JP3667530B2 - How to make stratum corneum - Google Patents

Description

[0001]
[Technical scope to which the invention belongs]
The present invention relates to a method for preparing a specimen for observing the skin such as the stratum corneum that is useful for skin diagnosis / analysis / differentiation and the like, and a specimen preparation kit useful for the preparation thereof.
[0002]
[Prior art]
A specimen for microscopic observation of the skin such as the stratum corneum is very important for knowing the skin state of a living body having the skin. For this reason, the method of creating these specimens has been devised. For example, the stratum corneum is collected with a film coated with an adhesive, and the film is transferred to a slide glass, and then the film is removed or attached with an adhesive. A transparent film is applied to the skin and the film is directly dyed to prepare a specimen, or the skin is observed under a stereomicroscope. Also, there has been a method in which an adhesive tape is applied to the skin, this is transferred onto another adhesive tape, and this transferred product is used as a stratum corneum specimen. The skin information obtained in this way is applied to preparation of a sample for counseling of cosmetics if the living body is a human, and such skin information makes it possible to select appropriate cosmetics. This is obvious when you go to the counter of the cosmetics department of a recent department store. However, the preparation of such a skin specimen has a problem that it takes a lot of time and manpower from the specimen collection to the preparation of the specimen and the end of observation by any method. The reason for this is that the former uses softening with an organic solvent for the film removal operation, which requires a lot of time and manpower, and because there is too much information in the specimen, this is organized and accurately identified. In order to achieve this, it is necessary to have knowledge and experience. In direct dyeing of films and dyeing of transferred materials from adhesive tape, a more stratum corneum sample that is more beautiful than the problem of application efficiency during sampling and transfer. Are difficult to obtain, a large amount of skin keratinocytes remain in the adhesive on the collection side, and it is impossible to observe these keratinocytes. Labor saving these is preferable in that skin information can be provided promptly, inexpensively and accurately, and the advent of such a technology has been awaited.
[0003]
[Problems to be solved by the invention]
The present invention has been made under such circumstances, and it is an object of the present invention to provide means for saving information quickly and saving information on living body skin.
[0004]
[Means for solving problems]
In view of the above situation, the present inventors have sought a means for saving and quickly obtaining information on the skin of a living body, and have taken the following two approaches.
1) To improve the conventional sampling method so that more useful information can be easily obtained. That is, since the conventional method is a sample in which unnecessary information is mixed, it is considered that it took time to analyze the information, and this unnecessary information is removed.
2) Reduce time and save labor by improving methods and methods of combination such as dyeing.
As a result, after applying the adhesive-applied disc to the skin to be collected, it is peeled off, and the adhesive part is made of a transparent material and the adhesive of the specimen material to which the adhesive is applied at least on one side By attaching the sample to be peeled off and transferring it to the specimen material, it is possible to shorten the time of 1) and save labor. Even specimens that could not be stained by conventional sampling can be stained even under the time-saving and labor-saving conditions studied in 2), and because it is a beautiful one-layer sample, it can be observed easily and appropriately. The discovery and the invention were completed. Furthermore, in the present invention, there is an advantage that the disk used and the specimen holder itself coated with the adhesive to which the keratinocytes are transferred can be used as a specimen for observation. Hereinafter, the present invention will be described in detail with a focus on embodiments.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
(1) Preparation method of the specimen of the present invention The preparation method of the specimen of the present invention is the method of preparing the specimen of the present invention, after sticking the adhesive part of the disc coated with the adhesive on at least one side to the skin of the sample to be collected, The material to be observed is peeled off from the skin and transferred to the specimen material. To do. Here, the disk is a thin plate material that exhibits some formability but exhibits shape maintenance against normal stress, and has a shape without corners, that is, a shape such as a circle or an ellipse. Means the material. Here, transfer means physical movement of a part or all of a sample such as a stratum corneum in the present invention. By performing such an operation, it is possible to collect a beautiful single stratum corneum sample. By making the stratum corneum one layer, it became possible to clearly observe each corneocyte one by one in the conventional method, which overlapped and could only see multiple perspective images of the stratum corneum. In addition, since the sample is a thin layer of keratinocytes, it is possible to detect the failure of the melanin distribution that causes darkness and spots. Furthermore, dyeing can be easily performed as compared with conventional sampling methods. Therefore, it is possible to increase the accuracy of discrimination of observations and cells, and to greatly shorten the specimen preparation time. Also, the adhesive part of the specimen material, which is a transparent material and coated with an adhesive, is applied to the skin to be collected and peeled off, and the adhesive is applied to the skin specimen adhering to the specimen material on at least one side. By applying and peeling the applied film, the method of removing the excess skin specimen and using it as a sample also increases the accuracy of observation and differentiation of cells, etc., as described above, and greatly shortens the specimen preparation time. Although this method also belongs to the technical scope of the present invention, the adhesive portion of the specimen material coated with at least one side of the adhesive, which was first described in terms of ease of identification and accuracy in the subsequent operation, is to be collected. The adhesive part is peeled off after being applied to the skin, and the adhesive part is attached to and peeled off from the adhesive part of the disc made of a transparent material and coated with an adhesive on at least one side. Use specimens as specimen materials Better way to copy is preferable. That is, the sample preparation method of the present invention is roughly represented by the following two types.
