KR101543181B1 - Biomarker for prediction of sensitive skin and test kit using the same - Google Patents

Abstract

A biomarker is disclosed in which the amount of gene expression is specifically increased or suppressed for sensitive skin. The biomarker specific to the sensitive skin can be used for screening the sensitive skin diagnosis kit and the stimulant relieving agent.

Description

TECHNICAL FIELD The present invention relates to a biomarker for sensitive skin diagnosis and a diagnostic kit using the same.

The present invention relates to a biomarker for sensitive skin diagnosis and a sensitive skin diagnosis kit using the same. The present invention also relates to a method for screening an emollient that effectively alleviates the sensation of irritation to sensitive skin.

The human genome consists of 3 x 10 ⁹ base pairs and is estimated to have about 50,000 genes, and the number of functional genes expressed in one cell is estimated to be about 10,000. In other words, only about 10,000 proteins are selectively produced from 50,000 genes, and the functional characteristics of the cells are determined by the protein expression. In addition, the difference in the gene expression characteristics of the cells does not appear only in different cells, and there are differences in gene expression characteristics between the pathological conditions of the same cells, for example, between young and aged cells.

In the field of life sciences, conventionally, we sought to find a change in gene expression from a one-to-one relationship. However, the action of a specific protein is not limited to a single protein but is involved in the expression of various proteins. In other words, the characteristics of cells are determined not by a specific protein but by the harmony of the characteristics of various proteins. Therefore, the conventional approach is very limited.

Sensitive skin is determined not only by external stimuli such as UV, ROS, etc., but also by the interactions among factors inherent in the cells, such as genes, proteins, etc. Therefore, it is necessary to observe these various factors as well.

However, studies on sensitive skin so far have been conducted by treating substances that cause an artificial stimulus to the skin, observing the subjective reaction of the subject, analyzing skin changes due to external factors, etc. through questionnaires I was doing. Thus, there was a limit to objectively understanding the cause or substance of sensitive skin.

In addition, since the relationship between the various phenomena of the sensitive skin and the actual skin condition is not clear, the concept of the sensitive skin itself has not been fully objectified. In addition, attempts to understand the characteristics of sensitive skin have also been carried out in order to understand the phenomenon in the human body by treating a substance expected to be efficacious or a variety of stimulant inducing substances, But it is limited.

For sensitive skin, most of the external and internal factors caused by a decrease or abnormalities in the physiological function of the skin, and various skin symptoms are said to appear. Generally, the causes of sensitive skin include increased neurosensory input, increased immunoreactivity, or reduced barrier function, and the deterioration of the barrier function is caused by two other factors It is said. Sensitive skin is manifested by a variety of different skin conditions, but it is known that the most unpleasant sensations (irritation) that are felt in the skin are most noticeable in relatively large or frequent feelings.

When the prominent feature of sensitive skin is called "irritation", people's skin can be divided into highly sensitive and non-sensitive. These differences are often due to distribution or number of skin nerves, differences in sensitivity, or differences in barrier function. The nerve fibers, which are presumed to be related to stimulation in the skin, are A? And C fibers, which are known to be involved in feeling mild discomfort in the skin, such as stinging and burning. In addition, the deterioration of the barrier function is one of the features observed in many sensitive skin. It is a characteristic that can be associated with irritation, flushing, allergy or irritation which is frequently observed in sensitive skin.

In the field of cosmetics or dermatology, the number of people who can be classified as sensitive skin is on the rise, and the importance of identifying the cause is becoming more important. However, there is insufficient research beyond the above-mentioned phenomenological studies, and there is insufficient research on whether sensitive skin is caused by inherited factors, acquired factors, or what mechanism It is true. In addition, there is a limitation in explaining the classification of sensitive skin or its academic meaning as well as the lack of objectivity in describing sensitive skin by the test result of the subject's own judgment or subjective sensation alone.

In addition, in several studies of sensitive skin, the likelihood of a family history of sensitive skin is evident, suggesting that sensitive skin may be inherited or genetic. In addition, since skin is susceptible to a certain period of time, changes in metabolism or gene expression are thought to be influential.

It is an object of one embodiment of the present invention to provide a biomarker for sensitive skin diagnosis.

