WO2018168768A1 - Biomarker for obstructive pulmonary disease - Google Patents

Biomarker for obstructive pulmonary disease Download PDF

Info

Publication number
WO2018168768A1
WO2018168768A1 PCT/JP2018/009502 JP2018009502W WO2018168768A1 WO 2018168768 A1 WO2018168768 A1 WO 2018168768A1 JP 2018009502 W JP2018009502 W JP 2018009502W WO 2018168768 A1 WO2018168768 A1 WO 2018168768A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
alpha
chain
subunit
region
Prior art date
Application number
PCT/JP2018/009502
Other languages
French (fr)
Japanese (ja)
Inventor
吉人 武田
淳 熊ノ郷
宗到 玄山
幸嗣 植田
Original Assignee
国立大学法人大阪大学
公益財団法人がん研究会
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人大阪大学, 公益財団法人がん研究会 filed Critical 国立大学法人大阪大学
Publication of WO2018168768A1 publication Critical patent/WO2018168768A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • 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
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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

Definitions

  • the present invention relates to a biomarker for obstructive pulmonary disease, use thereof, and the like.
  • Bronchial asthma (BA) is estimated to have 300 million patients, and the increase in prevalence is a social and economic burden.
  • the true form of bronchial asthma is chronic airway inflammation, but the elucidation of key molecules that control inflammation and airway remodeling is insufficient.
  • chronic obstructive pulmonary disease COPD
  • COPD chronic obstructive pulmonary disease
  • Obstructive pulmonary diseases such as bronchial asthma and COPD develop on the basis of dysregulation of the cellular molecular network formed by the interaction of individual genes and proteins rather than the mutations themselves. I think that. In fact, there are various phenotypes of asthma and COPD characterized by airflow obstruction and inflammation, and this seems to make it more difficult to elucidate the pathogenesis of these diseases. Furthermore, although there is a high rate of overlap syndrome in which both diseases coexist, the differentiation method and detailed pathology are unknown.
  • COPD chronic lung disease
  • asthma estimated to have 200 million patients worldwide, and 5.3 million in Japan, of which only 5% are being treated.
  • asthma estimated to have 200 million patients worldwide, has been declining due to the widespread use of inhaled steroids, but the number of affected cases continues to increase with aging.
  • ACO combined with COPD and asthma is attracting attention because of its poor prognosis and 20% of COPD patients.
  • asthma has various phenotypes (phenotypes) and end types, individualized medicine is required.
  • treatments targeting molecules such as IL-5, IL-13, and IgE that induce Th2 type inflammation have been developed one after another.
  • treatments targeting Th2-type inflammation are being developed one after another overseas, there are few indicators (companion biomarkers) for judging treatment selection and effectiveness.
  • Extracellular vesicles are membrane vesicles that are secreted from cells. By transporting intracellular proteins, lipids, and nucleic acids (mRNA, microRNA, etc.) to the outside of the cell, information transmission between cells in the local and whole body is performed. I'm in charge.
  • extracellular vesicles (exosomes) have attracted attention as test samples for cancer, but as test samples for inflammatory respiratory diseases, particularly obstructive pulmonary diseases such as bronchial asthma, which is the two major obstructive pulmonary diseases The usefulness of is not known.
  • An object of the present invention is to provide a biomarker for obstructive pulmonary diseases such as bronchial asthma and a method for using the same.
  • an object of the present invention is to provide a biomarker capable of differentiating between bronchial asthma and chronic obstructive pulmonary disease (COPD) and these complications, and a method for using the biomarker.
  • COPD chronic obstructive pulmonary disease
  • an object of the present invention is to provide a biomarker that reflects the pathological condition of obstructive pulmonary disease and a method for using the biomarker.
  • the present inventor has found that a specific protein group in extracellular vesicles of body fluid collected from a subject is useful as a biomarker for obstructive pulmonary disease.
  • these biomarkers can differentiate bronchial asthma from chronic obstructive pulmonary disease (COPD) and these complications.
  • COPD chronic obstructive pulmonary disease
  • a method for testing obstructive pulmonary disease comprising: (1) Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from the subject: (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig
  • the step (2) (2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value
  • (2c) When the amount or concentration of at least one protein selected from the protein group (A) is not less than a cut-off value, the subject suffers from bronchial asthma without chronic obstructive pulmonary disease.
  • the examination method according to Item 2 which is at least one step selected from the group consisting of the steps of determining that both lung diseases are affected.
  • the cut-off value is 2.5 to 6 times the value of the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from a subject not suffering from obstructive pulmonary disease, 3.
  • Item 5. The examination method according to any one of Items 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
  • the protein (A) group is (A1) 14-3-3 protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2 (VI) chain, Proteoglycan 4, Glycophorin- C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B) group is ( B1) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin -1, Alpha-2-macroglobulin, Collagen alpha-1 (I) chain, Platelet factor 4,
  • the protein (A1) group is a group consisting of (A2) Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein
  • the protein (B1 ) Group is a group consisting of (B2) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, and Complement C1q subcomponent subunit B.
  • Item 7. The inspection method according to Item 6.
  • Item 8 The examination method according to any one of Items 1 to 7, wherein the subject is a mouse.
  • Item 11 The screening method according to Item 10, comprising a step of selecting the animal as an obstructive pulmonary disease model animal when the index value is equal to or greater than a cutoff value.
  • Item 14 When the value of the indicator is lower than the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance, the test substance is used to prevent obstructive pulmonary disease or Item 14.
  • a biomarker for obstructive pulmonary disease can be provided.
  • testing for obstructive pulmonary disease, determination of the degree of eosinophil infiltration, screening for obstructive pulmonary disease model animals, etc. should be performed more simply, more efficiently and at lower cost. Can do.
  • COPD chronic obstructive pulmonary disease
  • the number of particles and the particle size of the extracellular vesicle fraction are shown (Test Example 3).
  • Control shows the result of extracellular vesicle fraction obtained from normal mice
  • Asthma shows the result of extracellular vesicle fraction obtained from bronchial asthma model mice
  • COPD from chronic obstructive pulmonary disease model mice
  • the result of the obtained extracellular vesicle fraction is shown. In both disease groups, no significant difference was observed in the number of particles and particle size compared to the control.
  • An electron microscopic observation image (immunoelectron microscopic result) of the extracellular vesicle fraction is shown (Test Example 3).
  • Beads labeled with CD9 which is an exosome marker, are attached to the vesicles, and the particle diameter is 100 nm or less, which is no different from previous reports.
  • Control shows the result of extracellular vesicle fraction obtained from normal mice
  • Asthma shows the result of extracellular vesicle fraction obtained from bronchial asthma model mice
  • COPD chronic obstructive pulmonary disease model mice The result of the obtained extracellular vesicle fraction is shown.
  • the present invention is a method for examining obstructive pulmonary disease, comprising: (1) Detect at least one protein selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C) in extracellular vesicles of a body fluid collected from a subject
  • the present invention relates to a test method (in this specification, sometimes referred to as “the method for testing pulmonary obstructive pulmonary disease of the present invention”) including a step (step 1). This will be described below.
  • the “obstructive pulmonary disease” to be examined is not particularly limited, and specific examples include bronchial asthma, COPD, diffuse panbronchiolitis, obstructive bronchiolitis and the like.
  • the subject is a target organism of the inspection method of the present invention, and its species is not particularly limited.
  • the biological species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably mice.
  • specimens are samples that are unclear whether they have obstructive pulmonary disease, for example, specimens that have already been determined by other methods to have bronchial asthma, but are unclear whether they have COPD, Samples that have already been determined by other methods to have COPD but are unclear whether they have bronchial asthma, samples that have already been determined by other methods to have bronchial asthma and COPD Samples that have already been determined by another method as not having obstructive pulmonary disease, samples being treated for obstructive pulmonary disease, and the like.
  • Body fluid is not particularly limited.
  • the body fluid include whole blood, serum, plasma, spinal fluid, saliva, joint fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and preferably whole blood, serum. , Plasma and cerebrospinal fluid, and more preferably whole blood, serum and plasma.
  • Body fluids may be used alone or in combination of two or more.
  • Body fluid can be collected from the subject by methods known to those skilled in the art.
  • whole blood can be collected by blood collection using a syringe or the like.
  • Serum is a portion obtained by removing blood cells and a specific blood coagulation factor from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood.
  • Plasma is a part obtained by removing blood cells from whole blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not coagulate whole blood.
  • Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted and released from cells.
  • Extracellular vesicles are usually defined as membrane vesicles that carry information between cells locally and throughout the body by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell.
  • the Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, and secretory microvesicles.
  • Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or according to known methods.
  • methods for purifying, separating and concentrating extracellular vesicles include, for example, ultracentrifugation (eg, pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), a method using an immunoaffinity carrier, gel filtration, Examples thereof include a flow fractionation method and a FACS method.
  • purification, separation, concentration and the like of extracellular vesicles can be performed using a commercially available kit. These methods may be employed singly or in combination of two or more.
  • the detection target of step (1) is at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) (in the present specification, these are collectively referred to as “ It may be indicated as “target protein”).
  • Protein group (A) is (A) IgIlambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8 , Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosHOtan-bindingHSPHO2 protein 2, Integrin beta-2, Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1 , Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha
  • Protein group (A) is a bronchial asthma-specific biomarker, and it can be used as an index to distinguish bronchial asthma without chronic obstructive pulmonary disease.
  • the protein group (A) from the viewpoint of more reflecting the disease state (higher correlation with the degree of eosinophil infiltration), preferably 14-3-314protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2 (VI) chain, Proteoglycan 4, Glycophorin-C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serumoid A-1 protein, Serum amyloid A-2 protein, etc. are mentioned.
  • Amyloid beta is preferable.
  • Examples include A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, and the like.
  • Protein group (B) is Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen ⁇ alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, alphamin-5 subunit , Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain-2 Integrin beta-2, F-box only protein 40, Thrombo
  • Protein group (B) is a biomarker specific to chronic obstructive pulmonary disease, and by using this as an index, it is possible to distinguish chronic obstructive pulmonary disease that is not associated with bronchial asthma.
  • the protein group (B) from the viewpoint of more reflecting the disease state (higher correlation with the average alveolar diameter), preferably Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r- A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin-1, Alpha-2-macroglobulin, Collagen alpha-1 (I) chain , Platelet factor 4, Vesicle-associated membrane membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing protein-bulbulin-like extracellular matrix protein 1, collagen-alpha-2 (I) chain, Multimerin-1, Ras-related protein Rab- 7a, Serine incorporator 3, Basigin, C4b-binding protein, etc.
  • Cytohesin-2 is preferable from the viewpoint that the amount ratio to the normal specimen is higher (including the case where the expression level of the control cannot be confirmed).
  • -antiplasmin Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV genly cancCol41 2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2script, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1
  • Erythrocyte band 7 integral membrane protein preferably Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan-2, Collagen alpha chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like tranminin protein 1 subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (
  • the protein group (B) from the viewpoint of more reflecting the disease state (higher correlation with the average alveolar diameter) and higher amount ratio to the normal specimen, preferably Alpha-2- antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B and the like.
  • EGF-containing fibulin-like extracellular matrix protein 1 is preferable from the viewpoint of being more promising as a common biomarker for mice and humans.
  • Protein group (C) is a protein group consisting of Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 is there.
  • Protein group (C) is a specific biomarker for both bronchial asthma and chronic obstructive pulmonary disease. By using this as an index, bronchial asthma (or bronchial asthma complication type) Chronic obstructive pulmonary disease) can be differentiated.
  • Interferon-induced transmembrane protein 3 Collagen alpha-1 (XVIII) chain, Fibulin-2, Adenylyl cyclase
  • Collagen alpha-1 (XVIII) chain Collagen alpha-1 (XVIII) chain
  • Complement C1q subcomponent subunit C and the like are preferable.
  • Collagen alpha-1 (XVIII) chain preferably Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Examples include Complement C1q subcomponent subunit C, Fibulin-2, Adenylyl cyclase-associated protein 1.
  • Collagen alpha-1 (XVIII) chain preferably Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2
  • Examples include Complement1C1q subcomponent subunit ⁇ C.
  • Proteins (A) to (C) are proteins specified by UniProtKB accession numbers shown in Tables 1 to 4 in Examples described later in the case of mice. In the case of other species, it is an ortholog of the protein specified by the UniProtKB accession numbers shown in Tables 1-5.
  • the number of target proteins may be only one type or a combination of two or more types.
  • more detection targets eg 2, 5, 10, 20, 40, 80, 120 or more
  • Detecting is usually performed by measuring the amount or concentration of the target protein.
  • the “density” is not limited to the absolute density, but may be a relative density, a weight per unit volume, or raw data measured to know the absolute density.
  • the method for detecting the target protein is not particularly limited as long as it is a method capable of specifically detecting a part or all of the target protein.
  • Specific examples of the detection method include a mass spectrometry method for detecting a peptide constituting the target protein, and an immunological measurement method using an antibody that specifically recognizes the target protein.
  • the amino acid sequence information of the target protein can be obtained by searching the EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) database based on the UniProtKB accession number.
  • an immunohistochemical staining method an ELISA method, an EIA method, an RIA method, a Western blotting method and the like can be preferably exemplified.
  • a peptide sample is turned into gaseous ions using an ion source (ionization), and the peptide sample ionized by moving it in a vacuum and using electromagnetic force or by a time-of-flight difference in the analysis section is mass-charged.
  • ionization ionization
  • This is a measurement method using a mass spectrometer that can be separated and detected according to the ratio.
  • Ionization using an ion source includes the EI method, CI method, FD method, FAB method, MALDI method, and ESI method.
  • the method of separating ionized peptide samples in the analysis section can be selected as appropriate.
  • a separation method such as an ion cyclotron resonance type can be appropriately selected.
  • tandem mass spectrometry (MS / MS) or triple quadrupole mass spectrometry combining two or more mass spectrometry methods can be used.
  • the sample contains a phosphorylated peptide
  • the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer.
  • the peptide which comprises target protein can be isolate
  • a detection part and a data processing method can also be selected suitably.
  • a peptide labeled with a stable isotope having a known amino acid sequence and having a known concentration is used as an internal standard. can do.
  • Such a stable isotope labeled peptide is a stable isotope labeled peptide in which at least one of the amino acids in the peptide constituting the target protein to be detected contains at least one of 15 N, 13 C, 18 O, and 2 H. If necessary, the type, position, number, etc. of amino acids can be appropriately selected.
  • Such stable isotope-labeled peptides can be obtained by F-moc method (Amblard., Et al. Methods) using amino acids labeled with stable isotopes.
  • Mol Biol. 298: 3-24 (2005) can be chemically synthesized by iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark). (Trademark) It can also produce using labeling reagents, such as a reagent.
  • the amount and / or concentration of the target protein which is a detection index of obstructive pulmonary disease can be provided, thereby assisting in the detection of obstructive pulmonary disease and the like. can do.
  • the amount and / or concentration of the target protein that is a detection index can be provided, thereby assisting in the evaluation of the degree of eosinophil infiltration. You can also.
  • eosinophil infiltration is involved in bronchial asthma activity (hypersensitivity, reversibility, etc.), acute exacerbation of COPD, etc.
  • the evaluation of eosinophil infiltration is based on the judgment of the possibility of acute aversion and the poor prognosis group It can be used for identification of In this way, eosinophil infiltration is an important factor involved in obstructive disease, but how to evaluate it. No method other than the method requiring a complicated test of measuring exhaled NO (FeNO) has been known. If the biomarker of the present invention is used, it is possible to evaluate eosinophil infiltration more simply and efficiently than before without requiring such a complicated operation.
  • test results of the test method of the present invention including the step (1) are as follows: elucidation of the pathological condition of obstructive pulmonary disease, prognosis prediction of obstructive disease (respiratory function decline group, acute exacerbation), stratification of subjects, treatment method It can be used for selection (individualized medicine, treatment responsiveness), refractory in obstructive pulmonary disease (particularly bronchial asthma), evaluation of remodeling, differentiation of histological type, phenotype, etc. of obstructive pulmonary disease.
  • BM correlates with various eosinophil infiltration found by the present invention is a companion BM of various phenotypes (clustering) and various molecular target drugs (th2 targeted) currently under development. It can also be.
  • the inspection method of the present invention further includes, as one aspect, (2) including a step of determining that the subject suffers from obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is equal to or higher than a cutoff value (step 2) It is preferable. According to the test method of the present invention including the step 2, it becomes possible to determine obstructive pulmonary disease.
  • step 2 is divided into the following (2a) to (2e), for example.
  • (2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value
  • (2c) When the amount or concentration of at least one protein selected from the protein group (A) is not less than a cut-off value, the subject suffers from bronchial asthma without chronic obstructive pulmonary disease.
  • Step (2) is more preferably at least one step selected from the group consisting of Steps 2a to 2e.
  • the cut-off value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc., for example, collected from a subject not suffering from obstructive pulmonary disease Based on the amount and / or concentration of the target protein in the extracellular vesicles of the body fluid, it can be a value determined each time or a predetermined value.
  • the cut-off value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles of body fluid collected from a subject not suffering from obstructive pulmonary disease (in the case of multiple subjects, the average value, the center Value), for example, 1 to 10 times, preferably 2 to 8 times, more preferably 2.5 to 6 times.
  • the amount or concentration of the protein detected in the step (1) is greater than or equal to a cut-off value by using a biomarker reflecting the pathology of obstructive pulmonary disease.
  • the cut-off value is a certain value or more by setting the value based on the amount and / or concentration of the target protein in the extracellular vesicles of a body fluid collected from a subject suffering from obstructive pulmonary disease, for example. It can be evaluated whether or not the disease is.
  • the cutoff value is set to a value based on the amount and / or concentration of the target protein in the past sample for the same sample, for example. The effect can be determined.
  • the amount or concentration of the protein detected in the step (1) is used as an index.
  • the degree of eosinophil infiltration can be determined. More specifically, for example, a calibration curve based on the correlation between the degree of eosinophil infiltration (the number of eosinophil infiltration within a certain area) and the amount or concentration of the biomarker is prepared and measured in advance. The degree of eosinophil infiltration can be determined from the amount or concentration of sucrose and the calibration curve.
  • the test method of the present invention further includes By combining the steps of applying the diagnosis by the doctor of obstructive pulmonary disease, the obstructive pulmonary disease can be diagnosed with higher accuracy.
  • the test method of the present invention can detect obstructive pulmonary disease more accurately, combining the above-described steps with the test method of the present invention makes it more efficient and more accurate to “be afflicted with obstructive pulmonary disease. Can be diagnosed.
  • test method of the present invention including the treatment step (2) for obstructive pulmonary disease, in addition to the test method of the present invention or the above “2
  • diagnosis as suffering from obstructive pulmonary disease as described in ". Diagnosis of obstructive pulmonary disease with higher accuracy” the test method of the present invention is combined with the process of applying diagnosis by a doctor.
  • (3) treating the disease of the subject by performing a step of treating the disease on the subject determined or diagnosed as suffering from obstructive pulmonary disease. It becomes possible.
  • test method of the present invention can detect obstructive pulmonary disease more accurately, a process for the test method of the present invention or a combination of the test method of the present invention and a process of applying a diagnosis by a doctor By combining 3, it is possible to treat a subject suffering from obstructive pulmonary disease more efficiently and more reliably.
  • the method for treating obstructive pulmonary disease is not particularly limited, but a typical example is medication.
  • the drug used for the drug treatment is not particularly limited, but for example, anticholinergic drugs such as Spiriva, Seeburi, Enclasse, Ecrila, Atrovent, Telcigan, etc .; ⁇ 2-stimulating drugs such as Serebent, Ombres, Auxis, Sultanol, Meptin; Ultimolo, Anolo And anti-cholinergic / ⁇ 2-stimulant combination drugs such as sport; steroid drugs such as cuvard, flutide, palmicoat, olvesco, and azmanex; steroid drugs / beta-stimulant combination drugs such as adair, simbicoat, and lerbea.
  • a pharmaceutical can be used 1 type, 2 types, or 3 or more types in combination.
  • the present invention provides an agent for detecting at least one protein selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C).
  • the present invention relates to a test agent for obstructive pulmonary disease (also referred to as “the test agent of the present invention” in the present specification). This will be described below.
  • Protein group (A), protein group (B), protein group (C), obstructive pulmonary disease and the like are the same as defined in “1. Method for examining obstructive pulmonary disease” above.
  • the detection agent is not particularly limited as long as it can specifically detect the target protein.
  • the detection agent include an antibody against the target protein.
  • the detection agent may be labeled. Examples of the label include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Further, the detection agent (particularly antibody) may be in a state of being adsorbed on a base material (for example, a plastic base material such as a microwell plate).
  • the antibody is not particularly limited as long as it recognizes the target protein selectively (specifically).
  • “selectively (specifically) recognize” means that the protein of interest can be specifically detected, for example, in Western blotting or ELISA, but is not limited thereto. Any substance can be used as long as it can be determined that the detected substance is derived from the target protein.
  • the antibody includes a part of the above antibody having antigen binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library.
  • the antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of at least continuous amino acid sequence of the subject protein, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)).
  • the antibody of the present invention is a polyclonal antibody
  • an oligopeptide having a partial amino acid sequence of the target protein is synthesized using a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or according to a conventional method.
  • a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method.
  • spleen cells obtained by immunizing a non-human animal such as a mouse with a target protein expressed and purified in Escherichia coli according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein and It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
  • the target protein used as an immunizing antigen for antibody production is based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of a transformant, and recovery of the protein from the culture. It can obtain by operation of. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
  • a recombinant DNA capable of expressing a gene encoding the target protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured.
  • the protein as the immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture.
  • the partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) in accordance with known gene sequence information.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has an immunogenic property similar to that of the target protein.
  • An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
  • Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other components as necessary. Examples of other components include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, and a fragrance. And chelating agents.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain a substance that can be used for detection of a target protein in extracellular vesicles of a body fluid of a subject.
  • reagents for example, secondary antibodies, buffers, etc.
  • instruments for example, instruments for purification, separation, and concentration of extracellular vesicles (for example, columns)).
  • the present invention provides a protein group (A) and a protein group (B) in an extracellular vesicle of a body fluid collected from an animal treated for induction of obstructive pulmonary disease. And a method for screening an obstructive pulmonary disease model animal using the amount or concentration of at least one protein selected from the group consisting of the protein group (C) as an index (hereinafter referred to as “model animal screening of the present invention”). Method "). This will be described below.
  • the animal is an animal to be screened, and its species is not particularly limited as long as it is a non-human animal.
  • the biological species of the animal include various mammalian animals such as monkeys, mice, rats, dogs, cats, rabbits and the like, preferably mice.
  • the obstructive pulmonary disease induction treatment is not particularly limited as long as it is a treatment capable of inducing obstructive pulmonary disease.
  • OVA ovalbumin
  • administration of an inducer such as mite, pollen, insect, etc.
  • the induction treatment is performed by air administration, intravenous administration, transdermal administration, etc. (preferably a combination of intraperitoneal administration and nasal administration) or by genetic modification treatment.
  • inducers such as elastase, smoking exposure, tobacco extract, LPS + tobacco extract (eg, nasal administration, oral administration, inhalation administration, transbronchial administration
  • the induction treatment is performed by intraperitoneal administration, intravenous administration, transdermal administration, etc. (preferably nasal administration) or by genetic modification treatment.
  • the model animal screening method of the present invention selects the animal as an obstructive pulmonary disease model animal (or an obstructive pulmonary disease model candidate animal) when the value of the index is not less than a cutoff value.
  • the process of carrying out is included.
  • the cut-off value can be appropriately set by those skilled in the art from the viewpoint of screening accuracy and the like, for example, the amount of the target protein in the extracellular vesicles of body fluid collected from an animal that has not been induced to induce obstructive pulmonary disease, and Based on the density, it can be a value determined each time or a predetermined value.
  • the cut-off value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles of body fluid collected from an animal that has not been induced to induce obstructive pulmonary disease (in the case of multiple subjects, the average value, the median value) For example, 1 to 10 times, preferably 2 to 8 times, and more preferably 2.5 to 6 times.
  • a prophylactic or therapeutic agent for obstructive pulmonary disease suppresses at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C).
  • the present invention relates to a prophylactic or therapeutic agent for obstructive pulmonary disease containing the agent (sometimes referred to as “the agent of the present invention”). This will be described below.
  • Protein group (A), protein group (B), protein group (C) and the like are the same as defined in “1. Method for examining obstructive pulmonary disease” above.
  • the inhibitor is preferably an expression inhibitor of the target protein.
  • the target protein expression inhibitor is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, etc.
  • gene-specific small interfering RNA (siRNA) of the target protein gene-specificity of the target protein MicroRNA (miRNA), gene-specific antisense nucleic acid of target protein, expression vectors thereof; gene-specific ribozyme of target protein; gene gene editing agent of target protein by CRISPR / Cas system.
  • the expression suppression refers to the expression level of the target protein, its mRNA, etc., for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100 , 1/200, 1/300, 1/500, 1/1000, 1 / 10,000 or less, and the expression level of these is set to 0.
  • the gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein.
  • the siRNA is preferably 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length, for example.
  • it is preferable that siRNA is 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less, for example. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined.
  • the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases, and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases A certain length; a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 Combinations of lengths that are bases are envisioned.
  • the siRNA may be shRNA (small hairpin RNA).
  • shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is a sequence a and the complementary strand to the sequence a is a sequence b, these sequences are present in a single RNA strand in the order of sequence a, spacer, and sequence b. And can be designed to be 45-60 bases in total.
  • the sequence a is a sequence of a partial region of the base sequence encoding the target protein to be targeted, and the target region is not particularly limited, and any region can be a candidate.
  • the length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.
  • the gene-specific siRNA of the target protein may have an additional base at the 5 'or 3' end.
  • the length of the additional base is usually about 2 to 4 bases.
  • the additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuuu-3'.
  • SiRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents deoxythymidine) is added. Further, it may be a blunt end (blunt end) without end addition.
  • the siRNA may have a different number of bases in the sense strand and the antisense strand. For example, the “asymmetrical interfering RNA” in which the antisense strand has an overhang at the 3 ′ end and the 5 ′ end ( aiRNA) ".
  • a typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases at each end of the antisense strand.
  • the position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, the target is from the 5′-UTR and the start codon to about 50 bases, and from a region other than the 3′-UTR. It is desirable to select the sequence.
  • the selected target sequence candidate group whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ It is preferable to check using a homology search software such as) to confirm the specificity of the selected target sequence.
  • a sense strand having a 3 'end overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 ′ end overhang of UU may be designed as an siRNA.
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are connected via the linker sequence. It can be designed by connecting.
  • siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include the following. SiRNA Target Finder provided by Ambion (http://www.ambion.com/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer® Expression Vector (http://www.ambion.com/ jp / techlib / misc / psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
  • the siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with an RNA-cleaving protein dicer.
  • the gene-specific miRNA of the target protein is arbitrary as long as the translation of the gene encoding the target protein is inhibited.
  • an miRNA may pair with the 3 'untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA like an siRNA.
  • the miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA.
  • the length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ⁇ 30 bases.
  • the gene-specific miRNA of the protein of interest is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA.
  • a gene-specific miRNA of the target protein may be synthesized by a known technique or purchased from a company that provides synthetic RNA.
  • the gene-specific antisense nucleic acid of the target protein is a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA encoding the target protein, or a part thereof, and specific to the mRNA. It is a nucleic acid having a function of suppressing target protein synthesis by forming and binding an ideal and stable duplex.
  • the antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera.
  • the antisense nucleic acid is DNA
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA.
  • the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of interest protein.
  • the intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the target protein gene using a homology search program such as BLAST or FASTA.
  • the length of the target region of the gene-specific antisense nucleic acid of the target protein is not limited as long as the antisense nucleic acid is hybridized and consequently translation into the target protein is inhibited.
  • the gene-specific antisense nucleic acid of the target protein may be the entire sequence or a partial sequence of mRNA encoding the target protein.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but is not limited thereto.
  • a 3 ′ end hairpin loop or the like can be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • the gene-specific antisense nucleic acid of the target protein not only hybridizes with the mRNA or initial transcription product of the target protein gene and inhibits translation into the protein, but also binds to these genes that are double-stranded DNA. It may be a substance capable of forming a triplex and inhibiting transcription to RNA (antigene).
  • the nucleotide molecules constituting the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein have stability (chemical and / or anti-enzyme) and specific activity ( Various chemical modifications may be included in order to improve (affinity with RNA).
  • a phosphate residue (phosphate) of each nucleotide constituting an antisense nucleic acid is selected from, for example, phosphorothioate (PS), methylphosphonate, phosphorodithio. It can be substituted with a chemically modified phosphate residue such as a phosphorodithioate.
  • the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R ⁇ CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , or CH 2 CH 2 CN) may be substituted.
  • the base moiety pyrimidine, purine
  • pyrimidine, purine may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. May be applied.
  • a part of nucleotide molecules constituting siRNA or miRNA may be replaced with natural DNA.
  • Gene-specific siRNA of the target protein, gene-specific miRNA of the target protein, and gene-specific antisense nucleic acid of the target protein are targets of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the target protein gene It can be prepared by determining the sequence and synthesizing a complementary sequence using a commercially available DNA / RNA automatic synthesizer.
  • any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.
  • the expression vector comprises a promoter sequence and a gene-specific siRNA of the protein of interest, a gene-specific miRNA of the protein of interest, or a coding sequence of a gene-specific antisense nucleic acid of the protein of interest (optionally further A polynucleotide containing a transcription termination signal sequence) and optionally other sequences.
  • the promoter is not particularly limited, and examples thereof include RNA-polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter; mouse and human U6-snRNA promoters, Examples include human H1-RNase P ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ RNA promoter, human valine-tRNA promoter, and other RNA polymerase III (polIII) promoters. Among these, polIII promoters can be used for accurate transcription of short RNAs. preferable.
  • Other sequences are not particularly limited, and various known sequences that can be included in the expression vector can be employed. Examples of such sequences include the origin of replication and drug resistance genes. Examples of the drug resistance gene and the vector include those described above.
  • RNA-specific ribozyme of the target protein is a gene-specific ribozyme of the target protein.
  • “Ribozyme” in a narrow sense means RNA having an enzyme activity for cleaving nucleic acid, but in the present application, it includes DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA.
  • ribozyme nucleic acid has an advantage that it does not attack genomic DNA because it uses only RNA as a substrate.
  • the target sequence is single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • ribozymes when used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • the application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats and deer. .
  • the form of the agent of the present invention is not particularly limited, and can take a form normally used in each application depending on the application of the agent of the present invention.
  • the form when the use is a medicine, health enhancer, nutritional supplement (eg supplement), etc., for example, tablets (inner disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.) ), Pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral intake Forms (oral dosage forms), nasal drops, inhalants, rectal suppositories, inserts, enemas, jellies, injections, patches, lotions, creams, etc. Oral dosage form).
  • tablets inner disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.
  • Pills granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral intake Forms (oral dosage forms), nasal drops,
  • a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula , Cake mix, powdered or liquid dairy products, bread, cookies and the like.
  • an oral composition for example, liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate agent, capsule, Film, kneaded material, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid toothpaste, liquid toothpaste, powder toothpaste etc.), mouthwash,
  • a coating agent for example, chewing gum, tablet candy, candy, gummi, film, troche, etc.
  • the agent of the present invention may further contain other components as necessary.
  • Other components are not particularly limited as long as they are components that can be blended in, for example, pharmaceuticals, food compositions, oral compositions, health enhancers, nutritional supplements (such as supplements), etc. , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.
  • the content of the inhibitor of the target protein of the agent of the present invention depends on the type of inhibitor, application, use mode, application target, application target state, and the like, and is not limited. For example, 0.0001 to 100 % By weight, preferably 0.001 to 50% by weight.
  • the amount of application (eg, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits a medicinal effect, and is generally 0.1 to 1000 per day as the weight of the active ingredient. mg / kg body weight.
  • the above dose is preferably administered once a day or divided into 2 to 3 times a day, and can be appropriately increased or decreased depending on age, disease state, and symptoms.
  • the screening method of the active ingredient of the preventive or therapeutic agent for obstructive pulmonary disease is a protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance, A screening method for an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease using the amount or concentration of at least one protein selected from the group consisting of protein group (B) and protein group (C) as an index (this book) In the specification, it may be referred to as “the active ingredient screening method of the present invention”. This will be described below.
  • Species of animal species are not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably mice.
  • test substance can be widely used regardless of whether it is a naturally occurring compound or a man-made compound.
  • composition which mixed not only the refined compound but various compounds, and the extract of animals and plants can also be used.
  • the compounds include not only low molecular compounds but also high molecular compounds such as proteins, nucleic acids, and polysaccharides.
  • the active ingredient screening method of the present invention is such that the value of the index is the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal that has not been treated with the test substance (control value). And a step of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease (or a candidate substance for an active ingredient of prophylactic or therapeutic agent for obstructive pulmonary disease).
  • Corresponding protein means the same protein as the target protein used as an index.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • the present invention in one aspect thereof, is a protein group (A), a protein group in an extracellular vesicle of a body fluid collected from an animal treated with a test substance. (B) and an evaluation method for inducing or malignancy of obstructive pulmonary disease using the amount or concentration of at least one protein selected from the group consisting of the protein group (C) as an index (in the present specification, It may be referred to as “the toxicity evaluation method of the present invention”. This will be described below.
  • obstructive lung disease For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive lung disease, measurement of the amount or concentration of the target protein, animal species, test substances, etc.
  • the definition is the same as in “1. Method for testing obstructive pulmonary disease” and “7. Screening method for active ingredient of prophylactic or therapeutic agent for obstructive pulmonary disease”.
  • the value of the above-mentioned index is the amount or concentration of the corresponding protein in the extracellular vesicles of body fluid collected from an animal that has not been treated with the test substance (control value).
  • the test substance is determined to be inducible or malignant for obstructive pulmonary disease.
  • Corresponding protein means the same protein as the target protein used as an index.
  • “High” means, for example, that the index value is 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value.
  • Test Example 1 Preparation of serum of bronchial asthma model mice 11-week-old C57BL6 / J male mice were used. 20 ⁇ g of OVA (Sigma, A5503, Grade V) and 2 mg of Alum (Thermo Imject Alum (# 77161 Lot NB168145)) were suspended in 400 ⁇ L of PBS and administered intraperitoneally to mice anesthetized with 4% isoflurane. Seven days after the first administration, a suspension of OVA (20 ⁇ g) and Alum (2 mg) in PBS (400 ⁇ L) was intraperitoneally administered again. 21, 22, and 23 days after the initial administration, a suspension of OVA (50 ⁇ g) in PBS (50 ⁇ L) was administered intranasally.
  • OVA Sigma, A5503, Grade V
  • Alum Thermo Imject Alum (# 77161 Lot NB168145)
  • Serum was collected 28 days after the first dose.
  • COPD chronic obstructive pulmonary disease
  • the extracellular vesicle fraction was prepared using an extracellular vesicle purification column (EV-Second, manufactured by GL Sciences).
  • the number and particle diameter of the extracellular vesicles were measured for the obtained extracellular vesicle fraction.
  • the measurement was carried out using a nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion speed for each particle size, tracking (tracking) each individual scattered light reflected on the screen, and using each moving speed (diffusion coefficient) in the liquid.
  • the particle diameter (hydrodynamic diameter) can be calculated. The results are shown in FIG.
  • extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was performed as follows. 5 to 8 ⁇ L of the fixed extracellular vesicle solution was placed on the grid and allowed to stand for 15 minutes to fix the extracellular vesicles to the form bar of the grid. After washing 3 times with PBS, blocking reaction (1% BSA / PBS, 10 minutes) followed by primary antibody reaction (BD Pharmingen 553758 Purified NA / LE Rat Anti-Mouse CD9 Clone: KMC8, 100 times dilution, 2 Half hour, room temperature).
  • Test Example 4 Proteomics analysis (non-label, LC-MS / MS) The protein in the extracellular vesicle fraction was quantified by LC-MS / MS analysis (non-label method). Specifically, it was performed as follows.
  • sample preparation The extracellular vesicle fraction was reduced with 5 mM TCEP for 30 minutes at 37 ° C. and alkylated with 25 mM iodoacetamide for 45 minutes at room temperature. The sample was then diluted 7-fold with 50 mM ammonium bicarbonate, placed in a 96 well filter plate and digested by shaking with 5 ⁇ L of immobilized trypsin (Thermo Fisher Scientific) at 37 ° C. for 6 hours. The trypsin digest was desalted using Oasis HLB 96-well ⁇ Elution Plate (Waters Corporation, USA) and subjected to LC-MS / MS analysis.
  • Oasis HLB 96-well ⁇ Elution Plate Waters Corporation, USA
  • the eluted peptides were ionized with a spray voltage of 2000 V and MS data was acquired by the data-dependent fragment method.
  • the measurement scan (survey scan) was performed at m / z 400 to 1600, resolution 60000, AGC target value 1.0 ⁇ 10 6 ion count.
  • the top 20 intensities of precursor ions in each measurement scan were subjected to low-resolution MS / MS acquisition using a normal CID scan mode with an AGC target value of 5000 ions count with a linear ion trap.
  • FIG. 1 shows the overall workflow of RefinerMS software.
  • Test Example 5 Detection of chronic obstructive pulmonary disease biomarker in lung tissue by immunostaining Lung tissue of normal mice (4 mice), bronchial asthma model mice (8 mice) and chronic obstructive pulmonary disease (COPD) model mice (4 mice) was immunostained with an antibody against a chronic obstructive pulmonary disease biomarker (Test Example 4).
  • Anti-EEA1 antibody (code: ab2900 / abcam), anti-VCAN antibody (code: ab19345 / abcam), anti-PRG4 antibody (code: LS-B8236 / LSBio), anti-decorin antibody (code: ab175404 / abcam) ), Anti-HABP2 antibody (code: ab174953 / abcam), anti-EFEMP1 antibody (code: GTX111657 / GeneTex), anti-fibulin2 antibody (code: bs-13160R / Bioss), anti-biglycan antibody (code: ab49701 / abcam), anti-TSP1 An antibody (code: ab85762 / abcam) and an anti-CAP1 antibody (code: ab182604 / abcam).
  • the expression level of the chronic obstructive pulmonary disease biomarker tends to be higher in the lung tissues of the bronchial asthma model mouse and the chronic obstructive pulmonary disease model mouse than in the lung tissue of the normal mouse. there were. Moreover, the examined chronic obstructive pulmonary disease biomarkers tended to be expressed in higher amounts in the lung tissue of bronchial asthma model mice than in the lung tissue of chronic obstructive pulmonary disease model mice.
  • the correlation with the amount of the extracellular vesicle fraction of the chronic obstructive pulmonary disease biomarker measured in Test Example 4 was analyzed. As a result, it was found that the biomarker for chronic obstructive pulmonary disease has a positive correlation between the amount in the extracellular vesicle fraction and the expression level in lung tissue.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Nutrition Science (AREA)
  • Biophysics (AREA)
  • Polymers & Plastics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pulmonology (AREA)