1) After sticking the adhesive part of the disc on which the adhesive is applied on at least one side to the skin to be collected, it is peeled off, and the adhesive part is made of a transparent material and the adhesive is applied on at least one side. Method of transferring the specimen to be observed on the skin peeled off from the skin by applying it to the adhesive part of the specimen material and transferring it to the specimen material (first method)
2) The adhesive part of the specimen material, which is a transparent material and coated with an adhesive, is applied to the skin to be collected and peeled off, and the adhesive is applied to the skin specimen adhering to the specimen material on at least one side. A method of removing excess skin specimens and applying samples by applying and peeling off the applied film (second method)
Since the specimen prepared by the specimen preparation method of the present invention is a beautiful single-layered stratum corneum sample that does not have an excess portion of the skin, it has better staining than the specimen prepared by the conventional sampling method. It can be enough staining at a concentration of about one-tenth of the staining solution, preferably for use with such a stain how. In the present invention, the following equipment can be combined to form a kit. The kit of the present invention comprises a specimen material having a specimen holding part that is transparent and coated with an adhesive, and a skin specimen collecting disk that is coated with an adhesive on at least one side. Sample preparation for skin observation, comprising a sample preparation kit and a sample material having a specimen holding part that is transparent and coated with an adhesive, and an adhesive-coated film coated with an adhesive on at least one side The kit can be preferably exemplified. The specimen preparation method and kit of the present invention are particularly suitable for preparing a specimen of the stratum corneum in the skin.
[0006]
(2) Equipment used in the specimen preparation method of the present invention The equipment used in the first method will be described below.
(Disk with adhesive applied on at least one side)
As a requirement of the disc that can be used, it is necessary to maintain the shape while maintaining flexibility, and to apply the adhesive uniformly. In the first method, since the disc is used only for collecting skin samples, the disc itself is not required to be transparent. Of course, it is possible to be transparent, but it is important to have shape maintenance while having flexibility so that the sampling of the skin can be performed efficiently. In this method, the stratum corneum can be collected by pressing the skin and deforming the skin somewhat to collect the stratum corneum. It is also necessary to have a strength that can be easily peeled off from the skin. Examples of such disk materials include lined cardboard, polyvinyl chloride plate, polyethylene terephthalate, acrylic acid ester, methacrylic acid ester, polymer thin plate such as cellulose derivatives such as celluloid and acetyl cellulose, and the like. A composite material etc. can be illustrated suitably. Among these, a cardboard or polyethylene terephthalate thin plate lined with polyethylene is preferable from the viewpoint of cost. The pressure-sensitive adhesive can be used without particular limitation as long as it is usually used as a pressure-sensitive adhesive for skin. For example, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, Rubbers such as vinylidene chloride, polyvinyl ether, acrylic, natural, SBR, NBR, butadiene-vinylpyridine, butyl, regenerated, cyanoacrylate and chloroprene and derivatives thereof, polysaccharides such as tragacanth gum and gum arabic A pressure-sensitive adhesive or the like can be preferably exemplified. In the disk coated with these adhesives, the adhesive part can be covered with release paper such as silicone-impregnated paper to prevent the adhesive force from being reduced by dust or the like. Further, the shape is preferably one that does not easily cause irritation when applied and has no corners. Specifically, a circular or elliptical shape can be preferably exemplified. Furthermore, it is preferable in terms of usability to have a small protrusion for peeling.