It is an object of another embodiment of the present invention to provide a sensitive skin diagnostic kit.

It is an object of another embodiment of the present invention to provide a method for screening stimulant relieving agents for sensitive skin.

The biomarker for sensitive skin diagnosis according to the present invention is a biomarker for sensitive skin diagnosis comprising at least one of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, PI3, PSPH, HORMAD1, SPRR2G and ACTR2 LBP10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, G0S2, NEB, H19, LIPE, PLIN, H19, GPAM, FABP4, CIDEC, LPL Wherein at least one gene selected from the group consisting of PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3, .

The biomarker for sensitive skin diagnosis according to the present invention can quickly diagnose sensitivity of the skin using bioinformatics technology. In addition, the biomarker for sensitive skin diagnosis according to the present invention and the screening method of the sensitive skin diagnosis kit and the stimulant relieving agent using the biomarker for sensitive skin diagnosis can be variously used in the field of beauty and skin medicine.

FIG. 1 shows the results of a promoter analysis for a gene whose expression is increased compared to the non-sensitive normal value in skin tissue derived from sensitive skin.
FIG. 2 shows the results of a promoter analysis for a gene whose expression is decreased in skin tissue derived from sensitive skin as compared with a non-sensitive normal value.

The present invention has found 19 kinds of genes whose expression is increased and 35 kinds of genes whose expression is decreased in skin tissues derived from sensitive skin compared to the non-sensitive normal values. Therefore, by measuring the degree of expression of the genes, it is possible to judge whether or not the skin is sensitive.

In Table 1, the expression level of the skin of the subject, which is judged to be sensitive skin, is increased compared to the normal level, and the expression level of the gene is shown in Table 2 below. For reference, GBAcc in the following table means accession ID of NCBI genebank.

GBAcc Gene name Official gene symbol XR_015693 Major histocompatibility complex, class I, C HLA-C BX538034 Transcription elongation factor A (SII), 1 TCEA1 AK128652 Immunoglobulin heavy constant alpha 1 IGHA1 BC001188 Transferrin receptor (p90, CD71) TFRC BG739729 S100 calcium binding protein A8 S100A8 BU838441 Similar to Ig heavy chain V-II region ARH-77 precursor LOC652128 AJ001696 Serpin peptidase inhibitor, clade B (ovalbumin), member 13 SERPINB13 NM_012153 Ets homologous factor EHF BC032508 Hypothetical protein FLJ10781 FLJ10781 NM_003722 Tumor protein p63 TP63 NM_004360 Cadherin 1, type 1, E-cadherin (epithelial) CDH1 NM_148957 Tumor necrosis factor receptor superfamily, member 19 TNFRSF19 AK127910 GM2 ganglioside activator GM2A AB209893 FK506 binding protein 5 FKBP5 CF596521 Peptidase inhibitor 3, skin-derived (SKALP) PI3 BX537439 Phosphoserine phosphatase PSPH BC047406 HORMA domain containing 1 HORMAD1 BG200844 Small proline-rich protein 2G SPRR2G BM466590 ARP2 actin-related protein 2 homolog (yeast) ACTR2

