WO2021177448A1 - Procédé de criblage - Google Patents

Procédé de criblage Download PDF

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Publication number
WO2021177448A1
WO2021177448A1 PCT/JP2021/008723 JP2021008723W WO2021177448A1 WO 2021177448 A1 WO2021177448 A1 WO 2021177448A1 JP 2021008723 W JP2021008723 W JP 2021008723W WO 2021177448 A1 WO2021177448 A1 WO 2021177448A1
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Prior art keywords
base sequence
bases
seq
test
gene expression
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PCT/JP2021/008723
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English (en)
Japanese (ja)
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明彦 田口
由紀子 竹内
由佳 沖中
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公益財団法人神戸医療産業都市推進機構
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Publication of WO2021177448A1 publication Critical patent/WO2021177448A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for screening a candidate substance that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among test substances.
  • the present invention also relates to a method for obtaining an index for selecting a target animal individual to be treated with an active substance for improvement, treatment and / or prevention of dementia or brain dysfunction from among the test animal individuals.
  • Alzheimer's disease has the largest number of patients, and it is reported that it accounts for more than 60% of the total.
  • donepezil hydrochloride (trade name: Aricept, etc.) for Alzheimer-type dementia (improved memory and cognitive impairment and suppressed the progression of the disease)
  • galantamine the same Reminyl
  • rivastigmine the same Ixeron patch, rivastigmine patch.
  • Memantine the same Memary
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • cognitive function tests such as the Hasegawa simple intelligence evaluation scale for discriminating dementia are known, but the cognitive function test is a test used after the patient himself / herself recognizes the progression of symptoms, and is used in the early stage of onset. It cannot be used to judge dementia and brain function. There is also the problem that the results of cognitive function tests are ambiguous.
  • a method for comprehensive analysis of blood microRNAs in subjects has also been developed for predicting the onset of dementia.
  • Patent Documents 1 to 3 can be exemplified as patent documents that disclose a method for determining brain dysfunction based on the gene expression level (biomarker) of a specific protein.
  • Patent Document 1 examines Alzheimer's disease, which comprises detecting in vitro a decrease in the level or function of at least one factor in the insulin / IGF signaling pathway, such as insulin, in a subject-derived central nervous system tissue. The method is described.
  • Patent Document 2 detects phosphorylation of at least one substrate protein such as MARCKS in a subject, and when the degree of phosphorylation is higher than that of a normal sample, the subject suffers from or is at risk of developing Alzheimer's disease. A method for determining that the substance has sex is described.
  • Patent Document 3 describes a biomarker for detecting a cognitive dysfunction disease.
  • the biomarker described in Patent Document 3 is described as a protein that depends on Complement C4, Prothrombin, Complement C3, Gelsolin, and the like.
  • a substance having an action of improving, treating and / or preventing dementia or brain dysfunction can be screened, it will be beneficial for the development of drugs and foods and drinks having the above action.
  • the substance since there are large individual differences in whether or not a substance having an action of improving, treating and / or preventing dementia or brain dysfunction exerts the desired action when actually administered, the substance is effective. It would be beneficial if the target individuals that act on the disease could be selected in advance.
  • One or more embodiments of the present invention is a method for screening a candidate substance among test substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction by a simple operation.
  • the purpose is to provide.
  • a target animal individual treated with an active substance for improving, treating and / or preventing dementia or brain dysfunction from among the test animal individuals by a simple operation.
  • the purpose is to provide a way to obtain indicators for selecting.
  • One or more embodiments of the present invention It is a method of screening candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among the test substances. Incubating cells isolated from animal body fluids or bone marrow in the presence of test material, Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response in the cells, and comparing the gene expression or enzymatic activity to the case where it is not incubated in the presence of the test substance.
  • the present invention relates to a method including determining the test substance as the candidate substance when the substance is suppressed.
  • Another embodiment of the present invention Incubating cells isolated from animal body fluids or bone marrow in the presence of test substances of unknown efficacy in ameliorating, treating and / or preventing dementia or brain dysfunction. Gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response in the cells, and gene expression of the one or more proteins in the cells not incubated in the presence of the test substance.
  • the present invention relates to a method including measuring enzyme activity.
  • Another embodiment of the present invention It is a method of screening candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among the test substances.
  • the test substance is suppressed as compared with the case where the test substance is not administered, the test substance is judged to be the candidate substance.
  • Regarding methods including.
  • Another embodiment of the present invention Genes of one or more proteins involved in the energy metabolism response in cells derived from body fluids or bone marrow of non-human animals administered with a test substance whose effectiveness in improving, treating and / or preventing dementia or brain dysfunction is unknown.
  • the present invention relates to a method comprising measuring expression or enzyme activity, and measuring gene expression or enzyme activity of one or more proteins in the cells of a non-human animal to which the test substance has not been administered.
  • Another embodiment of the present invention It is a method of screening candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among the test substances.
  • Another embodiment of the present invention To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in cells derived from body fluids or bone marrow isolated from humans to which the test substance was administered, and to administer the test substance.
  • the present invention relates to a method comprising measuring the gene expression or enzymatic activity of one or more proteins in the cells isolated from a non-human.
  • Another embodiment of the present invention It is a method for obtaining an index for selecting a target animal individual to be treated with an active substance for improvement, treatment and / or prevention of dementia or brain dysfunction from among individual test animals. Incubating cells isolated from the body fluids or bone marrow of a test animal in the presence of the active substance, Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response in the cells, and comparing the gene expression or enzymatic activity to the case where it is not incubated in the presence of the active substance. When the test animal is suppressed, the test animal is judged to be the target animal. Regarding methods including.
  • Another embodiment of the present invention It is a method of selecting a target animal individual to be treated with an active substance for improvement, treatment and / or prevention of dementia or brain dysfunction from among individual test animals. Incubating cells isolated from the body fluids or bone marrow of a test animal in the presence of the active substance, Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response in the cells, and comparing the gene expression or enzymatic activity to the case where it is not incubated in the presence of the active substance. When the test animal is suppressed, the test animal is judged to be the target animal. Regarding methods including.
  • the one or more proteins are preferably one or more proteins involved in glucose transport, lactic acid transport, or energy metabolism regulation.
  • the cells are preferably peripheral blood cells.
  • candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction can be screened.
  • an index for selecting a target animal individual to be treated with an active substance for amelioration, treatment and / or prevention of dementia or brain dysfunction. can.
  • an animal refers to a human or non-human animal.
  • non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats.
  • the body fluid includes blood, saliva, urine, sputum, sweat, pharyngeal swab, nasal swab and the like. Blood is particularly preferable as the body fluid, and peripheral blood is particularly preferable. Various forms of peripheral blood samples such as serum, plasma, and whole blood of blood such as peripheral fluid can be used. Bone marrow fluid is preferable as the bone marrow.
