JP4469762B2 - Sensitive skin evaluation method and evaluation kit - Google Patents

Description

  The present invention relates to a novel sensitive skin evaluation method and an evaluation kit thereof using a biochemical index capable of simply and reliably evaluating whether the skin is sensitive.

  Sensitive skin is perceived as skin that is prone to adverse and harmful reactions without obvious skin lesions. Or it can be said that sensitive skin is less susceptible to external stimuli than healthy skin and easily causes skin troubles. In recent years, there has been a tendency to increase the number of women who are conscious of sensitive skin, and there is a growing demand for cosmetics that are less irritating. In addition, when carrying out skin care and chemical peeling treatments with cosmetics, beauty specialists evaluated whether the user's skin was sensitive through interviews, etc., and matched the user's skin according to the results. Cosmetics, chemical peeling agents, etc. are used.

  Conventionally, interviews by beauty professionals have been required in this way, but if it is possible to evaluate whether the user's skin is sensitive or not, the cosmetics and chemicals that are more suitable for the user's skin It is possible to easily select a peeling agent, thereby avoiding skin troubles and side effects such as skin irritation, and it is possible to further enhance the benefits of cosmetic skin care and chemical peeling. For this reason, there is a need for a method for objectively determining whether or not the skin is sensitive, regardless of an inquiry.

  In addition, in order to give a burden to a user, it is not appropriate to include a method of surgically removing the skin as disclosed in Non-Patent Document 1 in the cosmetic purpose of evaluating sensitive skin.

  Patent Document 1 discloses a method for evaluating UV irradiation injury to skin using calprotectin as a marker gene. However, this method relates to UV irradiation injury and is not a method for evaluating sensitive skin. Further, this method cannot be said to be a simple method because UV irradiation is required for evaluation.

As a method for evaluating whether or not the skin is sensitive, Patent Document 2 collects keratinocytes of the face and horny cells of a part of the body surface where the number of exposures is low, and the ratio of these sizes is calculated. A method for identifying skin quality as an index is disclosed. Patent Document 3 discloses a skin discrimination method using as an index the difference between the standard keratinocyte area with respect to age and the average cell area of keratinocytes collected from a subject. However, these methods are for evaluating appearance such as size and area, and there are problems such as an evaluation method, a quantification method, and an error caused by individual differences of each examiner.
WO02 / 0909340 JP 2001-108674 A JP-A-11-304798 M. Grewe, J Invest Delmatol 2000, vol 114, pp1108-1112 Eckert RT et al., J Invest Dermatol 2004, vol.123, pp23-33.

  As described above, a method for objectively evaluating whether or not the skin is sensitive is desired. The evaluation method should not be a method that places a burden on the user, such as surgically removing the skin or irritating the skin. In addition, an evaluation method using a biochemical index is desirable instead of evaluating the appearance. Therefore, the present invention provides a novel sensitive skin evaluation method using a biochemical index and an evaluation kit thereof that can easily and reliably evaluate whether the skin is sensitive without burdening the user. Objective.

  As a result of intensive studies in view of the above problems, the presence of calprotectin of sensitive skin-derived keratinocytes in keratinocytes obtained by a simple method of collecting only the surface layer portion of the horny layer with a single keratin tape, such as tape stripping The amount was found to be significantly higher than that from healthy skin. The abundance of calprotectin can be evaluated from keratinocytes obtained without stimulating the skin with ultraviolet rays or chemical substances. This has led to the completion of the present invention in which sensitive skin is simply evaluated using calprotectin as an index. That is, the present invention is as follows.

  The method for evaluating sensitive skin according to claim 1 of the present invention is characterized in that the amount of calprotectin present in keratinocytes is used as an index.

  The method for evaluating sensitive skin according to claim 2 of the present invention is characterized in that the amount of calprotectin present in the keratinocytes other than the vicinity of the pores is used as an index.

  The method for evaluating sensitive skin according to claim 3 of the present invention is the method for evaluating sensitive skin according to claim 1 or 2, in which the corneocytes are collected and the corneocytes collected in the collecting step A measurement step for measuring the abundance of calprotectin and a comparison step for comparing the abundance of calprotectin measured in the measurement step with the abundance of calprotectin in keratinocytes of healthy skin It is characterized by.

