A method for estimating the facial condition of a subject and a system for doing so.

Abstract

In order to improve not only facial redness but also other facial conditions (such as edema), there is a need to develop new methods for accurately estimating these conditions. [Solution] The method according to the present disclosure is a method for estimating the facial condition of a subject, and includes estimating the facial condition of a subject by using the expression level of a VEGF-A signaling-related protein gene as an indicator for a skin sample of the subject's face.

Description

Technical Field

[0001] The present invention relates to a method for estimating the facial state of a subject and a system therefor.

Background Art

[0002] The face is the most important body part that affects a human's impression, and swelling and blushing greatly affect the impression given to others.

[0003] Swelling of the body such as facial swelling upon waking up or foot swelling during fatigue is not only unfavorable for health but also a major enemy of beauty. Furthermore, if this swelling is left untreated, the surrounding blood vessels may be compressed, causing further swelling and potentially leading to pathological swelling.

[0004] The cutaneous vascular system exists in the dermis and is composed of blood vessels and lymphatic vessels. In order to maintain homeostasis, the tissue fluid that has moved outside the blood vessels must return to the veins again. The veins in the skin efficiently send blood flow to the center. However, the ability of the veins themselves to take in tissue fluid is poor. From this, it has been understood that the tissue that takes in tissue fluid, that is, the lymphatic vessels, is also an essential structure in the skin.

[0005] Lymphatic vessels play an important role in maintaining a constant state of the microenvironment around cells by collecting waste substances present in the skin and water and proteins that constantly leak from blood vessels. When water and the like that have leaked outside the blood vessels are not smoothly collected into the veins or lymph fluid and accumulate excessively between cells, this state is generally called "swelling".

[0006] Although massages and the like for eliminating facial swelling are known (Patent Document 1), since there are almost no methods for quantitatively measuring swelling, it has not been possible to accurately grasp how effective such massage methods have been in eliminating swelling.

[0007] Rosacea is a symptom that affects the impression one makes on others and is a source of distress for the person experiencing it. Rosacea is a condition in which the redness of the face persists and is mainly caused by skin inflammation or dilation of capillaries beneath the skin. Skin inflammation is caused by conditions such as atopic dermatitis (AD), and the dilation of capillaries beneath the skin causes the skin to turn red.

[0008] Various markers are used to clinically assess the overall severity of atopic dermatitis (AD). However, few markers are known to efficiently indicate the severity of local lesions. Vascular endothelial growth factor (VEGF), a potent activator of vascular permeability, is known to increase in AD lesions, and among these, the VEGF content in the stratum corneum (scVEGF) is known to be usable as a marker to assess the severity of AD lesions (Non-Patent Literature 1). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2000-119157 [Non-patent literature]

[0010] [Non-Patent Document 1] Amarbayasgalan T., et al., Int Arch Allergy Immunol. 2012;157(3):251-8. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] However, the method described in Non-Patent Document 1, which targets AD patients, showed low expression levels of VEGF protein in the stratum corneum compared to non-AD patients (healthy individuals), and samples frequently exceeded the detection limit, making it difficult to use for comparisons between non-AD patients (healthy individuals). Furthermore, in order to improve not only facial redness but also other facial conditions (such as edema), there has been a need for the development of new methods to accurately estimate these conditions, but these methods have not yet been put into practical use. [Means for solving the problem]

[0012] As a result of diligent research by the present inventors, we investigated the gene expression status of facial skin samples regarding the facial condition of the subject and found that differences occurred in VEGF-A signaling-related proteins. We discovered that the facial condition of the subject can be estimated by using the expression levels of these genes as indicators, and thus completed the present invention. In other words, it encompasses the following inventions.

[0013] [1] A method for estimating the facial condition of a subject, The facial condition of the subject is estimated by using the expression level of VEGF-A signaling-related protein genes as an indicator for the facial skin sample of the subject. Methods that include... [2] The method according to item 1, wherein one or more VEGF-A signaling-related protein genes are selected from the genes listed in Table 1 below. [Table 1] [3] The method according to item 1, wherein the facial condition is redness. [4] The method according to item 3, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 2 below. [Table 2] [5] The method according to item 3, wherein one or more VEGF-A signaling-related protein genes are selected from the genes listed in Table 3 below. [Table 3] [6] The method according to item 1, wherein the facial condition is physiological edema. [7] The method according to item 6, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes described in Table 4 below.

