JPS63106558A - Method for forming sample of stratum corneum - Google Patents

Abstract

PURPOSE:To form a sharp sample easy to observe and having good preservability, by adhering a tape wherein a lining stratum corneum is adhered to an adhesive substance soluble in an org. solvent to a transparent plate coated with a fixing agent and immersing the whole in the org. solvent to peel off the tape. CONSTITUTION:A tape coated with a viscous substance composed of a resin or high-molecular compound soluble in org. solvent is closely contacted with a cortical layer and peeled off to sample a part of a stratum corneum on the tape. This tape is adhered to a transparent plate preliminarily coated with a fixing agent and the whole is immersed in the org. solvent for several hr to dissolve and swell the viscous substance and the tape is subsequently peeled off. The fixing agent applied to the transparent plate is set to a substance not modifying the cells of the stratum corneum and hardly soluble in the org. solvent and the tape is a narrow one and improved in the permeability of the solvent. This sample is stained and sealed in balsam to make it possible to obtain a sharp sample easy to observe and improved in preservability.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a specimen preparation method for forming a specimen of the stratum corneum of the skin, and is applicable to cosmetics, foods, medicines, skin quality testing, etc. It is used in the field.

[Conventional technology]

Essentially, when selling products such as cosmetics that are required to maintain a good skin condition, it is necessary to accurately understand the user's skin condition and instruct the user to apply makeup accordingly. Based on this idea, in recent years, it has become possible to know the skin type, skin type, skin condition, etc. by observing or measuring the skin condition.
It has become an important issue.

Conventionally, many methods have been proposed to observe these skin conditions, but it has been found that accurate skin examinations can be performed by closely observing the stratum corneum on the skin surface. Conventionally, methods for examining the surface condition of the skin include (a) a method of directly observing the skin with the naked eye;

(b) Thump method (a method in which an organic solvent such as amyl acetate is dropped onto a celluloid plate, the celluloid plate is brought into close contact with the skin, and then peeled off to create a skin replica and observed).

(C) A method in which an enlarged image of the skin is displayed on a monitor and observed using a device such as a videoscope.

Such methods have been used.

[Problem that the invention seeks to solve]

However, 1. Methods (a) and (C) are based on external findings, and are convenient for measuring skin color and luster, but cannot precisely examine the condition of the skin. Further, although method (b) is an advantageous method for examining the roughness and unevenness of the skin, it merely measures the surface shape.

Therefore, in order to examine the condition of the skin in more detail, the best method is to collect the stratum corneum and observe it.

The method for preparing this stratum corneum specimen is, for example, by applying a known adhesive to a transparent plate, bringing it into close contact with the skin, and then peeling it off to hold the stratum corneum on the transparent plate, which is then used as a specimen of the stratum corneum. There are possible ways. However, with this method, it is not possible to obtain a clear and well-preserved stratum corneum specimen, and the test subject's skin is often damaged.

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

[Means for solving problems]

The present invention was developed in view of the above-mentioned problems, and includes a tape coated with an adhesive substance soluble in an organic solvent, and a tape to which the stratum corneum of the skin is attached to the adhesive substance. The stratum corneum is fixed to the transparent plate by attaching it to a transparent plate coated with a fixing agent, and then removing the adhesive tape using an organic solvent. If necessary, the stratum corneum is stained to obtain a specimen of the stratum corneum. It is something to create.

[Effect]

In the method of the present invention, by taking the above-mentioned steps, the stratum corneum can be collected from the skin using a tape coated with an adhesive substance soluble in an organic solvent. By the above method, a clear and well-preserved specimen of the stratum corneum can be obtained, and by using this specimen to observe and measure the cells of the stratum corneum, the condition of the skin can be precisely and quickly investigated. Can be done.