  • the cells derived from body fluid or bone marrow may be in a form isolated from a human or non-human animal, or may be in a form existing in the body fluid or bone marrow in the living body of a human or non-human animal.
  • cells derived from body fluid or bone marrow are preferably in the form of a mononuclear cell-containing sample containing mononuclear cells (MONOCLEAR Cells; MNC) isolated from body fluid or bone marrow.
  • MNC mononuclear cell-containing sample containing mononuclear cells isolated from body fluid or bone marrow.
  • the term "mononuclear cell” refers to lymphocytes and / or monocytes.
  • a method for obtaining a mononuclear cell-containing sample from body fluid or bone marrow a method using a reagent and / or a column for separating mononuclear cells is preferable.
  • the reagent and / or column include Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Laboratories Technologies), and Human Peripheral Blood Monologic Laboratory (manufactured by Human Biological Blood Mononulus Technology).
  • CPT mononuclear cell separation blood collection tube manufactured by Becton Dickinson
  • purriMate manufactured by purriSelect
  • SepMate manufactured by STEMCELL Technologies
  • Example 1 in peripheral blood cells isolated from an aged mouse individual to which a drug known to have an action of relieving dementia was administered, and in peripheral blood cells isolated from an aged mouse individual and then contacted with the drug. It has been confirmed that the gene expression level of the protein described later is lower than that of the cell which has not been in contact with the drug.
  • Example 4 when the drug was brought into contact with peripheral blood cells isolated from humans having subjective symptoms regarding age-related decline in cranial nerve function, the gene expression level decreased as compared with the cells not in contact with the drug. It has been confirmed that
  • the term “dementia” may be any of Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, but Alzheimer's disease is particularly preferable. be.
  • Brain function refers to the function of brain tissue involved in memory, movement, sensation, sleep, language, and the like.
  • Brain dysfunction includes the occurrence or worsening of autonomic nervous system disorders, the occurrence or deterioration of memory disorders, the occurrence or deterioration of motor disorders, the occurrence or deterioration of cooperative movement disorders, the occurrence or deterioration of involuntary movement disorders, and sensation.
  • Occurrence or worsening of impairment Occurrence or worsening of impairment, occurrence or deterioration of visual impairment, occurrence or deterioration of olfactory sensation, occurrence or deterioration of hearing impairment, occurrence or deterioration of balance sensation, occurrence or deterioration of sleep disorder, occurrence of speech disorder Or worsening, emotional dysregulation, headache, spasm, tremor, illusion, delusion, illusion, or abnormal behavior Occurrence or exacerbation, outbreak or exacerbation of depression, etc. are included.
  • one or more proteins involved in energy metabolism reactions include glucose transport, lactic acid transport, energy metabolism regulation, mitochondrial biosynthesis, glycolysis, pentose phosphate pathway, TCA cycle, electron transport chain, fatty acid metabolism, and the like.
  • One or more proteins involved in gap binding or sodium-potassium pump can be exemplified, and one or more proteins involved in glucose transport, lactic acid transport, or energy metabolism regulation are particularly preferable.
  • proteins involved in glucose transport include proteins that constitute membrane proteins (glucose transporters) involved in glucose transport that transport glucose used for energy metabolism in cells from outside the cell to the inside of the cell.
  • proteins belonging to the Glucose transporter family Glucose 1 to 12.
  • gene expression or enzymatic activity of Glut1, Glut2, Glut3 and Glut4, particularly Glut1 and Glut3 is preferable.
  • the nucleotide sequence of mouse Glut1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.
  • the base sequence of human Glut1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
  • the nucleotide sequence of mouse Glut3 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.
  • the base sequence of human Glut3 is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.
  • the nucleotide sequences of SEQ ID NOs: 1, 3, 5 and 7 all have a nucleotide sequence encoding the amino acid sequence of a protein (CDS) and a nucleotide sequence of an untranslated region (UTR) located upstream and downstream of the CDS. include.
  • a protein constituting a membrane protein (lactic acid transporter) involved in lactic acid transport that transports lactic acid produced by energy metabolism in the cell from the inside of the cell to the outside of the cell can be exemplified.
  • examples include proteins belonging to the MCT (monocarboxylate transport protein) family.
  • MCT1 monocarboxylate transport protein
  • MCT2 monocarboxylate transport protein
  • MCT4 gene expression or enzymatic activity of MCT1, MCT2, MCT3 and MCT4, particularly MCT4, is preferable.
  • the nucleotide sequence of mouse MCT4 is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.
  • the nucleotide sequence of human MCT4 is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12.
  • the nucleotide sequences of SEQ ID NOs: 9 and 11 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS.
  • proteins involved in the regulation of energy metabolism include proteins belonging to the PHD (procollagen-contining protein) family.
  • PHD procollagen-contining protein
  • gene expression or enzymatic activity of PHD2 and PHD3, particularly PHD3, is preferable.
  • the nucleotide sequence of mouse PHD3 is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14.
  • the nucleotide sequence of human PHD3 is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16.
  • the nucleotide sequences of SEQ ID NOs: 13 and 15 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS.
  • CDS amino acid sequence of the protein
  • UTR untranslated region
  • HIF1a hyperoxia-inducible factoror 1 alpha
  • Sirtuin 1 sirtuin 1
  • PPARa peroxisome proliferator-activated receptor
  • PGC1a peroxisome proliferators-active receptor-ganma co-activator-1 alpha
  • proteins involved in glycolysis include LDHa (lactate dehydrogenase A), LDHb (lactate dehydrogenase B), HK1 (hexokinase 1), PFK (phosphofructase kinase), and PKR. Can also be exemplified.
  • Proteins involved in the pentose phosphate pathway include G6PD (glucose-6-phosphate dehydrange), RPI (ribose-5-phosphate isomerase), RPE (ribose-5-phosphate epimerase), and 6PGLose (6-PGLose. ) Can be exemplified.
  • proteins involved in the TCA cycle include proteins belonging to the PDK (pyruvate dehydogenesis kinase) family.
  • proteins belonging to the PDK family PDK1, PDK3 and PDK4, particularly PDK1 gene expression or enzymatic activity is preferable.
  • IDH2 isocitrate dehydrogenase 2
  • OGDH oxoglute dehydrogenase
  • CS citrate synthase
  • AMPK AMP-activated protein kinase
  • proteins involved in fatty acid metabolism include FABP1 (fatty acid binding protein 1), FABP4 (fatty acid binding protein 4), CD36 (fatty acid transit protein: FAT), CPT1 (carnitine palmitoyl), and CPT1 (carnitine palmitoyl).
  • -Transphase 1) and ACC protein-CoA fatty acid
  • proteins involved in gap junctions include Cx37 (connexin 37) and Cx43 (connexin 43).
