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WO2004040301A1 - Methode de diagnostic du diabete de type 2 - Google Patents

Methode de diagnostic du diabete de type 2 Download PDF

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Publication number
WO2004040301A1
WO2004040301A1 PCT/JP2002/011306 JP0211306W WO2004040301A1 WO 2004040301 A1 WO2004040301 A1 WO 2004040301A1 JP 0211306 W JP0211306 W JP 0211306W WO 2004040301 A1 WO2004040301 A1 WO 2004040301A1
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WIPO (PCT)
Prior art keywords
diabetes
irs
gene
type
capn10
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PCT/JP2002/011306
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English (en)
Japanese (ja)
Inventor
Hideyoshi Harashima
Hiroyuki Kamiya
Masamichi Kuwajima
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Hokkaido Technology Licensing Office Co Ltd
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Hokkaido Technology Licensing Office Co Ltd
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Priority to AU2002344611A priority Critical patent/AU2002344611A1/en
Priority to PCT/JP2002/011306 priority patent/WO2004040301A1/fr
Priority to JP2004547992A priority patent/JP4214235B2/ja
Publication of WO2004040301A1 publication Critical patent/WO2004040301A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for diagnosing type 2 diabetes and a diagnostic kit, a method for screening a substance having a preventive / therapeutic effect for type 2 diabetes, and a screenin.
  • Diabetes is broadly classified into type 1 and type 2 based on its etiology, but most of the diabetic patients are occupied by type 2 diabetic patients, and overcoming it is one of the major challenges for humankind.
  • Type 1 diabetes is a disease in which the islets of Langerhans are inflamed and the ability of jS cells to secrete insulin is significantly reduced, and the disease is an insulin-dependent disease that cannot survive without supplementing insulin.
  • Type 2 diabetes In type 2 diabetes, insufficient action of insulin appears due to other causes, resulting in hyperglycemia.Environmental factors such as obesity, overeating, lack of exercise, stress, etc. are greatly involved, and relatively elderly people after middle age It is easy to develop in obese people. Type 2 diabetes generally presents a non-insulin-dependent pathology, with diet and exercise therapies being the basis of treatment. Drug therapy is the next stage of diet and exercise therapy, and insulin therapy is used if treatment is still difficult.
  • Symptoms such as polydipsia, polyuria and nocturnal urine are seen at the onset of diabetes, but few patients are aware of these initial symptoms, and many patients are associated with complications Diabetes is imperceptible because symptoms cannot be realized until symptoms appear, and when it is discovered, complications appear and treatment is often extremely difficult. Therefore, early detection and early treatment are extremely important for overcoming diabetes.However, since the pathogenesis of type 2 diabetes is still unclear compared to type 1 diabetes, early detection and early treatment are difficult. May be. Therefore, various genetic approaches have been taken to elucidate the pathogenesis of type 2 diabetes. Recently, SNP s analysis in diabetic patients ⁇ Inherited nucleotide sequence abnormality has been found in genes by haplotype analysis Is becoming clearer.
  • Gene expression analysis analyzes the expression state (phenotype) of a gene, and is essentially different from SNP s analysis, etc., which analyzes congenital abnormalities of a gene (genotype). It is also advantageous in that the current situation of patients taking into account environmental factors can be ascertained.
  • the present invention firstly makes it possible to diagnose type 2 diabetes by analyzing gene expression in tissues that are easily sampled (especially before type 2 diabetes onset, is it possible to develop type 2 diabetes in the future? It is possible to provide a diagnostic method and a diagnostic kit for type 2 diabetes.
  • the present invention provides, secondly, a substance having a type 2 diabetes preventive / therapeutic effect, which can screen a substance having a type 2 diabetes preventive / therapeutic effect by analyzing gene expression in a tissue which is easily sampled.
  • An object of the present invention is to provide a screening method and a screening kit.
  • the present invention provides the following method for diagnosing type 2 diabetes and a diagnostic kit, and a method for screening a substance having a preventive / therapeutic effect for type 2 diabetes and a kit for screening.
  • a method for diagnosing type 2 diabetes which comprises diagnosing type 2 diabetes using the expression level of CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the subject as an index.
  • the expression level is determined based on the abundance of CAP N 10 or IRS-1.
  • a kit for diagnosing type 2 diabetes comprising an oligonucleotide or a polynucleotide capable of hybridizing to a nucleic acid encoding CAPN10 or IRS-1.
  • a kit for diagnosing type 2 diabetes comprising an antibody capable of reacting with CAPN10 or IRS-1 or a fragment thereof.