Abstract

The purpose of the present invention is to provide a biomarker for obstructive pulmonary diseases such as bronchial asthma, and a method for using the biomarker. The present invention involves extracellular-vesicle protein groups (A) through (C) which are in a body fluid collected from a subject.

Description

閉塞性肺疾患バイオマーカーBiomarker for obstructive pulmonary disease
　本発明は、閉塞性肺疾患バイオマーカー、その利用等に関する。 The present invention relates to a biomarker for obstructive pulmonary disease, use thereof, and the like.
　気管支喘息（BA）は患者数が3億人に及ぶと推測されており、その有病率の増加が社会的・経済的負担となっている。気管支喘息の本態は、慢性の気道炎症であるが、炎症を制御する鍵分子や気道リモデリングの解明は不十分である。一方、慢性閉塞性肺疾患（COPD）は、2020年には世界死因の第3位と推定されているものの、その診断は呼吸機能の評価のみに依存しているために、多くの患者は未診断のまま放置され、病態解明が不十分である。 Bronchial asthma (BA) is estimated to have 300 million patients, and the increase in prevalence is a social and economic burden. The true form of bronchial asthma is chronic airway inflammation, but the elucidation of key molecules that control inflammation and airway remodeling is insufficient. On the other hand, chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death in the world in 2020, but the diagnosis depends only on the evaluation of respiratory function, so many patients have not yet been diagnosed. It is left as diagnosed, and the pathophysiology is insufficient.
　気管支喘息、COPD等の閉塞性肺疾患は、個々の遺伝子やタンパク質の変異そのものが病態を形成するというよりも、それらが相互作用して形成された細胞分子ネットワークの調節不全が基礎となって発症すると考えられる。実際、気流閉塞と炎症を特徴とする喘息とCOPDには種々の表現型があり、このことがこれらの疾患の病態解明をより困難にしていると考えられる。さらに両疾患が混在するオーバーラップ症候群も高率に存在するものの、鑑別方法や詳細な病態は不明である。 Obstructive pulmonary diseases such as bronchial asthma and COPD develop on the basis of dysregulation of the cellular molecular network formed by the interaction of individual genes and proteins rather than the mutations themselves. I think that. In fact, there are various phenotypes of asthma and COPD characterized by airflow obstruction and inflammation, and this seems to make it more difficult to elucidate the pathogenesis of these diseases. Furthermore, although there is a high rate of overlap syndrome in which both diseases coexist, the differentiation method and detailed pathology are unknown.
　COPDは、世界に2億人の患者が推定され、日本では530万人、そのうち治療を受けている患者はわずか5%に過ぎない。一方、世界2億人の患者が推定されている喘息は、吸入ステロイドの普及により死亡者数は減少しているものの、罹患数は高齢化とともに増加し続けている。最近、COPDと喘息を合併するACOは、予後不良であることやCOPD患者の20%とも推定されているため注目されている。さらに、喘息は種々の表現型（フェノタイプ）やエンドタイプが存在するため、個別化医療が求められている。このような現状のなかで、Th2 typeの炎症を誘導するIL-5, IL-13, IgEなどの分子を標的とした治療が次々に開発されてきた。海外でも次々にTh2タイプの炎症を標的とした治療が開発されているものの、治療選択や有効性を判断する指標（コンパニオンバイオマーカー）はほとんど開発されていないのが現状である。 COPD is estimated to have 200 million patients worldwide, and 5.3 million in Japan, of which only 5% are being treated. On the other hand, asthma, estimated to have 200 million patients worldwide, has been declining due to the widespread use of inhaled steroids, but the number of affected cases continues to increase with aging. Recently, ACO combined with COPD and asthma is attracting attention because of its poor prognosis and 20% of COPD patients. Furthermore, since asthma has various phenotypes (phenotypes) and end types, individualized medicine is required. Under these circumstances, treatments targeting molecules such as IL-5, IL-13, and IgE that induce Th2 type inflammation have been developed one after another. Although treatments targeting Th2-type inflammation are being developed one after another overseas, there are few indicators (companion biomarkers) for judging treatment selection and effectiveness.
　マウスにおける疾患活動性を示すバイオマーカーの開発も急務である。なぜなら、創薬の開発にあたる大手製薬企業において、COPD（肺気腫）や喘息モデルの評価法は、マウス呼吸機能を一匹ずつ測定するという原始的方法に頼らざるを得ないため、ハイスループットスクリーニングが不可能である。 Developing biomarkers that indicate disease activity in mice is also urgent. This is because, in a major pharmaceutical company that develops drugs, COPD (pulmonary emphysema) and asthma model evaluation methods must rely on the primitive method of measuring mouse respiratory function one by one, which makes high-throughput screening difficult. Is possible.
　血清は、非侵襲的に繰り返し採取可能であるので理想的な検査試料と考えられるものの、そこに含まれる99%以上のタンパク質はバイオマーカーの観点から夾雑物とされる多量タンパク（アルブミン等）であるため、プロテオミクスの観点からは、検査資料としての有用性は低い。例えば、COPDと喘息を鑑別可能なバイオマーカーに関する最近の論文（非特許文献１）では、患者数十人の解析を試みるも、2次元電気泳動法による古い質量分析(MS)技術によるものであっただけでなく、大量夾雑物を含む血清を活用したため、見出されたバイオマーカーはプロトロンビンを含む血清夾雑物のみで、COPD特異的バイオマーカーとは言い難い。 Although serum is considered an ideal test sample because it can be repeatedly collected non-invasively, more than 99% of the protein contained therein is a large amount of protein (such as albumin) that is considered a contaminant from the viewpoint of a biomarker. Therefore, from the viewpoint of proteomics, its usefulness as a test material is low. For example, in a recent paper on biomarkers that can distinguish COPD from asthma (Non-Patent Document 1), an attempt was made to analyze dozens of patients, but it was based on the old mass spectrometry (MS) technique based on two-dimensional electrophoresis. In addition, since serum containing a large amount of contaminants was used, the only biomarkers found were serum contaminants containing prothrombin, which are difficult to say as COPD-specific biomarkers.
　細胞外小胞は、細胞から分泌される膜小胞であり、細胞内のタンパク質、脂質、核酸（mRNA, microRNA等）を細胞外に運搬することにより、局所や全身における細胞間の情報伝達を担っている。近年、がんの検査試料として細胞外小胞（エクソソーム）が注目を集めているものの、炎症性呼吸器疾患、特に二大閉塞性肺疾患である気管支喘息等の閉塞性肺疾患の検査試料としての有用性については知られていない。 Extracellular vesicles are membrane vesicles that are secreted from cells. By transporting intracellular proteins, lipids, and nucleic acids (mRNA, microRNA, etc.) to the outside of the cell, information transmission between cells in the local and whole body is performed. I'm in charge. In recent years, extracellular vesicles (exosomes) have attracted attention as test samples for cancer, but as test samples for inflammatory respiratory diseases, particularly obstructive pulmonary diseases such as bronchial asthma, which is the two major obstructive pulmonary diseases The usefulness of is not known.
　本発明は、気管支喘息等の閉塞性肺疾患のバイオマーカー及びその利用方法を提供することを課題とする。好ましくは、本発明は、気管支喘息と慢性閉塞性肺疾患（COPD）との鑑別やこれらの合併症の鑑別が可能なバイオマーカー及びその利用方法を提供することを課題とする。より好ましくは、閉塞性肺疾患の病態を反映するバイオマーカー及びその利用方法を提供することを課題とする。 An object of the present invention is to provide a biomarker for obstructive pulmonary diseases such as bronchial asthma and a method for using the same. Preferably, an object of the present invention is to provide a biomarker capable of differentiating between bronchial asthma and chronic obstructive pulmonary disease (COPD) and these complications, and a method for using the biomarker. More preferably, an object of the present invention is to provide a biomarker that reflects the pathological condition of obstructive pulmonary disease and a method for using the biomarker.
　本発明者は上記課題に鑑みて鋭意研究した結果、被検体から採取された体液の細胞外小胞における特定のタンパク質群が、閉塞性肺疾患のバイオマーカーとして有用であることを見出した。さらに研究を進めた結果、これらのバイオマーカーにより、気管支喘息と慢性閉塞性肺疾患（COPD）との鑑別やこれらの合併症の鑑別も可能であることを見出した。また、一部のバイオマーカーは、閉塞性肺疾患の病態を反映することをも見出した。これらの知見に基づいてさらに研究を進めた結果、本発明が完成した。即ち、本発明は、下記の態様を包含する。 As a result of intensive studies in view of the above problems, the present inventor has found that a specific protein group in extracellular vesicles of body fluid collected from a subject is useful as a biomarker for obstructive pulmonary disease. As a result of further research, it was found that these biomarkers can differentiate bronchial asthma from chronic obstructive pulmonary disease (COPD) and these complications. We have also found that some biomarkers reflect the pathology of obstructive pulmonary disease. As a result of further research based on these findings, the present invention was completed. That is, the present invention includes the following aspects.
　項１．　
閉塞性肺疾患を検査する方法であって、
（1）被検体から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
（B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
（C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質を検出する工程を含む、検査方法。 Item 1.
A method for testing obstructive pulmonary disease, comprising:
(1) Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from the subject:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A test method comprising a step of detecting at least one protein selected from the group consisting of a protein group consisting of Fibulin-2 and Adenylyl cyclase-associated protein 1.
　項２．　
さらに、（2）前記工程（1）で検出されたタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が閉塞性肺疾患に罹患していると判定する工程を含む、項１に記載の検査方法。 Item 2.
And (2) a step of determining that the subject suffers from obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is not less than a cutoff value. The inspection method according to 1.
　項３．　
前記工程（2）が、
（2a）前記タンパク質群（A）及び前記タンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息に罹患していると判定する工程、
（2b）前記タンパク質群（B）及び前記タンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が慢性閉塞性肺疾患に罹患していると判定する工程、
（2c）前記タンパク質群（A）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が慢性閉塞性肺疾患非合併型の気管支喘息に罹患していると判定する工程、
（2d）前記タンパク質群（B）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息非合併型の慢性閉塞性肺疾患に罹患していると判定する工程、並びに
（2e）前記タンパク質群（C）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息及び慢性閉塞性肺疾患の両方に罹患していると判定する工程
からなる群より選択される少なくとも1種の工程である、項２に記載の検査方法。 Item 3.
The step (2)
(2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value The process of determining that
(2b) When the amount or concentration of at least one protein selected from the group consisting of the protein group (B) and the protein group (C) is a cut-off value or more, the subject is chronic obstructive lung Determining that the patient is afflicted with a disease,
(2c) When the amount or concentration of at least one protein selected from the protein group (A) is not less than a cut-off value, the subject suffers from bronchial asthma without chronic obstructive pulmonary disease. A process of determining that
(2d) When the amount or concentration of at least one protein selected from the protein group (B) is equal to or higher than a cutoff value, the subject suffers from bronchial asthma non-complicated chronic obstructive pulmonary disease. And (2e) when the amount or concentration of at least one protein selected from the protein group (C) is equal to or higher than a cutoff value, the subject is bronchial asthma and chronic obstructive Item 3. The examination method according to Item 2, which is at least one step selected from the group consisting of the steps of determining that both lung diseases are affected.
　項４．　
前記カットオフ値が、閉塞性肺疾患に罹患していない被検体から採取された体液の細胞外小胞における対応タンパク質の量又は濃度の値の、2.5～6倍の値である、項２又は３に記載の検査方法。 Item 4.
The cut-off value is 2.5 to 6 times the value of the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from a subject not suffering from obstructive pulmonary disease, 3. The inspection method according to 3.
　項５．　
前記体液が全血、血漿、及び血清からなる群より選択される少なくとも1種である、項１～４のいずれかに記載の検査方法。 Item 5.
Item 5. The examination method according to any one of Items 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
　項６．　
前記タンパク質（A）群が（A1）14-3-3 protein theta、Thrombospondin-1、Platelet glycoprotein 4、Pyridoxal phosphate phosphatase PHOSPHO2、Integrin beta-2、Collagen alpha-2(VI) chain、Proteoglycan 4、Glycophorin-C、Versican core protein、Amyloid beta A4 protein、Ras-related protein Rap-2c、Decorin、Serum amyloid A-1 protein、及びSerum amyloid A-2 proteinからなる群であり、且つ前記タンパク質（B）群が（B1）Alpha-2-antiplasmin、Complement C1s-A subcomponent、Complement C1r-A subcomponent、Biglycan、Complement C1q subcomponent subunit B、Laminin subunit alpha-5、Interferon-induced transmembrane protein 2、Laminin subunit gamma-1、Serglycin、Thrombospondin-1、Alpha-2-macroglobulin、Collagen alpha-1(I) chain、Platelet factor 4、Vesicle-associated membrane protein 3、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Collagen alpha-2(I) chain、Multimerin-1、Ras-related protein Rab-7a、Serine incorporator 3、Basigin、及びC4b-binding proteinからなる群である、項１～５のいずれかに記載の検査方法。 Item 6.
The protein (A) group is (A1) 14-3-3 protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2 (VI) chain, Proteoglycan 4, Glycophorin- C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B) group is ( B1) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin -1, Alpha-2-macroglobulin, Collagen alpha-1 (I) chain, Platelet factor 4, Vesicle-associated membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Collagen alpha -2 (I) chain, Multimerin-1, Ras-related protein Rab- Item 6. The test method according to any one of Items 1 to 5, which is a group consisting of 7a, Serine incorporator 3, Basigin, and C4b-binding protein.
　項７．　
前記タンパク質（A1）群が（A2）Amyloid beta A4 protein、Ras-related protein Rap-2c、Decorin、Serum amyloid A-1 protein、及びSerum amyloid A-2 proteinからなる群であり、且つ
前記タンパク質（B1）群が（B2）Alpha-2-antiplasmin、Complement C1s-A subcomponent、Complement C1r-A subcomponent、Biglycan、及びComplement C1q subcomponent subunit Bからなる群である、
項６に記載の検査方法。 Item 7.
The protein (A1) group is a group consisting of (A2) Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B1 ) Group is a group consisting of (B2) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, and Complement C1q subcomponent subunit B.
Item 7. The inspection method according to Item 6.
　項８．　
前記被検体がマウスである、項１～７のいずれかに記載の検査方法。 Item 8.
Item 8. The examination method according to any one of Items 1 to 7, wherein the subject is a mouse.
　項９．　
タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
（B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
（C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の検出剤を含む、閉塞性肺疾患の検査薬。 Item 9.
Protein group (A), protein group (B), and protein group (C):
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A test agent for obstructive pulmonary disease, comprising a detection agent for at least one protein selected from the group consisting of protein group consisting of, Fibulin-2, and Adenylyl cyclase-associated protein 1.
　項１０．　
閉塞性肺疾患誘発処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
（B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
（C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患モデル動物のスクリーニング方法。 Item 10.
Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluids collected from animals treated with obstructive pulmonary disease induction:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A method for screening an obstructive pulmonary disease model animal using as an index the amount or concentration of at least one protein selected from the group consisting of a protein group consisting of, Fibulin-2, and Adenylyl cyclase-associated protein 1.
　項１１．　
前記指標の値がカットオフ値以上である場合に、前記動物を閉塞性肺疾患モデル動物として選択する工程を含む、項１０に記載のスクリーニング方法。 Item 11.
Item 11. The screening method according to Item 10, comprising a step of selecting the animal as an obstructive pulmonary disease model animal when the index value is equal to or greater than a cutoff value.
　項１２．　
タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
（B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
（C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の抑制剤を含有する、閉塞性肺疾患の予防又は治療剤。 Item 12.
Protein group (A), protein group (B), and protein group (C):
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A prophylactic or therapeutic agent for obstructive pulmonary disease, comprising an inhibitor of at least one protein selected from the group consisting of, Fibulin-2, and Adenylyl cyclase-associated protein 1.
　項１３．　
被検物質で処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
（B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
（C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患の予防又は治療剤の有効成分のスクリーニング方法。 Item 13.
Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from animals treated with the test substance:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 , Fibulin-2, and an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease, using as an index the amount or concentration of at least one protein selected from the group consisting of proteins consisting of Adenylyl cyclase-associated protein 1 Screening method.
　項１４．　
前記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞における対応タンパク質の量又は濃度よりも低い場合に、前記被検物質を閉塞性肺疾患の予防又は治療剤の有効成分として選択する工程を含む、項１３に記載のスクリーニング方法。 Item 14.
When the value of the indicator is lower than the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance, the test substance is used to prevent obstructive pulmonary disease or Item 14. The screening method according to Item 13, which comprises a step of selecting as an active ingredient of a therapeutic agent.
　項１５．　
被検物質で処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
（B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
（C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患の誘発性又は増悪性の評価方法。 Item 15.
Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from animals treated with the test substance:
(A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
(B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 , Fibulin-2, and Adenylyl cyclase-associated protein 1 A method for evaluating inducibility or malignancy of obstructive pulmonary disease, using as an index the amount or concentration of at least one protein selected from the group consisting of the protein group consisting of 1 .
　本発明によれば、閉塞性肺疾患のバイオマーカーを提供することができる。該バイオマーカーを利用することにより、閉塞性肺疾患の検査、好酸球浸潤の程度の判定、閉塞性肺疾患モデル動物のスクリーニング等を、より簡便、より効率的、且つより低コストで行うことができる。また、該バイオマーカーを利用することにより、気管支喘息と慢性閉塞性肺疾患（COPD）との鑑別やこれらの合併症の鑑別も可能である。さらに、該バイオマーカーを利用することにより、閉塞性肺疾患の予防又は治療、閉塞性肺疾患の予防又は治療剤の有効成分のスクリーニング等も可能になり得る。 According to the present invention, a biomarker for obstructive pulmonary disease can be provided. By using the biomarker, testing for obstructive pulmonary disease, determination of the degree of eosinophil infiltration, screening for obstructive pulmonary disease model animals, etc. should be performed more simply, more efficiently and at lower cost. Can do. Further, by using the biomarker, it is possible to differentiate bronchial asthma from chronic obstructive pulmonary disease (COPD) and to distinguish these complications. Furthermore, by using the biomarker, it may be possible to prevent or treat obstructive pulmonary disease, screen for active ingredients of preventive or therapeutic agents for obstructive pulmonary disease, and the like.
細胞外小胞画分の粒子数及び粒子径を示す（試験例3）。コントロールは正常マウスから得られた細胞外小胞画分の結果を示し、喘息は気管支喘息モデルマウスから得られた細胞外小胞画分の結果を示し、COPDは慢性閉塞性肺疾患モデルマウスから得られた細胞外小胞画分の結果を示す。両疾患群において、コントロールと比べると、粒子数や粒子径に有意差を目留めなかった。The number of particles and the particle size of the extracellular vesicle fraction are shown (Test Example 3). Control shows the result of extracellular vesicle fraction obtained from normal mice, Asthma shows the result of extracellular vesicle fraction obtained from bronchial asthma model mice, COPD from chronic obstructive pulmonary disease model mice The result of the obtained extracellular vesicle fraction is shown. In both disease groups, no significant difference was observed in the number of particles and particle size compared to the control. 細胞外小胞画分の電子顕微鏡観察像（免疫電顕結果）を示す（試験例3）。エクソソームのマーカーとされるCD9で標識されたビーズが小胞に付着しており、粒子径も100nm以下であり既報と相違ない。コントロールは正常マウスから得られた細胞外小胞画分の結果を示し、喘息は気管支喘息モデルマウスから得られた細胞外小胞画分の結果を示し、COPDは慢性閉塞性肺疾患モデルマウスから得られた細胞外小胞画分の結果を示す。An electron microscopic observation image (immunoelectron microscopic result) of the extracellular vesicle fraction is shown (Test Example 3). Beads labeled with CD9, which is an exosome marker, are attached to the vesicles, and the particle diameter is 100 nm or less, which is no different from previous reports. Control shows the result of extracellular vesicle fraction obtained from normal mice, Asthma shows the result of extracellular vesicle fraction obtained from bronchial asthma model mice, COPD from chronic obstructive pulmonary disease model mice The result of the obtained extracellular vesicle fraction is shown.
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.
　1．閉塞性肺疾患の検査方法
　本発明は、その一態様において、閉塞性肺疾患を検査する方法であって、
（1）被検体から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質を検出する工程（工程1）を含む、検査方法（本明細書において、「本発明の肺閉塞性肺疾患検査方法」と示すこともある。）に関する。以下、これについて説明する。 1． In one aspect, the present invention is a method for examining obstructive pulmonary disease, comprising:
(1) Detect at least one protein selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C) in extracellular vesicles of a body fluid collected from a subject The present invention relates to a test method (in this specification, sometimes referred to as “the method for testing pulmonary obstructive pulmonary disease of the present invention”) including a step (step 1). This will be described below.
　1-1．工程（1）
　検査対象である「閉塞性肺疾患」は、特に制限されないが、具体的には、例えば気管支喘息、COPD、びまん性汎細気管支炎、閉塞性細気管支炎等が挙げられる。 1-1. Process (1)
The “obstructive pulmonary disease” to be examined is not particularly limited, and specific examples include bronchial asthma, COPD, diffuse panbronchiolitis, obstructive bronchiolitis and the like.
　被検体は、本発明の検査方法の対象生物であり、その生物種は特に制限されない。被検体の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられ、好ましくはマウスが挙げられる。 The subject is a target organism of the inspection method of the present invention, and its species is not particularly limited. Examples of the biological species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably mice.
　被検体の状態は、特に制限されない。被検体としては、例えば閉塞性肺疾患に罹患しているかどうか不明な検体、気管支喘息に罹患していると既に別の方法により判定されているが、COPDに罹患しているかどうか不明な検体、COPDに罹患していると既に別の方法により判定されているが、気管支喘息に罹患しているかどうか不明な検体、気管支喘息及びCOPDに罹患していると既に別の方法により判定されている検体、閉塞性肺疾患に罹患していないと既に別の方法により判定されている検体、閉塞性肺疾患の治療中の検体等が挙げられる。 The state of the subject is not particularly limited. Examples of specimens are samples that are unclear whether they have obstructive pulmonary disease, for example, specimens that have already been determined by other methods to have bronchial asthma, but are unclear whether they have COPD, Samples that have already been determined by other methods to have COPD but are unclear whether they have bronchial asthma, samples that have already been determined by other methods to have bronchial asthma and COPD Samples that have already been determined by another method as not having obstructive pulmonary disease, samples being treated for obstructive pulmonary disease, and the like.
　体液は、特に制限されない。体液としては、例えば全血、血清、血漿、髄液、唾液、関節液、尿、組織液（気管支肺胞洗浄液を含む）、汗、涙、喀痰、鼻汁などが挙げられ、好ましくは全血、血清、血漿、髄液が挙げられ、より好ましくは全血、血清、血漿が挙げられる。体液は、1種単独で採用してもよいし、2種以上を組み合わせて採用してもよい。 Body fluid is not particularly limited. Examples of the body fluid include whole blood, serum, plasma, spinal fluid, saliva, joint fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and preferably whole blood, serum. , Plasma and cerebrospinal fluid, and more preferably whole blood, serum and plasma. Body fluids may be used alone or in combination of two or more.
