TW201514494A - Method of evaluating degree of skin stress accumulation - Google Patents

Abstract

The purpose of this invention is to provide a novel evaluation method, which is capable of evaluating the degree of skin stress accumulation and further of simply and reliably evaluating whether the skin is sensitive, without being burdensome to a user. According to the present invention, the degree of stress accumulation is evaluated by sampling the stratum corneum via tape stripping, etc., and by assaying the expression level of DJ-1 contained therein.

Description

Evaluation method of skin stress accumulation degree

The present invention relates to a method for evaluating the amount of stress accumulation in human skin.

Human skin is often exposed to the outside world and subjected to various stimuli. A representative example of such stimulation is ultraviolet light. Ultraviolet rays cause DNA rupture of skin cells or denaturation of collagen even if they do not cause too much change in the appearance of the skin, and cause skin aging, which often causes stress on the skin. In addition, for women, daily activities such as face washing and makeup can also cause irritation to the skin, and these stimuli accumulate in the skin as potential stress. Moreover, if the resistance to these stimuli (stress) is reduced for some reason, it is considered to be sensitive skin. The amount of accumulation of skin external stimuli caused by such ultraviolet rays or oxidation is referred to as the stress accumulation amount of the skin in the present invention. On the other hand, sensitive skin refers to a skin that has no obvious skin lesions but is likely to cause adverse and harmful reactions. Moreover, as a cause of such sensitive skin, it is said that the sensitivity of the skin is improved when the accumulation of stress reaches a certain level. In addition, sensitive skin is less resistant to external stimuli than healthy skin, and it is easy to produce skin. The skin of the problem. In recent years, there has been an increase in the number of women who are aware of sensitive skin, and there is an increasing expectation of products that are less stimulating in cosmetics.

At the sales site of cosmetics, a cosmetic expert conducts a consultation and evaluates the accumulation of skin stress and whether it is a sensitive skin, thereby selecting a cosmetic that causes less stress (stimulation) on the skin. However, in recent years, sales methods such as mail-order sales and the like are not widely available. Therefore, it is desirable to determine the stress state of the skin, and it is possible to easily evaluate the degree of sensitive skin even without an expert. If this is feasible, even if you do not rely on the expert's consultation, you can choose the appropriate cosmetics by evaluating the stress state of the user's skin and whether it is sensitive skin. In addition, it is possible to easily select a cosmetic or chemical peeling agent which is more suitable for the skin of the user, thereby avoiding skin problems and side effects such as inflammation of the skin, and further improving the skin care effect and chemistry of the cosmetic. The beneficial effects of skin rejuvenation. As described above, a method is being sought for evaluating the external stimuli exposure process of the skin (the amount of accumulation of skin stress) without relying on the consultation of a beauty expert, and objectively discriminating whether it is sensitive skin.

Patent Document 1 discloses a method of applying a cosmetic to a skin, and then recovering and observing the exfoliated skin stratum corneum to evaluate the sensitivity of the skin. However, this method requires examination of each cosmetic product, and in addition, the amount of stress accumulation of the user's skin cannot be evaluated.

Patent Document 2 discloses a method of applying a stimulating substance such as capsinoids to the skin to evaluate whether it is sensitive skin. However, the use of substances such as capsaicin esters Test methods for irritating substances are not good for the subject.

Patent Document 3 discloses a method of measuring the amount of calprotectin present in the stratum corneum of the face to evaluate whether it is sensitive skin.

The applicant has found that the DJ-1 protein (alias PARKK protein) having an antioxidant action is useful as a marker for atopic dermatitis, and thus a patent application (Patent Document 4) has been proposed. On the other hand, in recent years, it has been gradually shown that the DJ-1 protein can be utilized as a marker for various diseases. Patent Document 4 discloses an antibody that specifically recognizes an oxidized DJ-1 protein. Next, a method for diagnosing Alzheimer's disease and Parkinson's disease by measuring the oxidized DJ-1 using the antibody has been proposed. Further, Patent Document 5 discloses a method for diagnosing a brain injury disease by measuring DJ-1 protein such as cerebrospinal fluid or blood. Further, Patent Document 6 proposes a method and a reagent for diagnosing diabetic retinopathy by measuring DJ-1 protein.

