WO2012114932A1 - 皮膚のストレス蓄積度の評価方法 - Google Patents

皮膚のストレス蓄積度の評価方法 Download PDF

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Publication number
WO2012114932A1
WO2012114932A1 PCT/JP2012/053345 JP2012053345W WO2012114932A1 WO 2012114932 A1 WO2012114932 A1 WO 2012114932A1 JP 2012053345 W JP2012053345 W JP 2012053345W WO 2012114932 A1 WO2012114932 A1 WO 2012114932A1
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Prior art keywords
skin
hsp27
stratum corneum
expression level
measured
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PCT/JP2012/053345
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English (en)
French (fr)
Japanese (ja)
Inventor
石渡　潮路
奈帆子 大石
知永 安田
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Fancl Corp
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Fancl Corp
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Priority to KR1020137014928A priority Critical patent/KR20130140069A/ko
Priority to CN2012800090971A priority patent/CN103380375A/zh
Publication of WO2012114932A1 publication Critical patent/WO2012114932A1/ja
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
    • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
    • A61B5/443—Evaluating skin constituents, e.g. elastin, melanin, water
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/40—Disorders due to exposure to physical agents, e.g. heat disorders, motion sickness, radiation injuries, altitude sickness, decompression illness

Definitions

  • the present invention relates to a method for evaluating the degree of stress accumulation in human skin and a method for evaluating sensitive skin.
  • Ultraviolet rays cause skin aging by inducing DNA breakage of the skin cells, collagen degeneration, and the like even when the skin is not significantly changed. Ultraviolet rays are constantly stressing the skin. For women, daily acts such as face washing and makeup also stimulate the skin, and these stimuli accumulate as potential stress on the skin. And if resistance to these stimuli (stress) decreases for some reason, it will be recognized as sensitive skin. In the present invention, the accumulation degree of such external stimulation of the skin is referred to as skin stress accumulation degree.
  • Sensitive skin on the other hand, is perceived as skin that has no obvious skin lesions but is prone to adverse and harmful reactions. It is also said that when the accumulation of stress reaches a certain level as a cause of such sensitive skin, the sensitivity of the skin increases. Sensitive skin is less resistant to external stimuli than healthy skin, and can be said to be skin that 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.
  • Patent Document 1 discloses a method for applying skin care to the skin, and then collecting and observing the exfoliated skin stratum corneum to evaluate skin sensitivity. However, this method must be inspected for each cosmetic product, and the accumulation degree of the user's skin stress cannot be evaluated.
  • Patent Document 2 discloses a method for evaluating whether the skin is sensitive by applying an irritating substance such as capsinoid to the skin.
  • an irritating substance such as capsinoid
  • a test method using a strongly irritating substance such as capsinoid is not preferable for the subject.
  • Patent Document 3 discloses a method for evaluating whether there is sensitive skin by measuring the amount of carloprotectin present in the stratum corneum of the face.
  • Non-Patent Documents 1 and 2 describe that heat shock protein (HSP) 27 increases in the skin in response to skin irritation by sodium lauryl sulfate (SDS), which is a surfactant.
  • SDS sodium lauryl sulfate
  • Ingeborg L.A.Boxman et al., Experimental Dermatology, 2002, 11, 509-517. Ingeborg L.A.Boxman, et al., British Journal of Dermatology, 2002, 146, 777-785.
  • the evaluation method is not a method that imposes a burden on the user, such as surgically removing the skin or irritating the skin.
  • the present invention provides a novel evaluation method using a biochemical index that can evaluate the degree of accumulation of skin stress without burdening the user, and can easily and reliably evaluate whether the skin is sensitive or not. The task is to do.
  • the present invention has the following configuration. (1) A method for evaluating the degree of stress accumulation in human skin, characterized by comparing the expression level of heat shock protein (HSP) 27 in the stratum corneum with the expression level of HSP27 in normal human skin measured in advance or simultaneously . (2) A method for evaluating sensitive skin, characterized in that the expression level of HSP27 in the stratum corneum is compared with the expression level of HSP27 in normal human skin measured in advance or simultaneously.
  • HSP heat shock protein
  • a collecting step for collecting the skin stratum corneum a measuring step for measuring the expression level of HSP27 in the stratum corneum cells collected in the collecting step, and an expression level of HSP27 measured in the measuring step
  • a method for evaluating the degree of stress accumulation in human skin characterized by comparing the expression level of HSP27 in the skin stratum corneum with the distribution of expression level of HSP27 in the skin of a sample population measured in advance or simultaneously.
  • a method for evaluating sensitive skin wherein the expression level of HSP27 in the stratum corneum is compared with the distribution of expression level of HSP27 in the skin of a sample population measured in advance or simultaneously.
  • a comparison step of comparing the expression level distribution of HSP27 in the stratum corneum at the site to be evaluated of the sample population with an evaluation method according to (5) or (6).