The stratum corneum specimen provided by the present invention is a type of stratum corneum specimen that is made by fixing the stratum corneum, which is a thin film-like cell group that exists in the outermost layer of the epidermis that mainly covers the surface of human skin, on a transparent plate. This is a tissue specimen. The above-mentioned stratum corneum specimen allows detailed observation of stratum corneum cells by staining, etc., and is particularly suitable for microscopic observation. If the stained stratum corneum cells have nuclei, they can be clearly observed and the state of keratinization of the epidermal cells can be seen, making it possible to determine the metabolic rate of the epidermal cells and the state of rough skin. be. Furthermore, by measuring the area, circumference, aspect ratio, homogeneity, etc. of the stratum corneum cells, the degree of aging and skin quality of the skin can be objectively evaluated. Of course, it is also preferable to use a device such as a videoscope to display an enlarged view of the stratum corneum sample on a screen, and then observe and measure this.

Next, in the method for preparing a stratum corneum specimen according to the present invention, the adhesive substance used is a resin or a polymer compound that is soluble in an organic solvent, and specific examples include polyvinyl acetate, polyvinyl alcohol, Polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, polybutene, asphalt, natural rubber or its derivatives, SBR rubber, NBR rubber, butadiene-
Examples include rubber materials such as vinylpyridine rubber, butyl rubber, chloroprene rubber, recycled rubber, cyanoacrylate rubber, gum arabic, and rubber tragacanth, and one or a mixture of two or more of these can be used alone or with other base materials. It may be used if it is contained in Among the above adhesive substances, natural rubber or its derivatives, SBR rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber, chlorobutanone rubber, recycled rubber, cyanoacrylate rubber, etc. Rubber materials such as rubber, gum arabic, and gum tragacanth are preferably used in the method of the present invention because of their removability when removing the tape and their ability to not change the stratum corneum cells. Of course, in the method of the present invention, tapes coated with the above-mentioned adhesive substances, such as commercially available cellophane tapes, are suitable. This is used. However, tapes that use gum as an adhesive substance, such as duct tape,
Since the gum substance is insoluble in the above-mentioned 6-organic solvent, it cannot be used in the method of the present invention. The material of the above-mentioned tape itself is generally or industrially used, and in addition to those with uniform thickness, those with pores,
A mesh-like material can also be used.

Further, the six organic solvents used are those capable of dissolving the above-mentioned sticky substances, and examples of such organic solvents include methanol, ethanol, propatool, butanol, ether, acetone, and chloroform. , hexane, TI-IF1 ethyl acetate, Q-xylene, m-
Examples include xylene, p-kiln, methylbenzene, ethylbenzene, propylbenzene, benzene, toluene, amyl acetate, etc., and these may be used alone or in combination or in combination.

Next, the fixing agent used in the method of the present invention is one that fixes the stratum corneum to the transparent plate, does not degenerate the stratum corneum cells, and is not easily soluble in the above-mentioned organic solvents. . Examples of such a fixing agent include known adhesives, proteins, etc., but known adhesives are preferably used because the obtained stratum corneum specimen is clearer, and specific examples include: Polyvinyl adhesives such as polyvinyl acetate, polyvinyl alcohol, polyvinyl acecool, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, melamine resin adhesives, phenol resin adhesives, resorcinol adhesives, xylene resin adhesives, Furan resin adhesive, polyisocyanate resin adhesive, polyester resin adhesive, polyamide adhesive, polybutene adhesive, ethylene copolymer adhesive, thermoplastic polybutene adhesive, polyester acrylate adhesive, silicone rubber adhesive, Tuunol mixed adhesive, epoxy adhesive, cyanoacrylate adhesive, anaerobic polyester adhesive, polyhenraimidazole adhesive, polyimide adhesive, polyimide amide adhesive, Trenice adhesive, metalloxane adhesive Examples include adhesives, glue adhesives, casein adhesives, cellulose adhesives, viscose adhesives, etc., and these adhesives may be used alone or in combination of two or more. Among these adhesives, polyvinyl adhesives are most preferred because they are resistant to staining when dyed.

In addition, examples of the above-mentioned proteins include ovalbumin, milk albumin, myogen, serum albumin,
Albumins such as robin and leucosin, serum globulin, β-lactoglobulin, lysodeme, myonone, edesdin, insulin, fibrinogen, globulins such as thyroglobulin, soluble collagen, elastin, keratin, silk fibroin, spondin, gorgonin, histone, protamine , egg white, gelatin, peptone,
Vepdide, others, phosphoproteins, lipoproteins,
Examples include glycoproteins, and one or more of these
A mixture consisting of L alone or in another base material such as a polyhydric alcohol is used.

