JPH07209292A - Method for measuring extent of damage on cuticle cell of skin - Google Patents

Abstract

PURPOSE:To realize a noninvasive measurement of the extent of damage on the skin by sticking a cuticle cell of skin to a tape coated with a viscous substance, stripping the tape using a solvent and bonding the cuticle cell to a color plate and then observing the cuticle cell through a microscope. CONSTITUTION:A tape is coated with a viscous substance soluble to organic solvent, e.g. polyvinylacetate, polyvinylalcohol or polyvinylacetal. The tape is applied, on the viscous side thereof, to a skin and then it is peeled off thus sampling a cuticle cell. The tape is then applied, on the viscous side thereof, to a color plate coated with a polyvinyl-based, melamine resin-based or phenol resin-based bond. Subsequently, the viscous substance is dissolved using an organic solvent, e.g. methanol or ethanol, and the tape is peeled off. The cuticle cell is fixed to the color plate and dyed thus preparing a sample which is observed through a microscope thus measuring the extent of damage on the skin noninvasively.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the degree of damage to skin keratinocytes, and more particularly to measuring the degree of damage to skin surface keratinocytes in order to ensure the diagnosis of patients with atopic dermatitis. It is about the method.

[0002]

BACKGROUND OF THE INVENTION Atopic dermatitis is a skin disease whose main symptoms are itching, skin inflammation, and a feeling of dryness of the skin, and it usually develops from infancy and is often cured by childhood. It is a skin disease that has become more chronic and has not been cured even in adulthood in recent years. The fundamental treatment method has not been established, and most of them rely on symptomatic treatment.
The onset of this disease is allergic factors due to diet (rice, milk, eggs, etc.) and environmental factors (house dust, mites, molds, etc.), changes in the living environment (dry conditions, etc.), and skin damage due to scratches, etc. It is said that the factors that impair the barrier function are deeply involved.

As a method for testing the above-mentioned allergenic factors, regarding allergenicity, the method of measuring the IgE antibody level in serum or the method of contacting allergen directly on the skin, such as dropping, subcutaneous injection and sticking, and then contacting The method of measuring the skin reaction of is performed.

However, it is not clear whether the allergens selected by these methods are directly associated with the development of atopic dermatitis. At present, even if food allergens such as eggs and milk that have a high possibility of being selected are discovered, it is not easy to maintain a diet from which they are therapeutically removed, including nutritional problems. In addition, IgE antibody in blood is
It is considered to be a rough indication of the allergic strength of the patient, and even if the IgE antibody titer is high, atopic dermatitis may be completely cured, or if the antibody titer is not detected at all, inflammation of the skin may continue. is there. Therefore, therapeutically,
Too much sticking to antibody titers can cause nutritional and other problems.

On the other hand, regarding the reduction of the barrier function of the skin (degree of damage), (a) measurement of the amount of sebum, (b) measurement of transepidermal water loss (TWL), (c) measurement of the stratum corneum water content, etc. Is known, but it is difficult to use it in general medical care because it requires a dedicated device to set the measurement conditions quite strictly and takes a long time to measure. . In addition, (d) there is also a method of directly excising the skin to perform a histopathological examination, but due to the inability to evaluate the painful point and the state of the stratum corneum of the skin, most of the diagnosis by skin examination has been performed to date. Not done.

By the way, as a method for inspecting the stratum corneum of the skin, an adhesive substance soluble in an organic solvent is applied, and a tape having the stratum corneum of the skin adhered to this adhesive substance is applied with a fixing agent in advance. It was prepared by sticking to a transparent plate, then immersing it in an organic solvent, and then removing the tape to fix a part of the stratum corneum on the transparent plate, and dyeing it if necessary. The present applicant has disclosed a method for examining the condition of the stratum corneum using a stratum corneum specimen (Japanese Patent Laid-Open No. 63-113358). According to this method, the condition of the skin, the skin quality, the skin quality, the degree of aging of the skin, and the like can be inspected, but it is difficult to determine the deterioration (damage level) of the barrier function of the skin.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoint, and in order to make it possible to diagnose the condition of the skin of a patient with atopic dermatitis while judging the therapeutic effect, An object of the present invention is to provide a method for easily and non-invasively measuring a damage level.