Table 4

Table 5

[10] The method according to item 9, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes described in Table 6 below.

Table 6

[11] The method according to item 9, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes described in Table 7 below.

Table 7

[0014]

[12] A system for estimating the facial state of a subject, comprising a storage unit, an analysis measurement unit, a data processing unit, and an output unit, wherein the storage unit stores the expression level of the VEGF-A signaling-related protein gene and the facial state corresponding to the expression level; the analysis measurement unit quantifies the expression level of the VEGF-A signaling-related protein gene in the skin sample of the subject's face; the data processing unit estimates the facial state of the subject by applying the expression level of the VEGF-A signaling-related protein gene of the subject measured by the analysis measurement unit to the facial state corresponding to the expression level stored in the storage unit; The output unit outputs information about the facial condition estimated by the data processing unit.

[13] The system described in item 12, wherein one or more of the VEGF-A signaling-related proteins are selected from the genes listed in Table 1 below. [Table 8]

[14] The system according to item 12, wherein the facial condition is a rosacea.

[15] The method according to item 14, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 2 below. [Table 9]

[16] The system described in item 14, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 3 below. [Table 10]

[17] The system described in item 12, wherein the facial condition is physiological edema.

[18] The system described in item 17, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 4 below. [Table 11]

[19] The system according to item 17, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 5 below. [Table 12]

[20] The system described in item 12, wherein the facial condition is a state of facial edema.

[21] The system described in item 20, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 6 below. [Table 13]

[22] The system described in item 20, wherein one or more of the VEGF-A signaling-related protein genes are selected from the genes listed in Table 7 below. [Table 14] [Effects of the Invention]

[0015] According to this disclosure, it becomes possible to quantitatively estimate the facial condition of the subject, such as redness of the face or facial edema (e.g., physiological edema, edema due to disease or injury, etc.). [Brief explanation of the drawing]

[0016] [Figure 1] A block diagram illustrating an example of the system configuration of the present invention is shown. [Figure 2] This is a schematic diagram showing the sampling and color measurement sites of the facial samples used in the example. [Figure 3] This is a diagram illustrating the gravity bulge analyzed in the example. [Figure 4] This is a diagram illustrating the gravity-induced depression analyzed in the example. [Figure 5] This figure illustrates a series of events in a subject who experienced edema on one side of their face, as analyzed in the example. [Figure 6] This is an image (front view) showing the three-dimensional shape of the face of a subject who had edema on one half of their face, as analyzed in the example. [Figure 7] This is a data image (oblique) representing the three-dimensional shape of the face of a subject who had edema on one half of their face, as analyzed in the example. [Figure 8] This shows the changes over time in the amount of facial swelling in subjects who experienced edema on one side of their face, as analyzed in the examples. [Modes for carrying out the invention]

[0017] The following describes in detail one embodiment of the present invention, but the scope of the present invention is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the invention. Furthermore, if multiple upper and lower limits are given for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0018] In one embodiment, the present invention is a method for estimating the facial condition of a subject, The facial condition of the subject is estimated by using the expression level of VEGF-A signaling-related protein genes as an indicator for the facial skin sample of the subject. This provides a method that includes [something].

[0019] In this specification, "subject" refers to the subject whose facial condition is to be analyzed, and may be a human or a non-human mammal (e.g., a non-human primate), but is preferably a human.

[0020] In this specification, "skin sample" refers to a sample containing stratum corneum cells recovered from the skin. This may include, for example, a biopsy sample obtained by cutting out a small tissue piece from the skin, a swab sample obtained by rubbing the surface of the skin with a cotton swab to collect cells, or an exfoliated sample obtained by peeling stratum corneum cells from the surface of the skin using a method such as a tape strip. However, an exfoliated sample is preferred.

[0021] In this specification, "facial condition" refers to the appearance and health status of the face, and in one embodiment, the present invention can estimate a state of facial redness, physiological edema, or facial edema. In one embodiment, the information estimated about the subject's facial condition may be provided as supplementary information for use in therapeutic diagnosis, or as supplementary information for use in counseling, etc., in non-therapeutic cosmetic procedures.

[0022] The inventors of this application have found that the "facial condition" of a subject can be estimated by using the expression level of the "VEGF-A signaling-related protein gene" as an indicator.

