WO2004016783A1 - Method of examining atopic dermatitis - Google Patents

Method of examining atopic dermatitis 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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WO
WIPO (PCT)
Prior art keywords
gene
indicator gene
atopic dermatitis
indicator
expression level
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PCT/JP2003/005277
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French (fr)
Japanese (ja)
Inventor
Mikito Itoh
Akira Shinagawa
Hajime Sudo
Hideoki Ogawa
Chisei Ra
Kouichi Mitsuishi
Original Assignee
Genox Research, Inc.
Juntendo University
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Application filed by Genox Research, Inc., Juntendo University filed Critical Genox Research, Inc.
Priority to JP2004528829A priority Critical patent/JPWO2004016783A1/en
Priority to AU2003227362A priority patent/AU2003227362A1/en
Publication of WO2004016783A1 publication Critical patent/WO2004016783A1/en

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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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Abstract

A gene SK404 showing a difference in its expression level between a rash part and a no-rash part of a patient suffering from atopic dermatitis or between a rash part of the patient and a normal subject. Thus, it is clarified that this gene is useful as an indication in examining atopic dermatitis and screening a remedy therefor. Namely, a method of examining atopic dermatitis and a method of screening a compound for treating this disease by comparing the expression levels of the indication gene SK404 thus found out.

Description

ァトピー性皮膚炎の検査方法 技術分野  Testing method for atopic dermatitis
本発明は、 アトピー性皮膚炎の検査方法に関する。 背景技術  The present invention relates to a method for testing atopic dermatitis. Background art
ァトビー性皮膚炎等のアレルギー性疾患は、 多因子性の病気 (multifactorial diseases)と考えられている。 これらの病気は多くの異なる遺伝子の発現の相互 作用によって起こり、 これらの個々の遺伝子の発現は、 複数の環境要因によって 影響を受ける。 このため、 特定の病気を起こす特定の遺伝子を解明することは、 非常に困難である。  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.
またアレルギー性疾患には、 変異や欠陥を有する遺伝子の発現や、 特定の遺伝 子の過剰発現や発現量の減少が関わっていると考えられている。 病気に関して遺 伝子発現が果たしている役割を解明するためには、 遺伝子が発症にどのように関 わり、 薬剤などの外的な刺激が遺伝子発現をどのように変化させるのかを理解す る必要がある。  In addition, allergic diseases are thought to be related to the expression of mutated or defective genes, or overexpression or decreased expression of specific genes. To understand the role gene expression plays in disease, it is necessary to understand how genes are involved in pathogenesis and how external stimuli, such as drugs, alter gene expression. is there.
さて、 現在アレルギー性疾患の診断においては、 一般に、 問診、 家族歴、 そし て本人の既往症の確認が重要な要素となっている。 またアレルギーをより客観的 な情報に基づいて診断するために、 血液を試料とする試験方法や、 アレルゲンに 対する患者の免疫学的な応答を観察する方法も実施されている。 前者の例として、 アレルゲン特異的 IgE測定、 白血球ヒスタミン遊離試験、 あるいはリンパ球幼若 ィ匕試験等が挙げられる。 アレルゲン特異的 IgEの存在は、 そのアレルゲンに対す るアレルギー反応の証明である。 しかし患者によっては、 必ずしもアレルゲン特 異的な IgEを検出できるとは限らない場合もある。 また、 その測定原理上、 診断 に必要なアレルゲンの全てに対して、 試験を実施しなければならない。 白血球ヒ スタミン遊離試験やリンパ球幼若化試験は、 免疫システムのァレルゲンに対する 反応を ώ 'iroで観察する方法である。 これらの方法は、 操作が煩雑である。 一方、 患者を実際にアレルゲンに接触させたときに観察される免疫応答をァレ ルギ一の診断に役立てる方法 （後者） も公知である。 ブリック ·テスト、 スクラ ツチ 'テスト、 パッチ'テスト、 皮内反応、 あるいは誘発試験等が、 この種の試 験に含まれる。 これらの試験では、 患者のアレルギー反応を直接診断することが できる反面、 実際に被検者をァレルゲンに曝露する侵襲を伴う検査であると言う ことができる。 Now, in the diagnosis of allergic diseases, interviews, family histories, and confirmation of the patient's history are generally important factors. In order to diagnose allergies based on more objective information, test methods using blood samples and methods of observing patients' immunological responses to allergens are also being implemented. Examples of the former include 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. However, some patients may not always be able to detect allergen-specific IgE. Also, due to its measurement principle, 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 ώ'iro. These methods are complicated in operation. On the other hand, a method for utilizing the immune response observed when a patient is actually brought into contact with an allergen to diagnose allergens (the latter) 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.
この他、 アレルゲンに関わらず、 アレルギー反応の関与を証明するための試験 方法も試みられている。 たとえば、 血清 IgE値が高値である場合、 その患者には ァレルギ一反応が起きていると推定することができる。 血清 IgE値は、 ァレルゲ ン特異 IgEの総量に相当する情報である。 アレルゲンの種類に関わらず IgEの総 量を決定することは容易であるが、 非ァトピー型気管支炎喘息等の疾患を持つ患 者では、 IgEが低値となる場合がある。  In addition, test methods have been attempted to prove the involvement of allergic reactions regardless of the allergen. For example, 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.
従って、 アレルゲンに関わらず、 アレルギー患者の病態の把握や治療方針の決 定に役立てることができる診断指標が求められていた。 さらに、 患者に対する侵 襲が小さく、 しかも診断に必要な情報を容易に得ることができるアレルギー性疾 患のマーカーが提供されれば有用である。 このようなマーカーは、 アレルギー性 疾患の発症に深く関与していると考えられるので、 診断のみならず、 アレルギー 症状のコント口一ノレにおいても、重要な標的となる可能性がある。 発明の開示  Therefore, there has been a demand for a diagnostic index that can be used to understand the condition of an allergic patient and determine a treatment policy, regardless of the allergen. Furthermore, it would be useful if markers for allergic diseases could be provided that would be minimally invasive to the patient and that would readily provide the information needed for diagnosis. Since such markers are considered to be deeply involved in the development of allergic diseases, they may be important targets not only in diagnosis but also in controlling allergic symptoms. Disclosure of the invention
本発明はこのような状況に鑑みて為されたものであり、 その目的は、 アトピー 性皮膚炎の検査を可能とする新しい指標を提供することにある。 さらに本発明は、 該指標に基づくァトピー性皮膚炎の検査方法、 およぴァトピー性皮膚炎治療薬侯 補化合物のスクリ一ユング方法を提供することを目的とする。 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.
本発明者らは上記課題を解決するために鋭意研究を行った。 すなわち、 アトピ —性皮膚炎患者の皮疹部と同一の患者における無疹部、 または健常者の皮膚との 間で発現レベルに差が見られる遺伝子を明らかにし、 アトピー性皮膚炎反応との 関連性を明らかにすることにより、 アトピー性皮膚炎治療の新たな標的を見出す ことができると考えた。  The present inventors have intensively studied to solve the above problems. In other words, 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. By clarifying this, we thought that a new target for the treatment of atopic dermatitis could be found.
このような考えに基づき、 本発明者らは、 アトピー性皮膚炎患者の皮疹部と同 一の患者における無疹部、 および健常者の皮膚との間で発現状態の違う遺伝子を 探索した。 遺伝子の発現状態を比較する生体試料としては、 被検者の皮膚組織を 選択した。 実際に炎症が起きている皮膚組織検体には病態形成に重要なリンパ球 などが多く浸潤しており、 皮膚局所における遺伝子発現解析を行えばアトピー性 皮膚炎の病態の解明につながると考えられる。  Based on this idea, 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.
本発明者らは、 アトピ一性皮膚炎患者の皮疹部と同一の患者における無疹部、 並びに健常者の皮膚で発現している遺伝子について、 ジーンチップを用いて発現 プロファイルを比較した。 2倍以上の発現変動のあった遺伝子を選別し、 遺伝子 の発現レベルを測定した。 そして、 S K 4 0 4の発現が変動していることを確認 した。  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.
本発明における指標遺伝子 S K 4 0 4の塩基配列は既知である（W0200164835)。 S K 4 0 4については次のような情報があるが、 アレルギー性疾患との関係は示 唆されていない。  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.
—大腸癌で発現している大腸癌関連遺伝子である（W0200055351)  — Colorectal cancer-related gene expressed in colorectal cancer (W0200055351)
-分泌蛋白質である（TO9854206)  -A secreted protein (TO9854206)
一 2型樹状細胞前駆細胞（Type 2 dendritic cell precursor)由来の DNA(EP11 74502)  DNA derived from Type 2 dendritic cell precursor (EP11 74502)
ァトピー性皮膚炎患者の皮疹部と同一の患者における無疹部または健常者との 間において、 指標遺伝子の発現レベルに変動が観察されたという事実は、 本発明 の指標遺伝子とアトピー性皮膚炎症状との深 、結びつきを表している。 また、 ダ 二ァレルゲン感作マゥスの耳介皮膚とダニァレルゲン非感作マゥスの耳介皮膚に おいて、 上記指標遺伝子のマウスにおけるカウンターパートの発現レベルに差が 見られることも、 本発明の指標遺伝子とアトピー性皮膚炎症状との結びつきを裏 付けている。 以上の知見に基づいて、 本突明者らは、 指標遺伝子の発現レベル、 あるいは指標遺伝子によってコードされる蛋白質の活性を指標とすることにより、 ァトピー性皮膚炎の診断、 およぴ該疾患のための治療薬のスク.リ一ユングが可能 であることを見出し、 本発明を完成させた。 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. Based on the above-mentioned findings, 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.
すなわち本発明は、 以下のアトピー性皮膚炎の検査方法、 並びにアトピー性皮 膚炎治療薬候補化合物のスクリ一二ング方法に関する。  That is, 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.
〔1〕 次の工程 ( 1 ) 〜 （3 ) を含む、 アトピー性皮膚炎の検査方法であって、 指標遺伝子が S K 4 0 4である方法。  [1] A method for testing atopic dermatitis, comprising the following steps (1) to (3), wherein the indicator gene is SK404.
( 1 ) 被検者の皮疹部から採取された生体試料における指標遺伝子の発現レべ ルを測定する工程  (1) A step of measuring the expression level of an indicator gene in a biological sample collected from the rash on the subject
( 2 ) 工程 ( 1 ) で測定された皮疹部の発現レベルを、 対照として同じ被検者 の無疹部または健常者皮膚から採取された生体試料における指標遺伝子 の発現レベルと比較する工程、 および  (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; and
( 3 ) 工程 ( 2 ) の比較の結果、 対照と比較して発現レベルが高い場合に前記 被検者はァトピ一性皮膚炎を有すると判定する工程  (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);
〔2〕 遺伝子の発現レベルを、 cDNAの PCRによって測定する 〔1〕 に記載の検 查方法。  [2] The detection method according to [1], wherein the gene expression level is measured by cDNA PCR.
〔3〕 遺伝子の発現レベルを、 指標遺伝子によってコードされる蛋白質の検出に よって測定する 〔1〕 に記載の検查方法。  [3] The detection method according to [1], wherein the expression level of the gene is measured by detecting a protein encoded by the indicator gene.
〔4〕 指標遺伝子の塩基配列を含むポリヌクレオチド、 またはその相補鎖に相補 的な塩基配列を有する少なくとも 1 5塩基の長さを有するオリゴヌクレオ チドからなる、 アトピー性皮膚炎検査用試薬であって、 指標遺伝子が S K 4 0 4であるアトピー性皮膚炎検査用試薬。 (4) 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.
〔 5 指標遺伝子によってコードされる蛋白質のァミノ酸配列を含むペプチドを 認識する抗体からなる、 アトピー性皮膚炎検查用試薬であって、 指標遺伝 子が S K 4 0 4であるアトピー性皮膚炎検査用試薬。 (5) 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.
〔6〕 次の工程を含む、 アトピー性皮膚炎の治療薬のスクリーニング方法であつ て、 指標遺伝子が S K 4 0 4であるスクリ一二ング方法。 [6] A screening method for a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404.
( 1 ) 指標遺伝子を発現する細胞に候補化合物を接触させる工程、  (1) contacting a candidate compound with cells expressing the indicator gene,
( 2 ) 前記遺伝子の発現レベルを測定する工程、  (2) measuring the expression level of the gene,
( 3 ) 候補化合物を接触させない対照と比較して、 指標遺伝子の発現レベルを 低下させる化合物を選択する工程  (3) a step of selecting a compound that reduces the expression level of the indicator gene as compared to a control not contacted with the candidate compound
〔7〕 細胞が株化皮膚細胞である 〔6〕 に記載の方法。  [7] The method of [6], wherein the cells are established skin cells.
〔8〕 指標遺伝子の塩基配列を含むポリヌクレオチド、 またはその相補鎖に相補 的な塩基配列を有する少なくとも 1 5塩基の長さを有するオリゴヌクレオ チドと、 指標遺伝子を発現する細胞を含む、 アトピー性皮膚炎の治療薬候 補化合物をスクリーユングするためのキットであって、 指標遺伝子が S K 4 0 4であるキット。  [8] 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 treatment of dermatitis, wherein the indicator gene is SK404.
[ 9 ] 指標遺伝子によってコードされる蛋白質のァミノ酸配列を含むぺプチドを 認識する抗体と、 指標遺伝子を発現する細胞を含む、 アトピー性皮膚炎の 治療薬候補化合物をスクリーニングするためのキットであって、 指標遺伝 子が S K 4 0 4であるキット。  [9] 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.
〔1 0〕 指標遺伝子または指標遺伝子と機能的に同等な遺伝子の皮膚における発 現強度を上昇させたトランスジエニック非ヒト脊椎動物からなるアトピ 一性皮膚炎モデル動物であって、 指標遺伝子が S K 4 0 4またはその機 能的に同等な遺伝子であるモデル動物。  [10] 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 1〕 非ヒト脊椎動物がマウスである 〔1 0〕 に記載のモデル動物。  [11] The model animal according to [10], wherein the non-human vertebrate is a mouse.
〔1 2〕 次の i)または ii)に記載の成分をマウスに投与する工程を含む、 アレル ギ一性皮膚炎モデ/レ動物の製造方法。 [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.
i) SK404のホモログを構成する塩基配列を含むポリヌクレオチド、 ii) SK404のホモログを構成する塩基配列を含むポリヌクレオチドによ つてコードされるタンパク質  i) 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
〔13〕 〔12〕 における i)または ii)のいずれかに記載の成分を有効成分とし て含む、 マウスにァレノレギー性皮膚炎を誘導するための誘導剤。  [13] An inducer for inducing arenoleggic dermatitis in mice, comprising as an active ingredient the ingredient according to any of i) or ii) in [12].
〔14〕 次の工程を含む、 アトピー性皮膚炎の治療薬のスクリーニング方法であ つて、 指標遺伝子が SK404またはそのホモログであるスクリーニン グ方法。  [14] A screening method for a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404 or a homolog thereof.
(1) 被験動物に候補ィヒ合物を投与する工程、  (1) administering a candidate Eich compound to the test animal,
( 2 ) 前記被験動物の生体試料における指標遺伝子の発現強度を測定する工程、 (2) measuring the expression intensity of the indicator gene in the biological sample of the test animal,
(3) 候補ィ匕合物を接触させない対照と比較して、 前記遺伝子の発現レベルを 低下させるィ匕合物を選択する工程、 (3) a step of selecting a candidate conjugate that reduces the expression level of the gene, compared to a control that does not contact the candidate conjugate.
〔15〕 次の工程を含む、 アトピー性皮膚炎の治療薬のスクリーニング方法であ つて、 指標遺伝子が SK404であるスクリ一二ング方法。  [15] A screening method for a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404.
(1) 指標遺伝子の転写調節領域と、 この転写調節領域の制御下に発現するレ ポーター遺伝子とを含むベタターを導入した細胞と候補ィヒ合物を接触さ せる工程、  (1) contacting a candidate transgenic cell with a cell into which a betater containing the transcriptional regulatory region of the indicator gene and a reporter gene expressed under the control of the transcriptional regulatory region has been introduced;
(2) 前記レポーター遺伝子の活性を測定する工程、 および  (2) measuring the activity of the reporter gene, and
(3) 候補化合物を接触させない対照と比較して、 前記レポ一ター遺伝子の発 現レベルを低下させる化合物を選択する工程、  (3) selecting a compound that reduces the expression level of the reporter gene, as compared to a control not contacting the candidate compound,
〔16〕 次の工程を含む、 アトピー性皮膚炎の治療薬のスクリーニング方法であ つて、 指標遺伝子が SK404、 または SK404と機能的に同等な遺 伝子であるスクリー二ング方法。  [16] 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.
( 1 ) 指標遺伝子によってコードされる蛋白質と候補ィ匕合物を接触させる工程、 (1) contacting the protein encoded by the indicator gene with the candidate conjugate;
(2) 前記蛋白質の活性を測定する工程、 および ( 3 ) 候補ィ匕合物を接触させない対照と比較して、 前記活性を低下させる化合 物を選択する工程 (2) measuring the activity of the protein, and (3) a step of selecting a compound that reduces the activity compared to a control that does not contact the candidate conjugate
〔1 7〕 〔6〕 、 〔1 4〕 、 〔1 5〕 、 および 〔1 6〕 のいずれかに記載のスク リ一ユング方法によって得ることができる化合物を有効成分として含有 する、 アトピー性皮膚炎の治療薬。  [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.
〔1 8〕 指標遺伝子、 またはその一部のアンチセンス DNAを有効成分として含む ァトピー性皮膚炎の治療薬であって、 指標遺伝子が S K 4 0 4である治  [18] 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
〔1 9〕 指標遺伝子によってコードされる蛋白質のアミノ酸配列を含むペプチド を認識する抗体を有効成分とレて含む、 ァトピー性皮膚炎の治療薬であ つて、 指標遺伝子が S K 4 0 4である治療薬。 [19] 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.