[0008]

Means for Solving the Problems As a result of intensive studies aimed at solving the above problems, the present inventor has found that in order to easily and non-invasively measure the degree of skin damage, the keratin of the outermost layer of the skin can be easily measured. It was considered best to observe the cells histopathologically or morphologically. Then, the keratinocytes of the skin are peeled off and held on a transparent plate or a colored resin plate, and the keratinocytes are appropriately stained or fluorescently stained, observed with an optical microscope or a scanning electron microscope, and the keratinocytes are hypokeratotic, To evaluate histopathologically or morphologically the abnormalities of cell detachment, the presence of polysaccharides on the cell surface, the abnormal formation of the stratum corneum during the keratinization process, and the morphological abnormalities on the body surface side and back surface of keratinocytes. As a result, they have found that the degree of skin damage can be measured, and completed the present invention.

That is, according to the present invention, skin keratinocytes are attached to a tape coated with an organic solvent-soluble adhesive substance, and the adhesive surface of the tape is brought into contact with a colored plate coated with an adhesive agent in advance. Affixing, fixing the keratinocytes to the coloring plate by peeling the tape from the coloring plate by dissolving the adhesive substance with the organic solvent, and observing the morphological characteristics of the keratinocytes with a microscope using the obtained keratinocyte preparation. A method for measuring the degree of damage to skin keratinocytes, which is characterized in that

Further, as one aspect of the present invention, in the above-mentioned measuring method, the keratinocyte specimen is microscopically observed from the front side of the specimen, and further, the tape on which the skin keratinocytes are adhered is microscopically observed from the back side. A method of measuring the degree of damage to skin keratinocytes, further, sticking skin keratinocytes to a tape coated with an adhesive substance soluble in an organic solvent, and sticking this tape to a transparent plate pre-coated with an adhesive agent The tape is peeled off from the transparent plate by adhering them in contact with each other and dissolving the adhesive substance with the organic solvent to fix the corneocytes to the transparent plate, which is then stained with HE, PAS, D
Provided is a method for measuring the degree of damage of skin keratinocytes, which comprises observing a skin keratinocyte stained sample obtained by staining by a method selected from ACM staining with a microscope.

In summary, the measuring method of the present invention is a method of observing a keratinocyte sample (hereinafter referred to as "surface sample") prepared by using a colored plate from the front side (body surface side), and further using an adhesive surface-attached tape. A method of observing the collected keratinocytes (hereinafter referred to as "rear surface sample") from the back side, and in addition to these, a sample obtained by staining keratinocytes on a transparent plate (hereinafter referred to as "stained sample") It is a method of observing.

In the present specification, the degree of skin damage refers to the degree of reduction in skin barrier function, and specifically, the appearance of nucleated cells, the presence of multi-layered cell groups, and the appearance of PAS-positive cells. , The fluorescent intensity of SH groups on the surface of the corneal cell membrane, the appearance of sponge-like structures on the surface of the stratum corneum, and the fine villus-like projections on the rear surface of the stratum corneum, etc.

The present invention will be described in detail below.

<1> Corneal Cell Specimen Used in the Method for Measuring the Degree of Damage to Skin Keratinocytes of the Present Invention (1) Surface Specimen Surface specimens are sponge-like structures, groove-like structures and cells in which holes or wrinkles are observed on the cell surface. This is a sample for observing surface irregularities and the like.

The tape coated with the adhesive substance used in the present invention is used for pressing the adhesive surface of the tape against the skin and peeling the tape from the skin to collect the stratum corneum. As the adhesive substance, a resin or polymer compound soluble in an organic solvent is used, and examples thereof include polyvinyl acetate, 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, cyanoacrylate rubber, gum arabic, tragacanth rubber and the like. They are,
A single type or a mixture of two or more types contained alone or in another substrate is used.

Among the above-mentioned adhesive substances, natural rubber or its derivative, SBR rubber, NBR rubber, butadiene-vinylpyridine rubber, butyl rubber, chloroprene rubber, recycled rubber, cyanoacrylate rubber, arabic rubber, tragacanth rubber. A rubber substance such as is preferable because it does not change the peeling property when the tape is peeled off and keratinocytes are not changed. The tape may be one that is generally or industrially used, and examples of the material include plastic materials such as cellophane, polyethylene, polypropylene, polyvinyl chloride, polyester, and fluororesin.
As the shape, in addition to those having a uniform thickness, those having pores, those having a mesh shape, and the like can be used.