  • the type 2 of the candidate substance After administering the candidate substance to a type 2 diabetes model animal, using the index of the effect of improving the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the animal as an index, the type 2 of the candidate substance A method for screening a substance having a preventive and therapeutic effect on type 2 diabetes, which comprises determining the preventive and therapeutic effects of diabetes.
  • a kit for screening a substance having a type 2 diabetes preventive / therapeutic effect comprising an oligonucleotide or a polynucleotide capable of hybridizing to a nucleic acid encoding CAPN10 or IRS-1.
  • kits for screening a substance having a preventive / therapeutic effect for type 2 diabetes comprising an antibody or a fragment thereof that can react with CAPN10 or IRS-1.
  • Figure 1 shows changes in body weight of OLETF rats and LETO rats. is there.
  • FIG. 2 is a graph showing changes in glucose tolerance of OLETF rats and LET0 rats.
  • FIG. 3 shows IR (Fig. 3A), SHIP2 (Fig. 3B), PPARa (Fig. 3C), CAPN10 (Fig. 3D) and IRS-1 in leukocytes of OLETF rats and LETO rats.
  • FIG. 3E shows the results of mRNA expression analysis of (FIG. 3E), in which white (mouth) indicates the results of LETO rats, and black ( ⁇ ) indicates the results of OLE.
  • the method for diagnosing type 2 diabetes of the present invention is characterized in that diagnosis of type 2 diabetes is performed using the expression level of the CAPN10 gene or the IRS-1 gene in leukocytes collected from the blood of the subject as an index.
  • the method for collecting white blood cells from the blood of the subject is not particularly limited.
  • white blood cells can be collected by selectively dissolving red blood cells in blood and then centrifuging.
  • Leukocytes include neutrophils, eosinophils, basophils, lymphocytes and monocytes, and one or more of these may be used as a specimen. It may be a mixture of
  • the CAPN10 gene is a gene encoding CAPN10 (CalpainlO, calpain10).
  • CAPN10 is a cysteine protease that is non-specifically expressed.
  • CAPN10 does not have the calcium-binding domain that calpain normally has, but instead has a T domain (Horikawa, Y. et al., Nat Genet, 2000. 26 (2) p .163-7 5).
  • the function of CAPN 10 is, for example, involved in the degradation of IRS-1 (Smith, LK et al., Biochem Biophys Res Commun, 1993. 196 (2) p.
  • IRS-1 is a gene that encodes IRS-1 (I lin receptor substrate-1, insulin receptor substrate-1). IRS-1 is a protein involved in the mechanism of insulin action. In other words, when insulin binds to the insulin receptor on the cell surface, various proteins, including IRS-1 bound to the insulin receptor, are activated one after another, and their signals are transmitted. GLUT4) activates and transports glucose from outside to inside the cell.
  • the nucleotide sequence of the CAPN10 gene may differ among subjects due to polymorphisms, isoforms, etc. Contained in the CAPN10 gene. The same applies to the IRS-1 gene.
  • “Expression level of CAPN10 gene” This includes the level of transcription of the 1 ⁇ 10 gene into 111 RNA and the level of translation into protein.
  • the “expression level of the IRS_1 gene” includes the level of transcription of the IRS-1 gene into mRNA and the level of translation into protein. Therefore, the expression level of the CAPN10 gene or IRS-1 gene is determined by the amount of mRNA that encodes CAPN10 or IRS-1 in the sample, or the CAPN10 or IRS-1 gene in the sample. It can be measured based on the abundance of IRS-1.
  • DNA microarray method etc.
  • gene amplification technology eg, RT-PCR etc.
  • an oligonucleotide or polynucleotide capable of hybridizing to the nucleic acid encoding CAPN10 or IRS-1 can be used as a probe, and when using the gene amplification technique. Can use the oligonucleotide as a primer.
  • Nucleic acids encoding CAPN10 or IRS-1 include both DNA and RNA, including, for example, mRNA, cDNA, cRNA and the like.
  • the nucleotides constituting the oligonucleotide or the polynucleotide may be any of deoxyliponucleotides and liponucleotides, or unnatural nucleotides.
  • the base length of the oligonucleotide is usually 15 to 100 bases, preferably 18 to 40 bases, and the base length of the polynucleotide is usually 200 to 300 bases, preferably 500 to 100 bases. 100 bases.
