KR20210002200A - Composition for diagnosing skin damage caused by fine dust, and screening method for substances inhibiting or alleviating skin damage caused by fine dust - Google Patents

Abstract

Disclosed in the present invention is a method for screening a substance for inhibiting or alleviating skin cell damage caused by fine dust. Specifically, the present invention can effectively screen substances which inhibit or alleviate skin cell damage caused by fine dust, as the screening method of the present invention uses, as a marker, a gene which exhibits a change in the expression level specifically depending on fine dust while not responding to other external environmental stimuli.

Description

Composition for diagnosing skin damage caused by fine dust, and screening method for substances that inhibit or relieve skin damage caused by fine dust {COMPOSITION FOR DIAGNOSING SKIN DAMAGE CAUSED BY FINE DUST, AND SCREENING METHOD FOR SUBSTANCES INHIBITING OR ALLEVIATING SKIN DAMAGE CAUSED BY FINE DUST}

The present invention provides a biomarker that can be used to diagnose skin damage due to fine dust, a composition containing the same, a kit containing the same, a method for diagnosing skin damage due to fine dust using the same, and inhibiting skin damage due to fine dust or It relates to a method of screening for palliatives.

Among the constituent layers of the skin, the epidermis plays an important role in preventing water evaporation inside the human body. The epidermis is divided into the stratum corneum layer, granule layer, play layer, and basal layer in order from the outside, and the cells in the stratum corneum act like bricks, and the intercellular lipids between the keratinocytes act like mortar to form the skin barrier (J Invest.Dermatol. 80 (Suppl.), 44-49, 1983).

On the other hand, fine dust is a particulate matter floating in the atmosphere and is known to be mainly generated from exhaust gas of automobiles, combustion of fossil fuels, and dust of roads. In general, fine dust means having a size of less than an average particle diameter of 10 μm, and when exposed to fine dust, problems such as respiratory and cardiovascular diseases as well as skin and eye diseases may occur. In particular, it has been observed that pigmentation and nasolabial folds increase in the skin of people living in areas with a lot of yellow dust, dust or fine dust.

Since fine dust is very small in size, it is difficult to identify whether it exists with the naked eye, and it is an air pollutant that is directly connected to human health.There is an increasing demand for a method to clearly check the exposure to fine dust. to be.

Republic of Korea Patent Publication No. 10-2016-0119703

In one aspect, an object of the present invention is to provide a biomarker that can be used to diagnose skin damage caused by fine dust, and a composition containing the same.

In another aspect, an object of the present invention is to provide a method for diagnosing skin damage caused by fine dust.

In another aspect, an object of the present invention is to provide a method for screening a substance for suppressing or alleviating skin damage caused by fine dust.

To achieve the above object,

In one aspect, the present invention comprises an agent for measuring the expression level of the mRNA of at least one or more genes of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes. It provides a composition for diagnosing damage.

In another aspect, the present invention, from a skin cell sample of a subject,

1) measuring the expression level of the mRNA of at least one or more genes of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes; And

2) A method for diagnosing skin damage caused by fine dust, comprising comparing the expression level with the expression level of the mRNA of the gene or the protein encoded therefrom in a skin cell sample not damaged by fine dust Provides.

In another aspect, the present invention, the step of treating fine dust on skin cells;

Treating the skin cells treated with the fine dust with a test substance; And

In the skin cells treated with the test substance, measuring the expression level of the mRNA of at least one or more of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene before and after the test substance treatment or the protein encoded from the genes. It provides a method of screening for substances that inhibit or alleviate skin damage caused by fine dust.

In one aspect, when the biomarker for diagnosing skin cell damage due to fine dust and a composition comprising the same according to the present invention is used, the expression level of a gene whose expression level is changed depending on whether the skin cell is damaged by fine dust is confirmed. By doing so, it is possible to easily and quickly diagnose whether the skin cells are damaged, and by checking the expression level of the gene, it is possible to easily screen for substances that suppress or alleviate skin damage caused by fine dust.

1 shows a cell survival ratio (%) by treatment of a fine dust extract, where PM2.5 (Particulate matter 2.5) represents fine dust having a particle size of 2.5 μm.
2 shows that the expression level of a specific gene in skin cells stimulated by fine dust decreases.

Hereinafter, the present invention will be described in detail.

