WO2020262934A1 - Composition de marqueur biologique pour diagnostiquer une maladie du cerveau due à des lésions microvasculaires cérébrales - Google Patents

Composition de marqueur biologique pour diagnostiquer une maladie du cerveau due à des lésions microvasculaires cérébrales Download PDF

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WO2020262934A1
WO2020262934A1 PCT/KR2020/008162 KR2020008162W WO2020262934A1 WO 2020262934 A1 WO2020262934 A1 WO 2020262934A1 KR 2020008162 W KR2020008162 W KR 2020008162W WO 2020262934 A1 WO2020262934 A1 WO 2020262934A1
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brain
gene
damage
scgb3a
protein
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Korean (ko)
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박주영
이은희
한문
최효진
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재단법인 대구경북첨단의료산업진흥재단
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/35—Animals modified by environmental factors, e.g. temperature, O2
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention is a biomarker composition for diagnosing brain microvascular damage brain disease, more specifically, by precisely classifying a local area of the brain based on an MRI image to induce blood-brain barrier degeneration only in that local area, and brain disease due to brain microvascular damage
  • the blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain.
  • the blood-brain barrier blocks foreign substances from entering the brain and protects the brain by receiving substances necessary for metabolism. For example, it prevents the invasion of bacteria or viruses from the brain, and facilitates glucose supply and oxygen-carbon dioxide exchange. Blood is filtered through the blood-brain barrier, and as a result, very few substances that reach the brain are water or gas molecules, glucose, and certain fat-soluble substances.
  • the blood-brain barrier has the shape of enclosing blood vessels with endothelial cells of the brain, and since the endothelial cells are tight junctions, it is impossible for substances to pass between cells. For this reason, the blood-brain barrier prevents the passage of drugs not only for various imaging agents for diagnosis, but also for diseases in the brain such as tumors, Alzheimer's, and Parkinson's disease.
  • the present inventors have repeatedly researched to develop an animal model that damages the blood-brain barrier, and as a result, by irradiating ultrasound energy to the mouse to induce degeneration of the blood-brain barrier, a mouse model for brain disease due to brain microvascular damage was produced, and the mouse As a result of screening biomarkers related to brain microvascular damage brain disease due to degeneration of the blood-brain barrier by analyzing proteins isolated from the brain tissue of the model, Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, The present invention was completed by confirming that Scgb3a or Bst2 is a biomarker related to brain disease due to brain microvascular damage.
  • An object of the present invention is to provide a biomarker composition for diagnosing brain diseases due to brain microvascular damage, including the gene of Scgb3a.
  • Another object of the present invention is to provide a composition for diagnosing brain diseases due to brain microvascular injury, comprising a preparation for measuring the mRNA expression of the gene of Scgb3a or the level of protein activity thereof.
  • Another object of the present invention is to provide a kit for diagnosing brain diseases due to brain microvascular damage comprising the above-described composition.
  • Another object of the present invention is to provide a method for screening a pharmaceutical composition for the prevention or treatment of brain diseases caused by brain microvascular damage comprising the following steps. (a) treating the biological sample with any compound; (b) confirming the expression level or activity level of the gene or protein thereof of Scgb3a from the biological sample; And (c) comparing the level of the expression with the level of the gene or its expression protein or the level of activity in a normal control not treated with any compound.
  • Another object of the present invention is to provide a method for producing an optimal BBB denatured mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds.
  • Another object of the present invention is to provide a method of manufacturing a mild damaged BBB denatured mouse model comprising (a) treating the mouse with focused ultrasound of 0.42 to 0.44 MPa for 10 to 230 seconds.
  • Another object of the present invention is to provide a use of a composition comprising an agent for measuring the gene expression level of Scgb3a for diagnosing brain diseases caused by damage to brain microvessels.
  • the present invention can provide a biomarker composition for diagnosing brain diseases due to brain microvascular damage caused by brain microvascular damage including the gene of Scgb3a.