  • the nucleotide sequence of an oligonucleotide or polynucleotide that can hybridize to a nucleic acid encoding CAPN 10 or IRS-1 is designed based on the nucleotide sequence of CAPN 10 gene or IRS-1 gene. be able to. For example, a region containing CDS is selected from cDNAs encoding CAPN 10 or IRS-1, and the nucleotide sequence of the oligonucleotide or polynucleotide is designed so as to hybridize to the region.
  • the region consisting of the 24th to 24th nucleotides is the CDS region, and in the cDNA shown in SEQ ID NO: 2 it is comprised of the 1021 to 4749th nucleotides
  • the area is the CDS area.
  • a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 'end.
  • a label such as a fluorescent dye or a radioisotope can be added.
  • RT_PCR A specific method for measuring the abundance of mRNA that encodes CAPN 10 or IRS-1 will be described using RT_PCR as an example.
  • the synthesized cDNA is type II, and cDNA encoding CAP N10 or IRS-1 PCR is performed using a primer set capable of hybridizing to the above, and the amount of mRNA encoding CAPN10 or IRS-1 can be measured by quantifying the PCR-amplified fragment.
  • the PCR is performed under conditions such that the amount of PCR amplified fragment production reflects the amount of the initial type III cDNA (for example, the number of PCR cycles at which the PCR amplified fragment increases exponentially).
  • the method for quantifying the PCR-amplified fragment is not particularly limited.
  • a quantification method using a radioisotope (RI), a quantification method using a fluorescent dye, or the like can be used. it can.
  • Examples of the quantification method using RI include: (i) adding a RI-labeled nucleotide (for example, 32 P-labeled dCTP, etc.) as a substrate to a reaction solution, incorporating the nucleotide into an amplified PCR fragment, and PCR amplification. Fragments; RI-labeled and P Separating the CR amplified fragment by electrophoresis, etc., measuring the radioactivity to quantify the PCR amplified fragment, (ii) RI-labeling the PCR amplified fragment by using RI-labeled primers, Separating the amplified CR fragments by electrophoresis, etc. and measuring the radioactivity to quantify the amplified PCR fragments.
  • a RI-labeled nucleotide for example, 32 P-labeled dCTP, etc.
  • Quantitative methods using fluorescent dyes include (i) using a fluorescent dye that interacts with double-stranded DNA (eg, ethidium bromide (EtBr), SYBR GreenI, PicoGreen, etc.) A method for quantifying PCR amplified fragments by staining and measuring the intensity of fluorescence emitted by excitation light irradiation, and (ii) labeling PCR amplified fragments with a fluorescent dye by using a fluorescent dye-labeled primer. After the PCR amplified fragment is separated by electrophoresis or the like, the fluorescence intensity is measured to quantify the PCR amplified fragment. The fluorescence intensity can be measured using, for example, a CCD camera, a fluorescence scanner, a spectrofluorometer, or the like.
  • a fluorescent dye that interacts with double-stranded DNA eg, ethidium bromide (EtBr), SYBR GreenI, PicoGreen, etc.
  • RT-PCR When RT-PCR is used, the gene amplification process is monitored in real time using a commercially available device such as ABI PRISM 7700 (Applied Biosystems), so that the PCR-amplified fragment can be quantitatively determined. Analysis can be performed. ⁇
  • the measured value of the expression level of the CAPN10 gene or the IRS-1 gene is based on the measured value of the expression level of a gene whose expression level does not fluctuate greatly (for example, the housekeeping gene such as the / 3-actin gene and the GAPDH gene). It is preferable to make correction.
  • known protein analysis techniques for example, western blotting using an antibody or a fragment thereof capable of reacting with CAPN10 or IRS_1, Sedimentation, ELISA and the like can be used.
  • Antibodies capable of reacting with CAPN 10 or IRS-1 include both monoclonal antibodies and polyclonal antibodies, and “fragments thereof” include those that can react with CAPN 10 or IRS-1. , Any fragments. Examples of antibody fragments include Fab fragments, F (ab) ' 2 fragments, and single-chain antibodies (scFv).
  • the term "capable of reacting with CAPN10 or IRS-1” includes the case where it reacts with any part of CAPN10 or IRS-1. It is preferable that the antibody or a fragment thereof that can react with CAPN10 or IRS-1 reacts with CAPN10 or IRS-1, but does not react with other proteins contained in leukocytes.
  • An antibody capable of reacting with CAPN10 or IRS-1 can be obtained, for example, as follows.
  • CAPN 10 or IRS-1 can be used as the immunizing antigen.