[0023] VEGF-A protein is one of the vascular endothelial growth factors (VEGF). VEGF is known as a major angiogenic growth factor that regulates neovascularization. The biological effects of VEGF are mediated through specific VEGF receptors on the surface of endothelial cells. There are VEGF-A, VEGF-B, VEGF-C, and VEGF-D proteins, which mediate various reactions in the body by interacting with VEGF receptors such as VEGFR1, VEGFR2, and VEGFR3. Among these, VEGF-A / VEGFR2 signaling is known to significantly mediate cellular responses involved in angiogenesis.

[0024] In this specification, "VEGF-A signaling-related protein genes" refers to a group of genes that express proteins related to VEGF-A / VEGFR2 signaling, for example, genes that express human VEGF-A / VEGFR2 signaling-related proteins (154 types of proteins) as described at https: / / www.wikipathways.org / pathways / WP3888.html (accessed July 31, 2024). Among these, in the invention of this embodiment, preferably, the facial condition of the subject can be estimated by using the expression level of one or more genes selected from the genes that express the proteins listed in Table 15 below as an indicator.

[0025] [Table 15]

[0026] The specific amino acid sequences of VEGF-A signaling-related proteins or the sequences of the genes encoding them listed in this specification can be obtained, for example, by searching on UniProt (https: / / www.uniprot.org / ) by entering the abbreviation of the gene to be searched or the ID of the Uniprot Primary accession. The specific gene sequences that can be used in the present invention are not limited to the amino acid sequences or nucleic acid sequences of the genes encoding them that can be searched and obtained using the IDs listed in the Uniprot Primary accession in Table 8. For example, sequences that are 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identical to the amino acid sequences or nucleic acid sequences of the genes encoding them that can be obtained using the Primary accession in Table 8, as well as their splicing variants. Furthermore, the origin of the gene may be from any animal, but it is preferably from humans or other mammals (e.g., non-human primates), and most preferably from humans.

[0027] In this specification, "gene expression level" refers to a level that can be determined by detecting and measuring a transcript transcribed from any gene, or a translation product translated using that transcript as a template. A transcript refers to, for example, an RNA chain transcribed using the DNA of a gene as a template, i.e., an RNA chain synthesized by RNA polymerase, and an RNA chain modified in the cell after transcription. An example of an RNA chain included in a transcript is messenger RNA (mRNA). These RNA chains also include those processed in the cell after transcription. A translation product refers to, for example, a polypeptide chain translated using a transcript transcribed by a gene as a template, i.e., a polypeptide chain synthesized by ribosomes, and a protein formed by the folding of that polypeptide chain. Translation products also include polypeptide chains or protein fragments.

[0028] Gene expression levels can be measured using known methods. For example, when measuring gene transcripts to determine gene expression levels, appropriate probes can be designed based on the sequence of the target gene, and then measured using methods such as quantitative PCR (qPCR), in situ hybridization, Northern blotting, DNA microarrays, and next-generation sequencing.

[0029] Furthermore, for example, when measuring the translation product of a gene to measure the gene expression level, the measurement may be performed using Western blotting, flow cytometry (FACS), or ELISA, which utilize antibodies to detect the protein translated by the target gene. Alternatively, it can be measured using omics methods (e.g., proteomics) that comprehensively analyze proteins and other components. Omics methods are preferred.

[0030] In one embodiment, gene expression levels can be compared by normalizing them by the expression level of any housekeeping gene. Examples of housekeeping genes that can be used for comparison include GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2-microglobulin, and HPRT 1 (hypoxanthine phosphoribosyltransferase 1). The expression levels of housekeeping genes can be measured using the same method as described above for the genes used in the present invention, and then compared between different samples by normalizing them by the expression level of the said housekeeping gene.

[0031] In one embodiment, "using gene expression levels as an indicator" may mean detecting whether the expression level of a desired gene in a skin sample is above or below a predetermined level (for example, the signal level of a negative control sample, or the average signal value calculated from a group of healthy skin samples). By comparing with the reference value, it becomes possible to estimate the facial condition of the target sample. The threshold for determining the facial condition is difficult to determine uniquely because it is appropriately changed depending on the sample population, but a judgment criterion may be calculated by performing any necessary statistical processing and used as the standard.

