WO2023287269A1 - 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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WO2023287269A1
WO2023287269A1 PCT/KR2022/095112 KR2022095112W WO2023287269A1 WO 2023287269 A1 WO2023287269 A1 WO 2023287269A1 KR 2022095112 W KR2022095112 W KR 2022095112W WO 2023287269 A1 WO2023287269 A1 WO 2023287269A1
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brain
damage
cst7
srgn
tgfbi
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PCT/KR2022/095112
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Korean (ko)
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박주영
이은희
한문
최효진
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재단법인 대구경북첨단의료산업진흥재단
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Priority claimed from KR1020210093799A external-priority patent/KR102651116B1/ko
Priority claimed from KR1020210093802A external-priority patent/KR102635835B1/ko
Priority claimed from KR1020210093801A external-priority patent/KR102651118B1/ko
Priority claimed from KR1020210093800A external-priority patent/KR102635834B1/ko
Application filed by 재단법인 대구경북첨단의료산업진흥재단 filed Critical 재단법인 대구경북첨단의료산업진흥재단
Publication of WO2023287269A1 publication Critical patent/WO2023287269A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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 is a biomarker composition for diagnosing brain diseases caused by cerebral microvascular damage, and more specifically, brain diseases caused by cerebral microvascular damage by precisely classifying local areas of the brain based on MRI images and inducing degeneration of the blood-brain barrier only in those local areas.
  • 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 prevents foreign substances from entering the brain and protects the brain by accepting substances necessary for metabolism. For example, it prevents the invasion of bacteria or viruses in 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 reach the brain, such as water or gas molecules, glucose, and certain fat-soluble substances.
  • the blood-brain barrier has a shape that surrounds blood vessels with brain endothelial cells, and it is impossible for substances to pass between cells because the endothelial cells are in tight junctions with each other.
  • the blood-brain barrier prevents the passage of not only various imaging agents for diagnosis, but also drugs when diseases occur in the brain, such as tumors, Alzheimer's disease, and Parkinson's disease, thereby hindering the development of brain-related diagnosis and treatment technologies.
  • the inventors of the present invention irradiated ultrasonic energy to mice to induce degeneration of the blood-brain barrier to produce a mouse model of brain disease caused by brain microvascular damage, and the mouse Proteins isolated from the brain tissue of the model were analyzed to screen for biomarkers related to brain microvascular damage and encephalopathy due to blood-brain barrier degeneration. By confirming that it is a biomarker, the present invention was completed.
  • An object of the present invention is to provide a biomarker composition for diagnosing brain diseases caused by brain microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
  • Another object of the present invention is to provide a composition for diagnosing brain diseases caused by cerebral microvascular damage, comprising an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. is to do
  • Another object of the present invention is to provide a kit for diagnosing brain diseases caused by 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 preventing or treating 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 any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and (c) comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound.
  • Another object of the present invention is to provide a method for producing an optimal BBB degenerated 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 for producing a mildly damaged BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 to 230 seconds.
  • the present invention provides a biomarker composition for diagnosing brain diseases caused by cerebral microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. can do.
  • the present invention can also provide a composition for diagnosing brain diseases caused by cerebral microvascular damage comprising an agent for measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn. there is.
  • the agent may be a primer or probe that specifically binds to any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
  • the agent may be an antibody that specifically binds to a protein of one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
  • the present invention can also provide a kit for diagnosing brain diseases caused by brain microvascular damage comprising the above-described composition.
  • the present invention also relates to the use of a biomarker composition comprising at least one gene selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn for diagnosing brain diseases caused by brain microvascular damage and Tgfbi, Cst7 from biological samples. It is possible to provide a method for diagnosing brain diseases caused by damage to brain microvessels including; measuring the mRNA expression level or the protein activity level of the Angptl4 or Srgn gene.
  • the present invention is also directed to a method for diagnosing brain diseases caused by brain microvascular damage comprising measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample. How to provide information can be provided.
  • the present invention may 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 any one or more genes or proteins thereof selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from the biological sample; and (c) comparing the expression level with the level of the gene or its expressed protein or activity level in a normal control group not treated with any compound.
  • the present invention may provide a method for producing an optimal BBB degenerated 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 may provide a method for producing a mildly damaged BBB degenerated mouse model comprising the step of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 seconds to 230 seconds.
