WO2004016783A1 - Procede permettant d'examiner une dermatite atopique - Google Patents

Procede permettant d'examiner une dermatite atopique Download PDF

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Publication number
WO2004016783A1
WO2004016783A1 PCT/JP2003/005277 JP0305277W WO2004016783A1 WO 2004016783 A1 WO2004016783 A1 WO 2004016783A1 JP 0305277 W JP0305277 W JP 0305277W WO 2004016783 A1 WO2004016783 A1 WO 2004016783A1
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Prior art keywords
gene
indicator gene
atopic dermatitis
indicator
expression level
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PCT/JP2003/005277
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English (en)
Japanese (ja)
Inventor
Mikito Itoh
Akira Shinagawa
Hajime Sudo
Hideoki Ogawa
Chisei Ra
Kouichi Mitsuishi
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Genox Research, Inc.
Juntendo University
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Priority to JP2004528829A priority Critical patent/JPWO2004016783A1/ja
Priority to AU2003227362A priority patent/AU2003227362A1/en
Publication of WO2004016783A1 publication Critical patent/WO2004016783A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for testing atopic dermatitis.
  • Allergic diseases such as atby dermatitis are considered to be multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by several environmental factors. Therefore, it is very difficult to elucidate the specific genes that cause specific diseases.
  • allergic diseases are thought to be related to the expression of mutated or defective genes, or overexpression or decreased expression of specific genes.
  • allergen-specific IgE measurement, leukocyte histamine release test, and lymphocyte juvenile dung test.
  • the presence of allergen-specific IgE is evidence of an allergic reaction to the allergen.
  • some patients may not always be able to detect allergen-specific IgE.
  • diagnosis Testing must be performed for all allergens required for The leukocyte histamine release test and lymphocyte blastogenesis test are methods for observing the immune system's response to allergens using ⁇ 'iro. These methods are complicated in operation.
  • a method for utilizing the immune response observed when a patient is actually brought into contact with an allergen to diagnose allergens is also known. Brick tests, scratch 'tests, patches' tests, intradermal reactions, or provocation tests are included in this type of test. While these tests can directly diagnose a patient's allergic reaction, they can be described as tests that involve invasive exposure of the subject to allergens.
  • a high serum IgE level may indicate that the patient has an allergic reaction.
  • the serum IgE value is information corresponding to the total amount of allergen-specific IgE. Although it is easy to determine the total amount of IgE regardless of the type of allergen, IgE levels may be low in patients with diseases such as non-atopic bronchitis asthma.
  • the present invention has been made in view of such a situation, and an object of the present invention is to provide a new index that enables a test for atopic dermatitis. Further, the present invention provides a method for testing atopic dermatitis based on the index, and a method for treating atopic dermatitis. It is an object of the present invention to provide a method for screening complement compounds.
  • the present inventors have intensively studied to solve the above problems.
  • the genes whose expression levels are different between the eruption area of atopic dermatitis patients and the rash area of the same patient or the skin of healthy subjects were identified, and the relationship with the atopic dermatitis reaction was clarified.
  • the present inventors searched for genes whose expression status is different between the eruption part of a patient with atopic dermatitis, the eruption part of the same patient, and the skin of a healthy person.
  • the subject's skin tissue was selected as a biological sample for comparing gene expression states. Skin tissue specimens that are actually inflamed contain many infiltrating lymphocytes, etc., which are important for the pathogenesis. Analysis of gene expression in the local skin is thought to elucidate the pathology of atopic dermatitis.
  • the present inventors compared the expression profile of a gene expressed in the rash area of the same patient as the rash area of atopic dermatitis patient and the skin of a healthy person using a gene chip. Genes with expression fluctuations of two times or more were selected, and the expression levels of the genes were measured. Then, it was confirmed that the expression of SK404 was fluctuating.
  • the nucleotide sequence of the indicator gene SK404 in the present invention is known (W0200164835). The following information is available on SK404, but no relationship with allergic disease has been suggested.
  • the fact that a change was observed in the expression level of the indicator gene between the eruption site of atopic dermatitis patients and the eruption site or a healthy subject in the same patient is the present invention. It indicates the depth and association between the indicator gene and the atopic dermatitis symptom. Further, the difference in the expression level of the counterpart of the above-mentioned indicator gene in mice between the auricle skin of the sensitized mouse and the auricle skin of the non-sensitized mouse was also confirmed by the indicator gene of the present invention. Correlates with the symptoms of atopic dermatitis.
  • the present pioneers can use the expression level of the indicator gene or the activity of the protein encoded by the indicator gene as an indicator to diagnose atopic dermatitis and to diagnose the disease.
  • the present inventors have found that it is possible to use therapeutic Jung for remedy, and completed the present invention.
  • the present invention relates to the following methods for examining atopic dermatitis, and methods for screening a candidate compound for a therapeutic agent for atopic dermatitis.
  • a method for testing atopic dermatitis comprising the following steps (1) to (3), wherein the indicator gene is SK404.
  • step (2) comparing the expression level of the rash area measured in step (1) with the expression level of the indicator gene in a biological sample collected from the rash-free area or skin of a healthy subject of the same subject as a control;
  • step (3) a step of judging that the subject has atopic dermatitis when the expression level is higher than the control as a result of the comparison in step (2);
  • a reagent for testing atopic dermatitis comprising a polynucleotide containing a nucleotide sequence of an indicator gene or an oligonucleotide having a nucleotide sequence complementary to a complementary strand thereof and having a length of at least 15 bases; ,
  • the indicator gene is SK An atopic dermatitis test reagent which is 404.
  • a reagent for detecting atopic dermatitis comprising an antibody recognizing a peptide containing an amino acid sequence of a protein encoded by an indicator gene, wherein the indicator gene is SK404, atopic dermatitis test For reagents.
  • a screening method for a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is SK404.
  • an atopic compound comprising a polynucleotide containing the nucleotide sequence of the indicator gene, or an oligonucleotide having a nucleotide sequence complementary to the complementary strand thereof and having a length of at least 15 bases, and a cell expressing the indicator gene;
  • a kit for screening a candidate compound for a therapeutic agent for atopic dermatitis comprising an antibody that recognizes a peptide containing an amino acid sequence of a protein encoded by an indicator gene and a cell that expresses the indicator gene.
  • the kit wherein the indicator gene is SK404.
  • An atopic dermatitis model animal comprising a transgenic non-human vertebrate with an increased expression intensity in the skin of an indicator gene or a gene functionally equivalent to the indicator gene, wherein the indicator gene is SK A model animal that is 404 or a functionally equivalent gene.
  • [1 2] an allele comprising a step of administering the ingredient described in the following i) or ii) to a mouse Method for producing animal with dermatitis model.