The term "probe" as used herein refers to a polynucleotide having a base of a sequence capable of complementarily binding to a target site of a gene, a variant thereof, or a polynucleotide and a labeling substance bound thereto. do.

As used herein, the term "primer" has a base of a sequence capable of complementarily binding to the end of a specific region of a gene used to amplify a specific region corresponding to a target site of a gene using PCR. It means a polynucleotide or a variant thereof. The primer is not required to be completely complementary to the end of a specific region, and may be used as long as it is complementary enough to hybridize to the end to form a double chain structure.

The term "hybridization" as used herein means that two single-stranded nucleic acids form a duplex structure by pairing complementary nucleotide sequences. Hybridization can occur not only when the complementarity between single-stranded nucleic acid sequences is perfect, but also in the presence of some mismatch bases.

The term "fine dust" as used herein is a particulate matter that is invisible to the human eye and floats or scatters for a long time in the atmosphere, and may mean a material having a particle diameter of 10 μm or less. Particularly, particulate matter having a particle diameter of 2.5 μm or less is referred to as "ultra-fine dust", and "fine dust" is intended to include "ultra-fine dust" as used herein.

The present invention, in one aspect, relates to a biomarker for diagnosing skin damage caused by fine dust, comprising at least one selected from the group consisting of a specific gene and a protein encoded from the gene. In this case, the skin damage may be damage to skin cells, and the skin cells may be keratinocytes.

The specific gene may be at least one or more of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene.

One of the genes or all of the genes may be used as a biomarker for diagnosis of skin damage caused by fine dust.

In another aspect, the present invention relates to a composition for diagnosing skin damage caused by fine dust, comprising an agent measuring the expression level of mRNA or protein thereof of one or more genes selected from the group consisting of the genes.

In one embodiment, the agent is a polynucleotide or fragment thereof that is complementary to the mRNA of the gene, a primer or probe capable of amplifying the gene, or an antibody that specifically binds to the protein, for example, a monoclonal antibody ( monoclonal antibody) or polyclonal antibody.

In one aspect, the present invention relates to a kit comprising a composition for diagnosing skin damage caused by the fine dust. By using the kit according to the present invention, it is possible to quickly and conveniently diagnose whether the skin is damaged by fine dust.

In one embodiment, using the kit, the expression level of the mRNA of at least one or more of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene measured from the skin cells of the subject or the protein encoded therefrom is determined by fine dust. If it is lower than in the intact skin cell sample, it can be determined that the skin cells are damaged by fine dust.

In one embodiment, the kit may include one or more antibodies that specifically recognize a protein encoded by one of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or both of these genes, and the diagnosis target By measuring the amount of antigen bound to the antibody in skin cells, it is possible to determine whether the skin is damaged by fine dust.

In one aspect, the present invention relates to a method for diagnosing whether or not skin is damaged by fine dust.

Specifically, the method includes the steps of: 1) measuring the expression level of the mRNA of at least one or more genes of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes; And 2) comparing the level of expression with the level of expression of the mRNA of the gene or a protein encoded therefrom in a skin cell sample not damaged by fine dust.

In one embodiment, the method comprises the mRNA of at least one of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene, or the expression level of the protein encoded from the genes, measured from a skin cell sample of the subject. If it is lower than in the skin cell sample not damaged by the dust, the step of determining that the skin cells are damaged by fine dust may be further included.

In one embodiment, the method of measuring the expression level of the mRNA or protein of the gene is PCR (polymerase chain reaction), RT-PCR (reverse transcription polymerase chain reaction), northern blotting (northern blotting), western blotting (western blotting) and ELISA (enzyme-linked immunosorbent assay).

The term "normal level" such as the amount of gene expression used herein refers to the amount of gene expression in normal skin cells not stimulated by fine dust. In the present specification, by measuring the amount of one or more mRNAs or proteins thereof among the genes in the skin cell to be diagnosed, and comparing the measured value with the expression level in normal skin cells not stimulated by fine dust, whether the skin cells are damaged Will be evaluated.

As used herein, the terms "more or less" and "high or low" mean that there is a difference compared to the reference amount, and the amount is 1.5 times or more, twice or more, preferably, compared to the standard amount. It is a case where there is a difference in the amount by increasing or decreasing more than 2.2 times.