  • a mouse model of brain microvascular damage and brain disease caused by opening of the blood-brain barrier was prepared by irradiating a mouse with focused ultrasound.
  • the degree of patency of the blood-brain barrier was controlled by adjusting the intensity of the focused ultrasound, and the control caused optimal and moderate damage, thereby producing an optimal BBB degenerated mouse model and a mild BBB degenerated mouse model did
  • transcriptome sequencing analysis a biological analysis method, was performed to discover diagnostic markers for brain diseases caused by BBB degeneration. was confirmed to be diagnosable.
  • Tgfbi, Cst7, Angptl4 and Srgn were increased in the blood of optimal BBB degeneration animal models and mild BBB degeneration animal models. Based on the above results, there is an effect of diagnosing brain diseases such as dementia, brain cancer, Parkinson's, concussion (TBI) or stroke caused by brain microvascular damage using Tgfbi, Cst7, Angptl4 or Srgn.
  • brain diseases such as dementia, brain cancer, Parkinson's, concussion (TBI) or stroke caused by brain microvascular damage using Tgfbi, Cst7, Angptl4 or Srgn.
  • FIG. 1 is a schematic diagram of a method for manufacturing an animal model of BBB opening using focused ultrasound.
  • 5a to 5d show the results of confirming the expression levels of Tgfbi, Cst7, Angptl4, and Srgn in the blood of BBB animal models, respectively.
  • the present invention can provide a biomarker composition for diagnosing brain diseases caused by brain microvascular damage, including any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
  • the blood-brain barrier is a barrier that exists between the brain and blood vessels, preventing foreign substances from entering the brain.
  • Recent studies have shown that blood-brain barrier leakage is an independent risk factor. Therefore, in order to study brain diseases caused by brain microvascular damage, such as dementia and Parkinson's disease, blood-brain barrier damaged animal models are needed.
  • brain diseases caused by brain microvascular damage such as dementia and Parkinson's disease
  • blood-brain barrier damaged animal models are needed.
  • real-time observation is difficult and it is difficult to distinguish the functions of individual cellular elements. It is necessary. Accordingly, the present inventors irradiated mice with focused ultrasound to create a blood-brain barrier (BBB) patency model.
  • BBB blood-brain barrier
  • the degree of patency of the blood-brain barrier was controlled by adjusting the intensity of specific focused ultrasound, and optimal damage and mild damage were caused by the control, thereby creating an optimal BBB degeneration mouse model and a mild BBB degeneration mouse model.
  • a biological analysis method transciptome sequencing analysis, was performed to discover diagnostic markers for brain diseases caused by brain microvascular damage.
  • RNA sequencing analysis under the mild damage BBB condition where BBB damage can be expected to be moderate, about 518 genes were increased and 174 genes were decreased at 48 hours showed a tendency to become (Fig. 3). Based on the above results, blood biomarker candidates under mild damage BBB conditions were selected.
  • Tgfbi, Cst7, Angptl4 and Srgn 4 genes encoding water-soluble proteins detectable in blood and increased by more than 3 times compared to the control were selected as candidates.
  • Tgfbi, Cst7, Angptl4 and Srgn as a result of confirming in the blood of an optimal BBB degenerated mouse model and a mild BBB degenerated mouse model, the expression of Tgfbi, Cst7, Angptl4 and Srgn was increased over time after BBB opening under optimal and mild damaged conditions. It can be seen that this increases gradually over time. Through this, the possibility of discovering blood biomarkers for brain diseases caused by brain microvascular damage was confirmed (FIGS. 5a to 5d).
  • TGFBI Transforming growth factor beta induced
  • NM_000358.3 Human
  • NM_009369.5 Mae
  • Cst7 (Cystatin-F) is known to inhibit papain and cathepsin L, and is known to be involved in immune regulation by inhibiting targets in the hematopoietic system. There is no known correlation between brain diseases caused by cerebral microvascular damage. Information on Cst7 of the present invention is preferably disclosed in, for example, NCBI gene ID NM_003650.4 (Mouse, Human), but is not limited thereto.
  • Angptl4 (Angiopoietin like 4) is induced in hypoxic conditions in various cells and is a target of Peroxisome proliferator-activated receptor.