  • Proteins involved in the sodium-potassium pump include ATP1A1 (sodium / potassium-transporting TAPase subunit alpha-1), ATP1A2 (sodium / potassium-transporting TAPase subunitAtropa 3) can be exemplified.
  • the first embodiment of the present invention is It is a method of screening candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among the test substances. Incubating cells isolated from animal body fluids or bone marrow in the presence of test material, Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response in the cells, and comparing the gene expression or enzymatic activity to the case where it is not incubated in the presence of the test substance.
  • the present invention relates to a method including determining the test substance as the candidate substance when the substance is suppressed.
  • cells derived from body fluids such as peripheral blood, urine, saliva, or bone marrow of an animal suffering from dementia or an animal having decreased brain function are used to improve dementia or decreased brain function.
  • a drug effective for treatment and / or prevention or when the drug is administered to an animal suffering from dementia or an animal having decreased brain function, it is involved in an energy metabolism reaction in the cells 1
  • a test substance acts to reduce the gene expression or enzyme activity in cells derived from body fluid or bone marrow
  • the test substance is effective in improving, treating or preventing dementia or brain dysfunction. It becomes an index of that.
  • the "animal” may be a human or a non-human animal. Examples of non-human animals are as described above.
  • the "animal” is preferably an animal suffering from dementia or an animal whose brain function has deteriorated due to old age or the like.
  • the step of incubating cells isolated from animal body fluids or bone marrow in the presence of the test substance can be performed by immersing the test substance and cells in a suitable culture medium or buffer solution.
  • the concentration of the test substance and cells can be adjusted as appropriate.
  • the gene expression or the enzyme activity is suppressed as compared with the case where the test substance is not incubated in the presence of the test substance.
  • the test substance is incubated in the presence of the test substance.
  • the protein of the same species as measured by incubating in the presence of the test substance as compared with the gene expression or enzymatic activity of the protein in the absence of incubation in the presence of the test substance. It means that gene expression or enzyme activity is suppressed.
  • a comparative control "gene expression or enzymatic activity of the protein of cells of the same type as those incubated in the presence of the test substance in the absence of incubation in the presence of the test substance" , It may be a measured value measured each time, it may be a known measured value measured in advance, or it may be a reference value set from a known measured value measured in advance.
  • the measured value may be a measured value of the gene expression or the enzyme activity in a cell isolated from the same individual, or the gene expression or the enzyme activity in a cell isolated from another individual of the same species. It may be a measured value of.
  • only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured.
  • Gene expression of the one or more proteins was measured by detecting mRNA (including coding region and untranslated region) of the gene of the one or more proteins in cells incubated in the presence of the test substance. Alternatively, it may be measured by detecting the protein amount of the above-mentioned one or more proteins.
  • the measurement value of the gene expression of the one or more proteins is the relative expression level of the gene expression of the one or more proteins to the gene expression level of the one or more endogenous controls in the cells incubated in the presence of the test substance.
  • an endogenous control a housekeeping gene typified by 18S ribosomal RNA can be used.
  • the nucleotide sequence of mouse 18S ribosomal RNA is shown in SEQ ID NO: 17.
  • the nucleotide sequence of human 18S ribosomal RNA is shown in SEQ ID NO: 18.
  • Northern blotting Northern blotting, RT-PCR method, real-time RT-PCR method, DNA microarray method (method using DNA chip), dot blotting method, RNase protection for detection of mRNA of gene of amino acid sequence of one or more proteins.
  • An assay method or the like can be used. These methods can be performed by known methods. These methods can be performed by known methods.
  • the protein amount of the one or more proteins can be detected by an immunoassay method using an antibody that specifically recognizes and binds to the one or more proteins.
  • the antibody can be prepared by a known method.
  • the immunoassay method include a method using a solid-phase carrier on which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, and the like.
  • the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate and an agglutination method (immunoprecipitation method) using immobilized particles.
  • ELISA enzyme-linked immunosorbent assay
  • a known immunoassay can be used to detect the protein content of one or more of the above proteins. Further, the detection of the protein amount of the above-mentioned one or more proteins can also be performed by a method using LC-MS / MS MRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be carried out by a conventional protocol.
  • the enzymatic activity of the one or more proteins can be measured by a method according to the enzymatic activity of the protein to be measured.
  • a preferred example of a reagent that can be used to measure the gene expression or enzymatic activity of one or more proteins and the endogenous control protein is a nucleic acid (genomic DNA, mRNA or A primer pair for amplifying a cDNA prepared based on mRNA), a probe hybridizing with a nucleic acid containing the gene of the protein to be measured (genomic DNA, mRNA or a cDNA prepared based on mRNA), to the protein. Examples thereof include an antibody that specifically binds to the protein, a reaction substrate for measuring the enzymatic activity of the protein, and the like.
  • the nucleic acid may also include an untranslated region, an intron region, a signal sequence region, and the like.
  • the nucleic acid is mRNA or cDNA.
  • the mRNA or cDNA may contain at least a part of the base sequence encoding the amino acid sequence of the protein (CDS) and the base sequence of the untranslated region (UTR) located upstream and downstream thereof.
  • CDS amino acid sequence of the protein
  • UTR untranslated region
  • An example of the primer pair is a primer pair capable of amplifying at least a part of the base sequences of CDS and UTR.
  • An example of the probe is a probe that can hybridize to at least a part of the base sequences of CDS and UTR.
  • 3 is a primer pair for amplifying a nucleic acid containing the Glut1 gene, which has the same or homologous base sequence Af as any of the base sequences shown in (A1) to (A4) below.
  • An example includes a Glut1 primer pair containing.
  • (A1) Consecutive 10 or more base sequences contained in the base sequences of positions 101 to 180 among the base sequences of SEQ ID NO: 1
  • (A2) Bases of SEQ ID NO: 3 among the base sequences of SEQ ID NO: 3.
  • the base sequence corresponding to the base sequence of (A1) is changed to the base sequence of positions 256 to 336 among the base sequences of (A3) SEQ ID NO: 3.
  • Consecutive base sequence of 10 or more bases (A4) Of the base sequences of SEQ ID NO: 1, when the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 3 are aligned, the base sequence of (A3) is obtained.
  • the "base sequence at positions 101 to 180” is preferably the “base sequence at positions 111 to 170", and more preferably “base sequence at positions 121 to 160". It is a “base sequence”, more preferably “base sequence at positions 121 to 155”, and more preferably “base sequence at positions 126 to 150”.
  • the "base sequence at positions 256 to 336” is preferably the “base sequence at positions 266 to 326", and more preferably “base sequence at positions 276 to 316". It is a “base sequence”, more preferably “base sequence at positions 276 to 311”, and more preferably “base sequence at positions 281 to 306”.
  • the base sequence Af may be the same as or homologous to the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (A1), (A2), (A3) or (A4), and the rest of the base sequence Af is It is the same as or homologous to the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4).