あるいは本発明は、 〔6〕 、 〔1 4〕 、 〔1 5〕 および 〔1 6〕 のいずれかに 記載のスクリ一二ング方法によって得ることができる化合物を投与する工程を含 む、 アトピー性皮膚炎の治療方法に関する。 また本発明は、 〔6〕 、 〔1 4〕 、 〔1 5〕 および 〔1 6〕 のいずれかに記載のスクリーニング方法によって得るこ とができる化合物の、 アトピー性皮膚炎の治療用医薬組成物の製造における使用 に関する。  Alternatively, 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
加えて本発明は、 次の成分 (i)または（ii)を投与する工程を含む、 アトピー性 皮膚炎の治療方法に関する。 あるいは本発明は、 次の成分 (i)または (ii)の、 ァ トビ一性皮膚炎の治療用医薬組成物の製造における使用に関する。  In addition, the present invention relates to a method for treating atopic dermatitis, comprising a step of administering the following component (i) or (ii). Alternatively, 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.
(i) S K 4 0 4遺伝子、 またはその一部のアンチセンス DM、  (i) SK404 gene or a part thereof, antisense DM,
(ii) S K 4 0 4遺伝子によってコードされる蛋白質のァミノ酸配列を含むぺプ チドを認識する抗体  (ii) an antibody that recognizes a peptide containing an amino acid sequence of a protein encoded by the SK404 gene
本発明において、 アレルギー性疾患（allergic disease)とはアレルギー反応の 関与する疾患の総称である。 より具体的には、 アレルゲンが同定され、 アレルゲ ンへの曝露と病変の発症に深い結びつきが証明され、 その病変に免疫学的な機序 が証明されることと定義することができる。 ここで、 免疫学的な機序とは、 ァレ ルゲンの刺激によって白血球細胞が免疫応答を示すことを意味する。 アレルゲン としては、 ダュ抗原や花粉抗原等を例示することができる。 In the present invention, 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.
代表的なアレルギー性疾患には、 アトピー性皮膚炎、 気管支喘息、 アレルギー 性鼻炎、 花粉症、 あるいは昆虫アレルギー等を示すことができる。 アレルギー素 因（allergic diathesis)とは、 アレルギー性疾患を持つ親から子に伝えられる遺 伝的な因子である。 家族性に発症するアレルギー性疾患はァトピー性疾患とも呼 ばれ、 その原因となる遺伝的に伝えられる因子がアトピー素因である。 アトピー 性皮膚炎は、 ァトピー性疾患のうち、 特に皮膚炎症状を伴う疾患に対して与えら れた総称である。  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.
本発明において、 アトピー性皮膚炎の指標とすることができる遺伝子を、 指標 遺伝子という。 また指標遺伝子がコードするァミノ酸配列からなる蛋白質を指標 蛋白質という。 指標遺伝子は特に断らない限り、 S K 4 0 4または S K 4 0 4と 機能的に同等な遺伝子を示す用語として用いられる。 本発明における指標遺伝子 の塩基配列、 およびこの塩基配列によってコードされるァミノ酸配列は公知であ る。 ヒト S K 4 0 4の塩基配列おょぴそれによつてコードされるアミノ酸配列を、 配列番号： 1と配列番号： 2にそれぞれ示した。 またマウス S K 4 0 4の塩基配 列およびそれによつてコードされるアミノ酸配列を、 配列番号： 3と配列番号： 4にそれぞれ示した。  In the present invention, 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. Unless otherwise specified, 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.
本発明のァレルギ一性疾患の検査方法は、 被検者の生体試料における指標遺伝 子の発現レベルを測定し、 対照として同じ被検者の無疹部または健常者から採取 された生体試料における指標遺伝子の発現レベルと比較する工程を含む。 比較の 結果、 対照と比較して発現レベルが高い場合に被検者がアトピー性皮膚炎と判定 される。  The method for testing for an allergic disease according to the present invention 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.
発現レベルの比較のためには、 通常、 たとえば健常者における前記指標遺伝子 の発現レベルに基づいて、 標準値が設定される。 この標準値をもとに、 たとえば ± 2 S. D.の範囲が許容範囲とされる。 指標遺伝子の測定値に基づいて、 標準値や 許容範囲を設定する手法は公知である。 あるいは、 同一患者の無疹部との発現レ ベルの比較においては、 予め無疹部における指標遺伝子の発現レベルを測定して、 その患者における無疹部の標準値を決定することができる。 標準値を設定した後 には、 皮疹部の発現レベルのみを測定し、 予め決定されたその患者の無疹部の標 準値との比較に基づいて、 本発明の検査方法を実施することもできる。 For comparison of the expression level, 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. Alternatively, in comparing the expression level with the rash-free area of the same patient, 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.
被検者の指標遺伝子の発現レベルが許容範囲よりも高ければ、 被検者はァトピ 一性皮膚炎であると判定される。 被検者の指標遺伝子の発現レベルが許容範囲内 であれば、 アトピー性皮膚炎である可能性は低いと予想される。  If the expression level of the indicator gene in the subject is higher than the allowable range, 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.
本発明において、 指標遺伝子の発現レベルとは、 該指標遺伝子の mENAへの転 写、 並びに蛋白質への翻訳を含む。 従って本発明によるアトピー性皮膚炎の検查 方法は、 指標遺伝子に対応する mR Aの発現強度、 あるいは該指標遺伝子によつ てコードされる蛋白質の発現レベルの比較に基づいて行われる。  In the present invention, 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.
本発明におけるァトピー性皮膚炎の検查における指標遺伝子の発現レベルの測 定は、 公知の遺伝子解析方法にしたがって実施することができる。 具体的には、 例えばこの遺伝子にハイプリダイズする核酸をプロ一プとしたハイブリダイゼー ション技術、 または本発明の指標遺伝子にハイプリダイズする DNAをプライマ一 とした遺伝子増幅技術等を利用することができる。  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.
本発明め検査に用いられるプローブまたはプライマーは、 指標遺伝子の塩基配 列に基づいてデザインすることができる。 指標遺伝子の塩基配列、 およぴ該指標 遺伝子によってコードされるァミノ酸配列は公知である。  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.
なお一般に高等動物の遺伝子は、 高い頻度で多型を伴う。 また、 スプライシン グの過程で相互に異なるァミノ酸配列からなるアイソフォームを生じる分子も多 く存在する。 多型やアイソフォームによつて塩基配列が異なる遺伝子であつても、 指標遺伝子と同様の活性を持ち、 アトピー性皮膚炎に関与する遺伝子は、 いずれ も本発明の指標遺伝子に含まれる。 また、 本発明において、 指標遺伝子は、 ヒトのみならず、 他種におけるホモ口 グも含む。 従って、 ヒ ト以外の種における指標遺伝子とは、 特に断らないときに は、 その種に固有の指標遺伝子のホモログ、 あるいはその個体に導入されている 外来性の指標遺伝子を言う。 In general, higher animal genes are frequently associated with polymorphisms. Also, there are many molecules that produce isoforms composed of mutually different amino acid sequences during the splicing process. Even if the genes differ in base sequence depending on the polymorphism or isoform, all genes having the same activity as the indicator gene and involved in atopic dermatitis are included in the indicator gene of the present invention. In the present invention, 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.
本発明においてヒト指標遺伝子のホモログとは、 ヒト指標遺伝子をプロープと してストリンジェントな条件下でハイプリダイス 'することができる、 ヒト以外の 種に由来する遺伝子を言う。 ストリンジェントな条件とは、 一般的には以下のよ うな条件を示すことができる。 すなわち、 4X SSC、 65°Cでハイプリダイゼーショ ンさせ、 0. 1 X SSCを用いて 65°Cで 1時間洗浄する。 ストリンジエンシーを大き く左右するハイブリダイゼーションゃ洗浄の温度条件は、 融解温度 (Tm)に応じて 調整することができる。 はハイプリダイズする塩基対に占める構成塩基の割 合、 ハイプリダイゼーシヨン溶液組成 （塩濃度、 ホルムアミドゃドデシル硫酸ナ トリウム濃度） によって変動する。 従って、 当業者であればこれらの条件を考慮 して同等のストリンジエンシーを与える条件を実験または経験的に設定する と ができる。  In the present invention, 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.
プライマーあるいはプローブには、 指標遺伝子の塩基配列からなるポリヌクレ ォチド、 またはその相補鎖に相補的な少なくとも 15ヌクレオチドを含むポリヌ クレオチドを利用することができる。 ここで 「相補鎖」 とは、 A: T (RNAの場合 は U) 、 G:Cの塩基対からなる 2本鎖 DNAの一方の鎖に対する他方の鎖を指す。 また、 「相補的」 とは、 少なくとも 15個の連続したヌクレオチド領域で完全に 相補配列である場合に限られず、 少なくとも 70%、 好ましくは少なくとも 80%、 より好ましくは 90%、 さらに好ましくは 95%以上の塩基配列上の相同性を有すれ ばよい。 塩基配列の相同性は、 BLAST等のアルゴリズムにより決定することがで さる。  As the primer or the probe, a polynucleotide comprising the nucleotide sequence of the indicator gene or a polynucleotide containing at least 15 nucleotides complementary to its complementary strand can be used. Here, 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. Further, 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.
このようなポリヌクレオチドは、 指標遺伝子を検出するためのプロープとして、 また指標遺伝子を増幅するためのプライマーとして利用することができる。 ブラ イマ一として用いる場合には、 通常、 15bp〜； 100bp、 好ましくは 15bp〜35bpの鎖 長を有する。 また、 プローブとして用いる場合には、 指標遺伝子 （またはその相 補鎖） の少なくとも一部若しくは全部の配列を有し、 少なくとも 15bPの鎖長の DNAが用いられる。 プライマーとして用いる場合、 3'側の領域は相捕的である必 要があるが、 5'側には制限酵素認識配列ゃタグなどを付加することができる。 なお、 本発明における 「ポリヌクレオチド」 は、 DNAあるいは RNAであること ができる。 これらポリヌクレオチドは、 合成されたものでも天然のものでもよい。 また、 ハイプリダイゼーシヨンに用いるプローブ DNAは、 通常、 標識したものが 用いられる。 標識方法としては、 例えば次のような方法を示すことができる。 な お用語オリゴヌクレオチドは、 ポリヌクレオチドのうち、 重合度が比較的低いも のを意味している。 オリゴヌクレオチドは、 ポリヌクレオチドに含まれる。 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. When used as a primer, the 3'-side region needs to be complementary, but a restriction enzyme recognition sequence 酵素 tag or the like can be added to the 5'-side. The “polynucleotide” in the present invention can be DNA or RNA. These polynucleotides may be synthetic or natural. The probe DNA used for hybridization is usually labeled. As the labeling method, for example, the following method can be shown. The term oligonucleotide means a polynucleotide having a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides.
• DNAポリメラーゼ Iを用いるニックトランスレーションによる標識  • Labeling by nick translation using DNA polymerase I
•ポリヌクレオチドキナーゼを用いる末端標識  • End labeling using polynucleotide kinase
'クレノーフラグメントによるフィルィン末端標識 (Berger SL, Kimmel AR. (1 987) Guide to Molecular Cloning Techniques, Method in Enzpiology, Acaa emic Press ; Hames BD, Higgins SJ (1985) Genes Probes : A Practical Appr oach. IRL Press ; Sambrook J, Fritsch EF， Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory P ress)  'Filin end labeling with Klenow fragment (Berger SL, Kimmel AR. (1 987) Guide to Molecular Cloning Techniques, Method in Enzpiology, Acaa emic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach. IRL Press; Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press)
•藤ポリメラーゼを用いる転写による標識 (Melton DA, Krieg, PA, Rebagkiat i MR, Maniatis T, Zinn K， Green MR. (1984) Nucleic Acid Res. , 12, 7035- 7056)  Labeling by transcription using wisteria polymerase (Melton DA, Krieg, PA, Rebagkiat i MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12, 7035- 7056)
•放射性同位体を用いない修飾ヌクレオチドを DNAに取り込ませる方法 （Kricka LJ. (1992) Nonisotopic DNA Probing Techniques. Academic Press) ハイブリダイゼーション技術を利用したァトピー性皮膚炎の検査は、 例えば、 ノ—ザンハイプリダイゼ一ンョン法、 ドットプロット法、 DNAマイクロアレイを 用いた方法などを使用して行うことができる。 さらには、 RT - PCR法等の遺伝子 増幅技術を利用することができる。 RT- PCR法においては、 遺伝子の増幅過程に おいて PCR増幅モユタ一法を用いることにより、 本発明の指標遺伝子の発現につ いて、 より定量的な解析を行うことが可能である。 • Incorporation of modified nucleotides without radioisotopes into DNA (Kricka LJ. (1992) Nonisotopic DNA Probing Techniques. Academic Press) Testing for atopic dermatitis using hybridization techniques is, for example, Northern Hypride. Daition method, dot plot method, DNA microarray It can be performed using the method used. Furthermore, gene amplification techniques such as the RT-PCR method can be used. In the RT-PCR method, the expression of the indicator gene of the present invention can be more quantitatively analyzed by using the PCR amplification method in the gene amplification process.
PCR遺伝子増幅モニター法においては、 両端に互いの蛍光を打ち消し合う異な つた蛍光色素で標識したプローブを用い、 検出対象 （DNAもしくは RNAの逆転写 産物） にハイプリダイズさせる。 PCR反応が進んで Taqポリメラ一ゼの 5， -3'ェ キソヌクレアーゼ （exonuclease) 活性により同プローブが分解されると二つの 蛍光色素が離れ、 蛍光が検出されるようになる。 この蛍光の検出をリアルタイム に行う。 検出対象についてコピー数の明らかな標準試料について同時に測定する ことにより、 PCR増幅の直線'性のあるサイクル数で目的試料中の検出対象のコピ 一数を決定する （Holland, P. M. et al.， 1991, Proc. Natl. Acad. Sci. USA 8 8 : 7276-7280； Livak, K. J. et al. , 1995, PCR Methods and Applications 4 (6) :357-362； Heid, C. A. et al. , Genome Research 6 : 986—994; Gibson, E. M. U. et al. , 1996， Genome Research 6:995-1001) 。 PCR増幅モニター法におい ては、 例えば、 ABI PRISM7700 (Applied Biosystems社） を用いることができる。 また本発明のァトピー性皮膚炎の検査方法は、 指標遺伝子によりコードされる 蛋白質を検出することにより行うこともできる。 このような検査方法としては、 例えば、 指標蛋白質に結合する抗体を利用したウェスタンプロッティング法、 免 疫沈降法、 ELISA法などを利用することができる。  In the PCR gene amplification monitoring method, 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). As the PCR proceeds and the probe is degraded by the 5,3 'exonuclease activity of Taq polymerase, 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. USA 88: 7276-7280; Livak, KJ et al., 1995, PCR Methods and Applications 4 (6): 357-362; Heid, CA et al., Genome Research 6: 986-994; Gibson, EMU et al., 1996, Genome Research 6: 995-1001). In the PCR amplification monitoring method, for example, ABI PRISM7700 (Applied Biosystems) can be used. Further, the method for testing atopic dermatitis of the present invention can also be performed by detecting a protein encoded by an indicator gene. As such a test method, 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.
この検出に用いる指標蛋白質に結合する抗体は、 当業者に周知の技法を用いて 得ることができる。 本発明に用いる抗体は、 ポリクローナル抗体、 あるいはモノ クローナル抗体 (Milstein C, et al. , 1983， Nature 305 (5934)： 537-40) であ ることができる。 例えば、 指標蛋白質に対するポリクローナル抗体は、 抗原を感 作した哺乳動物の血液を取り出し、 この血液から公知の方法により血清を分離す る„ ポリクローナル抗体としては、 ポリクローナル抗体を含む血清を使用するこ とができる。 あるいは必要に応じてこの血清からポリクローナル抗体を含む画分 をさらに単離することもできる。 また、 モノクローナル抗体を得るには、 上記抗 原を感作した哺乳動物から免疫細胞を取り出して骨髄腫細胞などと細胞融合させ る。 こうして得られたハイプリドーマをクローニングして、 その培養物から抗体 を回収しモノクローナル抗体とすることができる。 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). For example, 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. In addition, to obtain a monoclonal antibody, 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.
指標蛋白質の検出には、 これらの抗体を適宜標識して用いればよい。 また、 こ の抗体を標識せずに、 該抗体に特異的に結合する物質、 例えば、 プロテイン Aや プロテイン Gを標識して間接的に検出することもできる。 具体的な検出方法とし ては、 例えば、 ELISA法を挙げることができる。  These 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.
抗原に用いる蛋白質もしくはその部分ぺプチドは、 例えば指標遺伝子もしくは その一部を発現ベクターに組込み、 これを適当な宿主細胞に導入して、 形質転換 体を作成し、 該形質転換体を培養して組み換え蛋白質を発現させ、 発現させた組 み換え蛋白質を培養体または培養上清から精製することにより得ることができる。 あるいは、 該遺伝子によってコードされるアミノ酸配列、 あるいは全長 cDNAに よってコードされるァミノ酸配列の部分ァミノ酸配列からなるオリゴぺプチドを 化学的に合成し、 免疫原として用いることもできる。  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. Alternatively, 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.
更に本発明においては、 指標遺伝子の発現レベルのみならず、 生体試料におけ る指標蛋白質の活性を指標として、 ァレルギ一性疾患の検査を行うこともできる。 指標蛋白質の活性とは、 該蛋白質が備える生物学的な活性を言う。  Further, in the present invention, 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.
本発明の検査方法においては、 被検者の皮膚組織を試料とする。 皮膚組織試料 の採取は、 被検者に多少の痛みを伴う。 一方で、 皮膚組織は容易に採取できるた め、 診断材料としては有用である。  In the test method of the present invention, 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.