　体液は、当業者に公知の方法で被検体から採取することができる。例えば、全血は、注射器などを用いた採血によって採取することができる。血清は、全血から血球及び特定の血液凝固因子を除去した部分であり、例えば、全血を凝固させた後の上澄みとして得ることができる。血漿は、全血から血球を除去した部分であり、例えば、全血を凝固させない条件下で遠心分離に供した際の上澄みとして得ることができる。 Body fluid can be collected from the subject by methods known to those skilled in the art. For example, whole blood can be collected by blood collection using a syringe or the like. Serum is a portion obtained by removing blood cells and a specific blood coagulation factor from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood. Plasma is a part obtained by removing blood cells from whole blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not coagulate whole blood.
　細胞外小胞は、細胞から分泌、放出等される膜小胞である限り特に制限されない。細胞外小胞は、通常は、細胞内のタンパク質や遺伝情報（mRNA, microRNA等）を細胞外に運搬することにより、局所や全身における細胞間の情報伝達を担っている膜小胞として定義される。細胞外小胞としては、例えばエクソソーム、微小小胞体、アポトーシス小体、エクトソーム、マイクロパーティクル、分泌マイクロベシクル等が挙げられる。 Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted and released from cells. Extracellular vesicles are usually defined as membrane vesicles that carry information between cells locally and throughout the body by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell. The Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, and secretory microvesicles.
　細胞外小胞は、体液から、公知の方法に従って又は準じて、精製、分離、濃縮等することができる。細胞外小胞を精製、分離、濃縮等する方法としては、例えば超遠心法（例えばペレットダウン法、スクロースクッション法、密度勾配遠心法等）、イムノアフィニティー担体を用いる方法、ゲルろ過法、フィールド・フロー分画法、FACS法等が挙げられる。また、細胞外小胞の精製、分離、濃縮等は、市販のキットを用いて行うことも可能である。これらの方法は、1種単独で採用してもよいし、2種以上を組み合わせて採用してもよい。 Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or according to known methods. Examples of methods for purifying, separating and concentrating extracellular vesicles include, for example, ultracentrifugation (eg, pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), a method using an immunoaffinity carrier, gel filtration, Examples thereof include a flow fractionation method and a FACS method. Further, purification, separation, concentration and the like of extracellular vesicles can be performed using a commercially available kit. These methods may be employed singly or in combination of two or more.
　工程（1）の検出対象は、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質（本明細書において、これらをまとめて「対象タンパク質」と示すこともある。）である。 The detection target of step (1) is at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) (in the present specification, these are collectively referred to as “ It may be indicated as “target protein”).
　タンパク質群（A）は、（A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群である。 Protein group (A) is (A) IgIlambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8 , Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosHOtan-bindingHSPHO2 protein 2, Integrin beta-2, Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1 , Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3-A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1, Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Plate12integra Platelet 2, Oncoprotein-induced transcriptase 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core-protein, Protein-unc-13-homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain V91 HP91 , Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig Vappagiochain n L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinos 3,4,5-trisphosphate5-5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cellProgramdeath protein 6, Cysteine-rich secretory protein 3, and Protein FAM177A1 It is a protein group.
　タンパク質群（A）は、気管支喘息特異的なバイオマーカーであり、これを指標とすることにより慢性閉塞性肺疾患非合併型の気管支喘息を鑑別可能である。 Protein group (A) is a bronchial asthma-specific biomarker, and it can be used as an index to distinguish bronchial asthma without chronic obstructive pulmonary disease.
　タンパク質群（A）の中でも、病勢をより反映している（好酸球浸潤の程度との相関がより高い）という観点からは、好ましくは、14-3-3 protein theta、Thrombospondin-1、Platelet glycoprotein 4、Pyridoxal phosphate phosphatase PHOSPHO2、Integrin beta-2、Collagen alpha-2(VI) chain、Proteoglycan 4、Glycophorin-C、Versican core protein、Amyloid beta A4 protein、Ras-related protein Rap-2c、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein等が挙げられる。 Among the protein group (A), from the viewpoint of more reflecting the disease state (higher correlation with the degree of eosinophil infiltration), preferably 14-3-314protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2 (VI) chain, Proteoglycan 4, Glycophorin-C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serumoid A-1 protein, Serum amyloid A-2 protein, etc. are mentioned.
　タンパク質群（A）の中でも、正常検体に対する量比がより高い（コントロールの発現量が確認できない場合も包含する）という観点からは、好ましくはIg kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、Protein FAM177A1等が挙げられ、より好ましくはRas-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、Protein FAM177A1等が挙げられ、さらに好ましくはSerum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、Protein FAM177A1等が挙げられる。 Among the protein group (A), from the viewpoint that the ratio of the amount to the normal sample is higher (including the case where the expression level of the control cannot be confirmed), preferably Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, ComplementAsubcomponent , Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region Lrum, Decoroid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein-2, Ig heavy chain V Vregion 6.96, Keratin, type II cutcut Hb1, Early endosome antigen 1, TBC domain-con taining protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5 Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, Protein FAM177A1 Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A c omplex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, Protein FAM177A1 etc., more preferably Serum amyloid A-1 protein, Serum amyloid A-2 protein, GrowthGfactor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antigen 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chai n-V-II region-MOPC-167, Programmed-cell-death-protein-6, Cysteine-rich secretory-protein-3, Protein-FAM177A1, and the like.
　タンパク質群（A）の中でも、正常検体に対する量比がより高い（コントロールの発現量が確認できない場合は包含しない）という観点からは、好ましくはIg kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein等が挙げられ、より好ましくはRas-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein等が挙げられ、さらに好ましくはSerum amyloid A-1 protein、Serum amyloid A-2 protein等が挙げられる。 Among the protein group (A), from the viewpoint that the ratio of the amount to the normal sample is higher (not included when the expression level of the control cannot be confirmed), preferably Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, ComplementAsubcomponent , Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1-syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region Lrum, Decoroid A-1 protein, Serum amyloid A-2 protein, etc., more preferably Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloi d A-2 protein, and the like, more preferably Serum amyloid A-1 protein, Serum amyloid A-2 protein and the like.
　タンパク質群（A）の中でも、病勢をより反映している（好酸球浸潤の程度との相関がより高い）という観点、及び正常検体に対する量比がより高いという観点からは、好ましくはAmyloid beta A4 protein、Ras-related protein Rap-2c、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein等が挙げられる。 Among the protein group (A), from the viewpoint of more reflecting the disease state (higher correlation with the degree of eosinophil infiltration), and from the viewpoint of higher dose ratio to normal specimen, Amyloid beta is preferable. Examples include A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, and the like.
　タンパク質群（B）は、Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群である。 Protein group (B) is Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen 、 alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, alphamin-5 subunit , Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain-2 Integrin beta-2, F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, faci litated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated proteinSyn23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine- protein phosphatase eta, Serine inco Protein group consisting of rporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain, Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 chain, Alpha-synuclein, and calcium-transporting ATPase type 2C member 1 It is.
　タンパク質群（B）は、慢性閉塞性肺疾患特異的なバイオマーカーであり、これを指標とすることにより気管支喘息非合併型の慢性閉塞性肺疾患を鑑別可能である。 Protein group (B) is a biomarker specific to chronic obstructive pulmonary disease, and by using this as an index, it is possible to distinguish chronic obstructive pulmonary disease that is not associated with bronchial asthma.
　タンパク質群（B）の中でも、病勢をより反映している（平均肺胞径との相関がより高い）という観点からは、好ましくは、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Complement C1r-A subcomponent、Biglycan、Complement C1q subcomponent subunit B、Laminin subunit alpha-5、Interferon-induced transmembrane protein 2、Laminin subunit gamma-1、Serglycin、Thrombospondin-1、Alpha-2-macroglobulin、Collagen alpha-1(I) chain、Platelet factor 4、Vesicle-associated membrane protein 3、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Collagen alpha-2(I) chain、Multimerin-1、Ras-related protein Rab-7a、Serine incorporator 3、Basigin、C4b-binding protein等が挙げられる。 Among the protein group (B), from the viewpoint of more reflecting the disease state (higher correlation with the average alveolar diameter), preferably Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r- A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospondin-1, Alpha-2-macroglobulin, Collagen alpha-1 (I) chain , Platelet factor 4, Vesicle-associated membrane membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing protein-bulbulin-like extracellular matrix protein 1, collagen-alpha-2 (I) chain, Multimerin-1, Ras-related protein Rab- 7a, Serine incorporator 3, Basigin, C4b-binding protein, etc.
　タンパク質群（B）の中でも、正常検体に対する量比がより高い（コントロールの発現量が確認できない場合も包含する）という観点からは、好ましくはCytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1等が挙げられ、より好ましくはCytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1等が挙げられ、さらに好ましくはCytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1等が挙げられる。 Among the protein group (B), Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2 are preferable from the viewpoint that the amount ratio to the normal specimen is higher (including the case where the expression level of the control cannot be confirmed). -antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV genly cancCol41 2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2script, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Alpha-synuclein, and calcium-transporting ATPase type 2C member 1, and more preferably Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2 -antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Alpha-synuclein, and calcium-transporting2ATPase type1 More preferably, Cytohesin-2, Erythrocyte band 7 integra membrane protein, Alpha-2-antiplasmin, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1 etc. are mentioned.
　タンパク質群（B）の中でも、正常検体に対する量比がより高い（コントロールの発現量が確認できない場合は包含しない）という観点からは、好ましくはErythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5等が挙げられ、より好ましくはErythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent等が挙げられ、さらに好ましくはErythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin等が挙げられる。が挙げられる。 Among the protein group (B), from the viewpoint that the ratio of the amount to the normal sample is higher (not included when the expression level of the control cannot be confirmed), preferably Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan-2, Collagen alpha chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E, Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like tranminin protein 1 subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2, F-box onl y protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane protein 8, CD81 antigen, Alpha-1-antitrypsin 1-5 and the like, more preferably Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r-A subcomponent and the like, more preferably Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin and the like. Is mentioned.
　タンパク質群（B）の中でも、病勢をより反映している（平均肺胞径との相関がより高い）という観点、及び正常検体に対する量比がより高いという観点からは、好ましくはAlpha-2-antiplasmin、Complement C1s-A subcomponent、Complement C1r-A subcomponent、Biglycan、Complement C1q subcomponent subunit B等が挙げられる。 Among the protein group (B), from the viewpoint of more reflecting the disease state (higher correlation with the average alveolar diameter) and higher amount ratio to the normal specimen, preferably Alpha-2- antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B and the like.
　タンパク質群（B）の中でも、マウスとヒトとの共通バイオマーカーとしてより有望であるという観点から、好ましくはEGF-containing fibulin-like extracellular matrix protein 1が挙げられる。 Among the protein group (B), EGF-containing fibulin-like extracellular matrix protein 1 is preferable from the viewpoint of being more promising as a common biomarker for mice and humans.
　タンパク質群（C）は、Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群である。 Protein group (C) is a protein group consisting of Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 is there.
　タンパク質群（C）は、気管支喘息及び慢性閉塞性肺疾患の両方に特異的なバイオマーカーであり、これを指標とすることにより慢性閉塞性肺疾患合併型の気管支喘息（或いは気管支喘息合併型の慢性閉塞性肺疾患）を鑑別可能である。 Protein group (C) is a specific biomarker for both bronchial asthma and chronic obstructive pulmonary disease. By using this as an index, bronchial asthma (or bronchial asthma complication type) Chronic obstructive pulmonary disease) can be differentiated.
　タンパク質群（C）の中でも、好酸球浸潤の程度との相関がより高いという観点からは、好ましくは、Interferon-induced transmembrane protein 3、Collagen alpha-1(XVIII) chain、Fibulin-2、Adenylyl cyclase-associated protein 1等が挙げられ、平均肺胞径との相関がより高いという観点からは、好ましくは、Collagen alpha-1(XVIII) chain、Complement C1q subcomponent subunit C等が挙げられる。 Among the protein group (C), from the viewpoint of higher correlation with the degree of eosinophil infiltration, Interferon-induced transmembrane protein 3, Collagen alpha-1 (XVIII) chain, Fibulin-2, Adenylyl cyclase From the viewpoint that the correlation with the average alveolar diameter is higher, Collagen alpha-1 (XVIII) chain, Complement C1q subcomponent subunit C and the like are preferable.
　タンパク質群（C）の中でも、正常検体に対する量比がより高い（コントロールの発現量が確認できない場合も包含する）という観点からは、好ましくはCollagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Fibulin-2、Adenylyl cyclase-associated protein 1等が挙げられる。 Among the protein group (C), from the viewpoint that the ratio of the amount to the normal sample is higher (including the case where the expression level of the control cannot be confirmed), preferably Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Examples include Complement C1q subcomponent subunit C, Fibulin-2, Adenylyl cyclase-associated protein 1.
　タンパク質群（C）の中でも、正常検体に対する量比がより高い（コントロールの発現量が確認できない場合も包含しない）という観点からは、好ましくはCollagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C等が挙げられる。 Among the protein group (C), from the viewpoint that the ratio to the normal sample is higher (not including the case where the expression level of the control cannot be confirmed), preferably Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Examples include Complement1C1q subcomponent subunit 等 C.
　タンパク質群（A）～（C）のタンパク質は、マウスの場合であれば、後述の実施例における表1～4に示されるUniProtKBアクセッション番号によって特定されるタンパク質である。他の生物種の場合であれば、表1～5に示されるUniProtKBアクセッション番号によって特定されるタンパク質のオーソログである。 Proteins (A) to (C) are proteins specified by UniProtKB accession numbers shown in Tables 1 to 4 in Examples described later in the case of mice. In the case of other species, it is an ortholog of the protein specified by the UniProtKB accession numbers shown in Tables 1-5.
　工程（1）における対象タンパク質の数は、1種のみでもよいが、2種以上の組み合わせであってもよい。より多く（例えば2種、5種、10種、20種、40種、80種、120種以上）の検出対象を組み合わせることにより、閉塞性肺疾患の検査、気管支喘息と慢性閉塞性肺疾患との鑑別、これらの合併症の鑑別等を、より性格に行うことが可能になる。 In the step (1), the number of target proteins may be only one type or a combination of two or more types. By combining more detection targets (eg 2, 5, 10, 20, 40, 80, 120 or more), it is possible to test for obstructive pulmonary disease, bronchial asthma and chronic obstructive pulmonary disease. Differentiation of these complications, differentiation of these complications, etc. can be performed more accurately.
　検出は、通常は、対象タンパク質の量又は濃度を測定することによって行われる。「濃度」とは、絶対濃度に限らず、相対濃度や、単位体積辺りの重量や、絶対濃度を知るために測定した生データなどでもよい。 Detecting is usually performed by measuring the amount or concentration of the target protein. The “density” is not limited to the absolute density, but may be a relative density, a weight per unit volume, or raw data measured to know the absolute density.
　対象タンパク質を検出する方法としては、対象タンパク質の一部又は全部を特異的に検出できる方法であれば特に制限されない。検出方法としては、具体的には、例えば、対象タンパク質を構成するペプチドを検出する質量分析法、対象タンパク質を特異的に認識する抗体を用いた免疫学的測定法等を挙げることができる。なお、対象タンパク質のアミノ酸配列情報は、UniProtKBアクセッション番号を基に、EBI（http://www.ebi.ac.uk/IPI/IPIhelp.html）のデータベースで検索することにより得ることができる。 The method for detecting the target protein is not particularly limited as long as it is a method capable of specifically detecting a part or all of the target protein. Specific examples of the detection method include a mass spectrometry method for detecting a peptide constituting the target protein, and an immunological measurement method using an antibody that specifically recognizes the target protein. The amino acid sequence information of the target protein can be obtained by searching the EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) database based on the UniProtKB accession number.
　免疫学的測定法としては、免疫組織化学染色法、ELISA法、EIA法、RIA法、ウェスタンブロッティング法等を好適に例示することができる。 As an immunological measurement method, an immunohistochemical staining method, an ELISA method, an EIA method, an RIA method, a Western blotting method and the like can be preferably exemplified.
　質量分析法とは、ペプチド試料を、イオン源を用いて気体状のイオンとし（イオン化）、分析部において、真空中で運動させ電磁気力を用いて、あるいは飛行時間差によりイオン化したペプチド試料を質量電荷比に応じて分離し、検出できる質量分析計を用いた測定方法のことをいい、イオン源を用いてイオン化する方法としては、EI法、CI法、FD法、FAB法、MALDI法、ESI法などの方法を適宜選択することができ、また、分析部において、イオン化したペプチド試料を分離する方法としては、磁場偏向型、四重極型、イオントラップ型、飛行時間（TOF）型、フーリエ変換イオンサイクロトロン共鳴型などの分離方法を適宜選択することができる。また、2以上の質量分析法を組み合わせたタンデム型質量分析（MS／MS）やトリプル四重極型質量分析を利用することができる。また、サンプルがリン酸化したペプチドを含む試料の場合、質量分析計へのサンプル導入前に、サンプルを鉄イオン固定化アフィニティークロマトグラフィー（Fe-IMAC）を用いて濃縮することができる。また、液体クロマトグラフ（LC）やHPLCにより、対象タンパク質を構成するペプチドを分離・精製してサンプルとすることができる。また、検出部やデータ処理方法も適宜選択することができる。なお、質量分析法を用いて対象タンパク質を構成するペプチドを質量分析法で検出・定量する場合、かかるペプチドと同一のアミノ酸配列からなる、濃度が既知の安定同位体で標識したペプチドを内部標準とすることができる。かかる安定同位体標識ペプチドとしては、検出する対象タンパク質を構成するペプチドにおけるアミノ酸の1個以上が、15N，13C，18O，及び2Hのいずれか1以上を含む安定同位体標識ペプチドであれば、アミノ酸の種類、位置、数などは適宜選択することができ、かかる安定同位体標識ペプチドは、安定同位元素により標識されたアミノ酸を用いてF-moc法（Amblard., et al. Methods Mol Biol.298:3-24(2005)）等の適当な手段で化学合成することができるが、iTRAQ（登録商標）試薬、ICAT（登録商標）試薬、ICPL（登録商標）試薬、NBS（登録商標）試薬などの標識試薬を用いて作製することもできる。 In mass spectrometry, a peptide sample is turned into gaseous ions using an ion source (ionization), and the peptide sample ionized by moving it in a vacuum and using electromagnetic force or by a time-of-flight difference in the analysis section is mass-charged. This is a measurement method using a mass spectrometer that can be separated and detected according to the ratio. Ionization using an ion source includes the EI method, CI method, FD method, FAB method, MALDI method, and ESI method. The method of separating ionized peptide samples in the analysis section can be selected as appropriate. Magnetic field deflection type, quadrupole type, ion trap type, time of flight (TOF) type, Fourier transform A separation method such as an ion cyclotron resonance type can be appropriately selected. In addition, tandem mass spectrometry (MS / MS) or triple quadrupole mass spectrometry combining two or more mass spectrometry methods can be used. When the sample contains a phosphorylated peptide, the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer. Moreover, the peptide which comprises target protein can be isolate | separated and refine | purified by a liquid chromatograph (LC) or HPLC, and it can be set as a sample. Moreover, a detection part and a data processing method can also be selected suitably. In addition, when detecting and quantifying peptides constituting the target protein by mass spectrometry using mass spectrometry, a peptide labeled with a stable isotope having a known amino acid sequence and having a known concentration is used as an internal standard. can do. Such a stable isotope labeled peptide is a stable isotope labeled peptide in which at least one of the amino acids in the peptide constituting the target protein to be detected contains at least one of 15 N, 13 C, 18 O, and 2 H. If necessary, the type, position, number, etc. of amino acids can be appropriately selected. Such stable isotope-labeled peptides can be obtained by F-moc method (Amblard., Et al. Methods) using amino acids labeled with stable isotopes. Mol Biol. 298: 3-24 (2005)) can be chemically synthesized by iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark). (Trademark) It can also produce using labeling reagents, such as a reagent.
　工程（1）を含む本発明の検査方法によれば、閉塞性肺疾患の検出指標である対象タンパク質の量及び／又は濃度を提供することができ、これにより閉塞性肺疾患の検出などを補助することができる。また、工程（1）を含む本発明の検査方法によれば、検出指標である対象タンパク質の量及び／又は濃度を提供することができ、これにより好酸球浸潤の程度の評価を補助することもできる。好酸球浸潤は、気管支喘息の活動性（過敏性、可逆性等）、COPDの急性増悪等に関与するので、好酸球浸潤の評価は、急性憎悪の可能性の判断や、予後不良群の鑑別等に利用し得る。また、このように、好酸球浸潤は閉塞性疾患に関与する重要なファクターであるものの、それを評価する方法は。呼気NO (FeNO)の測定という煩雑な検査を要する方法以外は知られていなかった。本発明のバイオマーカーを用いれば、このような煩雑な作業を要せず、従来より簡便且つ効率的に好酸球浸潤を評価することができる。 According to the test method of the present invention including the step (1), the amount and / or concentration of the target protein which is a detection index of obstructive pulmonary disease can be provided, thereby assisting in the detection of obstructive pulmonary disease and the like. can do. Moreover, according to the test method of the present invention including the step (1), the amount and / or concentration of the target protein that is a detection index can be provided, thereby assisting in the evaluation of the degree of eosinophil infiltration. You can also. Since eosinophil infiltration is involved in bronchial asthma activity (hypersensitivity, reversibility, etc.), acute exacerbation of COPD, etc., the evaluation of eosinophil infiltration is based on the judgment of the possibility of acute aversion and the poor prognosis group It can be used for identification of In this way, eosinophil infiltration is an important factor involved in obstructive disease, but how to evaluate it. No method other than the method requiring a complicated test of measuring exhaled NO (FeNO) has been known. If the biomarker of the present invention is used, it is possible to evaluate eosinophil infiltration more simply and efficiently than before without requiring such a complicated operation.