  The method for evaluating sensitive skin according to claim 4 of the present invention includes a collecting step of collecting a keratinocyte at a site to be evaluated by a subject, and a measurement for measuring the abundance of calprotectin in the horny cell collected in the collecting step. And a comparison step for comparing the abundance of calprotectin measured in the measurement step with the abundance of calprotectin in the keratinocytes of the site to be evaluated of a person with healthy skin. And

  The kit for evaluating sensitive skin according to claim 5 of the present invention comprises a collecting means for collecting keratinocytes and a measuring means for measuring the abundance of calprotectin in the keratinocytes. And

  The kit for evaluating sensitive skin according to claim 6 of the present invention comprises: a collecting means for collecting keratinocytes; and a measuring means for measuring the abundance of calprotectin in the keratinocytes other than the vicinity of the pores. It is characterized by having.

  According to the method for evaluating sensitive skin according to claim 1 of the present invention, since the sensitive skin has significantly higher abundance of calprotectin in keratinocytes than healthy skin, the abundance of calprotectin in keratinocytes. Whether or not the skin is sensitive skin can be easily evaluated by using the number of cues.

  According to the method for evaluating sensitive skin of claim 2 of the present invention, as the abundance of calprotectin in the keratinocytes, the abundance of calprotectin in the keratinocytes other than the vicinity of the pores is more reliably determined. Can evaluate sensitive skin.

  According to the method for evaluating sensitive skin according to claim 3 of the present invention, in the method for evaluating sensitive skin according to claim 1 or 2, the keratin collected based on the abundance of calprotectin in the keratinocytes of healthy skin. By evaluating the abundance of calprotectin in cells, sensitive skin can be evaluated more accurately.

  According to the method for evaluating sensitive skin according to claim 4 of the present invention, in the method for evaluating sensitive skin according to claim 1 or 2, calprotectin in keratinocytes collected from the same evaluation target site of a person having healthy skin. By evaluating the abundance of calprotectin in the corneocytes collected from the evaluation target site of the subject on the basis of the abundance, the sensitive skin can be evaluated more accurately.

  According to the evaluation kit for sensitive skin according to claim 5 of the present invention, the kit comprises a collecting means for collecting keratinocytes and a measuring means for measuring the abundance of calprotectin in the keratinocytes. Thus, it is possible to provide an evaluation kit for sensitive skin that can easily evaluate sensitive skin.

  According to the sensitive skin evaluation kit of claim 6 of the present invention, a collecting means for collecting keratinocytes, a measuring means for measuring the abundance of calprotectin in the keratinocytes other than the vicinity of the pores, With this, it is possible to provide a sensitive skin evaluation kit that can easily evaluate sensitive skin.

  Hereinafter, the present invention will be described in detail.

  The method for evaluating sensitive skin of the present invention is characterized in that the abundance of calprotectin in keratinocytes is used as an index.

  Calprotectin is a heterodimer produced by pairing S100 proteins S100A8 and S100A9, which are known as a series of calcium-responsive proteins, and is a mediator of signal transduction. Both S100A8 and S100A9 are known to exist in abundance in neutrophils and monocytes, and it is well studied that they are involved in inflammatory effects by binding to vascular endothelial cells and the like. Calprotectin in plasma and S100A8 and S100A9 are also used as markers for acute or homeostatic inflammation. On the other hand, in normal skin, although all molecules are slightly expressed in the hair follicle epithelium and granule layer, the expression level is generally considered to be small. However, it is also well known that S100A8 and S100A9 are highly expressed in the epidermis due to pathological conditions such as psoriasis and external stimuli such as light exposure and barrier destruction. Activin A-high-expressing mice are characterized by hyperproliferation but no inflammation is observed, suggesting that S100A8, S100A9 or both are involved in proliferative responses other than inflammation (Non-patent Document 2).

  Sensitive skin in the present invention is skin that is prone to adverse and harmful reactions without obvious skin lesions. In addition, it can be said that the skin is less susceptible to external stimuli than healthy skin and easily causes skin troubles. On the other hand, healthy skin is healthy and normal skin that does not exhibit the properties of sensitive skin as described above. In sensitive skin, it is known that there is a tendency that the transdermal moisture transpiration (TEWL) increases and the high-frequency conductivity (horny layer moisture) decreases. In sensitive skin, rash and rough skin may occur.