皮膚組織を採取する方法は公知である。 皮膚組織は、 例えば次のようにして採 取することができる。 すなわち、 まず、 局所麻酔薬により採取部位を麻酔する。 生検個所の周囲の皮膚を引っ張ってたるみの無い状態とした後、 パンチを皮膚の 中に埋め込み、 回転させて標本分の組織をパンチの中に入れる。 パンチを引き出 し、 切り取られたパンチ内の皮膚を回収する。 パンチは、 中空の皮膚組織採取用 の器具である。 たとえば直径 3 mmの皮膚組織の採取が可能な器具が一般に利用 されている。 Methods for collecting skin tissue are known. 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.
本発明において、 皮膚組織とは、 解剖学的には表皮および真皮を含む。 皮膚組 織には、 皮膚固有の細胞のみならず、 リンパ球、 ランゲルハンス細胞、 あるいは マスト細胞などの、 皮膚組織において見出される皮膚以外の細胞が含まれる場合 がある。 これらの皮膚細胞とともに採取される細胞も皮膚組織試料に含まれる。 本発明において、 皮疹部における指標遺伝子の発現レベルを決定するために、 皮疹部の皮膚組織試料が用いられる。 皮疹部とは、 急性病変を形成している皮膚 を言う。 たとえば曰本皮膚科学会誌 104： 1210 (1994) に報告されている診断基 準にしたがって、 急' [4病変部を判定することができる。 具体的には、 たとえば次 のような臨床所見が急性病変部の指標とされている。  In the present invention, 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. In the present invention, 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. For example, according to the diagnostic criteria reported in the Journal of the Dermatological Association 104: 1210 (1994), it is possible to determine a sudden lesion. Specifically, for example, the following clinical findings are used as indicators of acute lesions.
急性病変：紅斑、 湿潤性紅斑、 丘疹、 漿液性丘疹、 鱗屑、 痂皮  Acute lesions: erythema, wet erythema, papules, serous papules, scales, crusts
また患者の無疹部における指標遺伝子の発現レベルを決定するために、 無疹部 の皮膚組織試料が用いられる。 患者における無疹部とは、 上記病変を伴わない部 位の皮膚である。 更に、 健常者の皮膚における指標遺伝子の発現レベルを決定す るには、 健常者の皮膚組織が用いられる。 本発明における健常者とは、 アレルギ 一性疾患を有していないことが明らかなヒトを言う。 皮膚組織の採取は、 患者の 皮膚組織の採取と同様の手法によって採取することができる。 指標遺伝子の発現 レベルの比較においては、 できるだけ同じ部位の皮膚を比較するのが望ましい。 しかし同一患者の皮疹部 無疹部の比較においては、 同じ部位に無疹部の皮膚を 見出すのが困難であるケースも予想される。 このようなケースでは、 異なる部位 の皮膚を比較に用いることもできる。  To determine the expression level of the indicator gene in the non-rash area of the patient, 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. Further, to determine the expression level of the indicator gene in the skin of a healthy subject, 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. When comparing the expression levels of indicator genes, it is desirable to compare the skin at the same site as much as possible. However, when comparing the eruption area and the eruption area of the same patient, it may be difficult to find the eruption skin at the same site. In such cases, skin from different sites can be used for comparison.
皮膚組織試料の採取は容易であるため、 ベッドサイドにおける簡便な検査が可 能である。 例えば、 実施例に示す方法によって調製することができる。 調製され た皮膚組織を破壊してライセートとすれば、 指標蛋白質の免疫学的な測定のため の試料とすることができる。 Since 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.
上記の生体試料からライセートを調製すれば、 指標蛋白質の免疫学的な測定の ための試料とすることができる。 あるいはこのライセートから mRNAを抽出すれ ば、 指標遺伝子に対応する mRNAの測定のための試料とすることができる。 生体 試料のライセートまたは mRNAの抽出には、 市販のキットを利用すると便利であ る。 また指標蛋白質が血中に分泌されていれば、 被検者の血液や血清などの体液 試料に含まれる目的とする蛋白質の量を測定することによって、 それをコードす る遺伝子の発現レベルの比較が可能である。 上記試料は、 必要に応じて緩衝液等 で希釈して本発明の方法に使用することができる。  If a lysate is prepared from the above biological sample, it can be used as a sample for immunological measurement of the indicator protein. Alternatively, if mRNA is extracted from this lysate, it can be used as a sample for measuring mRNA corresponding to the indicator gene. For extraction of lysate or mRNA from biological samples, it is convenient to use commercially available kits. If 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.
mRNAを測定する場合には、 本発明における指標遺伝子の発現レベルの測定値 は、 公知の方法によって補正することができる。 補正により、 細胞における遺伝 子の発現レベルの変化を比較することができる。 測定値の補正は、 上記生体試料 における各細胞において、 発現レベルが大きく変動しない遺伝子 （例えば、 ハウ スキーピング遺伝子） の発現レベルの測定値に基づいて、 本発明において指標遺 伝子の発現レベルの測定値を補正することによって行われる。 発現レベルが大き く変動しない遺伝子の例としては、 β -ァクチン、 GAPDH等を挙げることができ る。  When measuring mRNA, 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 β-actin, GAPDH and the like.
更に本発明は、 本発明の検査方法のための試薬を提供する。 すなわち本発明は、 指標遺伝子の塩基配列を含むポリヌクレオチド、 またはその相補鎖に相補的な塩 基配列を有する少なくとも 1 5塩基の長さを有するオリゴヌクレオチドからなる、 アトピー性皮膚炎の検査用試薬に関する。 あるいは本発明は、 指標蛋白質のアミ ノ酸配列を含むぺプチドを認識する抗体からなる、 ァトピー性皮膚炎の検査用試 薬に関する。 本発明の試薬を構成するオリゴヌクレオチドや抗体は、 アツセィフ ォーマツトに応じて適当な標識を結合することができる。 あるいは本発明の試薬 を構成するオリゴヌクレオチドや抗体は、 アツセィフォーマツトに応じて適当な 支持体に固定ィ匕しておくこともできる。 また本発明の試薬は、 前記オリゴヌタレ ォチドまたは前記抗体の他に、 検査や保存に必要な付加的な要素と組み合せて検 查用キットとすることもできる。 キットを構成することができる付加的な要素を、 以下に示す。 これらの要素は、 必要に応じて予め混合しておくこともできる。 ま た、 必要に応じて、 保存剤や防腐剤を各要素に加えることができる。 Further, 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. Alternatively, 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. Alternatively, the oligonucleotides and antibodies constituting the reagent of the present invention can be immobilized on an appropriate support depending on the format. In addition, the reagent of the present invention is characterized in that: In addition to the peptide or the antibody, 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.
試薬や生体試料を希釈するための緩衝液  Buffer for diluting reagents and biological samples
陽性対照  Positive control
陰性対照  Negative control
標識を測定するための基質  Substrates for measuring labels
反応容器  Reaction vessel
アツセィプロトコルを記載した指示書  Instructions describing Atsushi protocol
本発明における指標遺伝子は、 アトピー性皮膚炎患者の皮疹部と同一の患者に おける無疹部との比較において、 各皮膚組織において発現量の変動が確認された。 従って、 指標遺伝子の発現レベルを指標として、 アトピー性皮膚炎の検査を行う ことができる。  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.
本発明におけるァトピー性皮膚炎の検査とは、 たとえば以下のような検査が含 まれる。 アトピー性皮膚炎が疑われる症状を示しながら、 一般的な検査ではアト ピー性皮膚炎と判定できない患者であっても、 本発明に基づく検査を行えばァト ピー性皮膚炎の患者であるか否かを容易に判定することができる。 より具体的に は、 アトピー性皮膚炎が疑われる症状を示す患者において、 指標遺伝子の発現の 上昇は、 その症状の原因がァトピー性皮膚炎である可能性が高いことを示してい る。  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.
あるいは、 アトピー '性皮膚炎が改善に向かっているのかどうかを判断するため の検査が可能となる。 つまり、 アトピー性皮膚炎に対する治療効果の判定に有用 である。 たとえばアトピー性皮膚炎と診断された患者において、 指標遺伝子の発 現の上昇は、 ァトピー 皮膚炎がさらに進行している可能性が高いことを示して いる。 更に、 発現レベルの違いに基づいてァトピー性皮膚炎の重症度を判定すること もできる。 すなわち、 指標遺伝子の発現の上昇の程度は、 アトピー性皮膚炎の重 症度に相関する。 Alternatively, tests can be done to determine if atopic dermatitis is improving. In other words, it is useful for judging the therapeutic effect on atopic dermatitis. For example, in patients diagnosed with atopic dermatitis, increased expression of the indicator gene indicates that atopic dermatitis is likely to be more advanced. Further, 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.
また本発明は、 指標遺伝子または指標遺伝子と機能的に同等な遺伝子の、 皮膚 における発現強度を上昇させたトランスジエニック非ヒト動物からなるアトピー 性皮膚炎モデル動物に関する。  Further, 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.
本発明によって、 指標遺伝子の発現強度が、 アトピー性皮膚炎患者の皮疹部に おいて上昇することが明らかとなった。 したがって、 皮膚において、 指標遺伝子 または指標遺伝子と機能的に同等な遺伝子の発現レベルを人為的に増強した動物 は、 ァトピー性皮膚炎のモデル動物として利用することができる。  According to the present invention, it has been clarified that 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.
本発明において機能的に同等な遺伝子とは、 指標遺伝子によってコードされる 蛋白質において明らかにされている活性と同様の活性を備えた蛋白質をコードす る遺伝子である。 機能的に同等な遺伝子の代表的なものとしては、 被験動物が本 来備えている、 その動物種における指標遺伝子のカウンターパートを挙げること ができる。 たとえばマウス S K 4 0 4は、 マウスにおける機能的に同等な遺伝子 である。 マウス S K 4 0 4の塩基配列は onz inという名称で登録されているが、 機能的な情報は伴っていない。 マウス S K 4 0 4遺伝子は、 本発明に基づくスク リーユングをマウスを用いて実施するときに望ましい指標遺伝子である。 マウス In the present invention, 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. For example, 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. mouse
S K 4 0 4は、 感作マゥス耳介皮膚と非感作マゥス耳介皮膚における遺伝子の発 現レベルを比較したときに、 2倍以上の差が見られた遺伝子である。 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.
したがって、 マウス指標遺伝子の発現レベルの調節や、 投与によって、 アトピ 一性皮膚炎モデル動物を作り出すことができる。 すなわち本発明は、 マウス指標 遺伝子の発現レベルの調節による、 ァトピー性皮膚炎モデル動物の製造方法に関 する。 あるいは本発明は、 マウス指標遺伝子によってコードされるタンパク質そ のものの投与による、 アトピー性皮膚炎モデル動物の製造方法に関する。  Therefore, 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.
本発明のマウス指標遺伝子は、 発現レベルの上昇によってアトピー性皮膚炎を 誘導することができる。 あるいはこの遺伝子や、 その遺伝子がコードするタンパ ク質の投与によって、 アトピー性皮膚炎モデル動物を作り出すことができる。 こ れらのカウンターパートはいずれもマウスの遺伝子であることから、 遺伝子ゃタ ンパク質を投与する場合には、 マウスに投与するのが望ましい。 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.
該ァトピー性皮膚炎モデル動物は、 ァトピー性皮膚炎における生体内の変化を 明らかにするために有用である。 更に、 該アトピー性皮膚炎モデル動物を使用す ることにより、 指標遺伝子のさらなる機能を解明すること、 およぴ該遺伝子を標 的とする薬剤を評価することには大きな意義がある。  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.
また本発明によるアトピー性皮膚炎モデル動物は、 アトピー性皮膚炎のメカ二 ズムの解明、 さらにはスクリ一ユングされた化合物の安全性の試験に有用である。 たとえば本発明によるアトピー性皮膚炎モデル動物が皮膚炎を発症したり、 何ら 力のァレルギ一性疾患に関連した測定値の変化を示せば、 それを回復させる作用 を持つた化合物を探索するスクリーニングシステムが構築できる。  In addition, 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.
本発明において、 発現レベルの上昇とは、 指標遺伝子が外来遺伝子として導入 され強制発現している状態、 あるいは被験動物が本来備えている指標遺伝子の転 写と蛋白質への翻訳が増強されている状態、 並びに翻訳産物である蛋白質の分解 が抑制された状態のいずれかを意味する。  In the present invention, 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.
遺伝子の発現レベルは、 たとえば実施例に示すような DNAチップにおけるシグ ナル強度の差により確認することができる。 また翻訳産物である蛋白質の活性は、 正常な状態と比較することにより確認することができる。  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.
代表的なトランスジエニック動物は、 指標遺伝子を導入し強制発現させた動物 等を示すことができる。 その他、 たとえば指標遺伝子のコード領域に変異を導入 し、 その活性を増強したり、 あるいは分解されにくいアミノ酸配列に改変した動 物などを示すことができる。 アミノ酸配列の変異として、 置換、 欠失、 挿入、 あ るいは付加を示すことができる。 その他、 遺伝子の転写調節領域を変異させるこ とにより、 本発明の指標遺伝子の発現そのものを調節することもできる。 特定の遺伝子を対象として、 トランスジエニック動物を得る方法は公知である。 すなわち、 遺伝子と卵を混合してリン酸カルシウムで処理する方 や、 位相差顕 微鏡下で前核期卵の核に、 微小ピペットで遺伝子を直接導入する方法 （マイクロ インジェクション法、 米国特許第 4873191号） 、 胚性幹細胞 （ES細胞） を使用 する方法などによってトランスジエニック動物を得ることができる。 その他、 レ トロウィルスベクターに遺伝子を揷入し、 卵に感染させる方法、 また、 ***を介 して遺伝子を卵に導入する***ベクター法等も開発されている。 ***ベクター法 とは、 ***に外来遺伝子を付着またはエレクトロポレーシヨン等の方法で***細 胞内に取り込ませた後に、 卵子に受精させることにより、 外来遺伝子を導入する 遺伝子組換え法である （M. Lavitranoet ら Cell, 57， 717， 1989) 。 Representative transgenic animals include animals into which an indicator gene has been introduced and forcibly expressed. In addition, for example, 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. In addition, 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. That is, a method in which a gene and an egg are mixed and treated with calcium phosphate, or a method in which a gene is directly introduced into a nucleus of a pronuclear stage egg with a micropipette under a phase contrast microscope (microinjection method, US Pat. No. 4,873,191). Transgenic animals can be obtained by methods using embryonic stem cells (ES cells). In addition, 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).
発現ベクターに使用するプロモーターとして、 適当な薬剤等の物質により転写 が調節されるプロモーターを用いれば、 該物質の投与によってトランスジェュッ ク動物における外来性の指標遺伝子の発現レベルを調整することができる。  When a promoter whose transcription is regulated by a substance such as an appropriate drug is used as the promoter used in the expression vector, the expression level of the exogenous indicator gene in the transgenic animal can be adjusted by administering the substance.
本発明のアトピー性皮膚炎のモデル動物として用いるトランスジエニック動物 は、 ヒ ト以外のあらゆる脊椎動物を利用して作成することができる。 具体的には、 マウス、 ラット、 ゥサギ、 ミニプタ、 ャギ、 ヒッジ、 あるいはゥシ等の脊椎動物 において様々な遺伝子の導入や発現レベルを改変されたトランスジェニック動物 が作り出されている。  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.
さらに本発明は、 ァトピー性皮膚炎治療薬候補化合物のスクリ一ユング方法に 関する。 本発明において、 指標遺伝子は S K 4 0 4である。 S K 4 0 4遺伝子は、 ァトピ一性皮膚炎患者の無疹部に比較して同一の患者の皮疹部において有意に発 現レベルが上昇している。 また S K 4 0 4遺伝子は、 健常者と比較してアトピー 性皮膚炎患者の皮疹部において有意に発現レベルが上昇している。  Further, the present invention relates to a method for screening a candidate compound for a therapeutic agent for atopic dermatitis. In the present invention, 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.
したがって、 前記指標遺伝子の発現レベルを低下させることができる化合物を 選択することによって、 アトピー性皮膚炎の治療薬を得ることができる。  Therefore, a therapeutic agent for atopic dermatitis can be obtained by selecting a compound that can reduce the expression level of the indicator gene.
本発明において遺伝子の発現レベルを低下させる化合物とは、 遺伝子の転写、 翻訳、 およおぴ蛋白質の活性発現のいずれかのステップに対して抑制的に作用す る作用を持つ化合物である。 In the present invention, 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.
本発明のアレルギー性疾患治療候補ィヒ合物のスクリーニング方法は、 in vivo で行うことも ώ vitroで行うこともできる。 このスクリ一ユングは、 例えば以 下のような工程にしたがって実施することができる。  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.
( 1 ) 被験動物に、 候補化合物を投与する工程、  (1) administering a candidate compound to a test animal,
( 2 ) 前記被検動物の生体試料における指標遺伝子の発現強度を測定する工程、 (2) measuring the expression intensity of the indicator gene in the biological sample of the test animal,
( 3 ) 候補化合物を接触させない対照と比較して、 前記遺伝子の発現レベルを低 下させる化合物を選択する工程 (3) a step of selecting a compound that reduces the expression level of the gene as compared to a control not contacted with a candidate compound
本発明のスクリ一二ング方法において、 指標遺伝子としては、 S K 4 0 4遺伝 子と機能的に同等な遺伝子を利用することができる。 本発明において機能的に同 等な遺伝子とは、 指標遺伝子によってコードされる蛋白質において明らかにされ ている活性と同様の活性を備えた蛋白質をコードする遺伝子である。 機能的に同 等な遺伝子の代表的なものとしては、 被験動物が本来備えている、 その動物種に おける指標遺伝子の力ゥンターパ一トを挙げることができる。  In the screening method of the present invention, a gene functionally equivalent to the SK404 gene can be used as the indicator gene. In the present invention, 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.
