JPH0820444B2 - Stratum corneum inspection method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for inspecting the stratum corneum of the skin, which can be used in the fields of cosmetics, foods, pharmaceuticals, skin quality inspection and the like. Is.

[Conventional technology]

Originally, when selling articles such as cosmetics that are required to keep the skin in good condition, it is necessary to accurately grasp the skin condition of the user and instruct makeup accordingly. Based on such an idea, in recent years, it has become an important subject to know the skin quality, skin property, skin condition, etc. by observing or measuring the skin condition.

Conventionally, as a method for observing these skin conditions, many methods have been proposed, but by observing the stratum corneum of the skin surface in detail, an accurate skin inspection can be performed,
I understand. Conventionally, as a method of examining the surface condition of the skin, (a) a method of directly observing the skin with the naked eye.

(B) Sump method (a method of dripping an organic solvent such as amyl acetate onto a celluloid plate to bring the celluloid plate into close contact with the skin, and then peeling the celluloid plate to prepare a skin replica, and observing the skin replica).

(C) A method of displaying a magnified image of the skin on a monitor and observing the image using a device such as a videoscope. Etc. have been carried out.

[Problems to be solved by the invention]

However, the method (a) or (c) is based on the appearance findings and is convenient for the measurement of the skin gloss and the like, but the condition of the skin could not be precisely investigated. Further, the method (b) is an advantageous method for examining the roughness and unevenness of the skin, but it merely measures the surface shape. Therefore, in order to investigate the skin condition in more detail,
The best way is to collect the stratum corneum and observe it.

As the method for examining the stratum corneum, for example, a method of applying a known adhesive to a transparent plate, peeling it off after adhering it to the skin, holding the stratum corneum on the transparent plate, and using this as a stratum corneum sample as it is Conceivable.

However, according to this method, a clear and well-preserved stratum corneum sample cannot be obtained, and the skin of the subject is often damaged.

Therefore, it is difficult to precisely observe or inspect the condition of the skin.

[Means for solving problems]

The present invention has been made in view of the above-mentioned matters, and a sticky substance soluble in an organic solvent is applied, and a tape having a skin stratum corneum adhered to the sticky substance is applied with a fixing agent in advance. We focused on the fact that a clear and well-preserved stratum corneum sample can be obtained by sticking it to a transparent plate and then peeling off this adhesive tape using an organic solvent and fixing the stratum corneum to the transparent plate. If you observe and measure the stratum corneum cells using this stratum corneum sample,
As a result, the condition of the skin can be examined precisely and quickly, and the above problems can be solved.

[Action]

According to the method of the present invention, by taking the above-mentioned means, the stratum corneum can be collected from the skin with a tape coated with an adhesive substance soluble in an organic solvent.

The stratum corneum sample applied by the present invention is a kind of the stratum corneum which is a thin-film-like group of cells existing in the outermost layer of the epidermis which mainly covers the surface of human skin and is fixed on a transparent plate. It is a tissue sample. The above-mentioned stratum corneum specimen is capable of observing the stratum corneum cells in detail by staining or the like, and is particularly suitable for microscopic observation. When there is a nucleus in the stained stratum corneum cells, the nucleus can be clearly observed, and the keratinized state of the epidermal cells can be understood, so it is also possible to know the metabolic rate of the epidermal cells and the state of rough skin. is there.
In addition, by measuring the area, circumference, flattening ratio, homogeneity, etc. of the stratum corneum cells, it is possible to objectively evaluate the degree of skin aging and skin properties. Of course, it is also preferable to project a magnified view on the screen of the above stratum corneum sample using a device such as a videoscope, and observe and measure this.

Thus, the method of the present invention provides a lot of knowledge about the condition of the skin, which allows the individual's condition of the skin to be accurately known. Based on this, it is necessary to provide makeup instruction according to the skin quality of the person. It is also possible.