  Rough skin means a state of skin deterioration in a broad sense, and includes rough skin caused by scratching due to pruritus and worsening deterioration of atopic dermatitis, rough skin due to drying, and the like. Therefore, the sensitive skin and rough skin referred to in the present invention are different.

  In the present invention, a keratinocyte is a cell constituting the keratin on the top of the skin. The corneocytes have a function of protecting the skin from foreign substances and irritation from outside the body.

  The site to be evaluated in the present invention may include any site as long as the corneocytes can be obtained, but examples of the main site include the cheek and forehead of the face. In accordance with conventional methods, skin-derived keratinocytes at these sites can be obtained. However, as described above, a method such as surgically removing the skin places a burden on the user, and therefore, a method that can easily obtain keratinocytes such as tape stripping and rubbing is preferable.

  The abundance of calprotectin in each sample thus prepared can be measured by a conventionally known method. For example, methods such as enzyme immunoassay, radioimmunoassay, and Western blotting based on a reaction with an antibody against calprotectin can be used.

  Calprotectin is measured from each sample after extracting calprotectin by a biochemical method known per se, for example, an extraction method in which a soluble fraction is prepared through freeze-thaw method, ultrasonic disruption method, etc. preferable.

  Sensitive skin has significantly higher abundance of calprotectin in keratinocytes than healthy skin, so it is easy to determine whether the skin is sensitive skin by using the amount of calprotectin in keratinocytes as an index. Can be evaluated. The keratinocytes used in the present invention can be collected by a simple method such as tape stripping, in which only the surface layer portion of the horny layer is collected with a single horny tape. In addition, the present invention can measure the abundance of calprotectin without giving the skin a stimulus such as ultraviolet rays or chemical substances. For this reason, it is possible to evaluate whether the user's skin is sensitive without burdening the user, so it is possible to easily select cosmetics and chemical peeling agents that are more suitable for the user's skin. Thus, skin troubles such as skin inflammation and side effects can be avoided, and the benefits of skin care and chemical peeling treatment with cosmetics can be further enhanced.

  In addition, the method for evaluating sensitive skin of the present invention uses the amount of calprotectin present in keratinocytes other than the vicinity of pores as an index. In healthy skin-derived keratinocytes, calprotectin can hardly be detected in keratinocytes other than those near the pores, whereas in sensitive skin-derived keratinocytes, calprotectin is also detected in keratinocytes other than those near the pores. it can. For example, a calprotectin antibody that recognizes calprotectin and a secondary antibody calprotectin labeled with a fluorescent dye that recognizes the calprotectin antibody are reacted with corneocytes and observed with a fluorescence microscope. Thus, the presence of calprotectin in the keratinocytes other than the vicinity of the pores is visually confirmed, and by using it as an index, sensitive skin can be more reliably evaluated.

  The method for evaluating sensitive skin of the present invention includes a collecting step for collecting keratinocytes, a measuring step for measuring the abundance of calprotectin in the horny cells collected in the collecting step, and a calculant measured in the measuring step. A comparison step of comparing the abundance of protectin with the abundance of calprotectin in the keratinocytes of healthy skin. This evaluation method will be described below.

  First, the abundance of calprotectin measured as described above is compared with the abundance of calprotectin in keratinocytes of healthy skin. Further, the abundance of calprotectin is measured in the keratinocytes at a certain evaluation target site of the subject, and the measured abundance of calprotectin is used to calculate the calprotectin in the keratinocytes at the same evaluation target site of a person with healthy skin. You may compare with the abundance of. The abundance of calprotectin in the keratinocytes of people with healthy skin can be evaluated more objectively by using the average value of data from a plurality of people with healthy skin.

  As a result of the comparison, if the measured abundance of calprotectin is significantly larger than the abundance of calprotectin in healthy skin, it is evaluated as having sensitive skin. The healthy skin may be collected from a person who measures calprotectin or may be collected from a person different from the person who measures calprotectin.

  Sensitive skin also occurs when the amount of calprotectin present in the keratinocytes of a subject's evaluation target site is significantly greater than the amount of calprotectin present in the horny cells of the same evaluation target region of a person with healthy skin. It is evaluated that it has.

