WO2008013324A1 - Use of diabetes-related, liver-derived secreted protein in diagnosis or treatment of type-2 diabetes or vascular disorder - Google Patents

Use of diabetes-related, liver-derived secreted protein in diagnosis or treatment of type-2 diabetes or vascular disorder Download PDF

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Publication number
WO2008013324A1
WO2008013324A1 PCT/JP2007/065221 JP2007065221W WO2008013324A1 WO 2008013324 A1 WO2008013324 A1 WO 2008013324A1 JP 2007065221 W JP2007065221 W JP 2007065221W WO 2008013324 A1 WO2008013324 A1 WO 2008013324A1
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selenoprotein
diabetes
type
vascular
vascular disorder
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PCT/JP2007/065221
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French (fr)
Japanese (ja)
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Shuichi Kaneko
Toshinari Takamura
Hirofumi Misu
Nobuyuki Takakura
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National University Corporation Kanazawa University
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Priority to JP2008526853A priority Critical patent/JP5299900B2/en
Publication of WO2008013324A1 publication Critical patent/WO2008013324A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • Diabetes-related liver-derived secretory protein used for diagnosis or treatment of type 2 diabetes or vascular disorders
  • the present invention relates to the use of selenoprotein P for the diagnosis and treatment of type 2 diabetes or vascular disorders.
  • Type 2 diabetes which continues to increase on a global scale, threatens human Q0L and life by promoting arteriosclerotic diseases such as retinal, renal, and neurological complications and ischemic heart disease.
  • arteriosclerotic diseases such as retinal, renal, and neurological complications and ischemic heart disease.
  • the development of treatment is urgent.
  • the liver not only plays a major role in glucose and lipid metabolism, but is also the largest in-vivo producing organ of various physiologically active substances such as angiogenic factors. There is a possibility that many are produced.
  • insulin resistance see Non-Patent Documents 1 and 2). Insulin resistance leads to hyperglycemia due to increased glucose release from the liver and hyperlipidemia due to increased lipid production, both of which promote arteriosclerotic diseases.
  • the liver is the largest organ that produces various biologically active substances, including angiogenic factors that lead to the risk of arteriosclerosis.
  • Non-Patent Document 1 Michael MD. Et al., Mol. Cel l 6: 87-97, 2000
  • Non-Patent Document 2 Salt iel AR. Et al., Ature 414: 799-806, 2001
  • the present invention relates to selenoprotein P which is a secretory protein derived from diabetes-related liver.
  • a method for assessing the risk of suffering from a disorder, and selenoprote The purpose is to provide markers for detecting vascular disorders such as diabetes and arteriosclerotic diseases, and markers for assessing the risk of vascular disorders such as type 2 diabetes and arteriosclerotic diseases. .
  • the present invention relates to a method for screening a medicament for preventing or treating vascular disorders such as type 2 diabetes and arteriosclerotic diseases using selenoprotein P, and the prevention or treatment of vascular disorders containing selenoprotein P as an active ingredient.
  • the purpose is to provide medicine.
  • liver biopsy samples from more than 100 diabetic / metabolic syndrome patients, and comprehensively analyzed liver expression genes exceeding 600,000 genes using DNA chips and the SAGE method. I have done it. From these liver-expressed gene information, 62 secreted protein groups that correlate with liver gene expression and clinical indicators of patients such as glycemic control, BMI, insulin resistance, etc. were identified (Gyoen & Tsuji Kaneko et al. Genes whose expression varies in relation to the Japanese patent application, Japanese Patent Application No. 2005-125689).
  • the present inventors conducted further diligent studies on a diabetes-related liver-derived secreted protein.
  • the present inventors identified selenoprotein P as a secretory protein in which liver gene expression correlates with insulin resistance and blood glucose level 2 hours after glucose load from a comprehensive gene expression profile in the liver of type 2 diabetic patients.
  • the function was analyzed. As a result, it was found that administration of selenoprotein P to mice induces systemic insulin resistance and induces hyperglycemia after glucose loading. As a mechanism, the insulin signal in hepatocytes is attenuated and the liver is decreased. It was shown to increase sugar release from cells.
  • studies using mouse fetal organ cultures have revealed that selenoprotein P suppresses the proliferation of vascular endothelial cells and strongly induces the proliferation of vascular wall cells.
  • type 2 diabetes can be diagnosed by using selenoprotein P expression as an index because the expression of selenoprotein P gene is associated with the pathology of type 2 diabetes.
  • selenoprotein P greatly affects the action of insulin. Therefore, we found that controlling the expression of selenoprotein P can control the pathology of type 2 diabetes.
  • selenoprotein P affects the generation and proliferation of vascular cells, it has been found that it can be used for the treatment of vascular disorders, and the present invention has been completed.
  • the present invention is as follows.
  • [I] A method for detecting type 2 diabetes or vascular disorder, comprising measuring selenoprotein P.
  • a method for assessing the risk of suffering from type 2 diabetes or vascular disorders comprising measuring selenoprotein P.
  • a method for evaluating a subject's insulin resistance or glycemic control comprising measuring selenoprotein P.
  • a method for evaluating insulin resistance in patients with type 2 diabetes comprising measuring selenoprotein P.
  • a method for assessing the risk of developing type 2 glycouremia in a person with normal glucose tolerance comprising measuring selenoprotein P.
  • a marker for detecting type 2 diabetes or vascular disorder comprising selenoprotein P.
  • a marker for assessing the risk of morbidity of type 2 diabetes or vascular disorders consisting of selenoprotein P.
  • [1 4] Use of selenoprotein P as a marker for evaluation of insulin resistance or glycemic control.
  • a method for screening for preventive or therapeutic agents for type 2 diabetes or vascular disorders, which is selected as a prophylactic or therapeutic agent based on the ability to suppress the expression or action of the candidate compound selenoprotein P A screening method comprising:
  • a preventive or therapeutic agent for a disease associated with proliferation of vascular endothelial cells or decreased proliferation of vascular wall cells comprising selenoprotein P or a nucleic acid encoding selenoprotein P as an active ingredient.
  • a preventive or therapeutic agent for type 2 diabetes or vascular disorder comprising anti-selenoprotein P antibody as an active ingredient.
  • a prophylactic or therapeutic agent for type 2 diabetes or vascular disorder comprising, as an active ingredient, a double-stranded RNA that suppresses the expression of a gene encoding selenoprotein P.
  • Sense RNA consisting of a sequence homologous to a base sequence consisting of 15 to 50 bases of the base sequence encoding selenoprotein P shown in SEQ ID NO: 1 and complementary to the sense RNA Double strand consisting of antisense RNA consisting of sequence
  • Figure 1 shows the relationship between selenoprotein P and insulin resistance and arteriosclerosis.
  • FIG. 2 shows a vicious circle associated with hyperglycemia and hyperselenoproteinemia.
  • Fig. 3 shows the correlation between selenoprotein P gene expression in human liver and MCR.
  • Figure 4 shows selenoprotein P gene expression in human liver and blood glucose level for 2 hours after glucose loading. It is a figure which shows correlation of these.
  • FIG. 5 shows the effect of insulin administration on selenoprotein P gene expression in hepatocytes.
  • FIG. 6 is a graph showing the effect of glucose administration on selenoprotein P gene expression in hepatocytes.
  • FIG. 7A shows the effect of selenoprotein P on glucose release from hepatocytes.
  • Figure 7B shows the effect of selenoprotein P on glucose release from hepatocytes (part 2).
  • FIG. 8 shows the effect of selenoprotein P on the phosphorylation of Akt by insulin administration in hepatocytes.
  • FIG. 8A is a photograph showing the results of SDS-PAGE
  • FIG. 8B is a graph showing the strength of effect under each condition.
  • Figure 9 is a photograph showing the effect of selenoprotein P on fetal mouse angiogenesis.
  • Figure 9A shows the negative control
  • Figure 9B shows the results when selenoprotein P was administered at 5 Z g / mL
  • Figure 9C shows the results when selenoprotein P was administered at 25 // g / mL.
  • FIG. 10 is a graph showing the results of a glucose tolerance test on mice subjected to selenoprotein P administration.
  • FIG. 11 is a diagram showing the results of an insulin tolerance test on mice subjected to selenoprotein P administration.
  • Figure 12 shows the correlation between serum selenoprotein P concentration and BMI / QUICKI in patients with type 2 diabetes.
  • Fig. 12 A shows the correlation with BMI
  • Fig. 12 B shows the correlation with QUICKI.
  • FIG. 13 is a diagram showing the results of comparison of serum selenoprotein concentrations with and without obesity in patients with type 2 diabetes.
  • FIG. 14 is a graph showing the correlation between serum selenoprotein P concentration and body weight ′ H0MA-IR in normal glucose tolerance individuals.
  • Fig. 14 A shows the correlation with body weight
  • Fig. 14 B shows the correlation with H0MA-IR.
  • Figure 15 shows the rapid administration of hepatic selenoprotein P gene expression s iRNA to KKAy mice injected with selenoprotein P s iRNA, and RNA from the liver 7 days later. It is a figure which shows the extracted result.
  • the gene expression level was measured by Realtime PCR. ⁇ p is 0.05.
  • Figure 16 is a photograph showing blood SeP protein expression in KKAy mice injected with siRNA against selenoprotein P. siRNA was rapidly administered to mice, and blood was collected 7 days later. Selenoprotein P protein expression was evaluated by Western blotting.
  • FIG. 17 shows fasting blood glucose levels in KKAy mice injected with siRNA against selenoprotein P.
  • Selenoprotein P (Selenoprotein P, SeP) is a protein containing 10 residues of selenocystine. Selenoprotein P acts as an enzyme with dartathoperoxidase-like activity that reduces hydrogen peroxide and lipid peroxides to detoxify and controls intracellular redox.
  • SEQ ID NO: 1 shows the nucleotide sequence of DNA encoding selenoprotein P
  • SEQ ID NO: 2 shows the amino acid sequence of selenoprotein P.
  • the selenoprotein P used in the present invention can be chemically synthesized based on the above sequence information, or can be obtained as a recombinant protein using a gene recombination technique.
  • Selenoprotein is also contained in human serum and can be isolated and purified from human serum according to the method described in Saito et al., J Biol Chem 274: 2866-2871, 1999. it can.
  • Selenoprotein P used in the present invention has the amino acid sequence shown in SEQ ID NO: 2. And a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having the above-mentioned daltathione peroxidase-like activity.
  • 1 or several is 1 to 9, preferably 1 to 5, and more preferably 1 or 2. Also excluded are those lacking any of the 10-residue selenocystines.
  • a fragment of selenophine tin P can also be used.
  • the fragment of selenoprotein p is not limited, and any fragment of selenoprotein P can be used as the best.
  • Preferred is a fragment peptide consisting of a partial partial sequence of the amino acid sequence of selenoprotein P, comprising a partial sequence consisting of 10 or more amino acids, preferably 15 or more, and more preferably 20 or more.
  • a fragment in the case of using selenoprotein P as a medicine is a fragment peptide having the above-described daltathione peroxidase-like activity. Examples of such selenoprotein P fragments include the C-terminal fragment of selenoprotein P.
  • the fragment which consists of is mentioned.
  • a fragment peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having the above-described dartathione peroxidase-like activity is also included.
  • Blood selenoprotein P levels reflect systemic insulin resistance and the risk of developing vascular disorders.
  • Selenoprotein P can be used as a marker for detecting diseases such as type 2 diabetes and vascular disorders.
  • Vascular disorders include arteriosclerotic diseases such as myocardial infarction, cerebral infarction, and obstructive arteriosclerosis.
  • arteriosclerotic diseases such as myocardial infarction, cerebral infarction, and obstructive arteriosclerosis.
  • insulin resistance or glycemic control can be assessed.
  • it can be a predictive marker for new insulin resistance in patients with type 2 diabetes, and a predictive marker for the onset of type 2 diabetes in patients with normal glucose tolerance.
  • selenoprotein P when selenoprotein P is high, it can be evaluated that the sensitivity to insulin is attenuated. For those with normal glucose tolerance When selenoprotein P is high, it can be judged that the risk of developing type 2 diabetes is high.
  • Selenoprotein P levels are also associated with obesity and can be a criterion for complications of obesity. That is, when selenoprotein P is high, it can be determined that the patient suffers from obesity or obesity complications. Furthermore, if selenoprotein P is high, it can be evaluated and determined that insulin resistance is increased due to obesity.
  • selenoprotein p When using selenoprotein p as a marker, selenoprotein p may be detected in a sample such as blood of a subject.
  • a sample such as blood of a subject.
  • whole blood, serum or plasma can be used as the specimen sample.
  • selenoprotein P may be detected directly, or the expression of selenoprotein P may be detected by detecting selenoprotein P mRNA.
  • an immunoassay method using an anti-selenoprotein P antibody that specifically recognizes and binds to selenoprotein P can be used.
  • Anti-selenoprotein P antibody can be prepared by a known method. Examples of the immunoassay include a method using a carrier on which an anti-selenobrotin P antibody is immobilized, Western blotting, and the like.
  • Examples of the method using a solid-phased carrier include ELISA using a solid-phased microtiter plate and agglutination method using solid-phased particles, but are not limited thereto, and known immunological measurement methods Can be used to detect serum selenoprotein P.
  • Selenoprotein P mRNA can be detected by Northern blotting, RT-PCR, DNA chip (DNA microarray) and other methods. These methods can also be performed by known methods.
  • selenoprotein P is upregulated in the liver of subjects suffering from diseases such as type 2 diabetes, it may be possible to detect the expression of selenoprotein P in the liver.
  • Detect or diagnose whether a subject suffers from type 2 diabetes or vascular disorder by measuring selenoprotein P in a sample collected from the subject or measuring the expression of selenoprotein P And the risk of the subject suffering from type 2 diabetes and vascular disorders can be assessed.
  • subject is subject to sugar and lipid metabolism It is also possible to determine whether or not you are suffering from abnormal or diabetic complications and to assess the risk of suffering from it.
  • the subject can be diagnosed as having type 2 diabetes or vascular disorder, or can be assessed as having a high risk of suffering from type 2 diabetes or vascular disorder in the subject.
  • the pathological condition of type 2 diabetes in the subject can be determined.
  • the subject's insulin resistance and blood glucose control can be evaluated.
  • the evaluation of insulin resistance refers to the determination of whether or not a subject has insulin resistance and the evaluation of the risk of becoming insulin resistance.
  • the evaluation of the blood glucose control means an evaluation of the quality of the subject's blood glucose control or an evaluation of the risk of deterioration of the blood glucose control of the subject.
  • selenoprotein P when selenoprotein P is measured in a sample collected from a subject and the selenoprotein P concentration is higher than the selenoprotein P concentration in a normal human sample sample, or the expression of selenoprotein P in the subject If is elevated, it can be assessed that the subject is resistant to insulin or that glycemic control has deteriorated. Alternatively, it can be evaluated that the subject has a high risk of becoming insulin resistant or has a high risk of worsening glycemic control. Serum protein P concentration or the expression level of selenoprotein P in specimens of normal subjects should be measured in advance, and abnormal values of serum selenoprotein p and abnormal expression cutoff values may be set by statistical analysis. Is possible.
  • the present invention also includes a test method for evaluating the risk and a test reagent for evaluating the risk.
  • Selenoprotein P can also be used as a screening marker for evaluating the above risks.
  • the present invention further includes a method for screening a drug for preventing or treating type 2 diabetes or vascular disorder using selenoprotein P expression as an index.
  • Selenoprotein P can be a therapeutic target for type 2 diabetes and arteriosclerotic lesions.
  • In diabetics there may be a vicious circle of hyperglycemia, increased secretion of selenoprotein P from the liver, worsening systemic insulin resistance, and further hyperglycemia
  • the medicament is a medicament that suppresses the expression of selenoprotein p in the body, suppresses the secretion of selenoprotein P, or inhibits the action of selenoprotein P.
  • the medicament can attenuate insulin resistance and improve glycemic control.
  • These drugs are selenobrotin secretion inhibitors or selenoprotein attenuating drugs, and can reduce blood selenoprotein P concentration to an appropriate level.
  • the medicament is, for example, a compound that can bind to the promoter of selenoprotein P and suppress the expression and secretion of selenoprotein P.
  • the medicament is a compound that binds to a selenoprotein P receptor, inhibits selenoprotein P from binding to the receptor, and can inhibit the action of selenoprotein P.
  • Such compounds include, for example, DNAs encoding selenoprotein P and selenoprotein P promoters introduced into animal cells, etc., constructing a selenoprotein P expression system, and contacting the animal cells with the candidate compound, that is, candidate compounds Animal cells can be cultured in the presence of, and screened using the expression of selenoprotein P in the cells as an indicator. When the expression of selenoprotein P is reduced by a candidate compound, the candidate compound can be selected as a pharmaceutical agent for the prevention and treatment of type 2 diabetes and vascular disorders.
  • selenoprotein P, selenoprotein P receptor, and candidate compounds coexist, and the binding of selenoprotein P to the selenoprotein P receptor is inhibited by the trapping compound, and the action of selenoprotein P is suppressed.
  • the compound can be used as a pharmaceutical for the prevention and treatment of type 2 diabetes and vascular disorders can be selected.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising, as an active ingredient, selenoprotein P or a fragment peptide of selenoprotein P, which has a peptide having dartathione peroxidase-like activity, and a nucleic acid encoding selenoprotein P. Or a fragment nucleotide thereof having dartathione peroxidase-like activity
  • a pharmaceutical composition containing a fragment nucleotide that codes for a peptide as an active ingredient is included.
  • Selenoprotein P can suppress the proliferation of vascular endothelial cells and induce the proliferation of vascular wall cells, so it can be used for the prevention or treatment of diseases related to the proliferation of vascular endothelial cells or the decreased proliferation of vascular wall cells .
  • the pharmaceutical composition acts directly on vascular cells and can be used as a preventive or therapeutic agent for the above-mentioned diseases.
  • the present invention further includes a pharmaceutical composition comprising an elenoprotein P agonist or an antagonist of selenoprotein P as an active ingredient.
  • the selenoprotein P antagonist refers to a substance that binds to the selenoprotein P receptor and exerts a physiological activity in the cell.
  • the selenoprotein P antagonist refers to the selenoprotein P receptor.
  • Selenoprotein p agonists or antagonists can be used to prevent or treat diseases associated with vascular endothelial cell proliferation or vascular wall cell proliferation reduction.
  • diseases related to the proliferation of vascular endothelial cells or the decrease in the proliferation of vascular wall cells include arteriosclerotic diseases such as diabetic retinopathy, myocardial infarction, cerebral infarction, and obstructive arteriosclerosis.