  • the present invention may also provide a composition for diagnosing brain diseases due to brain microvascular injury, including an agent for measuring the mRNA expression of the gene of Scgb3a or the level of protein activity thereof.
  • the agent may be a primer pair, probe or antisense nucleotide that specifically binds to the gene of Scgb3a.
  • the agent may be an antibody, an interaction protein, a ligand, nanoparticles, or aptamer that specifically binds to a protein of a gene of Scgb3a or higher.
  • the present invention can also provide a kit for diagnosing brain diseases due to brain microvascular damage comprising the composition described above.
  • the present invention may also provide a method for providing information for diagnosing brain diseases due to brain microvascular injury, including the step of measuring the mRNA expression or protein activity level of the gene of Scgb3a from a biological sample.
  • the present invention can provide a method for screening a pharmaceutical composition for preventing or treating brain diseases caused by brain microvascular damage comprising the following steps: (a) treating a biological sample with an arbitrary compound; (b) confirming the expression level or activity level of the gene or protein thereof of Scgb3a from the biological sample; And (c) comparing the expression level with the level of the gene or its expression protein in a normal control group not treated with any compound or the level of activity.
  • the present invention can provide a method of producing an optimal BBB denatured mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds.
  • the present invention can provide a method of manufacturing a mild damaged BBB denatured mouse model comprising (a) treating the mouse with focused ultrasound of 0.42 to 0.44 MPa for 10 to 230 seconds.
  • the present invention can provide a use of a composition comprising an agent for measuring the gene expression level of Scgb3a for diagnosing brain diseases caused by damage to brain microvessels.
  • BBB blood-brain barrier
  • a transcriptome sequencing analysis was performed, which is a biological analysis method, to find a diagnostic marker for brain disease due to degeneration of BBB, Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, It was confirmed that clcf 1, Scgb3a, or Bst2 can diagnose brain diseases caused by brain microvascular damage. Based on the test results, it was confirmed that Klk 6 and Lcn2 were confirmed and increased in the blood of the optimal BBB denatured animal model and the mild BBB denatured animal model.
  • FIG. 1 is a schematic diagram of a method of manufacturing a BBB opening animal model using focused ultrasound.
  • FIG. 3 is a result of confirming the change in gene expression in BBB denatured brain tissue through genomic analysis, (a) is a confirmation of gene expression in optimal BBB degenerated brain tissue, (b) gene expression in mild BBB denatured brain tissue Is confirmed.
  • the present invention provides a biomarker composition for diagnosing brain diseases due to brain microvascular injury comprising any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2. Can provide.
  • the blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain.
  • leakage of the blood-brain barrier is an independent risk factor. Therefore, in order to study brain diseases caused by brain microvascular damage such as dementia and Parkinson's disease, an animal model of blood-brain barrier damage is needed.
  • an animal model of blood-brain barrier damage is needed.
  • an animal model that is widely used as a model for the cerebrovascular barrier it is difficult to observe in real time, it is difficult to distinguish the functions of individual cellular elements, and as ethical issues for animal experiments continue to be raised, alternative animal research models are It is a necessary situation.
  • the present inventors produced an opening model of a blood-brain barrier (BBB) by irradiating focused ultrasound to the mouse.
  • BBB blood-brain barrier
  • the degree of patency of the blood-brain barrier was controlled by controlling the intensity of a specific focused ultrasound, and the adjustment caused optimal and mild damage, thereby creating an optimal BBB denatured mouse model and a mild BBB denatured mouse model.
  • transciptome sequencing analysis which is a biological analysis method, was performed to find a marker for diagnosis of brain disease due to brain microvascular injury.
  • KLK 6 and LCN2 were confirmed in the blood of the optimal BBB denatured mouse model and mild BBB denatured mouse model, and the expression of KlK6 in the blood gradually increased with time after BBB opening in the optimal condition and mild damaged condition. It was confirmed that the expression of LCN2 in the blood increased until 12 hours after the opening of the optimal condition BBB, and then decreased again. Through this, it was confirmed the possibility of discovering blood biomarkers for brain diseases caused by brain microvascular damage (FIG. 5).
