JP6397733B2 - Acne Skin Evaluation Method - Google Patents

Description

  The present invention relates to an index for use in evaluation of acne skin symptoms and determination of a therapeutic effect, and an inspection method thereof.

Acne is an inflammatory skin disease that occurs in sebaceous hair follicles and medically refers to acne vulgaris, also called acne or acne. Many occur during adolescence and heal naturally with growth, but it is known that there are large individual differences in the symptoms. Major factors involved in acne development include endocrine factors (hormones), keratinization of the follicular tube, bacteria in the follicular tube, and genetic factors.
Hair follicles in the skin are classified into three types: fluffy hair follicles, sebaceous hair follicles, and terminal hair follicles. Of these, sebaceous hair follicles are the most dense on the face, followed by the chest and the middle of the back. Acne is said to occur easily on the face, chest and back. In addition, acne such as comedones, papules, and pustules are mixed in the sites of acne.

  The so-called acne occurs at the site of acne, and it becomes malignant when triggered by the formation of comedones, a rash symptom. The main causes of comedone formation include abnormal keratinization of the follicular tube, increased sebum secretion, changes in sebum components, and bacterial flora of the follicular tube. The comedones are filled with stratum corneum, sebum, and soft hair, and Propionibacterium acnes are also growing. When the content of the comedones increases and the content increases, free fatty acids, which are triglyceride degradation products, and enzymes derived from bacteria grown in the comedone are inflamed, and those that are inflamed are called red papules. Red papules form pustules due to inflammation and bacterial growth, and as they progress further, they commit skin tissue and become cysts, and scars form during subsequent tissue repair processes. Scars and pigmentation remain after inflammation.

Cosmetics and quasi-drugs for preventing comedones and skin rashes and preventing deterioration are on the market, also called acne cosmetics. These products are generally those that remove excess sebum and those that suppress the excessive action of sebaceous glands, and are often mixed with anti-inflammatory agents and antibacterial agents. In addition, products range from face wash and lotion to makeup cosmetics. In addition, quasi-drugs that promote the effect of “preventing acne” are commercially available in the form of medicated soaps, facial cleansers, lotions, packs, creams, and emulsions.
However, even if such precautions are taken, acne may occur, and the progress of symptoms after the occurrence varies greatly from person to person. Proper treatment should be performed while predicting the occurrence of skin problems due to acne. This is very important.

In Patent Document 1, in order to achieve such an object, acne status is evaluated by measuring the bone type ALP (BAP) concentration in the blood of acne patients, and the proportion of neutrophils is further measured. When the ratio is high, it is proposed to identify the predisposition to acne as stress and give advice for acne improvement.
Patent Document 2 uses as an index one or more measured values selected from FreT4, 8-OHdG production rate in blood, iron concentration, ferritin amount, hemoglobin amount, hematocrit amount, cholinesterase activity, and free fatty acid amount. A method for evaluating the skin risk caused by acne is described.
The applicant has filed numerous patent applications for biomarkers for assessing skin condition. Particularly in Patent Document 3, a plurality of proteins such as Galectin-7, FABP-5, Annexin II, Apolipoprotein A1, Enolase 1, and squamous cell carcinoma antigen-2 (SCCA2) are useful as biomarkers for the risk of developing atopic dermatitis. It is clear that there is.

JP 2007-199027 A JP 2008-107275 A Japanese Patent No. 5282228 WO2007 / 046463 publication

The present inventors are conducting research on the prevention and treatment of acne. In the process, we observed focusing on the comedones, the main acne symptoms of acne, and found that there were various symptom changes such as when the comedones became malignant and resulted in cysts, and when they were in the state of comedones or stayed in red papules or pustules. Was confirmed by observation. He found that acne increases and the malignancy of acne are scientifically quantified.
An object of the present invention is to provide an index and an inspection method for accurately evaluating acne diagnosis and progress.

  The present inventor has paid attention to FABP-5 (Fatty acid binding protein-5) which is a marker of the above-mentioned atopic dermatitis. When the expression level of FABP-5 in the part was measured, it was found that the expression level of FABP-5 was higher in response to acne malignant transformation than in healthy individuals. Based on this finding, it has been found that if FABP-5 in a skin sample is measured, the malignancy of acne can be diagnosed and the course of treatment can be diagnosed, thereby completing the present invention.

That is, the configuration of the present invention is as follows.
(1) Measure FABP-5 in the non-invasively collected skin stratum corneum , and compare it with the measured value of FABP-5 in normal skin stratum corneum measured in advance or simultaneously. Acne skin inspection method as an index.
(2) The inspection method according to (1), wherein the stratum corneum is obtained from human skin by a peeling operation.

According to the inspection method of the present invention, it is possible to accurately determine the treatment progress, therapeutic effect, etc. of acne skin having various stages of comedones such as cysts, red papules and pustules. It is possible to predict the occurrence of acne on the skin or site where acne does not appear. Furthermore, it is easy to collect a skin sample to be examined, and the examination can be easily performed with less burden on the patient.
In addition to the above, a cultured cell can be used to screen for a substance having an action of suppressing FABP-5 expressed in the cultured cell. The screened substance can be expected as an agent for preventing and treating acne.