本発明のスクリ一ユング方法における被験動物としては、 例えばァトピー性皮 膚炎モデル動物を利用することができる。 アトピー性皮膚炎モデル動物は公知で ある。 たとえば人間のァトピ一性皮膚炎に近いモデルとして、 NC/Ngaマウスを 用いた皮膚炎自然発症モデルが報告されている。 このマウスの耳介にダニ抗原 As a test animal in the screening method of the present invention, for example, an atopic dermatitis model animal can be used. Atopic dermatitis model animals are known. For example, 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
( 5 /z g/耳） を 2 - 3日間隔で計 8回投与すると、 2週間以降にはヒトのアトピー 性皮膚炎に酷似した症状を誘発することができる。 この系に候補化合物を投与し、 本発明の指標遺伝子の発現レベルの変化を追跡することによって本発明によるス クリ一ユングを実施することができる。 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.
このようにして被験動物に薬剤候補ィ匕合物を接触させ、 被験動物由来の生体試 料における指標遺伝子の発現に対する化合物の作用をモニターすることにより、 指標遺伝子の発現レベルに与える薬剤候補ィ匕合物の影響を評価することができる。 被験動物の由来の生体試料における指標遺伝子の発現レベルの変動は、 前記本発 明の検査方法と同様の手法によってモニターすることができる。 更にこの評価の 結果に基づいて、 指標遺伝子の発現レベルを低下させる薬剤候補化合物を選択す れば、 薬剤候補化合物をスクリーニングすることができる。 In this way, by contacting the test animal with the drug candidate conjugate and monitoring the effect of the compound on the expression of the indicator gene in the biological sample derived from the test animal, 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.
より具体的には、 被験動物から、 皮膚組織試料を採取し、 指標遺伝子の発現レ ベルを候補化合物を接触させない対照と比較することにより、 本発明によるスク リーユングを実施することができる。 皮膚組織試料の採取方法、 および調製方法 は公知である。  More specifically, 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.
このようなスクリ一ユングにより、 指標遺伝子の発現に様々な形で関与する薬 剤を選択することができる。 具体的には、 たとえば次のような作用を持つ薬剤候 補化合物を見出すことができる。  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.
•指標遺伝子の発現をもたらすシグナル伝達経路の抑制  • Suppression of signaling pathways that lead to expression of indicator genes
•指標遺伝子の転写活 '性の抑制  • Repression of transcription activity of indicator gene
•指標遺伝子の転写産物の安定化阻害もしくは分解の促進等  • Inhibition of stabilization or degradation of indicator gene transcripts
また、 in りでのスクリーニングにおいては、 例えば、 指標遺伝子を発現 する細胞に候補化合物を接触させ、 指標遺伝子の発現レベルを低下させる化合物 を選択する方法が挙げられる。 このスクリーニングは、 例えば以下のような工程 に従って実施することができる。  In the screening in vitro, for example, there is a method in which 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.
( 1 ) 指標遺伝子を発現する細胞に候補ィヒ合物を接触させる工程  (1) a step of contacting a candidate Eik compound with cells expressing the indicator gene
( 2 ) 前記指標遺伝子の発現レベルを測定する工程、  (2) measuring the expression level of the indicator gene,
( 3 ) 候補化合物を接触させない対照と比較して、 指標遺伝子の発現レベルを低 下させる化合物を選択する工程  (3) Step of selecting a compound that reduces the expression level of the indicator gene as compared to a control not contacted with the candidate compound
本発明において、 指標遺伝子を発現する細胞は、 指標遺伝子を適当な発現べク ターに挿入し、 該ベクターを適当な宿主細胞に導入することにより得ることがで きる。 利用できるベクター、 および宿主細胞は、 本発明の指標遺伝子を発現し得 るものであればよい。 宿主一ベクター系における宿主細胞としては、 大腸菌、 酵 母、 昆虫細胞、 動物細胞等が例示でき、 それぞれ利用できるベクタ一を適宜選択 することができる。 In the present invention, 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.
ベクターの宿主への導入方法としては、 生物学的方法、 物理的方法、 化学的方 法などを示すことができる。 生物学的方法としては、 例えば、 ウィルスベクター を使用する方法、 特異的受容体を利用する方法、 細胞融合法 (HVJ (センダイゥ ィルス）、 ポリエチレングリコール （PEG) 、 電気的細胞融合法、 微少核融合法 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
(染色体移入） ) が挙げられる。 また、 物理的方法としては、 マイクロインジェ クシヨン法、 エレクトロポレーシヨン法、 ジーンパーティクルガン （gene gun) を用いる方法が挙げられる。 化学的方法としては、 リン酸カルシウム沈殿法、 リ ポソーム法、 DEAEデキストラン法、 プロトプラスト法、 赤血球ゴースト法、 赤 血球膜ゴースト法、 マイクロカプセル法が挙げられる。 (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.
本発明のスクリ一二ング方法においては、 指標遺伝子を発現する細胞として、 皮膚細胞に加えて、 ランゲルハンス細胞、 マスト細胞、 T細胞、 好酸球、 B細胞、 好中球、 あるいは好塩基球等の皮膚組織に見出される細胞を用いることができる。 ヒト表皮角化細胞 （ケラチノサイト） の初代培養細胞 HEK (Normal Human Epi dermal Keratinocyte)に対して TGF - /3あるいは sodium butyrateにより刺激を 加えることにより分化を誘導することができるという報告がある （例えば Geng Wangら、 EXPERIMENTAL CELL RESEARCH 198, 27-30 (1992) )0 分化に伴い cornif i ed envelope (CE)という細胞内構造物が形成される。 CEの形成あるいは CE構成 分子 (involucrin, loricrinなど）の遺伝子発現を指標として分化を確認するこ とができる。 こうして分化した細胞は、 本発明におけるスクリーニングに有用で あ o。 In the screening method of the present invention, as 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. There is a report that 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.
皮膚組織に見出される細胞として、 皮膚細胞、 T細胞、 好酸球、 マスト細胞、 好塩基球、 B細胞、 ランゲルハンス細胞、 好中球を利用することもできる。 株ィ匕 皮膚細胞は、 均質な細胞を大量に入手できること、 培養が容易なことなどの点で、 本発明のスクリーニング方法に好適である。 以下に本発明に利用できる皮膚細胞 株の例を示す。 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.
—株化皮膚細胞： HaCaT， A431 (ATCC CRL-1555)  —Established skin cells: HaCaT, A431 (ATCC CRL-1555)
一株化 T細胞： Jurkat (ATCC TIB- 152), Molt- 4 (ATCC CRL-1582) , H9 (ATCC HTB-1 76) One cell line T cell: Jurkat (ATCC TIB-152), Molt-4 (ATCC CRL-1582), H9 (ATCC HTB-176)
一株化好酸球： AML14. 3D10 Single eosinophil: AML14. 3D10
—株化マスト細胞： HMC - 1  —Mast cell line: HMC-1
—株化好塩基球： KU- 812  —Basic basophils: KU-812
一株化 B細胞： DND39， Raji (ATCC CCL-86) One cell line B cell: DND39, Raji (ATCC CCL-86)
一 （株化） ランゲルハンス細胞： MUTZ- 3 I (Established) Langerhans cells: MUTZ-3
一 （株化） 好中球： HL- 60 (ATCC CCL-240) Neutrophil: HL-60 (ATCC CCL-240)
株化に力ッコをつけたものは、 使用に先だつて上記株ィヒ細胞から分ィ匕させる。 ランゲルハンス細胞（Allan J. Masterson ら Blood 2002 Jul 15; 100(2) :701— 3)、 あるいは好中球（Santos - Beneit AMら J Leukoc Biol 2000 May ;67 (5) :712- 24) の分化は公知である。  Those with emphasis on the establishment are separated from the above-mentioned strain cells prior to use. Differentiation of Langerhans cells (Allan J. Masterson et al. Blood 2002 Jul 15; 100 (2): 701-3) or neutrophils (Santos-Beneit AM et al. J Leukoc Biol 2000 May; 67 (5): 712-24) Is known.
スクリーニングの方法は、 まず前記株化皮膚細胞に候補化合物を接触させる。 その後、 該株化皮膚細胞における指標遺伝子の発現レベルを測定し、 侯補化合物 を接触させない対照と比較して、 指標遺伝子の発現レベルを低下させる化合物を 選択する。  In the screening method, first, 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.
なお本発明のスクリーニング方法において、 指標遺伝子の発現レベルは、 該遺 伝子がコードする蛋白質の発現レベルのみならず、 対応する mRNAを検出するこ とにより比較することもできる。 mRNA によって発現レベルを比較するためには、 蛋白質試料の調製工程に代えて、 先に述べたような mRNA試料の調製工程を実施 する。 mRNAや蛋白質の検出は、 先に述べたような公知の方法によって実施する ことができる。  In the screening method of the present invention, 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. In order to compare expression levels by 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.
さらに本発明の開示に基づいて本発明の指標遺伝子の転写調節領域を取得し、 レポーターアツセィ系を構築することができる。 レポーターアツセィ系とは、 転 写調節領域の下流に配置したレポーター遺伝子の発現量を指標として、 該転写調 節領域に作用する転写調節因子をスタリー二ングするアツセィ系をいう。 Further, based on the disclosure of the present invention, 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.
すなわち本発明は、 次のェ麁を含む、 アトピー性皮膚炎の治療薬のスクリー二 ング方法であって、 指標遺伝子が S K 4 0 4遺伝子、 または S K 4 0 4遺伝子と 機能的に同等な遺伝子である方法に関する。  That is, 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.
( 1 ) 指標遺伝子の転写調節領域と、 この転写調節領域の制御下に発現するレポ 一ター遺伝子を含むべクターを導入した細胞と候補ィ匕合物を接触させるェ 程、  (1) contacting a cell into which a vector containing a transcriptional regulatory region of an indicator gene and a reporter gene expressed under the control of this transcriptional regulatory region has been introduced, and the candidate conjugate;
( 2 ) 前記レポーター遺伝子の活性を測定する工程、 および  (2) measuring the activity of the reporter gene, and
( 3 ) 候補ィヒ合物を接触させない対照と比較して、 前記レポーター遺伝子の発現 レベルを低下させる化合物を選択する工程  (3) a step of selecting a compound that reduces the expression level of the reporter gene as compared to a control that does not come into contact with the candidate Eich compound
転写調節領域としては、 プロモーター、 ェンハンサー、 さらには、 通常プロモ —ター領域に見られる CMTボックス、 TATAボックス等を例示することができる。 またレポーター遺伝子としては、 CAT (chloramphenicol acetyl transferase) m 伝子、 ルシフェラーゼ（lucif erase)遺伝子、 成長ホルモン遺伝子等を利用するこ とができる。  Examples of the transcription control region include a promoter, an enhancer, and a CMT box, a TATA box and the like usually found in a promoter region. Further, as the reporter gene, CAT (chloramphenicol acetyl transferase) m gene, luciferase (lucif erase) gene, growth hormone gene and the like can be used.
あるいは本発明における指標遺伝子の転写調節領域を、 次のようにして取得す ることもできる。 すなわち、 まず本発明で開示した cDNAの塩基配列に基づいて、 BACライブラリー、 YACライブラリ一等のヒトゲノム DNAライプラリーから、 PCR またはハイプリダイゼーションを用いる方法によりスクリーニングを行レ、、 該 c DNAの配列を含むゲノム DNAクローンを得る。 得られたゲノム DNAの配列を基に、 本発明で開示した cDNAの転写調節領域を推定し、 該転写調節領域を取得する。 得られた転写調節領域を、 レポーター遺伝子の上流に位置するようにクローニン グしてレポーターコンストラクトを構築する。 得られたレポーターコンストラク トを培養細胞株に導入してスクリーニング用の形質転換体とする。 この形質転換 体に候捕化合物を接触させ、 候補ィヒ合物を接触させない対照と比較して、 前記レ ポーター遺伝子の発現レベルを低下させるィ匕合物を選択することにより本発明の スクリーニングを行うことができる。 Alternatively, 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.
in vitroでの本発明によるスクリ一ユング方法として、 指標蛋白質の活性に 基づくスクリーニング方法を利用することもできる。 すなわち本発明は、 次のェ 程を含む、 アトピー性皮膚炎の治療薬のスクリーニング方法であって、 指標遺伝 子が S K 4 0 4遺伝子、 または S K 4 0 4遺伝子と機能的に同等な遺伝子である 方法に関する。  As an in vitro screening method according to the present invention, 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.
( 1 ) 指標遺伝子によってコードされる蛋白質と候補ィ匕合物を接触させる工程、 (1) contacting the protein encoded by the indicator gene with the candidate conjugate;
( 2 ) 前記蛋白質の活性を測定する工程、 および (2) measuring the activity of the protein, and
( 3 ) 候補ィヒ合物を接触させない対照と比較して、 前記活性を低下させる化合物 を選択する工程  (3) a step of selecting a compound that reduces the activity as compared to a control not contacted with the candidate compound
本発明における指標蛋白質が有する活性を指標として、 その活性を阻害する活 性を有する化合物をスクリーニングすることができる。 このようにして得ること ができる化合物は、 指標遺伝子の働きを抑制する。 その結果、 皮膚において発現 が誘導された指標蛋白質の阻害を通じて、 ァトピー性皮膚炎を制御することがで 含る。  Using 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. As a result, it is possible to control atopic dermatitis through inhibition of an indicator protein whose expression is induced in the skin.
これらスクリーニングに用いる被験候補物質としては、 ステロイド誘導体等既 存の化学的方法により合成された化合物標品、 コンビナトリァルケミストリーに より合成された化合物標品のほか、 動 ·植物組織の抽出物もしくは微生物培養物 等の複数の化合物を含む混合物、 またそれらから精製された標品などが挙げられ る。  The 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.
本発明による各種のスクリーニング方法に必要な、 ポリヌクレオチド、 抗体、 細胞株、 あるいはモデル動物は、 予め組み合わせてキットとすることができる。 これらのキットには、 標識の検出に用いられる基質化合物、 細胞の培養のための 培地や容器、 陽性や陰性の標準試料、 更にはキットの使用方法を記載した指示書 等をパッケージしておくこともできる。 本発明のスクリーニング方法によって選択される化合物は、 アトピー性皮膚炎 の治療薬として有用である。 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.
さらに、 指標遺伝子によってコードされる蛋白質のアミノ酸配列を含むぺプチ ドを認識する抗体も、 アトピー性皮膚炎の治療薬として有用である。 指標遺伝子 は、 アトピー性皮膚炎患者の皮疹部において発現が上昇する遺伝子である。 した がってこれらの遺伝子の発現、 あるいはこれら遺伝子によってコードされる蛋白 質の機能を抑制することによって、 アトピー性皮膚炎の治療効果を期待すること ができる。  Furthermore, 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.
本発明のアレルギー性疾患の治療薬は、 スクリーニング方法によって選択され た化合物を有効成分として含み、 生理学的に許容される担体、 賦形剤、 あるいは 希釈剤等と混合することによつて製造することができる。 本発明のァレルギ一性 疾患の治療剤は、 アレルギー症状の改善を目的として、 経口、 あるいは非経口的 に投与することができる。  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.
経口剤としては、 顆粒剤、 散剤、 錠剤、 カプセル剤、 溶剤、 乳剤、 あるいは懸 濁剤等の剤型を選択することができる。 注射剤には、 皮下注射剤、 筋肉注射剤、 あるいは腹腔内注射剤等を示すことができる。  As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, and suspensions can be selected. Injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections.
また、 投与すべき化合物がタンパク質からなる場合には、 それをコードする遺 伝子を遺伝子治療の手法を用いて生体に導入することにより、 治療効果を達成す ることができる。 治療効果をもたらすタンパク質をコードする遺伝子を生体に導 入し、 発現させることによって、 疾患を治療する手法は公知である。  When the compound to be administered consists of a protein, 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.
投与量は、 患者の年齢、 性別、 体重および症状、 治療効果、 投与方法、 処理時 間、 あるいは該医薬組成物に含有される活性成分の種類などにより異なるが、 通 常成人一人あたり、 一回につき 0. 1 mgから 500 mgの範囲で、 好ましくは 0. 5 m gから 20 mgの範囲で投与することができる。 し力 し、 投与量は種々の条件によ り変動するため、 上記投与量よりも少ない量で充分な場合もあり、 また上記の範 囲を超える投与量が必要な場合もある。 なお本明細書において引用された全ての先行技術文献は、 参照として本明細書 に組み入れられる。 以下、 本発明を実施例に基づいてより具体的に説明する。 図面の簡単な説明 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. Can be administered in the range of 0.1 mg to 500 mg, preferably in the range of 0.5 mg to 20 mg. However, since the dose varies depending on various conditions, a smaller dose may be sufficient in some cases, or a dose exceeding the above range may be necessary. All prior art documents cited in the present specification are incorporated herein by reference. Hereinafter, the present invention will be described more specifically based on examples. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 TARC遺伝子の患者皮膚組識における発現解析 （GAPDH補正） の結果を 表わすグラフである。 縦軸は遺伝子の発現強度 (copy/5ng total R A)、 横軸は試 料の種類を示す。  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.
図 2は、. CCR4遺伝子の患者皮膚組識における発現解析 （GAPDH補正） の結果を 表わすグラフである。 縦軸と横軸は図 1と同じ。  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.
図 3は、 RANTES遺伝子の患者皮膚組織における発現解析 （GAPDH補正） の結果 を表わすグラフである。 縦軸と横軸は図 1と同じ。  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.
図 4は、 CXCR3遺伝子の患者皮膚糸且識における発現解析 （GAPDH補正） の結果 を表わすグラフである。 縦軸と横軸は図 1と同じ。  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.