  • arteriosclerotic diseases such as diabetic retinopathy, myocardial infarction, cerebral infarction, and obstructive arteriosclerosis.
  • selenoprotein p can suppress angiogenesis
  • selenoprotein P or selenoprotein p agonist or antagonist can also be used for the prevention or treatment of cancer.
  • the protein or fragment peptide When selenoprotein P protein or fragment peptide is used as a medicine, the protein or fragment peptide may be administered to the subject.
  • the nucleic acid or nucleotide when a nucleic acid encoding selenoprotein p or a fragment nucleotide thereof is used as a medicine, the nucleic acid or nucleotide may be administered into the body by a known gene therapy technique.
  • Methods for introducing a gene into a subject include a method using a viral vector and a method using a non-viral vector, and various methods are known (separate experimental medicine, basic technology of gene therapy, Yodosha, 1996; Separate Experimental Medicine, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997; edited by Japanese Society of Gene Therapy, Gene Therapy Development Research Handbook, NTS, 1999).
  • the present invention also includes a pharmaceutical that targets selenoprotein P and can suppress the activity of selenoprotein P or the expression of selenoprotein P.
  • These medicaments can be used as preventive or therapeutic agents for type 2 diabetes or vascular disorders.
  • Examples of such a compound that can be used as a medicament include an anti-selenoprotein P antibody capable of neutralizing the action of selenoprotein P.
  • An anti-selenoprotein P antibody capable of neutralizing the action of selenoprotein P that is, an antagonistic antibody against selenoprotein P can be prepared by a known antibody production method.
  • These antibodies are preferably genetically modified antibodies that have been artificially modified for the purpose of reducing the heterologous antigenicity to humans, such as chimeric antibodies and humanized antibodies. These modified antibodies can be produced using known methods.
  • a chimeric antibody consists of a variable region of a mammal-derived antibody other than human and a constant region derived from a human antibody.
  • a humanized antibody consists of a complementarity determining region of a mammal-derived antibody other than human, a framework region derived from a human antibody, and a C region.
  • a humanized antibody is useful as an active ingredient of the therapeutic agent of the present invention because its antigenicity in the human body is reduced.
  • a humanized antibody is also called a reshaped human antibody, and the complementarity determining region (CDR) of a mammal other than human, for example, a mouse antibody, is transplanted to the complementarity determining region of the human antibody. It can be obtained by doing. It also includes a human antibody that can be obtained by introducing a human antibody locus and administering an antigen to a transgenic animal having the ability to produce a human-derived antibody.
  • the anti-selenoprotein P antibody of the present invention is a part (partial fragment) of the anti-selenoprotein P antibody, and includes a fragment of an antibody having an action on the antigen of the antibody. Specifically, F (ab ′) 2 , Fab ′, Fab, Fv, disulfide bond Fv, single chain Fv (scFv), and polymers thereof.
  • Target selenoprotein P and can suppress the activity of selenoprotein P or can be used as drugs that can suppress the expression of selenoprotein P bind to the receptor for selenoprotein p. Also included are dominant negative mutants that do not have the action of wild-type selenoprotein P and inhibit the binding of wild-type selenoprotein P to the receptor. Furthermore, compounds that can be used as pharmaceuticals that target selenoprotein P, suppress selenoprotein P activity, or suppress selenoprotein P expression include compounds that can suppress selenoprotein P expression in the body. .
  • RNAi RNA interference
  • siRNA short interfering RNA
  • siRNA is formed from double-stranded RNA (dsRNA) by being processed by Dicer in cells or in vivo.
  • dsRNA double-stranded RNA
  • Examples of the compound include double-stranded RNA having RNAi action.
  • Double-stranded RNA consists of a sense RNA consisting of a sequence homologous to the mRNA sequence of the target gene and an antisense RNA consisting of a complementary sequence thereto.
  • the sense strand or the antisense strand may have an overhang at the 3, terminal, and the type and number of bases of the overhang are not limited, for example, 1 to 5, preferably 1 to 3, More preferred is a sequence consisting of 1 or 2 bases, such as UU and TT.
  • the term “overhang” refers to a base added to the end of one strand of the shRNA and having no base capable of complementary binding to the corresponding position of the other strand.
  • the overhang may be a base constituting DNA.
  • the double-stranded part consists of an RNA strand (sense strand) having a sequence that can hybridize to the target sequence in the sequence of the target gene whose expression is to be suppressed by RNA interference, and an RNA strand (antisense strand) complementary to the sequence.
  • RNA strand sense strand
  • antisense strand complementary to the sequence.
  • the double-stranded RNA may be a short hairpin RNA (shRNA) having a stem-loop structure in which a sense strand and an antisense strand are linked via a loop sequence.
  • the specific target sequence of the gene encoding selenoprotein P which is the target gene of the double-stranded RNA of the present invention, is a sequence in RNA corresponding to the base sequence of DNA shown in SEQ ID NO: 1, and based on the sequence information Can be set appropriately.
  • the number of bases is not limited and is selected in the range of 15 to 500 bases. Preferably 15 to 50, 15 to 45, 15 to 40, 15 to 35 or 15 to 30 bases, more preferably 20 to 35 bases, more preferably 19 to 30 bases, particularly preferably 19 to 29 bases Or 28 bases.
  • an antisense nucleic acid is mentioned as a compound which can suppress the expression of selenoprotein P in the body.
  • An antisense nucleic acid is DNA or RNA that is complementary to and hybridizes to a target gene of interest, and can suppress the expression of the target gene.
  • the antisense nucleic acid that suppresses the expression of selenoprotein P of the present invention is a nucleic acid complementary to the nucleotide sequence of DNA encoding selenoprotein P shown in SEQ ID NO: 1 or a part of the RNA sequence corresponding to the DNA sequence. It is.
  • the nucleic acid has a length of 10 to 400 nucleotides, preferably has a length of 250 or less, more preferably has a length of 100 or less, more preferably has a length force of S 50 or less, particularly preferably It is between 12 and 28 nucleotides in length.
  • Examples of the method for introducing the double-stranded RNA or antisense nucleic acid of the present invention include a hydrodynamic method, a method using canoleum ion, an electroporation method, a squib mouth plast method, a lithium acetate method, a calcium phosphate method, Examples include the ribofusion method and the microinjection method.
  • the present invention includes a pharmaceutical composition for treating or preventing type 2 diabetes or vascular disorder comprising the above compound as an active ingredient.
  • the pharmaceutical composition of the present invention comprises a selenoprotein P protein or a fragment peptide thereof, a nucleic acid encoding selenoprotein P, or a vector containing the fragment nucleotide, and a pharmacologically acceptable carrier, diluent or excipient.
  • a pharmacologically acceptable carrier diluent or excipient.
  • composition of the present invention can be administered in various forms, orally by tablets, capsules, granules, powders, syrups, etc., or injections, drops, suppositories, sprays, eye drops, Examples include parenteral administration by nasal administration and patch.
  • the pharmaceutical composition of the present invention can be administered locally.
  • the effect can be exerted by administering the composition to the liver tissue site by injection.
  • the pharmaceutical composition of the present invention contains a carrier, a diluent, and an excipient that are commonly used in the pharmaceutical field.
  • lactose and magnesium stearate are used as carriers and excipients for tablets.
  • Aqueous solutions for injection include saline, glucose and Isotonic solutions containing other adjuvants are used, and may be used in combination with appropriate solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants and the like. Sesame oil, soybean oil and the like are used as the oily liquid, and benzyl benzoate, benzyl alcohol and the like may be used in combination as the solubilizing agent.
  • oral administration is about 0.001 mg to 100 mg per day, and may be administered once or divided into several times.
  • 0.001 mg to 100 mg may be administered by subcutaneous injection, intramuscular injection, or intravenous injection.
  • selenoprotein P-encoding nucleic acid or a fragment nucleotide thereof inserted into an expression vector or the like to be translated in a subject is injected subcutaneously at a dose of 0.001 mg to 100 mg once every several days, weeks or months. It can be administered by intramuscular injection or intravenous injection.
  • administration may be carried out so that at least one copy of double-stranded RNA or antisense nucleic acid is introduced per liver cell.
  • the present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
  • Rat hematoma-derived H4I IEC cells were purchased from American Type Culture Collection (ATCC). Human-derived selenoprotein P was provided by Professor Kazuhiko Takahashi (Hokkaido Pharmaceutical University, Japan) (Sai to Y. et al., J Biol Chem 274: 2866-2871, 1999). Serenoprotein P concentration was measured using the blood-fod method with ushi serum albumin as a standard. Human recombinant insulin was purchased from Sigma Chemicals. Anti-Akt antibody and anti-serine (473) phosphorylated Akt antibody were purchased from Cel Signal Technology. The anti-CD31 monoclonal antibody was purchased from Pharmingen, and the HRP-conjugated anti-human smooth muscle actin antibody was purchased from DAKO Cytomat ion.
  • mice Female C57BL / 6J mice were purchased from Sankyo Lab Service (Japan) at the age of 8 weeks and maintained in a standard light (12 hours light / dark) and temperature environment. These mice were housed in 5 cages per group, provided water and food in ad ibi tum, and used for experiments at about 10 weeks of age.
  • Example 1 Selenoprotein P gene expression and pathology of diabetes Relationship between selenoprotein P gene expression in human liver and insulin resistance Ginsulin resistance mainly in skeletal muscles, and glucose metabol ic c Quantified as learance rate (MCR).
  • MCR reflects insulin-dependent sugar uptake into skeletal muscle, and the lower the MCR, the stronger the insulin resistance.
  • Figure 3 As shown in Fig. 3, the expression level of selenoprotein P gene in human liver quantified using a DNA chip was negatively correlated with MCR, an insulin resistance index. In other words, it was found that selenoprotein P gene expression in human liver is increased in relation to insulin resistance.
  • H4I IEC Hepatocytes (H4I IEC) on a 6-well plate Dulbecco's Mod if ied Eagle medium (DMEM)
  • Biosys tems b (Assays-on-Demand gene express ion product) 0 16s rRNA was used as a control gene for normalization. PCR conditions were 50 ° C ⁇ 2 minutes for 1 cycle, 95 ° C ⁇ 10 minutes for 1 cycle, then 95 ° C for 15 seconds and 60 ° C for 1 minute for 40 cycles. To exclude the effect of osmotic pressure, mannitol at the same concentration as glucose was used as a control.
  • Hepatocytes H4IIEC were cultured on a 6-well plate to 80% confluent in DMEM medium, 20% horse serum, 5% FBS. After washing the cells once with PBS, the cells were incubated with gluconeogenic buffer (glucose-free DMEM, 3.7 g / L sodium bicarbonate) and 0.4% FBS for 6 hours for starvation. Thereafter, glucose was added to the medium and incubated for 6 hours, and total RNA was recovered using the Quick gene system (FUJI FILM). To exclude the effect of osmotic pressure, mannitol at the same concentration as glucose was used as a control.
  • gluconeogenic buffer glucose-free DMEM, 3.7 g / L sodium bicarbonate
  • Insulin controls blood glucose by inhibiting gluconeogenesis from the liver.
  • FIGS. 7A and B The results are shown in FIGS. 7A and B.
  • insulin inhibited gluconeogenesis from hepatocytes in a dose-dependent manner.
  • insulin inhibited the hepatic gluconeogenesis inhibitory effect. This indicates that Serenob Mouth Tin P attenuates the insulin action in the liver.
  • Hepatocytes H4IIEC were cultured on a 6-well plate to 80% confluent in DMEM medium, 20% horse serum, 5% FBS. After washing the cells once with PBS, the cells were cultured with serum-free DMEM medium for 6 hours in the presence of selenoprotein P 0, 1, 5, 10 / zg / mL.
  • PVDMs polyvinylidene difluoride membranes
  • selenoprotein P inhibited serine phosphorylation of Akt in a dose-dependent manner. Since selenoprotein P concentration in normal human blood is reported to be 5.3 / ig / mL (Takahashi et al., Journal of Health Science 47, Pages 346-352, 2001), selenoprotein P is normal human blood. It was found that concentrations close to medium levels can suppress the insulin action in the liver.
  • Prolonged diabetes causes retinal, renal, neuronal microvascular disorders, and arteriosclerotic diseases such as ischemic heart disease and cerebral infarction. Therefore, we first examined the effect of selenoprotein P on the pathogenesis of these vascular complications using vascular cells in vitro. In the experiment, a mouse fetal organ culture system (Takakura et al., Cell 102: 199-20, 2000) was used.
  • the stromal cell line 0P9 (Nakano T. et al., Science 265: 1098-1101, 1994) was added to alpha— modi 1 led minimum essential media (a—MEM Gibco BR, 20% Fetal-calf serum (FCS; The fetus was removed from the 8th and 5th day of gestation, and the paraaortic mesoderm (PAS) region, which is one of the origins of vascular stem cells, was isolated from the umphalomesenteric artery.
  • a—MEM Gibco BR 20% Fetal-calf serum
  • FCS Fetal-calf serum
  • Selenoprotein P 1 ⁇ g / gBW or vehicle was administered intraperitoneally twice a day at 7 am and 7 pm to five 10-week-old female C57BL / 6J mice per group. Mice were fasted after the first injection. Two hours after the second injection, 1.5 mg / gBW of glucose was intraperitoneally administered, and blood glucose levels were measured from 0 to 120 minutes. Blood glucose levels were measured using FreeStyle (Kitssey Japan).
  • P 1 ⁇ g / gBW or vehicle was administered intraperitoneally twice at 7 am and 7 pm. Mice were fasted 2 hours before the second injection. After a total of 4 hours of fasting, 0.5 mU / gBW human insulin (Humalin R (registered trademark), Lilly Japan) was administered intraperitoneally, and blood glucose levels were measured from 0 to 120 minutes. Blood glucose levels were measured using FreeStyle (Kitssey, Japan).
  • Insulin resistance in patients with normal glucose tolerance was evaluated by calculating the homeostasis model for insulin resistance (H0MA—IR) 1 as follows (Matthews DR et al., Diabetologia 28: 412-419, 1985). .
  • Insulin resistance in patients with type 2 diabetes was evaluated by calculating quantitative insulin sensitivity check index (QUICKI) as follows (Arie Katz et al., J Clin Endocrinol Metab 85: 2402-2410).
  • H0MA- IR Fasting blood glucose level (mg / dL)
  • X Fasting blood insulin concentration (// U / ml) / 405
  • QUICKI l / [log Fasting blood insulin concentration ( ⁇ U / ml) + log Fasting blood glucose level (mg / dL)]
  • the mean concentration was 5.28 ⁇ 1.3 ⁇ g / mL.
  • the patient's selenoprotein P level was positively correlated with BMI and negatively correlated with QUICKI (Figs. 12 and 3 ⁇ 4).
  • Serum selenoprotein P concentrations in patients with type 2 diabetes were compared and examined with and without obesity.
  • serum selenoprotein P level is a clinical marker that strongly reflects obesity and insulin resistance.
  • a correlation between serum selenoprotein P levels and obesity / insulin resistance was observed in both type 2 diabetics and normal glucose tolerance.
  • selenoprotein P is considered to be one of the insulin resistance-causing hormones. Therefore, human serum selenoprotein P level measurement is a novel marker for evaluating insulin resistance in patients with type 2 diabetes. In addition, it can be a predictive marker for the onset of type 2 diabetes in patients with normal glucose tolerance.
  • selenoprotein P is one of the hormones that cause insulin resistance and hyperglycemia in type 2 diabetes. Therefore, we examined whether suppression of selenoprotein P production in the liver using short interference RNA (siRNA) could be a new treatment for type 2 diabetes.
  • siRNA short interference RNA
  • siRNA specific to selenoprotein P was introduced using the hydrodynamic method. Zender, L. et al. Proc Natl Acad Sci U S A 100, 7797-802
  • SiRNA for animal introduction was purchased from Amb ion (Silencer® In
  • mice Seppl 5 '-GGUGUCAGAACACAUCGCAtt-3'
  • Negative control siRNA (sense) (The sequence of self-excluding tt is shown in SEQ ID NO: 3).
  • Negative control siRNA was purchased from Amb ion. The negative control siRNA does not have significant homology to any known gene in mouse, rat or human. After general anesthesia of the mice with Bent Barbitool, 2 nmol of siRNA was dissolved in 3. OmL of PBS and administered via the tail vein for about 15-20 seconds. s A 12-hour post-fasting glucose tolerance test and an insulin tolerance test were performed 2-7 days after siRNA administration. 0. 3 mg / g body weight of gnolecose or 4 U / kg body wei ght of the engine was administered intrathecally and blood glucose was measured 15, 30, 60 and 120 minutes later.
  • liver-derived selenoprotein P is an important therapeutic target for type 2 diabetes. Furthermore, it has been shown that liver-derived selenoprotein P may be a therapeutic target for other insulin resistance-related diseases such as arteriosclerosis and metabolic syndrome.
  • Selenoprotein P is one of the causative hormones for insulin resistance and vascular scab damage, and measurement of selenoprotein P concentration is useful as a new clinical marker that reflects the risk of systemic insulin resistance or the development of vascular disorders. is there. Using selenoprotein P as a marker, it is possible to determine the pathology of diseases such as type 2 diabetes and arteriosclerotic diseases.
  • drugs containing these selenoprotein P or selenoprotein P inhibitory therapy can be a breakthrough treatment for vascular disorders.
  • selenoprotein P may be a therapeutic target for type 2 diabetes and atherosclerotic lesions. Particularly in diabetic patients, there is a possibility that a vicious circle of hyperglycemia, increased secretion of selenoprotein P from the liver, deterioration of insulin resistance throughout the body, and further hyperglycemia may occur. Can be dramatically improved in the pathology associated with glycotoxicity.

Abstract

Disclosed is use of selenoprotein P in the diagnosis or treatment of type-2 diabetes or a vascular disorder. Specifically disclosed are: a method for detection of type-2 diabetes or a vascular disorder, comprising measuring selenoprotein P; a method for evaluation of the risk of being suffering from type-2 diabetes or a vascular disorder; a method for evaluation of the insulin resistance or blood glucose control in a subject; and a therapeutic agent for type-2 diabetes or a vascular disorder which is targeted to selenoprotein P.