  • a polynucleotide comprising a nucleotide sequence comprising a homolog of SK404 ii) a protein encoded by a polynucleotide comprising a nucleotide sequence comprising a homolog of SK404
  • An inducer for inducing arenoleggic dermatitis in mice comprising as an active ingredient the ingredient according to any of i) or ii) in [12].
  • a screening method for a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is SK404 or a homolog thereof.
  • a screening method for a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is SK404.
  • a screening method for a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is SK404 or a gene functionally equivalent to SK404.
  • [17] atopic skin, comprising, as an active ingredient, a compound obtainable by the screening method according to any one of [6], [14], [15], and [16]. Remedies for flame.
  • a therapeutic agent for atopic dermatitis containing an indicator gene or a part of the antisense DNA as an active ingredient, wherein the marker gene is SK404
  • a therapeutic agent for atopic dermatitis which comprises, as an active ingredient, an antibody that recognizes a peptide containing the amino acid sequence of the protein encoded by the indicator gene, wherein the indicator gene is SK404 medicine.
  • the present invention provides an atopic method comprising the step of administering a compound obtainable by the screening method according to any one of [6], [14], [15] and [16].
  • the present invention relates to a method for treating dermatitis.
  • the present invention also provides a pharmaceutical composition for treating atopic dermatitis, comprising a compound obtainable by the screening method according to any one of [6], [14], [15] and [16]. Use in the manufacture of
  • the present invention relates to a method for treating atopic dermatitis, comprising a step of administering the following component (i) or (ii).
  • the present invention relates to the use of the following component (i) or (ii) in the manufacture of a pharmaceutical composition for treating atopic dermatitis.
  • an allergic disease is a general term for diseases associated with allergic reactions. More specifically, allergens have been identified and demonstrated a deep link between exposure to allergens and the development of lesions; Can be defined as Here, the immunological mechanism means that white blood cells show an immune response by stimulation of allergen. Examples of the allergen include a du antigen and a pollen antigen.
  • Representative allergic diseases can include atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, or insect allergy.
  • Allergic diathesis is a genetic factor transmitted from a parent with an allergic disease to a child.
  • a familial allergic disease is also called atopic disease, and the genetic factors that cause it are predisposed to atopy.
  • Atopic dermatitis is a generic name given to atopic diseases, especially those accompanied by dermatological symptoms.
  • an indicator gene a gene that can be used as an indicator of atopic dermatitis is referred to as an indicator gene.
  • a protein consisting of an amino acid sequence encoded by an indicator gene is called an indicator protein.
  • the indicator gene is used as a term indicating SK404 or a gene functionally equivalent to SK404.
  • the nucleotide sequence of the indicator gene and the amino acid sequence encoded by this nucleotide sequence in the present invention are known.
  • the nucleotide sequence of human SK404 and the amino acid sequence encoded thereby are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the nucleotide sequence of mouse SK404 and the amino acid sequence encoded thereby are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the method for testing for an allergic disease measures the expression level of a marker gene in a biological sample of a subject, and measures the index in a biological sample collected from a rash-free part or a healthy person of the same subject as a control. Comparing the expression level of the gene. As a result of the comparison, if the expression level is higher than that of the control, the subject is determined to have atopic dermatitis.
  • a standard value is usually set based on the expression level of the indicator gene in a healthy person, for example. Based on this standard value, for example The range of ⁇ 2 SD is considered as the allowable range.
  • Techniques for setting a standard value and an allowable range based on a measured value of an indicator gene are known.
  • the standard value of the rash-free area in the patient can be determined by measuring the expression level of the indicator gene in the rash-free area in advance. After setting the standard value, the expression method of the skin eruption alone may be measured, and the test method of the present invention may be performed based on a comparison with a predetermined standard value of the rash-free area of the patient. it can.
  • the subject is determined to have atopic dermatitis. If the expression level of the subject's indicator gene is within an acceptable range, the likelihood of atopic dermatitis is expected to be low.
  • the expression level of the indicator gene includes transcription of the indicator gene into mENA and translation into a protein. Therefore, the method for detecting atopic dermatitis according to the present invention is performed based on a comparison of the expression intensity of mRNA corresponding to an indicator gene or the expression level of a protein encoded by the indicator gene.
  • the measurement of the expression level of the indicator gene in the detection of atopic dermatitis in the present invention can be performed according to a known gene analysis method. Specifically, for example, it is possible to use a hybridization technique using a nucleic acid that hybridizes to the gene as a probe, or a gene amplification technique using a DNA that hybridizes to the indicator gene of the present invention as a primer. it can.
  • the probe or primer used in the test of the present invention can be designed based on the base sequence of the indicator gene.
  • the nucleotide sequence of the indicator gene and the amino acid sequence encoded by the indicator gene are known.
  • the indicator gene includes not only human but also homologs of other species. Therefore, an indicator gene in a species other than human refers to a homologue of an indicator gene specific to the species or an exogenous indicator gene introduced into the individual, unless otherwise specified.
  • the homologue of the human indicator gene refers to a gene derived from a species other than human that can hybridize under stringent conditions using the human indicator gene as a probe.
  • Stringent conditions generally indicate the following conditions. That is, hybridization is performed at 4 ° SSC at 65 ° C, and the plate is washed with 0.1X SSC at 65 ° C for 1 hour.
  • the hybridization / washing temperature conditions that greatly affect the stringency can be adjusted according to the melting temperature (Tm). Varies depending on the ratio of constituent bases to the base pairs to be hybridized and the composition of the hybridization solution (salt concentration, sodium concentration of formamide-dodecyl sulfate). Therefore, those skilled in the art can experimentally or empirically set conditions that give equivalent stringency in consideration of these conditions.
  • a polynucleotide comprising the nucleotide sequence of the indicator gene or a polynucleotide containing at least 15 nucleotides complementary to its complementary strand
  • the “complementary strand” refers to one strand of a double-stranded DNA composed of A: T (U in the case of RNA) and G: C base pairs with respect to the other strand.
  • the term “complementary” is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95%. It is only necessary to have the above homology on the base sequence.
  • the homology of the nucleotide sequences can be determined by an algorithm such as BLAST.
  • Such a polynucleotide can be used as a probe for detecting the indicator gene and as a primer for amplifying the indicator gene.
  • bra When used as an animal, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. Further, if used as a probe, at least a portion or the whole sequence of a marker gene (or its phase co-chain), DNA chain length of at least 15b P is used.
  • the 3'-side region needs to be complementary, but a restriction enzyme recognition sequence ⁇ tag or the like can be added to the 5'-side.
  • the “polynucleotide” in the present invention can be DNA or RNA.
  • oligonucleotide means a polynucleotide having a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides.
  • probes that are labeled at both ends with different fluorescent dyes that cancel each other's fluorescence are used to hybridize to the detection target (DNA or RNA reverse transcript).
  • the detection target DNA or RNA reverse transcript.
  • the two fluorescent dyes separate and the fluorescence is detected. This fluorescence is detected in real time.