  • the range of "homology" is as described later.
  • the base sequence Af is the same as the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4).
  • the Glut1 forward primer may be one containing a polynucleotide containing the base sequence Af at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Af.
  • the number of bases of the polynucleotide contained in the Glut1 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (A1) the base sequence of SEQ ID NO: 21 can be exemplified.
  • the base sequence of SEQ ID NO: 21 can be exemplified.
  • the base sequence of SEQ ID NO: 31 can be exemplified.
  • the base sequence of SEQ ID NO: 31 can be exemplified.
  • the "base sequence at positions 213 to 292” is preferably the “base sequence at positions 223 to 282", and more preferably “base sequence at positions 233 to 272". It is a “base sequence”, more preferably “base sequence at positions 238 to 272”, and more preferably “base sequence at positions 243 to 267”.
  • the "base sequence at positions 339 to 418” is preferably the “base sequence at positions 349 to 408", and more preferably “base sequence at positions 359 to 398". It is a “base sequence”, more preferably “base sequence at positions 364 to 398”, and more preferably “base sequence at positions 369 to 393”.
  • the base sequence Ar may be the same as or homologous to the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8), but the base From the 3'end of the sequence Ar, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).
  • the rest of the base sequence Ar is the same as or homologous to the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).
  • the range of "homology” is as described later.
  • the base sequence Ar is the same as the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).
  • the Glut1 reverse primer may contain a polynucleotide containing the base sequence Ar at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Ar.
  • the number of bases of the polynucleotide contained in the Glut1 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 22 can be exemplified.
  • the base sequence of SEQ ID NO: 22 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 32 can be exemplified.
  • the base sequence of SEQ ID NO: 32 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing the Glut3 gene, which has the same or homologous base sequence Bf as any of the base sequences shown in (B1) to (B4) below.
  • An example includes a Glut3 primer pair containing.
  • (B1) Consecutive 10 or more base sequences contained in the base sequences of positions 250 to 329 of the base sequence of SEQ ID NO: 5 (B2) Base of SEQ ID NO: 7 among the base sequences of SEQ ID NO: 7.
  • the base sequence corresponding to the base sequence of (B1) (B3) is the base sequence of positions 224 to 303 among the base sequences of SEQ ID NO: 7.
  • Consecutive base sequence of 10 or more bases (B4) Among the base sequences of SEQ ID NO: 5, when the base sequence of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 7 are aligned, the base sequence of (B3) is obtained.
  • the "base sequence at positions 250 to 329” is preferably the “base sequence at positions 260 to 319", and more preferably “base sequence at positions 270 to 309". It is a “base sequence”, more preferably “base sequence at positions 270 to 304", and more preferably “base sequence at positions 275 to 299”.
  • the "base sequence at positions 224 to 303” is preferably the “base sequence at positions 234 to 293", and more preferably “base sequence at positions 244 to 283". It is a “base sequence”, more preferably “base sequence at positions 244 to 278”, and more preferably “base sequence at positions 249 to 273”.
  • the base sequence Bf may be the same as or homologous to the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (B1), (B2), (B3) or (B4), and the rest of the base sequence Bf is It is the same as or homologous to the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4).
  • the range of "homology" is as described later.
  • the base sequence Bf is the same as the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4).
  • the Glut3 forward primer may be one containing a polynucleotide containing the base sequence Bf at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Bf.
  • the number of bases of the polynucleotide contained in the Glut3 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (B1) the base sequence of SEQ ID NO: 23 can be exemplified.
  • the base sequence of SEQ ID NO: 23 can be exemplified.
  • the base sequence of (B3) the base sequence of SEQ ID NO: 33 can be exemplified.
  • the base sequence of SEQ ID NO: 33 can be exemplified.
  • the "base sequence at positions 375 to 454" is preferably the “base sequence at positions 385 to 444", and more preferably “base sequence at positions 395 to 434". It is a “base sequence”, more preferably “base sequence at positions 400 to 434", and more preferably “base sequence at positions 405 to 429”.
  • the "base sequence at positions 288 to 367” is preferably the “base sequence at positions 298 to 357”, and more preferably “base sequence at positions 308 to 347". It is a “base sequence”, more preferably “base sequence at positions 313 to 347”, and more preferably “base sequence at positions 318 to 342”.
  • the base sequence Br may be the same as or homologous to the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8), but the base From the 3'end of the sequence Br, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).
  • the rest of the base sequence Br is the same as or homologous to the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).
  • the range of "homology" is as described later.
  • the base sequence Br is the same as the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).
  • the Glut3 reverse primer may be one containing a polynucleotide containing the base sequence Br at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Br.
  • the number of bases of the polynucleotide contained in the Glut3 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 24 can be exemplified.
  • the base sequence Br the base sequence of SEQ ID NO: 24 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 34 can be exemplified.
  • the base sequence Br the base sequence of SEQ ID NO: 34 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing the MCT4 gene, which has the same or homologous base sequence Cf as any of the base sequences shown in (C1) to (C4) below.
  • An example includes an MCT4 primer pair containing.
  • (C1) Consecutive base sequence of 10 or more bases contained in the base sequence of positions 40 to 118 of the base sequence of SEQ ID NO: 9
  • the "base sequence at positions 40 to 118" is preferably the “base sequence at positions 50 to 108", and more preferably “base sequence at positions 60 to 98". It is a “base sequence”, more preferably a “base sequence at positions 60 to 93”, and more preferably a "base sequence at positions 65 to 88".
  • the "base sequence at positions 146 to 225” is preferably the “base sequence at positions 156 to 215", and more preferably “base sequence at positions 166 to 205". It is a “base sequence”, more preferably “base sequence at positions 166 to 200”, and more preferably “base sequence at positions 171 to 195”.
  • the base sequence Cf may be the same as or homologous to the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (C1), (C2), (C3) or (C4), and the rest of the base sequence Cf is It is the same as or homologous to the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4).
  • the range of "homology" is as described later.
  • the base sequence Cf is the same as the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4).
  • the MCT4 forward primer may be one containing a polynucleotide containing the base sequence Cf at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Cf.
  • the number of bases of the polynucleotide contained in the MCT4 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (C1) the base sequence of SEQ ID NO: 25 can be exemplified.
  • the base sequence of SEQ ID NO: 25 can be exemplified.
  • the base sequence of (C3) the base sequence of SEQ ID NO: 35 can be exemplified.
  • the base sequence of SEQ ID NO: 35 can be exemplified.
  • the "base sequence at positions 107 to 186” is preferably the “base sequence at positions 117 to 176", and more preferably “base sequence at positions 127 to 166". It is a “base sequence”, more preferably “base sequence at positions 132 to 166”, and more preferably “base sequence at positions 137 to 161".
