the stratum corneum obtained from the lips Fluorescence micrograph of the stratum corneum (outermost layer) obtained from the outer side of the upper arm Drawing showing correlation between expression state of digitized desmoglein and water content of stratum corneum Drawing showing the correlation between the expression state of numerical desmoglein and TEWL Fluorescence micrograph of involucrin in Example 5

Claims (4)

A method for detecting the location of a cell adhesion protein in a stratum corneum, comprising peeling and collecting the stratum corneum at the same site on the skin multiple times by tape stripping, and staining the collected plurality of stratum corneum samples by a fluorescent antibody method. The method for detecting the position of the cell adhesion protein in the stratum corneum according to claim 1, which is used in a method for evaluating epidermal turnover . The method for detecting the position of cell adhesion protein in the stratum corneum according to claim 1, which is used for a skin condition evaluation method. The method for detecting the position of the cell adhesion protein in the stratum corneum according to any one of claims 1 to 3, wherein the cell adhesion protein is desmoglein.