Patent literature

Patent Document 1: Japanese Laid-Open Patent Publication No. Hei 10-295648

Patent Document 2: Japanese Laid-Open Patent Publication No. 2005-296007

Patent Document 3: Japanese Patent No. 4469762

Patent Document 4: WO2007/046463

Patent Document 5: Japanese Patent Publication No. 2007-506086

Patent Document 6: Japanese Laid-Open Patent Publication No. 2009-168819

As mentioned above, it is desirable to evaluate ultraviolet light or oxidation. The amount of skin stress accumulated and objectively evaluate whether it is a sensitive skin. Preferably, the evaluation method is not a method of causing a burden on the user by surgically removing the skin or causing irritation to the skin. An object of the present invention is to provide a novel evaluation method using a biochemical index that does not burden a user and can evaluate the amount of stress accumulation of the skin.

The present invention consists of the following contents.

(1) A method for evaluating the amount of stress accumulation in human skin, characterized in that the DJ-1 expression amount of the skin stratum corneum is compared with the DJ-1 expression amount of normal human skin measured in advance or simultaneously.

(2) The evaluation method according to (1), comprising: an acquisition step of collecting a stratum corneum of the evaluation target portion of the subject; and a measuring step of measuring the keratin collected by the collecting step a DJ-1 expression amount in the layer; and a comparison step of comparing the DJ-1 expression amount measured in the measuring step with the DJ-1 expression amount in the stratum corneum of the evaluation subject site of a healthy skin person .

(3) The evaluation method according to (1) or (2), comprising the steps of: collecting a stratum corneum of the evaluation target site of the subject; and measuring a step of measuring the collection The DJ-1 expression amount in the stratum corneum collected in the step; and a comparison step of expressing the DJ-1 expression amount in the measuring step and the DJ-1 expression in the stratum corneum of the evaluation target portion of the sample group The quantity distribution is compared.

(4) The evaluation method according to any one of (1) to (3) wherein the step of collecting the stratum corneum of the skin is performed by a tape peeling method.

(5) The evaluation method according to any one of (1) to (4), wherein The stress accumulation amount of the skin-like skin is the amount of stress accumulation caused by ultraviolet damage or oxidation.

According to the evaluation method of the present invention, the amount of stress accumulation of the skin can be evaluated by measuring the amount of DJ-1 expression in the stratum corneum. Further, since the evaluation method of the present invention collects an evaluation sample by a simple operation method such as a tape peeling method, the burden on the subject is small and any person can easily evaluate it. In addition, since it is a biochemical test method, the same result can be obtained by any person, and therefore it is not necessary to consult with an expert as in the past.

Fig. 1 is a graph showing the results of measuring oxidative stress caused by hydrogen peroxide in HaCaT cells and measuring the amount of protein in cells.

Fig. 2 is a graph showing the results of measuring oxidative stress caused by hydrogen peroxide in HaCaT cells and measuring the amount of DJ-1 in cells.

Fig. 3 is a graph showing the results of measuring the amount of DJ-1 released into the culture solution when oxidative stress caused by hydrogen peroxide in HaCaT cells was measured.

Figure 4 is a graph showing the effects of protein formation by BSO.

Fig. 5 is a graph showing the concentration of glutathione produced by cultured human epidermal keratinocytes by BSO.

Fig. 6 is a graph showing the amount of LDH secreted into a cell culture fluid by human epidermal keratinocytes loaded with stress caused by glutathione production inhibitor (i.e., BSO) and hydrogen peroxide.

Figure 7 shows the load of glutathione production inhibitor (BSO) and A graph of the amount of intracellular protein in human epidermal keratinocytes induced by hydrogen peroxide.

Fig. 8 is a graph showing the amount of intracellular DJ-1 of human epidermal keratinocytes expressing stress induced by glutathione production inhibitor (i.e., BSO) and hydrogen peroxide.

Fig. 9 is a graph showing the amount of DJ-1 secreted into a cell culture fluid by human epidermal keratinocytes loaded with stress caused by glutathione production inhibitor (i.e., BSO) and hydrogen peroxide.

Fig. 10 is a graph showing the amount of intracellular protein after 24 hours of irradiation of UVB in cultured human epidermal keratinocytes, and showing a decrease in the amount of protein according to the amount of UVB irradiation.

Fig. 11 is a graph showing the amount of intracellular DJ-1 after 24 hours of irradiation of UVB in cultured human epidermal keratinocytes, and showing an increase in the amount of DJ-1 according to the amount of UVB irradiation.