　工程（1）を含む本発明の検査方法による検査結果は、閉塞性肺疾患の病態解明、閉塞性疾患の予後予測（呼吸機能低下群、急性憎悪）、被検体の層別化、治療方法の選択（個別化医療、治療反応性）、閉塞性肺疾患（特に、気管支喘息）における難治化や、リモデリングの評価、閉塞性肺疾患の組織型や表現型等の鑑別等に利用し得る。さらに、本発明により見出された種々の好酸球浸潤と相関するBMは、種々の表現型の鑑別（クラスタリング）や現在開発中の種々の分子標的薬（Th2を標的とした）のコンパニオンBMにもなりうる。 The test results of the test method of the present invention including the step (1) are as follows: elucidation of the pathological condition of obstructive pulmonary disease, prognosis prediction of obstructive disease (respiratory function decline group, acute exacerbation), stratification of subjects, treatment method It can be used for selection (individualized medicine, treatment responsiveness), refractory in obstructive pulmonary disease (particularly bronchial asthma), evaluation of remodeling, differentiation of histological type, phenotype, etc. of obstructive pulmonary disease. Furthermore, BM correlates with various eosinophil infiltration found by the present invention is a companion BM of various phenotypes (clustering) and various molecular target drugs (th2 targeted) currently under development. It can also be.
　1-2．工程（2）
　本発明の検査方法は、一態様として、さらに、
（2）前記工程（1）で検出されたタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が閉塞性肺疾患に罹患していると判定する工程（工程2）を含むことが好ましい。該工程2を含む本発明の検査方法によれば、閉塞性肺疾患を判定することが可能となる。 1-2. Process (2)
The inspection method of the present invention further includes, as one aspect,
(2) including a step of determining that the subject suffers from obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is equal to or higher than a cutoff value (step 2) It is preferable. According to the test method of the present invention including the step 2, it becomes possible to determine obstructive pulmonary disease.
　工程2は、より具体的には、例えば以下の（2a）～（2e）に分けられる。
（2a）前記タンパク質群（A）及び前記タンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息に罹患していると判定する工程、
（2b）前記タンパク質群（B）及び前記タンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が慢性閉塞性肺疾患に罹患していると判定する工程、
（2c）前記タンパク質群（A）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が慢性閉塞性肺疾患非合併型の気管支喘息に罹患していると判定する工程、
（2d）前記タンパク質群（B）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息非合併型の慢性閉塞性肺疾患に罹患していると判定する工程、並びに
（2e）前記タンパク質群（C）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息及び慢性閉塞性肺疾患の両方に罹患していると判定する工程。 More specifically, step 2 is divided into the following (2a) to (2e), for example.
(2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value The process of determining that
(2b) When the amount or concentration of at least one protein selected from the group consisting of the protein group (B) and the protein group (C) is a cut-off value or more, the subject is chronic obstructive lung Determining that the patient is afflicted with a disease,
(2c) When the amount or concentration of at least one protein selected from the protein group (A) is not less than a cut-off value, the subject suffers from bronchial asthma without chronic obstructive pulmonary disease. A process of determining that
(2d) When the amount or concentration of at least one protein selected from the protein group (B) is equal to or higher than a cutoff value, the subject suffers from bronchial asthma non-complicated chronic obstructive pulmonary disease. And (2e) when the amount or concentration of at least one protein selected from the protein group (C) is equal to or higher than a cutoff value, the subject is bronchial asthma and chronic obstructive Determining that you have both lung diseases.
　工程（2）は、より好ましくは上記工程2a～2eからなる群より選択される少なくとも1種の工程である。 Step (2) is more preferably at least one step selected from the group consisting of Steps 2a to 2e.
　カットオフ値は、感度、特異度、陽性的中率、陰性的中率などの観点から当業者が適宜設定することができ、例えば、閉塞性肺疾患に罹患していない被検体から採取された体液の細胞外小胞における対象タンパク質の量及び／又は濃度に基づいて、その都度定められた値、或いは予め定められた値とすることができる。カットオフ値は、例えば、閉塞性肺疾患に罹患していない被検体から採取された体液の細胞外小胞における対象タンパク質の量及び／又は濃度（被検体が複数の場合は、平均値、中央値など）の、例えば1～10倍、好ましくは2～8倍、より好ましくは2.5～6倍の値とすることができる。 The cut-off value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc., for example, collected from a subject not suffering from obstructive pulmonary disease Based on the amount and / or concentration of the target protein in the extracellular vesicles of the body fluid, it can be a value determined each time or a predetermined value. The cut-off value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles of body fluid collected from a subject not suffering from obstructive pulmonary disease (in the case of multiple subjects, the average value, the center Value), for example, 1 to 10 times, preferably 2 to 8 times, more preferably 2.5 to 6 times.
　工程（2）のより好ましい態様においては、閉塞性肺疾患の病勢を反映しているバイオマーカーを用いることにより、前記工程（1）で検出されたタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体がより病勢の強い閉塞性肺疾患に罹患していると判定することができる。この場合、カットオフ値を、例えば閉塞性肺疾患に罹患している被検体から採取された体液の細胞外小胞における対象タンパク質の量及び／又は濃度に基づいた値とすることにより、一定以上の病勢であるかどうかを評価できる。或いは、被検体が閉塞性肺疾患の治療中の検体である場合、カットオフ値を、例えば同一検体についての過去の試料における対象タンパク質の量及び／又は濃度に基づいた値とすることにより、治療効果を判定することができる。 In a more preferred embodiment of the step (2), the amount or concentration of the protein detected in the step (1) is greater than or equal to a cut-off value by using a biomarker reflecting the pathology of obstructive pulmonary disease. In this case, it can be determined that the subject suffers from a more aggressive obstructive pulmonary disease. In this case, the cut-off value is a certain value or more by setting the value based on the amount and / or concentration of the target protein in the extracellular vesicles of a body fluid collected from a subject suffering from obstructive pulmonary disease, for example. It can be evaluated whether or not the disease is. Alternatively, when the subject is a sample being treated for obstructive pulmonary disease, the cutoff value is set to a value based on the amount and / or concentration of the target protein in the past sample for the same sample, for example. The effect can be determined.
　さらに、工程（2）のより好ましい態様においては、好酸球浸潤の程度と相関しているバイオマーカーを用いることにより、前記工程（1）で検出されたタンパク質の量又は濃度を指標として、好酸球浸潤の程度を判定することができる。より具体的には、例えば、予め好酸球浸潤の程度（一定面積内の好酸球浸潤数）とバイオマーカーの量又は濃度との相関に基づいた検量線を作成し、測定されたバイオマーカーの量又は濃度と検量線から、好酸球浸潤の程度を判定することができる。 Furthermore, in a more preferred embodiment of the step (2), by using a biomarker correlated with the degree of eosinophil infiltration, the amount or concentration of the protein detected in the step (1) is used as an index. The degree of eosinophil infiltration can be determined. More specifically, for example, a calibration curve based on the correlation between the degree of eosinophil infiltration (the number of eosinophil infiltration within a certain area) and the amount or concentration of the biomarker is prepared and measured in advance. The degree of eosinophil infiltration can be determined from the amount or concentration of sucrose and the calibration curve.
　2．閉塞性肺疾患のより高い精度での診断
　工程（2）を含む本発明の検査方法により、被検体が閉塞性肺疾患に罹患していると判定された場合、本発明の検査方法に、さらに閉塞性肺疾患の医師による診断を適用する工程を組み合わせることによって、より高い精度で閉塞性肺疾患を診断することができる。また、本発明の検査方法はより正確に閉塞性肺疾患を検出できるので、本発明の検査方法に上記工程を組み合わせることによって、より効率的且つより正確に「閉塞性肺疾患に罹患している」と診断できる。 2． When it is determined that the subject suffers from obstructive pulmonary disease by the test method of the present invention including the diagnosis step (2) with higher accuracy of obstructive pulmonary disease, the test method of the present invention further includes By combining the steps of applying the diagnosis by the doctor of obstructive pulmonary disease, the obstructive pulmonary disease can be diagnosed with higher accuracy. In addition, since the test method of the present invention can detect obstructive pulmonary disease more accurately, combining the above-described steps with the test method of the present invention makes it more efficient and more accurate to “be afflicted with obstructive pulmonary disease. Can be diagnosed.
　3．閉塞性肺疾患の治療
　工程（2）を含む本発明の検査方法により被検体が閉塞性肺疾患に罹患していると判定された場合は本発明の検査方法に対してさらに、或いは上記「2．閉塞性肺疾患のより高い精度での診断」に記載の様に閉塞性肺疾患に罹患していると診断された場合は本発明の検査方法と医師による診断を適用する工程との組合せに対してさらに、（3）閉塞性肺疾患に罹患していると判定又は診断された被検体に対して、該疾患の治療を行う工程を行うことによって、被検体の該疾患を治療することが可能となる。また、本発明の検査方法はより正確に閉塞性肺疾患を検出できるので、本発明の検査方法に対して、或いは本発明の検査方法と医師による診断を適用する工程との組合せに対して工程3を組み合わせることによって、閉塞性肺疾患に罹患している被検体をより効率的に、より確実に治療できる。 3． When it is determined that the subject suffers from obstructive pulmonary disease by the test method of the present invention including the treatment step (2) for obstructive pulmonary disease, in addition to the test method of the present invention or the above “2 When diagnosed as suffering from obstructive pulmonary disease as described in ". Diagnosis of obstructive pulmonary disease with higher accuracy", the test method of the present invention is combined with the process of applying diagnosis by a doctor. On the other hand, (3) treating the disease of the subject by performing a step of treating the disease on the subject determined or diagnosed as suffering from obstructive pulmonary disease. It becomes possible. In addition, since the test method of the present invention can detect obstructive pulmonary disease more accurately, a process for the test method of the present invention or a combination of the test method of the present invention and a process of applying a diagnosis by a doctor By combining 3, it is possible to treat a subject suffering from obstructive pulmonary disease more efficiently and more reliably.
　閉塞性肺疾患の治療方法は、特に制限されないが、代表的には投薬治療が挙げられる。投薬治療に用いる医薬としては、特に制限されないが、例えばスピリーバ、シーブリ、エンクラッセ、エクリラ、アトロベント、テルシガン等の抗コリン薬；　セレベント、オンブレス、オーキシス、サルタノール、メプチン等のβ2刺激薬；　ウルティブロ、アノーロ、スピオルト等の抗コリン薬・β2刺激薬配合剤；　キュバール、フルタイド、パルミコート、オルベスコ、アズマネックス等のステロイド薬；　アドエア、シムビコート、レルベア等のステロイド薬・β2刺激薬配合剤等が挙げられる。医薬は、1種、2種、又は3種以上を組み合わせて用いることができる。 The method for treating obstructive pulmonary disease is not particularly limited, but a typical example is medication. The drug used for the drug treatment is not particularly limited, but for example, anticholinergic drugs such as Spiriva, Seeburi, Enclasse, Ecrila, Atrovent, Telcigan, etc .; β2-stimulating drugs such as Serebent, Ombres, Auxis, Sultanol, Meptin; Ultimolo, Anolo And anti-cholinergic / β2-stimulant combination drugs such as sport; steroid drugs such as cuvard, flutide, palmicoat, olvesco, and azmanex; steroid drugs / beta-stimulant combination drugs such as adair, simbicoat, and lerbea. A pharmaceutical can be used 1 type, 2 types, or 3 or more types in combination.
　4．閉塞性肺疾患の検査薬
　本発明は、その一態様において、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の検出剤を含む、閉塞性肺疾患の検査薬（本明細書において、「本発明の検査薬」と示すこともある。）に関する。以下、これについて説明する。 Four. In one aspect, the present invention provides an agent for detecting at least one protein selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C). The present invention relates to a test agent for obstructive pulmonary disease (also referred to as “the test agent of the present invention” in the present specification). This will be described below.
　タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、閉塞性肺疾患等については、上記「1．閉塞性肺疾患の検査方法」における定義と同様である。 Protein group (A), protein group (B), protein group (C), obstructive pulmonary disease and the like are the same as defined in “1. Method for examining obstructive pulmonary disease” above.
　検出剤は、対象タンパク質を特異的に検出できるものである限り特に制限されない。該検出剤としては、例えば対象タンパク質に対する抗体が挙げられる。検出剤は、標識されたものであってもよい。標識としては、例えば蛍光物質、発光物質、色素、酵素、金コロイド、放射性同位体などが挙げられる。また、検出剤（特に抗体）は、基材（例えばマイクロウェルプレート等のプラスチック基材）に吸着した状態であってもよい。 The detection agent is not particularly limited as long as it can specifically detect the target protein. Examples of the detection agent include an antibody against the target protein. The detection agent may be labeled. Examples of the label include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Further, the detection agent (particularly antibody) may be in a state of being adsorbed on a base material (for example, a plastic base material such as a microwell plate).
　抗体は、対象タンパク質を選択的に（特異的に）認識するものであれば、特に限定されない。ここで、「選択的に（特異的に）認識する」とは、例えばウェスタンブロット法やELISA法において、対象タンパク質が特異的に検出できることを意味するが、それに限定されることなく、当業者が上記検出物が対象タンパク質に由来するものであると判断できるものであればよい。 The antibody is not particularly limited as long as it recognizes the target protein selectively (specifically). Here, “selectively (specifically) recognize” means that the protein of interest can be specifically detected, for example, in Western blotting or ELISA, but is not limited thereto. Any substance can be used as long as it can be determined that the detected substance is derived from the target protein.
　抗体には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、またはFabフラグメントやFab発現ライブラリーによって生成されるフラグメントなどのように抗原結合性を有する上記抗体の一部が包含される。対象タンパク質のアミノ酸配列のうち少なくとも連続する、通常8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるポリペプチドに対して抗原結合性を有する抗体も、本発明の抗体に含まれる。 The antibody includes a part of the above antibody having antigen binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library. The antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of at least continuous amino acid sequence of the subject protein, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.
　これらの抗体の製造方法は、すでに周知であり、本発明の抗体もこれらの常法に従って製造することができる（Current protocols in Molecular Biology , Chapter 11.12～11.13(2000)）。具体的には、本発明の抗体がポリクローナル抗体の場合には、常法に従って大腸菌等で発現し精製した対象タンパク質を用いて、あるいは常法に従って当該対象タンパク質の部分アミノ酸配列を有するオリゴペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、常法に従って大腸菌等で発現し精製した対象タンパク質、あるいは対象タンパク質の部分アミノ酸配列を有するオリゴペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞の中から得ることができる（Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4～11.11）。 The methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of the target protein is synthesized using a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or according to a conventional method. Thus, it is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, spleen cells obtained by immunizing a non-human animal such as a mouse with a target protein expressed and purified in Escherichia coli according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein and It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
　抗体の作製に免疫抗原として使用される対象タンパク質は、公知の遺伝子配列情報に基づいて、DNAクローニング、各プラスミドの構築、宿主へのトランスフェクション、形質転換体の培養および培養物からのタンパク質の回収の操作により得ることができる。これらの操作は、当業者に既知の方法、あるいは文献記載の方法（Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)）などに準じて行うことができる。 The target protein used as an immunizing antigen for antibody production is based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of a transformant, and recovery of the protein from the culture. It can obtain by operation of. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
　具体的には、対象タンパク質をコードする遺伝子が所望の宿主細胞中で発現できる組み換えDNA（発現ベクター）を作製し、これを宿主細胞に導入して形質転換し、該形質転換体を培養して、得られる培養物から、目的タンパク質を回収することによって、本発明抗体の製造のための免疫抗原としてのタンパク質を得ることができる。また対象タンパク質の部分ペプチドは、公知の遺伝子配列情報に従って、一般的な化学合成法（ペプチド合成）によって製造することもできる。 Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding the target protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. The protein as the immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture. The partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) in accordance with known gene sequence information.
　また本発明の抗体は、対象タンパク質の部分アミノ酸配列を有するオリゴペプチドを用いて調製されるものであってよい。かかる抗体の製造のために用いられるオリゴ（ポリ）ペプチドは、機能的な生物活性を有することは要しないが、対象タンパク質と同様な免疫原特性を有するものであることが望ましい。好ましくはこの免疫原特性を有し、且つ対象タンパク質のアミノ酸配列において少なくとも連続する8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるオリゴ（ポリ）ペプチドを例示することができる。 The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein. The oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has an immunogenic property similar to that of the target protein. An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
　かかるオリゴ（ポリ）ペプチドに対する抗体の製造は、宿主に応じて種々のアジュバントを用いて免疫学的反応を高めることによって行うこともできる。限定はされないが、そのようなアジュバントには、フロイントアジュバント、水酸化アルミニウムのようなミネラルゲル、並びにリゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油乳剤、キーホールリンペットヘモシアニン及びジニトロフェノールのような表面活性物質、BCG（カルメット－ゲラン桿菌）やコリネバクテリウム-パルヴムなどのヒトアジュバントが含まれる。 Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
　本発明の検査薬は、組成物の形態であってもよい。該組成物には、必要に応じて他の成分が含まれていてもよい。他の成分としては、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。 The test agent of the present invention may be in the form of a composition. The composition may contain other components as necessary. Examples of other components include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, and a fragrance. And chelating agents.
　本発明の検査薬は、キットの形態であってもよい。該キットには、上記検出剤或いはこれを含む上記組成物のほかに、被検体の体液の細胞外小胞における対象タンパク質の検出に使用し得るものを含んでいてもよい。このようなものの具体例としては、各種試薬（例えば二次抗体、緩衝液等）、器具（例えば細胞外小胞の精製、分離、濃縮用器具（例えばカラム等））等が挙げられる。 The test agent of the present invention may be in the form of a kit. In addition to the above-described detection agent or the above-mentioned composition containing the same, the kit may contain a substance that can be used for detection of a target protein in extracellular vesicles of a body fluid of a subject. Specific examples of such materials include various reagents (for example, secondary antibodies, buffers, etc.), instruments (for example, instruments for purification, separation, and concentration of extracellular vesicles (for example, columns)).
　5．閉塞性肺疾患モデル動物のスクリーニング方法
　本発明は、その一態様において、閉塞性肺疾患誘発処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患モデル動物のスクリーニング方法（本明細書において、「本発明のモデル動物スクリーニング方法」と示すこともある。）に関する。以下、これについて説明する。 Five. Screening Method for Obstructive Pulmonary Disease Model Animal In one aspect, the present invention provides a protein group (A) and a protein group (B) in an extracellular vesicle of a body fluid collected from an animal treated for induction of obstructive pulmonary disease. And a method for screening an obstructive pulmonary disease model animal using the amount or concentration of at least one protein selected from the group consisting of the protein group (C) as an index (hereinafter referred to as “model animal screening of the present invention”). Method "). This will be described below.
　体液、細胞外小胞、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、閉塞性肺疾患、対象タンパク質の量又は濃度の測定等については、上記「1．閉塞性肺疾患の検査方法」における定義と同様である。 For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive lung disease, measurement of the amount or concentration of the target protein, etc., see “1. Obstructive lung disease” above. This is the same as the definition in “Inspection Method”.
　動物は、スクリーニング対象動物であり、その生物種はヒト以外の動物である限り特に制限されない。該動物の生物種としては、例えばサル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられ、好ましくはマウスが挙げられる。 The animal is an animal to be screened, and its species is not particularly limited as long as it is a non-human animal. Examples of the biological species of the animal include various mammalian animals such as monkeys, mice, rats, dogs, cats, rabbits and the like, preferably mice.
　閉塞性肺疾患誘発処理は、閉塞性肺疾患を誘発できる処理である限り、特に制限されない。例えば気管支喘息を誘発させる場合であれば、例えばOVA（卵白アルブミン）；　、ダニ、花粉、昆虫等のアレルゲン等の誘発剤を投与（例えば経鼻投与、経口投与、吸入投与、経気管支投与、腹空内投与、静脈投与、経皮投与等、好適には腹空内投与と経鼻投与との組み合わせ）することによって、或いは遺伝子改変処理をすることによって、誘発処理が行われる。例えば慢性閉塞性肺疾患を誘発させる場合であれば、例えばエラスターゼ、喫煙暴露、タバコ抽出液、LPS+タバコ抽出液等の誘発剤を投与（例えば経鼻投与、経口投与、吸入投与、経気管支投与、腹空内投与、静脈投与、経皮投与等、好適には経鼻投与）することによって、或いは遺伝子改変処理をすることによって、誘発処理が行われる。 The obstructive pulmonary disease induction treatment is not particularly limited as long as it is a treatment capable of inducing obstructive pulmonary disease. For example, in the case of inducing bronchial asthma, for example, OVA (ovalbumin); administration of an inducer such as mite, pollen, insect, etc. (eg, nasal administration, oral administration, inhalation administration, transbronchial administration, abdominal administration) The induction treatment is performed by air administration, intravenous administration, transdermal administration, etc. (preferably a combination of intraperitoneal administration and nasal administration) or by genetic modification treatment. For example, in the case of inducing chronic obstructive pulmonary disease, for example, inducers such as elastase, smoking exposure, tobacco extract, LPS + tobacco extract (eg, nasal administration, oral administration, inhalation administration, transbronchial administration, The induction treatment is performed by intraperitoneal administration, intravenous administration, transdermal administration, etc. (preferably nasal administration) or by genetic modification treatment.
　本発明のモデル動物スクリーニング方法は、より具体的には、上記指標の値がカットオフ値以上である場合に、前記動物を閉塞性肺疾患モデル動物（或いは閉塞性肺疾患モデル候補動物）として選択する工程を含む。 More specifically, the model animal screening method of the present invention selects the animal as an obstructive pulmonary disease model animal (or an obstructive pulmonary disease model candidate animal) when the value of the index is not less than a cutoff value. The process of carrying out is included.
　カットオフ値は、スクリーニングの精度などの観点から当業者が適宜設定することができ、例えば、閉塞性肺疾患誘発処理していない動物から採取された体液の細胞外小胞における対象タンパク質の量及び／又は濃度に基づいて、その都度定められた値、或いは予め定められた値とすることができる。カットオフ値は、例えば、閉塞性肺疾患誘発処理していない動物から採取された体液の細胞外小胞における対象タンパク質の量及び／又は濃度（被検体が複数の場合は、平均値、中央値など）の、例えば1～10倍、好ましくは2～8倍、より好ましくは2.5～6倍の値とすることができる。 The cut-off value can be appropriately set by those skilled in the art from the viewpoint of screening accuracy and the like, for example, the amount of the target protein in the extracellular vesicles of body fluid collected from an animal that has not been induced to induce obstructive pulmonary disease, and Based on the density, it can be a value determined each time or a predetermined value. The cut-off value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles of body fluid collected from an animal that has not been induced to induce obstructive pulmonary disease (in the case of multiple subjects, the average value, the median value) For example, 1 to 10 times, preferably 2 to 8 times, and more preferably 2.5 to 6 times.
　6．閉塞性肺疾患の予防又は治療剤
　本発明は、その一態様において、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の抑制剤を含有する、閉塞性肺疾患の予防又は治療剤（本明細書において、「本発明の剤」と示すこともある。）に関する。以下、これについて説明する。 6． In one aspect of the present invention, a prophylactic or therapeutic agent for obstructive pulmonary disease suppresses at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C). The present invention relates to a prophylactic or therapeutic agent for obstructive pulmonary disease containing the agent (sometimes referred to as “the agent of the present invention”). This will be described below.
　タンパク質群（A）、タンパク質群（B）、タンパク質群（C）等については、上記「1．閉塞性肺疾患の検査方法」における定義と同様である。 Protein group (A), protein group (B), protein group (C) and the like are the same as defined in “1. Method for examining obstructive pulmonary disease” above.
　抑制剤としては、好ましくは対象タンパク質の発現抑制剤が挙げられる。 The inhibitor is preferably an expression inhibitor of the target protein.