It is the graph which displayed the quantity of FABP-5 in the skin exfoliation stratum corneum of the normal human cheek and acne skin by the presence or absence of acne. It is clear that FABP-5 is increased by the appearance of acne. It is the graph which displayed the quantity of FABP-5 in the skin peeling stratum corneum of the forehead of normal human forehead and acne skin by the presence or absence of acne. It is clear that FABP-5 is increased by the appearance of acne. It is the graph which displayed the quantity of FABP-5 in the skin exfoliation stratum corneum of the normal human cheek and acne skin corresponding to the malignancy of acne. It is clear that FABP-5 increases as the acne malignancy progresses. It is the graph which displayed the quantity of FABP-5 in the skin exfoliation horny layer of the forehead of normal human forehead and acne skin corresponding to the malignancy of acne. It is clear that FABP-5 increases as the acne malignancy progresses.

The present invention measures FABP-5 in a skin sample collected non-invasively and compares it with the measured value of FABP-5 in a normal skin sample measured in advance or simultaneously, and an increase in expression is an index of acne malignant transformation. It is invention which concerns on the inspection method of acne skin.
In the present invention, acne skin is a skin rash that is medically diagnosed as acne vulgaris and has skin in any of various stages such as cysts, red papules, pustules, and comedones.

FABP-5 (Fatty acid binding protein-5) is an intracellular protein having a molecular mass of 15,033 Da. It was identified mainly by proteins present in the epidermis and increased expression in tissues of psoriasis (characterized by regular thickening and keratinization of the epidermis). An intracellular protein that binds to fatty acids (oleic acid) and hydrophobic ligands, and is involved in fatty acid uptake, transport, metabolism, and cell proliferation and differentiation. Although the expression of FABP-5 is low in proliferating epidermal keratinocytes, it increases about 2-fold by induction of differentiation. It also binds to S100A7 (calcium-dependent signaling protein) that is highly expressed in psoriasis, and localizes to a focal adhesion-like structure at the differentiation stage of epidermal keratinocytes. The gene sequence information is disclosed in Fatty acid binding protin 5, J. Invest. Dermatol. 99: 299-305 (1992). Amino acid sequence information is disclosed in Fatty acid-binding protein, epidermal, Biochem. J. 302: 363-371 (1994). Patent Document 4 discloses that FABP-5 can be a marker for atopic dermatitis.
In relation to acne skin, it is not known at all that FABP-5 according to the present invention is involved.

  In the test method of the present invention, the gene amount or protein amount of FABP-5 in the skin sample is measured. Here, the skin sample is preferably a skin sample containing a human epidermal horny layer, and can be collected from human skin by biopsy. Usually, a method called a tape stripping method is a method in which an adhesive tape is affixed to the skin and the stratum corneum adhering to the tape is used as a sample when the tape is peeled off.

  When measuring FABP-5 in a skin sample as a gene sample, for example, the RT-PCR method and the like can be mentioned. In addition, the FABP-5 gene can be quantified by real-time RT-PCR. As a means for measuring FABP-5 protein, for example, immunostaining can be employed. Commercially available products can be used for the anti-FABP-5 antibody and the labeled product thereof used for the measurement.

  In the case of immunostaining, it can be measured by fluorescent immunostaining, for example. FABP-5 can be quantified by, for example, quantifying the fluorescence intensity with a commercially available measuring device and software.

  When the present invention is used as a diagnostic agent, an ELISA kit containing the anti-FABP-5 antibody, a labeled body thereof, a second antibody, a labeled second antibody and the like is preferable. Furthermore, a test kit combined with a buffer for analysis or the like may be used. Commercially available kits can also be used. Examples of such kits include epithelial fatty acid binding protein (FABP5) chemiluminescence immunoassay kit (USCN Life Science Inc.), CircuLex Mouse FABP5 / E-FABP / mal1 ELISA Kit (Medical and Biological Laboratories). it can.

When this inventor measured FABP-5 in the skin of a healthy person, the skin of all the healthy persons was a low value. On the other hand, it was confirmed that FABP-5 was remarkably increased from the affected part of the patient diagnosed with acne skin and the skin in the peripheral part thereof as compared with the healthy person. It was confirmed that the skin of the site of the same patient who did not present a skin rash such as comedones had higher FABP-5 than the affected part, although it was lower than the affected part. Therefore, by observing the amount of change in FABP-5 around the skin, particularly around acne, it is possible to diagnose the progress of acne, the therapeutic effect, and the course of treatment.
Since FABP-5 increases with acne skin exacerbation, it is thought that FABP-5 is involved in some form of acne exacerbation and increases. Therefore, screening of FABP-5 inhibitory or promoting substances using known human cultured skin cells (human three-dimensional epidermis model) can be performed using conventional techniques. In the test, when the substance to be screened is added to the culture system, the amount of FABP-5 produced in the epidermis model is measured, and compared with the amount of FABP-5 in the epidermis model without control Is evaluated as having an FABP-5 inhibitory action, and when it is increased, it is evaluated as having an FABP-5 promoting action.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

1. Acne skin FABP-5 measurement test Method (1) Healthy test subjects (12 persons):
Those who did not observe skin diseases such as inflammatory skin rash and atopic skin inflammation on the face / body were selected by a doctor's diagnosis.
Patients with acne skin:
Patients who were diagnosed with acne vulgaris according to a doctor's diagnosis (14 mild cases, 11 moderate / severe cases) were included.

