JP6656946B2 - How to correct the amount of stratum corneum extracted protein - Google Patents

Description

  The present invention relates to a method for correcting the expression level of a protein to be measured in the skin stratum corneum of a subject, comprising correcting the expression level of the protein to be measured by the expression level of cystatin A.

  In order to discuss the amount of biological factors, it is necessary to make comparisons per unit or assign them using an appropriate internal standard. For example, the amount of a biological factor contained in blood, urine, or the like can be represented by the amount of a factor per liquid volume. The biological factor amount in a tissue such as an organ can be represented by the factor amount per tissue weight. In the case of passing through the extraction step, it can be expressed by the amount of factors per extracted total protein amount or the amount of extracted factors per internal standard amount. On the other hand, regarding the correction in the measurement of the amount of biological factors in the skin stratum corneum, when correcting with the weight of the stratum corneum, it is necessary to measure the weight with a METTLER at the time of sampling the stratum corneum, but since the stratum corneum is minute, It is extremely difficult to measure. In addition, when correction is performed based on the total amount of protein in the sample extracted from the stratum corneum, it is necessary to separately measure the total amount of protein (BCA method, Bradford method, Lowry method, etc.), and a device for measurement (for example, constant temperature) Tank, absorption spectrophotometer, etc.). On the other hand, when using a protein as an internal standard for correction, if the measurement target factor is a protein, the correction is performed by using the same method as that for the measurement target protein without using any additional equipment. It is possible to do. Conditions for using the factors in the stratum corneum as an internal standard include the fact that detection is possible (the expression level is high to some extent), that there is little variation in the expression level within the comparison target, and that processing such as decomposition is required. Not that.

  Tubulin, GAPDH, β-actin, and the like are known as internal standards generally used in living cells. These factors are known as housekeeping factors that are expressed in constant amounts in many tissues, and are often used as internal standards. However, during the formation of the stratum corneum, skin epidermal cells lose their nuclei during the differentiation process, and proteins also differentiate into the stratum corneum while undergoing in vivo processing such as hydrolysis, so they are used as internal standards in living cells. Not all factors can be used as internal standards in the stratum corneum. In fact, we examined the possibility of using tubulin, GAPDH, and β-actin as an internal standard in the measurement of factors in the stratum corneum extract sample, but it is also highly likely that the expression level varies and is degraded in the stratum corneum Therefore, when quantifying the protein in the stratum corneum sample, it cannot be used as an internal standard.

  Keratin 2e and keratin 3 are known as proteins whose expression level is constant in the skin stratum corneum (Patent Document 1). However, the correlation between the expression level of keratin 2e or keratin 3 in the skin stratum corneum and the total protein amount is not sufficient. Keratin 2e and keratin 3 extracted from the skin stratum corneum cannot be detected by Western blotting or the like, and therefore have a problem in detection sensitivity.

JP-A-2011-185811

Vasilopoulos Y et al., "Association analysis of the skin barrier gene cystatin A at the PSORS5 locus in psoriatic patients: evidence for interaction between PSORS1 and PSORS5.", European Journal of Human Genetics, 2008; 16 (8): 1002-9 Blaydon DC et al., "Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion.", The American Journal of Human Genetics, 2011; 89 (4): 564- 71 Vasilopoulos Y et al., "A nonsynonymous substitution of cystatin A, a cysteine protease inhibitor of house dust mite protease, leads to decreased mRNA stability and shows a significant association with atopic dermatitis.", Allergy. 2007; 62 (5): 514 -9

  An object of the present invention is to provide a novel method for simply and accurately quantifying the amount of protein in a sample extracted from a stratum corneum.

  The present inventors have conducted intensive studies and, surprisingly, surprisingly, cystatin A in the horny layer extracted sample has not been subjected to processing such as hydrolysis in the horny layer of the skin, and the variation in the expression amount due to individual differences and skin conditions. The present invention has been found to be extremely useful as an internal standard used for quantification of a protein in a sample extracted from the stratum corneum because of its low sensitivity and high detection sensitivity, and has completed the present invention.

The present invention includes the following inventions.
[1] A method for correcting the expression level of a protein of interest in the stratum corneum of a subject,
Preparing a stratum corneum extract sample from the skin stratum corneum collected from the subject,
Measuring the expression level of the protein of interest in the sample extracted from the stratum corneum,
Measuring the expression level of cystatin A in the stratum corneum extract sample, and correcting the expression level of the target protein using the expression level of cystatin A,
A method that includes
[2] The method according to [1], wherein the skin stratum corneum collected from the subject is collected by tape stripping.
[3] The measurement of the expression level of the target protein in the stratum corneum-extracted sample and / or the measurement of the expression level of cystatin A in the stratum corneum-extracted sample include enzyme immunoassays (including ELISA and EIA). ), Quantitative immunoassay using fluorescence immunoassay, luminescence immunoassay, western blot, immunochromatography, label-free immunosensor (biosensor based on surface plasmon resonance, surface acoustic wave, quartz crystal microbalance, etc.) The method according to [1] or [2], which is performed by an assay or the like.
[4] The method according to any one of [1] to [3], which is used for evaluating a skin condition or a skin disease caused by expression of a protein of interest in the skin stratum corneum.