(Sample material)
As a sample material that is a transparent material and has an adhesive applied on at least one side, a hole is provided in the center of a structural material such as polypropylene, and a transparent film in which the adhesive is uniformly applied is provided here. Examples include a type to be affixed, a type in which an adhesive is applied to a transparent plate-like material, and a portion for transferring the skin material is provided. As a material for such a film or transparent plate, transparency is required, and polystyrene, polyethylene terephthalate, polyester, polyacrylic acid ester, polymethacrylic acid ester, tetron and the like can be preferably exemplified. Of these, tetron is particularly preferred from the balance of flexibility and form maintenance. Moreover, as a material of the structural material provided with a hole in the center, polymer materials such as glass, wood, polyvinyl chloride, polystyrene, and polypropylene can be preferably exemplified. These need not be transparent, but may be transparent. Examples of pressure-sensitive adhesives applied to such films and transparent plates include polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, acrylic, natural, SBR, NBR, and butadiene-vinyl. Preferred examples include rubbers such as pyridine-based, butyl-based, regenerated, cyanoacrylate-based and chloroprene-based rubbers and derivatives thereof, polysaccharide adhesives such as tragacanth gum and gum arabic. When applying these, care must be taken that the solvent used does not damage the film material or the transparent plate. Further, the adhesive application part can be covered with release paper such as silicone-impregnated paper to prevent the adhesive force from being reduced by dust or the like. The adhesive force of the adhesive of this specimen material needs to be stronger than the adhesive force of the skin sample collecting film.
[0007]
Below is a description of the equipment used in the second method.
(Sample material)
As a sample material that is a transparent material and has an adhesive applied on at least one side, a hole is provided in the center of a structural material such as polypropylene, and a transparent film in which the adhesive is uniformly applied is provided here. Examples include a type to be affixed, a type in which an adhesive is applied to a transparent plate-like material, and a part for collecting skin material is provided. As a material for such a film or transparent plate, transparency and the ability to collect skin samples are required, and some flexibility is required, such as tetron, polystyrene, polyethylene terephthalate, polyester, polyacrylate, polymethacrylate, etc. Can be preferably exemplified. Among these, Tetron is particularly preferable from the balance of transparency, flexibility, and shape maintenance. Moreover, as a material of the structural material provided with a hole in the center, polymer materials such as glass, wood, polyvinyl chloride, polystyrene, and polypropylene can be preferably exemplified. These need not be transparent, but may be transparent. Examples of pressure-sensitive adhesives applied to such films and transparent plates include polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, acrylic, natural, SBR, NBR, and butadiene-vinyl. Preferred examples include rubbers such as pyridine-based, butyl-based, regenerated, cyanoacrylate-based and chloroprene-based rubbers and derivatives thereof, polysaccharide adhesives such as tragacanth gum and gum arabic. In terms of ease of use, a transparent and flexible adhesive-coated film is used alone for collecting skin samples, and this is affixed to a structural material with a hole in the center. It is preferable to remove the sticking. When applying these, care must be taken that the solvent used does not damage the film material or the transparent plate. Further, the adhesive application part can be covered with release paper such as silicone-impregnated paper to prevent the adhesive force from being reduced by dust or the like.