Genes used in the present invention, whose expression levels are reduced by fine dust, are shown in Table 1 below. In Table 1, Name means NCBI's genebank accession ID, Gene Symbol means an official gene symbol, and Gene title means the name of each gene.

Name Gene Symbol Gene title NM_004430 EGR3 early growth response 3 NM_001003407 ABLIM1 actin binding LIM protein 1

The polynucleotide used as a probe in the kit of the present invention includes the full length of a marker gene or a fragment thereof whose expression level is decreased by stimulation by fine dust. It is preferable that the length of the fragment includes 10 or more contiguous nucleotides, because if the length of the probe is 10bps or less, it binds non-specifically.

The length of the polynucleotide used as a primer in the kit of the present invention is not greatly limited, but it is advantageous to use a primer of preferably 18 to 22.

Monoclonal antibodies against a polypeptide encoded by a marker gene, included in the kit of the present invention, can be prepared by a general method for producing monoclonal antibodies.

In one aspect, the present invention relates to a method of screening for a substance that suppresses or alleviates skin cell damage caused by fine dust by controlling the expression level of a specific gene in skin cells damaged by fine dust to a normal level.

Specifically, the screening method comprises the steps of treating skin cells with fine dust; Treating the skin cells treated with the fine dust with a test substance; And measuring the expression level of the mRNA of at least one of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes before and after treatment of the test substance in the skin cells treated with the test substance. Can include.

In one embodiment, the screening method is, the expression level of the mRNA of at least one or more genes of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes after the test material treatment compared to before the test material treatment When is increased, it may further include the step of determining as a substance to suppress or alleviate skin damage caused by fine dust.

That is, when the test substance is treated, when the expression level of the gene whose expression level is reduced by fine dust increases to a normal level, the test substance may be determined as a substance to suppress or alleviate skin damage caused by fine dust.

In one embodiment, the skin cells may be keratinocytes.

In one embodiment, the expression level of the mRNA or protein of the gene is a polynucleotide or fragment thereof complementary to the mRNA of the gene, a primer or probe capable of amplifying the gene, or an antibody that specifically binds to the protein. One or more can be used to measure.

In one embodiment, the method of measuring the expression level of the mRNA or protein of the gene is PCR (polymerase chain reaction), RT-PCR (reverse transcription polymerase chain reaction), northern blotting (northern blotting), western blotting (western blotting) and ELISA (enzyme-linked immunosorbent assay).

Hereinafter, the content of the present invention will be described in more detail through the following examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art can be performed. It can be, and this is also included in the scope of the present invention.

[Example 1] Collection and extraction of fine dust

Fine dust was collected using a low volume air sampler (Sensidyne, Gillian, Low Volume Air Sampler, FL, USA), and the filter pack was sampled for about 24 hours by changing the filter and denude around 10 am on every measurement day. Was collected. From February 1, 2014 to February 28, 2014, fine dust was collected every day at Pungha Station in Seoul (located in Cheoin-gu, Yongin-si, Gyeonggi-do, on the 6th floor of the dormitory of Hankuk University of Foreign Studies Center for Foreign Studies). The timer was started while turning on and the time of the timer was recorded when the vacuum pump was turned off. The sampling flow rate was 16.7 L/min, and the flow rate was measured with a flow meter (Model 4143, TSI Inc.) at the beginning of the measurement, and the flow rate was measured again at the end of the measurement. The Teflon filter in the filter pack was weighed before and after sample collection. Before measuring the weight of the Teflon filter, put a constant weight in a desiccator (NIKKO, Japan) with a relative humidity of 40% for 24 hours, and then measure the weight twice on an electronic scale (DVG215CD, Ohaus) with 5 decimal places to calculate the average value. Recorded. Even after collecting the sample, the weight was measured twice in a desiccator for 24 hours before measuring the weight, and the mass concentration was calculated by comparing it with the weight measured before collection. The extraction of fine dust was carried out by soaking a Teflon filter in 1 mL of ethanol, adding 14 mL of DW, closing the lid while making the aerosol collection surface of the filter contact the water surface, and applying ultrasonic waves for 30 minutes with an ultrasonic cleaner. In order to minimize the error due to moisture in the filtration step, after completely removing moisture from the filter for 48 hours in a decicator, weigh the filter using an ultra-precise scale (Mettler Toledo Company) that can measure up to 0.1 mg. The weight was weighed before and after extraction of the filter.