  According to the present invention, it becomes possible to simply and accurately correct the amount of a protein to be measured in the skin stratum corneum. This makes it possible to evaluate the skin condition caused by the measurement target protein in the skin stratum corneum, regardless of the variation in the amount of the collected stratum corneum and the extraction efficiency.

It is a photograph which shows the western blot analysis result of a stratum corneum extraction sample. It is a graph which shows the correlation (N = 34) between the total protein amount in a horny layer extraction sample, and the expression level of cystatin A.

The present invention provides a method for quantifying a protein to be measured in the skin stratum corneum of a subject, comprising correcting the expression level of the protein to be measured by the expression level of cystatin A.
Cystatin A used as an internal standard in the method of the present invention is a cysteine protease inhibitor present in epidermal cells, gastrointestinal mucosal epithelium, and leukocytes, and is known to form a conified envelope in epidermis. Cystatin A is associated with psoriasis, exfoliative ichthyosis, and atopic dermatitis (Non-patent Documents 1-3). In the absence of cystatin A, cell-cell adhesion failure to physical stimulation of cultured keratinocytes is observed. It has been reported that it is confirmed (Non-Patent Document 2). However, it has not been known that there is a correlation between the expression level of cystatin A in the stratum corneum and the expression level of protein.

  The method for collecting the skin stratum corneum is not particularly limited, but in view of its simplicity and non-invasiveness, the tape stripping method is preferred. Tape stripping is a method in which a piece of adhesive tape is applied to the skin surface layer, peeled off, and a horny layer sample is collected by attaching the horny layer of the skin to the peeled adhesive tape. A preferred method of tape stripping is to first purify the surface layer of the skin with, for example, ethanol or water to remove sebum, dirt, etc., and cut an adhesive tape piece of an appropriate size (for example, about 2 × about 6 cm) on the skin surface. It is performed by placing it lightly on the top, applying a uniform force to the entire tape and pressing it flat, and then peeling off the adhesive tape with a uniform force. The adhesive tape may be a commercially available cellophane tape or the like, and for example, Scotch Super Strength Mailing Tape (manufactured by 3M), cellophane tape (Cellotape (registered trademark); Nichiban Co., Ltd.) or the like can be used. The number of repetitions may be 2, 3, 4, 5, or more times, but it is preferable that the collected skin stratum corneum does not reach the granular layer.

  To prepare a horny layer extract sample from the collected horny layer of the skin, a neutral to weakly alkaline buffer containing a nonionic surfactant, for example, the following extraction buffer [0.1 M Tris-HCl (pH 8. 0), 0.14M NaCl, 0.1% Tween 20], but is not limited thereto. The tape to which the skin stratum corneum adheres is cut into small pieces with scissors or the like, transferred to a container such as a centrifuge tube, immersed in a small amount of the above buffer, and subjected to upside down stirring or ultrasonic treatment to efficiently extract the soluble component. The expression level of the target protein and the expression level of cystatin A in the prepared horny layer extracted sample are measured. Such a measurement can be performed without any particular limitation by a method conventionally used in the art. Typically, an enzyme-linked immunosorbent assay (including an ELISA method and an EIA method) and a fluorescent immunoassay method are used. Quantitative immunoassay using luminescence immunoassay, western blot, immunochromatography, and label-free immunosensors (biosensors based on surface plasmon resonance, surface acoustic waves, quartz crystal microbalance, etc.) it can.

  By correcting the expression level of the target protein in the stratum corneum extract sample thus measured using the expression level of cystatin A in the stratum corneum extract sample, the specific protein amount in the skin stratum corneum can be easily obtained. And it becomes possible to measure accurately. Such correction is typically performed by dividing the expression level of the protein of interest in the sample extracted from the stratum corneum by the expression level of cystatin A in the sample extracted from the stratum corneum. Alternatively, the total protein amount in the stratum corneum extract sample and the expression amount of cystatin A in the stratum corneum extract sample are measured, and a correlation function of these measured amounts is prepared in advance, and by using the correlation function, From the expression level of cystatin A in the stratum corneum extract sample, the total protein amount in the stratum corneum extract sample can be calculated. Therefore, by dividing the expression level of the target protein in the stratum corneum extract sample by the calculated total amount of protein in the stratum corneum extract sample, the stratum corneum extraction as a relative amount per total protein amount in the stratum corneum extract sample is performed. It is also possible to correct the expression level of the target protein in the sample.