  • Angptl4 information of the present invention is preferably disclosed in, for example, NCBI ID NM_001039667.3 (Human), NM_016109, NM_139314.3 (Human) or NM_020581.2 (Mouse), but is not limited thereto.
  • Srgn is also known as hematopoietic proteoglycan core protein or secretory granule proteoglycan core protein, which is known as an intracellular proteoglycan mainly expressed in hematopoietic cells and endothelial cells.
  • Serglycin is also known as hematopoietic proteoglycan core protein or secretory granule proteoglycan core protein, which is known as an intracellular proteoglycan mainly expressed in hematopoietic cells and endothelial cells.
  • Srgn information of the present invention is disclosed in, for example, NCBI gene ID NM_002727.4 (Human), NM_001321053.2 (Human), NM_001321054.1 (Human), NM_001358965.1 (Mouse) or NM_011157.3 (Mouse) Preferred, but not limited thereto.
  • 'diagnosis' means confirming the presence or characteristics of a pathological condition.
  • diagnosis is to determine whether or not a brain disease is caused by damage to brain microvessels.
  • Brain diseases caused by damage to the cerebral microvessels may preferably include dementia, Alzheimer's, Parkinson's, mild cognitive impairment, and mild traumatic brain injury (MTBI), but are not limited thereto.
  • MTBI mild traumatic brain injury
  • the brain microvascular damage may be caused by the opening or leakage of the blood-brain barrier.
  • a 'biomarker' is a substance capable of diagnosing brain diseases caused by cerebral microvascular damage, that is, a substance capable of diagnosing brain diseases caused by cerebral microvascular damage by distinguishing whether or not they occur 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 diseases caused by brain microvascular damage molecules, etc.
  • nucleic acids e.g., mRNA, etc.
  • lipids lipids
  • glycolipids glycoproteins
  • sugars monosaccharides, disaccharides, oligosaccharides, etc.
  • the biomarker consists of Tgfbi, Cst7, Angptl4 and Srgn, as any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn increase over time in a mouse model with mild BBB degeneration.
  • the increased expression level of any one or more genes selected from the group supports the development of brain diseases due to cerebral microvascular damage.
  • the biomarker is any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn, and the group consisting of Tgfbi, Cst7, Angptl4 and Srgn whose expression is increased over time in animal models with mild BBB degeneration.
  • the increased expression level of any one or more genes selected from supports the development of brain diseases due to damage to brain microvessels.
  • the present invention can provide a composition for diagnosing brain diseases caused by brain microvascular damage, comprising an agent for measuring the mRNA expression of one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn or the protein activity level thereof. there is.
  • any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4, and Srgn of the present invention are significantly increased in a brain disease model caused by brain microvascular damage compared to healthy individuals.
  • it is possible to diagnose a degenerative brain disease by measuring its gene mRNA expression or its protein level.
  • the measurement of the mRNA expression level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is the presence of biomarker genes in biological samples for the diagnosis of brain diseases caused by brain microvascular damage.
  • the amount of mRNA is measured using the preparation used in the method of measuring the level of mRNA transcribed from the target gene.
  • the agent for measuring the mRNA level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is preferably an antisense oligonucleotide, primer pair or probe, and Tgfbi, Cst7, Angptl4 and Srgn Since the base sequences of one or more genes selected from the group consisting of are registered in the gene bank, those skilled in the art can design antisense oligonucleotides, primer pairs or probes that specifically amplify specific regions of these genes based on the sequences. there is.
  • measuring the protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn is the presence or absence of biomarker proteins in biological samples for the diagnosis of brain diseases caused by damage to brain microvessels.
  • the amount of the protein can be confirmed using an antibody that specifically binds to the protein of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn.
  • the antibody is a term known in the art and refers to a specific protein molecule directed against an antigenic site.
  • the antibody refers to an antibody that specifically binds to Tgfbi, Cst7, Angptl4 or Srgn, ,
  • These antibodies can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the gene, and from the obtained protein by a conventional method. This includes partial peptides that can be made from the protein.
  • the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or any antibody having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.
  • the present invention can provide a kit for diagnosing brain diseases caused by brain microvascular damage, including the above-described composition.
  • the kit of the present invention may include, in addition to agents for measuring the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn genes or the protein activity level thereof, one or more other component compositions, solutions or devices suitable for analysis methods, and may be taken in any form.
