WO2018168769A1 - Biomarqueur de fibrose tissulaire - Google Patents

Biomarqueur de fibrose tissulaire Download PDF

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WO2018168769A1
WO2018168769A1 PCT/JP2018/009503 JP2018009503W WO2018168769A1 WO 2018168769 A1 WO2018168769 A1 WO 2018168769A1 JP 2018009503 W JP2018009503 W JP 2018009503W WO 2018168769 A1 WO2018168769 A1 WO 2018168769A1
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protein
chain
alpha
subunit
complement
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PCT/JP2018/009503
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Japanese (ja)
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吉人 武田
淳 熊ノ郷
隆典 松木
幸嗣 植田
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国立大学法人大阪大学
公益財団法人がん研究会
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • 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 tissue fibrosis biomarker, use thereof, and the like.
  • Idiopathic pulmonary fibrosis is an intractable disease with a poor prognosis that begins as alveolitis and progresses to interstitial fibrosis with destruction of the alveolar region due to chronic inflammation.
  • Tissue damage in pulmonary fibrosis involves inflammatory cells such as macrophages, neutrophils, and lymphocytes, and various cells such as alveolar epithelium, vascular endothelium, and fibroblasts, but the mechanism is insufficiently analyzed.
  • many of the patients with IPF are not only complicated with various comorbidities because they are older, but they are also attracted to COPD (pulmonary fibrosis with emphysema: CPFE).
  • Non-Patent Document 1 a recent paper on biomarkers that can distinguish COPD and asthma (Non-Patent Document 1) tries to analyze dozens of patients, but the analysis is based on the old mass spectrometry (MS) technique based on two-dimensional electrophoresis.
  • MS mass spectrometry
  • Extracellular vesicles are membrane vesicles that are secreted from cells, and 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. ing. In recent years, extracellular vesicles have attracted attention as cancer test samples, but their usefulness as test samples for respiratory diseases and tissue fibrosis diseases is not known.
  • An object of the present invention is to provide a biomarker for tissue fibrosis and a method for using the same.
  • an object of the present invention is to provide a biomarker capable of differentiating chronic obstructive pulmonary disease (COPD) in tissue fibrosis and a method for using the same.
  • COPD chronic obstructive pulmonary disease
  • the present inventor has found that a specific protein group in extracellular vesicles of a body fluid collected from a subject is useful as a biomarker for tissue fibrosis disease. As a result of further research, it was found that these biomarkers can also be used to differentiate chronic obstructive pulmonary disease (COPD) in tissue fibrosis. We have also found that some biomarkers reflect the pathology of tissue fibrosis. As a result of further research based on these findings, the present invention was completed. That is, the present invention includes the following aspects.
  • Item 1 A method for examining tissue fibrosis, comprising: (1) Protein group (A) in extracellular vesicles of body fluid collected from the subject: (A) Ig heavy chain V region 6.96, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Decorin, Collagen alpha-1 (XVIII) chain, Galectin-related protein, Cytochrome c oxidase subunit 6B1, Carboxypeptidase N subunit 2, Amyloid beta A4 protein, Complement C1q subcomponent subunit C, Gamma-1-syntrophin, Biglycan, Collagen alpha-2 (IV) chain, Ig kappa chain VV region MOPC 41, Platelet-derived growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1, Myc target protein 1, Alpha-2-antiplasmin, Interferon-induced transmembrane protein 2,
  • Item 2 a step of determining that the subject is suffering from a tissue fibrosis disease when the amount or concentration of the protein detected in the step (1) is not less than a cutoff value.