In the present invention, such a tape coated with the above-mentioned adhesive substance is used, but a commercially available adhesive tape such as cellophane tape can be used as it is.

The organic solvent used to dissolve the adhesive substance is 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 in the present invention is used for fixing the stratum corneum of the skin to a coloring plate or a transparent plate described later, which does not deform keratinocytes, and The ones that are not easily soluble in the organic solvents mentioned above are used. Examples of such an adhesive include known adhesives, proteins and the like, but known adhesives and the like are preferably used because the resulting keratinocyte specimen is clearer, and specific examples include: Polyvinyl acetate such as polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, melamine resin adhesive, phenol resin adhesive, resorcinol adhesive, xylene resin adhesive, furan resin adhesive Agent, polyisocyanate resin adhesive, polyester resin adhesive, polyamide adhesive, polybutene adhesive, ethylene copolymer adhesive, thermoplastic polyester adhesive, polyester acrylate adhesive, silicone rubber adhesive, phenol mixed adhesive Agent, epoxy adhesive, cyanoacrylate Adhesives, anaerobic polyester adhesives, polybenzimidazole adhesives, polyimide adhesives, metalloxane adhesives, glue adhesives, casein adhesives, cellulose adhesives, and the like. One kind or a mixture of two or more kinds is used.

Among these adhesives, polyvinyl chloride adhesives are most preferable because they are less likely to be dyed when dyed. Examples of the above-mentioned protein include albumins such as ovalbumin, milk albumin, myogen, serum albumin, globin, and leucosine,
Serum globulin, β-lactoglobulin, lysozyme,
Globulins such as myosin, edestin, insulin, fibrinogen, thyroglobulin, soluble collagen, elastin, keratin, silk fibroin, sponge, gorgonin, histone, protamine, egg white, gelatin, peptone, peptide, others, phosphoprotein, lipoprotein, sugar Protein etc. are mentioned, and these 1
One kind or a mixture of two or more kinds is used alone or contained in another base material such as polyhydric alcohol.

The colored plate used in the present invention is necessary for obtaining a specimen for observing morphological abnormalities on the surface of skin cells, and is colored black, green, blue, etc., and easily scissored. It can be cut and is insoluble or hardly soluble in organic solvents. The reason why the resin plate needs to be colored is that the transferred keratinocytes are clearly confirmed before the preparation of the sample. With a transparent plate, it is difficult to clearly distinguish white keratinocytes, and it cannot be fixed on a sample stage as a scanning microscope specimen. Since there is a possibility that a scanning electron microscope specimen may be prepared with the surface on which the keratinocytes are not attached by mistake, it is preferable that the surface is colored. Specific examples thereof include plastic plates or resin plates such as polymethylpentene and polyethylene. Among them, fixed keratinocytes can be visually discerned, and a black plastic plate is most preferable from the viewpoints of cutting property and solvent resistance. Used.

Next, a method for producing a surface specimen using the above materials will be described. First, press the adhesive-coated surface of the tape against the skin, press the surface of the tape that is not coated with the adhesive material with your finger and rub it gently 2 or 3 times to bring the tape into close contact with the skin. , Peel the tape from a certain direction with a certain force. Next, this tape is attached to a colored plate to which the above-mentioned adhesive has been applied in advance. At this time, if the adhesive is not applied to the colored plate in advance, the stratum corneum is separated from the colored plate in the step of peeling the tape described below. Further, when the tape holding the stratum corneum is attached to the colored plate, it is preferable to cut the tape into tape widths of about 3 to 10 mm.

If the tape width is too wide, the permeability of the organic solvent is poor and it is inconvenient to handle. In addition, if you prepare a holder with a window hole with tape attached to correspond to this window hole, you can make the tape adhere to the skin together with the holder, and further use the holder frame as a lever By doing so, the tape can be peeled off from a certain direction with a certain force, and a certain amount of a part of the stratum corneum can be constantly collected on the tape.