GBAcc Gene name Official gene symbol NM_006063 Kelch repeat and BTB (POZ) domain containing 10 KBTBD10 NM_133378 Titin TTN BC012597 Actin, alpha 1, skeletal muscle ACTA1 BX649073 Myosin binding protein C, slow type MYBPC1 BF670653 Myoglobin MB NM_021245 Myozenine 1 MYOZ1 BX648171 Tropomyosin 1 (alpha) TPM1 NM_005181 Carbonic anhydrase III, muscle specific CA3 NM_001231 Calsequestrin 1 (fast-twitch, skeletal muscle) CASQ1 NM_001976 Enolase 3 (beta, muscle) ENO3 BQ064708 Retinol binding protein 4, plasma RBP4 NM_003918 Glycogenin 2 GYG2 AK123969 Protein phosphatase 1, regulatory (inhibitor) subunit 1A PPP1R1A NM_005609 Phosphorylase, glycogen; muscle (McArdle syndrome, glycogen storage disease type V) PYGM CD511787 G0 / G1switch 2 G0S2 NM_004543 Nebulin NEB BC040073 H19, imprinted maternally expressed untranslated mRNA H19 BC070041 Lipase, hormone-sensitive LIPE BC031084 Perilipin PLIN BC040073 H19, imprinted maternally expressed untranslated mRNA H19 AL833061 Glycerol-3-phosphate acyltransferase, mitochondrial GPAM CB999901 Fatty acid binding protein 4, adipocyte FABP4 NM_022094 Cell death-inducing DFFA-like effector c CIDEC NM_000237 Lipoprotein lipase LPL BX648510 Phosphoenolpyruvate carboxykinase 1 (soluble) PCK1 NM_004797 Adiponectin contains C1Q and collagen domains ADIPOQ AB209326 Phosphodiesterase 3B, cGMP-inhibited PDE3B BC022530 Activin A receptor, type IC ACVR1C NM_000237 Lipoprotein lipase LPL CB999901 Fatty acid binding protein 4, adipocyte FABP4 AK123640 Hypothetical protein DKFZp761N09121 DKFZP761N09121 BC034030 Tenomodulin TNMD BC063411 ATPase, H + transport, lysosomal 56 / 58kDa, V1 subunit B1 (Renal tubular acidosis with deafness) ATP6V1B1 AJ276240 Chromosome 8 open reading frame 22 C8orf22 X94323 Cysteine-rich secretory protein 3 CRISP3

The biomarker for sensitive skin diagnosis according to one embodiment comprises the genes of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, PI3, PSPH, HORMAD1, SPRR2G and ACTR2, wherein the expression level of the gene is increased in the sensitive skin as compared with the normal value. It is preferable that the expression level of the gene is increased twice or more as compared with the normal value in the sensitive skin.

The biomarker for sensitive skin diagnosis according to another embodiment comprises at least one gene selected from the group consisting of NFYA, MYOG and ATF4, which are transcription factor genes of the genes in Table 1, and the expression amount of the gene is normal to sensitive skin . It is preferable that the expression level of the gene is increased twice or more as compared with the normal value in the sensitive skin.

The biomarker for sensitive skin diagnosis according to another embodiment of the present invention is a biomarker for sensitive skin diagnosis comprising the genes of KBTBD10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, GOS2, A gene selected from the group consisting of NEB, H19, LIPE, PLIN, H19, GPAM, FABP4, CIDEC, LPL, PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3 And the expression level of the gene is reduced in the sensitive skin as compared with the normal value. It is preferable that the expression level of the gene is reduced to less than 50% of the normal value in the sensitive skin.

The biomarker for sensitive skin diagnosis according to another embodiment includes at least one gene selected from the group consisting of NFKB1, SRF, RELA, TCF3, POU2F1, USF and JUN, which are transcription factor genes of the genes in Table 2, The expression level of the gene is reduced in the sensitive skin as compared with the normal value. It is preferable that the expression level of the gene is reduced to less than 50% of the normal value in the sensitive skin.

In addition, the present invention provides a biomarker for sensitive skin diagnosis using proteins encoded from the genes of Tables 1 or 2 above.

The biomarker for sensitive skin diagnosis according to one embodiment is a biomarker for sensitive skin diagnosis comprising at least one of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, PI3, PSPH, HORMAD1, SPRR2G And ACTR2, wherein the amount of the protein is increased in the sensitive skin as compared with the normal value. It is preferable that the expression level of the protein is increased more than twice as much as the normal value in the sensitive skin.

The biomarker for sensitive skin diagnosis according to another embodiment comprises a protein encoded from one or more transcription factor genes selected from the group consisting of NFYA, MYOG and ATF4, characterized in that the amount of the protein is increased in the sensitive skin as compared with the normal value . It is preferable that the expression level of the protein is increased more than twice as much as the normal value in the sensitive skin.

The biomarker for sensitive skin diagnosis according to another embodiment is a biomarker for sensitive skin diagnosis, which comprises KBTBD10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, GOS2, NEB, H19, LIPE, PLIN , A protein encoded by one or more genes selected from the group consisting of H19, GPAM, FABP4, CIDEC, LPL, PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3 And the amount of the protein is reduced in the sensitive skin as compared with the normal value. Preferably, the expression level of the protein is reduced to less than 50% of the normal value in the sensitive skin.