  • the number of copies to be detected in the target sample is determined based on the number of cycles with a linear PCR amplification cycle by simultaneously measuring the standard sample whose copy number is clear for the target (Holland, PM et al., 1991). Natl. Acad. Sci.
  • PCR amplification monitoring method for example, ABI PRISM7700 (Applied Biosystems) can be used.
  • the method for testing atopic dermatitis of the present invention can also be performed by detecting a protein encoded by an indicator gene.
  • a Western plotting method using an antibody that binds to the indicator protein for example, a Western plotting method using an antibody that binds to the indicator protein, an immunoprecipitation method, an ELISA method, and the like can be used.
  • Antibodies that bind to the indicator protein used for this detection can be obtained using techniques well known to those skilled in the art.
  • the antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody (Milstein C, et al., 1983, Nature 305 (5934): 537-40).
  • a polyclonal antibody against the indicator protein is obtained by removing the blood of a mammal sensitized with the antigen and separating serum from the blood by a known method. Serum containing a polyclonal antibody may be used as the polyclonal antibody. Can be. Alternatively, if necessary, a fraction containing a polyclonal antibody can be further isolated from the serum.
  • immune cells are removed from a mammal sensitized with the above-mentioned antigen, and are fused with myeloma cells and the like.
  • the hybridoma thus obtained can be cloned, and the antibody can be recovered from the culture to obtain a monoclonal antibody.
  • antibodies may be appropriately labeled and used for detection of the indicator protein. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G can be labeled and detected indirectly. As a specific detection method, for example, an ELISA method can be mentioned.
  • a protein or a partial peptide thereof used as an antigen can be obtained by, for example, incorporating an indicator gene or a part thereof into an expression vector, introducing this into an appropriate host cell, preparing a transformant, and culturing the transformant.
  • the recombinant protein can be obtained by expressing the recombinant protein and purifying the expressed recombinant protein from a culture or a culture supernatant.
  • an oligopeptide comprising an amino acid sequence encoded by the gene or a partial amino acid sequence of an amino acid sequence encoded by a full-length cDNA can be chemically synthesized and used as an immunogen.
  • an allergic monogenic disease can be tested using not only the expression level of the indicator gene but also the activity of the indicator protein in a biological sample as an indicator.
  • the activity of the indicator protein refers to the biological activity of the protein.
  • the skin tissue of the subject is used as a sample. Taking a skin tissue sample is somewhat painful to the subject. On the other hand, since skin tissue can be easily collected, it is useful as a diagnostic material.
  • Skin tissue can be collected, for example, as follows. That is, the sampling site is first anesthetized with a local anesthetic. After pulling the skin around the biopsy site to make it slack-free, embed the punch in the skin and rotate to insert the tissue of the specimen into the punch. Pull out the punch Then, the skin inside the cut punch is collected.
  • a punch is a hollow skin tissue sampling device. For example, instruments that can collect skin tissue with a diameter of 3 mm are commonly used.
  • the skin tissue anatomically includes the epidermis and the dermis.
  • Skin tissue may include non-skin cells found in skin tissue, such as lymphocytes, Langerhans cells, or mast cells, as well as cells specific to the skin. Cells collected together with these skin cells are also included in the skin tissue sample.
  • a skin tissue sample from the rash is used to determine the expression level of the indicator gene in the rash.
  • the rash is the skin that forms the acute lesion.
  • the diagnostic criteria reported in the Journal of the Dermatological Association 104: 1210 (1994)
  • the following clinical findings are used as indicators of acute lesions.
  • Acute lesions erythema, wet erythema, papules, serous papules, scales, crusts
  • a skin tissue sample from the non-rash area is used.
  • a rash-free area in a patient is the skin at a site not accompanied by the above-mentioned lesion.
  • the skin tissue of a healthy subject is used.
  • the healthy subject in the present invention refers to a human who clearly has no allergic monogenic disease.
  • the skin tissue can be collected in the same manner as the patient's skin tissue.
  • the collection of skin tissue samples is easy, simple examination at the bedside is possible. For example, it can be prepared by the method shown in Examples. If the lysate is prepared by destroying the prepared skin tissue, it can be used for immunological measurement of the indicator protein. Sample.
  • a lysate is prepared from the above biological sample, it can be used as a sample for immunological measurement of the indicator protein.
  • mRNA is extracted from this lysate, it can be used as a sample for measuring mRNA corresponding to the indicator gene.
  • the indicator protein is secreted into the blood, the expression level of the gene encoding it can be compared by measuring the amount of the target protein contained in a body fluid sample such as blood or serum of the subject. Is possible.
  • the above sample can be diluted with a buffer or the like, if necessary, and used in the method of the present invention.
  • the measured value of the expression level of the indicator gene in the present invention can be corrected by a known method. With the correction, changes in the expression level of the gene in the cells can be compared. In the present invention, the measured value is corrected based on the measured value of the expression level of a gene whose expression level does not fluctuate greatly (eg, a housekeeping gene) in each cell in the biological sample. This is done by correcting the measured values. Examples of genes whose expression levels do not fluctuate significantly include ⁇ -actin, GAPDH and the like.
  • the present invention provides a reagent for the test method of the present invention. That is, the present invention provides a test reagent for atopic dermatitis, comprising a polynucleotide comprising a base sequence of an indicator gene or an oligonucleotide having a base sequence complementary to a complementary strand thereof and having a length of at least 15 bases. About.
  • the present invention relates to a test reagent for atopic dermatitis, comprising an antibody that recognizes a peptide containing an amino acid sequence of an indicator protein. Oligonucleotides and antibodies constituting the reagent of the present invention can be bound with an appropriate label depending on the format.
  • the oligonucleotides and antibodies constituting the reagent of the present invention can be immobilized on an appropriate support depending on the format.
  • the reagent of the present invention is characterized in that:
  • a test kit can be prepared in combination with additional components required for testing and storage. Additional components that can make up the kit are shown below. These components can be pre-mixed if necessary. Preservatives and preservatives can be added to each element as needed.
  • the expression level of the indicator gene in the present invention was confirmed to vary in each skin tissue in comparison with the rash area of a patient with atopic dermatitis and the rash area of the same patient. Therefore, an atopic dermatitis test can be performed using the expression level of the indicator gene as an indicator.
  • the test for atopic dermatitis in the present invention includes, for example, the following tests. Even if a patient who shows symptoms suspected of having atopic dermatitis but cannot be determined to be atopic dermatitis by a general test is a patient with atopic dermatitis by performing the test based on the present invention? Can be easily determined. More specifically, an increase in the expression of an indicator gene in a patient exhibiting symptoms suspected of atopic dermatitis indicates that the cause of the symptoms is likely to be atopic dermatitis.
  • tests can be done to determine if atopic dermatitis is improving.
  • it is useful for judging the therapeutic effect on atopic dermatitis.