Fig. 12 is a graph showing the amount of DJ-1 in the culture supernatant after the human epidermal keratinocytes were irradiated with UVB for 24 hours and showing an increase in the amount of DJ-1 according to the amount of UVB irradiation.

Fig. 13 is a view showing the measurement result of DJ-1 after the human body is irradiated with UVB (stress load), and means that the stress caused by the irradiation continues to remain in the stratum corneum of the skin.

Fig. 14 is a graph showing the results of the questionnaire survey of the sarcoplasm and the corresponding amount of DJ-1.

Fig. 15 is a graph showing the results of a questionnaire survey of the experience of habitually irradiating ultraviolet rays and the corresponding amount of DJ-1.

Figure 16 is a graph showing the distribution of skin DJ-1 of 205 women randomly selected. The histogram shows the concentration and number of stratum corneum proteins per unit of DJ-1. The line chart shows the cumulative number of people.

Hereinafter, the present invention will be described in detail.

The evaluation method of the present invention is characterized in that the amount of DJ-1 present in the stratum corneum of the skin is used as an index.

DJ-1 is an intracellular protein with a molecular weight of 21 kDa. As described above, it was identified as a protein associated with Parkinson's disease, which was later shown to be involved in re-epithelialization during skin wound healing. In addition, it can also function as a protease from its stereoscopic structure.

The present invention uses terms such as sensitive skin and healthy skin. Sensitive skin refers to a skin that has no obvious skin lesions but is prone to adverse and harmful reactions. In addition, its resistance to external stimuli is lower than that of healthy skin, and it is also a skin that is prone to skin problems such as rash and rough skin. On the other hand, healthy skin refers to healthy and normal skin that does not exhibit the sensitive skin properties as described above.

In addition, the stratum corneum refers to the tissue located at the uppermost layer of the skin. The stratum corneum has the function of protecting the skin from foreign bodies and stimuli from outside the body.

The evaluation target portion in the present invention may include any portion as long as it is a portion in which the stratum corneum layer is obtained. Examples of the main site include a face, a neck portion, and an upper arm portion, which can be obtained by a conventional method. The stratum corneum of the skin of the part. However, as described above, since the method of surgically removing the skin or the like imposes a burden on the user, a method of easily obtaining the stratum corneum such as tape peeling and rubbing is preferable.

The amount of DJ-1 expressed in each sample thus prepared can be measured by a conventionally known method. For example, based on the reaction with an antibody against DJ-1, a method such as an enzyme immunoassay, a radioimmunoassay, or a Western blot method can be used.

The DJ-1 was prepared from each sample by a biochemical method known per se, for example, by freeze-thaw method, ultrasonication method, homogenization method, etc., and was quickly measured after extraction.

Since the amount of DJ-1 in the stratum corneum that accumulates stressed skin or sensitive skin is significantly higher than that of healthy skin, the stress accumulation can be easily evaluated by using the amount of DJ-1 in the stratum corneum as an indicator. Whether the amount or skin is sensitive skin. The stratum corneum used in the present invention can be collected by a simple method of collecting only the surface layer portion of the stratum corneum using a keratin tape by a tape peeling method or the like. Further, the present invention can measure the amount of expression of DJ-1 without causing irritation to ultraviolet rays or chemicals or the like on the skin. Therefore, the amount of stress accumulation of the user's skin can be evaluated without burdening the user. In particular, it is possible to easily select a cosmetic or chemical rejuvenating agent that is more suitable for the skin of the user, thereby avoiding skin problems such as inflammation of the skin or side effects, and further exerting the skin care effect brought by the cosmetic.

The evaluation method of the present invention comprises the following steps: an acquisition step of collecting a stratum corneum; an assay step of determining a DJ-1 expression amount in the stratum corneum collected in the collection step; and a comparison step, which is The amount of DJ-1 measured in the measurement step was compared with the amount of DJ-1 present in the stratum corneum of healthy skin. Further, the evaluation method of the present invention comprises the following steps: an acquisition step of collecting a stratum corneum; an assay step of determining a DJ-1 expression amount in the stratum corneum collected in the collection step; and a comparison step The DJ-1 expression amount measured in the measurement step was compared with the DJ-1 expression amount distribution in the stratum corneum of the sample set. Hereinafter, the evaluation method will be described.