The biomarker for sensitive skin diagnosis according to another embodiment comprises a protein encoded from one or more transcription factor genes selected from the group consisting of NFKB1, SRF, RELA, TCF3, POU2F1, USF and JUN, And is reduced in the sensitive skin as compared with the normal value. Preferably, the expression level of the protein is reduced to less than 50% of the normal value in the sensitive skin.

The present invention also provides a sensitive skin diagnostic kit for diagnosing sensitive skin using polynucleotides of genes of Tables 1 or 2 above.

The sensitive skin test kit according to one embodiment may further comprise at least one of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, PI3, PSPH, HORMAD1, SPRR2G And ACTR2, wherein the fragment comprises at least one polynucleotide comprising at least 10 consecutive nucleotides, or a polynucleotide complementary to the polynucleotide and a fragment thereof; And a solid support on which the polynucleotide is bound to the surface. When the amount of the transcript hybridized to the polynucleotide is increased as compared with the normal value, it is diagnosed as a sensitive skin. It is preferable that the amount of the transcript hybridized to the polynucleotide increases by at least two times as compared with the normal value.

The sensitive skin test kit according to another embodiment is a polynucleotide or fragment thereof of one or more genes selected from the group consisting of NFYA, MYOG and ATF4, wherein the fragment comprises at least one polynucleotide comprising 10 or more consecutive nucleotides, or Polynucleotides complementary to said polynucleotides and fragments thereof; And a solid support on which the polynucleotide is bound to the surface. When the amount of the transcript hybridized to the polynucleotide is increased as compared with the normal value, it is diagnosed as a sensitive skin. It is preferable that the amount of the transcript hybridized to the polynucleotide increases by at least two times as compared with the normal value.

The sensitive skin test kit according to another embodiment of the present invention may further comprise at least one of KBTBD10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, GOS2, NEB, , Polynucleotides of one or more genes selected from the group consisting of PLIN, H19, GPAM, FABP4, CIDEC, LPL, PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3 Said fragment comprising at least one polynucleotide comprising at least 10 contiguous nucleotides, or a polynucleotide complementary to said polynucleotide and said fragment; And a solid support on which the polynucleotide is bound to the surface. When the amount of the transcript hybridized to the polynucleotide is decreased as compared with the normal value, the diagnosis is made to be a sensitive skin. It is preferable that the amount of the transcript hybridized to the polynucleotide decreases to 50% or less of the normal value.

According to another embodiment, the sensitive skin test kit is a polynucleotide or fragment thereof of one or more genes selected from the group consisting of NFYA, MYOG and ATF4, wherein the fragment is one or more genes comprising 10 or more consecutive nucleotides Polynucleotides, or polynucleotides complementary to said polynucleotides and fragments thereof; And a solid support on which the polynucleotide is bound to the surface. When the amount of the transcript hybridized to the polynucleotide is decreased as compared with the normal value, the diagnosis is made to be a sensitive skin. It is preferable that the amount of the transcript hybridized to the polynucleotide decreases to 50% or less of the normal value.

In the above-described sensitive skin diagnostic kit, the polynucleotide used as a probe includes a full length or a fragment thereof of the marker gene whose expression is increased or decreased in the sensitive skin as compared to the non-sensitive skin. Preferably, the length of the fragment comprises at least 10 contiguous nucleotides. This is because when the length of the probe is less than 10 bps, it is unspecifically combined.

The sensitivity skin test kit according to one embodiment of the present invention may further comprise at least one selected from the group consisting of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, , HORMAD1, SPRR2G, and ACTR2 as a fragment of a polynucleotide of one or more genes selected from the group consisting of: a polynucleotide comprising 18 to 22 consecutive nucleotides; And at least one polynucleotide comprising 18 to 22 consecutive nucleotides as a fragment of a polynucleotide complementary to the polynucleotide of the gene. When the amount of DNA amplified using the polynucleotides as a primer is higher than a normal value, Is diagnosed. It is preferable that the amount of DNA amplified using the polynucleotides as a primer is increased more than two times as compared with the normal value.