[0032] In one embodiment of the present invention, the presumable facial condition is rosacea. In this specification, "rosacea" refers to a condition in which the face becomes red, and can be temporary or chronic. Causes of rosacea include, for example, atopic dermatitis, rosacea, telangiectasia, seborrheic dermatitis, psoriasis, and allergies, and in one embodiment of the present invention, the state of rosacea in a subject having these symptoms can be estimated.

[0033] The inventors of this application have found a correlation between the expression level of the "VEGF-A signaling-related protein gene" and the "a-star" (also written as "a*"), an indicator of facial redness (Kikuchi K., et al., Long-term changes in Japanese women's facial skin color, COLOR Res. and Appl. Volume 43, Issue 1, February 2018, Pages 119-129). In particular, a positive correlation was found between the "a-star" indicator of facial redness and the expression level of the "VEGF-A signaling-related protein gene." Among these, the following gene group showed particularly high correlation coefficients (e.g., |R|>0.4) in the examples: [Table 16] These are some examples.

[0034] Furthermore, the following gene groups were identified as having high correlation coefficients in the examples (e.g., |R|>0.45): [Table 17] The following genes are selected. By using the expression levels of these genes in the target skin sample as indicators, the condition of facial redness can be quantitatively estimated by measuring one or more of the above genes, without having to measure the VEGF-A protein, which is usually present in amounts below the detection limit.

[0035] In one embodiment of the present invention, the presumptive facial condition may be physiological edema. In this specification, "physiological edema" refers to a state in which the face is swollen in a healthy state and is distinguished from a state in which edema occurs in the face due to disease or injury. In one embodiment of the present invention, the state of physiological edema in a subject can be estimated. Methods for evaluating the degree of physiological edema and facial edema described later will be explained in the examples described later.

[0036] The inventors of this application have found a correlation between the state of "physiological edema" and the expression level of "VEGF-A signaling-related protein genes." In particular, the following gene group was identified in the examples as having a high correlation coefficient with the state of "physiological edema" (e.g., |R|>0.4): [Table 18] These are some examples.

[0037] Furthermore, the following gene groups were identified as having high correlation coefficients in the examples (e.g., |R|>0.5): [Table 19] Selected from these. By using the expression levels of these genes in the target skin sample as indicators, the state of physiological edema can be quantitatively estimated by measuring one or more of the above genes, without having to measure the VEGF-A protein, which is usually present in amounts below the detection limit, for the target's facial condition.

[0038] The inventors of this application have found a correlation between the state of "facial edema" and the expression level of "VEGF-A signaling-related protein genes." In particular, the following gene group showed a high correlation coefficient with the state of "facial edema" in the examples: [Table 20] These are some examples.

[0039] Furthermore, the following gene groups have a high correlation coefficient with the condition of "facial edema": [Table 21] Selected from these. By using the expression levels of these genes in the target skin sample as indicators, the state of "facial edema" can be quantitatively estimated by measuring one or more of the above genes, without having to measure the VEGF-A protein, which is usually present in amounts below the detection limit.

[0040] In one embodiment, the present invention provides a system for estimating the facial condition of a subject. In one embodiment, the system of the present invention is: It includes a storage unit, an analysis and measurement unit, a data processing unit, and an output unit. The memory unit stores the expression level of the VEGF-A signaling-related protein gene and the facial state corresponding to the expression level; The analytical measurement unit quantifies the expression level of VEGF-A signaling-related protein genes in the facial skin sample of the subject; The data processing unit estimates the facial condition of the subject by applying the expression level of the target VEGF-A signaling-related protein gene measured by the analysis and measurement unit to the facial condition corresponding to the expression level stored in the memory unit; The output unit outputs information about the facial condition estimated by the data processing unit.

[0041] More specifically, in the system 10 of the present invention, the memory unit 11 is configured to store the expression level of any target VEGF-A signaling-related protein gene input from the input unit 12, and the facial state corresponding to the expression level. The analytical measurement unit 13 is configured to quantify the expression level of VEGF-A signaling-related protein genes in the facial skin sample being evaluated. The data processing unit 14 is configured to compare the expression level of the VEGF-A signaling-related protein gene in the facial skin sample to be evaluated with the facial condition corresponding to the expression level stored in the memory unit, and to estimate the facial condition to be evaluated. The output unit 15 is configured to output information about the estimated facial condition of the subject being evaluated.