  • the accumulation degree of skin stress can be evaluated by measuring the expression level of HSP27 in the stratum corneum. Further, it is possible to easily evaluate whether the skin is sensitive skin by using the abundance of HSP27 as an index. Furthermore, since the evaluation method of the present invention collects an evaluation sample by a simple operation method such as a tape stripping method, the burden on the subject is small, and anyone can easily evaluate. In addition, this is a biochemical test method, and the same result can be obtained regardless of who makes the measurement, so there is no need for conventional counseling by an expert interview method.
  • HSP27 is a protein with a molecular weight of 27 KD, which is known as one of a series of heat shock protein families, and has a function as a molecular chaperone. It is known that gene expression in the skin increases with immune stress.
  • Sensitive skin in the present invention refers to skin that has no obvious skin lesions but is disadvantageous and susceptible to harmful reactions. Moreover, 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. It is known that in sensitive skin, there is a tendency that the transdermal moisture transpiration (TEWL) increases and the high-frequency conductivity (horny layer water content) decreases. In sensitive skin, rash and rough skin may occur.
  • TEWL transdermal moisture transpiration
  • horny layer water content horny layer water content
  • the stratum corneum is the tissue on the top of the skin and has a function of protecting the skin from foreign substances and irritation from outside the body.
  • the evaluation target site in the present invention can include any site as long as the stratum corneum is available, but examples of the main site include the face, neck, and upper arm. According to the conventional method, the stratum corneum derived from the skin at these sites can be obtained. However, as described above, the method of surgically removing the skin places a burden on the user, and therefore, a method that can easily obtain the stratum corneum, such as tape stripping and rubbing, is preferable.
  • the expression level of HSP27 in each sample thus prepared can be measured by a conventionally known method.
  • methods such as enzyme immunoassay, radioimmunoassay, and Western blotting based on the reaction with an antibody against HSP27 can be used.
  • a soluble fraction of HSP27 is prepared through a biochemical method known per se, such as a freeze-thaw method, an ultrasonic disruption method, a homogenate method, or the like. Measure immediately after extraction.
  • the stratum corneum 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 stratum corneum is collected with a keratin tape.
  • the present invention can measure the expression level of HSP27 without stimulating the skin with ultraviolet rays or chemical substances. For this reason, it is possible to evaluate whether the user's skin is sensitive skin without imposing a burden on the user.
  • cosmetics and chemical peeling agents that are more suitable for the user's skin, thereby avoiding skin troubles and side effects such as skin irritation and making skin care and benefits of cosmetics more effective. it can.
  • the evaluation method of the present invention includes a sampling step of sampling the stratum corneum, a measurement step of measuring the expression level of HSP27 in the stratum corneum sampled in the sampling step, and a healthy expression level of HSP27 measured in the measurement step. Comparing with the expression level of HSP27 in the stratum corneum of the skin.
  • the evaluation method of the present invention includes a sampling step of sampling the stratum corneum, a measurement step of measuring the expression level of HSP27 in the stratum corneum sampled in the sampling step, and the expression level of HSP27 measured in the measurement step Is compared with the distribution of the expression level of HSP27 in the stratum corneum of the sample population. This evaluation method will be described below.
  • the expression level of HSP27 measured as described above is compared with the expression level of HSP27 in the stratum corneum of healthy skin.
  • the expression level of HSP27 in the stratum corneum of a subject's evaluation target site is measured, and the measured expression level of HSP27 is compared with the expression level of HSP27 in the stratum corneum of the same evaluation target portion of a person with healthy skin. May be.
  • the expression level of HSP27 in the stratum corneum 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.
  • the expression level of HSP27 in the stratum corneum of a subject's evaluation target site is measured, and the measured expression level of HSP27 is compared with the distribution of the expression level of HSP27 in the stratum corneum of the same evaluation target portion of the sample population. Also good. By using the distribution of the expression level of HSP27 in the sample population, a more objective evaluation can be performed.
  • the expression level of HSP27 is large If it is determined, it can be evaluated that the accumulation of stress is large, and that the possibility of sensitive skin is high.
  • the sample of healthy skin may be collected from a person who measures HSP27, or may be collected from a person different from the person who measures HSP27.
  • the degree of stress accumulation can be evaluated by the degree of expression of HSP27 measured. If the expression level of HSP27 to be measured is judged to be significantly higher than that of healthy skin or the expression level distribution of the sample population, the skin strongly exhibits the properties of sensitive skin, Resistance to external stimuli is significantly lower than the average of the sample population, and skin troubles are very likely to occur.
  • the measured expression level of HSP27 is increased by a small amount compared with that of healthy skin, or the measured expression level of HSP27 is compared with the expression level distribution of the sample population, If it is judged that the skin is a little larger, it can be evaluated that the skin does not reach the sensitive skin where the accumulation of stress is higher than the average value of the sample group but the resistance to external stimuli is low and skin trouble occurs.