Description

糖尿病関連肝臓由来分泌タンパク質の 2型糖尿病または血管障害の診断または治 療への利用 技術分野 Diabetes-related liver-derived secretory protein used for diagnosis or treatment of type 2 diabetes or vascular disorders
本発明は、 セレノプロテイン Pの 2型糖尿病または血管障害の診断、 治療への 利用に関する。 明 背景技術  The present invention relates to the use of selenoprotein P for the diagnosis and treatment of type 2 diabetes or vascular disorders. Background art
書  book
世界規模で増加し続ける 2型糖尿病は、 網膜 ·腎 ·神経の合併症ゃ虚血性心疾 患などの動脈硬化性疾患を促進することで人類の Q0Lと生命を脅かしており、 そ の新たな治療法の開発は急務である。 肝臓は糖 ·脂質代謝の主役を担うだけでは なく、 血管新生因子をはじめとする各種生理活性物質の生体内最大の産生臓器で あることから、 2型糖尿病の病靡を形作る未知の分泌蛋白を多数産生している可 能性がある。 2型糖尿病では、 インスリンによる肝臓からの糖放出抑制作用は減 弱しており、 この現象はインスリン抵抗性と呼ばれる （非特許文献 1および 2を 参照）。インスリン抵抗性は肝臓からの糖放出亢進による高血糖と、脂質の産生亢 進による高脂血症をもたらし、 ともに動脈硬化性疾患を促進する。 さらに肝臓は 動脈硬化のリスクにつながる血管新生因子をはじめとする各種生理活性物質の生 体内最大の産生臓器である。  Type 2 diabetes, which continues to increase on a global scale, threatens human Q0L and life by promoting arteriosclerotic diseases such as retinal, renal, and neurological complications and ischemic heart disease. The development of treatment is urgent. The liver not only plays a major role in glucose and lipid metabolism, but is also the largest in-vivo producing organ of various physiologically active substances such as angiogenic factors. There is a possibility that many are produced. In type 2 diabetes, the action of insulin to suppress glucose release from the liver is attenuated, and this phenomenon is called insulin resistance (see Non-Patent Documents 1 and 2). Insulin resistance leads to hyperglycemia due to increased glucose release from the liver and hyperlipidemia due to increased lipid production, both of which promote arteriosclerotic diseases. Furthermore, the liver is the largest organ that produces various biologically active substances, including angiogenic factors that lead to the risk of arteriosclerosis.
非特許文献 1 Michael MD. et al. , Mol. Cel l 6 : 87-97, 2000  Non-Patent Document 1 Michael MD. Et al., Mol. Cel l 6: 87-97, 2000
非特許文献 2 Salt iel AR. et al. , ature 414 : 799-806， 2001  Non-Patent Document 2 Salt iel AR. Et al., Ature 414: 799-806, 2001
発明の開示 Disclosure of the invention
本発明は、 糖尿病関連肝臓由来分泌タンパク質であるセレノプロテイン P The present invention relates to selenoprotein P which is a secretory protein derived from diabetes-related liver.
(Selenoprotein P) を利用して 2型糖尿病や心筋梗塞、 脳梗塞、 閉塞性動脈硬化 症などの動脈硬化性疾患等の血管障害を検出する方法および 2型糖尿病や動脈硬 化性疾患等の血管障害に罹患するリスクを評価する方法、 ならびにセレノプロテ イン Pからなる糖尿病や動脈硬化性疾患等の血管障害を検出するためのマーカー および 2型糖尿病や動脈硬化性疾患等の血管障害に罹患するリスクを評価するた めのマーカーの提供を目的とする。 (Selenoprotein P) to detect vascular disorders such as type 2 diabetes, myocardial infarction, cerebral infarction, arteriosclerotic diseases such as obstructive arteriosclerosis, and blood vessels such as type 2 diabetes and arteriosclerotic diseases A method for assessing the risk of suffering from a disorder, and selenoprote The purpose is to provide markers for detecting vascular disorders such as diabetes and arteriosclerotic diseases, and markers for assessing the risk of vascular disorders such as type 2 diabetes and arteriosclerotic diseases. .
さらに、 本発明はセレノプロテイン Pを用いた 2型糖尿病や動脈硬化性疾患等 の血管障害を予防または治療する医薬のスクリ一二ング方法、 セレノプロティン Pを有効成分として含む血管障害の予防または治療薬の提供を目的とする。  Furthermore, the present invention relates to a method for screening a medicament for preventing or treating vascular disorders such as type 2 diabetes and arteriosclerotic diseases using selenoprotein P, and the prevention or treatment of vascular disorders containing selenoprotein P as an active ingredient. The purpose is to provide medicine.
本発明者らは、 100 例を超える糖尿病 ·代謝症候群患者の肝生検サンプルを前 向きに蓄積し、 DNAチップおよび SAGE法を用いて 60万遺伝子を超える肝発現遺 伝子を包括的に解析してきた。 これらの肝発現遺伝子情報から、 血糖コントロー ル、 BMI、 インスリン抵抗性等の患者の臨床指標と肝遺伝子発現が相関する 62種 の分泌蛋白群を同定した（御簾 ·篁 ·金子ら、 糖尿病の病態と関連して発現変動す る遺伝子群、 日本国特許出願 2005-125689号）。  We have prospectively accumulated liver biopsy samples from more than 100 diabetic / metabolic syndrome patients, and comprehensively analyzed liver expression genes exceeding 600,000 genes using DNA chips and the SAGE method. I have done it. From these liver-expressed gene information, 62 secreted protein groups that correlate with liver gene expression and clinical indicators of patients such as glycemic control, BMI, insulin resistance, etc. were identified (Gyoen & Tsuji Kaneko et al. Genes whose expression varies in relation to the Japanese patent application, Japanese Patent Application No. 2005-125689).
本発明者らは、 糖尿病関連肝臓由来分泌タンパク質についてさらに鋭意検討を 行った。  The present inventors conducted further diligent studies on a diabetes-related liver-derived secreted protein.
本発明者らは、 2型糖尿病患者の肝臓での包括的遺伝子発現プロフアイルから、 インスリン抵抗性と糖負荷後 2時間血糖値に肝遺伝子発現が相関する分泌タンパ クとしてセレノプロテイン Pを同定し、 その機能解析を行った。 その結果、 マウ スへのセレノプロテイン P投与が全身でのィンスリン抵抗性を誘導し糖負荷後の 高血糖を惹起することを見出し、 その機序として、 肝細胞でのインスリンシグナ ルを減弱させ肝細胞からの糖放出を増加させることを明らかにした。 加えて、 マ ウス胎仔器官培養を用いた検討から、 セレノプロテイン Pは血管内皮細胞の増殖 を抑制し、 血管壁細胞の増殖を強力に誘導することを明らかにした。 以上の結果 から、 2型糖尿病患者あるいはィンスリン抵抗性を有する肥満患者においては、 肝臓からのセレノプロテイン P分泌が亢進し、 その結果全身でのインスリン抵抗 性および食後高血糖が誘導され、 さらには血管内皮細胞の増殖低下が生じること で、 血管障害を進行させている可能性が示唆された （図 1 )。  The present inventors identified selenoprotein P as a secretory protein in which liver gene expression correlates with insulin resistance and blood glucose level 2 hours after glucose load from a comprehensive gene expression profile in the liver of type 2 diabetic patients. The function was analyzed. As a result, it was found that administration of selenoprotein P to mice induces systemic insulin resistance and induces hyperglycemia after glucose loading. As a mechanism, the insulin signal in hepatocytes is attenuated and the liver is decreased. It was shown to increase sugar release from cells. In addition, studies using mouse fetal organ cultures have revealed that selenoprotein P suppresses the proliferation of vascular endothelial cells and strongly induces the proliferation of vascular wall cells. Based on the above results, in patients with type 2 diabetes or obesity patients with insulin resistance, selenoprotein P secretion from the liver is increased, resulting in systemic insulin resistance and postprandial hyperglycemia, and further It was suggested that the decrease in the proliferation of endothelial cells may have caused the progression of vascular injury (Fig. 1).
本発明者らは、 セレノプロテイン P遺伝子の発現が 2型糖尿病の病態と関連し ていることから、 セレノプロテイン Pの発現を指標に 2型糖尿病の診断を行うこ とができることを見出した。 また、 セレノプロテイン Pがインスリンの作用に大 きく影響することから、 セレノプロテイン Pの発現を制御することにより 2型糖 尿病の病態を制御し得ることを見出した。 さらに、 セレノプロテイン Pが血管細 胞の発生および増殖に影響を及ぼすことから、 血管障害の治療に用い得ることを 見出し、 本発明を完成させるに至った。 The present inventors have found that type 2 diabetes can be diagnosed by using selenoprotein P expression as an index because the expression of selenoprotein P gene is associated with the pathology of type 2 diabetes. In addition, selenoprotein P greatly affects the action of insulin. Therefore, we found that controlling the expression of selenoprotein P can control the pathology of type 2 diabetes. Furthermore, since selenoprotein P affects the generation and proliferation of vascular cells, it has been found that it can be used for the treatment of vascular disorders, and the present invention has been completed.
すなわち、 本発明は以下の通りである。  That is, the present invention is as follows.
[I] セレノプロテイン Pを測定することを含む、 2型糖尿病または血管障害の 検出方法。  [I] A method for detecting type 2 diabetes or vascular disorder, comprising measuring selenoprotein P.
[2] セレノプロテイン Pを測定することを含む、 2型糖尿病または血管障害に 罹患するリスクを評価する方法。  [2] A method for assessing the risk of suffering from type 2 diabetes or vascular disorders, comprising measuring selenoprotein P.
[3] 血管障害が動脈硬化性疾患である [1 ]または [2]の方法。  [3] The method of [1] or [2], wherein the vascular disorder is an arteriosclerotic disease.
[4] セレノプロテイン Pを測定することを含む、 被験体のインスリン抵抗性ま たは血糖コントロールを評価する方法。  [4] A method for evaluating a subject's insulin resistance or glycemic control, comprising measuring selenoprotein P.
[5] セレノプロテイン Pを測定することを含む、 2型糖尿病患者におけるイン スリン抵抗性を評価する方法。  [5] A method for evaluating insulin resistance in patients with type 2 diabetes, comprising measuring selenoprotein P.
[6] セレノプロテイン Pを測定することを含む、 耐糖能正常者における 2型糖 尿病発症のリスクを評価する方法。  [6] A method for assessing the risk of developing type 2 glycouremia in a person with normal glucose tolerance, comprising measuring selenoprotein P.
[7] セレノプロテイン Pからなる、 2型糖尿病または血管障害検出用マーカー。  [7] A marker for detecting type 2 diabetes or vascular disorder, comprising selenoprotein P.
[8] セレノプロテイン Pからなる、 2型糖尿病または血管障害の罹患リスク評 価用マーカ一。 [8] A marker for assessing the risk of morbidity of type 2 diabetes or vascular disorders, consisting of selenoprotein P.
[9] 血管障害が動脈硬化性疾患である [7]または [8]のマーカー。  [9] The marker according to [7] or [8], wherein the vascular disorder is arteriosclerotic disease.
[1 0] セレノプロテイン Pからなる、 インスリン抵抗性または血糖コントロー ル評価用マ一力一。  [1 0] Consisting of selenoprotein P, the best for insulin resistance or glycemic control evaluation.
[I I] セレノプロテイン Pの、 2型糖尿病または血管障害検出用マーカーとし ての使用。  [I I] Use of selenoprotein P as a marker for detecting type 2 diabetes or vascular disorders.
[1 2] セレノプロテイン Pの、 2型糖尿病または血管障害の罹患リスク評価用 マーカーとしての使用。  [1 2] Use of selenoprotein P as a marker for risk assessment of type 2 diabetes or vascular disorder.
[1 3] 血管障害が動脈硬化性疾患である [1 1 ]または [1 2]の使用。  [1 3] Use of [1 1] or [1 2], wherein the vascular disorder is atherosclerotic disease.
[1 4] セレノプロテイン Pの、 インスリ ン抵抗性または血糖コントロール評価 用マーカーとしての使用。 [1 5] 2型糖尿病または血管障害の予防または治療薬をスクリーニングする方 法であって、 候補化合物のセレノプロティン Pの発現または作用を抑制する能力 を指標に、 予防または治療薬として選択することを含むスクリーニング方法。 [1 4] Use of selenoprotein P as a marker for evaluation of insulin resistance or glycemic control. [1 5] A method for screening for preventive or therapeutic agents for type 2 diabetes or vascular disorders, which is selected as a prophylactic or therapeutic agent based on the ability to suppress the expression or action of the candidate compound selenoprotein P A screening method comprising:
[1 6] 血管障害が動脈硬化性疾患である [ 1 5]のスクリーニング方法。 [1 6] The screening method according to [15], wherein the vascular disorder is atherosclerotic disease.
[1 7] セレノプロテイン Pまたはセレノプロティン Pをコードする核酸を有効 成分として含む血管内皮細胞の増殖または血管壁細胞の増殖低下に関連する疾患 の予防または治療剤。 [17] A preventive or therapeutic agent for a disease associated with proliferation of vascular endothelial cells or decreased proliferation of vascular wall cells, comprising selenoprotein P or a nucleic acid encoding selenoprotein P as an active ingredient.
[1 8] 血管内皮細胞の増殖または血管壁細胞の増殖低下に関連する疾患が糖尿 病性網膜症、 動脈硬化性疾患または癌である [1 7]の予防または治療剤。  [1 8] The prophylactic or therapeutic agent for [1 7], wherein the disease associated with proliferation of vascular endothelial cells or decreased proliferation of vascular wall cells is diabetic retinopathy, arteriosclerotic disease or cancer.
[1 9] 抗セレノプロテイン P抗体を有効成分として含む 2型糖尿病または血管 障害の予防または治療剤。 [1 9] A preventive or therapeutic agent for type 2 diabetes or vascular disorder comprising anti-selenoprotein P antibody as an active ingredient.
[20] セレノプロテイン Pをコードする遺伝子の発現を抑制する 2本鎖 RNAを 有効成分として含む 2型糖尿病または血管障害の予防または治療剤。  [20] A prophylactic or therapeutic agent for type 2 diabetes or vascular disorder comprising, as an active ingredient, a double-stranded RNA that suppresses the expression of a gene encoding selenoprotein P.
[2 1 ] 2本鎖 RNAが、 配列番号 1に示すセレノプロテイン Pをコードする塩基 配列の連続する 15〜50 塩基からなる塩基配列と相同な配列からなるセンス RNA および該センス RNA に相補的な配列からなるアンチセンス RNA からなる 2本鎖 [2 1] Sense RNA consisting of a sequence homologous to a base sequence consisting of 15 to 50 bases of the base sequence encoding selenoprotein P shown in SEQ ID NO: 1 and complementary to the sense RNA Double strand consisting of antisense RNA consisting of sequence
RNAである、 [20]の 2型糖尿病または血管障害の予防または治療剤。 The preventive or therapeutic agent for type 2 diabetes or vascular disorder according to [20], which is RNA.
[22] センス RNAが配列番号 3で示される塩基配列からなる、 [2 1]の 2型糖 尿病または血管障害の予防または治療剤。  [22] The preventive or therapeutic agent for type 2 glycouric disease or vascular disorder according to [2 1], wherein the sense RNA comprises the base sequence represented by SEQ ID NO: 3.
本明細書は本願の優先権の基礎である日本国特許出願 2006-206747号の明細書 および/または図面に記載される内容を包含する。 図面の簡単な説明  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2006-206747, which is the basis of the priority of the present application. Brief Description of Drawings
図 1は、 セレノプロティン Pとインスリン抵抗性および動脈硬化との関係を示 す図である。  Figure 1 shows the relationship between selenoprotein P and insulin resistance and arteriosclerosis.
図 2は、 高血糖および高セレノプロティン血症に伴う悪循環を示す図である。 図 3は、 ヒ ト肝臓でのセレノプロテイン P遺伝子発現と MCRの相関を示す図で ある。  FIG. 2 shows a vicious circle associated with hyperglycemia and hyperselenoproteinemia. Fig. 3 shows the correlation between selenoprotein P gene expression in human liver and MCR.
図 4は、 ヒ ト肝臓でのセレノプロテイン P遺伝子発現と糖負荷後 2時間血糖値 の相関を示す図である。 Figure 4 shows selenoprotein P gene expression in human liver and blood glucose level for 2 hours after glucose loading. It is a figure which shows correlation of these.
図 5は、 インスリン投与が肝細胞におけるセレノプロテイン P遺伝子発現に与 える影響を示す図である。  FIG. 5 shows the effect of insulin administration on selenoprotein P gene expression in hepatocytes.
図 6は、 グルコース投与が肝細胞におけるセレノプロティン P遺伝子発現に与 える影響を示す図である。  FIG. 6 is a graph showing the effect of glucose administration on selenoprotein P gene expression in hepatocytes.
図 7 Aは、 肝細胞からの糖放出に及ぼすセレノプロティン Pの影響を示す図で ある。  FIG. 7A shows the effect of selenoprotein P on glucose release from hepatocytes.
図 7 Bは、 肝細胞からの糖放出に及ぼすセレノプロテイン Pの影響を示す図で ある （その 2 )。  Figure 7B shows the effect of selenoprotein P on glucose release from hepatocytes (part 2).
図 8は、 肝細胞におけるィンスリン投与による Aktのリ ン酸化に及ぼすセレノ プロテイン Pの影響を示す図である。図 8 Aは SDS- PAGEの結果を示す写真であり、 図 8 Bはそれぞれの条件での発効強度を示すグラフである。  FIG. 8 shows the effect of selenoprotein P on the phosphorylation of Akt by insulin administration in hepatocytes. FIG. 8A is a photograph showing the results of SDS-PAGE, and FIG. 8B is a graph showing the strength of effect under each condition.
図 9は、 セレノプロティン Pが仔胎マウス血管新生に与える影響を示す写真で ある。図 9 Aは陰性対照、図 9 Bはセレノプロテイン Pを 5 Z g/mL投与した場合、 図 9 Cはセレノプロテイン Pを 25 // g/mL投与した場合の結果を示す。  Figure 9 is a photograph showing the effect of selenoprotein P on fetal mouse angiogenesis. Figure 9A shows the negative control, Figure 9B shows the results when selenoprotein P was administered at 5 Z g / mL, and Figure 9C shows the results when selenoprotein P was administered at 25 // g / mL.
図 1 0は、 セレノプロテイン P投与マウスに対する糖負荷試験の結果を示す図 である。  FIG. 10 is a graph showing the results of a glucose tolerance test on mice subjected to selenoprotein P administration.
図 1 1は、 セレノプロテイン P投与マウスに対するインスリン負荷試験の結果 を示す図である。  FIG. 11 is a diagram showing the results of an insulin tolerance test on mice subjected to selenoprotein P administration.
