JPH11344489A - Method for determining melanine of horny layer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply, correctly detect melamine of the skin by transferring horny layer cells of the skin to a double-sided tape attached to one face of a transparent plate, dyeing and determining the cells. SOLUTION: An adhesive plate obtained by attaching a double-sided tape to one face of a transparent plate is brought in tight contact with a skin surface, whereby horny layer cells of the skin surface are transferred to an adhesive face. The transparent plate is a glass plate, a polyethylene plate or the like which is insoluble to an organic solvent. The glass plate having a superior transparency is preferred and formed in a size and a shape nearly close to a slide glass not obstructing a microscopic observation. An adhesive of the double-sided tape is preferably a rubber substance such as a natural rubber, a tragacanth gum or the like in terms of a separation easiness when the tape is separated from the skin. The double-sided tape is smaller than a plane of the transparent plate, has a size enabling a quantity of the horny layer cells sufficient for the microscopic observation to be transferred and is formed of cellophane, a fluororesin or the like. The horny layer cells transferred onto the transparent plate are dyed and a quantity of a dyed melamine per unit area of the horny layer cells is determined on the transparent plate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a method for quantifying a biological component in in sizu . More specifically, the invention relates to a method for quantifying melanin in the stratum corneum, in which the amount of melanin in the stratum corneum is quantified as the amount of melanin in stratum corneum cells.

[0002]

BACKGROUND OF THE INVENTION The amount of melanin in the skin is a factor closely related to the beauty of the skin. That is, it is a well-known fact that the amount of melanin in the skin increases due to the effect of ultraviolet rays in sunlight, aging, and the like, causing stains and freckles. For the purpose of controlling the amount of melanin in this skin and protecting against spots and freckles, so-called "whitening cosmetics" are used, and such "whitening cosmetics" are used to eliminate once-generated spots and freckles. Obviously, it is more preferable to use it prophylactically to prevent its occurrence.

In order to control the amount of melanin for the purpose of prevention, not only can the amount of melanin in the skin be accurately grasped, but also the amount of melanin can be grasped easily. is necessary. That is,
It is preferable to use the most appropriate whitening cosmetic in accordance with the amount of melanin that is sequentially grasped in order to control the amount of melanin in the skin. Further, it is very preferable to provide an accurate and simple means for quantifying the amount of melanin in the skin in developing an excellent whitening cosmetic.

[0004]

However, at present, it cannot be said that means for quantifying melanin in the skin, which has the above-mentioned accuracy and simplicity, has been provided.

The problem to be solved by the present invention is to provide a method for quantifying melanin in skin, which has such accuracy and simplicity.

[0006]

Means for Solving the Problems As a result of diligent studies for solving this problem, the present inventor applied a double-sided tape to one side of a transparent plate, and transferred and removed the stratum corneum cells of the skin to the double-sided tape. It is possible to easily and accurately grasp the melanin of the skin by quantifying the copied melanin of the stratum corneum, preferably while removing noise due to elements other than the stratum corneum by image processing. Heading,
The present invention has been completed.

That is, according to the present invention, a double-sided tape is adhered to one side of a transparent plate, the non-contact surface of the double-sided tape with the transparent plate is brought into close contact with the skin, and the horny cells on the skin surface are adhered to the adhesive surface. Transferred, the transferred keratinocytes are subjected to a staining treatment, and the amount of melanin in the keratinocytes is reduced on the transparent plate, preferably, by image processing, noise due to elements other than the keratinocytes is removed in the image. And a method for quantifying melanin in the stratum corneum while quantifying melanin in the stratum corneum.

[0008]

Embodiments of the present invention will be described below. In the method for quantifying melanin in the skin according to the present invention (hereinafter, referred to as the melanin quantifying method of the present invention),
First, it is necessary to bring a "adhesive plate" in which a double-sided tape is adhered to one surface of a transparent plate into close contact with the skin, and transfer keratinocytes on the skin surface to the adhesive surface.

The transparent plate used is not particularly limited as long as it is transparent in principle and does not hinder the determination of melanin described later and is insoluble or hardly soluble in an organic solvent. For example, a glass plate, a polymethylpentene plate, A polyethylene plate and the like are exemplified. Generally, it is preferable to select a glass plate having excellent transparency.