First, the amount of DJ-1 measured as described above was compared with the amount of DJ-1 present in the stratum corneum of healthy skin. In addition, the DJ-1 expression amount in the stratum corneum of a certain evaluation target site of the subject can also be measured, and the measured DJ-1 expression amount and the DJ- in the stratum corneum of the same evaluation target site of the healthy skin person can be measured. 1 The amount of performance is compared. By using the average value of data from a plurality of healthy skins, the DJ-1 expression level of the stratum corneum of healthy skin can be more objectively evaluated.

Alternatively, the DJ-1 expression amount in the stratum corneum of a certain evaluation target site of the subject may be measured, and the measured DJ-1 expression amount and the DJ-1 expression in the stratum corneum of the same evaluation target portion of the sample group may be measured. The quantity distribution is compared. The DJ-1 expression distribution by using the sample group can be evaluated more objectively.

The result of this comparison is that when the measured DJ-1 expression is significantly greater than the DJ-1 expression of healthy skin, or is compared to the DJ-1 expression distribution of the sample group, it is judged as DJ-1 expression. When the amount is large, it can be evaluated as a large accumulation of stress. The sample of the healthy skin can be collected from the person measuring DJ-1 or from the person different from the person measuring DJ-1. In addition, the sample set is preferably randomly sampled on a statistically effective scale, but is also preferably Sample groups are drawn according to different purposes, for example, by gender and age.

In addition, when the amount of DJ-1 in the stratum corneum of a certain evaluation target site of the subject is significantly larger than that of the DJ-1 in the stratum corneum of the same evaluation subject site of the healthy skin, or compared with the sample When the DJ-1 expression amount distribution in the stratum corneum of the same evaluation target site was determined and the DJ-1 expression amount was large, it was also evaluated that the accumulation of stress was large. In addition, when the value is significantly high, it is evaluated as having sensitive skin.

That is, the degree of stress accumulation can be evaluated based on the measured degree of DJ-1 expression. When the measured amount of DJ-1 is judged to be significantly more than that of healthy skin or the sample group, the skin exhibits a strong sensitive skin property and its resistance to external stimuli. It is significantly lower than the average value of healthy skin or sample group, and is highly likely to cause skin problems.

The invention will be described by way of Test Examples and Examples. The invention is not limited to the following examples.

Example

Test example 1

Changes in the amount of DJ-1 from skin cells caused by oxidative stress were measured. Cells for knocking down the DJ-1 gene for confirming DJ-1 function were prepared and subjected to an oxidative stress load test.

The relationship between oxidative stress and DJ-1 protein was confirmed using a human cell strain, that is, HaCaT cells (J. Cell Biol. 106: 761-771 (1988).) (purchased from DKFZ (Deutsches Krebsforschungszentrum)). In addition, inhibition of DJ-1 gene expression (weakening) by siRNA was confirmed Changes in the amount of DJ-1 protein expression.

(1) The load of oxidative stress caused by HaCaT cell culture and hydrogen peroxide (H ₂ O ₂ ) treatment and the weakening of DJ-1 gene caused by siRNA treatment

After HaCaT cells were washed with PBS(-), the cells were detached by trypsin treatment, and centrifuged at 1200 rpm for 3 minutes to obtain cells. The culture was carried out using DMEM medium containing 10% FBS, seeded into a culture flask at a density of 5000 cells/cm ² , and subcultured in a subconfluent state every 2 to 3 days.

HaCaT cells were seeded in a 12-well plate at a concentration of 2.1×10 ⁵ cells/well in DMEM medium containing 10% FBS and cultured for 24 hours, and then siRNA (final concentration 3 nM, Hs_PARK7_6 FlexiTube siRNA, SI02662107, manufactured by Qiagen), A transfection reagent (2 μl/well, HiPerFect Transfection Reagent, 301707, manufactured by Qiagen) was mixed with the medium and allowed to stand at room temperature for 10 minutes. Then, 100 μl/well of this mixture was added. Untreated cells served as a control group.

In addition, the same operation was performed using AllStars negative control siRNA (manufactured by Qiagen) as a negative control group for the weakening operation.

After culturing for 48 hours, H ₂ O ₂ (manufactured by Wako) was added at 0, 250, 500, 750, and 1000 μM, and after 24 hours, the culture supernatant was collected, and the cells were washed with PBS (-), and then 100 μl/well of cell lysis was added thereto. A solution (50 mM Tris-HCl, 1 mM Na ₃ VO ₄ , 0.4% NP-40, 120 mM NaCl) was obtained to obtain a cell lysate.