  • Lcn2 (Lipocalin 2) is a gene encoding a protein belonging to the lipocalin family and carries small hydrophobic molecules such as lipids, steroid hormones, and retinoids.
  • the presence of this protein in blood and urine is an early biomarker of acute kidney injury.
  • the information on Lcn2 of the present invention is preferably disclosed in, for example, NCBI gene ID NM 008491, but is not limited thereto.
  • SPP1 (Secreted Phosphoprotein 1) is involved in the binding of osteoclasts to mineralized bone matrix.
  • the protein of this gene is also a cytokine that upregulates the expression of interferon- ⁇ and interleukin-12.
  • the information on SPP1 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_001204201, but is not limited thereto.
  • Saa3 (Serum amyloid A 3) is a family of apolipoproteins related to high-density lipoproteins.
  • the information of Saa3 of the present invention is preferably disclosed in NCBI IDNM_011315, for example, but is not limited thereto.
  • Ptx3 (Pentraxin 3) is induced by inflammatory cytokines in response to inflammatory stimuli in several mesenchymal and epithelial cell types, particularly endothelial cells and mononuclear phagocytes.
  • the protein of this gene promotes fibroblast differentiation and is involved in the regulation of inflammation and complement activation. It is a biomarker of several inflammatory diseases. However, the correlation of brain diseases caused by brain microvascular damage of the gene is not disclosed at all.
  • Ptx3 information of the present invention is preferably disclosed in, for example, NCBI gene ID NM_008987, but is not limited thereto.
  • A2m Alpha-2-Macroglobulin
  • a cytokine transporter is used to inhibit a wide range of proteolytic enzymes, including trypsin, thrombin and collagenase. Inhibits inflammatory cytokines.
  • proteolytic enzymes including trypsin, thrombin and collagenase.
  • Inhibits inflammatory cytokines is not disclosed at all.
  • the information on A2m of the present invention is preferably disclosed in, for example, NCBI gene ID NM_175628, but is not limited thereto.
  • Lrg1 Leucine Rich Alpha-2-Glycoprotein
  • LRR leucine-rich repeat
  • Lrg1 is a leucine-rich repeat (LRR) family including Lrg1, which is involved in protein-protein interaction, signal transduction, and cell adhesion and development.
  • LRG1 is associated with hydrocephalus.
  • the information on Lrg1 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_029796, but is not limited thereto.
  • Klk 6 Kerlikrein Related Peptidase 6
  • Diseases associated with the gene include gastric esophageal adenocarcinoma and synuclavism. It is a common cancer and other disease biomarker. However, the correlation of brain diseases caused by brain microvascular damage of the gene is not disclosed at all.
  • the information of Klk 6 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_001164696, but is not limited thereto.
  • Clcf1 Cardiotrophin Like Cytokine Factor 1
  • Clcf1 Cardiotrophin Like Cytokine Factor 1
  • the Clcf1 information of the present invention is preferably disclosed in, for example, NCBI gene ID NM_001310038, but is not limited thereto.
  • SCGB3A1 (Secretoglobin Family 3A Member 1) is associated with cytokine activity.
  • the information on SCGB3A1 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_054037, but is not limited thereto.
  • Bst2 (Bone Marrow Stromal Cell Antigen 2) is involved in pre-2 cell growth and rheumatoid arthritis.
  • Diseases associated with BST2 include human immunodeficiency virus type 1 and Westile encephalitis.
  • the information of Bst2 of the present invention is preferably disclosed in NCBI gene ID NM_198095, for example, but is not limited thereto.
  • diagnosis means confirming the presence or characteristics of a pathological condition.
  • diagnosis is to determine whether a brain disease has occurred due to damage to the brain microvessels.