  • the antigen for immunization include: (i) a crushed cell or tissue expressing CAPN10 or IRS-1 or a purified product thereof;
  • cDNA encoding CAPN 10 or IRS-1 (for example, the nucleotide sequence of SEQ ID NO: 1 for CAPN 10 and SEQ ID NO: 2 for IRS-1) using DNA recombination technology
  • mammals such as rats, mice, guinea pigs, rabbits, sheep, horses, horses, and horses are first immunized with an antigen for immunization.
  • complete Freund's to induce antibody production
  • FCA adjuvant
  • FIA Freund's incomplete adjuvant
  • the antibody titer against the insulin signaling control protein is measured, and blood is collected after the antibody titer has increased to obtain an antiserum.
  • a mammal is immunized with an immunizing antigen in the same manner as in the case of the polyclonal antibody, and then the antibody-producing cells are collected.
  • the antibody-producing cells include spleen cells, lymph node cells, thymocytes, peripheral blood cells and the like, and spleen cells are generally used.
  • cell fusion between the antibody-producing cell and the myeloid cell is performed. After the cell fusion treatment, the cells are cultured using a selection medium to select the desired hybridoma. Next, the presence or absence of the target antibody is screened in the culture supernatant of the grown hybridoma.
  • hybridomas are cloned by a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, etc., and finally, a hybridoma producing a monoclonal antibody is obtained.
  • a method for collecting a monoclonal antibody from the obtained hybridoma an ordinary cell culture method or the like can be used.
  • ascites may be collected, and a monoclonal antibody may be obtained from the ascites.
  • polyclonal or monoclonal antibodies If purification of polyclonal or monoclonal antibodies is required, select an appropriate method such as salting out with ammonium sulfate, gel chromatography, ion exchange chromatography, affinity chromatography, etc. These can be used in combination.
  • RIA radioimmunoassay
  • E IA enzyme immunization Assay
  • CL IA Chemiluminescence Assay
  • FIA fluorescence immunoassay
  • An antibody in which the captured CAPN 10 or IRS-1 is immobilized on a solid support is a labeled antibody having a different antigen recognition site for CAPN 10 or IRS-1 (for example, peroxidase, alkaline
  • the measurement of the abundance of CAPN 10 or IRS-1 can also be performed by measuring the activity of CAPN10 or IRS-1.
  • the activity of CAPN10 or IRS-1 can be measured, for example, by a known method such as a Western plotting method or an ELISA method using an antibody or a fragment thereof capable of reacting with CAPN10 or IRS_1. it can.
  • type 2 diabetes is a slowly progressing disease
  • type 2 diabetes progresses slowly even before obvious symptoms of type 2 diabetes such as hyperglycemia and diabetes appear (that is, before the onset of type 2 diabetes). You may have.
  • the method for diagnosing type 2 diabetes of the present invention utilizes the fact that the expression level of the CAPN10 gene or IRS-1 gene in leukocytes changes differently from the normal expression level as the type 2 diabetes progresses. Diagnosis of type 2 diabetes is performed using the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of the subject as an index.
  • the CAP N10 gene or IRS-11 gene in leukocytes is lower than the normal expression level with the progression of type 2 diabetes
  • the CAP N10 gene or IRS 11 gene or IRS When the expression level of the RS-1 gene is lower than the expression level of the CAPN10 gene or the IRS-1 gene in leukocytes of a healthy subject, the subject will develop type 2 diabetes in the future May be diagnosed as being likely to have or have presently developed type 2 diabetes.
  • the tissue that needs to be sampled from the subject is blood, and blood is easier to sample than other tissues. Can be.
  • the expression levels of CAPN10 gene or IRS-1 gene in leukocytes between a subject and a healthy subject are measured and the It is preferable to set a normal range from the distribution and determine whether the expression level of the subject is higher than or lower than the normal range.
  • the healthy subject group can be constituted by arbitrarily selecting a plurality of healthy subjects, but is preferably constituted by healthy subjects of the same age or generation as the subject. This is to eliminate as much as possible the effect of the age difference on the expression level of the CAPN10 gene or IRS-1 gene.
  • the kit for diagnosing type 2 diabetes of the present invention comprises CAPN10 or IRS-1 as a reagent for measuring the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of a subject. It is characterized by containing an oligonucleotide or polynucleotide capable of hybridizing to the encoding nucleic acid, or an antibody or a fragment thereof capable of reacting with CAPN10 or IRS-1.
  • diagnosis of type 2 diabetes can be performed by measuring the expression level of the CAPN10 gene or the IRS-1 gene in leukocytes collected from the blood of the subject.