図 1 2は、 2型糖尿病患者における血清セレノプロテイン P濃度と BMI/QUICKI の相関を示す図である。 図 1 2 Aは BMI との相関、 図 1 2 Bは QUICKI との相関を 示す。  Figure 12 shows the correlation between serum selenoprotein P concentration and BMI / QUICKI in patients with type 2 diabetes. Fig. 12 A shows the correlation with BMI, and Fig. 12 B shows the correlation with QUICKI.
図 1 3は、 2型糖尿病患者における肥満の有無による血清セレノプロテイン濃 度の比較の結果を示す図である。  FIG. 13 is a diagram showing the results of comparison of serum selenoprotein concentrations with and without obesity in patients with type 2 diabetes.
図 1 4は、耐糖能正常者における血清セレノプロテイン P濃度と体重 ' H0MA-IR の相関を示す図である。 図 1 4 Aは体重との相関、図 1 4 Bは H0MA-IRとの相関を 示す。  FIG. 14 is a graph showing the correlation between serum selenoprotein P concentration and body weight ′ H0MA-IR in normal glucose tolerance individuals. Fig. 14 A shows the correlation with body weight, and Fig. 14 B shows the correlation with H0MA-IR.
図 1 5は、セレノプロテイン P s iRNAを注入した KKAyマウスにおける肝セレノ プロティン P遺伝子発現 s iRNAをマウスに急速投与し、 7日後に肝臓から RNAを 抽出した結果を示す図である。遺伝子発現量は Realtime PCR法によって測定した。 氺 pく 0. 05である。 Figure 15 shows the rapid administration of hepatic selenoprotein P gene expression s iRNA to KKAy mice injected with selenoprotein P s iRNA, and RNA from the liver 7 days later. It is a figure which shows the extracted result. The gene expression level was measured by Realtime PCR.氺 p is 0.05.
図 1 6は、セレノプロティン Pに対する s iRNAを注入した KKAyマウスにおける 血中 SePタンパク発現を示す写真である。 s iRNAをマウスに急速投与し、 7日後 に採血を施行した。 セレノプロティン Pタンパク発現は Western blotting法によ つて評価した。  Figure 16 is a photograph showing blood SeP protein expression in KKAy mice injected with siRNA against selenoprotein P. siRNA was rapidly administered to mice, and blood was collected 7 days later. Selenoprotein P protein expression was evaluated by Western blotting.
図 1 7は、セレノプロティン Pに対する s iRNAを注入した KKAyマウスにおける 空腹時血糖値を示す図である。  FIG. 17 shows fasting blood glucose levels in KKAy mice injected with siRNA against selenoprotein P.
図 1 8は、月干でのセレノプロティン Pをノックダウンした KKAyマウスにおける 糖負荷試験の結果を示す図である。 siRNAをマウスに急速投与し、 2〜7 日後に糖 負荷試験を施行した（n=6_8 per group)。 *pく 0. 05、 **pく 0. 01である。  Figure 18 shows the results of a glucose tolerance test in KKAy mice in which selenoprotein P was knocked down in the moon. SiRNA was rapidly administered to mice, and a glucose tolerance test was performed 2-7 days later (n = 6_8 per group). * p 0. 05, ** p 0. 01.
図 1 9は、肝でのセレノプロティン Pをノックダウンした KKAyマウスにおける インスリン負荷試験の結果を示す図である。 siRNA をマウスに急速投与し、 2〜7 日後にインスリン負荷試験を施行した（n=6-8 per group)。 *pく 0. 05である。 発明を実施するための最良の形態  FIG. 19 shows the results of an insulin tolerance test in KKAy mice in which selenoprotein P was knocked down in the liver. SiRNA was rapidly administered to mice, and an insulin tolerance test was performed 2 to 7 days later (n = 6-8 per group). * p is 0.05. BEST MODE FOR CARRYING OUT THE INVENTION
セレノプロテイン P (Selenoprotein P, SeP) は、 セレノシスティンを 10残基 含むタンパク質である。 セレノプロテイン Pは、 過酸化水素や過酸化脂質を還元 して無毒化し、 また細胞内の酸化還元を制御するダルタチォンペルォキシダーゼ 様活性を有する酵素として作用する。  Selenoprotein P (Selenoprotein P, SeP) is a protein containing 10 residues of selenocystine. Selenoprotein P acts as an enzyme with dartathoperoxidase-like activity that reduces hydrogen peroxide and lipid peroxides to detoxify and controls intracellular redox.
配列番号 1に、 セレノプロテイン Pをコードする DNAの塩基配列を、 配列番号 2にセレノプロティン Pのァミノ酸配列を示す。  SEQ ID NO: 1 shows the nucleotide sequence of DNA encoding selenoprotein P, and SEQ ID NO: 2 shows the amino acid sequence of selenoprotein P.
本発明において用いるセレノプロテイン Pは、 上記配列情報に基づいて、 化学 合成することができ、 また遺伝子組換え技術を利用して組換えタンパク質として 得ることができる。  The selenoprotein P used in the present invention can be chemically synthesized based on the above sequence information, or can be obtained as a recombinant protein using a gene recombination technique.
また、セレノプロテインは、ヒ ト血清中に含まれており、Saito Υ· et al . , J Biol Chem 274 : 2866 - 2871， 1999の記載の方法に従って、 ヒ ト血清より単離 .精製する ことができる。  Selenoprotein is also contained in human serum and can be isolated and purified from human serum according to the method described in Saito et al., J Biol Chem 274: 2866-2871, 1999. it can.
本発明に用いるセレノプロテイン Pは、 配列番号 2に示されるアミノ酸配列に おいて、 1個または数個のアミノ酸が欠失、 置換または付加したアミノ酸配列か らなるタンパク質であって、 上記のダルタチオンペルォキシダ一ゼ様活性を有す るタンパク質をも含む。 ここで、 1または数個とは 1〜9個、 好ましくは 1〜5 個、 さらに好ましくは 1もしくは 2個である。 また、 10残基のセレノシスティン のいずれかが欠失したものは除外される。 さらに、 本発明においては、 セレノプ 口ティン Pの断片を用いることもできる。 Selenoprotein P used in the present invention has the amino acid sequence shown in SEQ ID NO: 2. And a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having the above-mentioned daltathione peroxidase-like activity. Here, 1 or several is 1 to 9, preferably 1 to 5, and more preferably 1 or 2. Also excluded are those lacking any of the 10-residue selenocystines. Furthermore, in the present invention, a fragment of selenophine tin P can also be used.
セレノプロテイン Pを 2型糖尿病等の検出等のマーカーとして用いる場合、 セ レノプロテイン pの断片は、 限定されず、 セレノプロテイン Pの任意の断片をマ 一力一として用いることができる。 好ましくは、 セレノプロテイン Pのアミノ酸 配列の連続した一部配列であって、アミノ酸が 10個以上、好ましくは 15個以上、 さらに好ましくは 20個以上からなる一部配列からなる断片べプチドである。 一方、 セレノプロテイン Pを医薬として用いる場合の断片は、 上記のダルタチ オンペルォキシダーゼ様活性を有する断片べプチドである。 このようなセレノプ 口ティン Pの断片として、 セレノプロテイン Pの C末端側断片を挙げることがで き、 例えば、 配列番号 2に示されるアミノ酸配列の 260番目のアミノ酸から 362 番目までのアミノ酸を含む配列からなる断片が挙げられる。 また、 これらの断片 のアミノ酸配列において、 1個または数個のアミノ酸が欠失、 置換または付加し たアミノ酸配列からなる断片べプチドであって、 上記のダルタチオンペルォキシ ダーゼ様活性を有するペプチドをも含む。  When selenoprotein P is used as a marker for detection of type 2 diabetes and the like, the fragment of selenoprotein p is not limited, and any fragment of selenoprotein P can be used as the best. Preferred is a fragment peptide consisting of a partial partial sequence of the amino acid sequence of selenoprotein P, comprising a partial sequence consisting of 10 or more amino acids, preferably 15 or more, and more preferably 20 or more. On the other hand, a fragment in the case of using selenoprotein P as a medicine is a fragment peptide having the above-described daltathione peroxidase-like activity. Examples of such selenoprotein P fragments include the C-terminal fragment of selenoprotein P. For example, a sequence comprising amino acids 260 to 362 of the amino acid sequence shown in SEQ ID NO: 2. The fragment which consists of is mentioned. In addition, in the amino acid sequences of these fragments, a fragment peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and having the above-described dartathione peroxidase-like activity Is also included.
血中セレノプロテイン P濃度は、 全身のインスリン抵抗性や血管障害進展への リスクを反映している。 セレノプロテイン Pを 2型糖尿病や血管障害等の疾患の 検出用マ一カーとして用いることができる。 血管障害は、 心筋梗塞、 脳梗塞、 閉 塞性動脈硬化症等の動脈硬化性疾患を含む。 また、 血中セレノプロティン Pを測 定することにより被験体の 2型糖尿病または血管障害に罹患するリスクを評価す ることができる。 さらに、 インスリ ン抵抗性または血糖コントロールを評価する ことができる。 さらに、 2型糖尿病患者においては新規インスリン抵抗性評価マ 一力一に、耐糖能正常患者においては 2型糖尿病発症の予知マーカーになり うる。 すなわち、 2型糖尿病患者において、 セレノプロテイン Pが高値である場合、 ィ ンスリ ン感受性が減弱していると評価することができる。 また、 耐糖能正常者に おいて、 セレノプロテイン Pが高値である場合、 2型糖尿病を発症するリスクが 高いと評価 '判定することができる。 また、 セレノプロテイン P値は、 肥満と相 関しており、 肥満の合併症の判定基準ともなり得る。 すなわち、 セレノプロティ ン Pが高値である場合、 肥満症または肥満の合併症に罹患していると判定するこ とができる。 さらに、 セレノプロテイン Pが高値である場合、 肥満によりインス リン抵抗性が高まっていると評価 ·判定することもできる。 Blood selenoprotein P levels reflect systemic insulin resistance and the risk of developing vascular disorders. Selenoprotein P can be used as a marker for detecting diseases such as type 2 diabetes and vascular disorders. Vascular disorders include arteriosclerotic diseases such as myocardial infarction, cerebral infarction, and obstructive arteriosclerosis. In addition, by measuring blood selenoprotein P, the risk of suffering from type 2 diabetes or vascular disorder in a subject can be evaluated. In addition, insulin resistance or glycemic control can be assessed. Furthermore, it can be a predictive marker for new insulin resistance in patients with type 2 diabetes, and a predictive marker for the onset of type 2 diabetes in patients with normal glucose tolerance. That is, in patients with type 2 diabetes, when selenoprotein P is high, it can be evaluated that the sensitivity to insulin is attenuated. For those with normal glucose tolerance When selenoprotein P is high, it can be judged that the risk of developing type 2 diabetes is high. Selenoprotein P levels are also associated with obesity and can be a criterion for complications of obesity. That is, when selenoprotein P is high, it can be determined that the patient suffers from obesity or obesity complications. Furthermore, if selenoprotein P is high, it can be evaluated and determined that insulin resistance is increased due to obesity.
セレノプロテイン pをマーカーとして用いる場合、 被験体の血液等の試料中の セレノプロテイン pを検出すればよい。 検体試料としては、 全血、 血清または血 漿等を用いることができる。  When using selenoprotein p as a marker, selenoprotein p may be detected in a sample such as blood of a subject. As the specimen sample, whole blood, serum or plasma can be used.
検出は、 セレノプロテイン Pを直接検出してもよいし、 セレノプロテイン Pの mRNAを検出して、 セレノプロテイン Pの発現を検出してもよレ、。 セレノプロティ ン Pを直接検出する方法として、 セレノプロテイン Pを特異的に認識し結合する 抗セレノプロテイン P抗体を用いる免疫測定法により行うことができる。 抗セレ ノプロテイン P抗体は、 公知の方法により作製することができる。 免疫測定法と しては、 抗セレノブロティン P抗体を固相化した担体を用いる方法やウェスタン ブロッテイング等が挙げられる。 固相化担体を用いる方法として、 例えば、 固相 化マイクロタイタープレートを用いる ELISA、 固相化粒子を用いる凝集法等が挙 げられるが、 これらには限定されず、 公知の免疫学的測定法を採用して、 血中セ レノプロテイン Pを検出することができる。  For detection, selenoprotein P may be detected directly, or the expression of selenoprotein P may be detected by detecting selenoprotein P mRNA. As a method of directly detecting selenoprotein P, an immunoassay method using an anti-selenoprotein P antibody that specifically recognizes and binds to selenoprotein P can be used. Anti-selenoprotein P antibody can be prepared by a known method. Examples of the immunoassay include a method using a carrier on which an anti-selenobrotin P antibody is immobilized, Western blotting, and the like. Examples of the method using a solid-phased carrier include ELISA using a solid-phased microtiter plate and agglutination method using solid-phased particles, but are not limited thereto, and known immunological measurement methods Can be used to detect serum selenoprotein P.
セレノプロティン Pの mRNAは、 ノ一ザンブロッティング、 RT- PCR法、 DNAチッ プ （DNA マイクロアレイ） を利用した方法等により検出することができる。 これ らの方法も公知の方法で行うことができる。  Selenoprotein P mRNA can be detected by Northern blotting, RT-PCR, DNA chip (DNA microarray) and other methods. These methods can also be performed by known methods.
さらに、 セレノプロテイン Pは 2型糖尿病等の疾患に罹患している被験体の肝 臓において、 発現が亢進しているので、 肝臓におけるセレノプロテイン Pの発現 を検出してもよレ、。  Furthermore, since selenoprotein P is upregulated in the liver of subjects suffering from diseases such as type 2 diabetes, it may be possible to detect the expression of selenoprotein P in the liver.
被験体から採取した検体試料中のセレノプロティン Pを測定し、 またはセレノ プロティン Pの発現を測定することにより、 被験体が 2型糖尿病や血管障害に罹 患しているか否かを検出 ·診断することができ、 また被験体が 2型糖尿病や血管 障害に罹患するリスクを評価することができる。 さらに、 被験体が糖 ·脂質代謝 異常、 糖尿病性合併症に罹患しているかどうかの判定や罹患するリスクを評価す ることもできる。 Detect or diagnose whether a subject suffers from type 2 diabetes or vascular disorder by measuring selenoprotein P in a sample collected from the subject or measuring the expression of selenoprotein P And the risk of the subject suffering from type 2 diabetes and vascular disorders can be assessed. In addition, subject is subject to sugar and lipid metabolism It is also possible to determine whether or not you are suffering from abnormal or diabetic complications and to assess the risk of suffering from it.
被験体から採取した検体試料中のセレノプロテイン Pを測定し、 セレノプロテ イン P濃度が正常人の検体試料中のセレノプロテイン P濃度より高い場合に、 あ るいは被験体のセレノプロティン Pの発現が亢進している場合に、 被験体が 2型 糖尿病や血管障害に罹患していると診断することができ、 あるいは被験体の 2型 糖尿病や血管障害に罹患するリスクが高いと評価することができる。  Measure selenoprotein P in a sample collected from a subject, and if the selenoprotein P concentration is higher than the selenoprotein P concentration in a normal human sample sample, or the selenoprotein P expression in the subject is increased If so, the subject can be diagnosed as having type 2 diabetes or vascular disorder, or can be assessed as having a high risk of suffering from type 2 diabetes or vascular disorder in the subject.
さらに、 セレノプロテイン Pを測定し、 またはセレノプロテイン Pの発現を測 定することにより、被験体の 2型糖尿病の病態を判断することができる。例えば、 被験体のインスリン抵抗性や血糖コントロールを評価することができる。ここで、 インスリン抵抗性の評価とは、 被験体がィンスリン抵抗性となっているかどうか の判定およびインスリン抵抗性となるリスクの評価をいう。 また、 血糖コント口 ールの評価とは、 被験体の血糖コントロールの良悪の評価または被験体の血糖コ ントロールが悪化するリスクの評価をいう。  Further, by measuring selenoprotein P or measuring the expression of selenoprotein P, the pathological condition of type 2 diabetes in the subject can be determined. For example, the subject's insulin resistance and blood glucose control can be evaluated. Here, the evaluation of insulin resistance refers to the determination of whether or not a subject has insulin resistance and the evaluation of the risk of becoming insulin resistance. The evaluation of the blood glucose control means an evaluation of the quality of the subject's blood glucose control or an evaluation of the risk of deterioration of the blood glucose control of the subject.
例えば、 被験体から採取した検体試料中のセレノプロテイン Pを測定し、 セレ ノプロテイン P濃度が正常人の検体試料中のセレノプロティン P濃度より高い場 合に、 あるいは被検体のセレノプロテイン Pの発現が亢進している場合に、 被験 体がィンスリン抵抗性であると評価し、 または血糖コントロールが悪化している と評価することができる。 あるいは、 被験体がインスリン抵抗性となるリスクが 高く、または血糖コントロールが悪化するリスクが高いと評価することができる。 正常人における検体試料中のセレノプロティン P濃度またはセレノプロティン P の発現程度はあらかじめ測定しておき、 統計的解析により、 血中セレノプロティ ン pの異常値や異常発現のカッ トオフ値を設定することが可能である。  For example, when selenoprotein P is measured in a sample collected from a subject and the selenoprotein P concentration is higher than the selenoprotein P concentration in a normal human sample sample, or the expression of selenoprotein P in the subject If is elevated, it can be assessed that the subject is resistant to insulin or that glycemic control has deteriorated. Alternatively, it can be evaluated that the subject has a high risk of becoming insulin resistant or has a high risk of worsening glycemic control. Serum protein P concentration or the expression level of selenoprotein P in specimens of normal subjects should be measured in advance, and abnormal values of serum selenoprotein p and abnormal expression cutoff values may be set by statistical analysis. Is possible.
すなわち、 本発明は上記のリスクを評価するための検査方法および上記のリス クを評価するための検查用試薬をも包含する。 また、 セレノプロテイン Pは、 上 記のリスクを評価するための検查用マ一カーとして用いることができる。  That is, the present invention also includes a test method for evaluating the risk and a test reagent for evaluating the risk. Selenoprotein P can also be used as a screening marker for evaluating the above risks.