The sensitive skin test kit according to one embodiment of the present invention comprises at least one polynucleotide comprising 18 to 22 consecutive nucleotides as a fragment of a polynucleotide of at least one gene selected from the group consisting of NFYA, MYOG and ATF4 ; And at least one polynucleotide comprising 18 to 22 consecutive nucleotides as a fragment of a polynucleotide complementary to the polynucleotide of the gene. When the amount of DNA amplified using the polynucleotides as a primer is higher than a normal value, Is diagnosed. It is preferable that the amount of DNA amplified using the polynucleotides as a primer is increased more than two times as compared with the normal value.

The sensitivity skin test kit according to another embodiment of the present invention may further comprise at least one of KBTBD10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, GOS2, NEB, H19, LIPE, A fragment of a polynucleotide of at least one gene selected from the group consisting of PLIN, H19, GPAM, FABP4, CIDEC, LPL, PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3. One or more polynucleotides comprising 22 consecutive nucleotides; And at least one polynucleotide comprising 18-22 consecutive nucleotides as a fragment of a polynucleotide complementary to the polynucleotide of the gene. When the amount of DNA amplified using the polynucleotides as a primer is lower than a normal value, It is preferable that the amount of DNA amplified using the polynucleotides as a primer is reduced to 50% or less as compared with the normal value.

In another embodiment, the sensitive skin test kit comprises 18-22 consecutive nucleotides as a fragment of a polynucleotide of one or more genes selected from the group consisting of NFKB1, SRF, RELA, TCF3, POU2F1, USF and JUN One or more polynucleotides; And at least one polynucleotide comprising 18-22 consecutive nucleotides as a fragment of a polynucleotide complementary to the polynucleotide of the gene. When the amount of DNA amplified using the polynucleotides as a primer is lower than a normal value, It is preferable that the amount of DNA amplified using the polynucleotides as a primer is reduced to 50% or less as compared with the normal value.

In the sensitive skin test kit using the polynucleotide, the polynucleotide used as the primer preferably has a length of 18-22. This is because when the length of the primer is less than 18 bps, it binds non-specifically. When the length exceeds 22 bps, the primers themselves bind to each other and the efficiency and cost are reduced.

The present invention also provides a sensitive skin diagnostic kit comprising a monoclonal antibody to a polypeptide encoded by the genes of Tables 1 or 2 above.

In one embodiment, the sensitive skin diagnostic kit comprises at least one of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, PI3, PSPH, HORMAD1, SPRR2G And ACTR2, wherein the antigen is diagnosed as a sensitive skin when the amount of the antigen bound to the antibody is increased compared to the normal value . It is preferable that the amount of the antigen bound to the antibody is increased by at least two times as compared with the normal value.

In another embodiment, the sensitive skin diagnostic kit comprises a monoclonal antibody to a polypeptide encoded by one or more genes selected from the group consisting of NFYA, MYOG, and ATF4, wherein the amount of antigen bound to the antibody is And when it is increased compared to the normal value, it is diagnosed that it is a sensitive skin. It is preferable that the amount of the antigen bound to the antibody is increased by at least two times as compared with the normal value.

In another embodiment, the sensitive skin diagnostic kit comprises KBTBD10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, GOS2, NEB, H19, LIPE, A monoclonal antibody to a polypeptide encoded by one or more genes selected from the group consisting of PLIN, H19, GPAM, FABP4, CIDEC, LPL, PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3. Wherein the antigen is diagnosed as a sensitive skin when the amount of the antigen bound to the antibody is decreased as compared with the normal value. It is preferable that the amount of the antigen bound to the antibody is reduced to 50% or less as compared with the normal value.

In another embodiment, the sensitive skin diagnostic kit comprises a monoclonal antibody to a polypeptide encoded by one or more genes selected from the group consisting of NFKB1, SRF, RELA, TCF3, POU2F1, USF and JUN And when the amount of the antigen bound to the antibody is decreased as compared with the normal value, it is diagnosed that it is a sensitive skin. It is preferable that the amount of the antigen bound to the antibody is reduced to 50% or less as compared with the normal value.

The present invention also provides a method for screening an irritation-relieving agent using a protein encoded by the genes of Tables 1 or 2 above.