[0042] The storage unit 11 includes, for example, memory devices such as RAM, ROM, and flash memory, fixed disk devices such as hard disk drives, or portable storage devices such as flexible disks and optical disks. The storage unit 11 stores data measured by the analysis and measurement unit 13, data and instructions input from the input unit 12, calculation processing results performed by the data processing unit 14, as well as computer programs and databases used for various processes of the information processing device. The computer programs may be installed, for example, on computer-readable recording media such as CD-ROMs and DVD-ROMs, or via the Internet. The computer programs are installed in the storage unit 11 using known setup programs. The storage unit 11 stores data on judgment values ​​related to facial conditions that have been input in advance from the input unit 12.

[0043] The input unit 12 is an interface, and also includes an operation unit such as a keyboard or mouse. This allows the input unit to receive data measured by the analysis and measurement unit 13, instructions for calculations performed by the data processing unit 14, and so on. Furthermore, if the analysis and measurement unit 13 is located externally, the input unit 12 may also include an interface unit separate from the operation unit that allows measured data to be input via a network or storage medium.

[0044] The analytical measurement unit 13 is configured to obtain the expression level of the target VEGF-A signaling-related protein gene by analyzing and measuring information from the target. For example, the analytical measurement unit 13 can be configured to measure the expression level of at least the VEGF-A signaling-related protein gene in a facial skin sample. Therefore, the analytical measurement unit 13 only needs to have a configuration that enables the measurement of the expression level of the VEGF-A signaling-related protein gene. The VEGF-A signaling-related protein gene may be analyzed one type at a time, or some or all types of VEGF-A signaling-related protein gene may be analyzed together. For example, the instrument may be capable of performing quantitative PCR (qPCR), in situ hybridization, Northern blotting, DNA microarray, or next-generation sequencing. It may also be capable of performing Western blotting, flow cytometry (FACS), ELISA, or omics (e.g., proteomics). The analysis and measurement unit 13 may be configured separately from the system 10, and the measured data may be input via the input unit 12 using a network or storage medium.

[0045] The data processing unit 14 provides information about the facial condition of the subject (e.g., redness, physiological edema, and / or facial edema) by comparing an index of the measured expression level of VEGF-A signaling-related protein genes with a determination value stored in the memory unit.

[0046] The data processing unit 14 performs various calculations on the data measured by the analysis and measurement unit 13 and stored in the storage unit 11, according to a program stored in the storage unit. The calculations are performed by a CPU included in the data processing unit. This CPU includes a functional module that controls the analysis and measurement unit 13, the input unit 12, the storage unit 11, and the output unit 15, and can perform various controls. Each of these units may be composed of an independent integrated circuit, microprocessor, firmware, etc.

[0047] The output unit 15 is configured to output information about the facial condition of the target, which is the result of calculation processing performed by the data processing unit. The output unit 15 may be a display device such as a liquid crystal display that directly displays the results of the calculation processing, an output means such as a printer, or an interface unit for outputting to an external storage device or outputting via a network.

[0048] In another embodiment, the present invention may also be a computer program (as appropriate, referred to as "the program of the present invention") that causes an information processing device to execute a method for estimating the facial condition of the subject in the above-mentioned subject. Specifically, the program of the present invention can be configured as a program including computer instructions that, when installed and executed on a general-purpose information processing device, causes the information processing device and external devices connected thereto, such as input / output interfaces and analyzers, to function as a sample analysis system 10 including, for example, a storage unit 11, an input unit 12, an analysis and measurement unit 13, a data processing unit 14, and an output unit 15, as shown in Figure 1. Such a program of the present invention can be realized by computer programming knowledge well known to those skilled in the art. Such a program of the present invention, and recording media such as CD-ROMs containing such programs, are also included within the technical scope of the present invention. [Examples]

[0049] The present invention will be described in more detail below based on examples, but these examples are not intended to limit the present invention in any way.

[0050] 1. Experimental Method

[0051] 1-1. Extraction and pretreatment of stratum corneum proteins Three layers of stratum corneum were collected from the same area of ​​the subject's cheek using D-squame tape (Promotool), and the third layer was used as the proteomics sample. The reagents and equipment used for protein extraction and pretreatment were mainly those included in the EasyPep® 96 MS Sample Prep Kits (Thermo).