Next, the colored plate to which the above-mentioned tape is attached is dipped in the above-mentioned organic solvent for 2 to 4 hours to dissolve the adhesive substance adhering to the tape, and the tape is transparent and colored resin plate. It peels off more naturally. It is also suitable to immerse in a fresh organic solvent for the purpose of removing the remaining sticky substance after peeling. Then, the organic solvent is dried to obtain a surface specimen in which keratinocytes are fixed on the coloring plate.

When the specimen is observed with a scanning electron microscope, the colored plate on which the keratinocytes of the specimen are fixed is 1 cm ×
Cut it to a size of about 1 cm and cut it 1 cm in advance.
It is preferable to fix the sample on a scanning electron microscope sample stage to which a double-sided tape having a size of 1 cm or less is attached, and vapor-deposit gold.

(2) Backside Sample The backside sample is a sample for observing fine villus-like projections, irregular concave folds and villi, circular concave structures, and thin wrinkled ridges. This is the same as the above <1>, press the tape coated with the adhesive substance against the skin,
It can be obtained by pressing the surface of the tape which is not coated with the sticky substance with a finger and rubbing it lightly two or three times to bring the tape into close contact with the skin, and then peeling the tape from a certain direction with a certain force. When observing the back surface sample with a scanning electron microscope, it is preferable that the tape of the sample is cut into a size of about 1 cm × 1 cm, fixed on a scanning electron microscope sample stand with a double-sided tape or the like, and gold vapor deposition is performed.

(3) Stained specimen The stained specimen showed that parakeratosis of the keratinocytes and abnormal cell detachment, inflammation due to the trace of cell infiltration, conversion of SH groups to SS bonds on the cell membrane of keratinocytes. Is a sample for evaluating.

The stained sample can be obtained by fixing keratinocytes on the transparent plate and staining the keratinocytes in the same manner as the surface sample except that a transparent plate is used instead of the coloring plate. The transparent plate used here is transparent, does not interfere with the observation of the keratinocyte preparation, 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.

As the staining of keratinocytes fixed on a transparent plate, hematoxylin-eosin staining (HE) that stains the cytoplasm and the nucleus and PA that stains a polysaccharide showing a trace of cell infiltration
SCM, DACM ((N- (7-Dimethylamino-4-, to evaluate the degree of conversion of cell surface SH groups to SS bonds, which is involved in maturation of the horny layer during keratinization.
Methyl-3-coumarinyl) -maleimide) dyeing is most suitable. These can be evaluated alone, but it is preferable to perform two or three types of dyeing for comprehensive evaluation.

HE staining is not observed in healthy individuals,
Cells with nuclei (nucleated cells), which are often found in patients with atopic dermatitis, can be discriminated, and in healthy individuals, cells can be observed separately, but in patients with atopic dermatitis, cells overlap ( Can be observed. These observations provide information on parakeratosis and abnormalities of cell detachment.

PAS staining can stain neutrophils, mast cells, and the like, which are related to inflammation, and is hardly stained in healthy subjects, but in patients with atopic dermatitis, the cells become blue-purple as PAS-positive cells. Dye. These observations show evidence of cell infiltration, suggesting possible inflammation.

Further, it is known that the SH groups on the cell membrane are converted into SS bonds as the cells are keratinized. DACM staining shows that the SH groups and SS bonds are dyed by fluorescent staining, and SH It can be determined whether the conversion of the group into the SS bond is normally performed. This makes it possible to evaluate whether normal keratinization is performed. In healthy individuals, the SH group fluorescence was weak, and SS bond fluorescence was observed.
In the patient with atopic dermatitis, the fluorescence of SH group is quite strong, and the fluorescence of SS bond is observed as in the healthy person.

Specifically, after transferring the keratinocytes to a transparent plate, the cytoplasm and nucleus are stained with a staining solution such as HE, PAS or DACM, and balsam encapsulation is performed to obtain a stained specimen.

Observation of a specimen by visible light discrimination such as HE and PAS staining is carried out using an ordinary optical microscope, and a specimen stained fluorescently like DACM staining is observed using a fluorescence microscope. To do.