In one embodiment, the method for screening for stimulation-sensitive emollients comprises administering an effective amount of a compound selected from the group consisting of HLA-C, TCEA1, IGHA1, TFRC, S100A8, LOC652128, SERPINB13, EHF, FLJ10781, TP63, CDH1, TNFRSF19, GM2A, FKBP5, PI3, PSPH, Binding the test substance to a protein encoded by one or more genes selected from the group consisting of SPRR2G and ACTR2; And confirming whether the test substance inhibits the action of the protein. It is preferable that the level at which the action of the protein is inhibited is reduced to 50% or less as compared with the protein to which the test substance is not bound.

In another embodiment, the screening method of the stimulation-sensitive emollient agent may comprise the steps of: screening for a stimulant-modulating agent selected from KBTBD10, TTN, ACTA1, MYBPC1, MB, MYOZ1, TPM1, CA3, CASQ1, ENO3, RBP4, GYG2, PPP1R1A, PYGM, G0S2, NEB, , A protein encoded by one or more genes selected from the group consisting of PLIN, H19, GPAM, FABP4, CIDEC, LPL, PCK1, ADIPOQ, PDE3B, ACVR1C, LPL, FABP4, DKFZP761N09121, TNMD, ATP6V1B1, C8orf22 and CRISP3 Binding the substance; And confirming that the test substance enhances the action of the protein. It is preferable that the level at which the action of the protein is inhibited is increased by a factor of two or more as compared with the protein to which the test substance is not bound.

In addition, the present invention provides a method for screening an irritation-relieving agent using a transcription factor gene extracted from the gene.

In one embodiment, the method of screening for stimulus relieving agent comprises the steps of: a first plasmid comprising at least one transcription factor gene and a promoter selected from the group consisting of NFYA, MYOG and ATF4; and a DNA sequence and a reporter Transfecting a second plasmid containing the gene into the cell, treating the cell with the test substance, and confirming whether the test substance inhibits the expression of the reporter gene. The level at which the test substance is inhibited in expression of the reporter gene is preferably reduced to 50% or less as compared with the protein to which the test substance is not bound.

In still another embodiment, the screening method of the stimulus-sensitive emollient comprises a first plasmid comprising at least one transcription factor gene and a promoter selected from the group consisting of NFKB1, SRF, RELA, TCF3, POU2F1, USF and JUN, Transfecting a cell with a second plasmid comprising a DNA sequence to which the transcription factor binds and a reporter gene, treating the test substance with the cell, and determining whether the test substance increases the expression of the reporter gene . It is preferable that the level at which the test substance is inhibited in expression of the reporter gene is increased by a factor of two or more as compared with the protein to which the test substance is not bound.

Tables 3 and 4 below show transcription factors (Table 3), which are highly observed in promoters of genes that increase expression in sensitive skin, and transcription factors (Table 4), which are highly observed in promoters of genes whose expression is decreased.

GBAcc Gene name Official gene symbol NM_007122 CCAAT box / V $ CAAT_01 CEBPB NM_002505 NF-Y / V $ NFY_Q6 NFYA NM_002479 Myogenin / NF-1 / V $ MYOGNF1_01 MYOG NM_001675 Tax / CREB / V $ TAXCREB_02 ATF4

GBAcc Gene name Official gene symbol NM_003998 NF-kappaB / V $ NFKB_Q6 NFKB1 NM_003131 SRF / V $ SRF_Q6 SRF NM_021975 NF-kappaB (p65) / V $ NFKAPPAB65_01 RELA NM_003200 E47 / V $ E47_02 TCF3 NM_000545 HNF-1 / V $ HNF1_C HNF1A NM_002697 Oct-1 / V $ OCT1_05 POU2F1 AL832119 USF / V $ USF_C USF NM_007122 CCAAT box / V $ CAAT_01 CEBPB NM_002228 AP-1 / V $ AP1_Q2 JUN

The transcription factor genes are transcription factors such as NFYA, MYOG, and ATF4, which are over-expressed in the promoter of the gene whose expression is increased in the sensitive skin (Table 1), and transcripts The factors NFKB1, SRF, RELA, TCF3, POU2F1, USF, and JUN are available. In addition, CEBPB is a transcription factor that is over-observed simultaneously in the increased expression gene and the decreased expression gene. The transcriptional regulatory activity of the transcription factor gene may be measured to screen for the stimulatory effector, using a reporter gene vector prepared according to conventional molecular biology techniques.