  • the step (2) (2a) Protein to be detected is (Aa) Ig heavy chain V region 6.96, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Decorin, Galectin-related protein, Platelet-derived growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1, Oncoprotein-induced transcript 3 protein, Probable carboxypeptidase PM20D1, Properdin, Ig kappa chain V-IV region S107B, Src kinase-associated phosphoprotein 2, Pyridoxal phosphate phosphatase PHOSPHO2, [3-methyl-2-oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrial, Syntenin-1, Low-density lipoprotein receptor, Pituitary tumor-transforming gene 1 protein-interacting protein, Transferrin receptor protein 1, Platelet glycoprotein
  • the cut-off value is 2.5 to 3.5 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 a tissue fibrosis disease, or 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) Platelet factor 4, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, Complement C1r-A subcomponent, Vesicle-associated membrane protein 8, Kininogen-1, Protein unc-13 homolog B, Ig heavy chain V region 6.96, Complement C1s-A subcomponent, Complement C1q subcomponent subunit C, Solute carrier family 2, facilitated glucose transporter member 3, Collagen alpha-1 (XVIII) chain, Transferrin receptor protein 1, Collagen alpha-2 (I ) Chain, Collagen alpha-2 (VI) chain, Basigin, Interferon-induced transmembrane protein 3, Hippocalcin-like protein 1, Mucin-13, Platelet-derived growth factor subunit B, and Plasma protease C1 inhibitor.
  • Item 6. The inspection method according to any one of Items 1 to 5.
  • the protein (A1) group is (A2) Complement C1q subcomponent subunit A, Complement C1r-A subcomponent, Protein unc-13 homolog B, Complement C1s-A subcomponent, Complement C1q subcomponent subunit C, Collagen alpha-1 (XVIII) chain, Collagen alpha-2 (VI) chain, Interferon -induced transmembrane protein 3, Hippocalcin-like protein 1, and Platelet-derived growth factor subunit B, Item 7.
  • 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 a tissue fibrosis 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 tissue fibrosis or Item 14.
  • a biomarker for tissue fibrosis can be provided.
  • examination of tissue fibrosis disease, determination of the degree of fibrosis, screening of tissue fibrosis disease model animals, etc. can be performed more simply, more efficiently and at a lower cost.
  • COPD chronic obstructive pulmonary disease
  • the number of particles and the particle size of the extracellular vesicle fraction are shown (Test Example 3).
  • PBS group shows the result of extracellular vesicle fraction obtained from normal mice
  • BLM group shows the result of extracellular vesicle fraction obtained from tissue fibrosis disease model mice. In the BLM group, no significant difference was observed in the number of particles and the particle size compared to the control (PBS group).
  • 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.
  • PBS group shows the result of extracellular vesicle fraction obtained from normal mice
  • BLM group shows the result of extracellular vesicle fraction obtained from tissue fibrosis disease model mice.
  • the present invention is a method for examining a tissue fibrosis disease, comprising: (1) A test method (in this specification, including a step (step 1) of detecting at least one protein selected from the protein group (A) in an extracellular vesicle of a body fluid collected from a subject. This may also be referred to as “tissue fibrosis disease testing method of the present invention”. This will be described below.
  • tissue fibrosis disease to be examined is not particularly limited as long as it is a disease involving tissue fibrosis or tissue fibrosis.
  • tissue fibrosis examples include lung, skin, kidney, heart, liver, bladder, gastrointestinal tract, blood vessel and the like, and preferably lung.
  • pulmonary fibrosis include, for example, interstitial pneumonia, more specifically idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, idiopathic organized pneumonia, acute interstitial pneumonia, exfoliation Idiopathic interstitial pneumonia such as interstitial pneumonia, lymphocytic interstitial pneumonia, respiratory bronchiolitis-related interstitial pneumonia; other interstitial pneumonia of unknown origin; interstitial pneumonia due to collagen disease, Interstitial pneumonia due to occupation or environment, interstitial pneumonia due to drugs, interstitial pneumonia due to radiation, interstitial pneumonia due to infection, interstitial pneumonia due to other diseases, etc. have a clear cause; Etc.
  • interstitial pneumonia more specifically idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, idiopathic organized pneumonia, acute interstitial pneumonia, exfoliation Idiopathic interstitial pneumonia such as interstitial pneumonia, lymphocytic interstitial pneumonia, respiratory bronchiolitis-related interstitial pneumonia; other inter
  • 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.