  • the "base sequence at positions 225 to 304" is preferably the “base sequence at positions 235 to 294", and more preferably “base sequence at positions 245 to 284". It is a “base sequence”, more preferably “a base sequence at positions 250 to 284", and more preferably “a base sequence at positions 255 to 279”.
  • the base sequence Cr may be the same as or homologous to the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8). From the 3'end of the sequence Cr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).
  • the rest of the base sequence Cr is the same as or homologous to the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).
  • the range of "homology” is as described later.
  • the base sequence Cr is the same as the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).
  • the MCT4 reverse primer may contain a polynucleotide containing the base sequence Cr at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Cr.
  • the number of bases of the polynucleotide contained in the MCT4 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 26 can be exemplified.
  • the base sequence of SEQ ID NO: 26 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 36 can be exemplified.
  • the base sequence of SEQ ID NO: 36 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing a PHD3 gene, which has the same or homologous base sequence Df as any of the base sequences shown in (D1) to (D4) below.
  • An example includes a pair of PHD3 primers.
  • (D1) Consecutive 10 or more base sequences contained in the base sequences of positions 864 to 943 among the base sequences of SEQ ID NO: 13
  • (D2) Bases of SEQ ID NO: 15 among the base sequences of SEQ ID NO: 15.
  • the base sequence corresponding to the base sequence of (D1) is changed to the base sequence of positions 863 to 942 among the base sequences of (D3) SEQ ID NO: 15.
  • Consecutive base sequence of 10 or more bases (D4) Among the base sequences of SEQ ID NO: 13, when the base sequence of SEQ ID NO: 13 and the base sequence of SEQ ID NO: 15 are aligned, the base sequence of (D3) is obtained.
  • the "base sequence at positions 864 to 943” is preferably the “base sequence at positions 874 to 933", and more preferably “base sequence at positions 884 to 923". It is a “base sequence”, more preferably “base sequence at positions 884 to 918”, and more preferably “base sequence at positions 889 to 913".
  • the "base sequence at positions 863 to 942” is preferably the “base sequence at positions 873 to 932", and more preferably “base sequence at positions 883 to 922". It is a “base sequence”, more preferably “base sequence at positions 883 to 917”, and more preferably “base sequence at positions 888 to 912".
  • the base sequence Df may be the same as or homologous to the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (D1), (D2), (D3) or (D4), and the rest of the base sequence Df is It is the same as or homologous to the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4).
  • the range of "homology" is as described later.
  • the base sequence Df is the same as the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4).
  • the PHD3 forward primer may be one containing a polynucleotide containing the base sequence Df at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Df.
  • the number of bases of the polynucleotide contained in the PHD3 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (D1) the base sequence of SEQ ID NO: 27 can be exemplified.
  • the base sequence of SEQ ID NO: 27 can be exemplified.
  • the base sequence of (D3) the base sequence of SEQ ID NO: 37 can be exemplified.
  • the base sequence of SEQ ID NO: 37 can be exemplified.
  • the "base sequence at positions 991 to 1072” is preferably the “base sequence at positions 1001 to 1062", and more preferably “base sequence at positions 1011 to 1052". It is a “base sequence”, more preferably “base sequence at positions 1016 to 1047”, and more preferably “base sequence at positions 1021 to 1042”.
  • the "base sequence at positions 1004 to 1083” is preferably the “base sequence at positions 1014 to 1073”, and more preferably “base sequence at positions 1024 to 1063". It is a “base sequence”, more preferably “base sequence at positions 1029 to 1063”, and more preferably “base sequence at positions 1034 to 1058”.
  • the base sequence Dr may be the same as or homologous to the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8). From the 3'end of the sequence Dr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).
  • the rest of the base sequence Dr is the same as or homologous to the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).
  • the range of "homology” is as described later.
  • the base sequence Dr is the same as the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).
  • the PHD3 reverse primer may be one containing a polynucleotide containing the base sequence Dr at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Dr.
  • the number of bases of the polynucleotide contained in the PHD3 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 28 can be exemplified.
  • the base sequence of SEQ ID NO: 28 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 38 can be exemplified.
  • the base sequence of SEQ ID NO: 38 can be exemplified.
  • a primer pair for amplifying the nucleic acid of the endogenous control gene is a primer pair for amplifying the nucleic acid encoding 18S ribosome RNA, and any of the following (F1) to (F4).
  • An 18S ribosome RNA reverse primer containing the sequence Fr at the 3'end and an 18S ribosome RNA primer pair containing the sequence Fr can be exemplified.
  • (F1) Consecutive 10 or more base sequences contained in the base sequences of positions 1216 to 1297 of the base sequence of SEQ ID NO: 17 (F2) Base of SEQ ID NO: 18 among the base sequences of SEQ ID NO: 18.
  • the base sequence corresponding to the base sequence of (F1) is changed to the base sequence of positions 1216 to 1297 of the base sequence of (F3) SEQ ID NO: 18.
  • Consecutive base sequence of 10 or more bases (F4) Of the base sequences of SEQ ID NO: 17, when the base sequence of SEQ ID NO: 17 and the base sequence of SEQ ID NO: 18 are aligned, the base sequence of (F3) is obtained.
  • the "base sequence at positions 1216 to 1297” is preferably the “base sequence at positions 1226 to 1287”, and more preferably “base sequence at positions 1236 to 1277”. It is a “base sequence”, more preferably “base sequence at positions 1241 to 1272”, and more preferably “base sequence at positions 1241 to 1267”.
  • the "base sequence at positions 1216 to 1297” is preferably the “base sequence at positions 1226 to 1287”, and more preferably “base sequence at positions 1236 to 1277”. It is a “base sequence”, more preferably “base sequence at positions 1236 to 1272”, and more preferably “base sequence at positions 1241 to 1267”.
  • the base sequence Ff may be the same as or homologous to the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (F1), (F2), (F3) or (F4), and the rest of the base sequence Ff is It is the same as or homologous to the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4).
  • the range of "homology" is as described later.
  • the base sequence Ff is the same as the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4).
  • the 18S ribosomal RNA forward primer may contain a polynucleotide containing the base sequence Ff at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Ff. ..
  • the number of bases of the polynucleotide contained in the 18S ribosomal RNA forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (F1) the base sequence of SEQ ID NO: 19 can be exemplified.
  • the base sequence of SEQ ID NO: 19 can be exemplified.
  • the base sequence of SEQ ID NO: 29 can be exemplified.
  • the base sequence of SEQ ID NO: 29 can be exemplified.
  • the "base sequence at positions 1317 to 1396” is preferably the “base sequence at positions 1327 to 1386", and more preferably “base sequence at positions 1337 to 1376". It is a “base sequence”, more preferably “base sequence at positions 1342 to 1376”, and more preferably “base sequence at positions 1347 to 1371”.