  • agents for measuring the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn genes or the protein activity level thereof one or more other component compositions, solutions or devices suitable for analysis methods, and may be taken in any form.
  • the scope of the present invention is not limited.
  • the kit for measuring the mRNA expression level of the Tgfbi, Cst7, Angptl4 or Srgn gene in the present invention may be a kit including essential elements necessary for performing RT-PCR.
  • the RT-PCR kit contains, in addition to primer pairs specific for the marker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (usen), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC- It may include water (DEPC-water), sterile water, and the like.
  • the kit of the present invention may be a kit including essential elements required to perform a microarray chip.
  • the microarray chip kit includes a substrate to which a 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. It can be easily prepared by a manufacturing method commonly used in the art.
  • a micropipetting method using a piezoelectric method or a pin type is used to immobilize the searched marker as a probe DNA molecule on a substrate of a DNA chip. It is preferable to use a method using a spotter of, but is not limited thereto.
  • the substrate of the microarray chip is preferably coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde, but is not limited thereto.
  • the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon membrane and nitrocellulose membrane, but is not limited thereto.
  • the kit for measuring the activity level of the Tgfbi, Cst7, Angptl4 or Srgn protein includes a substrate, an appropriate buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. etc. may be included.
  • a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used as the substrate, and the coloring enzyme may be peroxidase, alkaline phosphatase Alkaline phosphatase, etc.
  • FITC FITC
  • RITC RITC
  • ABTS 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sul phonic acid)
  • OPD o-phenylenediamine
  • TMB tetramethyl benzidine
  • the present invention also relates to the diagnosis of brain diseases caused by damage to brain microvessels, which includes measuring the mRNA expression or protein activity level of any one or more genes selected from the group consisting of Tgfbi, Cst7, Angptl4 and Srgn from a biological sample. Information provision methods can be provided.
  • the method for providing information for diagnosis is a preliminary step for diagnosis, which provides objective basic information necessary for the diagnosis of brain diseases caused by damage to brain microvessels, and the clinical judgment or opinion of a doctor is excluded. .
  • 'biological sample' refers to a direct target for measuring the expression level of a target gene or protein separated from an individual, 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 a subject suspected of having a brain disease due to brain microvascular damage, and may preferably be blood.
  • the subject is applicable to any mammal, and the mammal includes humans and primates as well as livestock such as cattle, pigs, sheep, horses, dogs, and cats.
  • the 'analysis method for measuring mRNA expression level' includes polymerase reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase Reaction (Realtime RT-PCR), RNase protection assay (RPA; RNase protection assay), northern blotting, or DNA microarray analysis, but is not limited thereto.
  • PCR polymerase reaction
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Realtime RT-PCR real-time reverse transcription polymerase Reaction
  • 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 Oktero Ouchterlony immunodiffusion assay, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis method, etc., but is not limited thereto.
  • ELISA enzyme linked immunosorbent assay
  • radioimmunoassay radioimmunodiffusion
  • Oktero Ouchterlony immunodiffusion assay Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip ( protein chip) analysis method, etc., but is not limited thereto.
  • the method of the present invention may include comparing the measured level of mRNA expression or protein activity of the Tgfbi, Cst7, Angptl4 or Srgn gene with the level measured in a control group.
  • the control group was a healthy person who did not develop a brain disease due to brain microvascular damage. Accordingly, in the method of the present invention, when the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the protein activity level thereof is higher than that of the control group, it can be determined that a brain disease caused by damage to brain microvessels is in progress or has already occurred. .
  • screening relates to an operation of selecting a substance having a therapeutic effect on brain diseases caused by damage to brain microvessels.
  • the candidate substance in step (a) means a substance to be tested for therapeutic activity of brain diseases, such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and any molecule of a wide range of compounds.
  • brain diseases such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and any molecule of a wide range of compounds.
  • candidate materials include natural materials as well as synthetic materials.
  • the step of measuring the mRNA expression or protein activity level of the Tgfbi, Cst7, Angptl4 or Srgn gene in step (b) is a nucleic acid encoding Tgfbi, Cst7, Angptl4 or Srgn by treatment with a candidate substance in the sample. Alternatively, it is a process of confirming the presence and expression level of Tgfbi, Cst7, Angptl4 or Srgn protein.
