JP2007151899A - Horny layer cell discrimination method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for selectively dying a hydrophobic region of a horny layer cell and discriminating its hydrophobicity/hydrophilicity under an optical microscope. <P>SOLUTION: A horny layer cell discrimination method includes dying the horny layer cell taken from skin with a stain solution that contains 30-50% by mass of ethanol including Sudan Black B from a direction of a region contacting its surrounding and discriminating its hydrophobicity/hydrophilicity under the optical microscope. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for distinguishing skin using horny layer cells of skin, and more specifically, the present invention selectively distinguishes the hydrophobic-hydrophilicity of horny layer cells by selectively staining the hydrophobic region of horny layer cells. On how to do.

  In selecting a cosmetic, it is very important to know the characteristics of the skin to be applied. This is because, depending on the skin condition, even if the same cosmetic is used, there is a favorable effect, and on the contrary, an undesirable effect is also achieved. In other words, it can be said that selecting appropriate cosmetics for skin care is a common theme for securing effects and safety. Reflecting this situation, various skin discrimination methods and cosmetic selection methods have been devised. Among these methods, a method using corneal cells that can be easily collected, appropriately reflects the skin state, and has a lot of skin information has been intensively studied in recent years. As the technology related to the stratum corneum cells, mainly those relating to the shape or pathological characteristics of the stratum corneum cells such as whether or not it has a shape, an area, a nucleus, and how many melanin granules are dominant. . In addition to the shape, there are those in which a relatively macro uneven state of the skin is added as an auxiliary factor (for example, Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5 and Patent). Reference 6).

  In addition to these technologies, we focused on the stratum corneum barrier function, which is important as a skin function, and evaluated the hydrophobicity of the cornified cells in the corneum cells (hereinafter abbreviated as CE), which is closely related to the barrier function, by selective staining. Have been disclosed (see, for example, Patent Document 7 and Patent Document 8). One of the reasons for the need to stain the hydrophobic region of the stratum corneum cells using such a special dye is that the stratum corneum cells have already lost their lives and become flattened to prevent the entry of substances from the outside. In other words, it is difficult to uniformly penetrate the staining solution. In other words, even when a commonly known staining solution for staining a hydrophobic portion is used, the stratum corneum cells are not stained at all or the entire stratum corneum cells are stained, and only the hydrophobic portion can be selectively dyed. It can be said that there was no current situation. However, when Nile Red, which is a pigment that can selectively stain hydrophobic regions, is used, observation with a fluorescence microscope is unavoidable, and there are practical problems such as expensive fluorescence microscopes and light shielding equipment. That is, a technique for selectively staining the hydrophobicity-hydrophilicity of stratum corneum cells, which can be observed with a practical optical microscope at low cost, has never been known.

JP 2004-105700 A Japanese Patent Laid-Open No. 2004-53491 JP 2002-65616 A JP 2001-13138 A JP 2000-116623 A Japanese Patent Laid-Open No. 11-344489 JP 2001-91514 A No. 02/25272

  The present invention has been made under such circumstances, and an object of the present invention is to provide a technique for discriminating the hydrophobicity-hydrophilicity of stratum corneum cells under an optical microscope.

In view of such circumstances, the present inventors have sought for a technique for distinguishing the hydrophobicity-hydrophilicity of stratum corneum cells under an optical microscope. By staining, it was found that the hydrophobicity-hydrophilicity of stratum corneum cells can be differentiated by an optical microscope, and the present invention has been completed. That is, the present invention is as follows.
(1) A skin differentiation method, in which stratum corneum cells collected from the skin are stained with a dye of Sudan Black B from the outside contact portion direction, and the hydrophobicity-hydrophilicity of the stratum corneum cells by an optical microscope stained with the dye A method for differentiating horny layer cells, characterized in that
(2) The method for distinguishing stratum corneum cells according to (1), wherein the staining solution of Sudan Black B contains 30 to 50% by mass of ethanol in the staining solution.
(3) The method for distinguishing horny layer cells according to (1) or (2), which is performed according to the following steps.
(Step 1) Using the adhesive tape, horny layer cells are collected from the skin.
(Step 2) The pressure-sensitive adhesive of the pressure-sensitive adhesive tape is softened and dissolved with an organic solvent, and the stratum corneum cells are transferred onto a slide glass to prepare a stratum corneum cell specimen.
(Step 3) The stratum corneum cell sample is conditioned with an aqueous ethanol solution (about 40% by mass) and then left to stand in a Sudan black B staining solution at room temperature for 24-48 hours for staining.
(Step 4) The stained stratum corneum cell specimen is washed with an aqueous ethanol solution, air-dried, sealed in a balsam, and identified under an optical microscope.

  The differentiation method of the present invention makes it possible to differentiate the hydrophobicity-hydrophilicity of stratum corneum cells under an optical microscope, eliminating the need for expensive fluorescent microscopes, high-sensitivity cameras, and darkrooms such as dark rooms, thereby reducing costs and savings. More practical technologies such as space can be provided.