  • the state of the subject is not particularly limited. For example, it is unknown whether or not it is suffering from tissue fibrosis, it is already determined by another method that it is suffering from tissue fibrosis, but it is unknown whether or not it is suffering from COPD. Specimen, already determined to be affected by COPD by another method, but unknown if it is afflicted by tissue fibrosis, tissue fibrosis or COPD Samples that have been determined by the above, samples that have already been determined by other methods as not having a tissue fibrosis, samples that are being treated for tissue fibrosis, 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 in step (1) is at least one protein selected from the protein group (A) (in the present specification, these may be collectively referred to as “target protein”).
  • Protein group (A) consists of (A) Ig heavy chain V region 6.96, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Decorin, Collagen alpha-1 (XVIII) chain, Galectin-related protein, Cytochrome c oxidase subunit 6B1, Carboxypeptidase N subunit 2, Amyloid beta A4 protein, Complement C1q subcomponent subunit C, Gamma-1-syntrophin, Biglycan, Collagen alpha-2 (IV) chain, Ig kappa chain VV regionPlate-41 growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1, Myc target protein 1, Alpha-2-antiplasmin, Interferon-induced transmembrane protein 2 , Bridging integrator 2, Complement C1s-A subcomponent, Thrombospondin-1, Collagen alpha-2 (VI)
  • Protein group (A) is a tissue fibrosis disease-specific biomarker, and tissue fibrosis disease can be identified using this as an index.
  • Ig heavy ⁇ chain V region 6.96, Laminin subunit alpha-2, Actin-related protein are preferable from the viewpoint that differentiation of tissue fibrosis disease without chronic obstructive pulmonary disease is possible.
  • 2/3 complex subunit 5 Decorin, Galectin-related protein, Platelet-derived growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1
  • Oncoprotein-induced transcript 3 protein Probable carboxypeptidase PM20D1, Properdin, Ig kappa chain V-IV region S107B, Src kinase-associated phosphoprotein 2, Pyridoxal phosphate phosphatase PHOSPHO2, [3-methyl-2-oxobutanoateodeoenaseolipase , Syntenin-1, Low-density lipoprotein receptor, Pituitary tumor-transforming gene 1 protein-interacting protein
  • 4F2 cell-surface antigen heavy chain Alpha-1-antitrypsin 1-5, Alpha-2-antiplasmin, Alpha- 2-macroglobulin, Amyloid beta A4 protein, Disintegrin and metalloproteinase domain-containing protein 10, Beta-2-microglobulin, Basigin, BridgingBintegrator 2, Complement C1q subcomponent subunit A, Complement C1q subcomponent sub, Complement subunit C1q ⁇ subcomponent -A subcomponent, C4b-binding protein, Collagen alpha-1 (I) chain, Collagen alpha-2 (I) chain, Collagen alpha-2 (IV) chain, Collagen alpha-2 (VI) chain, Collagen alpha-1 ( XVIII) chain, Carboxypeptidase N subunit 2, Complement C1s-A subcomponent, Cytochrome c oxidase subunit 6B1, EGF-containing fibulin
  • Platelet factor 4 Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A , Complement C1r-A subcomponent, Vesicle-associated membrane protein 8, Kininogen-1, Protein unc-13 homolog B, Ig heavy chain V region 6.96, Complement C1s-A subcomponent, Complement C1q subcomponent subunit C, Solute carrier family 2, glucose transporter member 3, Collagen alpha-1 (XVIII) chain, Transferrin receptor protein 1, Collagen alpha-2 (I) chain, Collagen alpha-2 (VI) chain, Basigin, Interferon-induced transmembrane protein 3, Hippocalcin-like protein 1, Mucin-13, Platelet-derived growth factor subunit B, Plasma protease C1 inhibitor, etc., more preferably Platelet factor 4, Complement C1q subcomponent subunit B, Complement C1q subcomponent subunit A, etc.