The method for screening the stimulus-relieving agent using the transcription factor gene will be described in more detail as follows.

A universal promoter which is expressed even under general culture conditions, a first plasmid to which the genes of Tables 3 or 4 are bound, and a promoter having a transcription response element capable of binding transcription factors to activate transcription, A second plasmid to which a firefly luciferase gene is bound can be used.

The TRE sequence of the second plasmid can be used to predict and detect motifs such as Matlspector (Quandt et al., 1995) and Transfac (Knuppel et al., 1994) The sequence can be retrieved by entering the accession ID of the genes. COS7 cells were transfected with the first plasmid having the transcription factor gene and the corresponding TRE promoter-reporter second plasmid at the same time. After 24 hours, the skin irritation inhibitor candidate drug was treated and the cells were harvested to obtain luciferase activity . Thus, the effect of the candidate drug on the activity of the transcription factor can be confirmed.

In the case of the transcription factor, which is excessively observed in the promoter of the gene whose expression is increased in the sensitive skin, it can be judged that it is an effective skin sensitizer when the cell luciferase activity is decreased. Conversely, in the case of the transcription factor, which is excessively observed in the promoter of the gene whose expression is decreased, it may be said that it is an effective skin sensitizer when the activity of the cell luciferase is increased. In addition, transcription factors that are highly observed in the promoter of genes whose expression increases or decreases can be used as an auxiliary screening method.

1 and 2, the present invention will be described in more detail as follows.

The promoter sequence of the changed gene was collected by NCBI Accession No. Upstream gene sequences were obtained from the Ensembl genome database and genomic sequences were obtained containing a 2000 bp upstream region from the transcription start site. A transcription factor binding site (TFBS) was searched using a program to predict, detect and analyze motifs such as MatInspector (Quandt et al., 1995) and Transfac (Knuppel et al., 1994) . As a result of hyper - geometry statistical analysis, transcription factor binding sites were selected for overexpression or underexpression. Then, the linkage between the gene cluster showing the expression change and the regulatory network between the transcription factor predicted to regulate the gene cluster was extracted and visualized as a graph. FIG. 1 shows the results of a promoter analysis for a gene whose expression is increased compared to a non-sensitive normal value in skin tissue derived from a sensitive skin. FIG. 2 shows the expression of the gene in the skin tissue derived from the sensitive skin, This is the result of the promoter analysis for the gene. What is at the center of the network is the transcription factor and what is around it is the target gene under the control of its transcription factor.

Hereinafter, the present invention will be described in further detail with reference to examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not limited thereto.

[Example 1] Identification of a sensitive skin marker gene

1-1. Obtaining tissue samples and from tissue samples RNA  Extraction

Skin tissue samples were taken from 9 patients with sensitive symptoms and 9 from control patients. Tissue samples were each biopsied and tissues were collected in sets. To purify RNA from tissue, trypsin (Invitrogen, Carlsbad, CA, USA) was added to the cells for pulverization, and chloroform was added to separate the supernatant RNA. To concentrate the RNA of the obtained supernatant, an equal amount of isopropanol was added and centrifuged to obtain precipitated RNA. The precipitated RNA was washed with 70% ethanol for base removal.

1-2. Microarray  Experiment

Microarray experiments were performed with the gene chip HG U133 Plus 2.0 (Affymetrix ^® ), in which a total of 54,675 human transcripts probes were integrated. We selected genes with changes in expression according to skin sensitivity and confirmed their expression patterns. Biotinylated cRNA was synthesized by in vitro transcription using 10 ug of RNA for cDNA synthesis.

Specifically, cDNA containing a promoter was synthesized from RNA using T7 primer and reverse transcriptase according to the protocol proposed by Affymetrix. Then, in vitro transcription reaction was performed using double-stranded cDNA as a circular form to synthesize cRNA. In vitro transcription reactions used T7 RNA polymerase and biotinylated ribonucleotide analogs for cRNA amplification and labeling.