  • Brain diseases caused by damage to the brain microvessels are preferably mild cognitive impairment, mild traumatic brain injury (MTBI), transient ischemic attack, Lou Gehrig's disease dementia, Alzheimer's, Parkinson, etc. However, it is not limited thereto.
  • the brain microvascular damage may be caused by the opening or leakage of the cerebrovascular barrier.
  • the'biomarker' is a substance capable of diagnosing brain diseases due to brain microvascular damage, that is, a substance capable of distinguishing and diagnosing whether or not brain diseases due to brain microvascular damage in a biological sample,
  • Organic organisms such as polypeptides, nucleic acids (e.g. mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show significant differences between normal individuals and individuals with brain disease caused by brain microvascular damage Includes molecules and the like.
  • Biomarkers for the purposes of the present invention are any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 as time passes in a mild BBB denatured mouse model.
  • the increased expression level of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 is a brain disease caused by brain microvascular injury. Support the outbreak of
  • the biomarker is any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2, depending on time in a mild BBB denatured animal model.
  • the present invention also includes an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2. It is possible to provide a composition for diagnosing brain diseases due to damage to brain microvessels.
  • any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 of the present invention are brain microscopic compared to healthy normal people. Since it is significantly increased in a brain disease model due to damage to blood vessels, it is possible to diagnose degenerative brain disease by measuring its gene mRNA expression or its protein level.
  • measurement of the mRNA expression level of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 is due to damage to brain microvessels.
  • this is a process of checking the presence and expression of a biomarker gene in a biological sample.
  • the amount of mRNA is measured using a formulation used in a method of measuring the level of mRNA transcribed from a target gene.
  • the agent measuring the mRNA level of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 is preferably an antisense oligonucleotide.
  • a primer pair or a probe, and the base sequence of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 is registered in the gene bank. Based on the above sequences, antisense oligonucleotides, primer pairs, or probes that specifically amplify specific regions of these genes can be designed.
  • measurement of the protein activity level of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a, and Bst2 is performed in the brain due to damage to the brain microvascular
  • a process of checking the presence and expression of a biomarker protein in a biological sample for diagnosis of a disease preferably the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 It may be an antibody, an interaction protein, a ligand, a nanoparticle, or an aptamer that specifically binds to the protein of any one or more genes selected from.
  • the antibody is a term known in the art and refers to a specific protein molecule directed to an antigenic site.
  • antibodies are Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1 , Scgb3a or Bst2 refers to an antibody that specifically binds to, and such an antibody is cloned into an expression vector according to a conventional method to obtain a protein encoded by the gene, and prepared from the obtained protein by a conventional method Can be. This includes partial peptides that can be made from these proteins.
  • the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, monoclonal antibody, or any one having antigen binding properties is included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.
  • the present invention can provide a kit for diagnosing brain diseases due to damage to brain microvessels comprising the above-described composition.
  • kits of the present invention in addition to the agent measuring the mRNA expression or protein activity level of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene, one or more other components suitable for the assay method Component compositions, solutions or devices may be included and do not limit the scope of the invention in any form.
  • the kit for measuring the mRNA expression level of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene in the present invention is an essential element required for performing RT-PCR It may be a kit containing.
  • the RT-PCR kit includes a test tube or other suitable container, reaction buffer, enzymes such as deoxynucleotide (Usene), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC- Water (DEPC-water), sterilized water, and the like may be included.
  • the kit of the present invention may be a kit including essential elements necessary to perform a microarray chip.
  • the microarray chip kit includes a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof, using the marker of the present invention. It can be easily manufactured by a manufacturing method commonly used in the art. In order to manufacture a microarray chip, a micropipetting method or a pin form using a piezoelectric method to immobilize on the substrate of the DNA chip using the searched marker as a probe DNA molecule It is preferable to use a method using a spotter (spotter) of, but is not limited thereto.
  • spotter spotter
  • the substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-Llysine, and aldehyde, but is not limited thereto.
  • the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicone, nylon film, and nitrocellulose membrane, but is not limited thereto.