図 5は、 SK404のァトピー性皮膚炎患者および健常者皮膚組織における発現解 析の結果を表わすグラフである。 縦軸と横軸は図 1と同じ。  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.
図 6は、 ヒト SK404 (上） とマウス SK404M (下） のアミノ酸配列を比較した図 である。 Iはアミノ酸の一致、 . は類似のアミノ酸への置換、 そして：はより類 似度の高いアミノ酸への置換、 を意味する。 アミノ酸配列中のドット （· ) はギ ャップを示す。  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.
図 7は、 SK40 Mの NcNgaマウスモデルの耳介皮膚における発現解析 (GAPDH捕 正） の結果を表わすグラフである。 縦軸は遺伝子の発現強度 (copy/5ng total RN A)、 横軸は NcNgaモデルマウスの条件を示す。  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.
図 8は、 SK404遺伝子のヒト組織における発現を表わすグラフである。  FIG. 8 is a graph showing the expression of the SK404 gene in human tissues.
図 9は、 SK404遺伝子の血球細胞における発現を表わすグラフである。 健常人 (5名） 由来の血球細胞を用いた。  FIG. 9 is a graph showing the expression of the SK404 gene in blood cells. Blood cells from healthy persons (5) were used.
図 1 0は SK404の situ hybridization解析を表す写真である。 図 1 1はアトピー性皮膚炎と乾癬患者皮膚組織における SK404遺伝子の発現解 祈を表すグラフである。 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.
図 1 2は SK404蛋白質の発現を表す図おょぴ写真である。  FIG. 12 is a photograph showing the expression of SK404 protein.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
〔実施例 1〕 アトピー性皮膚炎患者の皮膚組織における TARC、 CCR4、 RANTES、 C XCR3遺伝子の発現解析  [Example 1] Expression analysis of TARC, CCR4, RANTES, C XCR3 gene in skin tissue of atopic dermatitis patient
ァトピー性皮膚炎患者の皮疹部ではケモカイン TARCの高発現や、 そのケモカ インレセプタ一 CCR4を発現する Th2細胞の皮膚組織浸潤が報告されており、 こ のようなリンパ球の浸潤が病態形成の一因であることが明らかになつている。 このような報告に基づき、 リンパ球の遊走に重要な役割を果たしているケモカ イン （TARC, RANTES) およびリンパ球上に発現しているケモカインレセプター In the skin eruption area of atopic dermatitis patients, high expression of the chemokine TARC and infiltration of Th2 cells, which express the chemokine receptor CCR4, into skin tissues have been reported. It is clear that it is. Based on these reports, chemokines (TARC, RANTES) play important roles in lymphocyte migration and chemokine receptors expressed on lymphocytes
(CCR4, CXCR3)についてァトピー性皮膚炎患者皮膚組織における発現を定量し、 患者の炎症皮膚組織におけるリンパ球の浸潤を評価した。 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.
( 1 ) 皮膚検体の入手  (1) Obtaining skin samples
アトピー性皮膚炎患者 14例、 健常者 4例から同意を得て、 患者からは無疹部 と急性病変部を、 健常者からは正常皮膚を、 各部位からそれぞれ 3個の皮膚検体 (直径 3讓） を採取した。 急性病変部位は日本皮膚科学会の診断基準に基づき判 定した。 皮膚を採取したァトピー性皮膚炎患者の臨床情報を表 1に示す。  With the consent of 14 patients with atopic dermatitis and 4 healthy subjects, the skin rash and acute lesions were obtained from the patients, normal skin from healthy subjects, and three skin specimens from each site (diameter 3讓) was collected. Acute lesions were determined based on diagnostic criteria of the Japanese Dermatological Association. Table 1 shows the clinical information of atopic dermatitis patients whose skin was collected.
表 1  table 1
症例 重症度 IgE IU/ml *特異的 IgE Eosino/ μ 1 LDH ECP ダニ、 ハウスダスト  Case Severity IgE IU / ml * Specific IgE Eosino / μ 1 LDH ECP Tick, house dust
1 重症 28000  1 severe 28000
5；力ンジダ 3  5; Power 3
ダニ、 ノヽウスダスト  Ticks, dust dust
2 重症 4500 6；カンジダ 4；ピチ 670  2 severe 4500 6; Candida 4; Pichi 670
イロ 2  Iro 2
全身慢性湿  Whole body chronic humidity
3  Three
疹像 スギ 3；カンジダ 3； Rash Sugi 3; Candida 3;
タニ 6 ； ゾヽウスタス卜  Thani 6;
4 重症 22000 260  4 Severe 22000 260
6 ;牛乳 2 ;卵白 4 ;  6; milk 2; egg white 4;
小麦 3；米 2  Wheat 3; Rice 2
タニ、 /、ウスタス  Thani, /, Ustus
6 重症 15000 30 ト、 ピチイロ 5  6 Severe 15000 30 G, Pichiiro 5
Q ハウスダスト 5；ダニ  Q house dust 5; tick
虽重症？ It 1丄41 ±0リ0^ 0リ ΛΛ() Δ.0 虽 Severe? It 1 丄 4 1 ± 0 bits 0 ^ 0 bits ΛΛ () Δ.0
6  6
マラセチア、 /ヽウス  Malassezia, / ヽ us
10 重症 9000 ダスト、 ダュ、 カン 1800 1100 40 ジダ 4；スギ 3  10 Severe 9000 Dust, Du, Can 1800 1100 40 Jida 4; Cedar 3
スギ、 カンジダ 4 ;ダ  Cedar, candida 4; da
11 3400 1400 - - 二、 ハウスダスト 6  11 3400 1400--2, House dust 6
12 重症 10000 900  12 Severe 10000 900
スギ、 ダュ、 ハウス  Cedar, du, house
13 重症 12000 ダスト 6；ダイズ、 コ 710 1100 100 ムギ、 コメ 3  13 Severe 12000 Dust 6; Soybean, Ko 710 1100 100 Wheat, Rice 3
スギ 3 .力ンジ 2 ·  Japanese cedar 3. Power 2
14 9700 720 610  14 9700 720 610
ダニ、 ハウスダスト 6  Ticks, house dust 6
スギ、 ダニ、 ハウス  Cedar, tick, house
15 重症 22000 1200 750  15 Severe 22000 1200 750
ダスト 4  Dust 4
16 中等症 1100 ダニ、 ハウスダスト 5 70  16 Moderate 1100 Mites, house dust 5 70
17  17
( 2 ) ヒ ト皮膚からの遺伝子発現解析用の RNA調製 (2) RNA preparation for gene expression analysis from human skin
採取した 3個の皮膚 biopsy (直径 3應） を Isogen (日本ジーン；和光純薬） に浸漬し、 ウルトラタックス T8ホモジナイザー （IKA社） を使用してホモジナ ィズした。 ホモジナイズ以降は Isogenのマニュアルに従い、 total R Aの抽出 を行った。 クロ口ホルムを加え、 攪拌遠心して水層を回収した。 次にイソプロパ ノールを加え、 攪拌遠心して沈殿を回収した。 沈殿は 75%エタノールでリンス、 遠心を行い、 沈殿を total RNAとして回収した。 回収した total RNAは RNeasy Mini kit (QIAGEN) を用い、 そのマニュアルに従って更に精製した。  Three skin biopsy samples (diameter 3 mm) were immersed in Isogen (Nippon Gene; Wako Pure Chemical Industries) and homogenized using an Ultratax T8 homogenizer (IKA). After homogenization, 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. Next, 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.
( 3 ) 定量的 PCR用の c DNA合成 前述の方法で抽出した total Aを Dnase (二ツボンジーン） 処理し、 oligo (dT) 12_18 (GIBCO BRL) をプライマー iして逆転写した cDNAを铸型とした。 同時 に、 コピー数を算出する標準曲線のために両プライマーで増幅される塩基配列領 域を含むプラスミドクローンを各々の遺伝子について準備し、 その段階希釈を鎳 型として反応を行つた。 定量的 PCRの反応液組成は表 2に示した。 (3) cDNA synthesis for quantitative PCR The total A extracted by the above-mentioned method was treated with Dnase (Nitsubon Gene), and the cDNA reverse-transcribed with oligo (dT) 12 _ 18 (GIBCO BRL) as primer i was used as type III. At the same time, 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 carried out using serial dilutions as 鎳. The composition of the reaction mixture for quantitative PCR is shown in Table 2.
表 2  Table 2
ABI-PRISM 7700の反応組成 ( 1ゥエルあたりの反応量）  Reaction composition of ABI-PRISM 7700 (reaction volume per 1 ゥ)
23.75 (μΐ)  23.75 (μΐ)
10x TaqManパッファー A 5  10x TaqMan Puffer A 5
25mM MgCl2 7 25mM MgCl 2 7
dATP(lOmM) 1.0  dATP (lOmM) 1.0
dCTP(lOmM) 1.0  dCTP (lOmM) 1.0
dGTP(lOmM) 1.0  dGTP (lOmM) 1.0
dUTP(20mM) 1.0  dUTP (20mM) 1.0
Forward Primer (10 μ M) 1.0 Reverse Primer (10 μΜ) 1.0 TaqManプローブ（2.0 μΜ) 2.5 AmpliTaq Gold (5U//iL) 0.25  Forward Primer (10 μM) 1.0 Reverse Primer (10 μΜ) 1.0 TaqMan probe (2.0 μΜ) 2.5 AmpliTaq Gold (5U // iL) 0.25
AmpErase UNG (lU/^L) 0.5  AmpErase UNG (lU / ^ L) 0.5
一ト溶液 5  One solution 5
50 50
(4) TARC, CCR4, RANTESおよび CXCR3遺伝子の皮膚組織における発現解析 TARC, CCR4, RANTESおよび CXCR3遺伝子の皮膚組織における発現量を定量的 に評価するために、 ΑΒΙ 7700 (Applied Biosystems) による定量的 PCRを行った。 ABI 7700による測定に用いたプライマーおよび TaqManプローブは、 各遺伝子の 配列情報に基づいて Primer Express (PEバイオシステムズ） により設計した。 使用した全ての TaqManプローブの 5，末端は FAM (6- carboxy- fluorescein) で、 また 3，末端は TAMRA (6-carboxy-N, N, N' , N， -tetramethylrhodamine) で標識した。 また、 試料中の cDNA濃度の差を補正するため、 補正用内部標準として ーァ クチン - actin) 遺伝子、 およぴグリセルアルデヒド 3リン酸脱水素酵素 （GA PDH) 遺伝子について同様の定 析を行い、 それら遺伝子のコピー数を基に補 正して、 目的遺伝子のコピー数を算出した。 各遺伝子のフォワードプライマー (F) 、 リパースプライマー （R) 、 および TaqManプローブ （TP) に用いた オリゴヌクレオチドの塩基配列は、 以下に示すとおりである。 測定した遺伝子の 塩基配列に対応する Genbankのァクセッション番号を、 遺伝子名に続けて表示し た。 (4) Expression analysis of TARC, CCR4, RANTES and CXCR3 genes in skin tissues To quantitatively evaluate the expression levels of TARC, CCR4, RANTES and CXCR3 genes in skin tissues, quantitative PCR using 7700 (Applied Biosystems) Was done. Primers and TaqMan probes used for measurement by ABI 7700 were designed by Primer Express (PE Biosystems) based on the sequence information of each gene. All TaqMan probes used were labeled at the 5 and 5 ends with FAM (6-carboxy-fluorescein) and at the 3 and ends with TAMRA (6-carboxy-N, N, N ', N, -tetramethylrhodamine). In addition, the 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.
TARC (GenBank Acc.： NM一 002987)  TARC (GenBank Acc .: NM-I-002987)
F： 5' -GGAGTGCTGCCTGGAGTACTTC-3' (配列番号： 5 )  F: 5'-GGAGTGCTGCCTGGAGTACTTC-3 '(SEQ ID NO: 5)
R： 5' -CCTGGAGCAGTCCTCAGATGTC-3' (配列番号： 6 )  R: 5'-CCTGGAGCAGTCCTCAGATGTC-3 '(SEQ ID NO: 6)
TP ： 5' -AGCCATTCCCCTTAGAAAGCTGAAGA-3' (配列番号： 7 ) TP: 5'-AGCCATTCCCCTTAGAAAGCTGAAGA-3 '(SEQ ID NO: 7)
CCR4 (GenBank Acc.： NHL005508) CCR4 (GenBank Acc .: NHL005508)
F： 5' -TGTGGTGGTTCTGGTCCTGTT-3' (配列番号： 8 )  F: 5'-TGTGGTGGTTCTGGTCCTGTT-3 '(SEQ ID NO: 8)
R： 5' - CCCACTGGTCTGCTGCATAGT - 3， (配列番号： 9 )  R: 5'-CCCACTGGTCTGCTGCATAGT-3, (SEQ ID NO: 9)
TP ： 5' -CAACCTTGCCATCTCGGATCTGCTCT-3' (配列番号： 1 0) TP: 5'-CAACCTTGCCATCTCGGATCTGCTCT-3 '(SEQ ID NO: 10)
RANTES (GenBank Acc.： NM_002985) RANTES (GenBank Acc .: NM_002985)
F： 5' -ACCCAGCAGTCGTCTTTGTCAC-3' (配列番号： 1 1)  F: 5'-ACCCAGCAGTCGTCTTTGTCAC-3 '(SEQ ID NO: 11)
R： 5' -TCCCGAACCCATTTCTTCTCT-3' (配列番号： 1 2)  R: 5'-TCCCGAACCCATTTCTTCTCT-3 '(SEQ ID NO: 12)
TP ： 5' -AAAGAACCGCCAAGTGTGTGCCAACC-3' (配列番号： 1 3) TP: 5'-AAAGAACCGCCAAGTGTGTGCCAACC-3 '(SEQ ID NO: 13)
CXCR3 (GenBank Acc.： NM一 001504) CXCR3 (GenBank Acc .: NM-001504)
F： 5' - CCGTCCAGTGGGTCTTTGG- 3， （配列番号： 1 4)  F: 5'-CCGTCCAGTGGGTCTTTGG-3, (SEQ ID NO: 14)
R： 5' -AGCGGTCAAAGCTGATGCA-3' (配列番号： 1 5)  R: 5'-AGCGGTCAAAGCTGATGCA-3 '(SEQ ID NO: 15)
TP ： 5' -CCCTCTTCAACATCAACTTCTACGCAGGAG-3' (配列番号： 1 6) TP: 5'-CCCTCTTCAACATCAACTTCTACGCAGGAG-3 '(SEQ ID NO: 16)
β了クチン (GenBank Acc.： NM— 001101) β-acting Kuching (GenBank Acc .: NM— 001101)
F： 5' -TCACCCACACTGTGCCCATCTACGA-3' (配列番号： 1 7)  F: 5'-TCACCCACACTGTGCCCATCTACGA-3 '(SEQ ID NO: 17)
R： 5' -CAGCGGAACCGCTCATTGCCAATGG-3' (配列番号： 1 8)  R: 5'-CAGCGGAACCGCTCATTGCCAATGG-3 '(SEQ ID NO: 18)
TP ： 5'— ATGCCCTCCCCCATGCCATCCTGCGT - 3， （配列番号： 1 9) GAPDH (GenBank Acc.： 應一 002046) TP: 5'— ATGCCCTCCCCCATGCCATCCTGCGT-3, (SEQ ID NO: 19) GAPDH (GenBank Acc .: Oichi 002046)
F： 5' -GAAGGTGAAGGTCGGAGT-3' (配列番号： 2 0 )  F: 5'-GAAGGTGAAGGTCGGAGT-3 '(SEQ ID NO: 20)
R： 5' -GAAGATGGTGATGGGATTTC-3' (配列番号： 2 1 )  R: 5'-GAAGATGGTGATGGGATTTC-3 '(SEQ ID NO: 21)
T P： 5' -CAAGCTTCCCGTTCTCAGCC-3' (配列番号： 2 2 ) TP: 5'-CAAGCTTCCCGTTCTCAGCC-3 '(SEQ ID NO: 22)
GAPDHにより補正した各遺伝子の発現量 （coPy/5ng R A) を図 1〜図 4に示す。 症例 4、 13、 16の 3例では無疹部と比較して皮疹部において TARC、 RANTES, CCR 4および CXCR3の発現量が著しく增カ卩していた。 皮疹部の無疹部に対する変動倍 率を表 3に示した。 Expression level of each gene was corrected by GAPDH the (co P y / 5ng RA) shown in FIGS. In three of Cases 4, 13, and 16, TARC, RANTES, CCR4 and CXCR3 expression levels were significantly higher in the rash than in the rash. Table 3 shows the rate of change of the rash area relative to the non-rash area.
表 3  Table 3
症例 4、 13、 16の皮膚組織、 無疹部と皮疹部間、 における TARC、 RANTES、 CCR4、 CXCR3の遺伝子発現変動倍率 （Fold Change)  Fold change of TARC, RANTES, CCR4, CXCR3 gene expression in skin tissue, between rash and rash in Cases 4, 13, and 16
Figure imgf000033_0001
これらの 3例では、 皮疹部におけるケモカインの発現が高く、 すなわちリンパ 球が皮膚に遊走しやすい環境にあり、 かつリンパ球自身のマーカーであるケモカ インレセプターの発現が高い、 すなわち既に皮膚に遊走してきているリンパ球数 が多いことが推定される。
Figure imgf000033_0001
In these three cases, the expression of 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.