  When evaluating that the skin has sensitive skin, the degree of sensitive skin can be evaluated by the degree of calprotectin to be measured. That is, when the abundance of calprotectin measured is significantly higher than that of healthy skin, the skin strongly exhibits the properties of sensitive skin and is significantly less resistant to external stimuli than healthy skin. It can be evaluated as sensitive skin that easily causes skin problems. In addition, when the abundance of calprotectin measured is increased by a small amount compared to that of healthy skin, the skin is sensitive skin that is less resistant to external stimuli than normal skin and causes skin problems. Although there is, it can be evaluated that it does not show its properties strongly.

  The evaluation kit for sensitive skin of the present invention comprises a collecting means for collecting keratinocytes and a measuring means for measuring the abundance of calprotectin in the keratinocytes. Instead of the measurement means for measuring the abundance of calprotectin in keratinocytes, the measurement means may be configured to measure the abundance of calprotectin in keratinocytes other than the vicinity of the pores. The collecting means for collecting keratinocytes is not particularly limited as long as it is a means capable of collecting keratinocytes. For example, as described above, keratin tape or the like may be used as a collecting means for collecting keratinocytes. In order to implement this means, the kit can contain, for example, keratin tape. The measuring means for measuring the abundance of calprotectin in the corneocytes is not particularly limited as long as it can measure the abundance of calprotectin. For example, a reagent or instrument for performing an enzyme immunoassay based on a reaction with an antibody against calprotectin as described above may be used as a measuring means for measuring the abundance of calprotectin. In order to carry out this means, the kit may contain, for example, a calprotectin antibody that recognizes calprotectin and a labeled secondary antibody that recognizes the calprotectin antibody as measurement means. Furthermore, the evaluation kit for sensitive skin of the present invention can include reagents (for example, buffering agents) and instruments (microtiter plates, pipettes, etc.) that facilitate the implementation of the above means.

  Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.

(Experiment 1) Measurement of transcutaneous water transpiration (TEWL) The subjects (10 persons) and healthy persons (9 persons) who feel sensitive skin wash their face, and 30 minutes later, transdermal transpiration (TEWL) Was measured with a Thebameter (TM210) made by Curage & Khazaka. Subjects (10 persons) self-reported as having sensitive skin were classified as sensitive skin groups, and healthy persons (9 persons) self-reported as having healthy skin were defined as healthy skin groups.

(Experiment 2) Calprotectin quantification in keratinocytes After the measurement in Experiment 1, keratinocytes were collected from the subjects and healthy subjects using keratin tape. A keratin tape (25 × 45 mm) from which the keratinocytes are collected is cut into 2 mm squares, dispersed in 750 μL of PBS, sonicated under ice-cooling, and centrifuged at 4 ° C. to obtain a supernatant for quantification. It was. The total protein concentration tested by the PROTEIN Assay, Bradford method (BIO-RAD) was about 693 ± 37 ng / mL. 100 μL of this solution was quantified using MRP8 / 14EIA (manufactured by Buhlmann Laboratories AG) according to the manual.

Results of (Experiment 1) and (Experiment 2) The amount of calprotectin obtained in Experiment 2 was compared between the sensitive skin group and the healthy skin group. FIG. 1 is a diagram showing the abundance of calprotectin in keratinocytes of sensitive skin and healthy skin. It was revealed that calprotectin content of facial keratinocytes was significantly higher in sensitive skin groups.

  Furthermore, TEWL obtained in Experiment 1 was compared between the sensitive skin group and the healthy skin group. The sensitive skin group had significantly more TEWL of facial keratinocytes (table in FIG. 2). As described above, it is known that in sensitive skin, TEWL measured as an index of the skin barrier function tends to increase, and the result of FIG. When the correlation between TEWL and calprotectin levels obtained from the subjects and healthy subjects was examined, a relatively good correlation was obtained (graph in FIG. 2).

  From the above, it was clarified that the amount of calprotectin present in keratinocytes derived from sensitive skin was significantly higher than that of healthy skin, and calprotectin could be associated with sensitive skin.