<2> Method for measuring the degree of damage to skin keratinocytes of the present invention The above surface specimen is observed using an optical microscope or preferably a scanning electron microscope, and a sponge-like structure, groove-like structure or cell surface By determining the presence or absence of irregularities, it is possible to determine the morphological change of cells. The cell surface of a healthy person has a smooth, sponge-like structure, groove-like structure, and cell surface irregularities, but in atopic dermatitis patients, the sponge-like structure, groove-like structure, cell surface irregularities are present. Etc. are strongly recognized.

Further, the back surface sample is similarly observed using an optical microscope or a scanning electron microscope to determine the presence or absence of fine villi protrusions, circular depressions, and wrinkle-like ridges, thereby morphologically changing the cells. Can be determined. The back surface of the cell surface of a healthy person is smooth, and there are almost no fine villiform protrusions, circular depressions, wrinkle-like protrusions, etc., but in atopic dermatitis patients, fine villiform protrusions, circular depressions,
Wrinkled ridges are strongly observed.

The morphological changes of cells peculiar to atopic dermatitis patients can be judged from the observation findings of only the surface preparation, but the cells are morphologically examined from both the front and back surfaces. This reveals the relationship between the cell surface structure and the cell back structure. For example, it can be explained that the cells or holes in the holes or wrinkles on the front surface and the fine villus-like projections on the back surface overlap each other. Also, it is possible to determine whether the overlap is orderly or disordered. Therefore,
From the observations on both sides, the damage degree of keratinocytes can be determined more accurately by examining the cell arrangement and the degree of stratification in detail.

Further, in addition to the above-mentioned observation findings, by adding the microscopic observation findings of the stained specimen, it becomes possible to more accurately determine the degree of damage of keratinocytes. Specifically, the stained specimen obtained is observed by an optical microscope, and the presence or absence of nucleated cells observed in keratinocytes, the presence or absence of multi-layered cells with stratified corneocyte detachment as an index, and the case of cell infiltration The presence or absence of parakeratosis and cell detachment, which is an indicator of abnormal keratinization, and the presence of cell infiltration, which is an indicator of inflammatory state, are examined from the presence or absence of PAS-positive cells using polysaccharides as an indicator. Furthermore, abnormal formation of keratinocytes in the keratinization process is examined based on the fluorescence intensity of SH groups and the presence or absence of fluorescence at the nucleus position, the fluorescence intensity of SS bonds, and the presence or absence of uneven patchy fluorescence observed in keratinocytes.

[0039]

EXAMPLES The present invention will be described in more detail below with reference to examples. Although the holder is used in this embodiment,
Even without using a holder, a sample can be similarly prepared and the degree of damage to keratinocytes can be measured.

[0040]

Example 1 A holder 1 (FIG. 1) having a rectangular shape of 7.6 cm × 2.6 cm and a rectangular window hole 2 of 3.5 cm × 2.0 cm at the end corresponds to the window hole 2. As described above, the one to which the 4.0 cm × 2.6 cm cellophane tape 3 (a commercially available adhesive cellophane tape was cut) was attached was prepared. The frame of the window hole 2 of the holder 1 is provided with a scale 4 every 1 mm.

The adhesive surface of the adhesive tape 2 together with the holder 1 was attached to the skin rash of an atopic dermatitis patient and pressed with a finger and rubbed lightly 2 to 3 times to bring the adhesive surface of the cellophane tape into close contact with the skin. Then, using the frame of the stratum corneum collection holder as a lever, peel off the tape 2 from the skin from a certain direction with a certain force, and attach it to the frame to measure the width 4 with the scale 4 as a guide.
mm and 5 mm in length were cut using a cutter and scissors, and this was attached to a slide glass to which a vinyl chloride resin adhesive had been evenly applied so that the adhesive surface of the tape was in contact.

Next, this slide glass is mixed with xylene.
After immersing for a time, the adhesive substance of the adhesive tape was dissolved, and after confirming that the cellophane tape was peeled off from the slide glass, it was immersed in new xylene for 30 minutes and again in new xylene for 30 minutes. If the cellophane tape is not peeled off after immersion for 3 hours, it may be soaked in xylene until it is naturally peeled off.