  • Ig heavy chain V region 6.96 Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Decorin, Collagen alpha-1 (XVIII) chain, Galectin-related protein, Cytochrome c oxidase subunit 6B1, Carboxypeptidase N subunit 2, Amyloid beta A4 protein, Complement C1q subcomponent subunit C, Gamma-1-syntrophin, Biglycan, Collagen alpha-2 (IV) chain, Ig kappa chain VV region MOPC 41, Platelet-derived growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1, Myc target protein 1, Alpha-2-antiplasmin, Interferon-induced transmembrane protein 2, Bridging integrat
  • the ratio of the amount to the normal sample is higher among the protein group (A) (not included when the expression level of the control cannot be confirmed).
  • Decorin Collagen alpha-1 (XVIII) chain, Galectin-related protein, Cytochrome c oxidase subunit 6B1, Carboxypeptidase N subunit 2, Amyloid beta A4 protein, Complement C1q subcomponent subunit C, Gamma-1-syntrophin, Biglycan, Collagen alpha -2 (IV) chain, Ig kappa chain VV region MOPC 41, Platelet-derived growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1 , Myc target protein 1, Alpha-2-antiplasmin, Interferon-induced transmembrane protein 2, Bridging integrator 2, Complement C1s-A subcomponent, Thrombospondin-1, Collagen alpha-2
  • the protein of the protein group (A) is a protein specified by UniProtKB accession numbers shown in Tables 1 and 2 in Examples described later. In the case of other species, it is an ortholog of the protein specified by the UniProtKB accession numbers shown in Tables 1-2.
  • the number of target proteins may be only one type or a combination of two or more types.
  • more detection targets for example, 2, 5, 10, 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 As the immunological measurement method, an immunohistochemical staining method, ELISA method, sandwich ELISA method, EIA method, RIA method, 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 that is a detection index of tissue fibrosis can be provided, thereby assisting in the detection of tissue fibrosis. can do.
  • the amount and / or concentration of the target protein that is a detection index can be provided, thereby assisting the evaluation of the degree of fibrosis. .
  • Evaluation of fibrosis can be used for acute exacerbation, discrimination of poor prognosis groups, and the like.
  • the test results of the test method of the present invention including the step (1) include the elucidation of the pathological condition of the tissue fibrosis disease, the prognosis prediction of the tissue fibrosis disease (reduced respiratory function group, acute exacerbation), stratification of patients, It can be used for selection (individualized medicine, treatment responsiveness), evaluation of tissue remodeling, differentiation of tissue type and phenotype of tissue fibrosis, and the like.
  • the inspection method of the present invention further includes, as one aspect, (2) including a step of determining that the subject suffers from a tissue fibrosis disease (step 2) when the amount or concentration of the protein detected in the step (1) is equal to or higher than a cutoff value. It is preferable. According to the test method of the present invention including the step 2, it becomes possible to determine a tissue fibrosis disease.
  • step 2 is divided into the following (2a) to (2e), for example.
  • (2a) Protein to be detected is (Aa) Ig heavy chain V region 6.96, Laminin subunit alpha-2, Actin-related protein 2/3 complex subunit 5, Decorin, Galectin-related protein, Platelet-derived growth factor subunit B, Ig kappa chain V-III region PC 7183, Calnexin, Protein unc-13 homolog B, Calcium-transporting ATPase type 2C member 1, Oncoprotein-induced transcript 3 protein, Probable carboxypeptidase PM20D1, Properdin, Ig kappa chain V-IV region S107B, Src kinase-associated phosphoprotein 2, Pyridoxal phosphate phosphatase PHOSPHO2, [3-methyl-2-oxobutanoate dehydrogenase [lipoamide]] kinase, mitochondrial, Syntenin-1, Low-density lipoprotein receptor, Pituitary tumor-transforming gene 1 protein
  • Step (2) is more preferably at least one step selected from the group consisting of the above steps 2a to 2b.
  • 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, and the like, for example, collected from a subject not suffering from tissue fibrosis 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 cutoff 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 a tissue fibrosis disease (in the case of multiple subjects, the average value, the center Value), for example, 1.5 to 8 times, preferably 2 to 5 times, more preferably 2.5 to 3.5 times.
  • the amount or concentration of the protein detected in step (1) is greater than or equal to the cut-off value by using a biomarker reflecting the pathology of tissue fibrosis disease.