(2) Determination of the amount of cellular protein

The BCA protein kit (manufactured by Thermo Scientific) was used to measure fine The amount of protein in the lysate. The assay was performed according to the set of experimental guidelines.

(3) Determination of the amount of DJ-1 expression

The amount of DJ-1 in the supernatant and in the cell lysate was measured using a DouSet Park7/DJ-1 ELISA kit (manufactured by R&D). The assay was performed according to the set of experimental guidelines.

(4) Results

The amount of cellular protein in each cell of the stress load caused by hydrogen peroxide (H ₂ O ₂ ) is shown in Fig. 1 . Further, the amount of DJ-1 in the culture supernatant and the amount of DJ-1 in the cell lysate (intracellular) are shown in Fig. 2 and Fig. 3, respectively. The measurement results are expressed by the mean value ± standard deviation (SD) of every 3 wells.

H ₂ O ₂ reduces the amount of intracellular protein in a concentration-dependent manner.

In addition, it was confirmed that H ₂ O _{2 has} an effect on cells as a strong stress.

Further, in the case where the concentration of H ₂ O ₂ in the culture solution was 750 μM, the amount of intracellular protein was significantly reduced in the weakened cells treated with DJ-1 siRNA as compared with the negative control group. (Figure 1).

Regardless of the cell, when the concentration of H ₂ O ₂ in the culture solution was 1000 μM, the amount of DJ-1 in the cells increased.

On the other hand, when the concentration of H ₂ O ₂ in the culture solution was low, the amount of DJ-1 in the culture supernatant also increased.

It was confirmed that oxidative stress caused by hydrogen peroxide increased DJ-1 both inside and outside the cell (Figs. 2 and 3).

Further, regardless of whether or not H ₂ O _{2 was present in the} culture solution, the cells of the negative control group (non-weakened cells) increased in the amount of DJ-1 in the cells as compared with the control group. This is considered to be the effect of transfection treatment.

The amount of DJ-1 in the cells was reduced by about 40% compared to the negative control group by DJ-1 siRNA treatment.

From the above test, it was found that in HaCaT cells derived from human skin, DJ-1 was produced in response to stress, and the amount of production was inhibited by siRNA interference.

Test example 2

According to Test Example 1, it was confirmed that the DJ-1 gene expresses DJ-1 in response to stress. Next, cells were cultured using human epidermal keratinocytes (keratinocytes), and changes in the amount of DJ-1 caused by oxidative stress were measured. Further, changes in the amount of DJ-1 of keratinocytes which have improved glutathione-producing ability and reduced stress resistance which are improved in cell damage caused by oxidation were measured.

(1) Preparation of cells with reduced oxidative stress resistance caused by glutathione synthesis inhibitor

Human epidermal keratinocytes (Lonza) were washed with PBS(-), and the cells were detached by trypsin treatment, and centrifuged at 1200 rpm for 3 minutes to obtain cells. The culture was carried out using EpiLife medium (Life Technologies), seeded in a culture flask at a density of 5000 to 7000 cells/cm ² , and subcultured every 4 to 7 days in a growing state.

Human epidermal keratinocytes were seeded into EpiLife medium (Gibco) at 1 × 10 ⁵ cells/35 mm disks, and cultured to a cell density of about 50%.

Using the glutathione synthesis inhibitor L-Buthionine-sulfoximine (BSO: Sigma-Aldrich), the optimum BSO concentration which first led to inhibition of glutathione synthesis was determined.

BSO was added to the cell culture solution, and after 24 hours, the cells were trypsinized and recovered for glutathione measurement. The BSO concentration of glutathione was not detected as the next oxidative stress test condition.

(2) Implementation of an oxidative stress load test caused by hydrogen peroxide (H ₂ O ₂ )

Human epidermal keratinocytes (Gibco) were inoculated into EpiLife medium (Gibco) at 1 × 10 ⁵ cells/35 mm, and cultured to a cell density of about 50%. Next, BSO was added to obtain the BSO concentration (50 μM or 100 μM) determined in the above (1), and after further 24 hours, hydrogen peroxide was added to make 0, 0.5, and 1 mM. After hydrogen peroxide was added, it was further cultured for 24 hours, and the culture supernatant was recovered. Further, the cells were washed with PBS(-) and recovered with 100 μl of cell lysate.

The amount of LDH (lactate dehydrogenase), the amount of protein, and the amount of DJ-1 were measured using the obtained culture supernatant and cells.