Next, a resin or polymer compound that is soluble in an organic solvent is used as the adhesive substance used when preparing the stratum corneum sample according to the present invention, and specific examples thereof include polyvinyl acetate and polyvinyl alcohol. , Polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, polybutene, asphalt, and natural rubber or its derivatives, SBR rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber, chloroprene rubber, recycled rubber Examples thereof include rubber materials such as cyanoacrylate rubber, gum arabic, and tragacanth rubber, and one or a mixture of two or more of them may be used alone or in another substrate. Among the above-mentioned adhesive substances, natural rubber or its derivative, SBR rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber,
Rubber materials such as chloroprene rubber, regenerated rubber, cyanoacrylate rubber, gum arabic, and tragacanth rubber are preferably used in the method of the present invention in that they do not change the peeling property and the stratum corneum cells when the tape is peeled off. Of course, in the method of the present invention, a tape coated with the above-mentioned adhesive substance, such as a commercially available cellophane tape, is also preferably used. However, a tape using a gum substance such as a gum tape as an adhesive substance is not used in the method of the present invention because the gum substance is insoluble in the above-mentioned organic solvent.
It is sufficient that the material of the tape itself is one that is generally or industrially used, and one having a uniform thickness, one having pores, and one having a corrugated shape can be used.

Further, the organic solvent used in the preparation of the stratum corneum sample according to the method of the present invention is capable of dissolving the adhesive substance, and as such an organic solvent, specifically, methanol , Ethanol, propanol, butanol, ether, acetone, chloroform, hexane,
THF, ethyl acetate, o-xylene, m-xylene, p-
Xylene, methylbenzene, ethylbenzene, propylbenzene, benzene, toluene, amyl acetate and the like can be mentioned, and one kind or a mixture of two or more kinds thereof is used.

Next, the adhesive used when preparing the stratum corneum specimen according to the method of the present invention is one that fixes the stratum corneum to the transparent plate and does not denature the stratum corneum cells, and the above organic It is not readily soluble in solvents. Examples of such an adhesive include known adhesives, proteins and the like, but known adhesives and the like are preferably used because the obtained stratum corneum sample is clearer, and specific examples include: Polyvinyl acetate such as polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, melamine resin adhesive, phenol resin adhesive, resorcinol adhesive,
Xylene resin adhesive, furan resin adhesive, polyisocyanate resin adhesive, polyester resin adhesive, polyamide adhesive, polybutene adhesive, ethylene copolymer adhesive, thermoplastic polyester adhesive, polyester acrylate adhesive, silicone rubber -Based adhesives, phenol-based adhesives, epoxy-based adhesives, cyanoacrylate-based adhesives, anaerobic polyester adhesives, polybenzimidazole-based adhesives, polyimide-based adhesives, polyimideamide-based adhesives, Trenis-based adhesives, Metalloxane-based adhesives, glue-based adhesives, casein-based adhesives, cellulose-based adhesives, viscose-based adhesives, etc.
One kind or a mixture of two or more kinds is used. Among these adhesives, the polyvinyl-based adhesive is most preferable because it is difficult to dye when dyed. Examples of the above proteins include albumin such as ovalbumin, milk albumin, myogen, serum albumin, globin, leucosine, serum globulin, β-lactoglobulin, lysozyme, myosin, edestin, insulin, fibrinogen, thyroglobulin, etc. Globulin, soluble collagen, elastin, keratin,
Silk fibroin, sponge, gorgonin, histone,
Examples include protamine, egg white, gelatin, peptone, peptides, phosphoproteins, lipoproteins, glycoproteins, etc., and one or a mixture of two or more of these is contained alone or in another base material such as polyhydric alcohol. The stuffed one is used.

Further, the transparent plate used for preparing the stratum corneum specimen according to the method of the present invention is transparent, does not interfere with observation of the stratum corneum specimen, and is insoluble or hardly soluble in an organic solvent. Specific examples thereof include glass plates or resins such as polymethylpentene and polyethylene. Among them, glass plates are most preferably used from the viewpoint of transparency and solvent resistance.

