JP2003202336A - Method for dyeing corneocyte and method for preparing corneocyte specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for dyeing corneocytes and a specimen preparing method capable of clearly dyeing the corneocytes in a short time and speedily grasping information useful for counseling. <P>SOLUTION: In the method for preparing the specimen obtained by dyeing a sample containing the corneocytes obtained by stripping skin by an adhesive body, the specimen dyed by a dyeing agent dissolved in a dyeing agent solution containing an organic solvent miscible with water is sealed by fat and oil constituents and/or oil and fat compositions which are liquids at 25°C and one atmospheric pressure. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for staining keratinocytes and a method for preparing a keratinocyte sample, which provides information useful for differentiation of skin condition and proper selection of cosmetics.

[0002]

2. Description of the Related Art The condition of the skin changes from moment to moment depending on factors such as the season and physical condition, and the maintenance of applying cosmetics. Therefore, it is possible to select an appropriate cosmetic according to the changes. , It is an important factor in preventing aging and keeping the skin's properties beautiful. For this reason, a method for objectively and scientifically distinguishing the nature and condition of the skin has been devised. As such a method, for example, keratinocytes are collected from the skin such as a face with an adhesive tape or an adhesive disc, stained, and the regularity of the arrangement of keratinocytes, the shape of keratinocytes,
For example, the skin condition and the like can be differentiated by using the size and the exfoliation of keratinocytes, the presence or absence of nuclei in keratinocytes and the like as indices. In such discrimination, a staining method is preferred in which the contrast between the bag and the cytoplasm can be clearly defined, and thus the shape of individual cells can be confirmed, and at the same time the presence or absence of nuclei can be confirmed.Therefore, gentian violet and brilliant The method of dyeing with green is taken. However, in this method, since dyeing takes time, it does not serve as an information source for immediate cuts in counseling and other situations, and cosmetics etc. are selected by skin differentiation using the shape of keratinocytes as an index. In this case, it takes several days from the skin analysis to the presentation of the cosmetic sample, which causes a time lag. Usually, in such a case, it is considered to increase the concentration of the dyeing agent, but even if the concentration of both gentian violet and brilliant green is increased to the solubility limit in water (gentian violet 1%, brilliant green 0.5%). However, the dyeing took 10 minutes or more, and the problem of the time required for dyeing remained.
In the staining, the use of an organic solvent is usually taboo because it impairs the staining specificity and causes the alteration and degeneration of cells, and is not used for staining such keratinocytes. Therefore, a method for dyeing a sample containing keratinocytes obtained by stripping skin with an adhesive, wherein the dyeing solution in the dyeing step contains an organic solvent miscible with water, No method for staining keratinocytes is known.

Further, normally stained keratinocyte samples are subjected to an encapsulation operation in order to adjust the refractive index of the entire specimen to be the same, and as such an encapsulating agent, balsam or an ultraviolet curable resin is usually used. However, these encapsulating agents often induce bleeding of the staining agent, which is not preferable because it causes a large error when automatically measuring the cell area and the like by binarization. In addition, when the adhesive tape is used for collecting keratinocytes, the organic solvent used for such encapsulation may dissolve the tape and the adhesive, and therefore cannot be used. When performing rapid keratinocyte analysis by countercounseling as described above, automation by binarization of area measurement is unavoidable, and the solution of this bleeding is a particularly important matter, and this solution is strongly recommended. Was wanted. The oil / fat component and / or oil / fat composition which is liquid at 1 atm and 25 ° C. has never been used for such encapsulation. This is because the liquid encapsulant is difficult to store for more than one month, and no such material has ever been used as an encapsulant.
Therefore, it has not been known at all that the use of such an encapsulating agent can produce a keratinocyte preparation capable of automatic measurement of the keratinocyte area by binarization with almost no bleeding of the staining agent.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a method for staining a keratinocyte and a method for preparing a keratinocyte specimen, which allows a keratinocyte to be clearly stained in a short time and information useful for counseling can be quickly grasped. Is an issue.

[0005]

In view of such a situation, the inventors of the present invention have a method for staining keratinocytes, in which keratinocytes can be clearly stained in a short time, and information useful for counseling can be quickly grasped. As a result of intensive research efforts to find a method for preparing a keratinocyte specimen, a method for staining a sample containing keratinocytes obtained by stripping skin with an adhesive body, wherein the stain solution in the staining step is By virtue of containing an organic solvent miscible with water, it becomes possible to perform clear staining in a short time by staining with a keratinocyte staining method. Furthermore, the stained specimen is liquid at 1 atm and 25 ° C. Oil and fat component and /
Alternatively, by encapsulating with an oil / fat composition, it is possible to enclose the dye without bleeding, and therefore, for keratinocytes from a specimen, it is possible to binarize and automatically measure the area of keratinocytes. It came to completion. That is, the present invention relates to the techniques described below. (1) A method for dyeing a sample containing keratinocytes obtained by stripping skin with an adhesive body, wherein the dyeing solution in the dyeing step contains an organic solvent miscible with water, Method for staining keratinocytes. (2) The stain is characterized by containing gentian violet and brilliant green.
The method for staining keratinocytes according to (1). (3) The method for staining keratinocytes according to (1) or (2), wherein the organic solvent miscible with water is ethanol. (4) The adhesive body is an adhesive tape,
(1) to (3) The method for staining keratinocytes according to any one of items. (5) A method for producing a specimen in which a sample containing keratinocytes obtained by stripping the skin with an adhesive material is stained, wherein the stained specimen is a liquid oil component and / or oil composition at 1 atm and 25 ° C. A method for preparing a keratinocyte specimen, which is characterized in that (6) The oil / fat component and / or oil / fat composition that is liquid at 1 atmosphere and 25 ° C. for encapsulation is one or more kinds selected from silicone oil, fatty acid triglyceride, ester of higher alcohol and fatty acid, and hydrocarbon. The method for producing a keratinocyte specimen according to (5), which is characterized in that (7) The sample containing keratinocytes is stained by the method for staining keratinocytes according to any one of (1) to (4), (5) or (6) The method for preparing a keratinocyte preparation according to. (8) One or more types selected from area measurement of keratinocytes, presence / absence of nucleated cells, appearance frequency of nucleated cells, regularity of keratinocyte array, keratinocyte shape, and keratinocyte detachment state Is for measuring or discriminating two or more kinds, (5) to (7) The method for preparing a keratinocyte specimen according to any one of (1) to (7). Hereinafter, the present invention will be described in more detail with a focus on the embodiments.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Method for Staining Corneal Cells of the Present Invention The method for staining keratinocytes of the present invention is a method for staining a sample containing keratinocytes obtained by stripping skin with an adhesive.
The dyeing solution in the dyeing step contains an organic solvent miscible with water. That is, the present invention is characterized in that a dye is dissolved in an organic solvent miscible with water, and a sample containing keratinocytes obtained by stripping the skin with an adhesive is dyed. Here, as the adhesive body for stripping the skin, for example, an adhesive transparent tape such as cellophane tape or an adhesive disk obtained by coating an adhesive on a polyethylene terephthalate plate can be preferably exemplified. Of these, preferred are adhesive tapes that are commercially available and are extremely easy to obtain. The sample obtained by stripping the skin with such an adhesive can be dyed as it is, or it can be used again by contacting it with the adhesive and transferring it. The preferred method is to use it as is,
This is because the labor can be reduced. According to the dyeing method of the present invention, since it does not affect the pressure-sensitive adhesive or the support portion of the pressure-sensitive adhesive body such as a tape or disk, it can be dyed as it is in the collected form. In the dyeing method of the present invention, any commonly used dyeing agent can be used without particular limitation, and examples thereof include eosin, hematoxylin, gentian violet, brilliant green, and malachite green. A combination of the above can also be used. Of these, it is particularly preferable to use gentian violet and brilliant green in combination. This is because gentian violet can stain the cytoplasm with a good contrast to the background, so that it is possible to clarify the shape of keratinocytes, and with this cytoplasm as the background, the nucleus can be clearly stained with brilliant green. Is. The dyeing method of the present invention is characterized by dissolving such a dyeing agent in a liquid medium containing water and an organic solvent which is soluble in the dyeing agent. The preferred content of the coloring agent is 2 to 7% by weight, and if the combination of gentian violet and brilliant green is 1.5 to 5% by weight of gentian violet, more preferably 2 to 4% by weight.
And the brilliant green content is 0.7 to 2% by weight, and more preferably 0.8 to 1.5% by weight. or,
Examples of the water-soluble organic solvent include alcohols such as methanol, ethanol, isopropanol, and 1,3-butanediol, ketones such as acetone and methyl ethyl ketone, nitriles such as acetonitrile, ethers such as tetrahydrofuran, and the like. Can be preferably exemplified. Of these, alcohols are preferable, and ethanol is particularly preferable. The content of the water-soluble organic solvent in the liquid medium is preferably 3 to 55% by weight, more preferably 5 to 50% by weight. This is because if the amount of the organic solvent is too large, the time is not shortened, and on the contrary, the pressure-sensitive adhesive or the support of the pressure-sensitive adhesive may be damaged, and if it is too small, the dye-sharpening effect may not be obtained. Under such conditions of the present invention, the time required for dyeing is 1
~ 5 minutes, the conventional method (gentian violet 0.5-1% by weight and brilliant green 0.2-
0.5% by weight), the time is remarkably shortened compared to 10 to 30 minutes, and the contrast is also made clear. or,
Such dyeing can be performed at room temperature.

(2) Method of preparing keratinocyte specimen of the present invention The method of preparing keratinocyte specimen of the present invention is a method of preparing a stained specimen containing keratinocytes obtained by stripping skin with an adhesive. The stained specimen at 1 atm and 25 ° C.
And encapsulating with a liquid oil component and / or oil composition. Here, as the stained sample of the sample containing keratinocytes obtained by stripping the skin with an adhesive body, any sample that is usually prepared in such a step can be applied, but particularly preferable one Is a sample stained by the method for staining keratinocytes of the present invention described in (1) above. The oil and fat component and / or oil and fat composition that is liquid at 1 atm and 25 ° C. and is used in the method for preparing a keratinocyte specimen of the present invention is an oil or fat component usually used in cosmetics and medicines, or a composition combining them. Therefore, as long as it is liquid at 1 atm 25 ° C., it can be used without particular limitation. As a preferable combination of such substances, one or more kinds selected from silicone oil, fatty acid triglyceride, ester of higher alcohol and fatty acid, and hydrocarbon which are liquid at 1 atm and 25 ° C. can be preferably exemplified. Usually, encapsulation with a liquid component does not usually give stability for one month or more, but there is no problem in the immediate use such as counseling in a counter, which is the object of the present invention. No bleeding of the staining agent was observed in the oil agent, and the microscopic image of the obtained specimen was captured as an image and divided into two values, whether stained or not (binarization treatment), the shape of keratinocytes. Can be accurately displayed, and thus the keratinocyte area can be accurately and automatically measured. In addition to this, the information obtained from the sample produced by the method for preparing a sample of the present invention includes the presence or absence of nucleated cells, the appearance frequency of nucleated cells and the state of keratinocyte detachment, the regularity of the arrangement of keratinocytes, Examples thereof include the shape of keratinocytes, and the sample of the present invention is preferably used for measuring or discriminating these numerical values. That is, the keratinocyte sample prepared by the method of the present invention is an area measurement of keratinocytes, the presence or absence of nucleated cells, the frequency of appearance of nucleated cells, the regularity of the arrangement of keratinocytes, the shape of keratinocytes, and It is preferable to use one or more kinds selected from the exfoliation status of keratinocytes for measurement or discrimination.

[0008]

The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples.

<Example 1> From the same panel, 3 types of keratinocytes were collected by adhesive tape stripping, and 2 types were stained by the staining method of the present invention. That is, 2% by weight of gentian violet 3% by weight and 1% by weight of brilliant green
It was dipped in a dyeing solution dissolved in a 0% ethanol aqueous solution for 2 minutes and thoroughly washed with water. One is encapsulated in UV curable resin,
It was irradiated with ultraviolet rays and cured (Comparative Example 1). The other was encapsulated in dimethicone (silicone) according to the method for preparing a keratinocyte preparation of the present invention (Example 1). One of the saws was immersed in a dyeing solution prepared by dissolving 1% by weight of gentian violet and 0.5% by weight of brilliant green in water for 10 minutes, thoroughly washed with water, and then enclosed in dimethicone (Comparative Example 2). Micrographs of these specimens are shown in FIGS. As shown in FIGS. 1 to 3, in Comparative Example 1, bleeding causes a decrease in contrast, and in Comparative Example 2, staining of keratinocytes becomes thin, whereas in Example 1, there is no bleeding (Comparative Example The contrast was larger than that of No. 1) and a good dyeing condition was obtained. From this, it can be seen that the method of preparing a keratinocyte specimen prepared by the method of the present invention can clearly and clearly identify keratinocytes.

<Example 2> Using the samples of Example 1, Comparative Example 1 and Comparative Example 2 prepared in Example 1, the average area of keratinocytes was calculated. Further, in Example 1, the average area (unit: μm 2) was calculated by binarizing manually. The number of cells used to calculate the average was 20. The results are shown in Table 1. From this, it can be seen that according to the present invention, the same result as the manual is obtained even if the automatic area calculation is performed. In addition, it can be seen that in the case of unclear cell specimens or specimens with bleeding, a value larger than the actual area is obtained in the automatic area calculation.

[0011]

[Table 1]

<Example 3> According to the measuring method of Example 1,
The study was conducted by changing the concentration of gentian violet.
Brilliant green remained at 1% by weight. Dimethicone was used for encapsulation. The sharpness of dyeing is "○: Vividly dyed", "△: Somewhat unclear", "X: Unclear"
It evaluated by the judgment standard of. The results are shown in Table 2. From this, the preferred content of gentian violet is 1.5 to 5
It can be seen that weight percent is preferred and 2-4 weight percent is even more preferred.

[0013]

[Table 2]

<Example 3> Similar to Example 3, the same examination was conducted by changing the concentration of brilliant green. As a sample, a keratinocyte specimen of a person with a poor skin condition in which nucleated cells were observed was used. The sharpness of the dyeing condition of the nucleus was determined in Example 3.
It judged by the same standard as. The results are shown in Table 3. Therefore, the preferred concentration of brilliant green is 0.7-2% by weight.
It is found that it is 0.8 to 1.5% by weight, and more preferably 0.8 to 1.5% by weight.

[0015]

[Table 3]

<Example 4> By the same method as in Example 1, the concentration of ethanol was changed and examined. At the same time, the presence or absence of precipitate (insoluble portion) of the stain was also confirmed. The clarity of staining is "○: Vividly stained", "△: Slightly unclear",
Evaluation was made according to the criteria of "x: unclear". In addition, the presence or absence of precipitation was judged by "yes", "slightly" and "no". The results are shown in Table 4. Therefore, the preferable content of the water-soluble organic solvent in the liquid medium is 3 to 40% by weight.
It is found that it is 5 to 30% by weight, and more preferably 5 to 30% by weight.

[0017]

[Table 4]

<Embodiment 5> As in Embodiment 1, the examination was conducted by changing the organic solvent to a 20% methanol solution.
A specimen with clear staining was obtained in the same manner as in.

<Example 6> As in Example 1, an examination was conducted by changing the organic solvent to a 10% acetonitrile solution, but a sharply stained sample was obtained as in Example 1.