  • the kit for measuring the protein activity level of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 is a substrate for immunological detection of an antibody, a suitable buffer solution, and color development. It may include a secondary antibody labeled with an enzyme or a fluorescent substance, and a chromogenic substrate.
  • the substrate may be a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass, and the coloring enzyme is peroxidase, alkaline force.
  • Fatase alkaline phosphatase
  • FITC alkaline phosphatase
  • RITC ribonulose-1-phosphate
  • ABTS 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfur) Phonic acid)
  • OPD o-phenylenediamine
  • TMB tetramethyl benzidine
  • the present invention also comprises the steps of measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a and Bst2 from a biological sample. It is possible to provide a method for providing information for diagnosing brain diseases due to damage to brain microvessels, including.
  • RNA sequencing analysis results showed that about 518 genes increased and 174 genes decreased at 48 hours when comparing gene expression changes over time with the control group.
  • Selected 10 genes (Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2) that are increased by more than 3 times compared to Control and are encoding soluble proteins that can be detected in blood.
  • the method of providing information for diagnosis is a preliminary step for diagnosis, and provides objective basic information necessary for diagnosis of brain diseases caused by damage to brain microvessels, and clinical judgment or findings of doctors are excluded. .
  • the term'biological sample' means a direct object that is isolated from an individual to measure the expression level of a target gene or protein, and includes samples such as tissues, cells, whole blood, serum, plasma, saliva or urine.
  • the sample of the present invention is a sample for diagnosing the onset of a brain disease in an individual suspected of having a brain disease due to damage to brain microvessels, and may preferably be blood.
  • the'analytical method for measuring the mRNA expression level' includes a polymerase reaction (PCR), a reverse transcription polymerase reaction (RT-PCR), a competitive reverse transcription polymerase reaction (Competitive RT-PCR), and a real-time reverse transcription polymerase.
  • PCR polymerase reaction
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Reaction Realtime RT-PCR
  • RNase protection assay RNase protection assay
  • northern blotting or DNA microarray analysis
  • the'analysis method for measuring the protein activity level' includes western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, and octero.
  • Ouchterlony immune diffusion method Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS) or protein chip ( protein chip) analysis, but is not limited thereto.
  • the method of the present invention comprises the step of comparing the measured mRNA expression or protein activity level of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene with the level measured in the control. can do.
  • the control group is a healthy person who has not invented a brain disease due to damage to the brain microvessels. Accordingly, the method of the present invention is used when the mRNA expression of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene or its protein activity level is higher than that of the control group, due to damage to the brain microvessel. It can be determined that the brain disease is in progress or has already occurred.
  • screening relates to an operation of selecting a substance having a therapeutic effect on a brain disease caused by damage to brain microvessels.
  • the candidate substance in step (a) refers to a substance to be tested for the therapeutic activity of brain diseases, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and arbitrary molecules of a wide range of compounds.
  • Brain diseases such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and arbitrary molecules of a wide range of compounds.
  • These candidates include natural as well as synthetic materials.
  • the step of measuring the mRNA expression or protein activity level of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene in step (b) is a candidate substance in the sample.
  • Nucleic acid encoding Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 by treatment or Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 This is the process of checking the presence and expression of proteins.
  • the activity of the mRNA of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene or its protein measured in the step (b) is the candidate substance. If the mRNA expression of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene or its protein activity of the control group not treated It may further include a step.
  • the mRNA expression of the Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene or the activity of its protein by the treatment of the candidate substance is determined by the treatment of the candidate substance Lcn2, SPP1,
  • the candidate substance can be judged as a treatment for brain diseases, and on the contrary,
  • Lcn2, SPP1, Saa3 , Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene mRNA expression or its protein activity is Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, Scgb3a or Bst2 gene mRNA expression or its protein activity is Lcn2, SPP1, Saa3, Ptx3, A2m, Lrg1, Klk 6, clcf 1, If the mRNA expression of the Scgb3a
  • the present invention can provide a method of producing a mild BBB denatured mouse model comprising the step of (a) treating the mouse with focused ultrasound of 0.42 to 0.44 MPa for 10 to 230 seconds.