  • increased expression of the indicator gene indicates that atopic dermatitis is likely to be more advanced.
  • the severity of atopic dermatitis can be determined based on the difference in expression level. That is, the degree of increase in the expression of the indicator gene correlates with the severity of atopic dermatitis.
  • the present invention relates to an atopic dermatitis model animal comprising a transgenic non-human animal having an increased expression level in the skin of an indicator gene or a gene functionally equivalent to the indicator gene.
  • the expression intensity of the indicator gene is increased in the rash of atopic dermatitis patient. Therefore, an animal in which the expression level of the indicator gene or a gene functionally equivalent to the indicator gene in the skin is artificially enhanced can be used as a model animal for atopic dermatitis.
  • a functionally equivalent gene is a gene that encodes a protein having an activity similar to the activity clarified in the protein encoded by the indicator gene.
  • a representative example of a functionally equivalent gene is a counterpart of the indicator gene in the animal species that the test animal originally has.
  • mouse SK404 is a functionally equivalent gene in mice. The nucleotide sequence of mouse SK404 is registered under the name onz in, but has no functional information. The mouse SK404 gene is a desirable indicator gene when the screening according to the present invention is performed using a mouse.
  • SK404 is a gene showing a difference of at least two-fold when the expression level of the gene is compared between the sensitized mouse auricle skin and the non-sensitized mouse auricle skin.
  • an atopic dermatitis model animal can be created by adjusting the expression level of the mouse indicator gene or by administration. That is, the present invention relates to a method for producing an atopic dermatitis model animal by regulating the expression level of a mouse indicator gene. Alternatively, the present invention relates to a method for producing an atopic dermatitis model animal by administering the protein itself encoded by the mouse indicator gene.
  • the mouse indicator gene of the present invention causes atopic dermatitis by increasing the expression level. Can be guided. Alternatively, an atopic dermatitis model animal can be produced by administering this gene or the protein encoded by the gene. Since all of these counterparts are mouse genes, it is desirable to administer the gene protein to mice when administering the protein.
  • the atopic dermatitis model animal is useful for clarifying changes in the body in atopic dermatitis. Furthermore, using the atopic dermatitis model animal to elucidate the further function of the indicator gene and evaluating a drug targeting the gene are of great significance.
  • the atopic dermatitis model animal according to the present invention is useful for elucidating the mechanism of atopic dermatitis and for testing the safety of screened compounds. For example, if a model animal of atopic dermatitis according to the present invention develops dermatitis or shows a change in measured values related to allergic allergic disease, a screening system for searching for a compound having an action to restore it Can be constructed.
  • an increase in the expression level refers to a state in which the indicator gene has been introduced as a foreign gene and is forcibly expressed, or a state in which the transcription and translation of the indicator gene originally provided in the test animal are enhanced. And the state in which the degradation of the protein as a translation product is suppressed.
  • the gene expression level can be confirmed, for example, by a difference in signal intensity in a DNA chip as shown in Examples.
  • the activity of the protein as a translation product can be confirmed by comparison with a normal state.
  • transgenic animals include animals into which an indicator gene has been introduced and forcibly expressed.
  • animals in which a mutation has been introduced into the coding region of the indicator gene to enhance its activity or have been modified to an amino acid sequence that is hardly decomposed can be shown. Mutations in the amino acid sequence can indicate substitutions, deletions, insertions, or additions.
  • the expression itself of the indicator gene of the present invention can be regulated by mutating the transcription regulatory region of the gene. Methods for obtaining transgenic animals for specific genes are known.
  • Transgenic animals can be obtained by methods using embryonic stem cells (ES cells).
  • ES cells embryonic stem cells
  • a method of introducing a gene into a retrovirus vector and infecting an egg, and a sperm vector method of introducing a gene into an egg via sperm have been developed.
  • the sperm vector method is a genetic recombination method in which a foreign gene is introduced into sperm cells by attaching a foreign gene to sperm or by electroporation or the like, and then fertilizing the egg to introduce the foreign gene ( M. Lavitranoet et al. Cell, 57, 717, 1989).
  • the expression level of the exogenous indicator gene in the transgenic animal can be adjusted by administering the substance.
  • the transgenic animal used as a model animal for atopic dermatitis of the present invention can be prepared using any vertebrate other than human. Specifically, transgenic animals in which various genes have been introduced and expression levels of which have been altered in vertebrates such as mice, rats, magpies, miniptas, goats, sheep, and maggots have been created.
  • the present invention relates to a method for screening a candidate compound for a therapeutic agent for atopic dermatitis.
  • the indicator gene is SK404.
  • the expression level of the SK404 gene is significantly increased in the rash area of the same patient as compared to the rash area of atopic dermatitis patients. Also, the expression level of the SK404 gene is significantly increased in the eruption area of atopic dermatitis patients as compared with healthy subjects.
  • a therapeutic agent for atopic dermatitis can be obtained by selecting a compound that can reduce the expression level of the indicator gene.
  • the compound that reduces the expression level of a gene includes gene transcription, It is a compound that has an inhibitory effect on any of the steps of translation and expression of protein activity.
  • the method for screening a candidate compound for treating an allergic disease according to the present invention can be performed in vivo or in vitro. This screening can be performed, for example, according to the following steps.
  • a gene functionally equivalent to the SK404 gene can be used as the indicator gene.
  • a functionally equivalent gene is a gene that encodes a protein having the same activity as that of the protein encoded by the indicator gene.
  • a representative example of a functionally equivalent gene is the power gene of the indicator gene in the animal species that the test animal originally has.
  • an atopic dermatitis model animal can be used as a test animal in the screening method of the present invention.
  • Atopic dermatitis model animals are known.
  • a spontaneous dermatitis model using NC / Nga mice has been reported as a model similar to human atopic dermatitis. Mite antigen on the pinna of this mouse
  • Eight doses of (5 / z g / ear) at 2-3 day intervals can induce symptoms very similar to human atopic dermatitis after 2 weeks.
  • the screening according to the present invention can be performed by administering a candidate compound to this system and tracking the change in the expression level of the indicator gene of the present invention.
  • the drug candidate conjugate can be given to the expression level of the indicator gene.
  • the effect of the compound can be evaluated. Fluctuations in the expression level of the indicator gene in a biological sample derived from a test animal can be monitored by a method similar to the test method of the present invention. Further, by selecting a drug candidate compound that reduces the expression level of the indicator gene based on the result of this evaluation, the drug candidate compound can be screened.
  • the screening according to the present invention can be performed by collecting a skin tissue sample from a test animal and comparing the expression level of the indicator gene with a control not contacting the candidate compound.
  • Methods for collecting and preparing skin tissue samples are known.
  • Such a screen allows selection of drugs that participate in the expression of the indicator gene in various forms. Specifically, for example, drug candidate compounds having the following actions can be found.
  • a candidate compound is brought into contact with a cell that expresses an indicator gene to select a compound that reduces the expression level of the indicator gene.