〔実施例 2〕 ジーンチップによるディファレンシャル発現解析  [Example 2] Differential expression analysis using gene chip
次に、 これらの 3例 （症例 4、 13、 16) の皮膚組織 （無疹部と皮疹部） につい て、 ァフィメトリックス社ジーンチップによるディファレンシャル発現解析を行 い、 リンパ球浸潤が多いあるいはリンパ球が遊走しやすい環境にあるァトピー性 皮膚炎皮膚組織において発現が誘導あるいは抑制される遺伝子の探索を行った。 この解析によりリンパ球の皮膚組織への遊走、 浸潤を促す遺伝子、 病変部皮膚浸 潤リンパ球で発現変動している遺伝子、 すなわちァトピー性皮膚炎の病態形成に 重要な遺伝子を選択することができる。 Next, differential expression analysis was performed on the skin tissue (rash and rash) of these three cases (cases 4, 13, and 16) using Affymetrix GeneChip to find that lymphocyte infiltration was high or lymphatic infiltration was high. We searched for genes whose expression is induced or suppressed in atopic dermatitis skin tissue in an environment where spheres can easily migrate. Based on this analysis, genes that promote lymphocyte migration and infiltration into skin tissue, Genes whose expression is fluctuated in lymphocytes, ie, genes important for the pathogenesis of atopic dermatitis, can be selected.
( 1 ) ジーンチップ用の cRNA合成  (1) cRNA synthesis for gene chip
Total R A 2— 5 g力 ら、 T7-(dT) 24 ( Amersham Pharmacia Biotech )をプラ ィマーとして、 Affymetrix社の Expression Analysis Technical Manualの方法 に従い、 Superscript II Reverse Transcriptase (Life Technologies社) を用 いて逆転写し、 1本鎖 cDNAを作製した。 Reverse transcription using Superscript II Reverse Transcriptase (Life Technologies) using T7- (dT) 24 (Amersham Pharmacia Biotech) as a primer according to the method of Affymetrix Expression Analysis Technical Manual. A single-stranded cDNA was prepared.
T7- (dT) 24プライマーは、 以下のように T7プロモーターの塩基配列に（dT) 24を 付カ卩した塩基配列からなる。 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.
T7 -（dT) 24プライマー： 5， -GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG - (dT) 2 4-3' (配列番号： 2 3 ) T7 - (dT) 24 primer: 5, -GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG - (dT) 2 4 -3 '( SEQ ID NO: 2 3)
次 ίこ、 Expression Analysis Technical Manual ίこ従 ヽ、 DNA Ligase, DNA pol ymerase Iおよび RNase Hを加え、 2本鎖 cDNAを合成した。 cDNAをフエノー ノレ .クロ口ホノレム抽出後、 Phase Lock Gelsに供し、 エタノール沈殿により精製 した。  Next, Expression Analysis Technical Manual, DNA Ligase, DNA polymerase I and RNase H were added to synthesize double-stranded cDNA. The cDNA was extracted with phenolic honolem, subjected to Phase Lock Gels, and purified by ethanol precipitation.
さらに、 BioArray High Yield RNA Transcription Labeling Kit 用い、 ビ ォチンラベルした cRNAを合成した。 RNeasy Spin column (QIAGEN) を用いて cR NAを精製し、 熱処理により断片化した。  Furthermore, 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.
そのつ 10 gの cRNAを Expression Analysis Technical Manual【こ従!ヽ Hyb ridization Cocktailに加えた。 これを DNAチップに入れ、 45°Cで 16時間ハイ プリダイゼーシヨンした。 DNAチップとしては GeneChip (R) Human Genome U95Av 2， B, C， D, E (Af f ymetrix社製） を用いた。  In addition, 10 g of cRNA was added to Expression Analysis Technical Manual [Hot !! Hybridization Cocktail]. This was placed in a DNA chip and hybridized at 45 ° C for 16 hours. GeneChip® Human Genome U95Av2, B, C, D, E (Affymetrix) was used as a DNA chip.
DNAチップを洗浄した後、 Streptavidin- Phycoerythrinを加え染色した。 洗浄 後、 normal ャギ IgGとピオチン化ャギ抗ストレプトァビジン IgG抗体の混合液 をアレイに加えた。 さらに、 蛍光強度を増強する目的で、 再度 Streptavidin-Ph ycoerythrinを加え染色した。 洗浄後、 スキャナーにセットし、 DNAチップ解析 ソフトにて解析した。 After washing the DNA chip, 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.
( 2 ) DNAチップ解析  (2) DNA chip analysis
DNAチップ解析ソフトである Suiteを用いて発現蛍光強度を測定し、 データ解 析を行った。 まず全てのチップについて Absolute analysisを行い、 用いたサン プル各々の遺伝子発現量を測定した。  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.
1 transcriptに対応するチップデータの解析は、 プローブセットのパーフエ クトマツチとミスマッチの蛍光強度を比較して、 positiveと negativeを決定し た。 Positive Fraction, Log Avg, Pos/Negの値から判定される Absolute Call である P (present) , A (absent) , およぴ M (marginal)の 3区分の判定を行った。 用語定義は以下に示した。  In the analysis of the chip data corresponding to 1 transcript, 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. Three classifications of P (present), A (absent), and M (marginal), which are absolute calls determined from the values of Positive Fraction, Log Avg, and Pos / Neg, were performed. Term definitions are shown below.
Positive Fraction; Positiveなペアの割合 Positive Fraction; Positive pair ratio
Log Avg; パーフエクトマツチとミスマッチのプローブセルの蛍光強度比の対数 の平均  Log Avg; Average of logarithm of fluorescence intensity ratio between perfect match and mismatched probe cell
Pos/Neg; Positiveペア数と Negativeペア数の比  Pos / Neg; Ratio of Positive pairs to Negative pairs
また、 パーフエクトマツチとミスマッチのプローブセルの蛍光強度の差の平均 値である Average Difference (Avg Diff)も計算した。  In addition, 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.
サンプル間の遺伝子発現を比較する場合には、 GeneChip Analysis Suite User Guideに従い Comparison analysisを行った。 チップ間の補正は、 各チップの 全プローブセットの蛍光強度の平均値が一定になるように捕正を行った。  When comparing gene expression between samples, Comparison analysis was performed according to the GeneChip Analysis Suite User Guide. Correction between chips was performed so that the average value of the fluorescence intensities of all probe sets of each chip was constant.
( 3 ) アトピー性皮膚炎患者の無疹部と急性病変部間で発現変動する遺伝子の解 析  (3) Analysis of genes whose expression fluctuates between non-rash and acute lesions in patients with atopic dermatitis
上記で選んだ 3例の各患者の無疹部における遺伝子発現と急性病変部における 遺伝子発現を Comparison analysisによって比較解析し、 急性病変部で無疹部と 比べて 2倍以上発現が増加している遺伝子、 1/2以下に発現が減少している遺伝 子を選択した。 患者ごとに選んできた遺伝子について、 今度は 3例の患者全てで 共通変動している遺伝子群を選択した。 共通変動遺伝子群の中から、 アトピー性 皮膚炎患者の急性病変部で発現が増加している SK404を選択した。 SK404遺伝子 について、 Comparison analysisにより算出された fold changeを表 4に示した c 表 4 The gene expression in the rash-free area and the gene expression in the acute lesion of each of the three patients selected above were compared and analyzed by Comparison analysis, and the expression was more than doubled in the acute lesion compared to the rash-free area Genes, genes whose expression was reduced to 1/2 or less, were selected. With regard to the genes selected for each patient, this time we selected a group of genes that fluctuate in common among all three patients. Atopic property from common variable genes SK404, whose expression was increased in acute lesions of dermatitis patients, was selected. For SK404 gene, c Table 4 the fold change calculated by Comparison analysis shown in Table 4
SK404遺伝子の GeneChipを用いた発現解析結果  Expression analysis results using GeneChip of SK404 gene
(Fold Change；皮疹部発現量/無疹部発現量）
Figure imgf000036_0001
(Fold Change; expression level of rash / expression level of rash)
Figure imgf000036_0001
SK404の全長塩基配列を配列番号： 1に、 全長アミノ酸配列を配列番号： 2に 示した。 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.
( 4 ) SK404遺伝子の定量的 PCR発現解析による皮膚組織での評価  (4) Evaluation of skin tissue by quantitative PCR expression analysis of SK404 gene
更に、 先に記載した方法に従って ABI7700を用いた定量的 PCRを行い、 チップ 解析を行った 3症例を含めたァトピー性皮膚炎患者 12例と健常者 例の皮膚組 織について SK404の遺伝子発現を評価した。 定量的 PCRに用いたフォヮ一ドプラ イマ一 （F) 、 リバースプライマー （R) 、 Taqmanプローブ （TP) の配列を以下 に示した。 サンプル間の発現量の補正は GAPDHの発現量に基づいて行つた。  Furthermore, quantitative PCR using ABI7700 was performed according to the method described above, and chip analysis was performed to evaluate the gene expression of SK404 in the skin tissues of 12 patients with atopic dermatitis, including 3 cases and healthy subjects did. The sequences of the primary primer (F), the reverse primer (R), and the Taqman probe (TP) used in the quantitative PCR are shown below. The expression level between samples was corrected based on the expression level of GAPDH.
SK404 SK404
F： 5'一 CMCTGGCAGACAGGCATGT - 3， （配列番号： 2 4 )  F: 5'-one CMCTGGCAGACAGGCATGT-3, (SEQ ID NO: 24)
： 5， -GTCCTCATTGCGACGCTTGT-3' (配列番号： 2 5 )  : 5, -GTCCTCATTGCGACGCTTGT-3 '(SEQ ID NO: 25)
TP： 5， -CCTTGGGTGTCAAGTTGCAGCTGATATG-3' (配列番号： 2 6 )  TP: 5, -CCTTGGGTGTCAAGTTGCAGCTGATATG-3 '(SEQ ID NO: 26)
解析の結果を図 5およぴ表 5に示した。 SK404は解析した 11例の患者皮膚の うち 9例において急性病変部で高発現というプロファイルを示した。  The results of the analysis are shown in FIG. 5 and Table 5. SK404 showed a profile of high expression in acute lesions in 9 of 11 patient skins analyzed.
表 5  Table 5
SK404遺伝子の定量的 PCRを用いた発現解析結果  Expression analysis results of SK404 gene using quantitative PCR
(Fold Change；皮疹部発現量 Z無疹部発現量） 症例番号 (Fold Change; expression level of skin eruption area Z expression level of rash area) Case number
2 3 4 6 9 10 12 13 15 16 17 2 3 4 6 9 10 12 13 15 16 17
SK404 0.3 2.8 5 2.8 0.8 3.3 2.3 9.6 1.1 17.8 1.7 SK404 0.3 2.8 5 2.8 0.8 3.3 2.3 9.6 1.1 17.8 1.7
( 5 ) 統計解析 (5) Statistical analysis
ABI7700を用いた定量的 PCRにより得られた SK404 mRNAのコピー数をもとに、 ウィルコクソン符号付順位和検定 （Wilcoxon signed-ranks test) を行った。 p 値は 0. 0262と算出され、 SK404のァトピー性皮膚炎患者急性病変部での高発現 (無疹部比較） が統計学的に有意であることが示された。  A Wilcoxon signed-ranks test was performed based on the copy number of SK404 mRNA obtained by quantitative PCR using ABI7700. The p-value was calculated to be 0.0262, indicating that the high expression of SK404 in the atopic lesions of atopic dermatitis (comparison with no eruption) was statistically significant.
〔実施例 3〕 マウス皮膚炎モデルにおける SK404遺伝子の発現解析  [Example 3] Expression analysis of SK404 gene in mouse dermatitis model
ヒトアトピー性皮膚炎の無疹部と皮疹部間で発現変動している遺伝子とマウス 皮膚炎モデルの非感作皮膚と感作皮膚間で発現変動している遺伝子を比較解析し、 共通変動している遺伝子群を選ぶことができれば、 共通変動している遺伝子につ いてノックァゥトマウスあるいはトランスジエニックマウスを作製することによ り、 その遺伝子の皮膚炎病態における重要性を評価することができる。 また、 共 通変動遺伝子が液性因子あるいは膜蛋白質であった場合には中和抗体、 液性因子 そのもの、 あるいは可溶性レセプターなどを投与することにより、 遺伝子改変マ ウスを利用した場合に比べて短期間で、 その遺伝子のヒト皮膚炎病態における重 要性を評価できる。 SK404についてマウスホモログを同定し、 マウス皮膚炎モデ ルでの遺伝子発現解析を行つた。  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. . In addition, when 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.
( 1 ) SK404マウスホモログ （SK404M) の同定  (1) Identification of SK404 mouse homologue (SK404M)
GenBank遺伝子データベースに対して相同性検索を行い、 SK404のマウスホモ ログ、 SK404Mを同定した。 SK404Mの全長塩基配列を配列番号： 3に、 全長アミ ノ酸配列を配列番号： 4に示した。 SK404と SK404Mのアミノ酸レベルでの配列 比較の結果を図 6に示した。  A homology search was performed on the GenBank gene database to identify a mouse homologue of SK404, SK404M. The full-length nucleotide sequence of SK404M is shown in SEQ ID NO: 3, and the full-length amino acid sequence is shown in SEQ ID NO: 4. FIG. 6 shows the results of sequence comparison at the amino acid level between SK404 and SK404M.
( 2 ) NC/Ngaマウス皮膚炎モデルの作製 SPF NC/Ngaマウス （c? · 6週齢） の耳介部と背部皮膚にダニ抗原 (Dermatopha goides pteronyssinus 5 gZ部位） を 3日間隔で計 9回皮内投与した。 耳浮腫 の測定と背部皮膚の症状観察は週 1回の割合で行った。 血中の total IgE濃度に ついては、 ダニ抗原投与前、 投与開始 14日後、 28 B後に採血を行い、 マウス Ig E測定キット （ャマサ醤油） にて測定した。 コントロールとして非感作群を取り、 各群 20匹 （10匹：ダニ抗原投与開始 14日後解剖、 10匹：ダニ抗原投与開始 28 日後解剖） で試験を行った。 (2) Preparation of NC / Nga mouse dermatitis model Mite antigen (Dermatopha goides pteronyssinus 5 gZ site) was intradermally administered to the auricle and back skin of SPF NC / Nga mice (c? 6 weeks old) 9 times at 3 day intervals in total. Measurement of ear edema and observation of symptoms on the back skin were performed once a week. The blood total IgE concentration was measured before the administration of the mite antigen, 14 days after the administration, and 28 B after the administration, and measured using a mouse IgE measurement kit (Yamasa soy sauce). As a control, a non-sensitized group was taken, and the test was carried out with 20 animals (10 animals: dissected 14 days after the start of mite antigen administration, 10 animals: 28 days after the start of mite antigen administration).
ダニ抗原投与 14日後解剖および 28 S解剖のいずれの試験においても非感作群 では耳浮腫は認められなかったが、 感作群ではダニ抗原投与 1週間後より明ら力 な耳の肥厚を示し、 2週間後以降高値を維持した （表 6 ) 。  No ear edema was observed in the non-sensitized group in both the dissection 14 days after mite antigen administration and the 28 S necropsy test, but in the sensitized group, apparent ear thickening was observed 1 week after mite antigen administration. After 2 weeks, it remained high (Table 6).
表 6 耳浮腫率（％)  Table 6 Ear edema rate (%)
感作期間（週間）  Sensitization period (week)
1 2 3 4 非感作群 Mean 102 107 106 109  1 2 3 4 Non-sensitized group Mean 102 107 106 109
±S.E. 3 4 3 3 感作群 Mean 189 271 286 278  ± S.E. 3 4 3 3 Sensitization group Mean 189 271 286 278
±S.E. 5 6 5 6 耳浮腫率 (%) =感作後の耳の厚さ/感作前の耳の厚さ X 100 また、 血中の total IgE濃度は、 感作群においてダニ抗原投与 2週間後より濃 度の上昇が認められた （表 7 ) 。  ± SE 5 6 5 6 Ear edema rate (%) = Ear thickness after sensitization / Ear thickness before sensitization x 100 In addition, the total IgE concentration in blood The concentration increased after a week (Table 7).
表 7  Table 7
血中全 IgE濃度 (ng/ml)  Total IgE concentration in blood (ng / ml)
感作期間 （週間）  Sensitization period (week)
感作前 2 4  Before sensitization 2 4
非感作群 Mean 0 23. 7 19. 3  Non-sensitized group Mean 0 23. 7 19.3
土 S. E. 0 12. 1 9. 2  S.S.E. 0 12.1 9.2
感作群 Mean 0 183 250. 4  Sensitization group Mean 0 183 250. 4
土 S. E. 0 33. 3 73. 5 これらの結果に加え、 病理学的検査の結果、 感作群の耳介皮膚では、 表皮の肥 厚、 真皮と皮下組織には炎症性細胞浸潤 （好中球、 好酸球ならびにリンパ球） 、 浮腫、 結合組織の増生およびマスト細胞の脱顆粒等が軽度から中等度の変化で観 察され、 皮膚炎病態を形成していることが確認できた。 Sat SE 0 33. 3 73. 5 In addition to these results, pathological examination showed that in the auricle skin of the sensitized group, the epidermis was thickened, the dermis and subcutaneous tissue were infiltrated with inflammatory cells (neutrophils, eosinophils and lymphocytes), Edema, connective tissue hyperplasia, and mast cell degranulation were observed in mild to moderate changes, confirming that dermatitis was formed.
( 3 ) マウス皮膚からの遺伝子発現解析用の腿調製  (3) Preparation of thighs for gene expression analysis from mouse skin
非感作マウスおよぴ感作マゥス （感作後 14日と 28日） より耳介皮膚およぴ背 部皮膚を回収し、 Isogen (日本ジーン；和光純薬） に浸漬し、 ホモジナイザー 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.