The diagnostic criteria were as follows according to the criteria of the Japanese Dermatological Association Acne Acne Treatment Guidelines 2008 shown below, and patients with symptoms of moderate or higher were classified as moderate / severe groups in this application.

Diagnosis criteria of the Japanese Dermatological Association Mild: 5 or less inflammatory rash on one face Moderate: 6 or more and 20 or less inflammatory rash on one face Severe: 21 or more and 50 or less inflammatory rash on one face Severe: 51 or more inflammatory eruptions on one face Age 18 years or older, regardless of gender

(2) Skin stratum corneum collection method Using a horny checker (2.5 cm x 2.5 cm: manufactured by Asahi Biomed) from the skin of the cheek and forehead, near the site where acne appears, One piece of the tape was stripped from the area without comedones, and the stratum corneum was collected.

(3) Extraction from the stratum corneum The stratum corneum sampler was collected in a tube containing glass beads and 500 μl of RIPA buffer (# 89901 / Thermo SCIETIFIC) and shaken for 25 minutes with a vortex mixer. Protein was extracted.

(4) Measurement of the amount of FABP-5 In 96 well plate (COSTAR / # 3590), a solidified antibody (Bio Vender / # RD181060100) diluted to 0.5 μg / ml with PBS (−) (WAKO / # 16219321) was added. 100 μl / well was dispensed and incubated overnight at 20 ° C. to immobilize.
After washing 3 times with 300 μl of wash buffer (PBS containing 0.05% Tween2), 200 μl / well of Reagent Diluent (R & D systems / # 890803) was added, and blocking operation was performed at 25 ° C. for 1 hour.
After washing again, 100 μl / well of stratum corneum extraction sample was added and reacted at 37 ° C. for 1.5 hours. In addition, a calibration curve was prepared by adding FABP-5 Human Recombinant protein (Cosmobio # FAB0805) for preparing a calibration curve at a concentration of 0 to 50 ng / ml.
Next, after washing, a solution obtained by diluting the detection antibody (R & D systems / # AF3077) with Wash Buffer so as to be 0.5 μg / ml was added and reacted at 37 ° C. for 1 hour.
After completion of the reaction, the well was washed, and then Streptavidin-HRP (R & D Systems / # 890803) was diluted 200-fold with a wash buffer, added with 100 μl / well, and reacted at 25 ° C. for 30 minutes.
After completion of the reaction, the well was washed, developed with TMB reagent (Promega / # G7431) 100 μl / well (25 ° C., 15 minutes), stopped with 1N sulfuric acid, and then SPECTRA MAX 190 (Molecular Device) was used. The absorbance at 450 nm was measured.
The amount of FABP-5 of each sample was calculated from the prepared calibration curve. The amount of FABP-5 in the stratum corneum was corrected with the total amount of protein in the stratum corneum extract and expressed as the amount of FABP-5 per unit protein.

(5) Measurement results and analysis 1) Analysis based on the presence or absence of acne The subjects were divided into two groups of 12 healthy subjects and 25 patients with acne skin, and the measured values of FABP-5 based on the presence or absence of acne were analyzed. FIG. 1 shows the measurement result of the cheek and FIG. 2 shows the measurement result of the forehead.
It was revealed that FABP-5 in the stratum corneum markedly increased with or without acne.

2) Analysis of acne symptom severity The above-mentioned subject's data is further classified into 3 groups of 12 healthy subjects, 14 mild subjects and 11 moderate / severe subjects, and FABP-5 measurement results Was re-analyzed. Similarly, the analysis results are shown as the cheek portion measurement result (FIG. 3) and the forehead measurement result (FIG. 4).
FABP-5 increased with acne in both cheeks and forehead.

From the above analysis, it was found that the degree of acne malignancy can be evaluated by measuring FABP-5. The method of the present invention increases FABP-5 corresponding to the degree of acne malignancy. Therefore, the method of the present invention can be used for determination of acne treatment effect and evaluation of recovery degree.

Claims (2)

FABP-5 in non-invasively collected stratum corneum is measured and compared with the measured value of FABP-5 in normal stratum corneum measured in advance or at the same time, and increased expression is used as an index of acne malignant transformation How to check for acne skin. The test method according to claim 1, wherein the stratum corneum is obtained from human skin by a peeling operation.