(Adhesive coated film)
The requirement of the film that can be used is that it has flexibility and allows uniform application of the pressure-sensitive adhesive. In the second method, the film itself is used only to remove excess portions of the skin sample, so the film itself does not require transparency. Of course, it is possible to be transparent, but it is important to have flexibility so that it can be in close contact with the adhesive portion of the specimen material so that the skin sample can be adjusted efficiently. It is also necessary to have an appropriate adhesive strength so as not to remove the skin sample too much, that is, an adhesive strength slightly weaker than the adhesive strength of the specimen material. As such a film material, for example, a non-woven fabric which may be lined, paper or cloth, a polymer film such as polyester, polyethylene terephthalate, acrylic acid ester or methacrylic acid ester, or a composite material thereof can be suitably exemplified. . Among these, preferred is a paper lined with polyethylene from the viewpoint of cost. The pressure-sensitive adhesive can be used without particular limitation as long as it is usually used as a pressure-sensitive adhesive. For example, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride. , Polyvinyl ether, acrylic, natural, SBR, NBR, butadiene-vinylpyridine, butyl, regenerated, cyanoacrylate and chloroprene rubbers and their derivatives, polysaccharide adhesives such as tragacanth gum and gum arabic Etc. can be preferably exemplified. In the film coated with these adhesives, the adhesive part can be covered with release paper such as silicone-impregnated paper, and the adhesive force can be prevented from being reduced by dust or the like.
[0008]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but it is needless to say that the present invention is not limited to these examples.
[0009]
<Example 1>
Prior to the implementation of Example 1, the following were prepared.
1. As shown in FIG. 1, a specimen material 1 provided with a cut portion (hole) 2 in the center and a transparent tape 4 coated with a transparent adhesive 3 on the entire surface of one side is used (a taper tape manufactured by Sumitomo 3M is used as the transparent tape 4) The specimen material is made of polypropylene.
2. As a skin sample collecting disk, a double-sided tape (1 inch wide) cut into a length of 2.5 cm was affixed to polyethylene terephthalate and punched out with a punch having a diameter of 1.5 cm to obtain a disk.
Using the above material, first, this disc was applied to the cheek and pressed with a finger twice, and then peeled off. The adhesive portion of this disc appeared at the excised portion of the specimen material 1 to which the transparent tape 4 was attached. The sample was applied to the adhesive portion of the transparent tape 4 and peeled off, and the skin sample was transferred onto the transparent tape to obtain a specimen. This sample was prepared by using a silver staining solution in which ammonia water was added dropwise to a 0.5% silver nitrate aqueous solution to adjust the pH to 10, treated at 45 ° C. for 10 minutes, silver-stained, washed with water three times, and 0.8% gentian violet. The solution was treated for 10 minutes. The specimen was washed with water and dried, and a transparent silicone agent was poured so as to cover the dyed portion, and a cover glass was put on to prepare a sample. This micrograph is shown in FIG.
As Comparative Example 1, a skin sample was collected by applying a tapia tape to the cheek, and this was applied to a specimen material provided with a cut portion at the center, followed by staining in the same manner as in Example 1 to obtain Comparative Example 1. This micrograph is shown in FIG.
As Comparative Example 2, a skin sample was collected by applying a tapia tape to the cheek, and this was applied and transferred onto an adhesive tape with a specimen material having a cut portion provided in the center, and transferred according to the conventional method. It dye | stained and was set as the comparative example 2. That is, ammonia water was added to a 5% silver nitrate aqueous solution to adjust the pH to 10, and this was immersed in this at room temperature for one day and night, silver staining was performed, and washing with water was repeated 10 times. Thereafter, the sample was immersed in a 10% gentian violet solution for 10 minutes, washed with water, filled with transparent silicone, and covered with a cover glass to obtain a specimen. This micrograph is shown in FIG.
As can be seen from FIGS. 2 to 4, the specimen prepared by the specimen preparation method of the present invention can stain melanin clearly in a short time. Furthermore, it is easy to analyze a microscopic image. This is because the analysis of the microscopic image is not hindered by the overlap of cells and staining. Furthermore, the dyeing time could be greatly reduced from 14 hours to 60 minutes.
[0010]
< Comparative Example 3 > Prior to the implementation of Comparative Example 3 , the following were prepared.