In the method of the present invention, first, the tape coated with the above-mentioned adhesive substance is brought into close contact with the skin, and then the tape is peeled off to collect a part of the stratum corneum on the tape. Next, this tape is attached to a transparent plate coated with the above-mentioned adhesive in advance. Here, it is important to apply the adhesive agent to the transparent plate in advance.If this pretreatment is not performed, the stratum corneum is separated from the transparent plate in the steps such as tape peeling described below. Therefore, it is inappropriate. When a tape holding a stratum corneum is attached to a transparent plate, the tape width is 3 to 10 mm.
It is preferably about the same. If the width of the tape is too wide, the permeability of the organic solvent is poor and it is inconvenient to handle. Next, the transparent plate to which the above-mentioned tape was attached was immersed in the above-mentioned organic solvent for 1 to 3 hours to dissolve or swell a part of the adhesive substance adhering to the tape to enhance the peelability of the tape. After that, carefully peel it off from the transparent plate. further,
Subsequent immersion in an organic solvent is also suitable for the purpose of removing the remaining sticky substance. After transferring the stratum corneum to a transparent plate, usually, the operations such as staining and balsam encapsulation are performed by a conventional method, and this is used as a stratum corneum sample, and the stratum corneum is inspected by the following methods (a) and (b) Use as a material for skin examination.

(A) The obtained stratum corneum sample is observed with an optical microscope,
The shape, size, homogeneity, homogeneity, degree of aggregation, the presence or absence of nuclei, etc. of the stratum corneum cells are used to examine the skin condition, skin properties, skin quality, etc.

(B) The obtained stratum corneum sample is displayed on a monitor using a videoscope, and the skin condition and skin texture are determined based on the shape, amount, size, homogeneity, homogeneity, aggregation, and presence of nuclei of stratum corneum cells. ,
By examining the skin quality and the like, and further measuring the area, circumference, flattening rate, etc. of the stratum corneum cells, the skin condition, skin quality, skin quality, skin aging degree, skin age, etc. are examined.

〔Example〕

 Next, examples of the method of the present invention will be described.

<Example-1> A cellophane tape having a width of 12 mm and a length of 20 mm (commercially available adhesive cellophane tape is cut and used) is stuck to the cheek, and after pressing for a while, gently peel it off and use scissors. The width is about 6 mm and the length is 20 mm, and this is attached to a slide glass on which a polyvinyl acetate adhesive has been uniformly applied.

Next, this slide glass is immersed in xylene for 2 hours. Next, the tape is gently peeled off using tweezers, and then dipped in xylene for another hour.

Then, after taking this out and drying the xylene, a gentian violet-brilliant green dyeing solution (gentian violet 1 g and brilliant green
It is immersed in 100 g of distilled water (0.5 g) for 5 to 10 minutes, washed with water, dried, and enclosed in balsam by a conventional method to obtain a stratum corneum sample. Next, the obtained stratum corneum sample is observed by an optical microscope, and the shape, amount, size, and uniformity of the stratum corneum cells,
The skin condition, skin quality, skin quality, etc. were examined from the homogeneity, the degree of aggregation, the presence or absence of nuclei, etc.

The obtained stratum corneum specimen is a clear stratum corneum specimen which has never been seen before, and as shown in FIG. 1, the stratum corneum cells are clearly observed, and particularly the normal or abnormal state of keratinization can be observed in detail. Therefore, I was able to examine the metabolism of the skin, the degree of aging, and the skin condition.

<Example-2> A cellophane tape having a width of 12 mm and a length of 20 mm (commercially available adhesive cellophane tape is cut and used) is attached to the cheek, and after pressing for a while, gently peel it off and use scissors. The width is about 6 mm and the length is 20 mm, and this is attached to a slide glass on which a polyvinyl acetate adhesive has been uniformly applied.

Next, after immersing this slide glass in xylene for 2 hours, the tape is gently peeled off using tweezers, and then further immersed in xylene for 1 hour. Next, after taking this out and drying the xylene, it was immersed in a gentian violet-brilliant green dyeing solution (gentian violet 1 g and brilliant green 0.5 g dissolved in 100 g of distilled water) for 5 to 10 minutes, washed with water and dried. rear,
Encapsulate balsam by a conventional method to obtain a stratum corneum specimen.