  • the kit for diagnosing type 2 diabetes of the present invention may be in any form as long as it contains the above-mentioned oligonucleotide or polynucleotide, or the above-mentioned antibody or fragment thereof, and may contain any reagent, instrument, etc. it can.
  • Type 2 diabetes diagnostic kit of the present invention when containing the oligonucleotide or polynucleotide may, P reagents required CR (e.g.
  • kits for diagnosing type 2 diabetes of the present invention contains the above-mentioned antibody or a fragment thereof, a solid phase carrier (eg, immunoplate, latex particles, etc.) for immobilizing the antibody or the fragment thereof may be used.
  • Labeling eg, enzymes, fluorescent substances, etc.
  • various reagents eg, enzyme substrates, buffers, diluents, etc.
  • Types or two or more types can be included.
  • the method for screening a substance having a preventive / therapeutic effect of type 2 diabetes comprises administering a candidate substance to a type 2 diabetes model animal, and then CAPN10 gene or IRS_1 in leukocytes collected from the blood of the animal.
  • the preventive and therapeutic effects of the candidate substance on type 2 diabetes are determined using the effect of improving the expression level of the gene as an index.
  • the expression level of the CAP N10 gene or IRS_1 gene in leukocytes decreases from the normal expression level with the progression of type 2 diabetes
  • the CAP N 10 gene or IRS_1 gene in the leukocytes of a type 2 diabetes model animal is By selecting a substance having an effect of improving the expression level of the IRS-11 gene, a substance having a preventive / therapeutic effect for type 2 diabetes can be screened.
  • the tissue that needs to be sampled from a type 2 diabetes model animal is blood, and blood is easier to sample than other tissues. Can be easily determined.
  • the “effect of improving the expression level of the CAPN 10 or IRS-1 gene” includes both the effect of returning the expression level of the CAPN 10 or IRS-1 gene to the normal expression level and the effect of approaching the normal expression level. And the effects exerted on any steps such as transcription and translation of the CAP N10 or IRS-1 gene, and expression of the activity of CAPN10 or IRS-1.
  • a type 2 diabetes model animal an animal in which the expression level of the CAPN10 gene or IRS_1 gene in leukocytes is lower than the normal expression level (for example, rat, mouse, etc.) , Guinea pigs, egrets). Such animals are those that are likely to develop type 2 diabetes in the future or that are currently developing diabetes.
  • a transgenic animal in which the expression level of the CAPN10 gene or the IRS-1 gene has been artificially reduced can also be used.
  • the decrease in the expression level of the CAP N10 gene or the IRS-1 gene in the transgenic animal includes a state in which the expression level of the CAPN10 gene or the IRS_1 gene is reduced, and the state of the CAPN10 or IRS-1 gene. Includes any state in which the degradation of _1 is enhanced. '
  • the screening method of the present invention utilizes the fact that a change in the expression level of the CAPN10 gene or IRS-1 gene in leukocytes is an indicator of the possibility of the onset of type 2 diabetes in the future or present,
  • the effect of improving the expression level of the CAP N10 gene or IRS-1 gene in leukocytes collected from the blood of the animal is used as an index to evaluate the preventive and therapeutic effects of the candidate substance on diabetes.
  • the effect of preventing and treating type 2 diabetes is determined, and based on the results, substances having the effect of preventing and treating diabetes are screened.
  • the normal expression level is preferably determined by measuring the expression levels of a plurality of healthy animals and determining the distribution of the values.
  • the screening kit for a substance having a cancer preventive / therapeutic effect of the present invention encodes CAPN10 or IRS-1 as a reagent for measuring the expression level of CAPN10 gene or IRS-1 gene in a sample. Or an oligonucleotide capable of hybridizing to a nucleic acid, or an antibody capable of reacting with CAPN10 or IRS-1, or a fragment thereof.
  • the screening kit of the present invention can be used to measure the expression level of the CAPN10 gene or IRS-1 gene in leukocytes collected from the blood of a subject, thereby screening for a substance having a cancer prevention / treatment effect. You can do one-two.
  • the screening kit of the present invention may be in any form as long as it contains the above-mentioned oligonucleotide or polynucleotide, or the above-mentioned antibody or a fragment thereof, and is exemplified in the type 2 diabetes diagnosis kit of the present invention.
  • various reagents and instruments, model animals and the like can be included.
  • OLETF rats are spontaneous diabetes mellitus model animals showing symptoms similar to type 2 diabetes (non-insulin-dependent diabetes) (Kawano, K. et al., Diabetes Res Clin Pract, 1994. 24 Suppl p. S317-20 ), LETO rats are control animals that do not develop diabetes at all.