The size and shape of the transparent plate are not particularly limited as long as they do not hinder direct observation with a microscope, and generally have a size and shape similar to a general slide glass.

The pressure-sensitive adhesive of the double-sided tape to be adhered to one side of the transparent plate can be a general pressure-sensitive adhesive that can be used as a pressure-sensitive adhesive of a generally known pressure-sensitive adhesive tape, and is not particularly limited. Vinyl, polyvinyl alcohol, polyvinyl acetal, polyvinyl chloride, polyvinylidene chloride, polyvinyl ether, polybutene, asphalt, natural rubber or its derivatives, SBR rubber,
Rubber materials such as NBR rubber, butadiene-vinyl pyridine rubber, butyl rubber, chloroprene rubber, recycled rubber, cyanoacrylate rubber, arabic rubber, and tragacanth rubber can be used alone or in combination.

[0012] Among these adhesive substances, it is preferable to select a rubber material that is equal to or less than natural rubber, in that it does not easily affect the releasability when the tape is peeled off from the skin and the keratinocytes themselves.

[0013] As a material of the tape itself of the double-sided tape,
The material is not particularly limited as long as it is excellent in transparency and is generally used as a material for an adhesive tape. For example, cellophane, polyethylene, polypropylene, polyvinyl chloride, polyester, fluororesin, etc. may be used. it can.

As the double-sided tape, a commercially available product can be used. For example, Scotch Double-Coa
Ted Tap (manufactured by 3M), Nystack (manufactured by Nichiban), hot melt (manufactured by HB Houra) and the like can be exemplified. The size of the double-sided tape to be attached to the transparent plate is at least smaller than the plane of the transparent plate, and it is possible to transfer a sufficient amount of horny cells on the skin surface from the skin to perform microscopic observation. Preferably, it is a size.

As described above, the "adhesive plate" in which the double-sided tape is adhered to one surface of the transparent plate is brought into close contact with the skin, and the keratinocytes on the skin surface can be transferred to the adhesive surface.

In the present invention, the use of a double-sided tape as the adhesive means makes it easier to produce an adhesive plate than to use an "adhesive plate" produced by directly applying an adhesive to a transparent plate, It is preferable in that it has excellent operability.

In the melanin quantification method of the present invention, as described above, the keratinocytes on the skin surface transferred to a transparent plate are subjected to a staining treatment, and the amount of melanin in the keratinocytes is quantified on the transparent plate. It is necessary to.

The staining treatment is not particularly limited as long as it can stain melanin in keratinocytes accurately. For example, a melanin having a silver-reducing property can be stained with a black-brown ammonia silver solution. As desired, by fixing silver with sodium thiosulfate or the like,
Melanin in keratinocytes can be stained in sizu .

Further, in order to clarify the morphology of keratinocytes and the presence of cell nuclei, the specimen is immersed in a hematoxylin solution, and the cell nuclei are stained and washed in blue-blue to pale-blue, and then treated with an eosin solution. And so on.

In order to improve the preservability of the keratinocyte specimen prepared in this manner, glycerin, balsam, or ultraviolet curable resin can be sealed.

The desired amount of melanin can be determined by specifying the amount of stained melanin per corneal cell area. This determination of the amount of melanin can be performed by observing the horny cell sample obtained as described above with an optical microscope. It is also possible to project the image on a monitor using a video scope for observation.

However, in order to quantify more desired melanin, a commercially available image analysis system [Mac As
pect: It is preferable to perform the image processing and quantify the melanin by using software (manufactured by Mitani Shoji Co., Ltd.) having a data storage system added to the Mac scope.

Further, in the melanin quantification method of the present invention, when specifying the amount of melanin, image processing is used to quantify melanin in horny cells while removing noise due to elements other than keratinocytes in the image. Is preferred.

By this image processing, the desired amount of melanin can be easily determined without passing through a step of applying a chemical means for removing noise such as dirt as in the prior art. Such image processing can be performed by using programmed software so that elements other than melanin can be removed from the image. It should be noted that software having such properties is already commercially available [for example, Adobe Photoshop].
(Adobe Systems Japan), etc.].

[0025]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the technical field of the present invention.