  • Example 1 Test of skin stress accumulation degree by ultraviolet irradiation (1) Stress load by ultraviolet ray A test was conducted with 10 healthy men as subjects. As an ultraviolet irradiation device, Multiple Solar Ultraviolet Simulator Model 601 (manufactured by Solarlight) was used, and a 150 W xenon lamp was used as a light source. The irradiation intensity was adjusted so that erythema that induces pigmentation appeared on the back skin of the subject, and ultraviolet rays (UVB) were irradiated for 170 seconds. In addition, the stress accumulation
  • horny layer checker manufactured by Asahi Biomed
  • the emission intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (Fuji Film). Evaluation was performed by reading the intensity of the luminescent signal as the amount of HSP27 protein using a calibration curve prepared in advance using a recombinant HSP27 protein as a standard.
  • Results Table 1 and FIG. 1 show the measurement results of the amount of HSP27 in the stratum corneum of subjects subjected to ultraviolet light stimulation (stress). It was confirmed that ultraviolet rays accumulate stress in the skin over a long period of time.
  • HSP27 Quantification of HSP27 by Western blotting After 1.5 ⁇ g of stratum corneum protein was separated by SDS-PAGE using XP PANTERA Gel (5-20% gradient gel: DRC), the protein was transferred to a PVDF membrane. Blocking was performed with StartingBlock Blocking Buffer (manufactured by Thermo Scientific). The primary antibody (HSP27 Polyclonal Antibody: manufactured by Stressgen) was diluted 1000 times, and the secondary antibody (Goat anti-mouse IgG: manufactured by invitrogen) was reacted at a 10,000 times dilution, and then ECLplus (manufactured by BD Bioscience) was used. Then, detection was performed with LAS-4000mini (Fuji Film).
  • the emission intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (Fuji Film). Evaluation was performed by reading the intensity of the luminescent signal as the amount of HSP27 protein using a calibration curve prepared in advance using a recombinant HSP27 protein as a standard.
  • Results Table 2 and FIG. 2 show the results of measurement of the stratum corneum of subjects given stimulation (stress) by SDS washing and the amount of HSP27 in the control. In addition, the change of moisture transpiration is also shown.
  • sampling was performed from the skin of the neck and upper arm of three healthy women as a control.
  • Results Table 3 and FIG. 3 show the measurement results of the amount of HSP27 in the stratum corneum of patients with atopic dermatitis and healthy individuals.
  • HSP27 of healthy people is hardly present in the stratum corneum, but HSP27 was detected at a high concentration from the affected skin of patients with atopic dermatitis. Moreover, HSP27 was detected from normal skin of patients with atopic dermatitis at a higher concentration than that of healthy individuals, although not as much as the site exhibiting dermatitis. From the above results, it can be seen that stress is accumulated in the skin of atopic dermatitis patients who are constantly stressed. It was also confirmed that HSP27 was relatively high in skin that was not affected by atopic dermatitis. From the above results, it was confirmed that it is useful to measure HSP27 as an index of sensitive skin.
  • Test method (1) Sample collection A stratum corneum sample was collected from 8 healthy women by a tape stripping method from the cheek, under the chin, forearm, and buttocks. Four samples were collected from one site.
  • HSP27 Quantification of HSP27 by ELISA
  • the amount of HSP27 contained in the stratum corneum extract was measured with Total human HSP27 DuoSet ELISA kit (manufactured by R & D systems). The results were analyzed by the amount of HSP27 per unit protein amount.
  • a frequency distribution table of HSP27 expression levels in the sample population is shown in Table 5, and a histogram is shown in FIG.
  • the expression level of HSP27 is distributed in a wide range from 0.4 pg / ⁇ g total protein to a value exceeding 50 pg / ⁇ g total protein.
  • the average value was 15.0 pg / ⁇ g total protein.
  • the standard for judging that the expression level of HSP27 is large compared to this frequency distribution can be freely set.
  • the expression value of HSP27 is an average value of 15.0 pg / ⁇ g total protein or more. Since the cumulative frequency of 10.0 pg / ⁇ g total protein may be about 50%, a value exceeding 10.0 pg / ⁇ g total protein may be determined to indicate that the expression level of HSP27 is large.
  • the expression level of HSP27 in Experiment 4 is 1/10 of the expression level of HSP27 in the cheek part of Experiment 3. This is because the protein extraction method in Experiment 4 extracts 1 HSP27-containing protein. This is because the protein in the stratum corneum was extracted using 50 mM Tris-HCl buffer containing% SDS, and the amount of total protein detected in Experiment 4 increased compared to Experiment 3. There is no difference in the process of extracting HSP27 in both Experiment 3 and Experiment 4, and HSP27 is highly soluble, so it is sufficiently extracted in this process.
  • Outdoor activity experience questionnaire The subjects were asked to select either “Yes” or “No” for outdoor activity experience. Examples of outdoor activities include sports and gardening. Those who answered “Yes” in outdoor activities were expected to have high stress accumulation in human skin, and those who answered “None” were expected to have low stress accumulation in human skin.
  • Table 7 and Fig. 7 show the relationship between questionnaire results of outdoor activity experience and HSP27 amount.
  • the person with lower than the standard value has low human skin stress accumulation, and the person with higher than the standard value has high human skin stress accumulation, It tends to correspond to the degree of stress accumulation due to outdoor ultraviolet rays.