The thus prepared biotinylated cRNAs were fragmented and then hybridized at 42 ° C for 17 hours on the gene chip. The gene chip was cleaned, stained and scanned, and data pre-processing was performed using Global scaling, a basic normalization algorithm of GenPlex software.

As a result, genes showing significant differences in gene expression between the sensitive skin sample and the control sample were obtained by comparing the p-value and the increase in gene expression.

Based on the gene expression patterns, genes that have been reduced to less than 50% in genes with reduced expression, and genes that have increased more than two fold in genes with increased expression can be selected and used as gene markers that can be used to diagnose sensitive skin. Respectively. The results are the genes listed in Tables 1 and 2 above.

Claims (11)

A polynucleotide of the SRF transcription factor gene or a fragment thereof,
Said fragment comprising one or more polynucleotides comprising 10 or more consecutive nucleotides,
Or polynucleotides complementary to said polynucleotides and fragments thereof; And
And a solid support on which the polynucleotide is bound to the surface,
Wherein the information that the amount of the transcript hybridized to the polynucleotide is decreased as compared with the normal value is used as one of information that judges that the skin is sensitive to extrinsic or intrinsic stimuli. Information kit for sensitive skin diagnosis of irritation.
The method according to claim 1,
The information providing kit for sensitive skin diagnosis comprises a polynucleotide or fragment thereof of one or more genes selected from the group consisting of RELA, TCF3, POU2F1, USF and JUN, or a polynucleotide complementary to the polynucleotide and the fragment thereof An information kit for sensitive skin diagnosis, comprising an extracellular or intradermal stimulation.
3. The method according to claim 1 or 2,
Wherein the information that the amount of the transcript hybridized to the polynucleotide is reduced to 50% or less as compared with the normal value is used as one of information for judging that it corresponds to a sensitive skin for extrinsic or intradermal stimulation Information kit for sensitive skin diagnosis for extrinsic or intradermal stimuli.
One or more polynucleotides comprising 18-22 contiguous nucleotides as a fragment of a polynucleotide of an SRF transcription factor gene; And
And at least one polynucleotide comprising 18-22 contiguous nucleotides as a fragment of a polynucleotide complementary to the polynucleotide of the gene,
Wherein the information that the amount of DNA amplified using the polynucleotides as a primer is decreased as compared with the normal value is used as one of information for judging that the skin is sensitive to extrinsic or intrinsic stimuli. Information kit for sensitive skin sensitivity to internal stimuli.
5. The method of claim 4,
Wherein the information providing kit for sensitive skin diagnosis comprises at least one polynucleotide comprising 18 to 22 consecutive nucleotides as a fragment of a polynucleotide of at least one gene selected from the group consisting of RELA, TCF3, POU2F1, USF and JUN; And one or more polynucleotides comprising 18-22 contiguous nucleotides as a fragment of a polynucleotide complementary to the polynucleotide of the gene. ≪ Desc / Clms Page number 13 >
The method according to claim 4 or 5,
Wherein information that the amount of DNA amplified using the polynucleotides as a primer is reduced to 50% or less with respect to a normal value is used as one of information for judging that it corresponds to a sensitive skin for extrinsic or intradermal stimulation An information kit for sensitive skin diagnosis of extrinsic or intradermal stimuli.
A monoclonal antibody to a polypeptide encoded by an SRF transcription factor gene,
Wherein the information that the amount of the antigen bound to the antibody is decreased as compared with the normal value is used as one of information for judging that the skin corresponds to the extrinsic or intrinsic stimulant sensitive skin, Information kit for sensitive skin diagnosis.
8. The method of claim 7,
Wherein the information providing kit for sensitive skin diagnosis further comprises a monoclonal antibody against a polypeptide encoded by one or more genes selected from the group consisting of RELA, TCF3, POU2F1, USF and JUN. Information kit for sensitive skin diagnosis of irritation.
9. The method according to claim 7 or 8,
Wherein the information that the amount of the antigen bound to the antibody is reduced to 50% or less as compared with the normal value is used as one of the information that judges that the skin corresponds to the extrinsic or intrinsic stimulant sensitive skin. Information kit for sensitive skin diagnosis for intradermal stimulation.
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