  • This screening can be performed, for example, according to the following steps.
  • cells that express the indicator gene can be obtained by inserting the indicator gene into an appropriate expression vector and introducing the vector into an appropriate host cell.
  • Usable vectors and host cells may be any as long as they can express the indicator gene of the present invention.
  • Host cells in the host-vector system include Escherichia coli, Mother cells, insect cells, animal cells, etc. can be exemplified, and one of the available vectors can be appropriately selected.
  • Examples of a method for introducing a vector into a host include a biological method, a physical method, and a chemical method.
  • Biological methods include, for example, a method using a virus vector, a method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene glycol (PEG), an electric cell fusion method, micronuclear fusion). Law
  • Chrosome transfer Chromosome transfer
  • Examples of the physical method include a microinjection method, an electroporation method, and a method using a gene particle gun.
  • Chemical methods include calcium phosphate precipitation, liposome method, DEAE dextran method, protoplast method, erythrocyte ghost method, erythrocyte membrane ghost method, and microcapsule method.
  • the cells expressing the indicator gene in addition to skin cells, Langerhans cells, mast cells, T cells, eosinophils, B cells, neutrophils, basophils, etc. Can be used.
  • differentiation can be induced by stimulating human primary keratinocyte (Keratinocyte) cultured cells HEK (Normal Human Epidermal Keratinocyte) with TGF- / 3 or sodium butyrate (eg, Geng Wang et al., EXPERIMENTAL cELL RESEARCH 198, 27-30 ( 1992)) intracellular structure that cornif i ed envelope (CE) with the 0 differentiation is formed.
  • Differentiation can be confirmed using CE formation or gene expression of CE constituent molecules (involucrin, loricrin, etc.) as an index.
  • the cells thus differentiated are useful for screening in the present invention.
  • Skin cells, T cells, eosinophils, mast cells, basophils, B cells, Langerhans cells, and neutrophils can also be used as cells found in skin tissue.
  • Strained skin cells are suitable for the screening method of the present invention in that a large amount of homogeneous cells can be obtained and that culturing is easy.
  • Skin cells that can be used in the present invention below Examples of strains are shown.
  • T cell Jurkat (ATCC TIB-152), Molt-4 (ATCC CRL-1582), H9 (ATCC HTB-176)
  • One cell line B cell DND39, Raji (ATCC CCL-86)
  • a candidate compound is brought into contact with the established skin cells. Thereafter, the expression level of the indicator gene in the established skin cells is measured, and a compound that reduces the expression level of the indicator gene is selected as compared with a control in which the candidate compound is not contacted.
  • the expression level of the indicator gene can be compared not only by the expression level of the protein encoded by the gene, but also by detecting the corresponding mRNA.
  • the mRNA sample preparation step described above is performed instead of the protein sample preparation step. Detection of mRNA and protein can be carried out by a known method as described above.
  • a transcriptional regulatory region of the indicator gene of the present invention can be obtained, and a reporter Atssei system can be constructed.
  • Reporter Atsushi It refers to an Atsushi system that uses a level of expression of a reporter gene located downstream of a transcription regulatory region as an index to star a transcription regulatory factor acting on the transcription regulatory region.
  • the present invention relates to a method of screening for a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is the SK404 gene or a gene functionally equivalent to the SK404 gene: On how to be.
  • transcription control region examples include a promoter, an enhancer, and a CMT box, a TATA box and the like usually found in a promoter region.
  • reporter gene CAT (chloramphenicol acetyl transferase) m gene, luciferase (lucif erase) gene, growth hormone gene and the like can be used.
  • the transcriptional regulatory region of the indicator gene in the present invention can be obtained as follows. That is, first, based on the nucleotide sequence of the cDNA disclosed in the present invention, screening from a human genomic DNA library such as a BAC library or YAC library by PCR or a method using hybridization is performed, and the sequence of the cDNA To obtain a genomic DNA clone containing Based on the sequence of the obtained genomic DNA, the transcription control region of the cDNA disclosed in the present invention is estimated, and the transcription control region is obtained. The obtained transcription regulatory region is cloned so as to be located upstream of the reporter gene to construct a reporter construct. The resulting reporter construct is introduced into a cultured cell line to obtain a transformant for screening. The transformant was contacted with a scavenger compound and compared to a control without the candidate compound.
  • the screening of the present invention can be performed by selecting a conjugate which reduces the expression level of the porter gene.
  • a screening method based on the activity of an indicator protein can also be used. That is, the present invention provides a method for screening a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is a SK404 gene or a gene functionally equivalent to the SK404 gene. There is a method.
  • the activity of the indicator protein in the present invention as an indicator, compounds having an activity of inhibiting the activity can be screened.
  • the compound thus obtained suppresses the function of the indicator gene.
  • test candidate substances used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, and extracts of animal and plant tissues or Examples thereof include a mixture containing a plurality of compounds such as a microorganism culture, and a sample purified therefrom.
  • kits Polynucleotides, antibodies, cell lines, or model animals required for various screening methods according to the present invention can be combined in advance to form a kit. These kits must be packaged with the substrate compound used to detect the label, culture media and containers for cell culture, positive and negative standard samples, and instructions describing how to use the kit. You can also.
  • the compound selected by the screening method of the present invention is useful as a therapeutic agent for atopic dermatitis.
  • an antibody that recognizes a peptide containing the amino acid sequence of the protein encoded by the indicator gene is also useful as a therapeutic agent for atopic dermatitis.
  • the indicator gene is a gene whose expression is increased in the rash area of atopic dermatitis patients. Therefore, a therapeutic effect on atopic dermatitis can be expected by suppressing the expression of these genes or the functions of the proteins encoded by these genes.
  • the therapeutic agent for an allergic disease of the present invention contains the compound selected by the screening method as an active ingredient, and is produced by mixing with a physiologically acceptable carrier, excipient, diluent, or the like. Can be.
  • the therapeutic agent for allergic diseases of the present invention can be administered orally or parenterally for the purpose of improving allergic symptoms.
  • dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, and suspensions can be selected.
  • Injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections.
  • a therapeutic effect can be achieved by introducing a gene encoding the protein into a living body using a gene therapy technique.
  • Techniques for treating a disease by introducing a gene encoding a protein having a therapeutic effect into a living body and expressing the gene are known.
  • the dosage varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, processing time, or the type of active ingredient contained in the pharmaceutical composition, but is usually once per adult.
  • FIG. 1 is a graph showing the results of expression analysis (GAPDH correction) of TARC gene in patient skin tissues.
  • the vertical axis indicates the gene expression intensity (copy / 5ng total RA), and the horizontal axis indicates the type of sample.
  • FIG. 2 is a graph showing the results of expression analysis (GAPDH correction) of the CCR4 gene in patient skin tissues.
  • the vertical and horizontal axes are the same as in Fig. 1.