(NS-310E ; (株） 日音医理科器械製作所） を使用して氷冷下にホモジナイズし た。 ホモジナイズ以降は Isogenのマ二ユア こ従い、 total RNAの抽出を行つ た。 クロ口ホルムを加え、 攪拌遠心して水層を回収した。 次にイソプロパノール を加え、 攪拌遠心して沈殿を回収した。 沈殿は 75%エタノールでリンス、 遠心 を行い、 沈殿を total RNAとして回収した。 回収した total RNAは RNeasy Mini kit (QIAGEN) を用い、 そのマニュアルに従って更に精製を行った。 (NS-310E; Nichion Medical Science Instrument Co., Ltd.) and homogenized under ice-cooling. After homogenization, total RNA was extracted according to the standard of Isogen. 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 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.
( 4 ) 定量的 PCR用の cDNA合成  (4) cDNA synthesis for quantitative PCR
1群 10匹の各個体の耳介から抽出した total RNAをまとめ、 その total RNA を Dnase (二ツボンジーン） 処理し、 oligo (dT) 12— 18 (GIBCO BRL) をプライマー として逆転写した cDNAを铸型とした。 同時に、 コピー数を算出する標準曲線の ために両プライマーで増幅される塩基配列領域を含むブラスミドクローンを各々 の遺伝子について準備し、 その段階希釈を铸型として反応を行った。 定量的 PCR の反応液組成は表 2に示した。 The total 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. Type. At the same time, 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.
( 5 ) SK404M遺伝子のマウス皮膚炎モデル皮膚組織における発現解析  (5) Expression analysis of SK404M gene in mouse dermatitis model skin tissue
SK404M遺伝子のマウス皮膚炎モデル耳介皮膚組織における発現量を定量的に 評価するために、 ABI 7700 (Applied Biosystems) による定量的 PCRを行った。 ABI 7700による測定に用いたプライマ一および TaqManプロープは、 SK404M遺伝 子の配列情報に基づいて Primer Express (PEバイオシステムズ) により設計し た。 使用した TaqManプローブの 5'末端は FAM (6- carboxy- fluorescein) で、 ま た 3，末端は TAMRA (6-carboxy-N, N， N' , N' -tetramethylrhodamine) で標識した。 また、 試料中の cDNA濃度の差を補正するため、 補正用内部標準としてグリセ ルアルデヒド 3リン酸脱水素酵素 （GAPDH) 遺伝子について同様の定量解析を行 レ、、 GAPDH遺伝子のコピー数を基に補正して、 目的遺伝子のコピー数を算出した。 In order to quantitatively evaluate the expression level of the SK404M gene in a mouse dermatitis model auricular skin tissue, quantitative PCR using ABI 7700 (Applied Biosystems) was performed. 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). In addition, in order to correct for differences in cDNA concentration in the sample, a similar quantitative analysis was performed on the glyceraldehyde triphosphate dehydrogenase (GAPDH) gene as an internal standard for correction, and based on the copy number of the GAPDH gene. After correction, the copy number of the target gene was calculated.
SK40 およびマウス GAPDH遺伝子のフォワードプライマー （F ) 、 リパース プライマー （R) 、 および TaqManプローブ （T P ) に用いたオリゴヌクレオチ ドの塩基配列は、 以下に示すとおりである。 指標遺伝子の塩基配列に対応する G enBankのァクセッション番号を、 遺伝子名に続けて表示した。  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. The GenBank accession number corresponding to the nucleotide sequence of the indicator gene is displayed following the gene name.
SK404M  SK404M
F： 5' -GCCTGTGTGATTGCTTCAGTGA-3' (配列番号： 2 7 )  F: 5'-GCCTGTGTGATTGCTTCAGTGA-3 '(SEQ ID NO: 27)
R： 5， -CTGCCACTTGACATCCAAGACA-3' (配列番号： 2 8 )  R: 5, -CTGCCACTTGACATCCAAGACA-3 '(SEQ ID NO: 28)
TP： 5， -AAAGGTCCCACAGAGGCAGACTCCG-3' (配列番号： 2 9 )  TP: 5, -AAAGGTCCCACAGAGGCAGACTCCG-3 '(SEQ ID NO: 29)
マウス GAPDH (GenBank Acc. 顧— 008084) Mouse GAPDH (GenBank Acc.
F： 5， - GCACCACCAACTGCTTAGCC-3' (配列番号： 3 0 )  F: 5,-GCACCACCAACTGCTTAGCC-3 '(SEQ ID NO: 30)
R： 5' - CTTTGGCATTGTGGAAGGGCTCATG-3' (配列番号： 3 1 )  R: 5'-CTTTGGCATTGTGGAAGGGCTCATG-3 '(SEQ ID NO: 31)
TP： 5' - GATGCAGGGATGATGTTCTGG - 3， （配列番号： 3 2 )  TP: 5'-GATGCAGGGATGATGTTCTGG-3, (SEQ ID NO: 32)
解析の結果を図 7に示した。 SK404Mは感作 14日後および 28日後の耳介皮膚 において非感作耳介皮膚と比べて高い発現を示した。 14日目では非感作マウス の 7. 5倍の、 28日目では非感作マウスの 17. 3倍の発現量が認められた。  The results of the analysis are shown in 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はァトピー性皮膚炎の急性期に発現が上昇してくることから、 アトピー 性皮膚炎の発症に重要な役割を果たしている可能性が考えられる。 また、 TARC や CCR4といったケモカイン、 ケモカインレセプターと似た患者発現プロフアイ ルを示すことから、 皮膚組織へのリンパ球浸潤に関与し、 病態形成の一因になつ ている可能性も考えられる。 SK404はその染色体上の位置 (4q21) やアミノ酸の —次構造から CXCケモカインである可能性があり、 リンパ球へ直接作用すること も充分考えられる。 マウス皮膚炎モデル病態においてもヒトのアトピー性皮膚炎 と同様の発現変動が認められたことから、 皮膚炎病態における重要性が裏付けら れた。 またこのようなマウスモデルにおいて様々な方法で遺伝子産物を阻害、 あ る ヽは強制発現させることにより SK404の重要性は容易に評価できる。 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 ヽ.
〔実施例 4〕 SK404遺伝子のヒト組織での発現分布  [Example 4] Expression distribution of SK404 gene in human tissues
SK404遺伝子のヒト組織における発現を Real Time PCR法にて解析した。 g の各種組織由来 total RNAを使用して定法により cDNAを合成し、 そのうちの 1 / 10量 （total RNA換算で lOOng相当） の cDNAを用いて Real Time PCR (LightCy cler: Roche社） を行った。 定量には 2本鎖 DNA特異的に結合する蛍光色素であ る SYBR Green Iを使用した。 発現解析には脂肪組織、 脳、 副腎、 骨髄、 大腸、 心臓、 腎臓、 胎児腎、 肝臓、 胎児肝、 肺、 リンパ節、 乳腺、 脾臓、 胎盤、 前立腺、 唾液腺、 骨格筋、 皮膚、 小腸、 脾臓、 胃、 精巣、 胸腺、 気管、 子宮、 Hela細胞、 腎皮質上皮細胞、 冠状動脈血管内皮細胞、 冠状動脈血管平滑筋細胞、 白血球、 PH A活性化白血球、 正常ヒト近位尿細管上皮細胞由来の total RMを使用した。 発現解析に使用した SK404遺伝子のフォワードプライマー （F) 、 リバースプ ライマー （R) の塩基配列は、 以下に示すとおりである。  Expression of the SK404 gene 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.
SK404 SK404
F： 5' -CAGGCATGTGTGACTGTTTC-3' (配列番号： 3 3 )  F: 5'-CAGGCATGTGTGACTGTTTC-3 '(SEQ ID NO: 33)
R： 5' -TTGTTCCACACAGACAGCATT-3' (配列番号： 3 4 ) R: 5'-TTGTTCCACACAGACAGCATT-3 '(SEQ ID NO: 34)
解析の結果を図 8に示した。 発現解析の結果は、 組織間の比較が可能なように GAPDHの発現量に基づいて補正を行った。 SK404遺伝子は大腸、 骨髄、 脾臓、 リ ンパ節、 肺、 気管、 脂肪組織、 白血球で高発現していた。  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.
〔実施例 5〕 SK404遺伝子の健常人由来血球細胞における発現  [Example 5] Expression of SK404 gene in healthy human-derived blood cells
ァレルギ一疾患の病態形成に重要な役割を果たしている T細胞、 B細胞などの 血球細胞における SK404遺伝子の発現を解析した。  We analyzed the expression of the SK404 gene in blood cells such as T cells and B cells that play an important role in the pathogenesis of allergic diseases.
( 1 ) 末梢血液からの細胞の分画  (1) Fractionation of cells from peripheral blood
健常人由来の末梢血液 （約 5 - 10ml) カゝら比重勾配法と磁気ビーズを用いた磁 気細胞分離システム（MACS - Miltenyi Biotec GmbH社）を用いて細胞の分画を行 つた。 まず末梢血液から遠心分離により血漿を除いた後、 残りの血液を比重勾配 液 Ficoll (Amersham Pharmacia Biotec社製） に重層して遠心分離を行い、 層 分離した中間層をリンパ球分画、 下層を顆粒球分画として分取した。 リンパ球分 画は CD3磁気ビーズと反応後、 ナイロンスチールカラムを用いた MACSにかけ、 C D3陽性細胞を T細胞として回収した。 CD3陰性細胞はさらに CD14磁気ビーズと 反応させ、 MACSによる CD14陽性細胞を単球、 CD14陰性細胞を B細胞とした。 顆 粒球分画は CD16磁気ビーズと反応させ、 CD16陽性細胞を好中球、 CD16陰性細胞 を好酸球として分取した。 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. 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.
( 2 ) 分画した細胞からの total RNAの調製、 cDNAの合成  (2) Preparation of total RNA and synthesis of cDNA from fractionated cells
分画した細胞は Isogen (日本ジーン；和光純薬） に溶解し、 そのマエュアル に従い、 total RNAの抽出を行った。 クロ口ホルムを加え、 攪拌遠心して水層を 回収した。 次にイソプロパノールを加え、 攪拌遠心して沈殿を回収した。 沈殿は 75%エタノーノレでリンス、 遠心を行い、 沈殿を total RNAとして回収した。  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.
調製した total RNAは DNase (二ッボンジーン）処理を行つた後、 oligo (dT) 12_18 (GIBCO BRL)をプフ マーとし、 Superscript II reverse transcriptase ( Invi trogen )を使用して逆転写を行い、 cDNAを合成した。 The prepared total 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.
( 3 ) SK404遺伝子の血球細胞における遺伝子発現解析  (3) Gene expression analysis of SK404 gene in blood cells
SK404遺伝子の分画した各細胞における発現量を定量的に評価するために、 前 述した方法に従い ABI 7700 ( Applied Biosystems )を使用して定量的 PCRを行 つた。 解析に使用した SK404のプライマーおよび Taqmanプロ一プは実施例 2に 載した。  In order to quantitatively evaluate the expression level of the SK404 gene in each fractionated cell, quantitative PCR was performed using ABI 7700 (Applied Biosystems) according to the method described above. SK404 primers and Taqman prop used for analysis are described in Example 2.
解析の結果を図 9に示した。 SK404は解析した 5種の細胞全てに発現しており、 発現量の高い順に B細胞、 T細胞、 単球、 好酸球、 好中球であった。  The results of the analysis are shown in 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.
〔実施例 6〕 NC/Ngaマウス皮膚炎モデノレの耳介皮膚を用いた SK404遺伝子の In situ hybridization (ISH)解析 SK404遺伝子はヒトのアトピー性皮膚炎の病変部位で高発現していることが明 らかになり、 マウス皮膚炎モデルにおいてもマウスホモログ （SK404M) が同様に 病変部位で高発現していることが明らかになった。 我々は病変部における SK404 発現細胞を同定する目的で、 NC/Ngaマウス皮膚炎モデルの耳介皮膚を用いた In situ hybridization解析 ¾：行つた。 [Example 6] In situ hybridization (ISH) analysis of SK404 gene using auricular skin of NC / Nga mouse dermatitis model It was revealed that the SK404 gene was highly expressed in the lesion site of human atopic dermatitis, and that the mouse homologue (SK404M) was also highly expressed in the lesion site in a mouse dermatitis model. It was revealed. We performed in situ hybridization analysis using auricle skin of NC / Nga mouse dermatitis model to identify SK404 expressing cells in the lesion.
感作 4週目のマウス耳介皮膚を 10%中性緩衝ホルマリンによる固定後、 パラ フィン包埋を行い、 パラフィンプロックの作製を行つた。 プローブは配列番号： 3 5に記載の配列を使用し、 SP6および T7 RNAポリメラーゼを用いてジコキシ ゲニン標識によるセンスプローブおよぴァンチセンスプローブを作製した。 シグ ナノレの検出は、 スライドグラスに貼り付けたパラフィン切片にプローブを反応さ せ、 更にアルカリフォスファターゼ標識した抗ジコキシゲニン抗体を反応させた 後、 NBT/BCIPを発色基質として反応させ、 発色させた。  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. For detection of signole, 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.
ISH染色により特異的なシグナルが検出できたスライドは水洗後、 Hematoxyli n-Eosin (HE)染色を行い、 SK404M遺伝子発現細胞の同定を行った。  Slides in which a specific signal was detected by ISH staining were washed with water and stained with Hematoxylin-Eosin (HE) to identify SK404M gene-expressing cells.
結果を図 1 0に示したが、 マウス皮膚炎病変部においては、 SK404Mは好中球 および T細胞に発現していた。 陽 細胞の多くは好中球であつたが、 シグナル強 度 （細胞内の発現量） としては T細胞の方が高かった。  The results are shown in FIG. 10, where 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).
〔実施例 7〕 SK40 遺伝子の乾癬患者皮膚組織における発現解析  [Example 7] Expression analysis of SK40 gene in skin tissue of psoriatic patients
SK40 はァトピー性皮膚炎患者の急性病変部において無疹部に比べて発現上昇 が認められた遺伝子として同定されたが、 別の皮膚炎症性疾患である乾癬の患者 皮膚組織における遺伝子発現解析を行うことにより、 アトピー性皮膚炎特異的な 発現変動を示すのか、 あるいは両疾患で共通した発現変動を示すのかが明らかに なる。 アトピー性皮膚炎特異的な発現変動を示した場合には、 アトピー性皮膚炎 病態を特徴づける遺伝子の一つとして診断および創薬ターゲットとしての価値が 高まると考えられる。  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.
( 1 ) 皮膚検体の入手 乾癬患者 6例から同意を得て、 無疹部と病変部を各部位からそれぞれ 3個の皮 膚検体 （直径 3mm) を採取した。 (1) Obtaining skin samples With consent from six patients with psoriasis, three skin specimens (3 mm in diameter) were collected from each of the rash-free and lesioned areas.
( 2 ) ヒト皮膚からの遺伝子発現解析用の腿調製  (2) Preparation of thighs for gene expression analysis from human skin
採取した 3個の皮膚 biopsy (直径 3讓） を Isogen (日本ジーン；和光純薬） に浸漬し、 ウルトラタックス T8ホモジナイザー （IKA社）を使用してホモジナイ ズした。 ホモジナイズ以降は Isogenのマニュアルに従い、 total RNAの抽出を 行った。 クロ口ホルムを加え、 攪拌遠心して水層を回収した。 次にイソプロパノ 一ノレを加え、 攪拌遠心して沈殿を回収した。 沈殿は 75%エタノールでリンス、 遠心を行い、 沈殿を total RNAとして回収した。 回収した total腿は RNeasy Mini kit (QIAGEN)を用い、 そのマニュアルに従って更に精製を行った。  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.
( 3 ) 定量的 PCR用の c DNA合成  (3) cDNA synthesis for quantitative PCR
前述の方法で抽出した total RNAを DNase (ュツボンジーン）処理し、 oligo (d T) 12_18 (GIBCO BRL)をプライマーとして逆転写した cDNAを鎵型とした。 同時に、 コピー数を算出する標準曲線のために両プライマーで増幅される塩基配列領域を 含むプラスミドクローンを各々の遺伝子について準備し、 その段階希釈を铸型と して反応を行つた。 定量的 PCRの反応液組成は表 2に示した。 Total 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鎵型. At the same time, 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.
( 4 ) SK404遺伝子の乾癬患者皮膚組織における発現解析  (4) Expression analysis of SK404 gene in skin tissue of psoriasis patients
SK404遺伝子の乾癬患者皮膚組織における発現量を定量的に評価するために、 先に記載した方法に従って ABI 7700 ( Applied Biosystems )による定量的 PCR を行った。 結果を図 1 1に示したが、 SK404遺伝子はアトピー性皮膚炎患者の皮 疹部でのみ無疹部と比べて発現充進を示し、 乾癬の病態においては無疹部と皮疹 部間で発現変動は認められなかった。 この結果から、 SK404遺伝子は単に皮膚の 炎症により発現が変動しているのではなく、 ァトピー性皮膚炎病態の特徴を反映 した発現変動を示していると考えられた。  In order to quantitatively evaluate the expression level of the SK404 gene in the skin tissue of psoriatic patients, quantitative PCR using ABI 7700 (Applied Biosystems) was performed according to the method described above. The results are shown in Fig. 11, where the SK404 gene was expressed and enhanced only in the rash area of atopic dermatitis patients compared to the rash area, and was expressed between the rash area and the rash area in psoriatic disease No change was observed. These results suggest that the expression of the SK404 gene does not fluctuate simply due to inflammation of the skin, but rather fluctuates in expression reflecting the characteristics of atopic dermatitis pathology.
〔実施例 8〕 SK404蛋白質の発現  Example 8 Expression of SK404 Protein
SK404遺伝子産物が細胞外に分泌される液性因子なのカゝ、 あるいは細胞内蛋白 質なのかを確かめるために、 HEK293細胞を使用して蛋白質の発現を行った。SK404 gene product is a humoral factor secreted extracellularly, or intracellular protein To confirm the quality, protein expression was performed using HEK293 cells.