　対象タンパク質の発現抑制剤としては、対象タンパク質、そのmRNAなどの発現量を抑制し得るものである限り特に制限されず、例えば対象タンパク質の遺伝子特異的small interfering RNA（siRNA）、対象タンパク質の遺伝子特異的microRNA（miRNA）、対象タンパク質の遺伝子特異的アンチセンス核酸、これらの発現ベクター；　対象タンパク質の遺伝子特異的リボザイム；　CRISPR/Casシステムによる対象タンパク質の遺伝子遺伝子編集剤などが挙げられる。 The target protein expression inhibitor is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, etc. For example, gene-specific small interfering RNA (siRNA) of the target protein, gene-specificity of the target protein MicroRNA (miRNA), gene-specific antisense nucleic acid of target protein, expression vectors thereof; gene-specific ribozyme of target protein; gene gene editing agent of target protein by CRISPR / Cas system.
　なお、発現抑制とは、対象タンパク質、そのmRNAなどの発現量を、例えば1/2、1/3、1/5、1/10、1/20、1/30、1/50、1/100、1/200、1/300、1/500、1/1000、1/10000以下に抑制することを意味し、これらの発現量を0とすることをも包含する。 The expression suppression refers to the expression level of the target protein, its mRNA, etc., for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100 , 1/200, 1/300, 1/500, 1/1000, 1 / 10,000 or less, and the expression level of these is set to 0.
　対象タンパク質の遺伝子siRNAは、対象タンパク質をコードする遺伝子の発現を特異的に抑制する二本鎖RNA分子である限り特に制限されない。一実施形態において、siRNAは、例えば、18塩基以上、19塩基以上、20塩基以上、又は21塩基以上の長さであることが好ましい。また、siRNAは、例えば、25塩基以下、24塩基以下、23塩基以下、又は22塩基以下の長さであることが好ましい。ここに記載するsiRNAの長さの上限値及び下限値は任意に組み合わせることが想定される。例えば、下限が18塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が19塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が20塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さ；下限が21塩基であり、上限が25塩基、24塩基、23塩基、又は22塩基である長さの組み合わせが想定される。 The gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein. In one embodiment, the siRNA is preferably 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length, for example. Moreover, it is preferable that siRNA is 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less, for example. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined. For example, the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases, and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases A certain length; a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 Combinations of lengths that are bases are envisioned.
　siRNAは、shRNA(small hairpin RNA)であっても良い。shRNAは、その一部がステムループ構造を形成するように設計することができる。例えば、shRNAは、ある領域の配列を配列aとし、配列aに対する相補鎖を配列bとすると、配列a、スペーサー、配列bの順になるようにこれらの配列が一本のRNA鎖に存在するようにし、全体で45～60塩基の長さとなるように設計することができる。配列aは、標的となる対象タンパク質をコードする塩基配列の一部の領域の配列であり、標的領域は特に限定されず、任意の領域を候補にすることが可能である。そして配列aの長さは19～25塩基、好ましくは19～21塩基である。 The siRNA may be shRNA (small hairpin RNA). shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is a sequence a and the complementary strand to the sequence a is a sequence b, these sequences are present in a single RNA strand in the order of sequence a, spacer, and sequence b. And can be designed to be 45-60 bases in total. The sequence a is a sequence of a partial region of the base sequence encoding the target protein to be targeted, and the target region is not particularly limited, and any region can be a candidate. The length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.
　対象タンパク質の遺伝子特異的siRNAは、5’又は3’末端に、付加的な塩基を有していてもよい。該付加的塩基の長さは、通常2～4塩基程度である。該付加的塩基は、DNAでもRNAでもよいが、DNAを用いると核酸の安定性を向上させることができる場合がある。このような付加的塩基の配列としては、例えばug-3’、uu-3’、tg-3’、tt-3’、ggg-3’、guuu-3’、gttt-3’、ttttt-3’、uuuuu-3’などの配列が挙げられるが、これらに限定されるものではない。 The gene-specific siRNA of the target protein may have an additional base at the 5 'or 3' end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuu-3'.
　siRNAは、3'末端に突出部配列（オーバーハング）を有していてもよく、具体的には、dTdT（dTはデオキシチミジンを表わす）を付加したものが挙げられる。また、末端付加がない平滑末端（ブラントエンド）であってもよい。siRNAは、センス鎖とアンチセンス鎖が異なる塩基数であってもよく、例えば、アンチセンス鎖が3'末端及び5'末端に突出部配列（オーバーハング）を有している「asymmetrical interfering RNA（aiRNA）」を挙げることができる。典型的なaiRNAは、アンチセンス鎖が21塩基からなり、センス鎖が15塩基からなり、アンチセンス鎖の両端で各々3塩基のオーバーハング構造をとる。 SiRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents deoxythymidine) is added. Further, it may be a blunt end (blunt end) without end addition. The siRNA may have a different number of bases in the sense strand and the antisense strand. For example, the “asymmetrical interfering RNA” in which the antisense strand has an overhang at the 3 ′ end and the 5 ′ end ( aiRNA) ". A typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases at each end of the antisense strand.
　対象タンパク質の遺伝子特異的siRNAの標的配列の位置は特に制限されるわけではないが、一実施形態において、5’-UTR及び開始コドンから約50塩基まで、並びに3’-UTR以外の領域から標的配列を選択することが望ましい。選択された標的配列の候補群について、標的以外のmRNAにおいて16-17塩基の連続した配列に相同性がないかどうかを、BLAST（http://www.ncbi.nlm.nih.gov/BLAST/）などのホモロジー検索ソフトを用いて調べ、選択した標的配列の特異性を確認することが好ましい。特異性が確認された標的配列について、AA（もしくはNA）以降の19－21塩基にTTもしくはUUの3’末端オーバーハングを有するセンス鎖と、該19－21塩基に相補的な配列及びTTもしくはUUの3’末端オーバーハングを有するアンチセンス鎖とからなる2本鎖RNAをsiRNAとして設計してもよい。また、siRNAの前駆体であるshRNAは、ループ構造を形成しうる任意のリンカー配列（例えば、5-25塩基程度）を適宜選択し、上記センス鎖とアンチセンス鎖とを該リンカー配列を介して連結することにより設計することができる。 The position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, the target is from the 5′-UTR and the start codon to about 50 bases, and from a region other than the 3′-UTR. It is desirable to select the sequence. For the selected target sequence candidate group, whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ It is preferable to check using a homology search software such as) to confirm the specificity of the selected target sequence. For the target sequence whose specificity has been confirmed, a sense strand having a 3 'end overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 ′ end overhang of UU may be designed as an siRNA. In addition, for the shRNA that is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) that can form a loop structure is appropriately selected, and the sense strand and the antisense strand are connected via the linker sequence. It can be designed by connecting.
　siRNA及び／又はshRNAの配列は、種々のwebサイト上に無料で提供される検索ソフトを用いて検索が可能である。このようなサイトとしては、例えば、以下を挙げることができる。
Ambionが提供するsiRNA Target Finder（http://www.ambion.com/jp/techlib/misc/siRNA_finder.html）pSilencer（登録商標）Expression Vector用インサートデザインツール（http://www.ambion.com/jp/techlib/misc/psilencer_converter.html）RNAi Codexが提供するGeneSeer（http://codex.cshl.edu/scripts/newsearchhairpin.cgi）。 The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include the following.
SiRNA Target Finder provided by Ambion (http://www.ambion.com/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer® Expression Vector (http://www.ambion.com/ jp / techlib / misc / psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
　siRNAは、mRNA上の標的配列のセンス鎖及びアンチセンス鎖をDNA/RNA自動合成機でそれぞれ合成し、適当なアニーリング緩衝液中、約90～約95℃で約1分程度変性させた後、約30～約70℃で約1～約8時間アニーリングさせることにより調製することができる。また、siRNAの前駆体となるshRNAを合成し、これを、RNA切断タンパク質ダイサー（dicer）を用いて切断することにより調製することもできる。 The siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with an RNA-cleaving protein dicer.
　対象タンパク質の遺伝子特異的miRNAは、対象タンパク質をコードする遺伝子の翻訳を阻害する限り任意である。例えば、miRNAは、siRNAのように標的mRNAを切断するのではなく、標的の3’非翻訳領域（UTR）に対合してその翻訳を阻害してもよい。miRNAは、pri-miRNA（primary miRNA）、pre-miRNA（precursor miRNA）、及び成熟miRNAのいずれでもよい。miRNAの長さは特に制限されず、pri-miRNAの長さは通常数百～数千塩基であり、pre-miRNAの長さは通常50～80塩基であり、成熟miRNAの長さは通常18～30塩基である。一実施形態において、対象タンパク質の遺伝子特異的miRNAは、好ましくはpre-miRNA又は成熟miRNAであり、より好ましくは成熟miRNAである。このような対象タンパク質の遺伝子特異的miRNAは、公知の手法で合成してもよく、合成RNAを提供する会社から購入してもよい。 The gene-specific miRNA of the target protein is arbitrary as long as the translation of the gene encoding the target protein is inhibited. For example, an miRNA may pair with the 3 'untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA like an siRNA. The miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ~ 30 bases. In one embodiment, the gene-specific miRNA of the protein of interest is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA. Such a gene-specific miRNA of the target protein may be synthesized by a known technique or purchased from a company that provides synthetic RNA.
　対象タンパク質の遺伝子特異的アンチセンス核酸とは、対象タンパク質をコードする遺伝子のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列又はその一部を含む核酸であって、該mRNAと特異的かつ安定した二重鎖を形成して結合することにより、対象タンパク質合成を抑制する機能を有する核酸である。アンチセンス核酸はDNA、RNA、DNA/RNAキメラのいずれでもよい。アンチセンス核酸がDNAの場合、標的RNAとアンチセンスDNAとによって形成されるRNA:DNAハイブリッドは、内在性リボヌクレアーゼH（RNase H）に認識されて標的RNAの選択的な分解を引き起こす。したがって、RNase Hによる分解を指向するアンチセンスDNAの場合、標的配列は、mRNA中の配列だけでなく、対象タンパク質の遺伝子の初期翻訳産物におけるイントロン領域の配列であってもよい。イントロン配列は、ゲノム配列と、対象タンパク質の遺伝子のcDNA塩基配列とをBLAST、FASTAなどのホモロジー検索プログラムを用いて比較することにより、決定することができる。 The gene-specific antisense nucleic acid of the target protein is a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA encoding the target protein, or a part thereof, and specific to the mRNA. It is a nucleic acid having a function of suppressing target protein synthesis by forming and binding an ideal and stable duplex. The antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of interest protein. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the target protein gene using a homology search program such as BLAST or FASTA.
　対象タンパク質の遺伝子特異的アンチセンス核酸の標的領域は、該アンチセンス核酸がハイブリダイズすることにより、結果として対象タンパク質への翻訳が阻害されるものであればその長さは制限されない。対象タンパク質の遺伝子特異的アンチセンス核酸は、対象タンパク質をコードするmRNAの全配列であっても部分配列であってもよい。合成の容易さや抗原性、細胞内移行性の問題などを考慮すれば、約10～約40塩基、特に約15～約30塩基からなるオリゴヌクレオチドが好ましいが、これらに限定されるものではない。より具体的には、対象タンパク質の遺伝子の5’端ヘアピンループ、5’端非翻訳領域、翻訳開始コドン、タンパク質コード領域、ORF翻訳終止コドン、3’端非翻訳領域、3’端パリンドローム領域又は3’端ヘアピンループなどをアンチセンス核酸の好ましい標的領域として選択しうるが、それらに限定されるものではない。 The length of the target region of the gene-specific antisense nucleic acid of the target protein is not limited as long as the antisense nucleic acid is hybridized and consequently translation into the target protein is inhibited. The gene-specific antisense nucleic acid of the target protein may be the entire sequence or a partial sequence of mRNA encoding the target protein. In view of easiness of synthesis, antigenicity, intracellular migration, and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but is not limited thereto. More specifically, the 5 ′ end hairpin loop, 5 ′ end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3 ′ end untranslated region, 3 ′ end palindromic region of the gene of the target protein Alternatively, a 3 ′ end hairpin loop or the like can be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
　対象タンパク質の遺伝子特異的アンチセンス核酸は、対象タンパク質の遺伝子のmRNAや初期転写産物とハイブリダイズしてタンパク質への翻訳を阻害するだけでなく、二本鎖DNAであるこれらの遺伝子と結合して三重鎖（トリプレックス）を形成し、RNAへの転写を阻害し得るもの（アンチジーン（antigene））であってもよい。 The gene-specific antisense nucleic acid of the target protein not only hybridizes with the mRNA or initial transcription product of the target protein gene and inhibits translation into the protein, but also binds to these genes that are double-stranded DNA. It may be a substance capable of forming a triplex and inhibiting transcription to RNA (antigene).
　上記した対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、及び対象タンパク質の遺伝子特異的アンチセンス核酸を構成するヌクレオチド分子は、安定性（化学的及び／又は対酵素）や比活性（RNAとの親和性）を向上させるために、種々の化学修飾を含んでもよい。例えば、ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンス核酸を構成する各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（phosphorothioate; PS)、メチルホスホネート（methylphosphonate）、ホスホロジチオネート（phosphorodithioate）などの化学修飾リン酸残基に置換することができる。また、各ヌクレオチドの糖（リボース）の2’位の水酸基を、-OR（R=CH3（2’-O-Me）、CH2CH2OCH3（2’-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、又はCH2CH2CNなど）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などを施してもよい。また、siRNAやmiRNAを構成するヌクレオチド分子の一部は、天然型のDNAに置換されていてもよい。 The nucleotide molecules constituting the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein have stability (chemical and / or anti-enzyme) and specific activity ( Various chemical modifications may be included in order to improve (affinity with RNA). For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting an antisense nucleic acid is selected from, for example, phosphorothioate (PS), methylphosphonate, phosphorodithio. It can be substituted with a chemically modified phosphate residue such as a phosphorodithioate. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R═CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , or CH 2 CH 2 CN) may be substituted. Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. May be applied. Moreover, a part of nucleotide molecules constituting siRNA or miRNA may be replaced with natural DNA.
　対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、及び対象タンパク質の遺伝子特異的アンチセンス核酸などは、対象タンパク質の遺伝子のcDNA配列もしくはゲノミックDNA配列に基づいてmRNAもしくは初期転写産物の標的配列を決定し、市販のDNA/RNA自動合成機を用いて、これに相補的な配列を合成することにより調製することができる。また、上記した各種修飾を含むアンチセンス核酸も、いずれも公知の手法により、化学的に合成することができる。 Gene-specific siRNA of the target protein, gene-specific miRNA of the target protein, and gene-specific antisense nucleic acid of the target protein are targets of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the target protein gene It can be prepared by determining the sequence and synthesizing a complementary sequence using a commercially available DNA / RNA automatic synthesizer. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.
　対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、又は対象タンパク質の遺伝子特異的アンチセンス核酸の発現ベクターについては、対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、又は対象タンパク質の遺伝子特異的アンチセンス核酸が発現可能な状態で組み込まれている限りにおいて特に限定されない。典型的には、該発現ベクターは、プロモーター配列、及び対象タンパク質の遺伝子特異的siRNA、対象タンパク質の遺伝子特異的miRNA、又は対象タンパク質の遺伝子特異的アンチセンス核酸のコード配列（必要に応じて、さらに転写終結シグナル配列）を含むポリヌクレオチド、必要に応じて他の配列を含む。プロモーターは、特に制限されず、例えばCMVプロモーター、EF1プロモーター、SV40プロモーター、MSCVプロモーター、hTERTプロモーター、βアクチンプロモーター、CAGプロモーターなどのRNA polymerase II（polII）系プロモーター；　マウス及びヒトのU6-snRNAプロモーター、ヒトH1-RNase P RNAプロモーター、ヒトバリン-tRNAプロモーターなどのRNA polymerase III（polIII）系プロモーターなどが挙げられ、これらの中でも、短いRNAの転写を正確に行うことができるという観点から、polIII系プロモーターが好ましい。他の配列としては、特に制限されず、発現ベクターが含み得る公知の配列を各種採用することができる。このような配列の一例としては、例えば複製起点、薬剤耐性遺伝子などが挙げられる。また、薬剤耐性遺伝子の種類及びベクターの種類は上述のものを例示できる。 For the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, or the gene-specific antisense nucleic acid expression vector of the target protein, the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, or the target There is no particular limitation as long as the gene-specific antisense nucleic acid of the protein is incorporated in an expressible state. Typically, the expression vector comprises a promoter sequence and a gene-specific siRNA of the protein of interest, a gene-specific miRNA of the protein of interest, or a coding sequence of a gene-specific antisense nucleic acid of the protein of interest (optionally further A polynucleotide containing a transcription termination signal sequence) and optionally other sequences. The promoter is not particularly limited, and examples thereof include RNA-polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter; mouse and human U6-snRNA promoters, Examples include human H1-RNase P プ ロ モ ー タ ー RNA promoter, human valine-tRNA promoter, and other RNA polymerase III (polIII) promoters. Among these, polIII promoters can be used for accurate transcription of short RNAs. preferable. Other sequences are not particularly limited, and various known sequences that can be included in the expression vector can be employed. Examples of such sequences include the origin of replication and drug resistance genes. Examples of the drug resistance gene and the vector include those described above.
　対象タンパク質の遺伝子発現抑制剤の別の例としては、対象タンパク質の遺伝子特異的リボザイムなどが挙げられる。「リボザイム」とは、狭義には、核酸を切断する酵素活性を有するRNAを意味するが、本願では配列特異的な核酸切断活性を有する限りDNAをも包含する。リボザイム核酸として最も汎用性の高いものは、ウイロイドやウイルソイドなどの感染性RNAに見られるセルフスプライシングRNAがあり、ハンマーヘッド型やヘアピン型などが知られている。ハンマーヘッド型は約40塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約10塩基程度）をmRNAの所望の切断部位と相補的な配列にすることにより、標的mRNAのみを特異的に切断することが可能である。このタイプのリボザイム核酸は、RNAのみを基質とするので、ゲノムDNAを攻撃することがないという利点を有する。対象タンパク質の遺伝子のmRNAが自身で二本鎖構造をとる場合には、RNAヘリカーゼと特異的に結合し得るウイルス核酸由来のRNAモチーフを連結したハイブリッドリボザイムを用いることにより、標的配列を一本鎖にすることができる［Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)］。さらに、リボザイムを、それをコードするDNAを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、tRNAを改変した配列をさらに連結したハイブリッドリボザイムとすることもできる［Nucleic Acids Res., 29(13): 2780-2788 (2001)］。 Another example of the gene expression inhibitor of the target protein is a gene-specific ribozyme of the target protein. “Ribozyme” in a narrow sense means RNA having an enzyme activity for cleaving nucleic acid, but in the present application, it includes DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has an advantage that it does not attack genomic DNA because it uses only RNA as a substrate. When the mRNA of the target protein gene has a double-stranded structure itself, the target sequence is single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-55772001 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
　本発明の剤の適用対象は特に限定されず、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ、ブタ、ウマ、ウシ、ヒツジ、ヤギ、シカなどの種々の哺乳類動物などが挙げられる。 The application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats and deer. .
　本発明の剤の形態は、特に限定されず、本発明の剤の用途に応じて、各用途において通常使用される形態をとることができる。 The form of the agent of the present invention is not particularly limited, and can take a form normally used in each application depending on the application of the agent of the present invention.
　形態としては、用途が医薬、健康増進剤、栄養補助剤（サプリメントなど）などである場合は、例えば錠剤（口腔内側崩壊錠、咀嚼可能錠、発泡錠、トローチ剤、ゼリー状ドロップ剤などを含む）、丸剤、顆粒剤、細粒剤、散剤、硬カプセル剤、軟カプセル剤、ドライシロップ剤、液剤（ドリンク剤、懸濁剤、シロップ剤を含む）、ゼリー剤などの経口摂取に適した製剤形態（経口製剤形態）、点鼻剤、吸入剤、肛門坐剤、挿入剤、浣腸剤、ゼリー剤、注射剤、貼付剤、ローション剤、クリーム剤などの非経口摂取に適した製剤形態（非経口製剤形態）が挙げられる。 As the form, when the use is a medicine, health enhancer, nutritional supplement (eg supplement), etc., for example, tablets (inner disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.) ), Pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral intake Forms (oral dosage forms), nasal drops, inhalants, rectal suppositories, inserts, enemas, jellies, injections, patches, lotions, creams, etc. Oral dosage form).
　形態としては、用途が食品組成物の場合は、液状、ゲル状あるいは固形状の食品、例えばジュース、清涼飲料、茶、スープ、豆乳、サラダ油、ドレッシング、ヨーグルト、ゼリー、プリン、ふりかけ、育児用粉乳、ケーキミックス、粉末状または液状の乳製品、パン、クッキーなどが挙げられる。 As a form, when the use is a food composition, a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula , Cake mix, powdered or liquid dairy products, bread, cookies and the like.
　形態としては、用途が口腔用組成物である場合は、例えば液体（溶液、乳液、懸濁液など）、半固体（ゲル、クリーム、ペーストなど）、固体（錠剤、粒子状剤、カプセル剤、フィルム剤、混練物、溶融固体、ロウ状固体、弾性固体など）などの任意の形態、より具体的には、歯磨剤（練歯磨、液体歯磨、液状歯磨、粉歯磨など）、洗口剤、塗布剤、貼付剤、口中清涼剤、食品（例えば、チューインガム、錠菓、キャンディ、グミ、フィルム、トローチなど）などが挙げられる。 As a form, when the use is an oral composition, for example, liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate agent, capsule, Film, kneaded material, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid toothpaste, liquid toothpaste, powder toothpaste etc.), mouthwash, Examples thereof include a coating agent, a patch, a mouth freshener, and a food (for example, chewing gum, tablet candy, candy, gummi, film, troche, etc.).
　本発明の剤は、必要に応じてさらに他の成分を含んでいてもよい。他の成分としては、例えば医薬、食品組成物、口腔用組成物、健康増進剤、栄養補助剤（サプリメントなど）などに配合され得る成分である限り特に限定されるものではないが、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤などが挙げられる。 The agent of the present invention may further contain other components as necessary. Other components are not particularly limited as long as they are components that can be blended in, for example, pharmaceuticals, food compositions, oral compositions, health enhancers, nutritional supplements (such as supplements), etc. , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.
　本発明の剤の対象タンパク質の抑制剤の含有量は、抑制剤の種類、用途、使用態様、適用対象、適用対象の状態などに左右されるものであり、限定はされないが、例えば0.0001～100重量％、好ましくは0.001～50重量％とすることができる。 The content of the inhibitor of the target protein of the agent of the present invention depends on the type of inhibitor, application, use mode, application target, application target state, and the like, and is not limited. For example, 0.0001 to 100 % By weight, preferably 0.001 to 50% by weight.
　本発明の組成物の適用（例えば、投与、摂取、接種など）量は、薬効を発現する有効量であれば特に限定されず、通常は、有効成分の重量として、一般に一日あたり0.1～1000 mg/kg体重である。上記投与量は1日1回又は2～3回に分けて投与するのが好ましく、年齢、病態、症状により適宜増減することもできる。 The amount of application (eg, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits a medicinal effect, and is generally 0.1 to 1000 per day as the weight of the active ingredient. mg / kg body weight. The above dose is preferably administered once a day or divided into 2 to 3 times a day, and can be appropriately increased or decreased depending on age, disease state, and symptoms.
　7．閉塞性肺疾患の予防又は治療剤の有効成分のスクリーニング方法
　本発明は、その一態様において、被検物質で処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患の予防又は治療剤の有効成分のスクリーニング方法（本明細書において、「本発明の有効成分スクリーニング方法」と示すこともある。）に関する。以下、これについて説明する。 7． The screening method of the active ingredient of the preventive or therapeutic agent for obstructive pulmonary disease The present invention, in one aspect thereof, is a protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance, A screening method for an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease using the amount or concentration of at least one protein selected from the group consisting of protein group (B) and protein group (C) as an index (this book) In the specification, it may be referred to as “the active ingredient screening method of the present invention”. This will be described below.
　体液、細胞外小胞、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、閉塞性肺疾患、対象タンパク質の量又は濃度の測定等については、上記「1．閉塞性肺疾患の検査方法」における定義と同様である。 For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive lung disease, measurement of the amount or concentration of the target protein, etc., see “1. Obstructive lung disease” above. This is the same as the definition in “Inspection Method”.
　動物の生物種は特に制限されない。動物の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられ、好ましくはマウスが挙げられる。 Species of animal species are not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably mice.
　被検物質としては、天然に存在する化合物又は人工に作られた化合物を問わず広く使用することができる。また、精製された化合物に限らず、多種の化合物を混合した組成物や、動植物の抽出液も使用することができる。化合物には、低分子化合物に限らず、タンパク質、核酸、多糖類等の高分子化合物も包含される。 The test substance can be widely used regardless of whether it is a naturally occurring compound or a man-made compound. Moreover, the composition which mixed not only the refined compound but various compounds, and the extract of animals and plants can also be used. The compounds include not only low molecular compounds but also high molecular compounds such as proteins, nucleic acids, and polysaccharides.
　本発明の有効成分スクリーニング方法は、より具体的には、上記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞における対応タンパク質の量又は濃度（対照値）よりも低い場合に、前記被検物質を閉塞性肺疾患の予防又は治療剤の有効成分（或いは、閉塞性肺疾患の予防又は治療剤の有効成分の候補物質）として選択する工程を含む。 More specifically, the active ingredient screening method of the present invention is such that the value of the index is the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal that has not been treated with the test substance (control value). And a step of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease (or a candidate substance for an active ingredient of prophylactic or therapeutic agent for obstructive pulmonary disease).