本発明は、 さらにセレノプロテイン Pの発現を指標にして、 2型糖尿病や血管 障害を予防または治療する医薬のスクリーニングを行う方法を包含する。 セレノ プロテイン Pは、 2型糖尿病および動脈硬化病変の治療標的となり得る。 特に、 糖尿病患者においては、 高血糖、 肝からのセレノプロテイン P分泌亢進、 全身の インスリン抵抗性悪化、 さらなる高血糖という悪循環が生じている可能性がありThe present invention further includes a method for screening a drug for preventing or treating type 2 diabetes or vascular disorder using selenoprotein P expression as an index. Selenoprotein P can be a therapeutic target for type 2 diabetes and arteriosclerotic lesions. In particular, In diabetics, there may be a vicious circle of hyperglycemia, increased secretion of selenoprotein P from the liver, worsening systemic insulin resistance, and further hyperglycemia
(図 2 )、 血中セレノプロテイン P濃度を適正レベルまで低下させることで、 糖毒 性に伴うこれらの病態を劇的に改善させうる可能性がある。 該医薬は、 体内での セレノプロテイン pの発現を抑制し、 セレノプロテイン Pの分泌を抑制し、 また はセレノプロテイン Pの作用を阻害する医薬である。 該医薬は、 インスリン抵抗 性を減弱させ、 また血糖コントロールを改善し得る。 これらの医薬は、 セレノブ ロティン分泌抑制薬またはセレノプロティン作用減弱薬であり、 血中セレノプロ ティン P濃度を適切なレベルに低下させることができる。 (Fig. 2) By reducing the blood selenoprotein P concentration to an appropriate level, it may be possible to dramatically improve these pathologies associated with glycotoxicity. The medicament is a medicament that suppresses the expression of selenoprotein p in the body, suppresses the secretion of selenoprotein P, or inhibits the action of selenoprotein P. The medicament can attenuate insulin resistance and improve glycemic control. These drugs are selenobrotin secretion inhibitors or selenoprotein attenuating drugs, and can reduce blood selenoprotein P concentration to an appropriate level.
該医薬は例えば、 セレノプロテイン Pのプロモーターに結合し、 セレノプロテ イン Pの発現、 分泌を抑制し得る化合物である。 また、 該医薬は、 セレノプロテ イン Pの受容体に結合し、 セレノプロテイン Pが受容体に結合するのを阻害し、 セレノプロテイン Pの作用を阻害し得る化合物である。  The medicament is, for example, a compound that can bind to the promoter of selenoprotein P and suppress the expression and secretion of selenoprotein P. The medicament is a compound that binds to a selenoprotein P receptor, inhibits selenoprotein P from binding to the receptor, and can inhibit the action of selenoprotein P.
このような化合物は例えば、動物細胞等にセレノプロテイン Pをコードする DNA およびセレノプロティン Pのプロモーターを導入し、 セレノプロティン P発現系 を構築し、 該動物細胞と候補化合物を接触させ、 すなわち候補化合物の存在下で 動物細胞を培養し、 該細胞におけるセレノプロティン Pの発現を指標にスクリ一 ユングすることができる。 候補化合物により、 セレノプロテイン Pの発現が低下 した場合、 該候補化合物を 2型糖尿病や血管障害の予防、 治療用医薬として選択 することができる。  Such compounds include, for example, DNAs encoding selenoprotein P and selenoprotein P promoters introduced into animal cells, etc., constructing a selenoprotein P expression system, and contacting the animal cells with the candidate compound, that is, candidate compounds Animal cells can be cultured in the presence of, and screened using the expression of selenoprotein P in the cells as an indicator. When the expression of selenoprotein P is reduced by a candidate compound, the candidate compound can be selected as a pharmaceutical agent for the prevention and treatment of type 2 diabetes and vascular disorders.
また、 セレノプロテイン P、 セレノプロテイン P受容体および候補化合物を共 存させ、 セレノプロテイン Pとセレノプロテイン Pの受容体との結合を候捕化合 物が阻害し、 セレノプロテイン Pの作用を抑制するか否かを指標にして、 2型糖 尿病や血管障害の予防、 治療用医薬として用い得る化合物を選択することができ る。  Whether selenoprotein P, selenoprotein P receptor, and candidate compounds coexist, and the binding of selenoprotein P to the selenoprotein P receptor is inhibited by the trapping compound, and the action of selenoprotein P is suppressed. Whether or not the compound can be used as a pharmaceutical for the prevention and treatment of type 2 diabetes and vascular disorders can be selected.
本発明は、 さらにセレノプロティン Pまたはセレノプロティン Pの断片べプチ ドであってダルタチオンペルォキシダーゼ様活性を有するぺプチドを有効成分と して含む医薬組成物、 ならびにセレノプロテイン Pをコードする核酸またはそれ らの断片ヌクレオチドであって、 ダルタチオンペルォキシダーゼ様活性を有する ぺプチドをコ一ドする断片ヌクレオチドを有効成分として含む医薬組成物を包含 する。 セレノプロテイン Pは、 血管内皮細胞の増殖を抑制し、 血管壁細胞の増殖 を誘導し得るので、 血管内皮細胞の増殖または血管壁細胞の増殖低下に関連した 疾患の予防または治療に用いることができる。 該医薬組成物は血管系細胞に直接 作用し、 上記疾患の予防、 治療薬として用いることができる。 The present invention further relates to a pharmaceutical composition comprising, as an active ingredient, selenoprotein P or a fragment peptide of selenoprotein P, which has a peptide having dartathione peroxidase-like activity, and a nucleic acid encoding selenoprotein P. Or a fragment nucleotide thereof having dartathione peroxidase-like activity A pharmaceutical composition containing a fragment nucleotide that codes for a peptide as an active ingredient is included. Selenoprotein P can suppress the proliferation of vascular endothelial cells and induce the proliferation of vascular wall cells, so it can be used for the prevention or treatment of diseases related to the proliferation of vascular endothelial cells or the decreased proliferation of vascular wall cells . The pharmaceutical composition acts directly on vascular cells and can be used as a preventive or therapeutic agent for the above-mentioned diseases.
本発明は、 さらにセレノプロテイン Pのァゴニストまたはセレノプロテイン P のアンタゴニストを有効成分として含む医薬組成物を包含する。 ここで、 セレノ プロテイン Pのァゴニス トとは、 セレノプロテイン Pの受容体に結合し、 細胞に 生理活性作用を発現させる物質をいい、 セレノプロテイン Pのアンタゴニス トと は、 セレノプロテイン Pの受容体に結合するが、 生体に対する作用を発現せず、 セレノプロテイン pの作用を阻害する物質をいう。 セレノプロテイン pのァゴニ ス トまたはアンタゴニストは、 血管内皮細胞の増殖または血管壁細胞の増殖低下 に関連した疾患の予防または治療に用いることができる。 血管内皮細胞の増殖ま たは血管壁細胞の増殖低下に関連した疾患として、 例えば糖尿病性網膜症、 心筋 梗塞、 脳梗塞、 閉塞性動脈硬化症等の動脈硬化性疾患等が挙げられる。 また、 セ レノプロティン pは血管新生を抑制することができ、 セレノプロティン Pまたは セレノプロテイン pのァゴニス トもしくはアンタゴニス トは、 癌の予防または治 療に用いることもできる。  The present invention further includes a pharmaceutical composition comprising an elenoprotein P agonist or an antagonist of selenoprotein P as an active ingredient. Here, the selenoprotein P antagonist refers to a substance that binds to the selenoprotein P receptor and exerts a physiological activity in the cell. The selenoprotein P antagonist refers to the selenoprotein P receptor. A substance that binds but does not exert an effect on the body and inhibits the action of selenoprotein p. Selenoprotein p agonists or antagonists can be used to prevent or treat diseases associated with vascular endothelial cell proliferation or vascular wall cell proliferation reduction. Examples of diseases related to the proliferation of vascular endothelial cells or the decrease in the proliferation of vascular wall cells include arteriosclerotic diseases such as diabetic retinopathy, myocardial infarction, cerebral infarction, and obstructive arteriosclerosis. In addition, selenoprotein p can suppress angiogenesis, and selenoprotein P or selenoprotein p agonist or antagonist can also be used for the prevention or treatment of cancer.
セレノプロテイン Pタンパク質または断片べプチドを医薬として用いる場合、 被験体にタンパク質または断片ペプチドを投与すればよい。 また、 セレノプロテ イン pをコードする核酸またはそれらの断片ヌクレオチドを医薬として用いる場 合、 公知の遺伝子治療の手法により、 核酸またはヌクレオチドを体内に投与すれ ばよい。 遺伝子を被験体へ導入する方法として、 ウィルスベクターを用いる方法 および非ウィルスベクターを用いる方法があり、種々の方法が公知である（別冊実 験医学、 遺伝子治療の基礎技術、 羊土社、 1996 ;別冊実験医学、 遺伝子導入 &発現 解析実験法、 羊土社、 1997 ; 日本遺伝子治療学会編、 遺伝子治療開発研究ハンド ブック、 ェヌ .ティー ' エス、 1999)。  When selenoprotein P protein or fragment peptide is used as a medicine, the protein or fragment peptide may be administered to the subject. In addition, when a nucleic acid encoding selenoprotein p or a fragment nucleotide thereof is used as a medicine, the nucleic acid or nucleotide may be administered into the body by a known gene therapy technique. Methods for introducing a gene into a subject include a method using a viral vector and a method using a non-viral vector, and various methods are known (separate experimental medicine, basic technology of gene therapy, Yodosha, 1996; Separate Experimental Medicine, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997; edited by Japanese Society of Gene Therapy, Gene Therapy Development Research Handbook, NTS, 1999).
さらに、 本発明はセレノプロテイン Pを標的とし、 セレノプロテイン Pの活性 を抑制する力、、またはセレノプロティン Pの発現を抑制し得る医薬をも包含する。 これらの医薬は、 2型糖尿病または血管障害の予防または治療薬として用いるこ とができる。 Furthermore, the present invention also includes a pharmaceutical that targets selenoprotein P and can suppress the activity of selenoprotein P or the expression of selenoprotein P. These medicaments can be used as preventive or therapeutic agents for type 2 diabetes or vascular disorders.
このような医薬として用い得る化合物として、 セレノプロテイン Pの作用を中 和し得る抗セレノプロテイン P抗体が挙げられる。 セレノプロテイン Pの作用を 中和し得る抗セレノプロテイン P抗体、 すなわち、 セレノプロテイン Pに対する アンタゴニスティック抗体は、公知の抗体作成方法により作製することができる。 これらの抗体は好ましくはヒ トに対する異種抗原性を低下させること等を目的と して人為的に改変した遺伝子組換え型抗体、 例えば、 キメ ラ抗体、 ヒ ト化 (Humanized) 抗体である。 これらの改変抗体は、 既知の方法を用いて製造するこ とができる。 キメラ抗体は、 ヒ ト以外の哺乳動物由来抗体の可変領域とヒ ト抗体 由来の定常領域とからなる。 ヒ ト化抗体は、 ヒ ト以外の哺乳動物由来抗体の相補 性決定領域と、 ヒ ト抗体由来のフレームワーク領域および C領域とからなる。 ヒ ト化抗体はヒ ト体内における抗原性が低下されているため、 本発明の治療剤の有 効成分として有用である。 ヒ ト化抗体は、再構成（reshaped) ヒ ト抗体ともいい、 ヒ ト以外の哺乳動物、例えばマウス抗体の相補性決定領域（CDR ; complementarity determining region) をヒ ト抗体の相補性決定領域へ移植することにより得るこ とができる。 ヒ ト抗体遺伝子座を導入し、 ヒ ト由来抗体を産生する能力を有する トランスジエニック動物に抗原を投与することにより得ることができるヒ ト抗体 も含む。 このような トランスジエニック動物としてマウスが挙げられ、 ヒ ト抗体 を産生し得るマウスの作出方法は、例えば、国際公開第 W002/43478号パンフレツ トに記載されている。 本発明の抗セレノプロテイン P抗体は、 抗セレノプロティ ン P抗体の一部分 （部分断片） であって、 抗体の抗原への作用を有する抗体の断 片も含み、 具体的には F (ab' ) 2 、 Fab'、 Fab、 Fv、 ジスルフィ ド結合 Fv、 一本 鎖 Fv (scFv)、 及びこれらの重合体等が挙げられる。 Examples of such a compound that can be used as a medicament include an anti-selenoprotein P antibody capable of neutralizing the action of selenoprotein P. An anti-selenoprotein P antibody capable of neutralizing the action of selenoprotein P, that is, an antagonistic antibody against selenoprotein P can be prepared by a known antibody production method. These antibodies are preferably genetically modified antibodies that have been artificially modified for the purpose of reducing the heterologous antigenicity to humans, such as chimeric antibodies and humanized antibodies. These modified antibodies can be produced using known methods. A chimeric antibody consists of a variable region of a mammal-derived antibody other than human and a constant region derived from a human antibody. A humanized antibody consists of a complementarity determining region of a mammal-derived antibody other than human, a framework region derived from a human antibody, and a C region. A humanized antibody is useful as an active ingredient of the therapeutic agent of the present invention because its antigenicity in the human body is reduced. A humanized antibody is also called a reshaped human antibody, and the complementarity determining region (CDR) of a mammal other than human, for example, a mouse antibody, is transplanted to the complementarity determining region of the human antibody. It can be obtained by doing. It also includes a human antibody that can be obtained by introducing a human antibody locus and administering an antigen to a transgenic animal having the ability to produce a human-derived antibody. Examples of such transgenic animals include mice, and a method for producing mice capable of producing human antibodies is described in, for example, International Publication No. W002 / 43478 pamphlet. The anti-selenoprotein P antibody of the present invention is a part (partial fragment) of the anti-selenoprotein P antibody, and includes a fragment of an antibody having an action on the antigen of the antibody. Specifically, F (ab ′) 2 , Fab ′, Fab, Fv, disulfide bond Fv, single chain Fv (scFv), and polymers thereof.
セレノプロテイン Pを標的と.し、 セレノプロテイン Pの活性を抑制する力 、 ま たはセレノプロテイン Pの発現を抑制し得る医薬として使用し得る化合物は、 セ レノプロティン pの受容体に結合するが、 野生型のセレノプロティン Pの作用は 有さず、 野生型のセレノプロテイン Pが受容体に結合することを阻害するドミナ ントネガティブ突然変異体をも包含する。 さらに、セレノプロテイン Pを標的とし、セレノプロテイン Pの活性を抑制し、 またはセレノプロテイン Pの発現を抑制し得る医薬として使用し得る化合物は、 体内においてセレノプロティン Pの発現を抑制し得る化合物を含む。 このような 化合物として、例えば RNAi (RNA干渉）により特定の配列を有する標的 mRNAを切断 し、 その mRNA に対応する遺伝子の発現を抑制し得る化合物が挙げられる。 RNAi においては、特定の標的 mRNAと実質的に同一な配列を有するセンス鎖と該センス 鎖に相補的なアンチセンス鎖からなる s iRNA (short interfering RNA)がガイ ドCompounds that target selenoprotein P and can suppress the activity of selenoprotein P or can be used as drugs that can suppress the expression of selenoprotein P bind to the receptor for selenoprotein p. Also included are dominant negative mutants that do not have the action of wild-type selenoprotein P and inhibit the binding of wild-type selenoprotein P to the receptor. Furthermore, compounds that can be used as pharmaceuticals that target selenoprotein P, suppress selenoprotein P activity, or suppress selenoprotein P expression include compounds that can suppress selenoprotein P expression in the body. . Examples of such a compound include a compound capable of cleaving a target mRNA having a specific sequence by RNAi (RNA interference) and suppressing the expression of a gene corresponding to the mRNA. In RNAi, siRNA (short interfering RNA) consisting of a sense strand having a sequence substantially identical to a specific target mRNA and an antisense strand complementary to the sense strand is a guide.
RNAとしてターゲッ ト配列を認識し、 ターゲッ ト mRNAを切断することにより、 遺 伝子の発現が抑制される。 s iRNAは細胞内または生体内でダイサー（Dicer)により プロセッシングを受けて 2重鎖 RNA (dsRNA)より形成される。 該化合物として、 例 えば RNAi作用を有する 2本鎖 RNAが挙げられる。 2本鎖 RNAは、 標的遺伝子の mRNA配列と相同な配列からなるセンス RNAおよびこれと相補的な配列からなるァ ンチセンス RNA とからなる。 また、 センス鎖またはアンチセンス鎖の 3，末端にォ 一バーハングを有していてもよく、 該オーバーハングの塩基の種類、 数は限定さ れず、 例えば、 1〜5、 好ましくは 1〜3、 さらに好ましくは 1もしくは 2塩基 からなる配列が挙げられ、 例えば、 UUや TTが挙げられる。 本発明において、 ォ 一バーハングとは、 shRNA の一方の鎖の末端に付加された塩基であって、 もう一 方の鎖の対応する位置に相補的に結合し得る塩基が存在しない塩基をいう。 ォー バーハングは DNAを構成する塩基であってもよい。 2本鎖部分は、 RNA干渉によ り発現を抑制しょうとする標的遺伝子の配列中の標的配列にハイブリダィズし得 る配列を有する RNA鎖 （センス鎖） および該配列に相補的な RNA鎖 （アンチセン ス鎖） が相補的に結合した構造を有する。 また、 2本鎖 RNAは、 センス鎖とアン チセンス鎖がループ配列を介して連結しているステムループ構造を有しているシ ョートヘアピン RNA (shRNA)であってもよい。 本発明の 2本鎖 RNAの標的遺伝子 であるセレノプロティン Pをコードする遺伝子の特定の標的配列は、 配列番号 1 に示す DNAの塩基配列に対応する RNA中の配列であり、 該配列情報に基づいて適 宜設定できる。 その塩基数は、 限定されず、 15〜500 塩基の範囲で選択される。 好ましくは 15〜50、 15〜45、 15〜40、 15〜35もしくは 15〜30塩基、 さらに好ま しくは 20〜35塩基、 さらに好ましくは 19〜30塩基、特に好ましくは 19〜29塩基 もしくは 28塩基である。 Recognizing the target sequence as RNA and cleaving the target mRNA suppresses gene expression. siRNA is formed from double-stranded RNA (dsRNA) by being processed by Dicer in cells or in vivo. Examples of the compound include double-stranded RNA having RNAi action. Double-stranded RNA consists of a sense RNA consisting of a sequence homologous to the mRNA sequence of the target gene and an antisense RNA consisting of a complementary sequence thereto. In addition, the sense strand or the antisense strand may have an overhang at the 3, terminal, and the type and number of bases of the overhang are not limited, for example, 1 to 5, preferably 1 to 3, More preferred is a sequence consisting of 1 or 2 bases, such as UU and TT. In the present invention, the term “overhang” refers to a base added to the end of one strand of the shRNA and having no base capable of complementary binding to the corresponding position of the other strand. The overhang may be a base constituting DNA. The double-stranded part consists of an RNA strand (sense strand) having a sequence that can hybridize to the target sequence in the sequence of the target gene whose expression is to be suppressed by RNA interference, and an RNA strand (antisense strand) complementary to the sequence. Have a structure of complementary binding. The double-stranded RNA may be a short hairpin RNA (shRNA) having a stem-loop structure in which a sense strand and an antisense strand are linked via a loop sequence. The specific target sequence of the gene encoding selenoprotein P, which is the target gene of the double-stranded RNA of the present invention, is a sequence in RNA corresponding to the base sequence of DNA shown in SEQ ID NO: 1, and based on the sequence information Can be set appropriately. The number of bases is not limited and is selected in the range of 15 to 500 bases. Preferably 15 to 50, 15 to 45, 15 to 40, 15 to 35 or 15 to 30 bases, more preferably 20 to 35 bases, more preferably 19 to 30 bases, particularly preferably 19 to 29 bases Or 28 bases.