  • the cut-off value is a certain value or more, for example, by setting the cut-off value to a value based on the amount and / or concentration of the target protein in the extracellular vesicle of body fluid collected from a subject suffering from a tissue fibrosis disease. It can be evaluated whether or not the disease is.
  • treatment is performed by setting the cutoff value 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, and fibrosis is detected.
  • the degree can be determined. More specifically, for example, a calibration curve based on the correlation between the fibrosis score and the amount or concentration of the biomarker is prepared in advance, and the fibrosis degree (from the measured amount or concentration of the biomarker and the calibration curve) ( For example, a fibrosis score or the like) can be determined or predicted.
  • the test method of the present invention including the diagnostic step (2) with higher accuracy of tissue fibrosis disease is determined that the subject suffers from the tissue fibrosis disease
  • the test method of the present invention further includes By combining the steps of applying a diagnosis by a doctor of tissue fibrosis, the tissue fibrosis can be diagnosed with higher accuracy.
  • the examination method of the present invention can detect a tissue fibrosis more accurately, the above-described steps are combined with the examination method of the present invention more efficiently and more accurately. Can be diagnosed.
  • test method of the present invention can detect a tissue fibrosis 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 diagnosis by a doctor By combining the three, a subject suffering from a tissue fibrosis can be treated more efficiently and more reliably.
  • the treatment method for the tissue fibrosis disease is not particularly limited, but a typical example is medication.
  • the drug used for the medication treatment is not particularly limited, and examples thereof include an antifibrotic agent such as pyrespa; a steroid; and an immunosuppressive agent.
  • antifibrotic agent such as pyrespa
  • a steroid such as a steroid
  • immunosuppressive agent such as a steroid
  • anticholinergic drugs such as Spiriva, Seeburi, Enclasse, Ecrila, Atrovent, Telcigan, etc .
  • ⁇ 2 such as Serebent, Ombres, Auxis, Sultanol, Meptin, etc.
  • Stimulant can also be used together.
  • Anticholinergic and ⁇ 2 stimulant combination such as Ultivolo, Anolo and Spiruto
  • Steroidal drug such as Cuvaru, Flutide, Palmicoat, Olvesco and Azumanex
  • Steroid and ⁇ 2 stimulant combination such as Adair, Simbicort and Lervea Etc.
  • a pharmaceutical can be used 1 type, 2 types, or 3 or more types in combination.
  • the present invention relates to a test for tissue fibrosis (in the present specification, comprising a detection agent for at least one protein selected from the protein group (A) (herein, “ It may be indicated as “the test agent of the present invention”. This will be described below.
  • Protein group (A), tissue fibrosis disease and the like are the same as defined in “1. Method for examining tissue fibrosis”.
  • 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 is at least one selected from the protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with tissue fibrosis disease induction.
  • the present invention relates to a tissue fibrosis disease model animal screening method (also referred to as “model animal screening method of the present invention” in the present specification) using the amount or concentration of a species protein as an index. This will be described below.
  • Body fluid, extracellular vesicles, protein group (A), tissue fibrosis disease, measurement of the amount or concentration of the target protein, etc. are the same as defined in “1. Method for examining tissue fibrosis disease” above.
  • 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 tissue fibrosis disease inducing treatment is not particularly limited as long as it is a treatment capable of inducing a tissue fibrosis disease.
  • an inducer such as bleomycin and silica (for example, nasal administration, oral administration, inhalation administration, transbronchial administration, intraperitoneal administration, intravenous administration, transdermal administration, etc., preferably nasal administration)
  • the induction treatment is performed by performing genetic modification treatment or radiation treatment.
  • the model animal screening method of the present invention selects the animal as a tissue fibrosis disease model animal (or tissue fibrosis disease model candidate animal) when the value of the index is equal to or higher than a cut-off 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.
  • the amount of the target protein in the extracellular vesicles of body fluid collected from an animal that has not been subjected to tissue fibrosis induction treatment 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 tissue fibrosis 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.
  • the present invention provides a preventive or therapeutic agent for tissue fibrosis disease (this book) containing an inhibitor of at least one protein selected from the protein group (A).