Next, the obtained stratum corneum sample is displayed on a monitor using a videoscope, and the skin condition and skin texture are determined based on the shape, amount, size, uniformity, homogeneity, degree of aggregation of the stratum corneum cells, and the presence or absence of nuclei. By examining the skin quality and the like, and further measuring the area, circumference, flattening rate, etc. of the stratum corneum cells using an image analysis device,
The skin condition, skin quality, skin quality, degree of skin aging, skin age, etc. were examined.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-3> A cellophane tape having a width of 12 mm and a length of 20 mm (commercially available adhesive cellophane tape is cut and used) is stuck to the cheek, and after pressing for a while, gently peel it off and use scissors. The width is about 6 mm and the length is 20 mm, and the mixture is gently attached to a slide glass on which the mixture of ovalbumin: glycerin = 1: 1 by weight is evenly applied, and left at a temperature of 60 ° C. for 2 hours.

Next, this slide glass is immersed in ethanol for 10 minutes, dried, and further immersed in xylene for 2 hours.
Gently remove the tape with tweezers and then soak in xylene for an additional hour. Next, after taking this out and drying the xylene, a gentian violet-brilliant green dyeing solution (gentian violet 1 g
And 0.5 g of brilliant green dissolved in 100 g of distilled water) for 5 to 10 minutes, washed with water, dried and then enclosed in balsam by a conventional method to obtain a stratum corneum sample.

Next, the obtained stratum corneum sample is displayed on a monitor using a videoscope, and the skin condition and skin texture are determined based on the shape, amount, size, uniformity, homogeneity, degree of aggregation of the stratum corneum cells, and the presence or absence of nuclei. By examining the skin quality and the like, and further measuring the area, circumference, flattening rate, etc. of the stratum corneum cells using an image analysis device,
The skin condition, skin quality, skin quality, degree of skin aging, skin age, etc. were examined.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-4> A paper tape having a width of 10 mm and a length of 20 mm was uniformly coated with an adhesive substance containing gum arabic, tragacanth or the like, and the adhesive tape was stuck to the cheek, pressed for a while, and then gently peeled off. Use scissors to make the width about 6 mm and the length 20 mm, and attach it to the slide glass on which the cellulosic adhesive has been uniformly applied.

Next, this slide glass is immersed in acetone for 2 hours, then the tape is gently removed using tweezers, and then further immersed in xylene for 1 hour. Next, this was taken out, xylene was dried, hematoxylin-eosin staining was performed, and after drying, it was enclosed in balsam by a conventional method to obtain a stratum corneum sample. Next, the obtained stratum corneum specimen is observed by an optical microscope, and the shape, amount, size, homogeneity, homogeneity, degree of aggregation of stratum corneum cells, the skin condition from the presence or absence of nuclei, skin texture,
The skin quality etc. were examined.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-5> An adhesive tape obtained by uniformly applying an adhesive substance containing natural rubber to a plastic tape having a width of 10 mm and a length of 20 mm was attached to the inner side of the upper arm, and gently peeled off. The same treatment as in 1 is performed and Giemsa staining is performed to obtain a stratum corneum specimen. Next, the obtained stratum corneum sample was projected on a monitor using a videoscope, and the shape, amount, and size of the stratum corneum cells,
Check the skin condition, skin quality, skin quality, etc. based on the uniformity, homogeneity, degree of aggregation, presence of nuclei, etc., and further measure the area, circumference, flattening rate, etc. of stratum corneum cells using an image analyzer. By
The skin condition, skin quality, skin quality, degree of skin aging, skin age, etc. were examined.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

<Example-6> Cellophane tape having a uniform distribution of pores and a width of 10 mm and a length of 20 mm (commercially available adhesive cellophane tape is processed and used.) It is attached to the inner side of the upper arm and gently peeled off. Then, the same procedure as in Example-2 is performed to obtain a stratum corneum sample. Next, the obtained stratum corneum sample was projected on a monitor using a videoscope, and the shape, amount, and size of the stratum corneum cells,
Check the skin condition, skin quality, skin quality, etc. based on the uniformity, homogeneity, degree of aggregation, presence of nuclei, etc., and further measure the area, circumference, flattening rate, etc. of stratum corneum cells using an image analyzer. By
The skin condition, skin quality, skin quality, degree of skin aging, skin age, etc. were examined.