  • FIG. 3 is a graph showing the results of expression analysis (GAPDH correction) of RANTES gene in patient skin tissue.
  • the vertical and horizontal axes are the same as in Fig. 1.
  • FIG. 4 is a graph showing the results of expression analysis (GAPDH correction) of the CXCR3 gene in the skin of a patient.
  • the vertical and horizontal axes are the same as in Fig. 1.
  • FIG. 5 is a graph showing the results of expression analysis of SK404 in skin tissues of patients with atopic dermatitis and healthy subjects.
  • the vertical and horizontal axes are the same as in Fig. 1.
  • FIG. 6 is a diagram comparing the amino acid sequences of human SK404 (top) and mouse SK404M (bottom).
  • I means an amino acid match,. Means a substitution for a similar amino acid, and: means a substitution for a more similar amino acid.
  • a dot ( ⁇ ) in the amino acid sequence indicates a gap.
  • FIG. 7 is a graph showing the results of expression analysis (GAPDH correction) of SK40M in the auricle skin of the NcNga mouse model.
  • the vertical axis shows the gene expression intensity (copy / 5ng total RNA), and the horizontal axis shows the conditions of the NcNga model mouse.
  • FIG. 8 is a graph showing the expression of the SK404 gene in human tissues.
  • FIG. 9 is a graph showing the expression of the SK404 gene in blood cells. Blood cells from healthy persons (5) were used.
  • FIG. 10 is a photograph showing a situ hybridization analysis of SK404.
  • Fig. 11 is a graph showing the expression of the SK404 gene in the skin tissue of patients with atopic dermatitis and psoriasis.
  • FIG. 12 is a photograph showing the expression of SK404 protein.
  • Example 1 Expression analysis of TARC, CCR4, RANTES, C XCR3 gene in skin tissue of atopic dermatitis patient
  • chemokines TARC, RANTES
  • CCR4, CXCR3 The expression of (CCR4, CXCR3) in the skin tissue of atopic dermatitis was quantified, and the infiltration of lymphocytes in the inflamed skin tissue of the patient was evaluated.
  • Isogen Natural Gene; Wako Pure Chemical Industries
  • IKA Ultratax T8 homogenizer
  • total RA was extracted according to the manual of Isogen.
  • a black-mouthed form was added, and the mixture was centrifuged with stirring to collect an aqueous layer.
  • isopropanol was added, and the mixture was centrifuged with stirring to collect the precipitate.
  • the precipitate was rinsed with 75% ethanol and centrifuged, and the precipitate was collected as total RNA.
  • the collected total RNA was further purified using the RNeasy Mini kit (QIAGEN) according to the manual.
  • actin-actin gene and glyceraldehyde triphosphate dehydrogenase (GA) were used as internal standards for correction to correct for differences in cDNA concentration in the sample.
  • the same analysis was performed for the (PDH) gene, and the copy number of the target gene was calculated by correcting based on the copy number of those genes.
  • the nucleotide sequences of the oligonucleotides used for the forward primer (F), reparse primer (R), and TaqMan probe (TP) for each gene are as shown below.
  • the accession number of Genbank corresponding to the nucleotide sequence of the measured gene is displayed following the gene name.
  • R 5'-CCCACTGGTCTGCTGCATAGT-3, (SEQ ID NO: 9)
  • RANTES GenBank Acc .: NM_002985
  • TP 5'— ATGCCCTCCCCCATGCCATCCTGCGT-3, (SEQ ID NO: 19) GAPDH (GenBank Acc .: Oichi 002046)
  • chemokines in the skin eruption area was high, that is, lymphocytes were in an environment where they could easily migrate to the skin, and the expression of chemokine receptors, markers of lymphocytes themselves, was high, that is, they had already migrated to the skin. It is estimated that the number of lymphocytes is large.
  • the T7- (dT) 24 primer consists of a base sequence obtained by adding (dT) 24 to the base sequence of the T7 promoter as follows.
  • DNA Ligase DNA polymerase I and RNase H were added to synthesize double-stranded cDNA.
  • biotin-labeled cRNA was synthesized using the BioArray High Yield RNA Transcription Labeling Kit.
  • the cDNA was purified using an RNeasy Spin column (QIAGEN) and fragmented by heat treatment.
  • Streptavidin-Phycoerythrin was added for staining. After washing, a mixture of normal goat IgG and a biotinylated goat anti-streptavidin IgG antibody was added to the array. Furthermore, for the purpose of enhancing the fluorescence intensity, Streptavidin-Phycoerythrin was added again for staining. After washing, set it on the scanner and analyze the DNA chip Analyzed with software.
  • the expression fluorescence intensity was measured using Suite, a DNA chip analysis software, and the data was analyzed. First, Absolute analysis was performed on all chips, and the gene expression level of each sample used was measured.
  • the positive and negative were determined by comparing the fluorescence intensity of the mismatch between the probe match and the probe match of the probe set.
  • Average Difference (Avg Diff), which is the average value of the difference in fluorescence intensity between the perfect match and the mismatched probe cell, was also calculated.
  • the full-length nucleotide sequence of SK404 is shown in SEQ ID NO: 1, and the full-length amino acid sequence is shown in SEQ ID NO: 2.
  • SK404 showed a profile of high expression in acute lesions in 9 of 11 patient skins analyzed.
  • Genes whose expression fluctuates between the rash and eruption areas of human atopic dermatitis and those whose expression fluctuates between unsensitized and sensitized skin of mouse dermatitis model are compared and analyzed. By selecting knockout or transgenic mice for commonly fluctuating genes, the importance of that gene in dermatitis pathology can be assessed. .
  • the common variable gene is a humoral factor or a membrane protein
  • administration of a neutralizing antibody, humoral factor itself, or a soluble receptor, etc. results in a shorter time than when a genetically modified mouse is used.
  • the importance of the gene in human dermatitis conditions can be assessed.
  • a mouse homolog was identified for SK404, and gene expression was analyzed in a mouse dermatitis model.
  • FIG. 6 shows the results of sequence comparison at the amino acid level between SK404 and SK404M.
  • Non-sensitized group Mean 0 23. 7 19.3
  • Sensitization group Mean 0 183 250. 4
  • Ear skin and back skin were collected from non-sensitized mice and sensitized mice (14 and 28 days after sensitization), immersed in Isogen (Nippon Gene; Wako Pure Chemical), and homogenized.
  • RNA extracted from the auricle of each individual of 10 animals per group is collected, the total RNA is treated with Dnase (two bonbon gene), and the cDNA obtained by reverse transcription using oligo (dT) 12 — 18 (GIBCO BRL) as a primer is obtained.
  • Dnase two bonbon gene
  • cDNA obtained by reverse transcription using oligo (dT) 12 — 18 GEBCO BRL
  • a plasmid clone containing a nucleotide sequence region amplified by both primers was prepared for each gene for a standard curve for calculating the copy number, and the reaction was performed using the serial dilution as type III.