  • Rats are animals in a light-dark environment with artificial lighting (light period 7: 00-21: 00, dark period 21: 0 0-7: 00), constant temperature (2rC ⁇ 0.5 ° C), relative humidity (58%) They were bred in the laboratory and had free access to chow and water.
  • GTT Glucose Tolerance Test
  • GTT glucose tolerance test
  • Blood glucose was measured by the glucose oxidase method using the glucose B test method (Trinder, P., J Clin Pathol, 1969. 22 (2) p. 158-61).
  • the blood sampled from the tail vein was allowed to stand on ice for 15 to 20 minutes, and then centrifuged at 100 ° C for 2 minutes at 4 ° C. After diluting 2 ⁇ l of the centrifuged supernatant with physiological saline, 20 ⁇ L was mixed with 1 mL of a color test solution of glucose B test protein. After incubating at 37 ° C for 1 hour, the absorbance at 505 nm was measured.
  • Glucose B Test Co. was purchased from Wako Pure Chemical Industries, Ltd. Glucose and other reagents were commercial grade products.
  • Figure 1 shows the change in body weight of 0LETF rats and LETO rats from 5 weeks to 24 weeks of age.
  • —Hataichi indicates the weight change of 0LETF rats
  • 110-1 indicates the weight change of LET0 rats.
  • Figure 2 shows the GTT results.
  • the band shows the results of 0LETF rats, and the mouth shows the results of LET0 rats.
  • the glucose tolerance at 5 weeks of age was not so different between 0LETF rats and LETO rats, but at 23 weeks of age, glucose tolerance of 0LETF rats was significantly deteriorated.
  • glucose tolerance of 0LETF rats was significantly deteriorated.
  • lg / kg BW glucose was administered intraperitoneally, if the blood glucose level after one hour exceeded 200 fflg / dL, it was considered to be a symptom of diabetes.
  • Four out of the 0 LETF rats exhibited diabetic symptoms.
  • OLETF rats and LET0 rats whose glucose tolerance was measured in the previous section were used for the next week (6 weeks and 24 weeks).
  • 24-week-old OLETF rats used were those whose blood glucose level was 200 mg / dL or more 1 hour after administration of 1 g / kg BW glucose.
  • OLETF rats and LET0 rats were fasted at 6 weeks of age (non-diabetic state) and 24 weeks of age '(oligodiabetic state) from 21:00 for 12 hours, after which whole blood was sampled, after blood removal, liver and muscle was sampled.
  • RNA is extracted from blood (white blood cells), liver and muscle J3 ⁇ 4, and insulin receptor (hereinafter, referred to as “IR”), SH2-containing inositol 5-phosphatase (SH2-containing inos i tol-5-phosphatase> “SHIP2”, Peroxisome Proliferat or -Activated Receptor (PPARa), Calpain 10 (CAM10) ),
  • IR insulin receptor
  • SH2-containing inositol 5-phosphatase SH2-containing inos i tol-5-phosphatase> “SHIP2”
  • Peroxisome Proliferat or -Activated Receptor (PPARa) Peroxisome Proliferat or -Activated Receptor
  • CAM10 Calpain 10
  • OLETF rats and LET0 rats were 6 weeks old (non-diabetic), 24 weeks old (sugar Urinary condition) and fasted for 12 hours from 21:00. After weighing, the rats were anesthetized with ether gas and laparotomy was performed, and sodium heparin (1 OOOU / mL) was injected into the abdominal vena cava using a 27G syringe. At this time, assuming that the whole blood volume of the rat was one-third of the body weight, the required amount of heparin sodium was estimated based on the attached text, and heparin sodium was injected twice as much as the estimated value.
  • a falcon tube in which 0.5 mL of a 0.5% EDTA / physiological saline solution had been previously collected was placed under the aorta, and the aorta was cut and blood was collected.
  • the obtained blood sample was immediately subjected to an RNA extraction operation from leukocytes.
  • physiological saline was perfused from the portal vein, and the liver and muscle were removed after blood removal. Liver and muscle were immediately immersed in liquid nitrogen after extraction and stored at _80 ° C.
  • RNA extraction from the blood sample obtained in 2.1.3 was performed by a spin column method using QIAamp RNA Blood Mini Kits from QIAGEN. After extracting RNA along the protocol attached to the kit, it was dissolved in DEPC water, the nucleic acid concentration and purity were measured, and stocked at -80 ° C.
  • RNA extraction at 24 weeks of age was performed using QIAGEN RNase-Free Dnase Digest Set together. Was.