Preparation of the adhesive plate Peel off one side of the double-sided tape (hot melt: H. B. Hula) cut to 2.0 × 2.0 cm, and take care not to allow air bubbles to enter the tape. Was attached to the center of the disinfected and dried slide glass. This was subjected to the following tests as an “adhesive plate” (hereinafter, simply referred to as an adhesive plate).

Determination of melanin in the stratum corneum (1) Peel off the double-sided tape on the stratum corneum collecting surface of the adhesive plate and lightly attach the double-sided tape to the cheek of a healthy panel (female: 40s) Then, the horny cells on the skin surface at that portion were transferred to the adhesive surface, and the horny cells were collected on the adhesive surface.

The adhesive plate to which the corneal cells had adhered was washed with distilled water, immersed in Fontana Masson silver solution (prepared at the time of staining) for one day, and stained with melanin. After the immersion, the adhesive plate was washed with distilled water and brought into contact with a sodium thiosulfate solution to stop the dyeing reaction.

Further, after the cytoplasm of the keratinocytes was stained by eosin staining, a dehydration operation was performed, and balsam was encapsulated to prepare a keratinocyte specimen. A CCD camera (DXD-950: manufactured by Sony) is connected to the optical microscope, the stratum corneum is projected, and an image analysis system [Adobe Photoshop] is used.
op (Adobe Systems Japan)], elements that are not directly related to the determination of melanin, such as stains, were deleted from the image.

FIG. 1 is an optical microscope image (20 ×) of the keratinocytes before the above image processing, and FIG. 2 is an optical microscope image (20 ×) of the keratinocytes after the image processing. .

From these photographs, skin-like keratinocytes are stained on the adhesive plate, and melanin stained with silver is observed in them. Then, it became clear that the image subjected to the image processing (FIG. 2) can provide a clearer melanin or keratinocyte staining image than the image not subjected to the image processing (FIG. 1).

(2) Determination of melanin in the stratum corneum (2) A panel of dark-skinned females (40's) and a panel of fair-skinned females (40's) were respectively treated in the same manner as described in (1) above. A sample of keratinocytes was prepared. Each keratinocyte specimen is subjected to image processing in the same manner as in the above (1) to remove elements not directly related to melanin quantification, and Mac Aspect (manufactured by Mitani Corporation) is used. By using the cells, the area of the stratum corneum and the melanin in the cells were quantified, and the melanin content in the stratum corneum was quantified.

In order to examine the reliability of the melanin quantification method of the present invention, the melanin index (mexameter MX16: Eurotech Japan Co., Ltd.), a melanin measuring instrument that has already been established, is used to measure Yes, indicating that the amount of melanin per unit is large) was examined for each panel. The result is
It is shown in Table 1.

Table 1 Panel Melanin Index Stratum corneum melanin content (%) ──────────────────────────────────── Dark-skinned women 501 17.9 Fair-skinned women 456 7.6────────────────────────────────────

According to this result, melanin was observed in dark-skinned women.
As the Index is higher than that of fair-skinned women, and in response, the melanin content of the stratum corneum is clearly higher in dark-skinned women than in fair-skinned women, and the information provided by the melanin quantification method of the present invention. Indicates that the information is highly reliable.

[0036]

According to the present invention, there is provided a means for quantifying melanin in skin, which has both accuracy and simplicity.

[Brief description of the drawings]

FIG. 1 is an optical microscope image (20 ×) of a keratinocyte before predetermined image processing.

FIG. 2 is an optical microscope image (× 20) of a keratinocyte after predetermined image processing.

Claims (3)

[Claims] 1. A double-sided tape is attached to one side of a transparent plate,
The non-contact surface of the double-sided tape with the transparent plate is brought into close contact with the skin, the keratinocytes on the skin surface are transferred to the adhesive surface, and the transferred keratinocytes are subjected to a staining treatment to melanin in the keratinocytes. A method for quantifying stratum corneum melanin, wherein the amount is quantified on the transparent plate. 2. The method according to claim 1, wherein the transparent plate is a glass plate. 3. The method for quantifying melanin in the stratum corneum according to claim 1 or 2, wherein the stratum corneum cells transferred onto a transparent plate and subjected to a staining treatment are subjected to image processing to determine the presence of elements other than stratum corneum. A method for quantifying melanin in the stratum corneum, wherein the melanin in the stratum corneum is quantified while removing noise in the image.