[0052] First, the stratum corneum tape was immersed in Lysis Buffer, then sonicated to extract the protein, and the supernatant was obtained by centrifugation. Next, Reduction Solution and Alkylation Solution were added to each sample and mixed, and incubated at 50°C for 10 minutes, followed by cooling at room temperature for 10 minutes. Then, Trypsin / Lys-C solution was added, and the mixture was incubated at 37°C for 3 hours to digest the peptide of the protein. Next, Digestion Stop solution was added and mixed to stop the digestion. Then, the peptide was purified using a Peptide Clean-Up Plate, and the resulting solution was dried using a centrifugal evaporator. Finally, the solution was redissolved in 0.1% formic acid solution and prepared as a sample for LC-MS / MS analysis.

[0053] 1-2. LC-MS / MS Measurement The UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) and Orbitrap Fusion Lumos (Thermo Fisher Scientific) were used as the LC-MS / MS system, along with a nano HPLC capillary column (ODS, inner diameter 75 μm × 120 mm, particle size: 3.0 μm, manufactured by Nikkyo Technos Co., Ltd.). A 0.1% formic acid solution was used as mobile phase A, and a 0.1% acetonitrile solution as mobile phase B. The gradient was performed under the following conditions.

[0054] 0 min, 2% B; 90 min, 30% B; 95 min, 60% B; 100 min, 60% B; 105 min, 2% B; 110 min, 2 % B

[0055] Data-dependent acquisition was used for data acquisition. The separated peptides were electrosprayed with a Nanospray Flex NG source under a 1.8kV voltage, and the ion transfer tube was set to 275°C. The RF level of the ion funnel was 45, the resolution of the MS1 Orbitrap was 120,000 (at m / z 200), and the MS1 AGC target and maximum injection time were 4 × 10⁻¹⁶. 5 The time was set to 50ms. Precursor ions with charges from +2 to +7 were isolated for MS2 sequencing. The MS2 isolation window was 1.6 Da, and the AGC target was 5 × 10⁻¹⁴. 4 The dynamic exclusion time was set to 20 seconds, and the mass accuracy to ±10 ppm. The resolution and maximum injection time of MS2 were 15000 (at m / z 120) and 22 ms, respectively, and the signal intensity threshold was 2.5 × 10⁻¹⁶. 4 The settings were adjusted. Precursor ions were dissociated by high-energy collision-induced dissociation (HCD) with a normalized collision energy of 30%. Mass calibration was performed automatically for each injection using a lock mass system with peaks at m / z = 391.2843 and 445.12.

[0056] 1-3. LC-MS / MS Data Analysis Database searches and protein / peptide identification and quantification were performed using Proteome Discoverer (version 2.5) and the Sequest and Amanda databases. MS / MS spectra were searched using the Swiss-Prot and TrEMBL human databases. Protein N-terminal acetylation and methionine oxidation were selected as dynamic modifications. Carbamide methylation of cysteine ​​residues was set as a static modification. The minimum peptide length was set to 7 amino acids, the maximum mass to 5000 Da, and up to two missed cleavages were set for each peptide. Filtering was performed with an FDR (False Discovery Rate) of 0.01 for both peptides and proteins. Both unique peptides and razor peptides were selected for label-free quantification (LFQ) calculations. Other unspecified parameters were set to the default settings of Proteome Discoverer.

[0057] 1-4. Data Analysis Database searches and protein / peptide identification and quantification were performed using Proteome Discoverer (version 2.3) and the Sequest and Amanda databases. MS / MS spectra were searched using the Swiss-Prot and TrEMBL human databases. Protein N-terminal acetylation and methionine oxidation were selected as dynamic modifications. Carbamide methylation of cysteine ​​residues was set as a static modification. The minimum peptide length was set to 7 amino acids, the maximum mass to 5000 Da, and up to two missed cleavages were set for each peptide. Filtering was performed with an FDR (False Discovery Rate) of 0.01 for both peptides and proteins. Both unique peptides and razor peptides were selected for label-free quantification (LFQ) calculations. Other unspecified parameters were left at the default settings of Proteome Discoverer. The extracted data were further processed and visualized in Microsoft Excel. Principal component analysis (PCA) using Proteome Discover was performed on the types and amounts of proteins obtained from the facial stratum corneum.

[0058] 2. Examples of each test <Example Test 1> Correlation analysis between facial redness and the expression level of VEGF-A signaling-related protein genes We investigated the correlation between the a-star ("a*"), an indicator of facial redness, and the protein levels of VEGF-A signaling-related proteins in 23 healthy Japanese female panelists aged 20 to 60.