  • Glyceralde yde 3-phosphate dehydrogenase [Tso, J, Y. et al., Nucleic Acids Res, 1985. 13 (7) p.2485-502] rat cDNA sequence was extracted and the following primers were designed.
  • the amount of mRNA of each gene was quantified by performing Realtime SYBR Green PCR using GAPDH as an internal standard.
  • cDNA sample 4 supplied enzyme buffer 25 / iL, sense and antisense primers 1 ⁇ each (6 ⁇ each) and sterile purified water 16 / iL: Prepare PCR reaction mixture and make final volume 50 50 L.
  • PCR was performed in the following cycle using ABI PRISM 7700. All samples were measured twice with 50 cycles of heat denaturation at 95 ° C for 15 seconds, annealing at 67 ° C for 5 seconds and amplification at 72 ° C for 10 seconds. Data were calculated using the standard curve method and expressed as relative amounts to GAPDH.
  • Table 1 shows the relative amounts of mRNA expression (%) of IR in muscle and muscle.
  • Fig. 3A shows the relative amount (%) of IR mMA expression in leukocytes.
  • the drawing shows the result of 0LETF rat, and the mouth shows the result of LET0 rat.
  • Muscle (n 6) 100 ⁇ 38 64 ⁇ 34 0.12 As shown in Table 1 and FIG. 3A, at the age of 6 weeks, in the leukocyte, liver and muscle tissues, between LET0 rats and 0LETF rats No significant difference was detected. At 24 weeks of age, no significant difference was detected between LET0 rats and 0LETF rats in leukocytes and muscles. Was also significantly reduced.
  • Table 2 shows the relative amounts of SHI mRNA expression () in leukocytes and liver of 6- and 24-week-old 0LETF rats and LETO rats. Analysis of SHI mRNA expression in muscle was too small to be analyzed. The relative expression (%) of SHIP2 mRNA expression in leukocytes is shown in FIG. 3B. In FIG. 3B, ⁇ indicates the result of 0LETF lad, and the mouth indicates the result of LET0 rat.
  • Liver (n 6) 100 ⁇ 25 87 ⁇ 36 0.49 As shown in Table 2 and FIG. 3B, at the age of 6 weeks and 24 weeks, LET0 rats and 0LETF No significant difference was detected between rats.
  • the expression levels of SHI mRNA in leukocytes and liver before the onset of type 2 diabetes are considered to be not significantly different from healthy animals.
  • Table 3 shows the relative expression (%) of PPART mRNA expression in skeletal muscle.
  • FIG. 3C shows the relative amount (%) of PMRT mRNA expression in leukocytes.
  • the image shows the result of the OLETF rat, and the mouth shows the result of the LETO rat.
  • Muscle (n 6) 100 ⁇ 38 50 ⁇ 19 0.0012 ** As shown in Table 3 and FIG. 3C, at the age of 6 weeks, LET0 rats and 0LETF rats in all tissues of leukocytes, liver and muscle No significant difference was detected between them. At 24 weeks of age, no significant difference was detected between LET0 rats and 0LETF rats in leukocytes and liver. It was significantly reduced.
  • Table 4 shows the relative amounts (%) of CAPN10 mRNA expression in leukocytes, liver and muscle of 6- and 24-week-old OLETF and LETO rats.
  • FIG. 3D shows the relative amount (%) of expression of CAPN10 niRNA in leukocytes.
  • the garden shows the results for OLETF rats, and the mouth shows the results for LETO rats.
  • Muscle (n 6) 100 ⁇ 22 39 ⁇ 18 0, .0004 ** As shown in Table 4 and Figure 3D, at the age of 6 weeks, between the LET0 and 0LETF rats in the liver and muscle Although no significant difference was detected, in leukocytes, the mRNA expression level of 0LETF rats was significantly lower than that of LETO rats. At 24 weeks of age, significant differences were detected between LET0 rats and 0LETF rats in all tissues of leukocytes, liver and muscle.
  • CAPN10 mRNA in liver and muscle before the onset is not considered to be significantly different from that in healthy animals
  • the amount of CAPN10 mA expression in leukocytes before onset of type 2 diabetes was significantly reduced compared to healthy animals. It is thought that there is. Further, it is considered that the amount of CAPN10 mRNA expression in leukocytes, liver and muscle is significantly reduced in animals developing type 2 diabetes compared to healthy animals.
  • Table 5 shows the relative amount (%) of IRS-1 mRNA expression in leukocytes, liver and muscle of 6- and 24-week-old 0LETF rats and LETO rats.