[Example 2] Culture of human normal keratinocytes

Human normal keratinocytes (Human normal epidermal keratinocyte cells) is Ron its (Lonza, Inc. Walkersville, Maryland material) 37 ℃ in a CO ₂ incubator (CO ₂ incubator) after subculture purchased from, 5% CO ₂ Cultured under conditions. The cell culture medium was in accordance with Lonza's guidelines. KGM-2 bullet kit CC-4152 (KGM ^TM -2 Bullet kit, CC-4152) (ingredient: BPE (Bovine pituitary extract)), human epidermis in 500 ml of KBM-2 (KBM ^TM -2, CC-3103) medium Human epidermal growth factor (hEGF), Insulin, Hydrocortisone, Transferrin, Epinephrine, and Gentamicin Sulfate + Amphofericin-B (Gentamycin Suflate + Amphofericin-B: GA-1000)) was added to the KGM-2 bullet kit CC-3107 (KGM ^TM -2 Bullet Kit, CC-3107).

[Example 3] Treatment of fine dust on normal human keratinocytes

In order to confirm the cytotoxicity through fine dust treatment, an MTT experiment using human normal keratinocytes was performed by the method of Mossman et al. (J. Immunol. Methods, 65, 55-63, 1983). Specifically, a 24-well plate was used and fine dust having a diameter of 2.5 μm obtained in Example 1 was dispersed in purified water to prepare a fine dust dispersion, and then 2.5 占10 ⁵ well cells were prepared under the cell culture conditions of Example 2. The cells cultured in water condition were treated with the fine dust dispersion and cultured for 24 hours, followed by mixing 5 mg/ml of 3-4,5-dimethylthiazol-2,5-diphenyltetra zolium bromide (MTT) at 37°C. Incubated for an additional 3 hours. Thereafter, the medium was removed and the formed formazan crystal was dissolved in 500 µl of DMSO. The lysate was transferred to a 96-well plate, aliquoted, and the OD value was measured at an absorbance of 540 nm. The measurement results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that the concentration (IC20) value showing 80% survival rate for cytotoxicity by the dispersion in which 2.5 μm diameter fine dust was dispersed in the cell line was 12.5 μg/ml.

[Example 4] Confirmation of skin cell gene change due to fine dust through Next Generation Sequencing (NGS)

For RNA-sequencing data processing and analysis, reference was made to the general analysis steps developed by Trapnell et al. (2012). RNA-seq data quality was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and FASTX (http://hannonlab.cshl.edu/fastx_toolkit/) was used. Thus, base and adapter sequences with poor accuracy were removed. Thereafter, alignment was performed using Tophat (Trapnell et al., 2009) and human genome (hg19), and the data amount of each sample was confirmed using EVER-seq, renamed RSeQC (Wang et al., 2012). ). In addition, the expression level of the transcript was quantified using Cufflinks, and the level of transcription was compared between the two samples treated with fine dust dispersion and the normal sample (Trapnell et al., 2010). FDR adjusted p-value<0.05, applying a strict cutoff of =2.0 fold-change, the gene that shows a significant difference in expression when treated with a dispersion of fine dust with a diameter of 2.5 μm and a dispersion of fine dust with a diameter of 10 μm. I decided. The measurement results are shown in Table 2 below.

Name Gene Symbol Drainage change NM_004430 EGR3 -1.261 NM_001003407 ABLIM1 -2.226

[Example 5] Real-time RT-PCR quantification

The fine dust with a diameter of 2.5 μm extracted in Example 1 was treated with 12.5 μg of human normal keratinocytes cultured in Example 2 in 1 ml of cell culture medium, and primers of the genes shown in Table 3 below (applied biosystems TaqMan ® Primers) was used to measure the relative amount of mRNA expression.