[0059] At 0, 1, 2, and 3 weeks post-test, the a* value was measured using a spectrophotometer CM-700d (KONICA MINOLTA) at the cheek area where the perpendicular line from the outer corner of the panelist's eye intersected with the horizontal line below the nose, as shown in Figure 2. On the same day as the a* measurement, three stratum corneum samples were collected from the cheek area at the same location as the perpendicular line from the outer corner of the panelist's eye, as also shown in Figure 2, and the third sample was used as the stratum corneum proteomics sample. Of the 23 panelists, one was deemed an outlier based on the a* value, and correlation analysis was performed between each panelist's a* and stratum corneum proteomics data for 22 panelists. VEGF-A signaling-related proteins with a high correlation to a* were extracted.

[0060] [Table 22]

[0061] The analysis revealed that the proteins listed in Table 22 showed particularly high correlation coefficients (|R|>0.4) between a*, an indicator of facial redness, and the protein levels of VEGF-A signaling-related proteins. Furthermore, the proteins of the genes listed in Table 17 from Table 22 showed even higher correlation coefficients (|R|>0.45).

[0062] <Example 2> Correlation analysis between physiological edema and the expression levels of VEGF-A signaling-related protein genes We investigated the correlation between physiological edema and VEGF-A signaling-related proteins in 23 healthy Japanese female panelists aged 20 to 60.

[0063] <Quantitative evaluation> At 0 weeks, 1 week, 2 weeks, and 3 weeks later, data representing the three-dimensional shape of the panelists' faces (three-dimensional facial images) was captured using a 3D image acquisition and analysis device (VECTRAHandy H2 (VEC-H2, manufactured by Canfield Scientific). At the same time as the capture, as described in "1. Experimental Method" above, three layers of stratum corneum were collected from the cheek area at the position where a perpendicular line is drawn from the outer corner of the eye, and the third layer was used as a sample for proteomics.

[0064] Three-dimensional facial images were obtained for each panelist's face when it was in a horizontal position (specifically, when the midline of the face was held perpendicular to the direction of gravity) and when it was in a vertical position (specifically, when the midline of the face was held parallel to the direction of gravity). Next, using a computer, the amount of gravitational bulge (VCswelling) and gravitational indentation (VCshrinking) were calculated based on the change in three-dimensional shape between the three-dimensional facial image of the panelist's face in the horizontal position and the three-dimensional facial image of the panelist's face in the vertical position. Then, based on the amount of gravitational bulge (VCswelling) and gravitational indentation (VCshrinking), the amount of facial swelling (Sc) was calculated as follows.

[0065] -Calculation of facial swelling (Sc)- Facial swelling (Sc) was calculated using the following formula (1). Three-dimensional facial images were obtained for each panelist three times at 0 weeks, 3 weeks, and 6 weeks, and the average value of these measurements was taken as the facial swelling (Sc) of that panelist.

[0066]

number

[0067] The gravitational bulge and gravitational indentation were calculated as follows.

[0068] <<Calculation of Gravitational Vulnerability (VCswelling)>> We compare the three-dimensional shape in the horizontal position with the three-dimensional shape in the vertical position, and define the difference in volume between the two positions (i.e., horizontal and vertical) of the part that has a larger volume in the vertical position (explained with reference to Figure 3) as the amount of gravitational bulge (VCswelling).

[0069] <<Calculation of Gravitational Indentation (VCshrinking)>> We compare the three-dimensional shape in the horizontal position with the three-dimensional shape in the vertical position, and define the difference in volume between the horizontal and vertical positions (i.e., the difference between the horizontal and vertical positions) of the part with a smaller volume in the vertical position (explained with reference to Figure 4) as the amount of gravity-induced indentation (VC shrinking).

[0070] The amount of facial swelling (Sc) from week 0 to week 6 of the study, and genes showing a correlation with VEGF-A signaling-related protein genes, as determined by omics analysis, were identified.

[0071] [Table 23]

[0072] <Example 3> Correlation analysis between facial edema and the expression levels of VEGF-A signaling-related protein genes In subjects who underwent the following clinical course, stratum corneum proteomics was performed on the edematous side of the face (hemifacial edema) and the non-edematous side of the face, as described above, and the gene expression levels of VEGF-A signaling-related proteins were compared and analyzed.