　対応タンパク質とは、指標としている対象タンパク質と同じタンパク質を意味する。 Corresponding protein means the same protein as the target protein used as an index.
　「低い」とは、例えば指標の値が、対照値の1/2、1/5、1/10、1/20、1/50、1/100であることを意味する。 “Low” means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
　8．閉塞性肺疾患の誘発性又は増悪性の評価方法
　本発明は、その一態様において、被検物質で処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患の誘発性又は増悪性の評価方法（本明細書において、「本発明の毒性評価方法」と示すこともある。）に関する。以下、これについて説明する。 8． In one aspect, the present invention, in one aspect thereof, is a protein group (A), a protein group in an extracellular vesicle of a body fluid collected from an animal treated with a test substance. (B) and an evaluation method for inducing or malignancy of obstructive pulmonary disease using the amount or concentration of at least one protein selected from the group consisting of the protein group (C) as an index (in the present specification, It may be referred to as “the toxicity evaluation method of the present invention”. This will be described below.
　体液、細胞外小胞、タンパク質群（A）、タンパク質群（B）、タンパク質群（C）、閉塞性肺疾患、対象タンパク質の量又は濃度の測定、動物の生物種、被検物質等については、上記「1．閉塞性肺疾患の検査方法」及び「7．閉塞性肺疾患の予防又は治療剤の有効成分のスクリーニング方法」における定義と同様である。 For body fluids, extracellular vesicles, protein group (A), protein group (B), protein group (C), obstructive lung disease, measurement of the amount or concentration of the target protein, animal species, test substances, etc. The definition is the same as in “1. Method for testing obstructive pulmonary disease” and “7. Screening method for active ingredient of prophylactic or therapeutic agent for obstructive pulmonary disease”.
　本発明の毒性評価方法は、より具体的には、上記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞における対応タンパク質の量又は濃度（対照値）よりも高い場合に、前記被検物質を閉塞性肺疾患の誘発性又は増悪性があると判定する工程を含む。 More specifically, in the toxicity evaluation method of the present invention, the value of the above-mentioned index is the amount or concentration of the corresponding protein in the extracellular vesicles of body fluid collected from an animal that has not been treated with the test substance (control value). The test substance is determined to be inducible or malignant for obstructive pulmonary disease.
　対応タンパク質とは、指標としている対象タンパク質と同じタンパク質を意味する。 Corresponding protein means the same protein as the target protein used as an index.
　「高い」とは、例えば指標の値が、対照値の2倍、5倍、10倍、20倍、50倍、100倍であることを意味する。 “High” means, for example, that the index value is 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
　試験例1．気管支喘息モデルマウスの血清の調製
　マウスとして、11週齢のC57BL6/J雄マウスを用いた。OVA（Sigma, A5503, GradeV）20μg及びAlum（Thermo Imject Alum (#77161　Lot NB168145)）2mgをPBS 400μLに懸濁して、4％イソフルランで吸入麻酔したマウスに、腹空内投与した。初回投与から7日後に、再度、OVA（20μg）及びAlum（2mg）のPBS（400μL）懸濁液を腹空内投与した。初回投与から21、22及び23日後に、OVA（50μg）のPBS（50μL）懸濁液を経鼻投与した。初回投与から28日後に血清を回収した。病理評価を行い、気管支喘息状態にあるマウス（n＝8）の血清を、気管支喘息モデルマウスの血清として選別した。なお、病理評価においては、公知の方法に従って、気管支周囲の血管周辺領域における一定面積当たりの好酸球浸潤数を測定した。この好酸球浸潤数は喘息の活性化指標となる値である。 Test Example 1. Preparation of serum of bronchial asthma model mice 11-week-old C57BL6 / J male mice were used. 20 μg of OVA (Sigma, A5503, Grade V) and 2 mg of Alum (Thermo Imject Alum (# 77161 Lot NB168145)) were suspended in 400 μL of PBS and administered intraperitoneally to mice anesthetized with 4% isoflurane. Seven days after the first administration, a suspension of OVA (20 μg) and Alum (2 mg) in PBS (400 μL) was intraperitoneally administered again. 21, 22, and 23 days after the initial administration, a suspension of OVA (50 μg) in PBS (50 μL) was administered intranasally. Serum was collected 28 days after the first dose. Pathological evaluation was performed, and the sera of mice with bronchial asthma (n = 8) were selected as sera of bronchial asthma model mice. In the pathological evaluation, the number of eosinophil infiltration per certain area in the blood vessel peripheral region around the bronchus was measured according to a known method. This eosinophil infiltration number is a value that serves as an activation index for asthma.
　試験例2．慢性閉塞性肺疾患（COPD）モデルマウスの血清の調製
　マウスとして、11週齢のC57BL6/J雄マウスを用いた。4％イソフルランで吸入麻酔したマウスに、ブタエラスターゼ（Elastase Products Company社製）7.5 Uを含有する滅菌PBS 40μLを、2回に分けて、20μLずつ投与した。より具体的には、一回目に20μLを滴下して吸引させ、しばらく休ませてから再度滴下して吸引させた。投与から21日後に、血清を回収した。病理評価を行い、慢性閉塞性肺疾患の状態にあるマウス（n＝8）の血清を、慢性閉塞性肺疾患モデルマウスの血清として選別した。なお、病理評価においては、公知の方法に従って、平均肺胞径を測定した。この平均肺胞径は肺気腫（COPD）の指標となる値である。 Test Example 2. Preparation of serum of chronic obstructive pulmonary disease (COPD) model mouse An 11-week-old C57BL6 / J male mouse was used. Mice anesthetized by inhalation with 4% isoflurane were administered with 20 μL of sterile PBS 40 μL containing 7.5 U of porcine elastase (Elastase Products Company) in two portions. More specifically, 20 μL was dropped and aspirated for the first time, and after resting for a while, it was dropped again and aspirated. Serum was collected 21 days after administration. Pathological evaluation was performed, and sera from mice with chronic obstructive pulmonary disease (n = 8) were selected as sera from chronic obstructive pulmonary disease model mice. In the pathological evaluation, the average alveolar diameter was measured according to a known method. This average alveolar diameter is a value indicative of emphysema (COPD).
　試験例3．細胞外小胞画分の調製
　試験例1及び2で得られた血清、並びに正常マウス（11週齢のC57BL6/J雄マウス、n＝8）の血清から細胞外小胞画分を調製した。細胞外小胞画分の調製は、細胞外小胞精製カラム（EV-Second、ジーエルサイエンス社製）を用いて行った。 Test Example 3. Extracellular vesicular fractions serum obtained in Preparation Test Example 1 and 2, as well as normal mice (11 weeks old C57BL6 / J male mice, n = 8) were prepared extracellular vesicular fractions from the serum of. The extracellular vesicle fraction was prepared using an extracellular vesicle purification column (EV-Second, manufactured by GL Sciences).
　続いて、得られた細胞外小胞画分について、細胞外小胞の粒子数及び粒子径を測定した。具体的には、ナノサイト（日本カンタム・デザイン株式会社、Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025）を使用して測定した。これは、粒子径ごとのブラウン運動速度の違いをもとに解析するものであり、画面に映る散乱光１つ１つの動きを追尾（トラッキング）し、各々の移動速度（拡散係数）から液中における粒子径（流体力学径）を算出することができる。結果を図１に示す。 Subsequently, the number and particle diameter of the extracellular vesicles were measured for the obtained extracellular vesicle fraction. Specifically, the measurement was carried out using a nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion speed for each particle size, tracking (tracking) each individual scattered light reflected on the screen, and using each moving speed (diffusion coefficient) in the liquid. The particle diameter (hydrodynamic diameter) can be calculated. The results are shown in FIG.
　さらに、細胞外小胞を免疫電子顕微鏡法によって観察した。具体的には、次のようにして行った。固定済み細胞外小胞溶液を5～8μLずつグリッドへ載せ15分静置して、グリッドのフォルムバールへ細胞外小胞を定着させた。PBSで3回洗浄した後、ブロッキング反応（1%BSA/PBS、10分）を行い、続いて一次抗体反応（BD Pharmingen 553758 Purified NA/LE Rat Anti-Mouse CD9 Clone: KMC8、100倍希釈、2時間半、室温）を行った。PBSで6回洗浄した後、二次抗体反応（CRL, , EMGAT10 Anti-IgG (H＋L), Rat, Goat-Poly, Gold 10 nm, EM、200倍希釈、1時間、室温）を行った。PBSで6回洗浄した後、1%GA/PBSで15分間処理し、さらにPBSで3回洗浄した。水洗し、0.5% UA（酢酸ウラニル）で処理して、乾燥させて、電子顕微鏡で観察した。得られた結果を図２に示す。 Furthermore, extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was performed as follows. 5 to 8 μL of the fixed extracellular vesicle solution was placed on the grid and allowed to stand for 15 minutes to fix the extracellular vesicles to the form bar of the grid. After washing 3 times with PBS, blocking reaction (1% BSA / PBS, 10 minutes) followed by primary antibody reaction (BD Pharmingen 553758 Purified NA / LE Rat Anti-Mouse CD9 Clone: KMC8, 100 times dilution, 2 Half hour, room temperature). After washing 6 times with PBS, secondary antibody reaction (CRL,, EMGAT10 Anti-IgG (H + L), Rat, Goat-Poly, Gold 10 nm, EM, 200-fold dilution, 1 hour, room temperature) was performed. After washing 6 times with PBS, it was treated with 1% GA / PBS for 15 minutes and further washed 3 times with PBS. Washed with water, treated with 0.5% UA (uranyl acetate), dried and observed with an electron microscope. The obtained results are shown in FIG.
　試験例4．プロテオミクス解析（ノンラベル、LC-MS/MS）
　細胞外小胞画分中のタンパク質の定量を、LC-MS/MS分析（ノンラベル法）により行った。具体的には以下のようにして行った。 Test Example 4. Proteomics analysis (non-label, LC-MS / MS)
The protein in the extracellular vesicle fraction was quantified by LC-MS / MS analysis (non-label method). Specifically, it was performed as follows.
　（サンプル調製）
　細胞外小胞画分を、5mMのTCEPを用いて37℃で30分間還元し、25mMのヨードアセトアミドを用いて室温で45分間アルキル化した。その後、サンプルを50mM重炭酸アンモニウムで7倍希釈し、96ウェルフィルタープレートに入れ、5μLの固定化トリプシン（Thermo Fisher Scientific社製）と共に37℃で6時間振とうして、消化させた。トリプシン消化物を、Oasis HLB 96-well μElution Plate（Waters Corporation社製、米国）を用いて脱塩し、LC-MS/MS分析に供した。 (Sample preparation)
The extracellular vesicle fraction was reduced with 5 mM TCEP for 30 minutes at 37 ° C. and alkylated with 25 mM iodoacetamide for 45 minutes at room temperature. The sample was then diluted 7-fold with 50 mM ammonium bicarbonate, placed in a 96 well filter plate and digested by shaking with 5 μL of immobilized trypsin (Thermo Fisher Scientific) at 37 ° C. for 6 hours. The trypsin digest was desalted using Oasis HLB 96-well μElution Plate (Waters Corporation, USA) and subjected to LC-MS / MS analysis.
　（LC／MS／MS分析）
　乾燥したペプチドサンプルを、0.1％のトリフルオロ酢酸を含む、2％のアセトニトリル溶液に再懸濁し、Ultimate 3000 RSLC nano-flow HPLC system（DIONEX社製、米国）を備えるLTQ-Orbitrap-Velos mass spectrometer（Thermo Fisher Scientific社製）を用いて分析した。75μm × 150 mm C18 tip-column（日京テクノス社製）において、溶媒A（0．1％のギ酸）および溶媒B（アセトニトリル中の0．1％ギ酸）を用い、流速250nL／分において、95分間で溶媒Bを6.4～30％、その後10分間で溶媒Bを30～95％とする多段階直線勾配で、サンプルを分離した。 (LC / MS / MS analysis)
The dried peptide sample is resuspended in a 2% acetonitrile solution containing 0.1% trifluoroacetic acid and an LTQ-Orbitrap-Velos mass spectrometer equipped with an Ultimate 3000 RSLC nano-flow HPLC system (DIONEX, USA) ( Analysis was performed using Thermo Fisher Scientific). In a 75 μm × 150 mm C18 tip-column (manufactured by Nikyo Technos), solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) were used at a flow rate of 250 nL / min. Samples were separated with a multi-step linear gradient with 6.4-30% solvent B in minutes and then 30-95% solvent B in 10 minutes.
　溶出したペプチドを、2000 Vのスプレー電圧でイオン化し、MSデータをデータ依存型フラグメント法で取得した。測定スキャン（survey scan）は、m／z 400～1600、分解度60000、AGCターゲット値1.0×106イオンカウントで行った。各測定スキャンにおける上位20の強度の前駆イオンを、リニアイオントラップでAGCターゲット値5000イオンカウントのノーマルCIDスキャンモードを用いた低分解能MS/MS取得に供した。 The eluted peptides were ionized with a spray voltage of 2000 V and MS data was acquired by the data-dependent fragment method. The measurement scan (survey scan) was performed at m / z 400 to 1600, resolution 60000, AGC target value 1.0 × 10 6 ion count. The top 20 intensities of precursor ions in each measurement scan were subjected to low-resolution MS / MS acquisition using a normal CID scan mode with an AGC target value of 5000 ions count with a linear ion trap.
　Proteome Discoverer 1.3 software（Thermo Fischer Scientific社製）を用いてSEQUESTデータベースサーチを行い、タンパク質の同定を試みた。MS/MSスペクトルをマウスタンパク質データベースUniProtに対して検索した。検索パラメーターは次のとおりである：Enzyme Name = Semitrypsin、Precursor Mass Tolerance = 3 ppm、Fragment Mass Tolerance = 0.8 Da、Dynamic Modification = Oxidation (Met)、Static Modification = Carbamidomethyl (Cys)。SEQUEST Decoy Database SearchにおけるPeptide Validator activityによるFDR（false discovery rate）が1％未満である場合に、そのペプチドと同定した。 Protein identification was attempted by performing a SEQUEST database search using Proteome Discoverer 1.3 software (Thermo Fischer Scientific). MS / MS spectra were searched against the mouse protein database UniProt. The search parameters are: Enzyme Name = Semitrypsin, Precursor Mass Tolerance = 3 ppm, Fragment Mass Tolerance = 0.8 Da, Dynamic Modification = Oxidation (Met), Static Modification = Carbamidomethyl (Cys). When the FDR (false discovery rate) by Peptide Validator activity in SEQUEST Decoy Database Search was less than 1%, the peptide was identified.
　（Expressionistサーバーにおけるラベルフリー定量分析）
　LC-MS/MSのデータセットを、Expressionist RefinerMS module（Genedata AG社製、スイス）にロードし、データ処理およびフリー定量分析を行った。RefinerMS softwareの全体的なワークフローを図2に示す。2D MSクロマトグラム面（x = m/z 、y =RT）上の10個のデータポイント毎にSpectrum Gridをセッティングした。次いで、第1および第2のChemical Noise Subtractionにおいて、RT = 2 scansおよびm/z = 6 pointsで、Structure Removalを順次行った。続いて、第3のChemical Noise Subtractionにおいて、RT Window = 500 scansおよびQuantile = 90%でStructure Removalを行った。第4のChemical Noise Subtractionにおいて、RT = 2 scansでStructure Removalを行った。その後、2000未満の強度のシグナルを、Intensity Thresholdingを用いて排除した。最後に、第5および第6のChemical Noise Subtractionにおいて、ぞれぞれRT = 2 scansおよびm/z = 5 pointsでStructure Removal行った。 (Label-free quantitative analysis on Expressionist server)
LC-MS / MS dataset was loaded into Expressionist RefinerMS module (Genedata AG, Switzerland) for data processing and free quantitative analysis. Figure 2 shows the overall workflow of RefinerMS software. A Spectrum Grid was set for every 10 data points on the 2D MS chromatogram plane (x = m / z, y = RT). Next, in the first and second chemical noise subtraction, structure removal was sequentially performed at RT = 2 scans and m / z = 6 points. Subsequently, in the third chemical noise subtraction, structure removal was performed with RT Window = 500 scans and Quantile = 90%. In the fourth Chemical Noise Subtraction, Structure Removal was performed at RT = 2 scans. Thereafter, signals of intensity less than 2000 were excluded using Intensity Thresholding. Finally, Structure Removal was performed at RT = 2 scans and m / z = 5 points in the fifth and sixth Chemical Noise Subtraction, respectively.
　次いで、Chromatogram Gridをノイズ減算データ上の10スキャン毎にセットし、次のパラメーターを用いてChromatogram RT Alignmentを行った：m/z Window = 11 points、RT Window = 11 scans、Gap Penalty = 1、RT Search Interval = 2 minutes、Alignment Scheme = pairwise alignment based tree。 Next, Chromatogram Grid was set every 10 scans on the noise subtraction data, and Chromatogram RT Alignment was performed using the following parameters: m / z Window = 11 points, RT Window = 11 scans, Gap Penalty = 1, RT Search Interval = 2 minutes, Alignment Scheme = pairwise alignment based tree.
　続いて、一時的平均化クロマトグラムにおけるピークを、次のパラメーターを用いて、Summed Peak Detection Activityによって検出した：Summation Window = 20 scans、Overlap = 10、Minimum Peak Size = 6 scans、Maximum Merge Distance = 1 data points、Gap/Peak Ratio = 5、Method = curvature-based peak detection、Peak Refinement Threshold = 5、Consistency Filter Threshold = 1。最後に、Summed Isotope Clustering Activityを用いて、1つの分子に由来する同位体ピークを同位体クラスターにグループ化した。このときのパラメーターは次のとおりである：Minimum Charge = 1、Maximum Charge = 6、Maximum Missing Peaks = 0、First Allowed Gap Position = 10、Ionization =protonation、RT Tolerance = 0.1 minute、m/z Tolerance = 0.01 Da、Minimum Cluster Size Ratio = 0.5。 Subsequently, peaks in the temporal averaged chromatogram were detected by Summed Peak Detection Activity using the following parameters: Summation Window = 20 scans, Overlap = 10, Minimum Peak Size = 6 scans, Maximum Merge Distance = 1 data points, Gap / Peak Ratio = 5, Method = curvature-based peak detection, Peak Refinement Threshold = 5, Consistency Filter Threshold = 1. Finally, we used SummedotoIsotope Clustering Activity to group the isotope peaks from one molecule into isotope clusters. The parameters at this time are as follows: Minimum Charge = 1, Maximum Charge = 6, Maximum Missing Peaks = 0, First Allowed Gap Position = 10, Ionization = protonation, RT Tolerance = 0.1 minute, m / z Tolerance = 0.01 Da, Minimum Cluster Size Ratio = 0.5.
　（Expressionist Analystにおける統計学的解析）
　組織線維化疾患モデルマウス群と正常マウス群とを比較し、t-検定により、2つの群間で有意に（P<0.05）離れた発現レベルを示す候補を抽出した。同様に、慢性閉塞性肺疾患モデルマウス群と正常マウス群とを比較し、t-検定により、2つの群間で有意に（P<0.05）離れた発現レベルを示す候補を抽出した。次に、バイオマーカー候補の最小の組合せを、Expressionist Analyst module （Genedata AG）におけるSupport Vector Machine-Recursive Feature Elimination（SVM-RFE）アルゴリズムまたはSVM-SVMアルゴリズムによって、取扱説明書に従い、見積もった。 (Statistical analysis in Expressionist Analyst)
A tissue fibrosis disease model mouse group and a normal mouse group were compared, and a candidate showing an expression level significantly (P <0.05) apart between the two groups was extracted by t-test. Similarly, the chronic obstructive pulmonary disease model mouse group was compared with the normal mouse group, and a candidate showing an expression level significantly (P <0.05) apart between the two groups was extracted by t-test. Next, the minimum combination of biomarker candidates was estimated according to the instruction manual by the Support Vector Machine-Recursive Feature Elimination (SVM-RFE) algorithm or SVM-SVM algorithm in Expressionist Analyst module (Genedata AG).
　（結果）
　各種タンパク質の定量データを得た。気管支喘息モデルマウスの細胞外小胞画分における量の平均値（気管支喘息群）が、正常マウスの細胞外小胞画分における量の平均値（コントロール群）の5倍以上であったタンパク質、及びコントロール群では発現を確認できないが気管支喘息群では発現が確認できたタンパク質、並びに慢性閉塞性肺疾患モデルマウスの細胞外小胞画分における量の平均値（COPD群）が、正常マウスの細胞外小胞画分における量の平均値（コントロール群）の3倍以上であったタンパク質、及びコントロール群では発現を確認できないがCOPD群では発現が確認できたタンパク質を、閉塞性肺疾患バイオマーカーとして一定程度の有効性があると判定し、選択した。さらに、各気管支喘息群モデルマウスにおける好酸球浸潤数とタンパク質量との相関、及び各慢性閉塞性肺疾患モデルマウスにおける平均肺胞径とタンパク質量との相関を解析し、相関係数（r値）を算出した。選択されたタンパク質、コントロール群に対する量比、及び相関係数を表１～４に示す。 (result)
Quantitative data for various proteins was obtained. A protein whose average value in the extracellular vesicle fraction of bronchial asthma model mice (bronchial asthma group) was more than 5 times the average value in the extracellular vesicle fraction of normal mice (control group), In addition, the average value (COPD group) of the protein that could not be confirmed in the control group but could be confirmed in the bronchial asthma group and the extracellular vesicle fraction of the chronic obstructive pulmonary disease model mouse (COPD group) Proteins that were more than 3 times the average amount in the outer vesicle fraction (control group) and proteins that could not be confirmed in the control group but could be confirmed in the COPD group were used as biomarkers for obstructive pulmonary disease. It was judged that there was a certain degree of effectiveness and was selected. Furthermore, we analyzed the correlation between the number of eosinophils infiltrated in each bronchial asthma model mouse and the amount of protein, and the correlation between the average alveolar diameter and protein amount in each chronic obstructive pulmonary disease model mouse. Value). Tables 1 to 4 show the selected proteins, the quantitative ratios to the control group, and the correlation coefficients.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表１～４のタンパク質群の内、Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1については、気管支喘息群及びCOPD群の両方において、コントロール群に比べて一定以上の量であったタンパク質である。 Among the proteins in Tables 1 to 4, Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3, Fibulin-2, and Adenylyl cyclase-associated protein 1 In both the bronchial asthma group and the COPD group, the amount of the protein was a certain amount or more compared to the control group.
　試験例5．免疫染色による肺組織における慢性閉塞性肺疾患バイオマーカーの検出
　正常マウス（4匹）、気管支喘息モデルマウス（8匹）、及び慢性閉塞性肺疾患（COPD）モデルマウス（4匹）それぞれの肺組織を、慢性閉塞性肺疾患バイオマーカー（試験例4）に対する抗体を用いて免疫染色した。使用した一次抗体は、抗EEA1抗体（code：ab2900／abcam）、抗VCAN抗体（code：ab19345／abcam）、抗PRG4抗体（code：LS-B8236／LSBio）、抗decorin抗体（code：ab175404／abcam）、抗HABP2抗体（code：ab174953／abcam）、抗EFEMP1抗体（code：GTX111657／GeneTex）、抗fibulin2抗体（code：bs-13160R／Bioss）、抗biglycan抗体（code：ab49701／abcam）、抗TSP1抗体（code：ab85762／abcam）、及び抗CAP1抗体（code：ab182604／abcam）である。 Test Example 5. Detection of chronic obstructive pulmonary disease biomarker in lung tissue by immunostaining Lung tissue of normal mice (4 mice), bronchial asthma model mice (8 mice) and chronic obstructive pulmonary disease (COPD) model mice (4 mice) Was immunostained with an antibody against a chronic obstructive pulmonary disease biomarker (Test Example 4). Primary antibodies used were anti-EEA1 antibody (code: ab2900 / abcam), anti-VCAN antibody (code: ab19345 / abcam), anti-PRG4 antibody (code: LS-B8236 / LSBio), anti-decorin antibody (code: ab175404 / abcam) ), Anti-HABP2 antibody (code: ab174953 / abcam), anti-EFEMP1 antibody (code: GTX111657 / GeneTex), anti-fibulin2 antibody (code: bs-13160R / Bioss), anti-biglycan antibody (code: ab49701 / abcam), anti-TSP1 An antibody (code: ab85762 / abcam) and an anti-CAP1 antibody (code: ab182604 / abcam).
　各マウスについて、ランダムに選ばれた3つの視野について、染色の程度を5段階（0点：なし、1点：極軽度、2点：軽度、3点：中等度、4点：高度）で評価して、合計点数を算出することによって、半定量解析した。 For each mouse, three randomly selected fields of view were evaluated in 5 grades (0 points: none, 1 point: very mild, 2 points: mild, 3 points: moderate, 4 points: advanced) Then, semi-quantitative analysis was performed by calculating the total score.
　得られた半定量解析結果に基づいて、気管支喘息モデルマウスの方が正常マウスよりも染色の程度が高いことの有意差（BA-Cont）、慢性閉塞性肺疾患モデルマウスの方が正常マウスよりも染色の程度が高いことの有意差（COPD-Cont）、及び気管支喘息モデルマウスの方が慢性閉塞性肺疾患モデルマウスよりも染色の程度が高いことの有意差（BA-COPD）を、t検定によって算出した。結果を表５に示す。 Based on the semi-quantitative analysis results obtained, a significant difference (BA-Cont) in the degree of staining in bronchial asthma model mice is higher than in normal mice, chronic obstructive pulmonary disease model mice are more normal than normal mice The significant difference in the degree of staining (COPD-Cont) and the significant difference in the degree of staining in the bronchial asthma model mice (BA-COPD) compared to the chronic obstructive pulmonary disease model mice, t Calculated by test. The results are shown in Table 5.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表５に示されるように、慢性閉塞性肺疾患バイオマーカーは、気管支喘息モデルマウス及び慢性閉塞性肺疾患モデルマウスそれぞれの肺組織において、正常マウスの肺組織におけるよりも、発現量が高い傾向にあった。また、調べた慢性閉塞性肺疾患バイオマーカーは、気管支喘息モデルマウスの肺組織において、慢性閉塞性肺疾患モデルマウスの肺組織におけるよりも、発現量が高い傾向にあった。 As shown in Table 5, the expression level of the chronic obstructive pulmonary disease biomarker tends to be higher in the lung tissues of the bronchial asthma model mouse and the chronic obstructive pulmonary disease model mouse than in the lung tissue of the normal mouse. there were. Moreover, the examined chronic obstructive pulmonary disease biomarkers tended to be expressed in higher amounts in the lung tissue of bronchial asthma model mice than in the lung tissue of chronic obstructive pulmonary disease model mice.
　さらに、上記で得られた半定量解析結果に基づいて、試験例4で測定された慢性閉塞性肺疾患バイオマーカーの細胞外小胞画分における量との相関を解析した。その結果、慢性閉塞性肺疾患バイオマーカーは、細胞外小胞画分における量と、肺組織における発現量との間に、正の相関があることが分かった。 Furthermore, based on the semi-quantitative analysis result obtained above, the correlation with the amount of the extracellular vesicle fraction of the chronic obstructive pulmonary disease biomarker measured in Test Example 4 was analyzed. As a result, it was found that the biomarker for chronic obstructive pulmonary disease has a positive correlation between the amount in the extracellular vesicle fraction and the expression level in lung tissue.