( 1 ) SK404遺伝子発現べクタ一の作製 (1) Preparation of SK404 gene expression vector
SK404遺伝子全長 348bpから終止コドンを除いて 5'側に EcoRIサイト、 3'側に BamHIサイトを付加した SK404遺伝子断片を PCRにより合成し、 C末側に FLAGタ グを付加した形で発現可能な p3XFLAG- CMV- 14ベクター （sigma) の EcoRI- BamHI サイト間に連結した。 終止コドンを含まない SK404遺伝子断片の合成に用いたプ ライマーの配列を以下に示した。  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.
SK404全長遺伝子増幅用プライマー SK404 full length gene amplification primer
F： 5'-CGCGAATTCACCATGCAAGCTCAGGCGCCG-3' (配列番号： 3 6 )  F: 5'-CGCGAATTCACCATGCAAGCTCAGGCGCCG-3 '(SEQ ID NO: 36)
R： 5， - CCGGGATCCGAAAGTACGCATGGCTCTCCT - 3， （配列番号： 3 7 ) R: 5, -CCGGGATCCGAAAGTACGCATGGCTCTCCT-3, (SEQ ID NO: 37)
( 2 ) HEK293細胞発現系を用いた SK404遺伝子産物の発現  (2) Expression of SK404 gene product using HEK293 cell expression system
直径 10cmの tissue culture plateあたり 2X106個の HEK293細胞を遺伝子導 入の前日に播種した。 翌日、 1枚の plateに対して ΙΟ/i gの SK404遺伝子発現べ クタ一を TransIT LT試薬 （Mirus社） を使用し、 そのマニュアルに従って導入 した。 遺伝子導入開始 8時間後に培地を交換し、 48時間後まで 5%C02インキュ ベータ一、 37°Cで培養した。 48時間後に培養上清を回収した後、 plateを PBSで 洗净し、 1mlの M - PER Mammalian Protein Extraction Reagentを加えて細胞を 破砕し、 細胞破碎液を回収した。 2 × 10 6 HEK293 cells per 10 cm diameter tissue culture plate were seeded on the day before gene transfer. The next day, ΙΟ / 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.
回収した培養上清 200mlを 500 z lの Ant i- Flag M2- Affinity column (sigma) に通塔し、 0. 1M Glycine/HCl buffer (pH3)で溶出を行った。 溶出時の分画は 50 Ομ ΐごとに行った。  200 ml of the collected culture supernatant was passed through a 500 zl Anti-Flag M2-Affinity column (sigma) and eluted with 0.1 M Glycine / HCl buffer (pH 3). Fractionation at the time of elution was performed every 50 μm.
細胞破砕液は 10 μ 1、 培養上清精製後の各フラクシヨンは 200 μ 1分を SDS -ポ リアクリルアミドゲル電気泳動に供した。 泳動後、 ゲルはナイロン膜上にのせ、 トランスファー緩衝液 （0. lM Tris, 0. 192M Glycine, 20% Methanol) に浸した ろ紙で挟み、 トランスファー装置上でプロッティングを行った （140mA/gel, lh r) 。 プロッティングした膜は Blocking溶液 （lOmM Tris， 150mM NaCl, 0. 1% Tw een20, 2%BSA) 中で 30分間振とうし、 未反応の活性基をブロックした。 次に、 膜を Blocking溶液 (10mM Tris, 150mM NaCl, 0. 1% T een20, 2%BSA) で 10 /i g/ mlに希釈した Anti-FLAG M2 Monoclonal Antibody ( sigma )溶液に浸し、 室温で 2時間反応させた。 Wash溶液 (120mM NaCl, lOmM NaH2P04, 30mM K2HP04) にて膜 を 3回洗浄した後、 Blocking溶液 (10mM Tris, 150mM NaCl, 0. 1% Tween20, 2% BSA) で希釈したペルォキシダ一ゼ標識ラビット抗マウスィムノグロプリン溶液 に浸し、 室温で 1時間反応させた。 反応後、 膜を Wash溶液 (120mM NaCl, lOmM NaH2P04， 30mM K2HP04) にて 3回洗浄し、 ECL Western blotting detection reag ents (Amersham lfc)を用いて、 抗体が結合したパンドを X線フィルムに感光させ て、 検出した。 10 μl of the cell lysate and 200 μl of each fraction after purification of the culture supernatant were subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the gel was placed on a nylon membrane, sandwiched between filter papers immersed in transfer buffer (0.1M Tris, 0.12M Glycine, 20% Methanol), and plotted on a transfer device (140mA / gel, lh r). The plotted membrane is blocked with Blocking solution (10 mM Tris, 150 mM NaCl, 0.1% Tw een20, 2% BSA) for 30 minutes to block unreacted active groups. Next, 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. 1% Tween20, 2% BSA) The cells were immersed in a peroxidase-labeled rabbit anti-mouse immunoglobulin solution and reacted at room temperature for 1 hour. After the reaction, the membrane Wash solution (120mM NaCl, lOmM NaH 2 P0 4, 30mM K 2 HP0 4) was washed three times with, using the ECL Western blotting detection reag ents (Amersham lfc), the Pando bound by the antibody Exposure to X-ray film detected.
結果を図 1 2に示す。 HEK293細胞発現系では SK404 は細胞外には分泌されず、 細胞内に存在していることが明らかになった。 産業上の利用の可能性  The results are shown in FIG. In the HEK293 cell expression system, it was revealed that SK404 was not secreted out of the cell but was present in the cell. Industrial potential
本発明は、 ァレルギ一性皮膚炎の指標遺伝子 S K 4 0 4を提供する。 S K 4 0 4は、 皮疹部における発現レベルがアトピー性皮膚炎患者の無疹部に比べて高い 指標遺伝子として同定された遺伝子である。 また S K 4 0 4は、 アトピー性皮膚 炎患者の皮疹部における発現レベルが健常者の発現レベルに比べて高い指標遺伝 子でもあった。  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.
更に本発明は、 マウス S K 4 0 4の指標遺伝子としての用途を提供した。 マウ ス S K 4 0 4遺伝子は、 ダニアレルゲン感作マウスの耳介皮膚における発現レべ ルがダニァレルゲン非感作マウスの耳介皮膚に比べて高い指標遺伝子である。 遺伝子の発現レベルを指標とすることにより、 アトピー性皮膚炎の検査方法、 ァレルギ一性疾患モデル動物の作成、 およぴ該疾患の治療のための化合物をスク リーユングすることが可能となつた。  Further, the present invention has provided use of mouse SK404 as an indicator gene. The 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. By using the gene expression level as an index, it has become possible to test atopic dermatitis, to prepare an animal model for allergic disease, and to screen compounds for the treatment of the disease.
本発明の指標遺伝子は、 その発現の変化が病態と結びついている。 したがって 指標遺伝子の発現や、 指標遺伝子によってコードされる蛋白質の活性を調節する ことによってァレルギ一性皮膚炎の治療が可能となる。 例えば患部や患者の皮膚 において発現が上昇する遺伝子の場合には、 発現やその活性を阻害することが、 ァトピー性皮膚炎の治療戦略のターゲットとなる。 また本発明の指標遺伝子には、 ァトピー性皮膚炎の治療におけるモニタリングのための新しい臨床診断指標とし ての有用性が期待できる。 In 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. In addition, 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.
本発明によって提供された指標遺伝子は、 アレルゲンの種類に関わらず、 簡便 にその発現レベルを知ることができる。 従って、 アレルギー反応の病態を総合的 に把握することができる。  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.
また本発明によるアトピー性皮膚炎の検查方法は、 生体試料を試料としてその 発現レベルを解析することができるので、 患者に対する侵襲性が低い。 しかも遺 伝子発現解析に関しては、 微量サンプルによる高感度な測定が可能である。 遺伝 子解析技術は、 年々ハイスループット化、 低価格ィ匕が進行している。 従って本発 明によるアトピー性皮膚炎の検査方法は、 近い将来、 ベッドサイドにおける重要 な診断方法となることが期待される。 この意味で本発明の指標遺伝子の診断的価 値は高い。  Moreover, 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. In addition, with regard to gene expression analysis, highly sensitive measurement using a small amount of sample is possible. In gene analysis technology, 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.

Claims

請求の範囲 The scope of the claims
1. 次の工程 (1) 〜 （3) を含む、 アトピー性皮膚炎の検查方法であって、 指標遺伝子が S K 404である方法。 1. A method for detecting atopic dermatitis, comprising the following steps (1) to (3), wherein the indicator gene is SK404.
( 1 ) 被検者の皮疹部から採取された生体試料における指標遺伝子の発現レべ ルを測定する工程  (1) A step of measuring the expression level of an indicator gene in a biological sample collected from the rash on the subject
(2) 工程 (1) で測定された皮疹部の発現レベルを、 対照として同じ被検者 の無疹部または健常者皮膚から採取された生体試料における指標遺伝子 の発現レベルと比較する工程、 および  (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 healthy skin of the same subject as a control, and
(3) 工程 (2) の比較の結果、 対照と比較して発現レベルが高い場合に前記 被検者はァトピー性皮膚炎を有すると判定する工程  (3) a step of determining that the subject has atopic dermatitis when the expression level is higher than the control as a result of the comparison in step (2);
2. 遺伝子の発現レベルを、 cDNAの PCRによつて測定する請求項 1に記載の 検査方法。  2. The test method according to claim 1, wherein the gene expression level is measured by cDNA PCR.
3. 遺伝子の発現レベルを、 指標遺伝子によってコードされる蛋白質の検出に よって測定する請求項 1に記載の検査方法。  3. The test method according to claim 1, wherein the expression level of the gene is measured by detecting a protein encoded by the indicator gene.
4. 指標遺伝子の塩基配列を含むポリヌクレオチド、 またはその相補鎖に相捕 的な塩基配列を有する少なくとも 15塩基の長さを有するオリゴヌクレオチ ドからなる、 アトピー性皮膚炎検査用試薬であって、 指標遺伝子が SK40 4であるアトピー性皮膚炎検査用試薬。  4. A reagent for testing atopic dermatitis, comprising a polynucleotide containing the nucleotide sequence of the indicator gene or an oligonucleotide having a nucleotide sequence complementary to its complementary strand and having a length of at least 15 bases, An atopic dermatitis test reagent whose indicator gene is SK404.
5. 指標遺伝子によってコードされる蛋白質のァミノ酸配列を含むぺプチドを 認識する抗体からなる、 アトピー性皮膚炎検査用試薬であって、 指標遺伝子 が SK404であるアトピー性皮膚炎検査用試薬。  5. An atopic dermatitis test reagent comprising an antibody that recognizes a peptide containing an amino acid sequence of a protein encoded by an indicator gene, wherein the indicator gene is SK404.
6. 次の工程を含む、 ァトピー性皮膚炎の治療薬のスクリ一ユング方法であつ て、 指標遺伝子が SK404であるスクリーニング方法。  6. A screening method for a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404.
( 1 ) 指標遺伝子を発現する細胞に侯補ィヒ合物を接触させる工程、  (1) contacting a cell expressing the indicator gene with a candidate compound;
(2) 前記遺伝子の発現レベルを測定する工程、 ( 3 ) 候補ィヒ合物を接触させない対照と比較して、 指標遺伝子の発現レベルを 低下させる化合物を選択する工程 (2) measuring the expression level of the gene, (3) Step of selecting a compound that reduces the expression level of the indicator gene as compared to a control that is not contacted with the candidate compound
. 細胞が株化皮膚細胞である請求項 6に記載の方法。7. The method according to claim 6, wherein the cells are established skin cells.
. 指標遺伝子の塩基配列を含むポリヌクレオチド、 またはその相補鎖に相補 的な塩基配列を有する少なくとも 1 5塩基の長さを有するオリゴヌクレオチ ドと、 指標遺伝子を発現する細胞を含む、 アトピー性皮膚炎の治療薬候補ィ匕 合物をスクリーニングするためのキットであって、 指標遺伝子が S K 4 0 4 であるキット。 Atopic dermatitis, 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 therapeutic drug candidate according to claim 1, wherein the indicator gene is SK404.
. 指標遺伝子によってコードされる蛋白質のァミノ酸配列を含むぺプチドを 認識する抗体と、 指標遺伝子を発現する細胞を含む、 アトピー性皮膚炎の治 療薬候補ィ匕合物をスクリーニングするためのキットであって、 指標遺伝子が S K 4 0 4であるキット。 An antibody recognizing a peptide containing an amino acid sequence of a protein encoded by an indicator gene, and a kit for screening a candidate for a therapeutic agent for atopic dermatitis, comprising cells expressing the indicator gene. A kit wherein the indicator gene is SK404.
0 . 指標遺伝子または指標遺伝子と機能的に同等な遺伝子の皮膚における発 現強度を上昇させたトランスジエニック非ヒト脊椎動物からなるアトピー 性皮膚炎モデル動物であって、 指標遺伝子が S K 4 0 4またはその機能的 に同等な遺伝子であるモデル動物。0. 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 SK404. Or a model animal whose gene is functionally equivalent.
1 . 非ヒト脊椎動物がマウスである請求項 1 0に記載のモデル動物。1. The model animal according to claim 10, wherein the non-human vertebrate is a mouse.
2 . 次の i)または ii)に記載の成分をマウスに投与する工程を含む、 アレル ギ一性皮膚炎モデル動物の製造方法。 2. A method for producing an animal model of allergic allergic dermatitis, comprising the step of administering the following component i) or ii) to a mouse.
i) S K 4 0 4のホモログを構成する塩基配列を含むポリヌクレオチド、 ii) S K 4 0 4のホモログを構成する塩基配列を含むポリヌクレオチドによつ てコードされるタンパク質 i) a polynucleotide comprising a nucleotide sequence constituting a homologue of SK404, ii) a protein encoded by a polynucleotide comprising a nucleotide sequence constituting a homologue of SK404
3 . 請求項 1 2における i)または ii)のいずれかに記載の成分を有効成分と して含む、 マウスにァレルギ一性皮膚炎を誘導するための誘導剤。 3. An inducer for inducing allergic monodermatitis in mice, comprising the ingredient according to any one of i) or ii) in claim 12 as an active ingredient.
4 . 次の工程を含む、 ァトピー性皮膚炎の治療薬のスクリ一ユング方法であ つて、 指標遺伝子が S K 4 0 4またはそのホモログであるスクリーニング 方法。 4. A method for screening a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404 or a homolog thereof. Method.
( 1 ) 被験動物に候補化合物を投与する工程、  (1) administering a candidate compound to a test animal,
( 2 ) 前記被験動物の生体試料における指標遺伝子の発現強度を測定する工程、 (2) measuring the expression intensity of the indicator gene in the biological sample of the test animal,
(3) 候補化合物を接触させない対照と比較して、 前記遺伝子の発現レベルを 低下させる化合物を選択する工程、 (3) selecting a compound that reduces the expression level of the gene, as compared to a control not contacting the candidate compound,
15. 次の工程を含む、 ァトピー性皮膚炎の治療薬のスクリ一エング方法であ つて、 指標遺伝子が SK404であるスクリーニング方法。  15. A screening method for a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404.
(1) 指標遺伝子の転写調節領域と、 この転写調節領域の制御下に発現するレ ポーター遺伝子とを含むベクターを導入した細胞と候補ィ匕合物を接触さ せる工程、  (1) a step of contacting a candidate conjugate with a cell into which a vector containing a transcription regulatory region of an indicator gene and a reporter gene expressed under the control of the transcription regulatory region has been introduced;
(2) 前記レポーター遺伝子の活性を測定する工程、 および  (2) measuring the activity of the reporter gene, and
(3) 候補化合物を接触させない対照と比較して、 前記レポーター遺伝子の発 現レベルを低下させる化合物を選択する工程、  (3) selecting a compound that reduces the expression level of the reporter gene, as compared to a control not contacting the candidate compound,
16. 次の工程を含む、 ァトピー性皮膚炎の治療薬のスクリ一ユング方法であ つて、 指標遺伝子が SK404、 または SK404と機能的に同等な遺伝 子であるスクリーユング方法。  16. A method for screening a therapeutic agent for atopic dermatitis, comprising the following steps, wherein the indicator gene is SK404 or a gene functionally equivalent to SK404.
( 1 ) 指標遺伝子によってコードされる蛋白質と候補化合物を接触させる工程、 (1) contacting a protein encoded by the indicator gene with a candidate compound,
(2) 前記蛋白質の活性を測定する工程、 および (2) measuring the activity of the protein, and
(3) 候補化合物を接触させない対照と比較して、 前記活性を低下させる化合 物を選択する工程  (3) selecting a compound that reduces the activity compared to a control that does not come into contact with the candidate compound
17. 請求項 6、 請求項 14、 請求項 15、 および請求項 16のいずれかに記 载のスタリーニング方法によって得ることができる化合物を有効成分とし て含有する、 ァトピー性皮膚炎の治療薬。  17. A therapeutic agent for atopic dermatitis, comprising, as an active ingredient, a compound obtainable by the stalling method according to any one of claims 6, 14, 15, and 16.
18. 指標遺伝子、 またはその一部のアンチセンス DNAを有効成分として含む アトピー性皮膚炎の治療薬であって、 指標遺伝子が SK404である治療 指標遺伝子によってコードされる蛋白質のアミノ酸配列を含むペプチド を認識する抗体を有効成分として含む、 ァトピー性皮膚炎の治療薬であつ て、 指標遺伝子が S K 4 0 4である治療薬。 18. A remedy for atopic dermatitis containing an indicator gene or a part of the antisense DNA as an active ingredient, wherein the indicator gene is SK404 A therapeutic agent for atopic dermatitis, comprising, as an active ingredient, an antibody that recognizes a peptide containing an amino acid sequence of a protein encoded by an indicator gene, wherein the indicator gene is SK404.
PCT/JP2003/005277 2002-08-06 2003-04-24 Method of examining atopic dermatitis WO2004016783A1 (en)

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Publication number Priority date Publication date Assignee Title
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