  • the present invention can also provide a use of a composition comprising an agent for measuring the expression level of the Scgb3a gene for diagnosing brain diseases due to damage to brain microvessels.
  • Redundant content is omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the field to which the present invention belongs.
  • the system consists of an ultrasonic control system and an ultrasonic irradiation system (compatible with MR environments).
  • the ultrasonic control system consists of a fuction generator that can transmit RF power to the ultrasonic transducer and an RF amplifier, and is composed of a power meter that can measure the power delivered to the ultrasonic transducer in real time.
  • the ultrasonic irradiation system consists of an ultrasonic transducer, a three-axis control unit that can precisely control the ultrasonic transducer in three dimensions, and a water tank, and is designed to be driven even in the environment of MRI equipment.
  • the MR image-guided focused ultrasound system is linked with the Bruker 9.4T MRI (BioSpec 94/20 USR-9.4T, Bruker, MA, USA) system.
  • microbubbles DEFINITY®
  • a contrast agent for ultrasound imaging are diluted in physiological saline at a ratio of 1:50 and injected into the vein of the animal at a rate of 0.012ml/s.
  • focused ultrasound with a burst length of 10 ms per 1 s was irradiated to the desired brain region for 2 minutes based on MR images.
  • the BBB opening and closing can be confirmed by comparing the images before and after the injection of the MR contrast agent.
  • ultrasound energy under optimal damage (0.25 ⁇ 0.27 MPa) and mild damage BBBD (0.42-0.44 MPa) conditions was irradiated to the thalmus area of the brain of ICR mice to degenerate BBB.
  • Ultrasound irradiation conditions were irradiated for 120 seconds at 1 Hz for RFP using a center frequency of 1.1 MHz (Fig. 1).
  • MR images of mice under optimal damage (0.25 ⁇ 0.27 MPa) conditions and mild damage BBBD (0.42-0.44 MPa) conditions were confirmed (FIG. 2).
  • a contrast agent was administered after the opening of the BBB, and the degree of enhancement over time was checked, and images were acquired in the coronal, axial and sagittal directions.
  • the experiment was conducted under optimal ( ⁇ 50%) and mild ( ⁇ 100%) BBB conditions through intensity measurement according to the degree of penetration of the MR contrast agent.
  • BBB denatured brain tissue was obtained according to time (1 hour, 6 hours, 12 hours, 24 hours, 48 hours), and blood from mice of each group About 1 ml of was collected and serum was separated.
  • RNA isolation kit Qiagen
  • Total RNA was treated with DNA to prevent DNA contamination, and random fragmentation was prepared for library construction.
  • the RNA fragment was reacted with reverse transcriptase to produce cDNA, and sequencing was performed by attaching an adpator to both ends of the cDNA.
  • the quality of raw data obtained through sequencing was analyzed, and expression levels were extracted as FPKM (Fragments per kilobase of transcript per milion mapped reads) through transcript quantification of each sample (Fig. 3).
  • the animal model that induced the BBB opening using focused ultrasound was sacrificed over time, and about 1 ml of blood was taken from the inferior vena cava, coagulated at room temperature for about 30 minutes, and then centrifuged at 6000 rpm for 30 minutes to separate only the supernatant. Thus, serum was obtained.
  • LCN2 ELISA kit BosterBio
  • KLK6 LS bio

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Abstract

La présente invention concerne une composition de marqueur biologique pour diagnostiquer une maladie du cerveau due à des lésions microvasculaires cérébrales ; et, plus particulièrement, un procédé de fabrication d'un modèle animal présentant une maladie du cerveau due à des lésions microvasculaires cérébrales par induction d'une dégénérescence de la barrière hémato-encéphalique uniquement dans une zone locale correspondante grâce à la classification précise de zones locales d'un cerveau sur la base d'une image IRM, un procédé de criblage d'un marqueur biologique pour diagnostiquer une maladie du cerveau due à une lésion microvasculaire du cerveau provoquée par la dégénérescence de la barrière hémato-encéphalique à l'aide du modèle animal, et une composition de marqueur biologique pour diagnostiquer une maladie du cerveau due à une lésion microvasculaire du cerveau provoquée par la dégénérescence de la barrière hémato-encéphalique.