  • the "base sequence at positions 1317 to 1396” is preferably the “base sequence at positions 1327 to 1386", and more preferably “base sequence at positions 1337 to 1376". It is a “base sequence”, more preferably “base sequence at positions 1342 to 1376”, and more preferably “base sequence at positions 1347 to 1371”.
  • the base sequence Fr may be the same as or homologous to the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8), but the base From the 3'end of the sequence Fr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).
  • the rest of the base sequence Fr is the same as or homologous to the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).
  • the range of "homology" is as described later.
  • the base sequence Fr is the same as the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).
  • the 18S ribosomal RNA reverse primer may contain a polynucleotide containing the base sequence Fr at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Fr. ..
  • the number of bases of the polynucleotide contained in the 18S ribosomal RNA reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 20 can be exemplified.
  • the base sequence Fr the base sequence of SEQ ID NO: 20 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 30 can be exemplified.
  • the base sequence of SEQ ID NO: 30 can be exemplified.
  • base sequence Y homologous to base sequence X or "base sequence X and base sequence Y are homologous"
  • a polynucleotide consisting of a complementary sequence of base sequence X and a base sequence As long as the polynucleotide consisting of Y is a combination capable of hybridizing under the annealing conditions of the nucleic acid amplification reaction and forming a hydrogen bond sufficient to form a stable double strand, the nucleotide sequences X and Y May be partially different.
  • a polynucleotide consisting of a complementary sequence of the base sequence X and a polynucleotide consisting of the base sequence Y have several mismatches such as 1 mismatch in 10 nucleotides, 1 mismatch in 20 nucleotides, or 1 mismatch in 30 nucleotides. There may be a mismatch.
  • the base sequence Y is "homologous" to the base sequence X, it means that the base sequences X and Y satisfy any of the following relationships.
  • the base sequence Y is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the base sequence X.
  • the base sequence Y is a base sequence having 70% or more identity with the base sequence X.
  • the polynucleotide consisting of the base sequence Y can hybridize with the polynucleotide consisting of the base sequence complementary to SEQ ID NO: X under stringent conditions.
  • “1 or several” preferably refers to 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2, and most preferably. It is one.
  • “1 or several” refers to the total number of deleted, substituted, added and / or inserted bases.
  • the identity value indicates a value calculated with default settings using software (for example, FASTA, DNASIS, and BLAST) that calculates the identity between a plurality of base sequences.
  • software for example, FASTA, DNASIS, and BLAST
  • the base sequence identity value the number of matching bases when the pair of base sequences are aligned so as to maximize the degree of matching is calculated, and the total number of bases in the compared base sequence of the number of matching bases is calculated. Calculated as a percentage of the number.
  • the total number of bases described above is the number of bases counted with one gap as one base.
  • the identity is more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more. , More preferably 99% or more identity.
  • the "stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • stringent conditions can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, in the hybridization step, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 40 to 68 ° C, preferably 40 to 65 ° C.
  • hybridization can be performed at 1 to 7 ⁇ SSC, 0.02 to 3% SDS, and a temperature of 40 ° C to 60 ° C.
  • a washing step may be performed after hybridization, and the washing step can be performed, for example, at 0.1 to 2 ⁇ SSC, 0.1 to 0.3% SDS, and a temperature of 50 to 65 ° C.
  • One or both of the above primer pairs may be modified with another substance such as a labeling substance.
  • the probe is a nucleotide sequence of a nucleic acid containing the gene of one or more proteins, for example, the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of the Glut1 gene (particularly, positions 131 to 262 of the nucleotide sequence of SEQ ID NO: 1).
  • the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 1 are aligned among the nucleotide sequence of position 3 or the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 1 is located at positions 131 to 262.
  • the base sequence corresponding to the base sequence at position), the base sequence of SEQ ID NO: 5 or SEQ ID NO: 7 of the Glut3 gene (particularly, the base sequence of positions 280 to 424 of the base sequence of SEQ ID NO: 5, or the base sequence number Of the 7 base sequences, when the base sequence of SEQ ID NO: 7 and the base sequence of SEQ ID NO: 5 are aligned, the base sequence corresponding to the base sequences of positions 280 to 424 of the base sequence of SEQ ID NO: 5 ),
  • the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 11 of the MCT4 gene (particularly, the nucleotide sequence of positions 70 to 156 of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 11 When the base sequence of 11 and the base sequence of SEQ ID NO: 9 are aligned, the base sequence corresponding to the base sequence of positions 70 to 156 of the base sequence of SEQ ID NO: 9), S
  • the probe can contain a nucleic acid fragment containing a continuous partial base sequence of bases or more, more preferably 20 bases or more, or a base sequence homologous to the complementary base sequence thereof.
  • the upper limit of the length of the partial base sequence is not particularly limited, but may be, for example, 50 bases or less, 40 bases or less, or 30 bases or less.
  • the probe may be one in which the nucleic acid fragment is modified with another substance such as a labeling substance.
  • the antibody that specifically binds to the one or more proteins may be a polyclonal antibody or a monoclonal antibody.
  • the antibody can also be used as a fragment as long as it can specifically bind to the protein to be measured.
  • Examples of the antibody fragment include a Fab fragment, an F (ab') 2 fragment, a single chain antibody (scFv), and the like.
  • the antibody may be immobilized on a solid-phase carrier such as a microtiter plate or particles.
  • the reaction substrate for measuring the enzyme activity of the one or more proteins can be appropriately selected according to the activity of the protein to be measured.
  • a second embodiment of the present invention It is a method of screening candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among the test substances.
  • the test substance is suppressed as compared with the case where the test substance is not administered, the test substance is judged to be the candidate substance.
  • Regarding methods including.
  • the second embodiment of the present invention also uses cells derived from body fluids such as peripheral blood, urine, saliva, or bone marrow of an animal suffering from dementia or an animal having decreased brain function to cause dementia or decreased brain function. It is involved in the energy metabolism response in the cells when treated with a drug effective for improvement, treatment and / or prevention, or when the drug is administered to an animal suffering from dementia or an animal having decreased brain function. It is based on the unexpected finding that the gene expression or enzymatic activity of one or more proteins is reduced as compared to the case without treatment with the agent. Examples of "non-human animals" in this embodiment are as described above.
  • the "non-human animal” is preferably a non-human animal suffering from dementia or a non-human animal whose brain function has deteriorated due to old age or the like.
  • the "non-human animal to which the test substance has been administered” may be any non-human animal to which the test substance has been administered by a route such as intravenous administration or oral administration.
  • the method according to the present embodiment may further include administering the test substance to a non-human animal as a prior step.
  • the "cells derived from body fluid or bone marrow” may be cells isolated from a non-human animal, or may be cells existing in the body fluid or bone marrow in the body of a non-human animal.