The obtained stratum corneum sample, the stratum corneum cells are clearly observed in the same manner as in Example-1, skin metabolism and aging degree,
I was able to check the skin condition.

〔The invention's effect〕

According to the present invention, by configuring as described above, since the stratum corneum is inspected using a specimen that is clear and easy to observe, and has good storage stability, the stratum corneum can be surely and easily inspected, and the skin quality etc. Can be effectively used for inspection.

<Example-7> A cellophane tape having a width of 12 mm and a length of 30 mm (commercially available adhesive cellophane tape is processed and used) is a subject with atopic dermatitis (at the first examination of a 5-year-old boy), which is likely to have symptoms. (Wrist, elbow fossa and patella), hold it down for a while, gently peel it off, and use scissors to make it 10 mm wide and 20 mm long, and then evenly apply polyvinyl acetate adhesive to it. Stick it on the slide glass.

Next, this slide glass was immersed in xylene for 3 hours, then this slide glass was taken out and dried with cold air. Furthermore, after immersing this slide glass in an aqueous solution of hematoxylin for 20 minutes and washing with running water for 5 minutes,
After immersing in 0.25% eosin for 2 minutes, rinsed with running water for 5 minutes, dried well with cold air, and enclosed in balsam by a conventional method to obtain a stratum corneum sample.

Next, the obtained stratum corneum sample was observed with an optical microscope to examine the presence and number of nuclei in the stratum corneum. In the obtained stratum corneum specimen, the stratum corneum nuclei were clearly observed, and the number of stratum corneum nuclei could be easily examined. (Atopic dermatitis patients have a large number of nuclei in the stratum corneum.) Atopic dermatitis patients (5-year-old boy) are continuously rubbed with a topical steroid on the five test sites to start treatment. After 2 weeks and 1 month, a stratum corneum sample was obtained in the same manner as above, and the presence and number of nuclei in the stratum corneum were observed. The results are shown in Table 1.

As is clear from the above results, a decrease in the number of nuclei of the affected stratum corneum is observed as the treatment is continued, and the stratum corneum inspection method of the present invention evaluates the efficacy of an external preparation such as atopic dermatitis and its treatment progress. It turns out that it is effective as a means of observation.

[Brief description of drawings]

FIG. 1 is a stratum corneum sample applied to the method of the present invention (Example-
It is an optical micrograph of 1) (magnification about 700 times).

Front page continuation (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location G01N 33/48 S 7638-2J A61B 5/10 300 Q

Claims (5)

[Claims] 1. A sticky substance which is soluble in an organic solvent is applied, and a tape to which the stratum corneum of the skin is adhered is attached to a transparent plate which has been applied with a sticking agent in advance. After immersing this in an organic solvent, the tape is removed to fix a portion of the stratum corneum on a transparent plate, and if necessary, this is dyed to form a stratum corneum sample and the state of the stratum corneum. A method for inspecting stratum corneum, which comprises: 2. The adhesive material is natural rubber or its derivative, SB.
R type rubber, NBR type rubber, butadiene-vinyl pyridine type rubber, butyl rubber, chloroprene type rubber, recycled rubber, cyanoacrylate type rubber, arabic rubber, tragacanth rubber, etc. The method for inspecting stratum corneum according to claim 1, wherein 3. The method for inspecting stratum corneum according to claim 1, wherein the tape coated with an adhesive substance soluble in an organic solvent is cellophane tape. 4. The fixing agent is a single or a mixture of two or more selected from polyvinyl adhesives such as polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride and polyvinyl ether. The method for inspecting the stratum corneum according to claim 1, wherein the method is contained in another base material. 5. The method for inspecting stratum corneum according to any one of claims 1 to 4, wherein the transparent plate is a glass plate.