Further, in the method of the present invention, the transparent plate used is transparent and does not obstruct observation of the stratum corneum specimen, and is insoluble or sparingly soluble in the organic solvent. Specific examples include resins such as glass plates, polymethylbentene, and polyethylene, among which glass plates are most preferably used due to their transparency and solvent resistance.

In the method of the present invention, first, a tape coated with the above-mentioned adhesive substance is brought into close contact with the skin, and then a part of the stratum corneum can be collected on the tape by peeling it off. Next, this tape is attached to a transparent plate that has been previously coated with the above-mentioned adhesive. The important thing here is to apply a fixing agent to the transparent plate in advance. If this pretreatment is not done, the stratum corneum will be removed from the transparent plate during the steps such as peeling off the tape described below. It is unsuitable because it will be released. Also, when attaching a tape holding the stratum corneum to a transparent plate, the width of the tape should be 3 to 1 O.
It is preferable that it is about xx. If the tape is too wide, it has poor permeability to organic solvents and is inconvenient to handle. Next, the transparent plate to which the above tape was pasted was immersed in the above-mentioned organic solvent for 1 to 3 hours to dissolve or swell a portion of the adhesive substance adhering to the tape, thereby increasing the releasability of the tape. After that, carefully remove it from the transparent plate.

Furthermore, it is also suitable to continue immersion in an organic solvent for the purpose of removing residual sticky substances.

As described above, the method of the present invention is characterized in that the stratum corneum collected on a tape is transferred to a transparent plate and used as a specimen.

After the stratum corneum is transferred to a transparent plate, operations such as staining and balm encapsulation are usually performed using conventional methods, and this is used as a stratum corneum specimen.

〔Example〕

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

<Example-1> Paste cellophane tape (cut commercially available adhesive cellophane tape and use it) with a width of 12 m x length of 20 mm on the cheek, press it for a while, then gently peel it off and use scissors. It has a width of approximately 6 mm and a length of 20 xi, and is attached to a glass slide that has been uniformly coated with polyvinyl acetate adhesive.

Next, this glass slide is immersed in xylene for 2 hours. Next, after gently peeling off the tape using a bottle set, the sample was further immersed in xylene for 1 hour.

After that, take it out, dry the xylene, and then use the gentian violet-brilliant green staining solution (1 g of gentian violet and 0 g of brilliant green).
．． 5g dissolved in distilled water 1009)
After immersing for 0 minutes, washing with water, drying, and enclosing in balm using a conventional method, a stratum corneum specimen was obtained.

The obtained stratum corneum specimen is an unprecedentedly clear specimen of the stratum corneum, and as shown in Figure 1, stratum corneum lines and follicles can be clearly observed, and the state of normal or abnormal keratinization can be observed in detail. Since it can be observed, it is possible to examine skin metabolism, the degree of aging, skin condition, etc.

<Example-2> A piece of cellophane tape with a width of 12cm and a length of '2Qmz' (used by cutting commercially available adhesive cellophane tape) was pasted on the cheek, pressed for a while, then gently peeled off and cut with scissors. It is made to have a width of approximately 6 ffJv and a length of 20 zR, and is attached to a slide glass that has been uniformly coated with a polyvinyl acetate adhesive in advance.

Next, this glass slide is immersed in xylene for 2 hours, and then the tape is gently removed using a bottle set, and then immersed in xylene for an additional 1 hour. Next, take it out, dry the xylene, and then add gentian violet-brilliant green staining solution (1 g of gentian violet and 0.59 g of brilliant green to 1 g of distilled water.
009) for 5 to 10 minutes, washed with water, dried, and then sealed in balsam by a conventional method to obtain a stratum corneum specimen.

In the obtained stratum corneum specimen, stratum corneum cells were clearly observed as in Example 1, and skin metabolism, degree of aging, skin condition, etc. could be investigated.