さらに、 体内においてセレノプロテイン Pの発現を抑制し得る化合物として、 アンチセンス核酸が挙げられる。 アンチセンス核酸は、 目的の標的遺伝子に相補 的でありハイブリダイズする配列を持つ DNAまたは RNAであり、 その標的遺伝子 の発現を抑制し得る。 本発明のセレノプロテイン Pの発現を抑制するアンチセン ス核酸は、 配列番号 1に示すセレノプロテイン Pをコードする DNAの塩基配列ま たは該 DNA配列に対応する RNA配列の一部に相補的な核酸である。 該核酸は、 長 さが 10から 400ヌクレオチドであり、好ましくは長さが 250以下のヌクレオチド、 さらに好ましくは長さカ 100 以下のヌクレオチド、 さらに好ましくは長さ力 S 50 以下のヌクレオチド、 特に好ましくは長さが 12〜28の間のヌクレオチドである。 本発明の 2本鎖 RNA またはアンチセンス核酸の導入方法と しては、 Hydrodynamic 法、 カノレシゥムイオンを用いる方法、 エレク トロポレーシヨン法、 スフヱ口プラス ト法、 酢酸リチウム法、 リ ン酸カルシウム法、 リボフヱクシヨン 法、 マイクロインジェクショ ン法等が挙げられる。  Furthermore, an antisense nucleic acid is mentioned as a compound which can suppress the expression of selenoprotein P in the body. An antisense nucleic acid is DNA or RNA that is complementary to and hybridizes to a target gene of interest, and can suppress the expression of the target gene. The antisense nucleic acid that suppresses the expression of selenoprotein P of the present invention is a nucleic acid complementary to the nucleotide sequence of DNA encoding selenoprotein P shown in SEQ ID NO: 1 or a part of the RNA sequence corresponding to the DNA sequence. It is. The nucleic acid has a length of 10 to 400 nucleotides, preferably has a length of 250 or less, more preferably has a length of 100 or less, more preferably has a length force of S 50 or less, particularly preferably It is between 12 and 28 nucleotides in length. Examples of the method for introducing the double-stranded RNA or antisense nucleic acid of the present invention include a hydrodynamic method, a method using canoleum ion, an electroporation method, a squib mouth plast method, a lithium acetate method, a calcium phosphate method, Examples include the ribofusion method and the microinjection method.
本発明は上記の化合物を有効成分として含む 2型糖尿病または血管障害の治療 または予防のための医薬組成物を包含する。  The present invention includes a pharmaceutical composition for treating or preventing type 2 diabetes or vascular disorder comprising the above compound as an active ingredient.
本発明の医薬組成物は、 セレノプロテイン Pタンパク質もしくはその断片ぺプ チドまたはセレノプロティン Pをコードする核酸またはそれらの断片ヌクレオチ ドを含むベクターならびに薬理学的に許容され得る担体、 希釈剤もしくは賦形剤 を含む。  The pharmaceutical composition of the present invention comprises a selenoprotein P protein or a fragment peptide thereof, a nucleic acid encoding selenoprotein P, or a vector containing the fragment nucleotide, and a pharmacologically acceptable carrier, diluent or excipient. Contains agent.
本発明の医薬組成物は、種々の形態で投与することができ、錠剤、カプセル剤、 顆粒剤、 散剤、 シロップ剤等による経口投与、 あるいは注射剤、 点滴剤、 座薬、 スプレー剤、 点眼剤、 経鼻投与剤、 貼付剤などによる非経口投与を挙げることが できる。  The pharmaceutical composition of the present invention can be administered in various forms, orally by tablets, capsules, granules, powders, syrups, etc., or injections, drops, suppositories, sprays, eye drops, Examples include parenteral administration by nasal administration and patch.
本発明の医薬組成物は、 局所投与することも可能であり、 例えば肝臓組織部位 に注射により投与することによりその効果を発揮し得る。  The pharmaceutical composition of the present invention can be administered locally. For example, the effect can be exerted by administering the composition to the liver tissue site by injection.
本発明の医薬組成物は、 製剤分野において通常用いられる担体、 希釈剤、 賦形 剤を含む。 たとえば、 錠剤用の担体、 賦形剤としては、 乳糖、 ステアリン酸マグ ネシゥムなどが使用される。 注射用の水性液としては、 生理食塩水、 ブドウ糖や その他の補助薬を含む等張液などが使用され、 適当な溶解補助剤、 たとえばアル コール、 プロピレングリコールなどのポリアルコール、 非イオン界面活性剤など と併用してもよい。 油性液としては、 ゴマ油、 大豆油などが使用され、 溶解補助 剤としては安息香酸ベンジル、 ベンジルアルコールなどを併用してもよい。 The pharmaceutical composition of the present invention contains a carrier, a diluent, and an excipient that are commonly used in the pharmaceutical field. For example, lactose and magnesium stearate are used as carriers and excipients for tablets. Aqueous solutions for injection include saline, glucose and Isotonic solutions containing other adjuvants are used, and may be used in combination with appropriate solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants and the like. Sesame oil, soybean oil and the like are used as the oily liquid, and benzyl benzoate, benzyl alcohol and the like may be used in combination as the solubilizing agent.
その投与量は、 症状、 年齢、 体重などによって異なるが、 タンパク質またはべ プチドの場合、 経口投与では、 1 日約 0. 001mg〜100mgであり、 1回または数回に 分けて投与すればよい。 また、 非経口投与では、 1回あたり、 0. 001mg〜100mgを 皮下注射、 筋肉注射、 または静脈注射によって投与すればよい。 また、 被験体内 で翻訳させる発現ベクター等に挿入されたセレノプロテイン Pをコードする核酸 またはそれらの断片ヌクレオチドは、 数日または数週間または数ケ月おきに 1回 あたり、 0. 001mg〜100mgを皮下注射、 筋肉注射、 または静脈注射によって投与す ればよい。 RNAiの作用を有する 2本鎖 RNAまたはアンチセンス核酸の場合、 肝臓 の細胞当たり少なく とも 1 コピーの 2本鎖 RNAまたはアンチセンス核酸が導入さ れるように投与してもよい。 本発明を以下の実施例によって具体的に説明する が、 本発明はこれらの実施例によって限定されるものではない。  The dose varies depending on symptoms, age, body weight, etc., but in the case of protein or peptide, oral administration is about 0.001 mg to 100 mg per day, and may be administered once or divided into several times. In parenteral administration, 0.001 mg to 100 mg may be administered by subcutaneous injection, intramuscular injection, or intravenous injection. In addition, selenoprotein P-encoding nucleic acid or a fragment nucleotide thereof inserted into an expression vector or the like to be translated in a subject is injected subcutaneously at a dose of 0.001 mg to 100 mg once every several days, weeks or months. It can be administered by intramuscular injection or intravenous injection. In the case of double-stranded RNA or antisense nucleic acid having the action of RNAi, administration may be carried out so that at least one copy of double-stranded RNA or antisense nucleic acid is introduced per liver cell. The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
一連の実施例において、 材料、 試薬および動物は以下のものを用いた。  In the series of examples, the following materials, reagents and animals were used.
ラッ トへノ トーマ由来 H4I IEC細胞は、 American Type Culture col lect ion (ATCC) より購入した。 ヒ ト由来セレノプロテイン Pは高橋和彦教授（北海道薬科大学， 日 本）から供与を受けた（Sa i to Y. et al . , J Biol Chem 274 : 2866- 2871， 1999)。 セ レノプロテイン P濃度はゥシ血清アルブミンをスタンダードとしてブラッドフォ 一ド法を用いて測定した。 ヒ ト組み換えインスリ ンは S igma Chemicalsから購入 した。抗 Akt抗体と抗セリ ン（473)リ ン酸化 Akt抗体は Cel l S ignal ing technology から購入した。 抗 CD31 モノクローナル抗体は Pharmingen、 HRP結合 Ant i- Human Smooth Muscle Act in抗体は DAKO Cytomat ion力 ら購入した。  Rat hematoma-derived H4I IEC cells were purchased from American Type Culture Collection (ATCC). Human-derived selenoprotein P was provided by Professor Kazuhiko Takahashi (Hokkaido Pharmaceutical University, Japan) (Sai to Y. et al., J Biol Chem 274: 2866-2871, 1999). Serenoprotein P concentration was measured using the blood-fod method with ushi serum albumin as a standard. Human recombinant insulin was purchased from Sigma Chemicals. Anti-Akt antibody and anti-serine (473) phosphorylated Akt antibody were purchased from Cel Signal Technology. The anti-CD31 monoclonal antibody was purchased from Pharmingen, and the HRP-conjugated anti-human smooth muscle actin antibody was purchased from DAKO Cytomat ion.
雌 C57BL/6Jマウスは三共ラボサービス （日本） より 8週齢の時点で購入し、 標 準的な光 （12 時間の光/闇） および温度の環境で維持した。 これらのマウスは 1 グループあたり 5匹のケージで飼育し、 水と食料を ad l ibi tumで提供し、 約 10 週齢で実験に使用した。  Female C57BL / 6J mice were purchased from Sankyo Lab Service (Japan) at the age of 8 weeks and maintained in a standard light (12 hours light / dark) and temperature environment. These mice were housed in 5 cages per group, provided water and food in ad ibi tum, and used for experiments at about 10 weeks of age.
実施例 1 セレノプロテイン P遺伝子発現と糖尿病の病態 ヒ ト肝臓におけるセレノプロテイン P遺伝子発現とインスリン抵抗性の関連 主に骨格筋におけるィンスリン抵抗性を、 人工帱臓を用いた正常血糖下高ィン スリンクランプ法を用レヽて、 glucose metabol ic c learance rate (MCR)として定 量化した。 MCRは骨格筋へのィンスリン依存性糖取り込みを反映し、 MCRが低いほ どインスリ ン抵抗性は強い。 結果を図 3に示す。 図 3に示すように、 DNA チップ を用いて定量したヒ ト肝臓でのセレノプロテイン P遺伝子発現量は、 インスリン 抵抗性指標である MCRと負に相関した。 すなわち、 ヒ ト肝でのセレノプロテイン P遺伝子発現がィンスリン抵抗性に関連して亢進することが判明した。 Example 1 Selenoprotein P gene expression and pathology of diabetes Relationship between selenoprotein P gene expression in human liver and insulin resistance Ginsulin resistance mainly in skeletal muscles, and glucose metabol ic c Quantified as learance rate (MCR). MCR reflects insulin-dependent sugar uptake into skeletal muscle, and the lower the MCR, the stronger the insulin resistance. The results are shown in Figure 3. As shown in Fig. 3, the expression level of selenoprotein P gene in human liver quantified using a DNA chip was negatively correlated with MCR, an insulin resistance index. In other words, it was found that selenoprotein P gene expression in human liver is increased in relation to insulin resistance.
同様に、 ヒ ト肝臓におけるセレノプロテイン P遺伝子発現と高血糖の関連を調 ベた。 結果を図 4に示す。 図 4に示すように、 ヒ ト肝臓でのセレノプロテイン P 遺伝子発現量は、 糖負荷後 2時間血糖値と正の相関を示した。 すなわち、 ヒ ト肝 でのセレノプロテイン P遺伝子発現が糖負荷後高血糖に関連して亢進することが 判明した。  Similarly, we investigated the relationship between selenoprotein P gene expression and hyperglycemia in human liver. The results are shown in Fig. 4. As shown in Fig. 4, the expression level of selenoprotein P gene in human liver was positively correlated with blood glucose level for 2 hours after glucose loading. In other words, it was found that selenoprotein P gene expression in human liver increases in relation to hyperglycemia after glucose load.
実施例 2 細胞レベルでの発現制御 ·機能解析 Example 2 Expression control and functional analysis at the cellular level
( 1 ) インスリン投与が肝細胞におけるセレノプロテイン P遺伝子発現に与え る影響  (1) Effects of insulin administration on selenoprotein P gene expression in hepatocytes
ヒ ト肝臓におけるセレノプロティン P遺伝子発現がィンスリ ン抵抗性や高血糖 で亢進することがわかったので、 インスリンおよびグルコースがセレノプロティ ン P遺伝子発現に及ぼす作用を試験管内で検討した。  Since it was found that selenoprotein P gene expression in human liver was enhanced by insulin resistance and hyperglycemia, the effects of insulin and glucose on selenoprotein P gene expression were examined in vitro.
肝細胞（H4I IEC)を 6穴プレート上で Dulbecco' s Mod if ied Eagle培地 （DMEM， Hepatocytes (H4I IEC) on a 6-well plate Dulbecco's Mod if ied Eagle medium (DMEM,
Gibco BRL)、 20%ゥマ血清（Gibco BRU、 5 %FBSにて 80%コンフルェント状態ま で培養した。 PBSにて一回細胞を洗つた後、 血清なしの DMEM培地で 6時間培養し starvat ionをおこなった。 その後、 インスリンを培地に添加し 6時間インキュべ ートし、 Quick gene system (FUJI FILM)を用いて Total RNAを回収した。 1サン プノレ（こっき 100ngの RNAを High-capac i ty cDNA Archive Kit (Appl ied Biosystems) にて cDNA に変換し、 ABI Prism 7700 Sequence Detect ion Sys tem (Appl i edGibco BRL), 20% horse serum (Gibco BRU, cultured to 5% FBS until 80% confluent. Wash cells once with PBS, then culture in serum-free DMEM medium for 6 hours. After that, insulin was added to the medium, incubated for 6 hours, and total RNA was collected using the Quick gene system (FUJI FILM) 1Sampnore (100 ng of RNA was high-capacity) Converted to cDNA using cDNA Archive Kit (Applied Biosystems) and ABI Prism 7700 Sequence Detection System (Applied ed
Biosystems)を用いてセレノプロティン P遺伝子発現の変化を Real- t ime PCR法に て観察した。 セレノプロテイン P のプライマーと TaqMan プロ一ブは Apl l i edChanges in selenoprotein P gene expression were observed by real-time PCR using Biosystems). Selenoprotein P primer and TaqMan probe are Apl l i ed
Biosys tems の bのを使用した (Assays- on- Demand gene express ion product) 0 normalizationのためのコントロール遺伝子として、 16s rRNA を用いた。 PCR条 件は 50°C · 2分を 1サイクル、 95°C · 10分を 1サイクル、 その後 95°C . 15秒と 60°C . 1分を 40サイクルとした。 浸透圧の影響を除外するため、 グルコースと等 濃度のマンニトールをコントロールに用いた。 Biosys tems b (Assays-on-Demand gene express ion product) 0 16s rRNA was used as a control gene for normalization. PCR conditions were 50 ° C · 2 minutes for 1 cycle, 95 ° C · 10 minutes for 1 cycle, then 95 ° C for 15 seconds and 60 ° C for 1 minute for 40 cycles. To exclude the effect of osmotic pressure, mannitol at the same concentration as glucose was used as a control.
結果を図 5に示す。 図 5に示すように、 インスリンは用量依存性に肝セレノプ 口ティン P遺伝子発現を低下させることが明らかとなった。  The results are shown in FIG. As shown in Fig. 5, insulin was found to decrease hepatic selenoptine P gene expression in a dose-dependent manner.
(2) グルコース投与が肝細胞におけるセレノプロティン P遺伝子発現に与え る影響  (2) Effect of glucose administration on selenoprotein P gene expression in hepatocytes
肝細胞（H4IIEC)を 6穴プレート上で DMEM培地、 20%ゥマ血清、 5%FBSにて 80% コンフルェント状態まで培養した。 PBS にて一回細胞を洗った後、 糖新生バッフ ァ一 (glucose-free DMEM, 3.7g/L sodium bicarbonate)、 0.4%FBSで 6時間培養 し starvationをおこなった。 その後、 グルコースを培地に添加し 6時間インキュ ベートし、 Quick gene system (FUJI FILM)を用いて Total RNAを回収した。 浸透 圧の影響を除外するため、 グルコースと等濃度のマンニトールをコントロールに 用レヽた。 1サンプノレ【こっき 100ng の RNA を High-capacity cDNA Archive Kit (Applied Biosystems)にて cDNAに変換し、 ABI Prism 7700 Sequence Detection System (Applied Biosystems)を用いてセレノプロテイン P 遺伝子発現の変化を Real-time PCR法にて観察した。 セレノプロティン Pのプライマーと TaqManプロ "~フは Apllied Biosystems のものを使用した (Assays— on—Demand gene expression product)。 normalization の 7こめのコノトロ1 ~ノレ退 ΐ:κナと して、 16s rRNAを用いた。 PCR条件は 50°C ' 2分を 1サイクル、 95°C · 10分を 1サイクノレ、 その後 95°C · 15秒と 60°C · 1分を 40サイクルとした。 Hepatocytes (H4IIEC) were cultured on a 6-well plate to 80% confluent in DMEM medium, 20% horse serum, 5% FBS. After washing the cells once with PBS, the cells were incubated with gluconeogenic buffer (glucose-free DMEM, 3.7 g / L sodium bicarbonate) and 0.4% FBS for 6 hours for starvation. Thereafter, glucose was added to the medium and incubated for 6 hours, and total RNA was recovered using the Quick gene system (FUJI FILM). To exclude the effect of osmotic pressure, mannitol at the same concentration as glucose was used as a control. 1Sampnole [100 ng of RNA is converted to cDNA using High-capacity cDNA Archive Kit (Applied Biosystems) and real-time PCR is used to detect changes in selenoprotein P gene expression using ABI Prism 7700 Sequence Detection System (Applied Biosystems). Observed by the method. Selenoprotein P primer and TaqMan pro "~" were from Apllied Biosystems (Assays—on-Demand gene expression product). Normalization 7th Conotro 1 ~ Nore ΐ: 16s rRNA PCR conditions were 50 ° C 'for 2 minutes for 1 cycle, 95 ° C · 10 minutes for 1 cycle, and then 95 ° C · 15 seconds and 60 ° C · 1 minute for 40 cycles.