  The present invention distinguishes the hydrophobicity-hydrophilicity of stratum corneum cells under an optical microscope by staining the stratum corneum cells collected from the skin with a staining solution containing a dye of Sudan Black B from the outside contact portion direction. It is characterized by. A more detailed description will be given below.

The steps of collecting and staining the stratum corneum cells are preferably performed as follows.
(Step 1) Using the adhesive tape, horny layer cells are collected from the skin.
(Step 2) The pressure-sensitive adhesive of the pressure-sensitive adhesive tape is softened and dissolved with an organic solvent, and the stratum corneum cells are transferred onto a slide glass to prepare a stratum corneum cell specimen.
(Step 3) The stratum corneum cell sample is conditioned with an aqueous ethanol solution (about 40% by mass) and then left to stand in a Sudan Black B staining solution at room temperature for 12 to 48 hours for staining.
(Step 4) The stained stratum corneum cell specimen is washed with an aqueous ethanol solution, air-dried, sealed in a balsam, and identified under an optical microscope. At this time, when the dyed result is too dark or a precipitate is adhered, it is preferable to fractionate with an ethanol solution.
If the room temperature conditions for dyeing are changed at 37 to 60 ° C. in the above step 3, the dyeing time can be shortened. In the step 2, the stratum corneum cells collected with the living body facing upwards are arranged so that this surface is in contact with the slide glass, thereby allowing staining from the direction of the external contact portion.

  Sudan Black B is a non-fluorescent fat-soluble dye (lysochrome) having a structural formula of 2,3-dihydro-2,2-dimethyl-6-[[1-naphthyl-4 -(Phenylazo)] azo] -1H-perimidine (2,3-dihydro-2,2-dimethyl-6-[[1-naphthyl-4- (phenylazo)] azo] -1H-perimidine) with a molecular weight of 456 .54. Since Sudan Black B is insoluble in water and soluble in ethanol, it can be used as a water-ethanol mixed solution to dye various complex lipids as well as neutral fats by the mechanism of physical staining. Compared to other Sudan dyeings, it has a stable dyeability and does not have an OH group, so it works as a basic dye. Due to its properties as a hydrophobic and hydrophilic dye, it has medium polarity such as phospholipids and glycolipids. It has excellent characteristics such as the ability to stain lipids (Sudan staining method: Monthly Medical Technology, separate volume P.47, published by dentistry).

  Nile red, Nile blue, Oil red O, and Sudan III are well known as pigments that can selectively stain the hydrophobicity-hydrophilicity of stratum corneum cells. These dyes are used for fat staining in pathological examinations, and have a very good staining property for fat, but the staining property is not sufficient for stratum corneum cells mainly composed of proteins. There is a problem that differentiation of stratum corneum cells is very difficult. For these dyes, it is difficult to selectively stain the hydrophobic portion existing on the living body contact side of the stratum corneum cells even by staining from the direction of the external contact portion. For this reason, it is necessary to observe the fluorescence of the stratum corneum cells stained with Nile Red with a fluorescence microscope under light-shielding conditions, but in addition to the equipment cost increase, it is necessary to distinguish the fluorescence from the stratum corneum cells themselves. New issues such as these also arise. For this reason, it is extremely preferable to use Sudan Black B as a stain and stain from the direction of the external contact portion to distinguish stratum corneum cells under an optical microscope.

  The preferable content of ethanol in the staining solution of the present invention is 30 to 50% by mass, more preferably 35 to 45% by mass, based on the total amount of the staining solution. In fat staining using non-fluorescent Sudan Black B or the like, an aqueous solution of about 70% by mass ethanol is generally used. However, in the staining of stratum corneum cells of the present invention, such ethanol is highly concentrated. When it is blended with the dye, sufficient dyeability cannot be obtained (see FIG. 2). This is because, in a high-concentration ethanol blending system such as fat staining conditions, the properties of Sudan Black B as a hydrophobic-hydrophilic pigment cannot be fully exhibited. It is presumed that the pigment distribution to the stratum corneum cells as the component is promoted and the dyeability is improved (see FIG. 1).

  The discrimination between the hydrophobicity and the hydrophilicity of the stratum corneum cells can be performed according to the degree of intensity of the staining. For example, if the staining property is uniform and good, the CE is in a mature state, and the basic evaluation items of the stratum corneum cells are the regularity of the stratum corneum cells, the presence or absence of delamination, the stratum corneum cell area In addition, the presence or absence of nucleated cells shows relatively good values, and it can be judged that the barrier function is sufficient and the skin state is good. On the other hand, if the stainability is uneven and poor, the above evaluation items of the stratum corneum cells are relatively bad values, and it is determined that the skin condition is insufficient due to insufficient barrier function. Such discrimination results can be used for skin condition counseling and selection of cosmetics suitable for each individual. Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited only to these examples.