<Example 3> Paste cellophane tape (cut commercially available adhesive cellophane tape and use it) with a width of 12 mm and a length of 20 mm onto the cheek, press it for a while, then gently peel it off and use scissors. The sample was made to have a width of approximately 611 cm and a length of 20 x m, and was gently pasted onto a slide glass to which a mixture of ovalbumin:glycerin in a weight ratio of 1:1 was evenly coated in advance, and left at a temperature of 60°C for 2 hours.

Next, this glass slide was immersed in ethanol for 10 minutes, dried, and further immersed in xylene for 2 hours.
Gently remove the tape using a bottle set and then soak in xylene for an additional hour. Next, after taking it out and drying the xylene, gentian violet-brilliant green staining solution (gentian violet I
g and Brilliant Green 0.59 in distilled water 100g
After 5 to 10 minutes of washing and drying, the sample was encapsulated in balsam using a conventional method to obtain a stratum corneum sample.

In the obtained stratum corneum specimen, stratum corneum cells were clearly observed as in Example 1, and skin metabolism, degree of aging, skin condition, etc. could be investigated.

<Example-4> Gum arabic on paper tape with width LOmx and length 20mm.
Apply an adhesive substance containing Lagant rubber, etc. evenly, apply adhesive tape to the cheek, press it down for a while, then gently peel it off and use scissors to remove the tape approximately 6 cm in width.
The length is 20 mm, and this is attached to a glass slide that has been uniformly coated with cellulose adhesive in advance.

Next, the slide glass is immersed in acetone for 2 hours, the tape is gently removed using a bottle set, and then the glass slide is further soaked in xylene for 1 hour. Next, the pieces were taken out, dried with xylene, and then stained with hematoxylin and eosin. After drying, they were sealed in balsam using a conventional method to obtain a stratum corneum specimen.

In the obtained stratum corneum specimen, stratum corneum cells were clearly observed as in Example 1, and skin metabolism, degree of aging, skin condition, etc. could be investigated.

<Example-5> A plastic tape with a width LOx* and a length of 20 ytm evenly coated with an adhesive substance containing natural rubber was pasted on the inner side of the upper arm, and gently peeled off.
Thereafter, it was treated in the same manner as in Example 1 and stained with Giemsa to obtain a stratum corneum specimen.

In the obtained stratum corneum specimen, stratum corneum cells were clearly observed as in Examples 1 to 1 above, and skin metabolism, degree of aging, skin condition, etc. could be investigated.

<Example-6> Width 1011 m, length 20 H with uniformly distributed pores
Cellophane tape (commercially available adhesive cellophane tape was processed and used) was applied to the outer part of the upper arm, gently peeled off, and treated in the same manner as in Example 2 to obtain a stratum corneum specimen.

In the obtained stratum corneum specimen, stratum corneum cells were clearly observed as in Example 2, and skin metabolism, degree of aging, skin condition, etc. could be investigated.

〔Effect of the invention〕

By configuring the present invention as described above, it is possible to create specimens that are clear, easy to observe, and have good storage stability.

[Brief explanation of the drawing]

FIG. 1 is an optical micrograph (approximately 700x magnification) of a stratum corneum specimen obtained by the method of the present invention (Example 1). Patent applicant: Pola Chemical Industries, Ltd. Figure 1

Claims (5)

[Claims] (1) A tape coated with an adhesive substance soluble in an organic solvent and with the stratum corneum of the skin attached to this adhesive substance is attached to a transparent plate coated with an adhesive in advance, and then The stratum corneum is immersed in an organic solvent, and then the tape is removed to substantially fix a part of the stratum corneum on a transparent plate, and if necessary, this is stained to prepare a specimen of the stratum corneum. How to prepare specimens. (2) The adhesive substance is a rubber material such as natural rubber or its derivatives, SBR rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber, chloroprene rubber, recycled rubber, cyanoacrylate rubber, gum arabic, rubber tragacanth, etc. 2. The method for preparing a stratum corneum specimen according to claim 1, wherein the method is one or a mixture of two or more selected from the following. (3) The method for preparing a stratum corneum specimen 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 one 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. 2. The method for preparing a stratum corneum specimen according to claim 1, wherein the stratum corneum specimen is contained in a base material. (5) The method for preparing a stratum corneum specimen according to any one of claims 1 to 4, wherein the transparent plate is a glass plate.