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PCT/JP2012/053345 2011-02-25 2012-02-14 皮膚のストレス蓄積度の評価方法 Ceased WO2012114932A1 (ja)

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CN2012800090971A CN103380375A (zh) 2011-02-25 2012-02-14 皮肤的应激积累度的评价方法

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JP2017009427A (ja) * 2015-06-22 2017-01-12 株式会社ファンケル 頭皮の圧縮応力の評価方法
TWI595235B (zh) * 2012-11-02 2017-08-11 芳珂股份有限公司 粗糙肌膚的評估方法
TWI622382B (zh) * 2014-01-09 2018-05-01 芳珂股份有限公司 肌理的評價方法

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CN103808940B (zh) * 2012-11-02 2017-06-06 株式会社芳珂 粗糙肌肤的评价方法
JP5775540B2 (ja) * 2013-02-22 2015-09-09 株式会社ファンケル 皮膚の紫外線照射により負荷される紫外線ストレスの評価方法
JP2015072226A (ja) * 2013-10-03 2015-04-16 住友ベークライト株式会社 検査方法

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JP2009174867A (ja) * 2008-01-21 2009-08-06 Shiseido Co Ltd 角層タンパク質の可溶性タンパク質の量比を指標とした肌質の評価方法

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
TWI595235B (zh) * 2012-11-02 2017-08-11 芳珂股份有限公司 粗糙肌膚的評估方法
TWI622382B (zh) * 2014-01-09 2018-05-01 芳珂股份有限公司 肌理的評價方法
JP2017009427A (ja) * 2015-06-22 2017-01-12 株式会社ファンケル 頭皮の圧縮応力の評価方法

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