  • the composition of the reaction mixture for quantitative PCR is shown in Table 2.
  • ABI 7700 Applied Biosystems
  • the primer and TaqMan probe used for the measurement with ABI 7700 were designed by Primer Express (PE Biosystems) based on the sequence information of the SK404M gene.
  • the 5 'end of the TaqMan probe used was FAM (6-carboxy-fluorescein) and 3.
  • the terminal was labeled with TAMRA (6-carboxy-N, N, N ', N'-tetramethylrhodamine).
  • GAPDH glyceraldehyde triphosphate dehydrogenase
  • the nucleotide sequences of the oligonucleotides used for the forward primer (F), the lipase primer (R), and the TaqMan probe (TP) of the SK40 and mouse GAPDH genes are as follows.
  • GenBank accession number corresponding to the nucleotide sequence of the indicator gene is displayed following the gene name.
  • FIG. SK404M showed higher expression in the auricle skin 14 days and 28 days after sensitization than in the non-sensitized auricle skin. On the 14th day, the expression level was 7.5 times higher than that of the non-sensitized mice, and on the 28th day, the expression level was 17.3 times higher than that of the non-sensitized mice.
  • SK404 Since the expression of SK404 increases in the acute phase of atopic dermatitis, it is possible that SK404 may play an important role in the development of atopic dermatitis. In addition, it shows a profile similar to that of chemokines and chemokine receptors such as TARC and CCR4, and may be involved in the infiltration of lymphocytes into skin tissues and may contribute to the pathogenesis. SK404 may be a CXC chemokine based on its position on the chromosome (4q21) and the amino acid secondary structure, and it is conceivable that it directly acts on lymphocytes. Human atopic dermatitis in mouse dermatitis model The same fluctuations in expression were observed, supporting the importance of the disease in dermatitis conditions. In addition, the importance of SK404 can be easily evaluated by inhibiting gene products in such mouse models by various methods and forcibly expressing ⁇ .
  • SK404 gene expression in human tissues was analyzed by Real Time PCR. g of various tissues from total tissues, cDNA was synthesized by a standard method, and Real Time PCR (LightCycler: Roche) was performed using 1/10 of the cDNA (equivalent to 100 ng in terms of total RNA). . For the quantification, SYBR Green I, a fluorescent dye that specifically binds to double-stranded DNA, was used.
  • Expression analysis includes adipose tissue, brain, adrenal gland, bone marrow, large intestine, heart, kidney, fetal kidney, liver, fetal liver, lung, lymph nodes, mammary gland, spleen, placenta, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen , Stomach, testis, thymus, trachea, uterus, Hela cells, renal cortical epithelial cells, coronary artery vascular endothelial cells, coronary artery vascular smooth muscle cells, leukocytes, PH A activated leukocytes, derived from normal human proximal tubular epithelial cells Total RM was used.
  • the nucleotide sequences of the forward primer (F) and reverse primer (R) of the SK404 gene used for expression analysis are as shown below.
  • Figure 8 shows the results of the analysis.
  • the results of expression analysis were corrected based on the expression level of GAPDH so that comparison between tissues was possible.
  • the SK404 gene was highly expressed in colon, bone marrow, spleen, lymph nodes, lung, trachea, adipose tissue, and leukocytes.
  • Peripheral blood from healthy humans (approximately 5-10 ml) Magnetic density using capillary density gradient method and magnetic beads Cells were fractionated using a gas cell separation system (MACS-Miltenyi Biotec GmbH). After removing plasma from peripheral blood by centrifugation, the remaining blood is overlaid on a specific gravity gradient solution Ficoll (manufactured by Amersham Pharmacia Biotec) and centrifuged.The separated middle layer is fractionated into lymphocytes and the lower layer is separated. It was fractionated as a granulocyte fraction. After reacting with CD3 magnetic beads, the lymphocyte fraction was subjected to MACS using a nylon steel column, and CD3-positive cells were collected as T cells.
  • MACS-Miltenyi Biotec GmbH After removing plasma from peripheral blood by centrifugation, the remaining blood is overlaid on a specific gravity gradient solution Ficoll (manufactured by Amersham Pharmacia Biotec) and centrifuged. The separated middle layer is fractionated into lymphocytes and
  • CD3-negative cells were further reacted with CD14 magnetic beads.
  • CD14-positive cells by MACS were monocytes, and CD14-negative cells were B cells.
  • the granulocyte fraction was reacted with CD16 magnetic beads, and CD16-positive cells were sorted as neutrophils and CD16-negative cells as eosinophils.
  • the fractionated cells were dissolved in Isogen (Nippon Gene; Wako Pure Chemical), and total RNA was extracted according to the manual. A black-mouthed form was added, and the mixture was centrifuged with stirring to collect an aqueous layer. Next, isopropanol was added, and the mixture was stirred and centrifuged to collect the precipitate. The precipitate was rinsed with 75% ethanol and centrifuged, and the precipitate was collected as total RNA.
  • RNA was treated with DNase (Nibbon Gene), reverse transcribed using oligo (dT) 12 _ 18 (GIBCO BRL) as a primer, and Superscript II reverse transcriptase (Invitrogen), Was synthesized.
  • FIG. SK404 was expressed in all of the five types of cells analyzed, with B cells, T cells, monocytes, eosinophils, and neutrophils in descending order of expression level.
  • ISH In situ hybridization
  • Sensitization The auricle skin of the mouse at 4 weeks was fixed with 10% neutral buffered formalin, embedded in paraffin, and paraffin blocks were prepared.
  • the probe used was the sequence described in SEQ ID NO: 35, and dicoxygenin-labeled sense and antisense probes were prepared using SP6 and T7 RNA polymerase.
  • a probe was reacted with a paraffin section stuck on a slide glass, and further reacted with an anti-dicoxygenin antibody labeled with alkaline phosphatase, followed by reaction with NBT / BCIP as a chromogenic substrate to develop color.
  • SK404M was expressed in neutrophils and T cells in mouse dermatitis lesions. Most of the positive cells were neutrophils, but T cells were higher in signal intensity (intracellular expression).
  • SK40 was identified as a gene with increased expression in atopic lesions in patients with atopic dermatitis compared to the eruption site, but we will analyze gene expression in skin tissues of patients with another skin inflammatory disease, psoriasis This makes it clear whether the expression changes are specific to atopic dermatitis or are common to both diseases. If atopic dermatitis-specific expression fluctuations are shown, it is thought that its value as a diagnostic and drug discovery target will increase as one of the genes that characterize the pathology of atopic dermatitis.