結果を図 6に示す。 図 6に示すように、 グルコースはコントロールであるマン 二トールに比較して有意に肝セレノプロテイン P遺伝子発現を亢進させた。  The result is shown in FIG. As shown in FIG. 6, glucose significantly enhanced hepatic selenoprotein P gene expression compared to mannitol, which is a control.
(3) 肝細胞におけるィンスリン作用におよぼすセレノプロティン Pの影響 次に、 セレノプロテイン Pの機能を解析した。  (3) Effect of selenoprotein P on the insulin action in hepatocytes Next, the function of selenoprotein P was analyzed.
インスリンは肝からの糖新生を抑制することで血糖を制御する。そこで、まず、 • セレノプロティン pが肝におけるインスリン作用に及ぼす効果を評価した。  Insulin controls blood glucose by inhibiting gluconeogenesis from the liver. First, we evaluated the effect of selenoprotein p on insulin action in the liver.
糖新生系を有するラッ ト hepatome由来培養肝細胞である H4IIEC細胞を、 6穴 プレート上で DMEM培地、 20%ゥマ血清、 5 %FBSにて 80%コンフルェント状態ま で培養した。 PBSにて二回細胞を洗った後、糖新生バッファー（glucose-free DMEM， sodium Dicarbonate 3. / g/L, sodium pyruvate 5mM, sodium lactate 50mM, cAMP ImM, dexamethasone 250nM)とセレノプロティン Pまたは対照ならびにィンスリン と共に 24時間培養した。培養液を回収し、上清中に遊離したグルコースの濃度を Glucose Oxidation kit (Sigma)にて測疋 した (Fukuhara A. et al. , Science 307:426-430, 2005)。 培養液回収後、 細胞を RIPAバッファ一にて回収し、 タンパ ク濃度を Lowry法にて測定した。 グルコース濃度をタンパク濃度で除した値を、 肝細胞からのグルコース放出として計算した。 6 wells of rat hepatome-derived cultured hepatocytes with gluconeogenic system The plate was cultured in DMEM medium, 20% horse serum, 5% FBS until 80% confluent. After washing the cells twice with PBS, gluconeogenesis buffer (glucose-free DMEM, sodium dicarbonate 3./g/L, sodium pyruvate 5mM, sodium lactate 50mM, cAMP ImM, dexamethasone 250nM) and selenoprotein P or control and Incubated with insulin for 24 hours. The culture solution was collected, and the concentration of glucose released in the supernatant was measured with a Glucose Oxidation kit (Sigma) (Fukuhara A. et al., Science 307: 426-430, 2005). After recovering the culture solution, the cells were recovered with RIPA buffer and the protein concentration was measured by the Lowry method. The glucose concentration divided by the protein concentration was calculated as glucose release from hepatocytes.
結果を図 7Aおよび Bに示す。図 7Aおよび Bに示すように、インスリンは用量 依存性に肝細胞からの糖新生を抑制した。 10/xg/mLのセレノプロティン P存在下 では、 インスリンによる肝糖新生抑制作用を減弱させた。 このことは、 セレノブ 口ティン Pは肝におけるィンスリン作用を減弱させることを示している。  The results are shown in FIGS. 7A and B. As shown in FIGS. 7A and B, insulin inhibited gluconeogenesis from hepatocytes in a dose-dependent manner. In the presence of 10 / xg / mL selenoprotein P, insulin inhibited the hepatic gluconeogenesis inhibitory effect. This indicates that Serenob Mouth Tin P attenuates the insulin action in the liver.
(4) 肝細胞内ィンスリンシグナルに対するセレノプロティン Pの影響 セレノプロテイン pが、 肝臓におけるインスリン作用を減弱することがわかつ たので、 次に、 肝細胞内インスリンシグナルへの影響を検討した。  (4) Effect of selenoprotein P on hepatic intracellular insulin signal Since selenoprotein p was found to attenuate the insulin action in the liver, we next investigated the effect on hepatocyte insulin signal.
肝細胞（H4IIEC)を 6穴プレート上で DMEM培地、 20%ゥマ血清、 5%FBSにて 80% コンフルェント状態まで培養した。 PBSにて一回細胞を洗った後、 無血清 DMEM培 地とともに、セレノプロテイン P 0、 1、 5、 10/zg/mL存在下で 6時間培養した。 その後、 ヒ トインスリン（ 1 ng/mL)で 37°C15 分間処置し、 SOO^uL/dish の Cell lysis buffer (Tris 20mM, EDTA 5mM, Na4P207 lOmM, NaF lOOmM, NP-40 1 %, Protease inhibitor cocktail (Sigma Chemicals), Na3V04 2 mM)を用いて細胞を回収した。 超音波破砕装置（BI0RUPT0R COSMO BI0)を用いて細胞を破砕した後、 15000rpm でHepatocytes (H4IIEC) were cultured on a 6-well plate to 80% confluent in DMEM medium, 20% horse serum, 5% FBS. After washing the cells once with PBS, the cells were cultured with serum-free DMEM medium for 6 hours in the presence of selenoprotein P 0, 1, 5, 10 / zg / mL. Thereafter, treatment with human insulin (1 ng / mL) at 37 ° C for 15 minutes, Cell lysis buffer of SOO ^ uL / dish (Tris 20mM, EDTA 5mM, Na 4 P 2 0 7 lOmM, NaF lOOmM, NP-40 1 %, Protease inhibitor cocktail (Sigma Chemicals), Na 3 V0 4 2 mM). After disrupting the cells using an ultrasonic disrupter (BI0RUPT0R COSMO BI0),
10分間遠心分離し上清を新しいチューブへ回収した。等量のタンパクを 4〜20%Centrifugation was performed for 10 minutes, and the supernatant was collected in a new tube. 4-20% equivalent protein
SDS-PAGE にて分離し、 polyvinylidene difluoride membranes (PVDMs)の上へSeparate by SDS-PAGE and onto polyvinylidene difluoride membranes (PVDMs)
Bio-Rad Transblot 装置を用いて転写した。 メンブレンをブロッキングバッファ 一（Tris 50mM， NaCl 150mM, 0.1% Tween 20, 5 % non-fat milk)で室温 1時間 プロッキングし、その後それぞれの一次抗体で室温 1時間もしくは 4°C16時間プ ローブした。 メンブレンをバッファー （Tris 50mM, NaCl 150mM, 0.1% Tween 20) で洗浄した後、 horseradish peroxidase 結合二次抗体とインキュベートし、 enhanced chemi luminescence reagent (ECL plus Amershamノを用！/ヽ"！ィ匕^"^ e光反 応を評価した。 Transcription was performed using a Bio-Rad Transblot instrument. The membrane was probed with blocking buffer (Tris 50 mM, NaCl 150 mM, 0.1% Tween 20, 5% non-fat milk) for 1 hour at room temperature, and then probed with each primary antibody for 1 hour at room temperature or 4 ° C for 16 hours. Membrane as buffer (Tris 50mM, NaCl 150mM, 0.1% Tween 20) After washing with horseradish peroxidase-conjugated secondary antibody, enhanced chemi luminescence reagent (using ECL plus Amersham! /！ "! 匕 ^" ^ e photoreaction was evaluated.
結果を図 8に示す。図 8に示すように、セレノプロティン Pは用量依存性に Akt のセリンリン酸化を阻害した。ヒ ト正常血中セレノプロテイン P濃度は 5.3/ig/mL と報告されていることから (Takahashi et al. , Journal of Health Science 47, Pages 346-352， 2001)、 セレノプロテイン Pはヒ ト正常血中濃度に極めて近い濃 度で、 肝におけるィンスリン作用を抑制しうることが判明した。  The results are shown in FIG. As shown in FIG. 8, selenoprotein P inhibited serine phosphorylation of Akt in a dose-dependent manner. Since selenoprotein P concentration in normal human blood is reported to be 5.3 / ig / mL (Takahashi et al., Journal of Health Science 47, Pages 346-352, 2001), selenoprotein P is normal human blood. It was found that concentrations close to medium levels can suppress the insulin action in the liver.
(5) セレノプロテイン Pが仔胎マウス血管新生に与える影響  (5) Effects of selenoprotein P on neonatal mouse neovascularization
長期間にわたる糖尿病の持続は網膜、 腎臓、 神経の細小血管障害、 および虚血 性心疾患や脳梗塞といった動脈硬化性疾患を引き起こす。 そこで、 これらの血管 合併症の病態に及ぼすセレノプロテイン Pの作用をまず試験管内の血管系細胞を 用いて検討した。 実験には、 マウス胎仔器官培養系（Takakura et al. , Cell 102： 199-20, 2000)を用いた。  Prolonged diabetes causes retinal, renal, neuronal microvascular disorders, and arteriosclerotic diseases such as ischemic heart disease and cerebral infarction. Therefore, we first examined the effect of selenoprotein P on the pathogenesis of these vascular complications using vascular cells in vitro. In the experiment, a mouse fetal organ culture system (Takakura et al., Cell 102: 199-20, 2000) was used.
ス トローマ細胞株である 0P9(Nakano T. et al. , Science 265: 1098-1101, 1994) を alpha— modi 1 led minimum essential media ( a—MEM Gibco BRい、 20% Fetal-calf serum (FCS; JRH Bioscience)にて培養した。 妊娠 8· 5 日目のマウスから胎仔を摘 出し、血管系幹細胞の起源のひとつとされるパラ大動脈臓側中胚葉（PAS)領域を臍 腸管動脈（omphalomesenteric artery)を含むように取り出し、 10%FCS、 10"5M 2 -メルカプトエタノール（2 - ME; Gibco BRL)、 RPMI 1640 (Gibco BRL)の中の 0P9 細胞上で 14日間培養した（Takakura Τ· et al. , Cell 102:199-209, 2000)。 培養 7日目からセレノプロテイン Pを 5、 25/ g/mLで投与し、 血管系細胞の発生を検 討した。血管内皮細胞を CD31抗体（MEC13.3 rat anti mouse monoclonal antibody; Pharmingen, San Diego, CA)を用いて青色に、 血管壁細胞を alpha- SMA 抗体 (Anti-Human Smooth Muscle Actin/HRP Dako Cytomation)を用レヽて茶色に免疫染 色した。 The stromal cell line 0P9 (Nakano T. et al., Science 265: 1098-1101, 1994) was added to alpha— modi 1 led minimum essential media (a—MEM Gibco BR, 20% Fetal-calf serum (FCS; The fetus was removed from the 8th and 5th day of gestation, and the paraaortic mesoderm (PAS) region, which is one of the origins of vascular stem cells, was isolated from the umphalomesenteric artery. And cultured for 14 days on 0P9 cells in 10% FCS, 10 " 5 M 2 -mercaptoethanol (2-ME; Gibco BRL), RPMI 1640 (Gibco BRL) (Takakura Τ et al , Cell 102: 199-209, 2000) From day 7 of culture, selenoprotein P was administered at 5 and 25 / g / mL to examine the development of vascular cells. .3 rat anti mouse monoclonal antibody; Pharmingen, San Diego, Calif.) In blue and vascular wall cells with alpha-SMA antibody (Anti-Human Smooth Muscle Actin / HRP Dako Cytomation) After use, it was immunostained brown.
結果を図 9に示す。 図 9に示すように、 セレノプロテイン Pは血管内皮細胞の 発生 ·増殖を有意に抑制し、 一方で血管壁細胞の発生 ·増殖を強力に誘導した。 実施例 3 個体レベルでの機能解析 ( 1 ) セレノプロテイン P投与がマウス耐糖能に与える影響 The results are shown in FIG. As shown in Fig. 9, selenoprotein P significantly suppressed the development and proliferation of vascular endothelial cells, while strongly inducing the development and proliferation of vascular wall cells. Example 3 Functional analysis at the individual level (1) Effect of selenoprotein P administration on glucose tolerance in mice
セレノプロテイン pが試験管内で、 肝細胞におけるインスリン作用を障害する ことがわかったので、 次に個体レベルでの作用を検討した。  Since selenoprotein p was found to impair insulin action in hepatocytes in vitro, we next examined its action at the individual level.
1グループにっき 5匹の 10週齢雌 C57BL/6Jマウスに対し、 セレノプロテイン P 1 μ g /gBW もしくはべヒクル （vehicle) を午前 7時と午後 7時の二回腹腔内 投与した。 マウスは一回目の注射後から絶食とした。 二回目の注射の 2時間後、 1.5m g /gBWのグルコースを腹腔内投与し、血糖値を 0から 120分において測定し た。 血糖値は FreeStyle (キツセィ 日本）を用いて測定した。  Selenoprotein P 1 μg / gBW or vehicle was administered intraperitoneally twice a day at 7 am and 7 pm to five 10-week-old female C57BL / 6J mice per group. Mice were fasted after the first injection. Two hours after the second injection, 1.5 mg / gBW of glucose was intraperitoneally administered, and blood glucose levels were measured from 0 to 120 minutes. Blood glucose levels were measured using FreeStyle (Kitssey Japan).
結果を図 1 0に示す。 図 1 0に示すように、 コントロールマウスと比較して、 セレノプロテイン P投与マウスでは、糖負荷後 15、 30分後の血糖が有意に高値を 示した。 すなわち、 個体レベルで、 セレノプロテイン Pが糖負荷後高血糖を誘導 しうることが明らかとなった。  The results are shown in FIG. As shown in FIG. 10, selenoprotein P-administered mice showed significantly higher blood glucose levels at 15 and 30 minutes after glucose loading as compared to control mice. That is, it became clear that selenoprotein P can induce hyperglycemia after glucose load at the individual level.
( 2) セレノプロテイン P投与がマウスのインスリン感受性に与える影響 セレノプロテイン Pがマウスに糖負荷後高血糖をもたらした機序を探るため、 本タンパクがマウスのィンスリン感受性に与える影響を検討した。  (2) Effect of selenoprotein P administration on insulin sensitivity in mice To investigate the mechanism of selenoprotein P causing hyperglycemia after glucose loading in mice, the effect of this protein on insulin sensitivity in mice was examined.
1グループにっき 5匹の 10週齢雌 C57BL/6Jマウスに対し、 セレノプロテイン Serenoprotein for 5 10-week-old female C57BL / 6J mice per group
P 1 μ g /gBW もしくはべヒクル （vehicle) を午前 7時と午後 7時の二回腹腔内 投与した。 マウスは二回目の注射の 2時間前から絶食とした。 合計 4時間の絶食 の後、 0.5mU/gBWのヒ トインスリン（Humalin R (登録商標）、 Lilly Japan)を腹腔 内投与し、 血糖値を 0から 120分において測定した。 血糖値は FreeStyle (キツセ ィ、 日本）を用いて測定した。 P 1 μg / gBW or vehicle was administered intraperitoneally twice at 7 am and 7 pm. Mice were fasted 2 hours before the second injection. After a total of 4 hours of fasting, 0.5 mU / gBW human insulin (Humalin R (registered trademark), Lilly Japan) was administered intraperitoneally, and blood glucose levels were measured from 0 to 120 minutes. Blood glucose levels were measured using FreeStyle (Kitssey, Japan).
結果を図 1 1に示す。 図 1 1に示すように、 コントロールマウスと比較して、 セレノプロテイン P投与マウスでは、糖負荷後 15、 30分後の血糖降下率が有意に 高値であった。 したがって、 セレノプロテイン Pはマウスにインスリン抵抗性を 誘導することにより、 糖負荷後高血糖をもたらすことが明らかとなった。  The results are shown in Figure 11. As shown in Fig. 11, the blood glucose lowering rate was significantly higher in selenoprotein P-administered mice at 15 and 30 minutes after glucose loading compared to control mice. Thus, selenoprotein P has been shown to induce hyperglycemia after glucose load by inducing insulin resistance in mice.
実施例 4 ヒ ト血中セレノプロテイン P濃度測定 Example 4 Measurement of serum selenoprotein P concentration
金沢大学医学部附属病院代謝内科に通院または入院した 2型糖尿病患者 16人 16 patients with type 2 diabetes who were admitted to or admitted to the Department of Metabolism, Kanazawa University Hospital
(年齢 59±14歳， BMI 25.2±4.6 kg/m2, 空腹時血糖 184±50 mg/dL， HbAlc 10.4(Age 59 ± 14 years, BMI 25.2 ± 4.6 kg / m 2 , fasting blood glucose 184 ± 50 mg / dL, HbAlc 10.4
±3.2%)、耐糖能正常者 8人（年齢 42±15歳， BMI 23.2±3.5 kg/m 2， 空腹時血糖 93 ±9 mg/dL)を対象とした。 空腹状態にて採血を施行し、 採取した血清のセレノプ ロティン P濃度を 2種のセレノプロティン Pモノクローナル抗体によるサンドィ ツチ ELISA法を用いて測定した（Saito Y et al. , J Health Sci 2001 ;47:346; Akira Andoh et al. , Nutrition 21 (2005) 574-579)。 抗体は北海道薬科大学高橋和彦 教授より供与を受けた。 耐糖能正常患者のインス リ ン抵抗性を、 下記のごとく homeostasis model for insulin resistance (H0MA— IR)を算出 1—ることによって 評価した（Matthews DR et al.， Diabetologia 28:412-419， 1985)。 2型糖尿病患 者のィンスリン抵抗性は、下記のごとく quantitative insulin sensitivity check index (QUICKI)を算出することによって評価した（Arie Katz et al.， J Clin Endocrinol Metab 85:2402-2410)。 ± 3.2%), normal glucose tolerance 8 people (age 42 ± 15 years, BMI 23.2 ± 3.5 kg / m 2 , fasting blood glucose 93 ± 9 mg / dL). Blood was collected in the fasted state, and the selenoprotein P concentration of the collected sera was measured using a sandwich ELISA method with two selenoprotein P monoclonal antibodies (Saito Y et al., J Health Sci 2001; 47: 346; Akira Andoh et al., Nutrition 21 (2005) 574-579). The antibody was provided by Professor Kazuhiko Takahashi, Hokkaido Pharmaceutical University. Insulin resistance in patients with normal glucose tolerance was evaluated by calculating the homeostasis model for insulin resistance (H0MA—IR) 1 as follows (Matthews DR et al., Diabetologia 28: 412-419, 1985). . Insulin resistance in patients with type 2 diabetes was evaluated by calculating quantitative insulin sensitivity check index (QUICKI) as follows (Arie Katz et al., J Clin Endocrinol Metab 85: 2402-2410).