Then, this was taken out, and xylene was dried without applying heat, and then two types of hematoxylin-eosin (HE) staining for staining the cytoplasm and nucleus and PAS staining for staining the polysaccharide were carried out by a conventional method. After drying and drying, balsam was encapsulated by a conventional method to prepare a stained sample of keratinocytes, which was observed with an optical microscope.

Furthermore, DACM staining was performed using the method already reported. That is, for the staining of SH group, the tape on which the keratinocytes were fixed was subjected to TAS (sodium chloride-
0.01 mMD after washing with Tris-acetate buffer
The ACM / TAS solution was dropped on the surface of the sample, reacted at room temperature for 3 minutes, washed again with TAS at 4 ° C., and observed using an epifluorescence microscope. On the other hand, SS bond staining was performed as follows. Specimen at 4 ℃ TAS
After washing with 0.15 M NEM (N-ethylmaleimide) / TAS solution for 45 minutes at 37 ° C. to block free SH groups, and then thoroughly wash with 4 ° C. TAS,
40 mM DTT (dithiothreol) / 0.5 mM ED
A TA (ethylene-diaminetetraacetic acid) / TAS solution wash was reacted at 37 ° C. for 15 minutes to reduce the SS bond in the sample to an SH group, and after sufficiently washing with 4 ° C. TAS, the SH group staining described above was performed. It was observed using an epifluorescence microscope.

A surface sample of keratinocytes was prepared by sticking a part (5 × 4 mm) of cellophane tape from which the stratum corneum was collected on a transfer resin plate on which a black vinyl chloride resin adhesive was spread, and the same procedure as above. Then, it was immersed in xylene, the cellophane tape was peeled off, and the xylene was dried. This sample was fixed to a sample stand (15 mmφ) for a scanning electron microscope with double-sided tape, and gold was vapor-deposited using an E-101 type ion sputter (manufactured by Hitachi Ltd.).

For the back surface sample, a part of the cellophane tape from which the stratum corneum was sampled was used as it was, fixed on a sample stand (15 mmφ) for a scanning electron microscope with a double-sided tape, and gold vapor deposition was carried out in the same manner.

These front surface sample and back surface sample were observed with a scanning electron microscope at a magnification of about 2,500 and photographed. The observation items of each specimen are as follows.

(Light Microscopic Findings) HE Staining (FIG. 2): Extent of Nucleated Cells and Existence of Multilayer Cell Group PAS Staining (FIG. 3): Extent of Nucleated Cells and PAS
Presence / absence of positive cells DACM staining (FIG. 4): SH group, SS compared to healthy subjects
Fluorescence intensity of binding, fluorescence at the nuclear position of SH group as a unique finding, and mottled fluorescence of SS binding

(Scanning Electron Microscope Findings) Surface sample (FIG. 5): Sponge-like structure Back surface sample (FIG. 6): Fine villiform projections

Each of the above observation items was evaluated by 4 grades (negative-, weakness ±, strength +, considerably strength ++), and each grade was digitized as 0, 0.5, 1.0 and 2.0. The average value of the scores of each observation finding was calculated. However, for the appearance of nucleated cells, the average of the observation results of two samples, HE staining and PAS staining, was used. The results are shown in Table 1. The stratum corneum was collected from healthy children and children with atopic dermatitis to examine keratinocyte parakeratosis, abnormal cell detachment, presence of cell infiltration, and keratinocyte malformation during keratinization. .

The obtained keratinocyte specimens are clear specimens that have never existed in the past, and as shown in FIGS. 2 to 6, keratinocytes are clearly observed, and keratinocytes of healthy children and pediatric atopic dermatitis patients. Since the histopathological and morphological differences of the cells can be observed in detail, it was possible to investigate the abnormality of keratinocytes due to atopic dermatitis.

As shown in Table 1, in pediatric patients with atopic dermatitis, the value of the horny layer test findings decreased as the symptom improved, and the atopic skin was measured by measuring the degree of skin damage by this method. Follow-up of clinical signs of inflammation could be evaluated.

Further, as shown in Table 1 and FIG. 5, the skin damage can be measured only by observing the surface specimen, but the observations of the back specimen and the stained specimen should be added. As a result, more reliable measurement can be performed.