1. A specimen material 5 (made of polypropylene) provided with a cut portion (hole) 6 in the center as shown in FIG.
2. As a skin sample collection disk, Sumitomo 3M tapia tape.
3. As a skin sample adjustment tape, Sumitomo 3M surgical tape (1 inch wide) cut to 2.5 cm in length.
A tapia tape was affixed to the cheek, and it was peeled by pressing twice with a finger. This was affixed to the specimen material 5 so as to block the excised part 6. A specimen was obtained by applying the surgical tape to the adhesive part of the tape and removing it twice. This sample was prepared by using a silver staining solution in which ammonia water was added dropwise to a 0.5% silver nitrate aqueous solution and the pH was adjusted to 10. The sample was treated with silver staining at 45 ° C. for 10 minutes, washed with water three times, and then washed with 0.8% gentian violet. The solution was treated for 10 minutes. The specimen was washed with water and dried, and a transparent silicone agent was poured so as to cover the dyed portion, and a cover glass was put on to prepare a sample. Although this did not reach Example 1, it was a very clear microscope specimen.
[0011]
< Comparative Example 4 > Prior to the implementation of Comparative Example 4 , the following were prepared.
1. As shown in FIG. 6, a specimen material 7 (made of polyethylene terephthalate, the pressure-sensitive adhesive is a polyacrylic ester) provided with a pressure-sensitive adhesive application portion 9 having a pressure-sensitive adhesive 8 applied at the center.
2. Surgical 3M surgical tape (1 inch wide) cut into 2.5 cm length as an adjustment tape for skin samples.
The adhesive-applied portion 9 of the specimen material 7 was pressed against the cheek and peeled off. Surgical tape was affixed to the adhesive portion and peeled off, and this operation was performed twice. When the specimen material was stained in the same manner as in Example 2, a clear specimen was obtained, which was not as good as Example 1.
[0012]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the means which saves the labor information of a biological body quickly and can be provided can be provided.
[Brief description of the drawings]
1 is a view showing a specimen material used in Example 1. FIG.
FIG. 2 is a photomicrograph of the specimen prepared in Example 1. (Drawing substitute photo)
3 is a photomicrograph of a specimen prepared in Comparative Example 1. FIG. (Drawing substitute photo)
4 is a photomicrograph of the specimen prepared in Comparative Example 2. FIG. (Drawing substitute photo)
5 is a view showing a specimen material used in Example 2. FIG.
6 is a view showing a specimen material used in Example 3. FIG.

Claims (6)

For specimens that have been peeled off after being applied to the skin to be collected, and the adhesive part of the disc with adhesive applied to at least one side is made of a transparent material and has adhesive applied to at least one side A method for preparing a specimen for observing skin, characterized in that it is applied to an adhesive part of a material and peeled off, and a specimen to be observed of the skin peeled off from the skin is transferred to the specimen material,
The disc is coated with an adhesive on at least one side of a thin sheet of a cardboard, polyvinyl chloride plate, polyethylene terephthalate, acrylic acid ester, methacrylic acid ester, celluloid, acetylcellulose or a composite material which may be lined. A method of creating a specimen that is a disk 2. The method for preparing a specimen for observing a skin according to claim 1, wherein the disc is a disc formed by applying an adhesive to at least one side of a polyethylene-lined cardboard or a polyethylene terephthalate thin plate. . The method for preparing a specimen for observing skin according to claim 1 or 2 , characterized in that a stratum corneum is included in the specimen to be observed on the skin. Wherein the adhesive strength of the disc of the pressure-sensitive adhesive coated on at least one side of the adhesive for collection is less than the adhesive force of the adhesive agent applied to the specimen material, in any one of claims 1 to 3 A method for preparing a specimen for observing the skin as described. A specimen preparation kit for observing the skin, consisting of a specimen material having a specimen holding part that is transparent and coated with an adhesive, and a disk for collecting a skin specimen coated with an adhesive on at least one side There,
The disc is coated with an adhesive on at least one side of a cardboard, polyvinyl chloride plate, or polyethylene terephthalate, acrylic ester, methacrylic ester, celluloid, acetyl cellulose, or a composite material that may be lined. A kit for specimen preparation, which is a disc. The adhesive for skin observation according to claim 5 , wherein the adhesive applied to the specimen holding portion of the specimen material is stronger than the adhesive applied to the disk for collecting the skin specimen. Sample preparation kit.

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