  • the collected three skin biopsy (3 diameter in diameter) were immersed in Isogen (Nippon Gene; Wako Pure Chemical) and homogenized using Ultratax T8 homogenizer (IKA). After homogenization, total RNA was extracted according to the manual of Isogen. A black-mouthed form was added, and the mixture was centrifuged with stirring to collect an aqueous layer. Next, isopropano was added, and the mixture was centrifuged with stirring to collect the precipitate. The precipitate was rinsed with 75% ethanol and centrifuged, and the precipitate was collected as total RNA. The collected total thigh was further purified using the RNeasy Mini kit (QIAGEN) according to the manual.
  • Isogen Natural Gene
  • IKA Ultratax T8 homogenizer
  • RNA extracted by the method described above was DNase (Yutsubonjin) process, an oligo (d T) 12 _ 18 (GIBCO BRL) was reverse transcribed as primers cDNA was ⁇ .
  • a plasmid clone containing a nucleotide sequence region to be amplified by both primers was prepared for each gene for a standard curve for calculating the copy number, and the reaction was carried out with the serial dilution as type III.
  • the composition of the reaction mixture for quantitative PCR is shown in Table 2.
  • SK404 gene product is a humoral factor secreted extracellularly, or intracellular protein To confirm the quality, protein expression was performed using HEK293 cells.
  • a SK404 gene fragment with the EcoRI site at the 5 'end and the BamHI site at the 3' end added by PCR, excluding the termination codon, from 348 bp of the full length SK404 gene, can be expressed by PCR, with the FLAG tag added at the C-terminal end. It was ligated between EcoRI-BamHI site of p3XFLAG-CMV-14 vector (sigma). The sequence of the primer used for the synthesis of the SK404 gene fragment containing no stop codon is shown below.
  • ⁇ / ig SK404 gene expression vector was introduced into one plate using TransIT LT reagent (Mirus) according to the manual.
  • the medium was changed after the gene introduction after 8 hours, 5% C0 2 incubator beta one to 48 hours, and cultured at 37 ° C. After 48 hours, the culture supernatant was recovered, the plate was washed with PBS, and 1 ml of M-PER Mammalian Protein Extraction Reagent was added to disrupt the cells, and a cell lysate was recovered.
  • the membrane was immersed in an Anti-FLAG M2 Monoclonal Antibody (sigma) solution diluted to 10 / ig / ml with Blocking solution (10 mM Tris, 150 mM NaCl, 0.1% Teen20, 2% BSA) and diluted at room temperature. Allowed to react for hours. Wash solution (120mM NaCl, lOmM NaH 2 P0 4, 30mM K 2 HP0 4) after membrane was washed three times with diluted with Blocking solution (10mM Tris, 150mM NaCl, 0.
  • the present invention provides an indicator gene SK404 of allergic dermatitis.
  • SK404 is a gene identified as an indicator gene whose expression level in the rash area is higher than that in the non-rash area of patients with atopic dermatitis.
  • SK404 was also an indicator gene whose expression level in the rash area of patients with atopic dermatitis was higher than that in healthy subjects.
  • mouse SK404 gene is an indicator gene whose expression level in the auricle skin of mite allergen-sensitized mice is higher than that in the mite allergen non-sensitized mice.
  • the indicator gene of the present invention a change in its expression is linked to a disease state. Therefore By controlling the expression of the indicator gene and the activity of the protein encoded by the indicator gene, it becomes possible to treat allergic dermatitis. For example, in the case of a gene whose expression is increased in the affected area or in the skin of a patient, inhibition of the expression and its activity is a target of a therapeutic strategy for atopic dermatitis.
  • the index gene of the present invention is expected to be useful as a new clinical diagnostic index for monitoring in the treatment of atopic dermatitis.
  • the expression level of the indicator gene provided by the present invention can be easily determined regardless of the type of allergen. Therefore, the pathology of allergic reactions can be comprehensively grasped.
  • the method for detecting atopic dermatitis according to the present invention can analyze the expression level using a biological sample as a sample, and therefore has low invasiveness to patients.
  • highly sensitive measurement using a small amount of sample is possible.
  • high throughput and low price are increasing year by year. Therefore, the test method for atopic dermatitis according to the present invention is expected to be an important bedside diagnostic method in the near future. In this sense, the diagnostic value of the indicator gene of the present invention is high.

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  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Dermatology (AREA)
  • Pulmonology (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)

Abstract

L'invention concerne un gène SK404 présentant un niveau d'expression différent entre une zone corporelle présentant un érythème et une zone corporelle ne présentant aucun érythème chez un patient souffrant de dermatite atopique, ou entre une zone corporelle d'un patient présentant un érythème et un sujet sain. Ce gène permet de fournir une indication, lors de l'examen de la dermatite atopique; il permet également de sélectionner un traitement. Cette invention concerne un procédé permettant d'examiner une dermatite atopique et un procédé permettant de sélectionner un composé pour traiter cette maladie, lequel procédé consiste à comparer les niveaux d'expression du gène SK404 susmentionné.
PCT/JP2003/005277 2002-08-06 2003-04-24 Procede permettant d'examiner une dermatite atopique WO2004016783A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2004528829A JPWO2004016783A1 (ja) 2002-08-06 2003-04-24 アトピー性皮膚炎の検査方法
AU2003227362A AU2003227362A1 (en) 2002-08-06 2003-04-24 Method of examining atopic dermatitis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002229320 2002-08-06
JP2002-229320 2002-08-06

Publications (1)

Publication Number Publication Date
WO2004016783A1 true WO2004016783A1 (fr) 2004-02-26

Family

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PCT/JP2003/005277 WO2004016783A1 (fr) 2002-08-06 2003-04-24 Procede permettant d'examiner une dermatite atopique

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JP (1) JPWO2004016783A1 (fr)
AU (1) AU2003227362A1 (fr)
WO (1) WO2004016783A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9095523B2 (en) 2006-06-29 2015-08-04 Merz Pharma Gmbh & Co. Kgaa High frequency application of botulinum toxin therapy

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004302A1 (fr) * 1999-07-09 2001-01-18 Mitsubishi Chemical Corporation Gene d'atopie
WO2001064835A2 (fr) * 2000-02-28 2001-09-07 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
JP2002095500A (ja) * 2000-09-25 2002-04-02 Jenokkusu Soyaku Kenkyusho:Kk アレルギー性疾患の検査方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004302A1 (fr) * 1999-07-09 2001-01-18 Mitsubishi Chemical Corporation Gene d'atopie
WO2001064835A2 (fr) * 2000-02-28 2001-09-07 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
JP2002095500A (ja) * 2000-09-25 2002-04-02 Jenokkusu Soyaku Kenkyusho:Kk アレルギー性疾患の検査方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9095523B2 (en) 2006-06-29 2015-08-04 Merz Pharma Gmbh & Co. Kgaa High frequency application of botulinum toxin therapy

Also Published As

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JPWO2004016783A1 (ja) 2006-01-19
AU2003227362A8 (en) 2004-03-03
AU2003227362A1 (en) 2004-03-03

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