[0073] <Subject Profile> (1) Since 2017, the subject had a benign cyst on the right side of his neck (Figure 5). (See a). (2) On January 23, 2020, the cyst was removed by surgical incision, and the doctor determined that it was completely cured (Figure 5). (See b). (3) Two days after surgery, following drain removal (Day-19), swelling and edema occurred on the right side of the face (Figure 6). (See b). (4) The swelling on the right side of the face subsided on February 12th (Day 0) (Figure 8b). (5) The edema on the right side of the face subsided on April 6 (Day 54), 54 days after February 12 (Figure 8c).

[0074] Figure 6 is an image (front view) showing the three-dimensional shape of a subject's face according to one embodiment.

[0075] The left side of Figure 6 shows an image representing the three-dimensional shape of the subject's face, taken from the front on January 23rd, after surgery. The middle side of Figure 6 shows an image representing the three-dimensional shape of the subject's face, taken from the front on February 14th. The right side of Figure 6 shows an image representing the three-dimensional shape of the subject's face, taken from the front on April 6th. In the image taken on January 23rd (left side of Figure 6), only the right side of the face is clearly swollen and edema. However, in the image taken on February 14th (middle side of Figure 6), there is no obvious swelling on the right side of the face, but it is edema. In the image taken on April 6th (right side of Figure 6), neither swelling nor edema is observed. Therefore, it can be seen that swelling and edema can be distinguished in images representing the three-dimensional shape of the face.

[0076] Figure 7 is a diagram illustrating the facial swelling of the subject described above. Figure 7 is an image representing the three-dimensional shape of the face (specifically, the subject's face is represented by three-dimensional data, and this data is reproduced as an image). Significant swelling is observed in the right cheek area.

[0077] Regarding the swelling of the right cheek, it was quantified using the following formula based on the three-dimensional data of the subject's face, with February 12th, the day the swelling subsided, as the baseline.

[0078] ΔΔ volume = (Volume of the right cheek on each day - Volume of the right cheek on 2 / 12) - (Volume of the left cheek on each day - Volume of the left cheek on 2 / 12)

[0079] Regarding the swelling of the right cheek, it was quantified using the following formula based on the three-dimensional data of the subject's face, with February 12th, the day the swelling subsided, as the baseline.

[0080] ΔΔSc = (Sc of the right cheek on each day - Sc of the right cheek on 2 / 12) - (Sclerosis of the left cheek on each day - Sclerosis of the left cheek on 2 / 12)

[0081] In the subject, facial swelling was quantitatively measured over time from January 23rd, after surgery, using the same method as in Example 2. Furthermore, stratum corneum proteomics was performed on January 24th (D1), February 14th (D2), immediately after the edema subsided, and April 6th (D3), when swelling was no longer detectable, to compare and analyze the gene expression levels of VEGF-A signaling-related proteins.

[0082] The results are shown below. [Table 24-1] [Table 24-2]

Claims

1. A method for estimating the facial condition of a subject, The facial condition of the subject is estimated by using the expression level of the VEGF-A signaling-related protein gene as an indicator for the facial skin sample of the subject. Methods that include...

2. The method according to claim 1, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 1 below. Table 1

3. The method according to claim 1, wherein the facial condition is a red face.

4. The method according to claim 3, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 2 below. Table 2

5. The method according to claim 3, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 3 below. Table 3

6. The method according to claim 1, wherein the facial condition is physiological edema.

7. The method according to claim 6, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 4 below. Table 4

8. The method according to claim 6, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 5 below. Table 5

9. The method according to claim 1, wherein the facial condition is a state of facial edema.

10. The method according to claim 9, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 6 below. Table 6

11. The method according to claim 9, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 7 below. Table 7

12. A system for estimating the facial condition of a subject, It includes a storage unit, an analysis and measurement unit, a data processing unit, and an output unit. The memory unit stores the expression level of the VEGF-A signaling-related protein gene and the facial state corresponding to the expression level; The analytical measurement unit quantifies the expression level of VEGF-A signaling-related protein genes in the facial skin sample of the subject; The data processing unit estimates the facial condition of the subject by applying the expression level of the target VEGF-A signaling-related protein gene measured by the analysis and measurement unit to the facial condition corresponding to the expression level stored in the memory unit; The output unit outputs information about the facial condition estimated by the data processing unit.

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