  As used herein, the term "protein to be measured" refers to a protein to be quantified, which is present in the skin stratum corneum, and typically means a protein that can be involved in skin conditions and skin diseases. Examples of such a protein include, but are not limited to, SCCA1, bleomycin hydrolase, caspase, IL-1α, IL-1RA, and the like.

  According to the method of the present invention, it is possible to simply and accurately correct the amount of a protein to be measured in the skin stratum corneum, regardless of individual differences between subjects and skin conditions. Therefore, the method of the present invention can be used in the evaluation of a skin condition or skin disease caused by the expression of a protein of interest in the skin stratum corneum.

  Hereinafter, the present invention will be described more specifically with reference to specific examples. Note that the present invention is not limited to this.

(1) Preparation of horny layer extracted sample The skin horny layer was collected from the forearm of the subject by the tape stripping method. Specifically, a piece of cellophane tape (Nichiban Co., Ltd.) cut into an appropriate size (about 2 × about 6 cm) is gently placed on the skin surface of the subject's forearm, and a flat force is applied to the entire tape by applying an even force. After pressing down, the adhesive tape was peeled off with a uniform force to attach the skin stratum corneum to the cellophane tape.
Then, the cellophane tape to which the skin stratum corneum adhered was cut, immersed in an extraction buffer [0.1 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.1% Tween 20, 1 ml], and subjected to ultrasonic treatment ( This was performed for 30 seconds × 4) to prepare a horny layer extracted sample. The horny layer extracted sample was subjected to the following Western blot analysis, ELISA analysis, and total protein amount measurement.

(2) Western blot analysis Anti-β-actin antibody (Sigma Aldrich Japan), anti-GAPDH antibody (Sigma Aldrich Japan), anti-β-microglobulin antibody (Sigma Aldrich Japan), anti-cystatin antibody (Sigma Aldrich) Western blot analysis was performed using an anti-tubulin antibody (Santa Cruz Biotechnology), an anti-HPRT antibody (Abcam), an anti-keratin 2e antibody (Progen), and an anti-ribosomal protein antibody (Aviva). Specifically, the horny layer extracted sample (n = 11) obtained above was subjected to SDS electrophoresis, and then transferred to a membrane using an iBlot Dry Blotting system (Invitrogen), and the transferred membrane was washed and blocked. After that, the cells were reacted with various antibodies at 4 ° C. overnight. Further, after removing the antibody by washing, the HRP-bound secondary antibody was reacted. After washing, each protein band emitted by the ECL Plus Western Blotting Detection Kit (GE Healthcare) was baked on an X-ray film, and a candidate factor corresponding to the antibody was detected based on the presence or absence of the band.
As a result, signals of candidate factors other than cystatin A and GAPDH could not be detected. Among the proteins from which signals were detected, GAPDH showed a single band of about 37 kDa, but it could not be used as an internal standard because the expression level was not constant (data not shown). ). On the other hand, with respect to cystatin A, a single band having a molecular weight of about 11 kDa was observed in all samples, and it was confirmed that it was useful as an internal standard because there was little variation in the expression level (FIG. 1). The results for each candidate factor are shown in the table below.

(3) ELISA analysis The expression level of cystatin A in the horny layer extracted sample obtained above was measured using Human Cystatin-A (CSTA / STF1 / STFA) ELISA Kit (Cusabio Biotech) according to the recommended protocol. .

(4) Measurement of Total Protein Amount Using the RC DC protein assay kit (Bio-Rad Laboratories), the total protein amount in the horny layer extracted sample obtained above was measured according to the recommended protocol.

(5) Correlation between expression amount of cystatin A and total protein amount Total protein amount (y: mg / ml) and cystatin A (x: ng / ml), the following formula:
y = 0.002x + 0.00212
Was obtained (FIG. 2).
The correlation coefficient between the expression level of cystatin A in the sample extracted from the stratum corneum and the total protein level was 0.9178 (R ² = 0.8425). Therefore, it was confirmed that the expression level of cystatin A in the skin stratum corneum had an extremely high correlation with the protein level.

Claims (4)

A method for correcting the expression level of a protein of interest in the stratum corneum of a subject,
Preparing a stratum corneum extract sample from the skin stratum corneum collected from the subject,
Measuring the expression level of the protein of interest in the sample extracted from the stratum corneum,
Measuring the expression level of cystatin A in the stratum corneum extract sample, and correcting the expression level of the target protein using the expression level of cystatin A,
A method that includes   The method according to claim 1, wherein the skin stratum corneum collected from the subject is collected by a tape stripping method.   The measurement of the expression level of the target protein in the stratum corneum extract sample and / or the measurement of the expression level of cystatin A in the stratum corneum extract sample are performed by enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, The method according to claim 1, wherein the method is performed by a Western blotting method, an immunochromatography method, or a quantitative immunoassay using a label-free immunosensor.   The method according to any one of claims 1 to 3, which is used for evaluating a skin condition or a skin disease caused by expression of a protein to be measured in the skin stratum corneum.