  • the method according to this embodiment is: Isolating body fluid or bone marrow-derived cells from non-human animals administered the test substance, May be further included.
  • only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured.
  • the method for measuring gene expression and enzyme activity, and the reagents (primer pair, probe, antibody, etc.) used for the measurement are as described with respect to the first embodiment of the present invention.
  • the "gene expression or enzymatic activity of the protein in cells of the same species in non-human animals not administered with the test substance" which is a comparative control here, may be a measured value measured each time.
  • the measured value may be a measured value of the gene expression or the enzyme activity in the cell in the same individual before administration of the test substance, or in a cell isolated from another individual of the same species. It may be a measured value of the gene expression or the enzyme activity.
  • a third embodiment of the present invention It is a method of screening candidate substances that may be effective in improving, treating and / or preventing dementia or brain dysfunction from among the test substances.
  • To measure the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction in the body fluid or bone marrow-derived cells isolated from the human to which the test substance was administered, and the gene expression or the enzyme activity When the test substance is suppressed as compared with the case where the test substance is not administered, the test substance is judged to be the candidate substance.
  • cells derived from body fluids such as peripheral blood, urine, saliva, or bone marrow of an animal suffering from dementia or an animal having decreased brain function can be used for dementia or decreased brain function. It is involved in the energy metabolism response in the cells when treated with a drug effective for improvement, treatment and / or prevention, or when the drug is administered to an animal suffering from dementia or an animal having decreased brain function. It is based on the unexpected finding that the gene expression or enzymatic activity of one or more proteins is reduced as compared to the case without treatment with the agent.
  • the "human” is preferably a human suffering from dementia or a human whose brain function has deteriorated due to old age or the like.
  • the "human to whom the test substance has been administered” may be a non-human animal to which the test substance has been administered by a route such as intravenous administration or oral administration.
  • only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured.
  • the method for measuring gene expression and enzyme activity, and the reagents (primer pair, probe, antibody, etc.) used for the measurement are as described with respect to the first embodiment of the present invention.
  • the gene expression or enzymatic activity of the protein in the body fluid or bone marrow-derived cells isolated from the human to whom the test substance was administered is suppressed as compared with the gene expression or enzymatic activity of the protein in. Means.
  • the "gene expression or enzymatic activity of the protein in cells of the same type in humans not administered with the test substance" which is a comparative control here, may be a measured value measured each time, or may be a measured value in advance.
  • the measured value may be a measured known measured value, or may be a reference value set from a known measured value measured in advance.
  • the measured value may be a measured value of the gene expression or the enzyme activity in the cell isolated from the same human individual before administration of the test substance, or isolated from another human individual. It may be a measured value of the gene expression or the enzyme activity in the cells.
  • a fourth embodiment of the present invention A method for obtaining an index for selecting a target animal individual to be treated with an active substance for improvement, treatment and / or prevention of dementia or brain dysfunction from among the test animal individuals, or a test animal individual. It is a method of selecting a target animal individual to be treated with an active substance for improvement, treatment and / or prevention of dementia or brain dysfunction.
  • test animal Incubating cells isolated from the body fluids or bone marrow of a test animal in the presence of the active substance, Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response in the cells, and comparing the gene expression or enzymatic activity to the case where it is not incubated in the presence of the active substance. When the test animal is suppressed, the test animal is judged to be the target animal. Regarding methods including.
  • cells derived from body fluids such as peripheral blood, urine, saliva, or bone marrow of an animal suffering from dementia or an animal having decreased brain function can be used for dementia or decreased brain function. It is involved in the energy metabolism response in the cells when treated with a drug effective for improvement, treatment and / or prevention, or when the drug is administered to an animal suffering from dementia or an animal having decreased brain function. It is based on the unexpected finding that the gene expression or enzymatic activity of one or more proteins is reduced as compared to the case without treatment with the agent.
  • test animal individual When cells derived from body fluid or bone marrow isolated from an individual test animal are incubated in the presence of an active substance for improving, treating and / or preventing dementia or brain dysfunction, the gene expression or When the enzyme activity decreases, the test animal individual is an individual sensitive to the active substance, and is an indicator that treatment with the active substance is effective.
  • the "animal” may be a human or a non-human animal. Examples of non-human animals are as described above.
  • the "animal” is preferably an animal suffering from dementia or an animal whose brain function has deteriorated due to old age or the like.
  • the step of incubating the cells isolated from the body fluid or bone marrow of the individual test animal in the presence of the active substance can be carried out by immersing the active substance and the cells in an appropriate culture solution or buffer solution.
  • the concentration of the active substance and cells can be adjusted as appropriate.
  • only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured.
  • the method for measuring gene expression and enzyme activity, and the reagents (primer pair, probe, antibody, etc.) used for the measurement are as described with respect to the first embodiment of the present invention.
  • the gene expression or the enzyme activity is suppressed as compared with the case where the enzyme is not incubated in the presence of the active substance
  • the gene expression or enzyme of the protein measured by incubating in the presence of the active substance as compared with the gene expression or enzyme activity of the protein in the cells of the cell in the absence of incubation in the presence of the active substance. It means that the activity is suppressed.
  • a comparative control "gene expression or enzymatic activity of the protein of the same type of cells as incubated in the presence of the active substance in the absence of incubation in the presence of the active substance” is the same.
  • the measured value may be a measured value measured each time, a known measured value measured in advance, or a reference value set from a known measured value measured in advance.
  • the measured value may be a measured value of the gene expression or the enzyme activity in a cell isolated from the same individual, or the gene expression or the enzyme activity in a cell isolated from another individual of the same species. It may be a measured value of.
  • Example 1 Cells in the peripheral blood of young mice (5 weeks old) and old mice (55 weeks old) were washed with PBS and then suspended in glucose-free RPMI1640 medium.
  • a drug known to have an effect of relieving dementia was added to the obtained peripheral blood cell suspension at a concentration of 1 mM.
  • RNA extraction kit Manton (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210B (A ⁇ 4)
  • a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)).
  • a nucleic acid amplification reagent PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)
  • Agilent AliaMx Real-Time PCR System Agilent AliaMx Real-Time PCR System (Agilent) was used.
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C.
  • the 18S ribosomal RNA gene which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method.
  • the Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration.
  • Nucleic Acids Res. 2001; 29 (9): e45 The method described in can be used. The test was carried out 5 times or more, and the average value (average relative expression level) of their relative expression levels was calculated. Table 1 shows the base sequences of primers for each gene.
  • the expression level of each gene decreased by the addition of the drug, which is known to have an action of relieving dementia.
  • the drug which is known to have an action of recovering dementia
  • the average relative expression level of PHD3 is higher than that when the drug is not added. It decreased by 0.32, the average relative expression level of Glut1 decreased by 0.31, the average relative expression level of Glut3 decreased by 0.57, and the average relative expression level of MCT4 decreased by 0.76.