  • the agent of the present invention This will be described below.
  • the protein group (A) and the like are the same as defined in “1. Method for examining tissue fibrosis”.
  • the target protein 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 the target protein, expression vectors thereof; gene-specific ribozyme of the target protein; gene editor of the target protein by the CRISPR / Cas system, genome editor, and the like.
  • 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 the tissue fibrosis disease is based on a protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance.
  • a method for screening an active ingredient of a prophylactic or therapeutic agent for tissue fibrosis using the amount or concentration of at least one selected protein as an index (in the present specification, the “active ingredient screening method of the present invention” is indicated) There is also. This will be described below.
  • Body fluid, extracellular vesicles, protein group (A), tissue fibrosis disease, measurement of the amount or concentration of the target protein, etc. are the same as defined in “1. Method for examining tissue fibrosis disease” above.
  • 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).
  • the test substance is selected as an active ingredient of a prophylactic or therapeutic agent for tissue fibrosis (or a candidate substance for an active ingredient of a prophylactic or therapeutic agent for tissue fibrosis).
  • 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 is selected from the protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance.
  • the present invention relates to a method for evaluating the induction or malignancy of tissue fibrosis using the amount or concentration of at least one protein 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.
  • tissue fibrosis disease For the body fluid, extracellular vesicle, protein group (A), tissue fibrosis disease, measurement of the amount or concentration of the target protein, animal species, test substance, etc., see “1. Test method for tissue fibrosis disease” above. And “7. Screening method for active ingredient of preventive or therapeutic agent for tissue fibrosis 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 of a tissue fibrosis 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.
  • COPD chronic obstructive pulmonary disease
  • 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 exosome fraction was quantified by LC-MS / MS analysis (non-label method). Specifically, it was performed as follows.
  • sample preparation The exosome 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 tissue fibrosis disease biomarkers in lung tissue by immunostaining Lungs from normal mice (4 mice), tissue fibrosis disease model mice (8 mice), and chronic obstructive pulmonary disease (COPD) model mice (4 mice) The tissue was immunostained with an antibody against a tissue fibrosis biomarker (Test Example 4).
  • the primary antibodies used were anti-fibulin2 antibody (code: bs-13160R / Bioss), anti-biglycan antibody (code: ab49701 / abcam), anti-decorin antibody (code: ab175404 / abcam), anti-Fragilis antibody (code: ab15592 / abcam ), Anti-CD147 antibody (code: ab108317 / abcam), anti-Col VI a2 antibody (code: ab180855 / abcam), anti-PDGF ⁇ antibody (code: ab23914 / abcam), and anti-PTTG1IL antibody (code: ab128040 / abcam).
  • the tissue fibrosis disease model mice showed a significant difference in the degree of staining higher than normal mice (Cont vs ⁇ ⁇ BLM), and the chronic obstructive pulmonary disease model mice A significant difference (Cont vs COPD) that the degree of staining was higher than that of normal mice was calculated by t-test.
  • the results are shown in Table 3.
  • the examined tissue fibrosis disease biomarker was significantly expressed in the lung tissue of the tissue fibrosis disease model mouse than in the lung tissue of the normal mouse.

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Abstract

L'invention fournit un biomarqueur des maladies de type fibrose tissulaire, et un procédé de mise en œuvre de celui-ci. Plus précisément, l'invention concerne un groupe de protéines (A) se trouvant dans une vésicule extracellulaire de fluide corporelle prélevée chez un sujet.
PCT/JP2018/009503 2017-03-14 2018-03-12 Biomarqueur de fibrose tissulaire WO2018168769A1 (fr)

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WO2023027186A1 (fr) * 2021-08-26 2023-03-02 国立大学法人大阪大学 Biomarqueur de fibrose pulmonaire

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WO2020162441A1 (fr) * 2019-02-04 2020-08-13 国立大学法人大阪大学 Biomarqueur de maladie granulomateuse
WO2023027186A1 (fr) * 2021-08-26 2023-03-02 国立大学法人大阪大学 Biomarqueur de fibrose pulmonaire

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