  • FIG. 3E shows the relative amount (%) of CAPN10 mRNA expression in leukocytes.
  • the drawing shows the result of 0LETF rat and the mouth shows the result of LET0 rat.
  • Muscle (n 6) 100 ⁇ 79 50 ⁇ 46 0.22
  • Table 5 and Figure 3E As shown in Table 5 and Figure 3E, at the age of 6 weeks, a significant difference was detected between LET0 rats and 0LETF rats in liver and muscle. However, in leukocytes, the mRNA expression level of 0LETF rats was lower than that of LET0 rats. Was significantly reduced. At 24 weeks of age, no significant difference was detected between LET0 rats and 0LETF rats in liver and muscle. Was also significantly reduced.
  • the amount of IRS-1 mRNA expression in liver and muscle before the onset of type 2 diabetes is not considered to be significantly different from that in healthy animals, but before the onset of type 2 diabetes. It is considered that the expression level of IRS-1 mRNA in leukocytes is significantly lower than that in healthy animals. In animals with type 2 diabetes, the amount of IRS-1 mRNA expression in liver and muscle is considered to be not significantly different from that in healthy animals, but the amount of IRS-1 mRNA expression in leukocytes is It is thought that it is significantly reduced compared to healthy animals.
  • CAPN10 and IRS-1 mRNA expression levels in leukocytes before (6 weeks) and after (24 weeks) the onset of diabetes was lower than that in healthy animals (LET0 rats), indicating that the expression levels of CAPN10 and IRS-1 genes in leukocytes were lower than the normal expression levels as diabetes progressed .
  • diabetes can be diagnosed by using the expression levels of the CAPN10 gene and the IRS-1 gene in leukocytes as indices. That is, when the expression levels of the CAPN10 gene and the IRS-1 gene in leukocytes are lower than the normal expression levels, it is possible that diabetes will develop in the future or that diabetes is currently occurring. It is considered possible to diagnose.
  • type 2 diabetes can be diagnosed by analyzing gene expression in tissues that can be easily sampled (especially, before the onset of type 2 diabetes, whether or not it is possible to develop type 2 diabetes in the future) Can be diagnosed to enable early detection of type 2 diabetes), and a method and kit for diagnosing type 2 diabetes are provided.
  • a substance having a preventive / therapeutic effect on type 2 diabetes can be screened by analyzing gene expression in a tissue that is easily sampled, thereby preventing / treating the type 2 diabetes.

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Abstract

L'invention concerne une méthode de diagnostic du diabète du type 2 par l'analyse de l'expression d'un gène présent dans un tissu pouvant être facilement échantillonné (en particulier, le risque de déclenchement de diabète de type 2 peut être prédit pour une détection précoce de la maladie). Ladite méthode est caractérisée en ce qu'elle utilise comme indicateur le niveau d'expression du gène CAPN10 ou du gène IRS-1 dans des leucocytes prélevés dans le sang du sujet.
PCT/JP2002/011306 2002-10-30 2002-10-30 Methode de diagnostic du diabete de type 2 Ceased WO2004040301A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2002344611A AU2002344611A1 (en) 2002-10-30 2002-10-30 Method of diagnosing type 2 diabetes
PCT/JP2002/011306 WO2004040301A1 (fr) 2002-10-30 2002-10-30 Methode de diagnostic du diabete de type 2
JP2004547992A JP4214235B2 (ja) 2002-10-30 2002-10-30 ２型糖尿病診断用キット

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PCT/JP2002/011306 WO2004040301A1 (fr) 2002-10-30 2002-10-30 Methode de diagnostic du diabete de type 2

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WO2004040301A1 true WO2004040301A1 (fr) 2004-05-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007082458A (ja) * 2005-09-21 2007-04-05 Hokkaido Univ ２型糖尿病の診断方法
WO2008047824A1 (fr) * 2006-10-19 2008-04-24 Dna Chip Research Inc. Procédé pour déterminer la présence ou l'absence de diabète

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
SE534386C2 (sv) 2009-10-29 2011-08-02 Alfa Laval Corp Ab Centrifugalseparator samt metod för separering av fasta partiklar

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CARUALHO E. ET AL., FASEB JOURNAL, vol. 13, no. 15, 1999, pages 2173 - 2178, XP002231943 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007082458A (ja) * 2005-09-21 2007-04-05 Hokkaido Univ ２型糖尿病の診断方法
WO2008047824A1 (fr) * 2006-10-19 2008-04-24 Dna Chip Research Inc. Procédé pour déterminer la présence ou l'absence de diabète

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