(3) Protein quantification

The amount of protein in the cell lysate was measured using a BCA protein kit (manufactured by Thermo Scientific).

(4) Determination of LDH

The LDH in the culture supernatant was measured using a cytotoxicity detection kit ^PLUS (LDH) (Roche Applied Science). The measurement was carried out according to the kit experimental guide and expressed by the absorbance at 490 nm.

(5) Determination of the amount of DJ-1 expression

The amount of DJ-1 in the supernatant and in the cell lysate was measured using a DuoSet Park7/DJ-1 ELISA kit (manufactured by R&D). According to the set of experimental guides Line measurement.

(6) Determination of glutathione

The cells recovered by trypsin treatment were washed with PBS(-) and resuspended in 100 μl of MES buffer. The amount of glutathione was measured by ultrasonic treatment for 1 minute as a cell lysate, using a glutathione test kit (Cayman Chemical Co., Ltd.). The assay was performed according to the set of experimental guidelines.

(7) Results

The amount of protein in the cells to which BSO was added is shown in Fig. 4, and the amount of glutathione is shown in Fig. 5.

Further, the amount of LDH in the cell culture supernatant of the stress load caused by the addition of hydrogen peroxide is shown in Fig. 6, and the amount of DJ-1 is shown in Fig. 7.

Similarly, the amount of protein in the cells is shown in Fig. 8, and the amount of DJ-1 is shown in Fig. 9.

The concentration of BSO indicating no effect on protein production and inhibition of glutathione production was 50 μM or 100 μM. Next, it was revealed that the release of glutathione-induced cells at this concentration caused the release of LDH due to oxidative stress caused by hydrogen peroxide (Fig. 6) and the amount of protein decreased (Fig. 7).

Further, it was confirmed that DJ-1 also increased depending on the concentration of hydrogen peroxide (Fig. 8 and Fig. 9).

From the results of Test Example 1 and Test Example 2 above, it was revealed that DJ-1 reflects stress accompanying oxidation of human epidermis, and the amount of production is affected by glutathione-producing ability and gene-induced interference. In addition, it can be seen that the more cells that are not resistant to oxidative stress, the more DJ-1 is produced. That is, it is judged that DJ-1 is increased when the resistance of the cells is lowered.

Test Example 3

In the same manner as in Test Example 2, human epidermal keratinocytes (keratinocytes) were used to primary culture the cells, and the change in the amount of DJ-1 by ultraviolet irradiation was measured.

(1) Culture of human epidermal keratinocytes and irradiation of ultraviolet rays

Human epidermal keratinocytes (Gibco) were inoculated into EpiLife medium (Gibco) at 1 × 10 ⁵ cells/35 mm, and cells were washed with PBS (-) on the 3rd day after culture, and then 1 ml of PBS (-) was added. using a Sankyo electric lamp system, the UVB irradiation intensity 0.2mW / cm ² of 10,20,30mJ / cm ^2. After the irradiation, the medium was changed, and after 24 hours of culture, the culture supernatant was collected, and the cells were collected with 200 μl of the cell lysate, and protein quantitation and DJ-1 amount were measured in the same manner as in Test Example 1 and Test Example 2.

(2) Results

The amount of protein in the culture supernatant is shown in Fig. 10. Further, the amount of DJ-1 in the cultured cells was as shown in Fig. 11, and the amount of DJ-1 in the supernatant was as shown in Fig. 12.

Human epidermal keratinocytes were irradiated with UVB, and the amount of DJ-1 after 24 hours was damaged depending on the amount of UVB irradiation light and the amount of protein was decreased. On the other hand, the amount of DJ-1 in the cells and extracellular cells increased, especially in the culture supernatant.

According to Test Examples 1 to 3 above, it was revealed that the DJ-1 value of human epidermal keratinocytes reflects the level of stress.

Test Example 4

Since Test Examples 1 to 3 show that DJ-1 reflects stress in human epidermal keratinocytes, it is tested whether or not the actual skin stress is reflected. which is, The human skin is directly irradiated with ultraviolet rays as a stress to observe and measure the change in the amount of DJ-1 expression.