H0MA- IR=空腹時血糖値 （mg/dL) X空腹時の血中インスリ ン濃度 （ // U/ml) /405 QUICKI=l/[log 空腹時の血中イ ンスリ ン濃度（ μ U/ml)+log 空腹時血糖値 (mg/dL) ]  H0MA- IR = Fasting blood glucose level (mg / dL) X Fasting blood insulin concentration (// U / ml) / 405 QUICKI = l / [log Fasting blood insulin concentration (μ U / ml) + log Fasting blood glucose level (mg / dL)]
( 1 ) ヒ ト血清におけるセレノプロテイン P濃度測定  (1) Measurement of selenoprotein P concentration in human serum
(i) 2型糖尿病患者における血清セレノプロテイン P濃度  (i) Serum selenoprotein P concentration in patients with type 2 diabetes
2型糖尿病患者 16人において血清セレノプロテイン P濃度を測定した結果、そ の平均濃度は 5.28±1.3μ g/mLであった。 患者のセレノプロテイン P値は BMI と 正に、 QUICKI と負に相関した（図 1 2 及び¾。 As a result of measuring serum selenoprotein P concentration in 16 patients with type 2 diabetes, the mean concentration was 5.28 ± 1.3 μg / mL. The patient's selenoprotein P level was positively correlated with BMI and negatively correlated with QUICKI (Figs. 12 and ¾).
(ii) 2型糖尿病患者における肥満の有無による血清セレノプロテイン P濃度の比 較  (ii) Comparison of serum selenoprotein P concentration with and without obesity in patients with type 2 diabetes
2型糖尿病患者における血清セレノプロティン P濃度を、 肥満の有無にて比較 検討した。 BMI 25以上を肥満、 25未満を非肥満群としたところ、 血清セレノプロ ティン P値は肥満群で 5.9±1. l g/mL、 非肥満群で 4.4 // g/mLであり、 肥満群に て有意に高値であった（Ρ=0· 029) (図 1 3)。  Serum selenoprotein P concentrations in patients with type 2 diabetes were compared and examined with and without obesity. Serum selenoprotein P levels were 5.9 ± 1 lg / mL in the obese group and 4.4 // g / mL in the non-obese group. The value was significantly high (Ρ = 0 · 029) (Fig. 13).
(iii) 耐糖能正常者における血清セレノプロティン P濃度と体重 · H0MA-IR の相 1*1  (iii) Serum Selenoprotein P Concentration and Body Weight in Normal Glucose Tolerant Phase 1 of H0MA-IR
耐糖能正常者 8人において血清セレノプロテイン P濃度を測定した結果、 その 平均濃度は 6. l±0.8/zg/mLであった。耐糖能正常者のセレノプロティン P値も糖 尿病患者のそれと同様に、体重および H0MA-I Rと正に相関する傾向を示した（図 1 4 Aおよび B)。 As a result of measuring serum selenoprotein P concentration in 8 subjects with normal glucose tolerance, the average concentration was 6. l ± 0.8 / zg / mL. The selenoprotein P value of normal glucose tolerance showed a tendency to positively correlate with body weight and H0MA-IR, similar to that of patients with glucoseuria (Fig. 1). 4 A and B).
以上の結果から、 血清セレノプロテイン P値は肥満 ' インスリン抵抗性を強く 反映する臨床マーカーであることが判明した。 さらに、 血清セレノプロテイン P 値と肥満 · インスリン抵抗性との相関が 2型糖尿病患者と耐糖能正常者の両者に おいて認められた。 細胞および動物実験の結果からセレノプロティン Pがィンス リン抵抗性原因ホルモンのひとつであると考えられることより、 ヒ ト血清セレノ プロテイン P値測定は 2型糖尿病患者においては新規インスリン抵抗性評価マ一 カーに、 耐糖能正常患者においては 2型糖尿病発症の予知マーカーになり うる。 実施例 5 肝臓由来セレノプロテイン Pをターゲッ トとした 2型糖尿病に対する 新規遺伝子治療  From the above results, it was found that serum selenoprotein P level is a clinical marker that strongly reflects obesity and insulin resistance. In addition, a correlation between serum selenoprotein P levels and obesity / insulin resistance was observed in both type 2 diabetics and normal glucose tolerance. Based on the results of cell and animal experiments, selenoprotein P is considered to be one of the insulin resistance-causing hormones. Therefore, human serum selenoprotein P level measurement is a novel marker for evaluating insulin resistance in patients with type 2 diabetes. In addition, it can be a predictive marker for the onset of type 2 diabetes in patients with normal glucose tolerance. Example 5 Novel gene therapy for type 2 diabetes targeting liver-derived selenoprotein P
これまでの投与実験の結果から、 セレノプロティン Pが 2型糖尿病におけるィ ンスリン抵抗性 ·高血糖の原因ホルモンのひとつであることが明らかとなった。 そこで、 short interference RNA (s iRNA)を用いた肝臓におけるセレノプロティ ン P産生の抑制が 2型糖尿病に対する新たな治療になり うるかを検討した。  From the results of previous administration experiments, it was revealed that selenoprotein P is one of the hormones that cause insulin resistance and hyperglycemia in type 2 diabetes. Therefore, we examined whether suppression of selenoprotein P production in the liver using short interference RNA (siRNA) could be a new treatment for type 2 diabetes.
自然発症肥満 2型糖尿病モデル動物である KKAyマウス（ 7〜8週令、 体重 31〜 Spontaneously obese type 2 diabetes model animal KKAy mice (7-8 weeks old, weight 31-
33 g)に対し、 hydrodynamic法を用いてセレノプロティン Pに特異的な s iRNAを 遺伝子導入した。方法は、 Zender, L. et al. Proc Natl Acad Sc i U S A 100, 7797-802For 33 g), siRNA specific to selenoprotein P was introduced using the hydrodynamic method. Zender, L. et al. Proc Natl Acad Sci U S A 100, 7797-802
(2003)および McCaffrey, A. P. et al . Nature 418, 38-9 (2002)に記載の方法 に従った。 動物導入用の s iRNAは Amb ionから購入した（Si l encer (登録商標） In(2003) and McCaffrey, A. P. et al. Nature 418, 38-9 (2002). SiRNA for animal introduction was purchased from Amb ion (Silencer® In
Vivo Ready Pre— des i gned s iRNA)。 セレノプロティン Pをノックダウンする s iRNA は以下の配列のごとく合成した ： Mice Seppl : 5 ' - GGUGUCAGAACACAUCGCAtt - 3 'Vivo Ready Pre—des i gned s iRNA). The s iRNA that knocks down selenoprotein P was synthesized as follows: Mice Seppl: 5 '-GGUGUCAGAACACAUCGCAtt-3'
(sense) (ttを除いた酉己列を配列番号 3に示す）。ネガティブコントロールの s iRNA は Amb ionより購入した。 ネガティブコントローノレの s iRNAはマウス、 ラッ トおよ びヒ トにおけるいずれの既知遺伝子に対しても有意な相同性を有さない。 ベント バルビツールによってマウスに全身麻酔をほどこした後、 2nmolの s iRNAを 3. OmL の PBSに溶解し、 尾静脈を介して約 15〜20秒間で投与した。 s iRNA投与 2〜7 日 後に 12 時間の絶食後糖負荷試験およびインスリ ン負荷試験をおこなった。 0. 3 mg/g body weight のグノレコースもしくは 4 U/kg body wei ght のィンスジ ンを月复 腔内投与し、 15、 30、 60および 120分後に血糖値を測定した。 セレノプロテイン P遺伝子特異的な s iRNAを経尾静脈から急速投与した結果、 肝でのセレノプロテイン P遺伝子発現は約 80%に減弱した（図 1 5 )。 血中セレノ プロテイン P濃度も同様に有意に低下した （図 1 6 )。 肝でのセレノプロテイン P 遺伝子をノックダウンした KKAy マウスでは空腹時血糖値には変動を認めなかつ たが（図 1 7 )、 糖負荷試験にて耐糖能異常の改善を（図 1 8 )、 インスリ ン負荷試 験にてィンスリン抵抗性の改善を認めた（図 1 9 )。 (sense) (The sequence of self-excluding tt is shown in SEQ ID NO: 3). Negative control siRNA was purchased from Amb ion. The negative control siRNA does not have significant homology to any known gene in mouse, rat or human. After general anesthesia of the mice with Bent Barbitool, 2 nmol of siRNA was dissolved in 3. OmL of PBS and administered via the tail vein for about 15-20 seconds. s A 12-hour post-fasting glucose tolerance test and an insulin tolerance test were performed 2-7 days after siRNA administration. 0. 3 mg / g body weight of gnolecose or 4 U / kg body wei ght of the engine was administered intrathecally and blood glucose was measured 15, 30, 60 and 120 minutes later. As a result of rapid administration of selenoprotein P gene-specific siRNA from the tail vein, selenoprotein P gene expression in the liver was attenuated to about 80% (Fig. 15). The blood selenoprotein P concentration was also significantly decreased (Fig. 16). KKAy mice knocked down the selenoprotein P gene in the liver showed no change in fasting blood glucose levels (Fig. 17), but the glucose tolerance test improved the glucose tolerance test (Fig. 18). In the load test, an improvement in insulin resistance was observed (Fig. 19).
hydrodynami c法を用いた遺伝子導入によって、肝臓におけるセレノプロティン P産生の抑制が全身のィンスリン抵抗性を改善し、 2型糖尿病における負荷後高血 糖を軽減させることが明らかとなった。 以上の結果から、 肝臓由来セレノプロテ イン Pは 2型糖尿病の重要な治療標的であることが証明された。 さらに肝臓由来 セレノプロテイン Pは、 動脈硬化症、 メタボリックシンドロームに代表されるそ の他のインスリ ン抵抗性関連疾患に対しても治療標的となり うる可能性があるこ とが示された。  Gene transfer using the hydrodynami c method revealed that suppression of selenoprotein P production in the liver improved systemic insulin resistance and reduced post-load hyperglycemia in type 2 diabetes. These results demonstrate that liver-derived selenoprotein P is an important therapeutic target for type 2 diabetes. Furthermore, it has been shown that liver-derived selenoprotein P may be a therapeutic target for other insulin resistance-related diseases such as arteriosclerosis and metabolic syndrome.
産業上の利用可能性 Industrial applicability
セレノプロテイン Pがィンスリン抵抗性および血管內皮障害の原因ホルモンの ひとつであり、 セレノプロテイン P濃度の測定は、 全身のインスリン抵抗性また は血管障害進展へのリスクを反映する新たな臨床マーカーとして有用である。 セ レノプロテイン Pをマーカーとして、 2型糖尿病や動脈硬化性疾患等の疾患の病 態を判定することが可能である。  Selenoprotein P is one of the causative hormones for insulin resistance and vascular scab damage, and measurement of selenoprotein P concentration is useful as a new clinical marker that reflects the risk of systemic insulin resistance or the development of vascular disorders. is there. Using selenoprotein P as a marker, it is possible to determine the pathology of diseases such as type 2 diabetes and arteriosclerotic diseases.
また、 セレノプロテイン Pは、 血管系細胞にも直接作用を有することから、 こ れらのセレノプロティン Pを含む医薬、あるいはセレノプロティン P抑制療法は、 血管障害に対する画期的な治療法となり得る。  In addition, since selenoprotein P has a direct action on vascular cells, drugs containing these selenoprotein P or selenoprotein P inhibitory therapy can be a breakthrough treatment for vascular disorders.
さらに、 セレノプロテイン Pは、 2型糖尿病および動脈硬化病変の治療標的と なり得る。 特に、 糖尿病患者においては、 高血糖、 肝からのセレノプロテイン P 分泌亢進、 全身のインスリ ン抵抗性悪化、 さらなる高血糖という悪循環が生じて いる可能性があり、 血中セレノプロテイン P濃度を適正レベルまで低下させるこ とで、 糖毒性にともなうこれらの病態を劇的に改善させ得る。  In addition, selenoprotein P may be a therapeutic target for type 2 diabetes and atherosclerotic lesions. Particularly in diabetic patients, there is a possibility that a vicious circle of hyperglycemia, increased secretion of selenoprotein P from the liver, deterioration of insulin resistance throughout the body, and further hyperglycemia may occur. Can be dramatically improved in the pathology associated with glycotoxicity.
本明細書で引用した全ての刊行物、 特許および特許出願をそのまま参考として 本明細書にとり入れるものとする。  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

請求の範囲 The scope of the claims
1. セレノプロテイン Pを測定することを含む、 2型糖尿病または血管障害 の検出方法。 1. A method for detecting type 2 diabetes or vascular disorder, comprising measuring selenoprotein P.
2. セレノプロテイン Pを測定することを含む、 2型糖尿病または血管障害 に罹患するリスクを評価する方法。  2. A method of assessing the risk of suffering from type 2 diabetes or vascular disorders, including measuring selenoprotein P.
3. 血管障害が動脈硬化性疾患である請求項 1または 2に記載の方法。  3. The method according to claim 1 or 2, wherein the vascular disorder is an arteriosclerotic disease.
4. セレノプロテイン Pを測定することを含む、 被験体のインス.リン抵抗性 または血糖コントロールを評価する方法。  4. A method for assessing a subject's insulin resistance or glycemic control, comprising measuring selenoprotein P.
5. セレノプロテイン Pを測定することを含む、 2型糖尿病患者におけるィ ンスリン抵抗性を評価する方法。  5. A method for assessing insulin resistance in patients with type 2 diabetes, comprising measuring selenoprotein P.
6. セレノプロテイン Pを測定することを含む、 耐糖能正常者における 2型 糖尿病発症のリスクを評価する方法。  6. A method for assessing the risk of developing type 2 diabetes in a person with normal glucose tolerance, comprising measuring selenoprotein P.
7. セレノプロテイン Pからなる、 2型糖尿病または血管障害検出用マーカ  7. Marker for detecting type 2 diabetes or vascular disorder, consisting of selenoprotein P
8. セレノプロテイン Pからなる、 2型糖尿病または血管障害の罹患リスク 評価用マーカー。 8. A marker for evaluating the risk of morbidity of type 2 diabetes or vascular disorders, comprising selenoprotein P.
9. 血管障害が動脈硬化性疾患である請求項 7または 8に記載のマーカー。 9. The marker according to claim 7 or 8, wherein the vascular disorder is an arteriosclerotic disease.
1 0. セレノプロテイン Pからなる、 インスリン抵抗性または血糖コント口 ール評価用マーカー。 1 0. A marker for evaluating insulin resistance or glycemic control, comprising selenoprotein P.
1 1. セレノプロテイン Pの、 2型糖尿病または血管障害検出用マーカーと しての使用。  1 1. Use of selenoprotein P as a marker for detecting type 2 diabetes or vascular disorders.
1 2. セレノプロテイン Pの、 2型糖尿病または血管障害の罹患リスク評価 用マーカーとしての使用。  1 2. Use of selenoprotein P as a marker for risk assessment of type 2 diabetes or vascular disorder.
1 3. 血管障害が動脈硬化性疾患である請求項 1 1または 1 2に記載の使用。 1 3. Use according to claim 1 1 or 12 wherein the vascular disorder is an arteriosclerotic disease.
1 4. セレノプロテイン Pの、 インスリン抵抗性または血糖コントロール評 価用マーカーとしての使用。 1 4. Use of selenoprotein P as a marker for insulin resistance or glycemic control evaluation.
1 5. 2型糖尿病または血管障害の予防または治療薬をスク リーニングする 方法であって、 候補化合物のセレノプロテイン Pの発現または作用を抑制する能 力を指標に、 予防または治療薬として選択することを含むスクリーニング方法。1 5. A method for screening a prophylactic or therapeutic agent for type 2 diabetes or vascular disorder, which is capable of suppressing the expression or action of the candidate compound selenoprotein P A screening method comprising selecting as a preventive or therapeutic agent using force as an index.
1 6 . 血管障害が動脈硬化性疾患である請求項 1 5に記載のスクリーユング 方法。 16. The screening method according to claim 15, wherein the vascular disorder is an arteriosclerotic disease.
1 7 . セレノプロテイン Pまたはセレノプロテイン Pをコードする核酸を有 効成分として含む血管内皮細胞の増殖または血管壁細胞の増殖低下に関連する疾 患の予防または治療剤。  1 7. A prophylactic or therapeutic agent for a disease associated with vascular endothelial cell proliferation or vascular wall cell proliferation reduction comprising selenoprotein P or a nucleic acid encoding selenoprotein P as an active ingredient.
1 8 . 血管内皮細胞の増殖または血管壁細胞の増殖低下に関連する疾患が糖 尿病性網膜症、 動脈硬化性疾患または癌である請求項 1 7記載の予防または治療 剤。  18. The prophylactic or therapeutic agent according to claim 17, wherein the disease associated with the proliferation of vascular endothelial cells or the decrease in the proliferation of vascular wall cells is glycouria retinopathy, arteriosclerotic disease or cancer.
1 9 . 抗セレノプロテイン P抗体を有効成分として含む 2型糖尿病または血 管障害の予防または治療剤。  1 9. A preventive or therapeutic agent for type 2 diabetes or vascular disorder containing anti-selenoprotein P antibody as an active ingredient.
2 0 . セレノプロテイン Pをコードする遺伝子の発現を抑制する 2本鎖 RNA を有効成分として含む 2型糖尿病または血管障害の予防または治療剤。  2 0. A prophylactic or therapeutic agent for type 2 diabetes or vascular disorder containing double-stranded RNA as an active ingredient that suppresses the expression of a gene encoding selenoprotein P
2 1 . 2本鎖 RNAが、 配列番号 1に示すセレノプロテイン Pをコードする塩 基配列の連続する 15〜50塩基からなる塩基配列と相同な配列からなるセンス RNA および該センス RNA に相補的な配列からなるアンチセンス RNA からなる 2本鎖 RNAである、請求項 2 0に記載の 2型糖尿病または血管障害の予防または治療剤。  2 1. Double-stranded RNA is a sense RNA consisting of a sequence homologous to a base sequence consisting of 15 to 50 bases of the base sequence encoding selenoprotein P shown in SEQ ID NO: 1, and complementary to the sense RNA. The preventive or therapeutic agent for type 2 diabetes or vascular disorder according to claim 20, which is a double-stranded RNA consisting of an antisense RNA consisting of a sequence.
2 2 . センス RNAが配列番号 3で示される塩基配列からなる、 請求項 2 1記 載の 2型糖尿病または血管障害の予防または治療剤。  2 2. The preventive or therapeutic agent for type 2 diabetes or vascular disorder according to claim 21, wherein the sense RNA comprises the base sequence represented by SEQ ID NO: 3.
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