PCT/KR2020/008162 2019-06-24 2020-06-23 Composition de marqueur biologique pour diagnostiquer une maladie du cerveau due à des lésions microvasculaires cérébrales WO2020262934A1 (fr)

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WO2020262935A1 (fr) * 2019-06-24 2020-12-30 재단법인 대구경북첨단의료산업진흥재단 Composition de biomarqueur pour le diagnostic d'une maladie du cerveau provoquée par une lésion microvasculaire cérébrale
KR102100588B1 (ko) * 2019-06-24 2020-04-13 재단법인 대구경북첨단의료산업진흥재단 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물
WO2020262937A1 (fr) * 2019-06-24 2020-12-30 재단법인 대구경북첨단의료산업진흥재단 Composition de marqueur biologique pour le diagnostic d'une maladie du cerveau provoquée par un dommage microvasculaire cérébral
CN117051102B (zh) * 2023-10-12 2024-01-26 上海爱谱蒂康生物科技有限公司 生物标志物组合在制备预测帕金森病的产品中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006517181A (ja) * 2002-09-11 2006-07-20 フランクゲン・ビオテクノロギー・アーゲー Ｂｂｂ−特異的タンパク質およびその断片を同定する方法
JP2009020049A (ja) * 2007-07-13 2009-01-29 Univ Of Tokyo 脳血管疾患の診断方法
KR20190031072A (ko) * 2017-09-15 2019-03-25 경북대학교 산학협력단 리포칼린-2를 이용한 혈관성 치매의 진단방법
KR102100588B1 (ko) * 2019-06-24 2020-04-13 재단법인 대구경북첨단의료산업진흥재단 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6472140B1 (en) * 1997-09-05 2002-10-29 The General Hospital Corporation α-2- macroglobulin therapies and drug screening methods for Alzheimer's disease.
US20060263783A1 (en) * 2003-07-03 2006-11-23 Podhajcer Osvaldo L Methods and systems for diagnosis of non-central nervous system (cns) diseases in cns samples
JP4667372B2 (ja) * 2004-02-25 2011-04-13 株式会社ペルセウスプロテオミクス 血管障害の程度の判定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006517181A (ja) * 2002-09-11 2006-07-20 フランクゲン・ビオテクノロギー・アーゲー Ｂｂｂ−特異的タンパク質およびその断片を同定する方法
JP2009020049A (ja) * 2007-07-13 2009-01-29 Univ Of Tokyo 脳血管疾患の診断方法
KR20190031072A (ko) * 2017-09-15 2019-03-25 경북대학교 산학협력단 리포칼린-2를 이용한 혈관성 치매의 진단방법
KR102100588B1 (ko) * 2019-06-24 2020-04-13 재단법인 대구경북첨단의료산업진흥재단 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHU PO-CHUN, CHAI WEN-YEN, TSAI CHIH-HUNG, KANG SHIH-TSUNG, YEH CHIH-KUANG, LIU HAO-LI: "Focused Ultrasound-Induced Blood-Brain Barrier Opening: Association with Mechanical Index and Cavitation Index Analyzed by Dynamic Contrast-Enhanced Magnetic-Resonance Imaging", SCIENTIFIC REPORTS, vol. 6, no. 1, 1 December 2016 (2016-12-01), pages 1 - 13, XP055773327, DOI: 10.1038/srep33264 *
JANG; JINU: "The Present and Future of the Treatment of Brain Diseases Using Focused Ultrasound", CONVERGENCE RESEARCH REVIEW,, vol. 5, no. 2, pages 4 - 36, XP009525519 *

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