(1) Test method

<UV irradiation>

The back of 10 men with healthy skin was irradiated with ultraviolet rays of 20, 40, 60, 80, and 100 mJ/cm ² using an ultraviolet irradiation device (DERMARAY, manufactured by Terumo Clinical Supply Co., Ltd.). The minimum erythema dose (Minimum Erythema Dose: MED) was determined according to the erythema on the next day of irradiation. Using this value, ultraviolet rays corresponding to 0, 0.5, and 1.0 MED were irradiated on the back, and after 7 and 14 days of irradiation, the stratum corneum was collected by a tape peeling method using a stratum corneum test piece (ASAHIBIOMED).

<DJ-1 determination in the stratum corneum>

In a test tube in which glass beads and 500 μl of T-PER buffer (Thermo Scientific) were placed, a stratum corneum test piece in which a stratum corneum was collected was placed, and the stratum corneum protein was extracted by shaking with a vortex mixer for 25 minutes. The protein amount of each sample was measured using a BCA protein test kit (Thermo Scientific). The measurement was carried out by adding 200 μl of a liquid mixed with reagent A: reagent B = 50:1 to 10 μl of the stratum corneum sample, and culturing at 60 ° C for 30 minutes, and measuring the absorbance at 562 nm. At the same time, a standard curve was prepared using fetal bovine serum albumin (BSA). The amount of protein was calculated from the value of the standard curve and absorbance.

The amount of DJ-1 contained in the stratum corneum extract was quantified using Human Park7/DJ-1 DuoSet (R&D systems).

(2) Results

The DJ-1 measurement results per unit protein are shown in Fig. 13 of Fig. 1. After 7 and 14 days of ultraviolet irradiation, the amount of DJ-1 in the stratum corneum of the site irradiated with 0.5 and 1.0 MED of ultraviolet rays was significantly increased as compared with the non-irradiated portion.

It is indicated that the state of human skin stress can be confirmed by measuring DJ-1.

Test Example 5

Since Test Examples 1 to 3 show that DJ-1 reflects stress in human epidermal keratinocytes, it is tested whether or not the actual skin stress is reflected. That is, the sarcoplasm (sensitivity) is asked through the questionnaire, and the answer and the amount of DJ-1 in the cheek are compared.

(1) Test method

<Questionnaire>

Ask 575 women with healthy skin about sarcoplasm (sensitivity) to answer any of "insensitive", "slightly sensitive", and "sensitive".

<Skins collection>

The stratum corneum was collected by a tape peeling method using a stratum corneum test piece (ASAHIBIOMED) from the cheeks of 575 women with healthy skin who had a questionnaire.

<DJ-1 determination in the stratum corneum>

In a test tube in which glass beads and 500 μl of T-PER buffer (Thermo Scientific) were placed, a stratum corneum test piece in which a stratum corneum was collected was placed, and the stratum corneum protein was extracted by shaking with a vortex mixer for 25 minutes. The protein amount of each sample was measured using a BCA protein test kit (Thermo Scientific). The measurement was carried out by adding 200 μl of a liquid mixed with reagent A: reagent B=50:1 to 10 μl of the stratum corneum sample, and culturing at 60 ° C for 30 minutes. The absorbance at 562 nm was measured. At the same time, a standard curve was prepared using fetal bovine serum albumin (BSA). The amount of protein was calculated from the value of the standard curve and absorbance.

The amount of DJ-1 contained in the stratum corneum extract was quantified using Human Park7/DJ-1 DuoSet (R&D systems).

(2) Results

The results of the questionnaire are as follows, non-sensitive: 281, slightly sensitive: 193, sensitive: 101. The DJ-1 measurement results of the unit protein of each questionnaire respondent are shown in Fig. 14. It can be seen that the more sensitive the DJ-1 is, the higher the amount.

Test Example 6

Since Test Examples 1 to 3 show that DJ-1 reflects stress in human keratinocytes, it is tested whether or not the actual skin stress is reflected. That is, the following questions were asked through the questionnaire, whether there was a habitual exposure to ultraviolet light such as sea sports, and the answer and the amount of DJ-1 in the cheek were compared.

(1) Test method

<Questionnaire>

For 386 women with healthy skin, the following question was asked as to whether or not there was a habitual exposure to ultraviolet rays such as sea sports, and any one of "none" and "yes" was answered.

<Skins collection>

The stratum corneum was collected by a tape peeling method using a stratum corneum test piece (ASAHIBIOMED) from the cheeks of 386 women with healthy skin who had a questionnaire.