(Experiment 3) Calprotectin immunostaining in corneocytes 15 minutes after washing the face, a small amount (about 3 mm in diameter) of medical Aron Alpha A (Sankyo Pharmaceutical) was put on a silane-coated slide and immediately lightly applied to the target evaluation site. After adhering and holding for 3 minutes, the keratinocytes were collected by slowly peeling off. This was ethanol 2 minutes, washed (3 minutes in PBS solution), 3% PFA-containing PBS solution 10 minutes, washed (5-10 minutes in PBS solution 3 times), 0.1% Triton X-100-containing PBS solution 10 minutes, washing (repeat 5-10 minutes in PBS solution 3 times), blocking with 10% goat serum (Nichirei) for 1 hour, mouse anti-human MRP8 / 14 monoclonal antibody T-1023 (BMA Biomedicals AG) 1 μg / mL PBS dilution was allowed to react overnight at 4 ° C. After washing (5-10 minutes in PBS solution 3 times), a 200-fold diluted PBS solution of Alexa Fluor 488-labeled anti-mouse IgG antibody was reacted for 1 hour.

Results of (Experiment 3) FIG. 3 shows a photo of the microscope. The upper row is a photograph of the result of observing the same site of keratinocytes of sensitive skin with a fluorescence microscope (upper left) and an optical microscope (upper right). The lower row is a photograph of the result of observing the same site of keratinocytes in healthy skin with a fluorescence microscope (lower left) and an optical microscope (lower right). Where calprotectin is present, fluorescence is emitted by Alexa Fluor 488-labeled anti-mouse IgG antibody. Representative sites where calprotectin is present are indicated by arrows (keratinocytes in the vicinity of the pores and in the vicinity of the pores).

  Comparing the photographs of the fluorescence microscope, it was confirmed that calprotectin was present in the vicinity of the pores in both healthy and sensitive skin. On the other hand, in the keratinocytes other than the vicinity of the pores, calprotectin was observed only in sensitive skin. Considering that it is difficult to collect proteins deep in the pores with keratin tape, it is considered that calprotectin collected and detected by keratin tape exists outside the vicinity of the pores. It was estimated that calprotectin levels in keratinocytes other than those near the pores were increased.

It is the figure which showed the abundance of calprotectin in the keratinocytes of sensitive skin and healthy skin. The point of a graph shows the abundance of calprotectin of a test subject and a healthy person, respectively. It is the figure which showed the result of the comparison between the groups of TEWL in the keratinocytes of sensitive skin and healthy skin, and the correlation of calprotectin amount and TEWL in the keratinocytes of sensitive skin and healthy skin. It is the figure which showed the microscope picture of the keratinocyte of sensitive skin and healthy skin. The upper row is a photograph of the result of observing the same site of keratinocytes of sensitive skin with a fluorescence microscope (upper left) and an optical microscope (upper right). The lower row is a photograph of the result of observing the same site of keratinocytes in healthy skin with a fluorescence microscope (lower left) and an optical microscope (lower right). Representative sites where calprotectin is present are indicated by arrows (keratinocytes in the vicinity of the pores and in the vicinity of the pores).

Claims (6)

A method for evaluating sensitive skin, characterized by using as an index the amount of calprotectin present in keratinocytes. A method for evaluating sensitive skin, characterized by using as an index the amount of calprotectin present in keratinocytes other than in the vicinity of pores. A collecting step for collecting keratinocytes, a measuring step for measuring the abundance of calprotectin in the keratinocytes collected in the collecting step, and an abundance of calprotectin measured in the measuring step as a keratin in healthy skin The method for evaluating sensitive skin according to claim 1, further comprising a comparison step of comparing with the abundance of calprotectin in the cells. A collecting step for collecting the keratinocytes of the evaluation target site of the subject, a measuring step for measuring the abundance of calprotectin in the keratinocytes collected in the collecting step, and the presence of the calprotectin measured in the measuring step The method for evaluating sensitive skin according to claim 1, further comprising a comparison step of comparing the amount with the amount of calprotectin present in the keratinocytes of the evaluation target site of a person having healthy skin. A kit for evaluating sensitive skin, comprising: a collecting means for collecting keratinocytes; and a measuring means for measuring the abundance of calprotectin in the keratinocytes. A kit for evaluating sensitive skin, comprising: a collecting means for collecting keratinocytes; and a measuring means for measuring abundance of calprotectin in keratinocytes other than the vicinity of the pores.