Claims (15)

  1. 閉塞性肺疾患を検査する方法であって、
    （1）被検体から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
    （A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
    （B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
    （C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質を検出する工程を含む、検査方法。     A method for testing obstructive pulmonary disease, comprising:
    (1) Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from the subject:
    (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
    (B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A test method comprising a step of detecting at least one protein selected from the group consisting of a protein group consisting of Fibulin-2 and Adenylyl cyclase-associated protein 1.
  2. さらに、（2）前記工程（1）で検出されたタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が閉塞性肺疾患に罹患していると判定する工程を含む、請求項１に記載の検査方法。 And (2) including a step of determining that the subject suffers from obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is not less than a cutoff value. Item 2. The inspection method according to Item 1.
  3. 前記工程（2）が、
    （2a）前記タンパク質群（A）及び前記タンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息に罹患していると判定する工程、
    （2b）前記タンパク質群（B）及び前記タンパク質群（C）からなる群より選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が慢性閉塞性肺疾患に罹患していると判定する工程、
    （2c）前記タンパク質群（A）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が慢性閉塞性肺疾患非合併型の気管支喘息に罹患していると判定する工程、
    （2d）前記タンパク質群（B）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息非合併型の慢性閉塞性肺疾患に罹患していると判定する工程、並びに
    （2e）前記タンパク質群（C）から選択される少なくとも1種のタンパク質の量又は濃度がカットオフ値以上である場合に、前記被検体が気管支喘息及び慢性閉塞性肺疾患の両方に罹患していると判定する工程
    からなる群より選択される少なくとも1種の工程である、請求項２に記載の検査方法。     The step (2)
    (2a) The subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value The process of determining that
    (2b) When the amount or concentration of at least one protein selected from the group consisting of the protein group (B) and the protein group (C) is a cut-off value or more, the subject is chronic obstructive lung Determining that the patient is afflicted with a disease,
    (2c) When the amount or concentration of at least one protein selected from the protein group (A) is not less than a cut-off value, the subject suffers from bronchial asthma without chronic obstructive pulmonary disease. A process of determining that
    (2d) When the amount or concentration of at least one protein selected from the protein group (B) is equal to or higher than a cutoff value, the subject suffers from bronchial asthma non-complicated chronic obstructive pulmonary disease. And (2e) when the amount or concentration of at least one protein selected from the protein group (C) is equal to or higher than a cutoff value, the subject is bronchial asthma and chronic obstructive The test method according to claim 2, wherein the test method is at least one process selected from the group consisting of determining that the patient suffers from both lung diseases.
  4. 前記カットオフ値が、閉塞性肺疾患に罹患していない被検体から採取された体液の細胞外小胞における対応タンパク質の量又は濃度の値の、2.5～6倍の値である、請求項２又は３に記載の検査方法。 The cutoff value is 2.5 to 6 times the value of the amount or concentration of the corresponding protein in extracellular vesicles of body fluid collected from a subject not suffering from obstructive pulmonary disease. Or the inspection method of 3.
  5. 前記体液が全血、血漿、及び血清からなる群より選択される少なくとも1種である、請求項１～４のいずれかに記載の検査方法。 The test method according to any one of claims 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
  6. 前記タンパク質（A）群が（A1）14-3-3 protein theta、Thrombospondin-1、Platelet glycoprotein 4、Pyridoxal phosphate phosphatase PHOSPHO2、Integrin beta-2、Collagen alpha-2(VI) chain、Proteoglycan 4、Glycophorin-C、Versican core protein、Amyloid beta A4 protein、Ras-related protein Rap-2c、Decorin、Serum amyloid A-1 protein、及びSerum amyloid A-2 proteinからなる群であり、且つ前記タンパク質（B）群が（B1）Alpha-2-antiplasmin、Complement C1s-A subcomponent、Complement C1r-A subcomponent、Biglycan、Complement C1q subcomponent subunit B、Laminin subunit alpha-5、Interferon-induced transmembrane protein 2、Laminin subunit gamma-1、Serglycin、Thrombospondin-1、Alpha-2-macroglobulin、Collagen alpha-1(I) chain、Platelet factor 4、Vesicle-associated membrane protein 3、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Collagen alpha-2(I) chain、Multimerin-1、Ras-related protein Rab-7a、Serine incorporator 3、Basigin、及びC4b-binding proteinからなる群である、請求項１～５のいずれかに記載の検査方法。 The protein (A) group is (A1) 14-3-3 protein theta, Thrombospondin-1, Platelet glycoprotein 4, Pyridoxal phosphate phosphatase PHOSPHO2, Integrin beta-2, Collagen alpha-2 (VI) chain, Proteoglycan 4, Glycophorin- C, Versican core protein, Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B) group is ( B1) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, Complement C1q subcomponent subunit B, Laminin subunit alpha-5, Interferon-induced transmembrane protein 2, Laminin subunit gamma-1, Serglycin, Thrombospond -1, Alpha-2-macroglobulin, Collagen alpha-1 (I) chain, Platelet factor 4, Vesicle-associated membrane protein 3, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Collagen al The test method according to any one of claims 1 to 5, which is a group consisting of pha-2 (I) chain, Multimerin-1, Ras-related protein Rab-7a, Serine incorporator 3, Basigin, and C4b-binding protein. .
  7. 前記タンパク質（A1）群が（A2）Amyloid beta A4 protein、Ras-related protein Rap-2c、Decorin、Serum amyloid A-1 protein、及びSerum amyloid A-2 proteinからなる群であり、且つ
    前記タンパク質（B1）群が（B2）Alpha-2-antiplasmin、Complement C1s-A subcomponent、Complement C1r-A subcomponent、Biglycan、及びComplement C1q subcomponent subunit Bからなる群である、
    請求項６に記載の検査方法。     The protein (A1) group is a group consisting of (A2) Amyloid beta A4 protein, Ras-related protein Rap-2c, Decorin, Serum amyloid A-1 protein, and Serum amyloid A-2 protein, and the protein (B1 ) Group is a group consisting of (B2) Alpha-2-antiplasmin, Complement C1s-A subcomponent, Complement C1r-A subcomponent, Biglycan, and Complement C1q subcomponent subunit B.
    The inspection method according to claim 6.
  8. 前記被検体がマウスである、請求項１～７のいずれかに記載の検査方法。 The examination method according to any one of claims 1 to 7, wherein the subject is a mouse.
  9. タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
    （A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
    （B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
    （C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の検出剤を含む、閉塞性肺疾患の検査薬。     Protein group (A), protein group (B), and protein group (C):
    (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
    (B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A test agent for obstructive pulmonary disease, comprising a detection agent for at least one protein selected from the group consisting of protein group consisting of, Fibulin-2, and Adenylyl cyclase-associated protein 1.
  10. 閉塞性肺疾患誘発処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
    （A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
    （B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
    （C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患モデル動物のスクリーニング方法。     Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluids collected from animals treated with obstructive pulmonary disease induction:
    (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
    (B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A method for screening an animal model of obstructive pulmonary disease, using as an index the amount or concentration of at least one protein selected from the group consisting of proteins consisting of, Fibulin-2, and Adenylyl cyclase-associated protein 1.
  11. 前記指標の値がカットオフ値以上である場合に、前記動物を閉塞性肺疾患モデル動物として選択する工程を含む、請求項１０に記載のスクリーニング方法。 The screening method according to claim 10, comprising a step of selecting the animal as an obstructive pulmonary disease model animal when the index value is equal to or greater than a cut-off value.
  12. タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
    （A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
    （B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
    （C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の抑制剤を含有する、閉塞性肺疾患の予防又は治療剤。     Protein group (A), protein group (B), and protein group (C):
    (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
    (B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 A prophylactic or therapeutic agent for obstructive pulmonary disease, comprising an inhibitor of at least one protein selected from the group consisting of, Fibulin-2, and Adenylyl cyclase-associated protein 1.
  13. 被検物質で処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
    （A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
    （B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
    （C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患の予防又は治療剤の有効成分のスクリーニング方法。     Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from animals treated with the test substance:
    (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
    (B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 , Fibulin-2, and an active ingredient of a prophylactic or therapeutic agent for obstructive pulmonary disease, using as an index the amount or concentration of at least one protein selected from the group consisting of proteins consisting of Adenylyl cyclase-associated protein 1 Screening method.
  14. 前記指標の値が、被検物質で処理されていない動物から採取された体液の細胞外小胞における対応タンパク質の量又は濃度よりも低い場合に、前記被検物質を閉塞性肺疾患の予防又は治療剤の有効成分として選択する工程を含む、請求項１３に記載のスクリーニング方法。 When the value of the indicator is lower than the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance, the test substance is used to prevent obstructive pulmonary disease or The screening method according to claim 13, comprising a step of selecting as an active ingredient of a therapeutic agent.
  15. 被検物質で処理された動物から採取された体液の細胞外小胞における、タンパク質群（A）、タンパク質群（B）、及びタンパク質群（C）：
    （A）Ig lambda-1 chain C region、Alpha-actinin-1、14-3-3 protein theta、H-2 class I histocompatibility antigen, K-B alpha chain、Vesicle-associated membrane protein 8、Alpha-2-macroglobulin、Thrombospondin-1、Zinc finger protein 536、Complement C1q subcomponent subunit B、Histone H3.3C、Clusterin、Condensin-2 complex subunit G2、Platelet glycoprotein 4、Serglycin、Pyridoxal phosphate phosphatase PHOSPHO2、Hyaluronan-binding protein 2、Integrin beta-2、Complement C1q subcomponent subunit A、Histone H1t、Myc target protein 1、Platelet-derived growth factor subunit B、Alpha-1-antitrypsin 1-5、Ig kappa chain C region、Laminin subunit gamma-1、Hippocalcin-like protein 1、Src kinase-associated phosphoprotein 2、Collagen alpha-2(VI) chain、Proteoglycan 4、4F2 cell-surface antigen heavy chain、Immunoglobulin J chain、CD5 antigen-like、Histone H4、RNA-binding protein MEX3B、Histone H2B type 3-A、Plasma protease C1 inhibitor、Ig kappa chain V-II region 7S34.1、Serine protease inhibitor A3N、Ig kappa chain V-III region PC 2880/PC 1229、Platelet factor 4、Bridging integrator 2、Oncoprotein-induced transcript 3 protein、Glycophorin-C、Alpha-2-antiplasmin、Versican core protein、Protein unc-13 homolog B、Calnexin、Ig kappa chain V-III region PC 7183、Ig kappa chain V-V region MOPC 41、Ig kappa chain V-III region PC 4050、Glucose-6-phosphate isomerase、Galectin-related protein、Lymphocyte antigen 6E、Amyloid beta A4 protein、Biglycan、Ig kappa chain V-III region PC 2154、Ig kappa chain V-V region HP 91A3、Complement C1r-A subcomponent、Inter-alpha-trypsin inhibitor heavy chain H1、Complement C1s-A subcomponent、Gamma-1-syntrophin、Hemopexin、Cytochrome c oxidase subunit 6B1、Ras-related protein Rap-2c、Ig kappa chain V-V region L6、Decorin、Serum amyloid A-1 protein、Serum amyloid A-2 protein、Growth factor receptor-bound protein 2、Ig heavy chain V region 6.96、Keratin, type II cuticular Hb1、Early endosome antigen 1、TBC domain-containing protein kinase-like protein、Platelet glycoprotein VI、Catenin delta-1、60S acidic ribosomal protein P2、Glycophorin-A、BRCA1-A complex subunit RAP80、Laminin subunit alpha-2、Actin-related protein 2/3 complex subunit 5、Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1、RAC-gamma serine/threonine-protein kinase、Ig kappa chain V-II region MOPC 167、Programmed cell death protein 6、Cysteine-rich secretory protein 3、及びProtein FAM177A1からなるタンパク質群、
    （B）Cytohesin-2、Erythrocyte band 7 integral membrane protein、Alpha-2-antiplasmin、Complement C1s-A subcomponent、Amyloid beta A4 protein、Gamma-1-syntrophin、Cytochrome c oxidase subunit 6B1、Fermitin family homolog 3、Complement C1r-A subcomponent、Ig kappa chain V-V region MOPC 41、Biglycan、Collagen alpha-2(IV) chain、Complement C1q subcomponent subunit B、Complement C1q subcomponent subunit A、Hippocalcin-like protein 1、Laminin subunit alpha-5、Lymphocyte antigen 6E、Myc target protein 1、Interferon-induced transmembrane protein 2、Trem-like transcript 1 protein、Laminin subunit gamma-1、Bridging integrator 2、Serglycin、Plasma protease C1 inhibitor、Collagen alpha-2(VI) chain、Integrin beta-2、F-box only protein 40、Thrombospondin-1、Alpha-2-macroglobulin、Disintegrin and metalloproteinase domain-containing protein 10、Solute carrier family 2, facilitated glucose transporter member 3、Collagen alpha-1(I) chain、Vesicle-associated membrane protein 8、CD81 antigen、Alpha-1-antitrypsin 1-5、Actin-related protein 2/3 complex subunit 3、Platelet glycoprotein IX、Choline transporter-like protein 2、Syntaxin-4、Tetraspanin-9、Platelet factor 4、Integrin alpha-6、Beta-2-microglobulin、Transthyretin、Vesicle-associated membrane protein 3、Platelet glycoprotein Ib beta chain、4F2 cell-surface antigen heavy chain、EGF-containing fibulin-like extracellular matrix protein 1、Integrin alpha-IIb、Collagen alpha-2(I) chain、Synaptosomal-associated protein 23、Alpha-1-antitrypsin 1-3、Mucin-13、Sodium-dependent serotonin transporter、Multimerin-1、Alpha-1-antitrypsin 1-4、Ras-related protein Rab-7a、Ras-related protein Rap-2b、Ubiquitin-60S ribosomal protein L40、Ig kappa chain V-V region L6、Receptor-type tyrosine-protein phosphatase eta、Serine incorporator 3、H-2 class I histocompatibility antigen, Q10 alpha chain、Basigin、C4b-binding protein、Signaling lymphocytic activation molecule、Tropomyosin alpha-4 chain、Alpha-synuclein、及びCalcium-transporting ATPase type 2C member 1からなるタンパク質群、並びに
    （C）Collagen alpha-1(XVIII) chain、Carboxypeptidase N subunit 2、Complement C1q subcomponent subunit C、Interferon-induced transmembrane protein 3、Fibulin-2、及びAdenylyl cyclase-associated protein 1からなるタンパク質群からなる群より選択される少なくとも1種のタンパク質の量又は濃度を指標とする、閉塞性肺疾患の誘発性又は増悪性の評価方法。      Protein group (A), protein group (B), and protein group (C) in extracellular vesicles of body fluid collected from animals treated with the test substance:
    (A) Ig lambda-1 chain C region, Alpha-actinin-1, 14-3-3 protein theta, H-2 class I histocompatibility antigen, KB alpha chain, Vesicle-associated membrane protein 8, Alpha-2-macroglobulin, Thrombospondin-1, Zinc finger protein 536, Complement C1q subcomponent subunit B, Histone H3.3C, Clusterin, Condensin-2 complex subunit G2, Platelet glycoprotein 4, Serglycin, Pyridoxal phosphate phosphatase PHOSPHO2, Hyaluronan-binding protein 2, Integrin beta-2 , Complement C1q subcomponent subunit A, Histone H1t, Myc target protein 1, Platelet-derived growth factor subunit B, Alpha-1-antitrypsin 1-5, Ig kappa chain C region, Laminin subunit gamma-1, Hippocalcin-like protein 1, Src kinase-associated phosphoprotein 2, Collagen alpha-2 (VI) chain, Proteoglycan 4, 4F2 cell-surface antigen heavy chain, Immunoglobulin J chain, CD5 antigen-like, Histone H4, RNA-binding protein MEX3B, Histone H2B type 3- A, Plasma protease C1 inhibitor, Ig kappa chain V-II region 7S34.1 Serine protease inhibitor A3N, Ig kappa chain V-III region PC 2880 / PC 1229, Platelet factor 4, Bridging integrator 2, Oncoprotein-induced transcript 3 protein, Glycophorin-C, Alpha-2-antiplasmin, Versican core protein, Protein unc- 13 homolog B, Calnexin, Ig kappa chain V-III region PC 7183, Ig kappa chain VV region MOPC 41, Ig kappa chain V-III region PC 4050, Glucose-6-phosphate isomerase, Galectin-related protein, Lymphocyte antigen 6E, Amyloid beta A4 protein, Biglycan, Ig kappa chain V-III region PC 2154, Ig kappa chain VV region HP 91A3, Complement C1r-A subcomponent, Inter-alpha-trypsin inhibitor heavy chain H1, Complement C1s-A subcomponent, Gamma-1 -syntrophin, Hemopexin, Cytochrome c oxidase subunit 6B1, Ras-related protein Rap-2c, Ig kappa chain VV region L6, Decorin, Serum amyloid A-1 protein, Serum amyloid A-2 protein, Growth factor receptor-bound protein 2, Ig heavy chain V region 6.96, Keratin, type II cuticular Hb1, Early endosome antig en 1, TBC domain-containing protein kinase-like protein, Platelet glycoprotein VI, Catenin delta-1, 60S acidic ribosomal protein P2, Glycophorin-A, BRCA1-A complex subunit RAP80, Laminin subunit alpha-2, Actin-related protein 2 / 3 complex subunit 5, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, RAC-gamma serine / threonine-protein kinase, Ig kappa chain V-II region MOPC 167, Programmed cell death protein 6, Cysteine-rich secretory protein 3, and a protein group consisting of Protein FAM177A1,
    (B) Cytohesin-2, Erythrocyte band 7 integral membrane protein, Alpha-2-antiplasmin, Complement C1s-A subcomponent, Amyloid beta A4 protein, Gamma-1-syntrophin, Cytochrome c oxidase subunit 6B1, Fermitin family homolog 3, Complement C1r -A subcomponent, Ig kappa chain VV region MOPC 41, Biglycan, Collagen alpha-2 (IV) chain, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Hippocalcin-like protein 1, Laminin subunit alpha-5, Lymphocyte antigen 6E , Myc target protein 1, Interferon-induced transmembrane protein 2, Trem-like transcript 1 protein, Laminin subunit gamma-1, Bridging integrator 2, Serglycin, Plasma protease C1 inhibitor, Collagen alpha-2 (VI) chain, Integrin beta-2 , F-box only protein 40, Thrombospondin-1, Alpha-2-macroglobulin, Disintegrin and metalloproteinase domain-containing protein 10, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (I) chain, Vesicle-associated membrane prote in 8, CD81 antigen, Alpha-1-antitrypsin 1-5, Actin-related protein 2/3 complex subunit 3, Platelet glycoprotein IX, Choline transporter-like protein 2, Syntaxin-4, Tetraspanin-9, Platelet factor 4, Integrin alpha-6, Beta-2-microglobulin, Transthyretin, Vesicle-associated membrane protein 3, Platelet glycoprotein Ib beta chain, 4F2 cell-surface antigen heavy chain, EGF-containing fibulin-like extracellular matrix protein 1, Integrin alpha-IIb, Collagen alpha-2 (I) chain, Synaptosomal-associated protein 23, Alpha-1-antitrypsin 1-3, Mucin-13, Sodium-dependent serotonin transporter, Multimerin-1, Alpha-1-antitrypsin 1-4, Ras-related protein Rab-7a, Ras-related protein Rap-2b, Ubiquitin-60S ribosomal protein L40, Ig kappa chain VV region L6, Receptor-type tyrosine-protein phosphatase eta, Serine incorporator 3, H-2 class I histocompatibility antigen, Q10 alpha chain , Basigin, C4b-binding protein, Signaling lymphocytic activation molecule, Tropomyosin alpha-4 Protein group consisting of chain, Alpha-synuclein, and Calcium-transporting ATPase type 2C member 1, and (C) Collagen alpha-1 (XVIII) chain, Carboxypeptidase N subunit 2, Complement C1q subcomponent subunit C, Interferon-induced transmembrane protein 3 , Fibulin-2, and Adenylyl cyclase-associated protein 1 A method for evaluating inducibility or malignancy of obstructive pulmonary disease, using as an index the amount or concentration of at least one protein selected from the group consisting of the protein group consisting of 1 .
PCT/JP2018/009502 2017-03-14 2018-03-12 Biomarker for obstructive pulmonary disease WO2018168768A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017048164A JP2020079707A (en) 2017-03-14 2017-03-14 Obstructive pulmonary disease biomarker
JP2017-048164 2017-03-14