  In accordance with the steps of collecting and staining the horny layer cells of the present invention, the horny layer cells are collected from the cheek of a 35 year old female subject, and using the following Sudan Black B staining solution formulations 1 and 2, the horny layer cells are collected. An optical microscope image of the specimen was obtained (see FIGS. 1 and 2). At the same time, as a comparative example, staining liquid formulation 3 in which Sudan black B was replaced with Nile red was prepared and the optical microscope was changed to a fluorescence microscope, and an image was similarly obtained (see FIG. 3). As apparent from FIGS. 1 to 3, the image of the stratum corneum of the present invention has the same impression as that observed with a fluorescence microscope with Nile Red staining, which is a conventional method, but is more stained than this method. It is clear that the hydrophobicity-hydrophilicity of stratum corneum cells can be distinguished.

(Prescription of staining solution 1)
Sudan Black B 0.1% by mass
Ethanol 40.0% by mass
Pure water 59.9% by mass

(Staining solution prescription 2)
Sudan Black B 0.1% by mass
Ethanol 70.0% by mass
Pure water 29.9% by mass

(Staining solution prescription 3)
Nile Red 0.1% by mass
Ethanol 70.0% by mass
Pure water 29.9% by mass

  In Example 1, the number of test subjects was increased, and stained images of stratum corneum cells were obtained using staining prescription 1 for 30 women aged 18 to 60 years. From the stratum corneum cell-stained image, the hydrophobicity-hydrophilicity level (five levels with the larger hydrophobicity being 1) was evaluated, and the correlation with the TEWL (transepidermal water permeation amount) of the cheek was examined.

  4 and 5 show images of the maximum (deep dyed state) and the minimum (lightly dyed state) of hydrophobicity, and FIG. 6 shows a scatter diagram of the hydrophobicity-hydrophilicity level evaluation value and TEWL. 4 and 5 that the intensity of staining in the hydrophobic region is clearly shown. Moreover, the rank correlation coefficient of the scatter diagram of FIG. 6 shows a significant correlation of 0.615, suggesting a relationship with the barrier function and the degree of maturity of CE. From these, it can be seen that the hydrophobicity-hydrophilicity of the stratum corneum of the present invention can be distinguished with high accuracy.

  According to the present invention, it becomes possible to distinguish the hydrophobicity-hydrophilicity of stratum corneum cells under an optical microscope, eliminating the need for expensive fluorescent microscopes, high-sensitivity cameras and light-shielding equipment such as a dark room, reducing costs and saving space. Can contribute to the development. Furthermore, useful information such as counseling and advice on skin conditions can be provided at department stores and stores.

It is a figure which shows the image of the optical microscope of the stratum corneum cell sample of the dye liquid formulation 1 in Example 1 (drawing substitute photograph). It is a figure which shows the image of the optical microscope of the stratum corneum cell sample of the dye liquid formulation 2 in Example 1 (drawing substitute photograph). It is a figure which shows the image of the fluorescence microscope of the stratum corneum cell sample of the staining liquid prescription 3 as a comparative example (drawing substitute photograph). It is a figure which shows the dyeing | staining image with high hydrophobicity in the stratum corneum cell sample in Example 2 (drawing substitute photograph). It is a figure which shows the dyeing | staining image with low hydrophobicity in the stratum corneum cell sample in Example 2 (drawing substitute photograph). It is a scatter diagram which shows the relationship between the hydrophobicity-hydrophilicity level (1-5) in Example 2, and TEWL (g / m < ² > / h).

Claims (3)

A method of skin differentiation, in which stratum corneum cells collected from the skin are stained with Sudan Black B dye from the outside contact area direction, and the hydrophobicity-hydrophilicity of the stratum corneum cells is differentiated by an optical microscope stained with the dye. A method for distinguishing horny layer cells, characterized by comprising: The method for distinguishing stratum corneum cells according to claim 1, wherein the staining liquid of Sudan Black B contains 30 to 50% by mass of ethanol in the staining liquid. The method for distinguishing horny layer cells according to claim 1 or 2, wherein the method is performed according to the following steps.
(Step 1) Using the adhesive tape, horny layer cells are collected from the skin.
(Step 2) The pressure-sensitive adhesive of the pressure-sensitive adhesive tape is softened and dissolved with an organic solvent, and the stratum corneum cells are transferred onto a slide glass to prepare a stratum corneum cell specimen.
(Step 3) The stratum corneum cell sample is conditioned with an aqueous ethanol solution (about 40% by mass) and then left to stand in a Sudan black B staining solution at room temperature for 24-48 hours for staining.
(Step 4) The stained stratum corneum cell specimen is washed with an aqueous ethanol solution, air-dried, sealed in a balsam, and identified under an optical microscope.

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