[0054]

[Table 1]

[0055]

[Example 2] Each sample was prepared in the same manner as in Example 1, but the horny layer was collected twice at the first visit and after 2 days in one patient with atopic dermatitis. The efficacy of the drug used for treatment was evaluated. The results of each of the two stratum corneum examinations were totaled, and the overall scores were compared. If the difference exceeds 2, it is markedly improved, and if it exceeds 1 and 2 or less, it is slightly improved, 0.5
Slightly less than 1 and less than 1, slightly less than 0.5-
The case of 0.5 or more was determined to be unchanged, and the case of less than −0.5 was determined to be worse. The results are shown in Tables 2 and 3.

[0056]

[Table 2]

[0057]

[Table 3]

From these results, it was found that the skin lesions of patients with atopic dermatitis were considerably improved, indicating that the treatment was effective.

[0059]

According to the present invention, the degree of skin damage can be easily measured non-invasively. By using the colored plate for the preparation of the surface sample of keratinocytes, white keratinocytes can be clearly discriminated and it is possible to prevent erroneous recognition of the front and back when fixing the keratinocytes on the electron microscope sample stage. In addition, the specimen used in the present invention can be stably stored, and several kinds of staining can be performed from one sample, and clear observation can be performed, so that the progress of skin diseases can be grasped and can be effectively used for skin diagnosis.

[Brief description of drawings]

FIG. 1 is a perspective view showing an example of a holder.

FIG. 2 is an optical micrograph (magnification about 350 times) of an HE-stained specimen (Example 1) used in the method of the present invention (morphology of organism). The left is a sample of a healthy child, and the right is a sample of a patient with atopic dermatitis.

FIG. 3 is an optical micrograph (magnification: about 350 times) of a PAS-stained specimen (Example 1) used in the method of the present invention (morphology of organism). The left is a sample of a healthy child, and the right is a sample of a patient with atopic dermatitis.

FIG. 4 is an optical micrograph (magnification about 350 times) of a DACM-stained specimen (Example 1) used in the method of the present invention (morphology of organism). The left is a sample of a healthy child, and the right is a sample of a patient with atopic dermatitis.

FIG. 5 is a scanning electron micrograph (magnification: about 1250 times) of a keratinous surface preparation (Example 1) used in the method of the present invention (morphological form). The left is a sample of a healthy child, and the right is a sample of a patient with atopic dermatitis.

FIG. 6 is a scanning electron micrograph (magnification: 1250 times) of a keratinous back surface sample (Example 1) used in the method of the present invention (morphological form). The left is a sample of a healthy child, and the right is a sample of a patient with atopic dermatitis.

[Explanation of symbols]

 1. Holder 2. Window 3. 3. Tape coated with adhesive material scale

Claims (4)

[Claims] 1. A skin keratinocyte is affixed to a tape coated with an adhesive substance soluble in an organic solvent, and the tape is attached to a colored plate coated with an adhesive in advance so that the adhesive surface of the tape contacts. The tape is peeled from the colored plate by dissolving the adhesive substance with an organic solvent to fix the keratinocytes on the colored plate, and the morphological characteristics of the keratinocytes are microscopically observed using the obtained keratinocyte specimen. A method for measuring the degree of damage to skin keratinocytes. 2. The degree of damage of skin keratinocytes according to claim 1, wherein the keratinocyte specimen is microscopically observed from the front side of the specimen, and the tape on which the skin keratinocytes are adhered is also microscopically observed from the back side. Measurement method. 3. The method according to claim 1, wherein the skin keratinocytes are attached to a tape coated with an adhesive substance soluble in an organic solvent, and the tape is attached to a transparent plate coated with an adhesive agent in advance. The tape is affixed so that the surfaces are in contact with each other, and the tape is separated from the transparent plate by dissolving the adhesive substance with the organic solvent to fix the corneocytes to the transparent plate, which is selected from HE staining, PAS staining, and DACM staining. A method for measuring the degree of damage to skin keratinocytes, which comprises observing a skin keratinocyte stained sample obtained by staining with a microscope. 4. The tape coated with an adhesive substance soluble in the organic solvent is attached to a holder having a window hole so as to correspond to the window hole. 4. The method for measuring the degree of damage to skin keratinocytes according to any one of 3 to 3.