  • no significant change in gene expression was observed by the addition of the drug, which is known to have an effect of relieving dementia.
  • the average relative expression level of PHD3 is compared with the case where the drug is not added. Increased by 0.23, the average relative expression level of Glut1 decreased by 0.09, the average relative expression level of Glut3 increased by 0.10, and the average relative expression level of MCT4 increased by 0.09.
  • Example 2 The drug, which is known to have an effect of relieving dementia, was intravenously administered to aged mice (55 weeks old). At this time, assuming that the mouse blood was 2 ml, the drug was prepared by intravenous injection in a liquid volume of 100 ⁇ l so that the blood concentration immediately after administration was 10 mM with PBS. In addition, as a control test, 100 ⁇ l of PBS was intravenously injected into aged mice (55 weeks old).
  • Peripheral blood was collected 3 hours after administration of the drug or PBS to prepare a peripheral blood cell suspension in the same manner as in Example 1.
  • Quantitative RT-PCR was performed using the same method and primer set as in Example 1.
  • the expression level of each gene was represented by the average value (average relative expression level) of the relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene calculated by the Pfaffl method.
  • Volunteer A is a human who has no subjective or objective symptoms regarding age-related deterioration of cranial nerve function and memory, with MMSE (Mini-Mental State Examination), which is a cognitive function test, scoring 30 out of 30 points. be.
  • the MMSE is a cognitive function test consisting of a total of 11 questions in the form of questions. Time orientation, location orientation, immediate recall, attention and calculation ability, delayed reproduction (short-term memory), linguistic ability, and graphic. Cognitive functions such as ability (spatial cognition) can be evaluated.
  • the maximum score of the MMSE score is 30 points, and a case of 27 points or less can be determined to be mild cognitive impairment (MCI), and a case of 23 points or less can be determined to be dementia.
  • MCI mild cognitive impairment
  • Peripheral blood was collected from Volunteer A using a blood collection tube containing an anticoagulant (EDTA) (Venoject II: manufactured by Terumo), and cells in the peripheral blood were washed with PBS and then suspended in RPMI1640 medium.
  • EDTA anticoagulant
  • the drug used in Examples 1 and 2 and known to have an action of relieving dementia was added to a concentration of 1 mM.
  • the peripheral blood cell suspension to which the drug was added and the peripheral blood cell suspension to which the drug was not added were incubated in vitro at 37 ° C. and 5% CO 2 for 3 hours, respectively. After incubation, quantitative RT-PCR was used to verify changes in the expression of energy metabolism-related genes, Glut1, Glut3, MCT4, and PHD3.
  • the average relative expression level of PHD3 increased by 0.03 and the average relative expression level of Glut1 was 0, as compared with the case where the drug was not added. It increased by .12, the average relative expression level of Glut3 increased by 0.31, the average relative expression level of MCT4 decreased by 0.09, and an increasing tendency was observed in the majority of the target genes. This tendency was the same as the responsiveness to the drug observed in the young mice with normal cranial nerve function shown in Example 1.
  • Volunteer B is a human who has subjective symptoms regarding age-related decline in cranial nerve function, and a physician has pointed out the possibility.
  • Peripheral blood was collected from Volunteer B, cells in the peripheral blood were washed with PBS, and then suspended in RPMI1640 medium.
  • the drug used in Examples 1 and 2 and known to have an action of relieving dementia was added to a concentration of 1 mM.
  • the peripheral blood cell suspension to which the drug was added and the peripheral blood cell suspension to which the drug was not added were incubated in vitro at 37 ° C. and 5% CO 2 for 3 hours, respectively. After incubation, quantitative RT-PCR was used to verify changes in the expression of energy metabolism-related genes, Glut1, Glut3, MCT4, and PHD3.
  • RNA was extracted from the sample according to the conditions and operations described in Example 3, and cDNA was prepared from the total RNA. Using the prepared cDNA as a template, quantitative real-time PCR was performed according to the conditions and operations described in Example 5 described later, and the target genes Glut1, Glut3, MCT4 and PHD3, and the housekeeping gene 18S as an endogenous control were performed. The expression level of the ribosomal RNA gene was determined, and the average relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method.
  • the average relative expression level of PHD3 decreased by 0.39 and the average relative expression level of Glut1 decreased by 0.39 as compared with the case where the drug was not added. It decreased by 0.25, the average relative expression level of Glut3 decreased by 0.04, the average relative expression level of MCT4 decreased by 0.54, and a downward tendency was observed in the majority of the target genes. This tendency was the same as the responsiveness to the drug observed in the aged cognitively impaired mice shown in Example 1.
  • Example 5 Volunteer A took 4 g of the drug and observed changes in gene expression before and after taking the drug. Blood was collected before and 2 hours after administration, and changes in the relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene were observed.
  • RNA isolation PAXgene (registered trademark) RNA collection tube
  • the blood collection tube was gently inverted and mixed, and then allowed to stand at room temperature to obtain total RNA derived from the peripheral blood (whole blood) of the volunteer donor.
  • Total RNA was frozen and stored at ⁇ 20 to ⁇ 80 ° C.
  • cDNA synthesis kit PrimeScript (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210B (A ⁇ 4)) according to the manufacturer's protocol.
  • CDNA was synthesized (reverse transcription) from the total RNA of.
  • a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)).
  • a nucleic acid amplification reagent PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)
  • Agilent AliaMx Real-Time PCR System Agilent AliaMx Real-Time PCR System (Agilent) was used.
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C.
  • the 18S ribosomal RNA gene which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method.
  • the Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration.
  • Nucleic Acids Res. 2001; 29 (9): e45 The method described in can be used. Table 2 shows the base sequences of primers for each gene.

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Abstract

La présente invention concerne un procédé de criblage de substances candidates ayant la possibilité d'être efficace dans la thérapie de la démence, et un procédé de sélection d'un sujet animal humain ou non humain à traiter avec une substance active pour ladites thérapie.　Le procédé de criblage comprend l'incubation de cellules à partir d'un fluide corporel d'un être humain ou d'un animal non humain avec une substance d'essai, et, lorsque l'expression génique ou l'activité enzymatique d'au moins une protéine impliquée dans des réactions de métabolisme énergétique est supprimée, déterminer que la substance d'essai est une substance candidate. Le procédé de sélection de l'individu humain ou animal non humain sujet comprend l'incubation de cellules provenant d'un fluide corporel d'un individu animal testé avec la substance active, et, lorsque l'expression génique ou l'activité enzymatique d'au moins une protéine impliquée dans les réactions du métabolisme énergétique est supprimée, la détermination que l'individu testé est un individu sujet.
PCT/JP2021/008723 2020-03-06 2021-03-05 Procédé de criblage WO2021177448A1 (fr)

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