  • the screening method of the present invention (c) the activity of the mRNA or protein of the Tgfbi, Cst7, Angptl4 or Srgn gene measured in step (b) is the Tgfbi, Cst7, Angptl4 or Srgn gene of the control group not treated with the candidate substance.
  • a step of determining the candidate substance as a therapeutic agent for brain disease may be further included.
  • the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of the protein thereof by the treatment of the candidate substance is lower than the mRNA expression of the Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of the protein of the candidate substance untreated group.
  • the candidate substance can be judged as a brain disease treatment, and on the contrary, the mRNA expression of Tgfbi, Cst7, Angptl4 or Srgn gene or the activity of its protein is the mRNA of Tgfbi, Cst7, Angptl4 or Srgn gene in the candidate substance untreated group. If the expression or its protein activity is similar to or higher than that, it can be determined that it cannot be used as a treatment for brain diseases caused by damage to brain microvessels.
  • a method for producing an optimal BBB degenerated mouse model comprising the step of treating the mouse with focused ultrasound at 0.25 to 0.27 MPa for 10 to 230 seconds may be provided.
  • the present invention can provide a method for producing a mouse model with mild BBB degeneration, comprising the steps of (a) treating the mouse with focused ultrasound at 0.42 to 0.44 MPa for 10 to 230 seconds.
  • Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification 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 environment).
  • the ultrasonic control system consists of a function generator and an RF amplifier that can deliver RF power to the ultrasonic transducer, and a power meter that can measure the power delivered to the ultrasonic transducer in real time.
  • the ultrasonic irradiation system is composed of an ultrasonic transducer, a 3-axis controller that can precisely control the ultrasonic transducer in three dimensions, and a water tank, and is designed to be operable within the MRI equipment environment.
  • 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 0.012 ml/s.
  • focused ultrasound with a burst length of 10 ms per 1 s was irradiated to the desired brain region based on MR images for 2 minutes.
  • MR imaging it is possible to confirm opening and closing of the BBB by comparing images before and after MR contrast agent injection.
  • MR images after opening the BBB, a contrast agent was administered, the degree of enhancement over time was confirmed, and images were acquired in the coronal, axial, and sagittal directions.
  • Experiments were conducted under optimal ( ⁇ 50%) and mild ( ⁇ 100%) BBB conditions through intensity measurement according to the degree of penetration of the MR contrast agent.
  • BBB-modified brain tissue was obtained over time (1 hour, 6 hours, 12 hours, 24 hours, 48 hours), and blood samples were collected from each group of mice. About 1 ml was collected and serum was separated.
  • RNA was isolated using an RNA isolation kit (Qiagen). . Total RNA was treated with Dnase to prevent DNA contamination, and random fragmentation was produced for library production. The RNA fragment was reacted with reverse transcriptase to prepare cDNA, and adpators were attached to both ends of the cDNA to perform sequencing. The quality of raw data obtained through sequencing was analyzed, and the expression level was extracted as FPKM (Fragments per kilobase of transcript per milion mapped reads) value through transcript quantification of each sample (FIG. 3).
  • FPKM Frragments per kilobase of transcript per milion mapped reads
  • Example 3 In order to confirm that the sequencing results of the candidate genes in Example 3 were significant, the expression of the candidate genes was checked using real-time PCR testing technique to confirm the correlation.
  • the tissue of the optimal damage BBB animal model and the mild damage BBB animal model prepared by the method of Example 1 were rapidly frozen in liquid nitrogen, and RNA was isolated using the Quiagne RNA isolation kit (Quiagen). did Total RNA was treated with Dnase to prevent DNA contamination, and random fragmentation was produced for library production. RNA fragments were reacted with reverse transcriptase to prepare cDNA.
  • qRT-q using the SYBR® Green real-time PCR kit in a 20 ⁇ L reaction volume containing 10 ⁇ L of SYBR® Green master PCR mix, 5 pmol each of forward and reverse primers, 1 ⁇ L of diluted cDNA template, and an appropriate amount of sterile distilled water.
  • PCR was performed. Gene amplification conditions were as follows. initial denaturation at 95 °C for 3 min; 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, and elongation at 72°C for 60 seconds; and a final extension at 72° C. for 5 minutes.