<Example 7> In the same manner as in Example 1, an examination was conducted by changing only the encapsulant. The non-bleeding of the finished sample was judged by the criteria of "○: no bleeding", "△: slightly bleeding", and "x: clear bleeding".
The results are shown in Table 5. From this, it is understood that it is preferable to use a fat and oil component and / or a fat and oil composition which is liquid at 1 atm and 25 ° C. as an encapsulating agent for the preparation of each layer sample of the present invention.

[0021]

[Table 5]

[0022]

EFFECTS OF THE INVENTION According to the present invention, it is possible to provide a method for staining a keratinocyte and a method for preparing a keratinocyte specimen, which allows the keratinocyte to be clearly stained in a short time, and information useful for counseling can be quickly grasped. .

[Brief description of drawings]

FIG. 1 is a micrograph of a keratinocyte specimen prepared by the production method of the present invention in Example 1. (Drawing substitute photograph)

FIG. 2 is a micrograph of a keratinocyte specimen prepared by the method of Comparative Example 1 of Example 1. (Drawing substitute photograph)

FIG. 3 is a micrograph of a keratinocyte specimen prepared by the preparation method of Comparative Example 2 of Example 1. (Drawing substitute photograph)

[Procedure amendment]

[Submission date] March 18, 2003 (2003.3.1)
8)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is a micrograph of a keratinocyte specimen prepared by the production method of the present invention of Comparative Example 1 of Example 1 . (Drawing substitute photograph)

FIG. 2 is a micrograph of a keratinocyte specimen prepared by the production method of the present invention in Example 1. (Drawing substitute photograph)

FIG. 3 is a micrograph of a keratinocyte specimen prepared by the preparation method of Comparative Example 2 of Example 1. (Drawing substitute photograph)

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toshikazu Yagi             1234 Aino, Fukuroi City, Shizuoka Prefecture             Fukuroi factory F term (reference) 2G045 AA24 BA14 BB02 BB23 BB25                       CB09 FA16 FA20 GB01 HA06                       JA01                 2G052 AA33 AD32 AD52 BA23 DA07                       DA27 FA02 FA09 GA32 HA17                       HA18 HB06 JA06 JA07 JA09                       JA11                 4H011 BC19 CA02 CB08 CD02 CD13                       DH07

Claims (8)

[Claims] 1. A method for dyeing a sample containing keratinocytes obtained by stripping skin with an adhesive, wherein the dyeing solution in the dyeing step contains an organic solvent miscible with water. A method for staining keratinocytes. 2. The method for staining keratinocytes according to claim 1, wherein the staining agent contains gentian violet and brilliant green. 3. The method for staining keratinocytes according to claim 1, wherein the organic solvent miscible with water is ethanol. 4. The method for staining keratinocytes according to claim 1, wherein the adhesive body is an adhesive tape. 5. A method for preparing a specimen in which a sample containing keratinocytes obtained by stripping the skin with an adhesive material is stained, wherein the stained specimen is a liquid oil and fat component at 1 atm and 25 ° C.
Alternatively, a method for preparing a keratinocyte specimen, which comprises encapsulating with an oil composition. 6. An oil and fat component and / or oil composition which is liquid at 1 atmosphere and 25 ° C. for encapsulation and is selected from one or more kinds selected from silicone oil, fatty acid triglyceride, esters of higher alcohols and fatty acids, and hydrocarbons. The method for producing a keratinocyte specimen according to claim 5, wherein 7. The keratin according to claim 5, wherein the sample containing keratinocytes is stained by the method for staining keratinocytes according to any one of claims 1 to 4. How to make a cell specimen. 8. The measurement of the area of keratinocytes, the presence or absence of nucleated cells, the frequency of appearance of nucleated cells, the regularity of the arrangement of keratinocytes,
The keratinocyte specimen according to any one of claims 5 to 7, which is for measuring or discriminating one or more kinds selected from the shape of keratinocytes and the detachment state of keratinocytes. How to make.