Name Gene Symbol TaqMan® primer NM_004430 EGR3 Hs04935588_m1 NM_001003407 ABLIM1 Hs01046520_m1

After 24 hours, the culture medium was removed, the cells were washed with 2 ml of phosphate buffered saline (PBS), and then RNA in the cells was removed using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). Separated. The isolated RNA was purified once more with QIAGEN's RNA kit (QIAGEN RNeasy kt, QIAGEN, Valencia, CA), and then Agilent's Bioanalyzer 2100 model instrument (Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, CA, USA) Was used to check the quality of RNA. CDNA was synthesized from the RNA using Invitrogen's Superscript Reverse Transcriptase (RT) kit, Invitrogen, Carlsbad, CA, and this was performed using the primers of Table 3 in real time reverse transcription polymerase chain reaction (Q-RT -PCR: real time-reverse transcription polymerase chain reaction). Changes in gene expression patterns were evaluated by real-time PCR using Applied Biosystems' TaqMan gene expression system (TaqMan gene expression assay kit, Applied Biosystems, Foster City, CA), and the results are shown in FIG. Done. Both Q-RT-PCR and real-time PCR used were performed according to the standard protocol distributed by Life Technology, and specifically, after treatment at 95°C for 20 seconds, processing at 95°C for 3 seconds and 60°C for 30 seconds Was performed for 40 cycles.

As shown in FIG. 2, it was confirmed that both genes of Table 3 were significantly reduced in skin cells stimulated by fine dust.

Claims (13)

EGR3 (NM_004430) gene and ABLIM1 (NM_001003407) comprising an agent for measuring the expression level of the mRNA of at least one or more genes or proteins encoded from the genes of the gene, a composition for diagnosis of skin damage caused by fine dust. The method of claim 1,
The agent for measuring the expression level of the mRNA or protein of the gene is a polynucleotide or fragment thereof complementary to the mRNA of the gene, a primer or probe capable of amplifying the gene, or an antibody that specifically binds to the protein, A composition for diagnosing skin damage caused by fine dust. A kit for diagnosing skin damage caused by fine dust, comprising the composition according to claim 1 or 2. From the subject's skin cell sample,
1) measuring the expression level of the mRNA of at least one or more genes of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes; And
2) A method for diagnosing skin damage caused by fine dust, comprising comparing the expression level with the expression level of the mRNA of the gene or the protein encoded therefrom in a skin cell sample not damaged by fine dust . The method of claim 4, wherein the method comprises:
A skin cell sample in which the expression level of the mRNA of at least one or more of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes measured from the skin cell sample of the subject is not damaged by the fine dust If it is lower than the inner, the method further comprising the step of determining that the skin cells are damaged by fine dust, whether the skin is damaged by fine dust. The method of claim 4,
Methods for measuring the expression level of the mRNA or protein of the gene include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), northern blotting, western blotting, and ELISA ( enzyme-linked immunosorbent assay), which is selected from the group consisting of a method for diagnosing skin damage caused by fine dust. Treating the skin cells with fine dust;
Treating the skin cells treated with the fine dust with a test substance; And
In the skin cells treated with the test substance, measuring the expression level of the mRNA of at least one or more of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene before and after the test substance treatment or the protein encoded from the genes. A method for screening substances that inhibit or relieve skin damage caused by fine dust. The method of claim 7, wherein the method,
After the test substance treatment compared to before the test substance treatment,
When the expression level of the mRNA of at least one or more of the EGR3 (NM_004430) gene and the ABLIM1 (NM_001003407) gene or the protein encoded from the genes increases, the step of determining as a substance to suppress or alleviate skin damage by fine dust is further performed. Containing, a method for screening a substance to suppress or relieve skin damage by fine dust. The method of claim 7,
The skin cells are keratinocytes, a method of screening for substances that inhibit or relieve skin damage by fine dust. The method of claim 7,
The level of expression of the mRNA or protein of the gene is determined by using one or more of a polynucleotide or fragment thereof complementary to the mRNA of the gene, a primer or probe capable of amplifying the gene, or an antibody that specifically binds to the protein. That is to measure, a method for screening substances that inhibit or alleviate skin damage by fine dust. The method of claim 7,
Methods for measuring the expression level of the mRNA or protein of the gene include polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), northern blotting, western blotting, and ELISA ( enzyme-linked immunosorbent assay), which is selected from the group consisting of fine dust-induced skin damage inhibition or amelioration material screening method. The method of claim 7,
The fine dust is fine dust having an average particle diameter of 10 μm or less, a method of screening for substances that inhibit or relieve skin damage caused by fine dust. The method of claim 7,
The fine dust is an ultrafine dust having an average particle diameter of 2.5 μm or less, a method of screening for substances that inhibit or relieve skin damage caused by fine dust.

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