  • qRT-PCR was performed on an ABI Step One Plus real-time PCR system. All quantifications were performed with U6 as an internal standard. PCR primer sequences are shown in Table 1 below. As a result, it was confirmed that the gene sequencing results and the qRT-PCR results according to time were similar (FIG. 4).
  • Biotin-labeled antibody solution was dispensed by 100ul, incubated at 37 °C for 1 hour, and washed three times with wash buffer for 5 minutes each. After dispensing 100ul of ABC solution, incubating at 37 °C for 30 minutes, washing with wash buffer, adding 90ul of TMB substrate solution, incubating at 37 °C for 30 minutes, adding 100ul of stop solution and using a plate reader (Tecan) The absorbance was measured at a wavelength of 450 nm and the results were analyzed. The expression of Tgfbi in blood was more than doubled at 48 hours at 0.42 MPa.

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Abstract

La présente invention concerne les éléments suivants : 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 la classification précise de zones locales d'un cerveau sur la base d'une image IRM et l'induction de la dégénérescence de la barrière hémato-encéphalique uniquement dans une zone locale correspondante; un procédé de criblage d'un bio-marqueur pour diagnostiquer une maladie du cerveau due à des lésions microvasculaires cérébrales 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 des lésions microvasculaires cérébrales provoquée par la dégénérescence de la barrière hémato-encéphalique.
PCT/KR2022/095112 2021-07-16 2022-07-18 Composition de marqueur biologique pour diagnostiquer une maladie du cerveau due à des lésions microvasculaires cérébrales WO2023287269A1 (fr)

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KR10-2021-0093801 2021-07-16
KR10-2021-0093799 2021-07-16
KR1020210093802A KR102635835B1 (ko) 2021-07-16 2021-07-16 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물
KR1020210093801A KR102651118B1 (ko) 2021-07-16 2021-07-16 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물
KR1020210093800A KR102635834B1 (ko) 2021-07-16 2021-07-16 뇌미세혈관 손상 뇌질환 진단용 바이오 마커 조성물
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US10809271B2 (en) * 2015-03-27 2020-10-20 The Translational Genomics Research Institute Biomarkers and methods of diagnosing and prognosing mild traumatic brain injuries

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10809271B2 (en) * 2015-03-27 2020-10-20 The Translational Genomics Research Institute Biomarkers and methods of diagnosing and prognosing mild traumatic brain injuries

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "2021 Brain Science Source Technology Development Project", 1 March 2012 (2012-03-01), KR, pages 1 - 51, XP009544540, Retrieved from the Internet <URL:http://click.ndsl.kr/servlet/OpenAPIFullTextView?keyValue=00894471&dbt=REPORT&cn=TRKO202100010476> *
CHAKRABORTY ANANYA, KAMERMANS ALWIN, VAN HET HOF BERT, CASTRICUM KITTY, AANHANE ED, VAN HORSSEN JACK, THIJSSEN VICTOR L, SCHELTENS: "Angiopoietin like-4 as a novel vascular mediator in capillary cerebral amyloid angiopathy", BRAIN, vol. 141, no. 12, 1 December 2018 (2018-12-01), GB , pages 3377 - 3388, XP093024546, ISSN: 0006-8950, DOI: 10.1093/brain/awy274 *
KONOFAGOU ELISA E: "Optimization of the Ultrasound-Induced Blood-Brain Barrier Opening", THERANOSTICS, vol. 2, no. 12, 1 January 2012 (2012-01-01), AU , pages 1223 - 1237, XP093024551, ISSN: 1838-7640, DOI: 10.7150/thno.5576 *
LYNCH MADELAINE, HEINEN STEFAN, MARKHAM-COULTES KELLY, O'REILLY MEAGHAN, VAN SLYKE PAUL, DUMONT DANIEL J., HYNYNEN KULLERVO, AUBER: "Vasculotide restores the blood-brain barrier after focused ultrasound-induced permeability in a mouse model of Alzheimer's disease", INTERNATIONAL JOURNAL OF MEDICAL SCIENCE, vol. 18, no. 2, 1 January 2021 (2021-01-01), AU , pages 482 - 493, XP093024550, ISSN: 1449-1907, DOI: 10.7150/ijms.36775 *

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