<DJ-1 determination in the stratum corneum>

In a test tube in which glass beads and 500 μl of T-PER buffer (Thermo Scientific) were placed, a stratum corneum test piece in which a stratum corneum was collected was placed, and the stratum corneum protein was extracted by shaking with a vortex mixer for 25 minutes. The protein amount of each sample was measured using a BCA protein test kit (Thermo Scientific). The measurement was carried out by adding 200 μl of a liquid mixed with reagent A: reagent B = 50:1 to 10 μl of the stratum corneum sample, and culturing at 60 ° C for 30 minutes, and measuring the absorbance at 562 nm. At the same time, a standard curve was prepared using fetal bovine serum albumin (BSA). The amount of protein was calculated from the value of the standard curve and absorbance.

The amount of DJ-1 contained in the stratum corneum extract was quantified using Human Park7/DJ-1 DuoSet (R&D systems).

(2) Results

The results of the questionnaire are as follows, no: 223, there are: 163. The DJ-1 measurement results of the unit protein of each questionnaire respondent are shown in Fig. 15. It can be seen that the more the respondent DJ-1 has an experience of habitually irradiating ultraviolet rays, the higher the amount.

Test Example 7

In order to grasp the state of stress accumulation in human skin, DJ-1 of the stratum corneum was measured for a large number of subjects.

(1) Test method

1) Collection of test samples

A random sample of 205 women was taken as a subject, and a stratum corneum sample was taken from the cheek by a tape peeling method. The sample is collected for 1 part.

2) Method for determining DJ-1 in the stratum corneum of the skin

This was carried out in the same manner as in Test Example 4.

In a test tube in which glass beads and 500 μl of T-PER buffer (Thermo Scientific) were placed, a stratum corneum test piece in which a stratum corneum was collected was placed, and the stratum corneum protein was extracted by shaking with a vortex mixer for 25 minutes. The protein amount of each sample was measured using a Pierce BCA protein test kit (Thermo Scientific). The measurement was carried out by adding 200 μl of a liquid mixed with reagent A: reagent B = 50:1 to 10 μl of the stratum corneum sample, and culturing at 60 ° C for 30 minutes, and measuring the absorbance at 562 nm. At the same time, a standard curve was prepared using fetal bovine serum albumin (BSA). The amount of protein was calculated from the value of the standard curve and absorbance.

The amount of DJ-1 contained in the stratum corneum extract was quantified using Human Park7/DJ-1 DuoSet (R&D systems, DY3995E).

(2) Results

The measurement results are shown in Fig. 16.

As shown in Fig. 16, the expression amount of DJ-1 was distributed over a wide range from 0.3 pg/μg of total protein to a value exceeding 5.0 pg/μg of total protein. The average value was 2.6 pg/μg total protein. A criterion for determining that the amount of expression of DJ-1 is large compared to the frequency distribution can be freely set. For example, a value of 2.6 pg/μg or more of the total protein is judged to be a large amount of expression of DJ-1. Alternatively, since the cumulative frequency of 2.3 pg/μg of total protein is about 50%, the value of more than 2.3 pg/μg of total protein can also be judged to be a large amount of expression of DJ-1. Next, it can be judged that the greater the amount of expression of DJ-1, the greater the amount of stress accumulation of human skin.

Claims (5)

A method for evaluating the amount of stress accumulation in human skin, characterized in that the DJ-1 expression amount of the stratum corneum of the skin is compared with the DJ-1 expression amount of normal human skin measured in advance or simultaneously. The evaluation method according to claim 1, comprising the steps of: collecting a stratum corneum of the evaluation target portion of the subject; and measuring a step of measuring the keratin collected by the collecting step a DJ-1 expression amount in the layer; and a comparison step of comparing the DJ-1 expression amount measured in the measuring step with the DJ-1 expression amount in the stratum corneum of the evaluation subject site of a healthy skin person . The evaluation method according to the first or second aspect of the patent application, comprising the steps of: collecting a stratum corneum of a subject to be evaluated, and measuring a step of measuring the collecting step a DJ-1 expression amount in the stratum corneum collected; and a comparison step of distributing the DJ-1 expression amount measured in the measuring step and the DJ-1 expression amount in the stratum corneum of the evaluation target portion of the sample group Compare. The evaluation method according to any one of claims 1 to 3, wherein the step of collecting the stratum corneum of the skin is carried out by a tape peeling method. The evaluation method according to any one of claims 1 to 4, wherein the stress accumulation amount of the human skin is an amount of stress accumulation caused by ultraviolet damage or oxidation.