Publications (1)

Publication Number Publication Date
WO2018168768A1 true WO2018168768A1 (en) 2018-09-20

Family

ID=63522084

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/009502 WO2018168768A1 (en) 2017-03-14 2018-03-12 Biomarker for obstructive pulmonary disease

Country Status (2)

Country Link
JP (1) JP2020079707A (en)
WO (1) WO2018168768A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100184046A1 (en) * 2008-11-12 2010-07-22 Caris Mpi, Inc. Methods and systems of using exosomes for determining phenotypes
WO2012048372A1 (en) * 2010-10-11 2012-04-19 Medsaic Pty Ltd Assay for disease detection
CN103065065A (en) * 2012-11-18 2013-04-24 浙江大学 Active tuberculosis differential expression protein profile model and building method thereof
US20130288233A1 (en) * 2010-12-24 2013-10-31 Map Diagnostics Pty Ltd. Selective Reaction Monitoring (SRM) Derived Protein Profiles for Cancer and other Pathologic Entities

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100184046A1 (en) * 2008-11-12 2010-07-22 Caris Mpi, Inc. Methods and systems of using exosomes for determining phenotypes
WO2012048372A1 (en) * 2010-10-11 2012-04-19 Medsaic Pty Ltd Assay for disease detection
US20130288233A1 (en) * 2010-12-24 2013-10-31 Map Diagnostics Pty Ltd. Selective Reaction Monitoring (SRM) Derived Protein Profiles for Cancer and other Pathologic Entities
CN103065065A (en) * 2012-11-18 2013-04-24 浙江大学 Active tuberculosis differential expression protein profile model and building method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KADOTA, T ET AL.: "EXTRACELLULAR VESICLES IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 17, no. 11, 27 October 2016 (2016-10-27), pages E1801, XP055556096, Retrieved from the Internet <URL:doi:10.3390/ijms17111801> *
SUN Y ET AL.: "Systematic comparison of exosomal proteomes from human saliva and serum for the detection of lung cancer", ANALYTICA CHIMICA ACTA, vol. 982, 17 June 2017 (2017-06-17), pages 84 - 95, XP055556161, Retrieved from the Internet <URL:https://doi.org/10.1016/j.aca.2017.06.005> *
vol. 16, no. 2, June 2015 (2015-06-01), pages 61 - 68 *

Also Published As

Publication number Publication date
JP2020079707A (en) 2020-05-28

Similar Documents

Publication Publication Date Title
Barman et al. VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites
Melo et al. Glypican1 identifies cancer exosomes and facilitates early detection of cancer
Xu et al. Surfaceome of exosomes secreted from the colorectal cancer cell line SW480: Peripheral and integral membrane proteins analyzed by proteolysis and TX114
WO2007061922A2 (en) Methods to predict and prevent resistance to taxoid compounds
WO2019173799A1 (en) Oligonucleotide probes and uses thereof
KR20190122105A (en) Biomarker for diagnosing overactive bladder disease and screening method of therapeutic agents using the same
Andersen et al. Plasma and urine proteomic profiles in childhood idiopathic nephrotic syndrome
JP6588893B2 (en) Autoantibody biomarkers for ovarian cancer
JP2009538415A (en) Detection and treatment of steroid receptors in circulating cancer cells
EP2605017A1 (en) Marker sequences for gynaecological malignoma and use of same
TW202223104A (en) Method of predicting progressive covid-19 severity by using protein markers in blood exosomes
WO2018168769A1 (en) Tissue fibrosis biomarker
JP2024010173A (en) Method of testing for urothelial cancer
JP7175526B2 (en) Preventive/therapeutic agents for diseases related to cell migration regulation and disease activity assessment/prognostic evaluation for pulmonary interstitial diseases
JP2018151276A (en) Aging biomarker
WO2018168779A1 (en) Biomarker for chronic obstructive pulmonary disease
WO2018168768A1 (en) Biomarker for obstructive pulmonary disease
WO2020004557A1 (en) Obstructive pulmonary disease biomarkers
JP2010014689A (en) Identification of melanoma marker using proteomics analysis
KR20150020392A (en) Method for screening cancer marker based on de-glycosylation of glycoproteins and marker for HCC
JP2019158753A (en) Biomarkers for lymphangioleiomyomatosis
WO2020162441A1 (en) Granulomatous disease biomarker
WO2023027186A1 (en) Pulmonary fibrosis biomarker
Wang et al. Proteomic profiling of heat acclimation in cerebrospinal fluid of rabbit
JP5688829B2 (en) Differential Diabetes Prediction / Diagnosis Method and Diabetes Prediction / Diagnosis Kit

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18768637

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18768637

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP