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WO2013009146A2 - Marqueur pour le diagnostic d'une rétinopathie diabétique - Google Patents

Marqueur pour le diagnostic d'une rétinopathie diabétique Download PDF

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Publication number
WO2013009146A2
WO2013009146A2 PCT/KR2012/005626 KR2012005626W WO2013009146A2 WO 2013009146 A2 WO2013009146 A2 WO 2013009146A2 KR 2012005626 W KR2012005626 W KR 2012005626W WO 2013009146 A2 WO2013009146 A2 WO 2013009146A2
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diabetic retinopathy
protein
expression level
gene
kit
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Korean (ko)
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WO2013009146A3 (fr
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김영수
유형곤
김경곤
김상진
유지영
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SNU R&DB Foundation
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SNU R&DB Foundation
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy

Definitions

  • the present invention is a composition, kit for diagnosing diabetic retinopathy, comprising a preparation for measuring the level of gene or protein that can be used for diagnosis and diagnosis of diabetic retinopathy patients, method for providing information for diagnosing diabetic retinopathy using the same and diabetes It relates to a method for diagnosing retinopathy.
  • Diabetes is a serious disease with increased blood sugar due to abnormalities in the production or use of insulin and accompanying various acute and chronic complications.
  • 9.6% (over 20 million people) of the population 20 years of age or older have diabetes, and an estimated 50 million pre-diabetes patients at high risk are estimated (2005 national diabetes facts in the United States. sheet).
  • $ 132 billion was spent on direct and indirect medical expenses related to diabetes.
  • diabetes Since diabetes has a long morbidity, it is accompanied by various complications of the whole system. Representative examples include cardiovascular disease, diabetes mellitus, diabetes neuropathy, and diabetic retinopathy. Among them, diabetic retinopathy (DR) occurs in over 60% of diabetic patients within 10 years of diabetic diagnosis and over 90% of diabetic patients in 20 years.
  • DR diabetic retinopathy
  • Diabetic retinopathy is a microangiopathy of diabetes characterized by changes in permeability of retinal vessels, vascular obstruction, ischemia, neovascularization, and thus fibrovascular proliferation.
  • Diabetic retinopathy is the largest cause of acquired blindness in adults, and in the United States, 12,000 to 24,000 people die from diabetes every year. In the United States, the prevalence of diabetic retinopathy is estimated to be about 40% of diabetic patients and 8% is reported to be a serious condition that can lead to blindness.
  • Diabetic retinopathy is divided into early non-proliferative diabetic retinopathy (NPDR) and late proliferative diabetic retinopathy (PDR) according to the degree of progression (FIG. 1).
  • NPDR non-proliferative diabetic retinopathy
  • PDR late proliferative diabetic retinopathy
  • Non-proliferative diabetic retinopathy is characterized by retinal bleeding, microaneurysm, exudate, retinal edema, etc. due to obstruction of the retinal capillaries and changes in permeability. have.
  • macular edema DME, diabetic macular edema
  • DME diabetic macular edema
  • Proliferative diabetic retinopathy is a stage in which neovascularization proliferates due to an ischemia condition following extensive vessel occlusion of the retina. This proliferation progresses from the retina to the vitreous and fibrovascular proliferation causes complications such as vitreous hemorrhage, tractional retinal detachment, and neovascular glaucoma, in which the retina falls from its original attachment site. This is the stage of blindness.
  • diabetic retinopathy has been mainly focused on biochemical and molecular biological studies of individual proteins in the vitreous.
  • proteomics study of diabetic retinopathy is a profiling (discovery) step of the vitreous protein bodies that identify proteins in patient vitreous by 2-DE and mass spcectrometry. Very little verification and validation studies have been conducted to determine whether these vitreous proteins are expressed in the blood or can be used as clinical biomarkers.
  • the present inventors have made diligent efforts to develop markers useful for early diagnosis of diabetic retinopathy. As a result, we have discovered a diabetic retinopathy-specific protein and using the expression pattern of the specific protein that increases or decreases in diabetic retinopathy. It was confirmed that can be easily diagnosed to complete the present invention.
  • One object of the present invention is for diagnosing diabetic retinopathy in which at least one selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF), which are new diabetic retinopathy markers for early diagnosis of diabetic retinopathy.
  • AGP alpha-1 acid glycoprotein
  • PEDF pigment epithelium-derived factor
  • Still another object of the present invention is to provide a composition for diagnosing diabetic retinopathy comprising an agent for measuring the level of mRNA or protein thereof of at least one gene selected from the above markers.
  • Another object of the present invention to provide a kit for diagnosing diabetic retinopathy comprising the composition.
  • Still another object of the present invention is to provide a method for providing information necessary for diagnosing diabetic retinopathy by using the composition or kit for diagnosing diabetic retinopathy.
  • Another object of the present invention to provide a method for diagnosing diabetic retinopathy using the composition or kit for diagnosing diabetic retinopathy.
  • the present invention provides a marker for diagnosing diabetic retinopathy, and by measuring and comparing the expression level of a gene or a protein whose expression is increased or decreased in a diabetic retinopathy, the early diagnosis and the degree of disease of the diabetic retinopathy is significant. Can be predicted or identified.
  • the marker of the present invention enables a non-invasive diagnosis can be a simple and effective early diagnosis of diabetic retinopathy by blood, urine tests and the like.
  • 1 is an eye photograph of a patient with non-proliferative diabetic retinopathy and proliferative diabetic retinopathy.
  • FIG. 2 is a flow chart schematically showing the implementation process of the first to sixth embodiments.
  • FIG. 3 is a graph illustrating an ROC curve and an interactive plot of AGP according to Example 6.
  • Figure 4 is a graph showing the ROC curve and interactive plot of PEDF according to Example 6.
  • FIG. 5 is a graph showing an ROC curve and an interactive plot of APOC1 according to Example 6.
  • FIG. 6 is a graph showing an ROC curve and an interactive plot of APOB100 according to Example 6.
  • FIG. 7 is a graph illustrating an ROC curve and an interactive plot of APOC3 according to Example 6.
  • FIG 8 is a graph illustrating an ROC curve and an interactive plot of the VTN according to the sixth embodiment.
  • FIG. 9 is a graph illustrating an ROC curve and an interactive plot of PLG according to Example 6.
  • FIG. 10 is a graph showing an ROC curve and an interactive plot of HRP according to Example 6.
  • FIG. 10 is a graph showing an ROC curve and an interactive plot of HRP according to Example 6.
  • FIG. 11 is a graph illustrating ROC curves and interactive plots of AFMs according to Example 6.
  • FIG. 11 is a graph illustrating ROC curves and interactive plots of AFMs according to Example 6.
  • FIG. 12 is a graph illustrating an ROC curve and an interactive plot of CP according to Example 6.
  • FIG. 12 is a graph illustrating an ROC curve and an interactive plot of CP according to Example 6.
  • FIG. 13 is a graph illustrating an ROC curve and an interactive plot of CFB according to Example 6.
  • the present invention provides a marker for diagnosing one or more diabetic retinopathy selected from Alpha-1 acid glycoprotein (AGP) and Pigment epithelium-derived factor (PEDF).
  • AGP Alpha-1 acid glycoprotein
  • PEDF Pigment epithelium-derived factor
  • the term "diagnostic" means identifying the presence or characteristic of a pathological condition.
  • the diagnosis is to determine whether diabetic retinopathy develops. Preferably it is to determine whether the early stage of diabetic retinopathy non-proliferative diabetic retinopathy.
  • diagnosis marker refers to significant levels of gene expression or protein expression levels in individuals with non-proliferative diabetic retinopathy compared to normal controls (non-diabetic retinopathy) or proliferative diabetic retinopathy.
  • Organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), etc.
  • the marker for diagnosing diabetic retinopathy for the purposes of the present invention may be AGP (Alpha-1 acid glycoprotein) or PEDF (Pigment epithelium-derived factor).
  • AGP Alpha-1 acid glycoprotein
  • PEDF Porture epithelium-derived factor
  • the marker according to the present invention may be used in combination of AGP, PEDF alone, or AGP and PEDF.
  • the diabetic retinopathy diagnostic marker is APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM (Afamin) It may further include one or more selected from Ceruloplasmin (CP) and Complement factor B (CFB). Preferably, at least one selected from nine markers, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine may be used in combination. For example, APOC1; APOC1 and VTN; AFM, CP and CFB; APOC1, APOC3, PLG and HRP; And the like, but are not limited thereto.
  • the combination of one or more selected from the nine markers and AGP or PEDF or AGP and PEDF may be used as a diagnostic marker for diabetic retinopathy.
  • Diabetic retinopathy is divided into early stage of non-proliferative diabetic retinopathy (NPDR) and late stage proliferative diabetic retinopathy (PDR).
  • NPDR non-proliferative diabetic retinopathy
  • PDR proliferative diabetic retinopathy
  • Non-proliferative diabetic retinopathy is characterized by not developing blood vessels, and proliferative diabetic retinopathy is different in its mechanism, such as the development of blood vessels. Since non-proliferative diabetic retinopathy does not necessarily progress to proliferative diabetic retinopathy, a marker known as a diagnostic marker for proliferative diabetic retinopathy cannot necessarily be used as a diagnostic marker for non-proliferative diabetic retinopathy.
  • AGP or PEDF can be used as a diagnostic marker for diabetic retinopathy through the following verification.
  • biomarkers that overexpress or underexpress diabetic retinopathy by analyzing plasma samples from MH (macular hole), PDR (proliferative diabetic retinopathy), and NPDR (non-proliferative diabetic retinopathy) individuals Excavated.
  • samples of PDR and MH individuals were analyzed to identify PDR-specific candidate markers, and based on these, 11 NPDR-specific markers (AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB). ) was finally unearthed.
  • the plasma samples of the normal control group (non-diabetic retinopathy) and NPDR (non-proliferative diabetic retinopathy) subjects were analyzed to identify the 11 types of NPDR (non-proliferative diabetic retinopathy) identified above. The validity of the enemy marker was established.
  • the present invention provides a composition for diagnosing diabetic retinopathy comprising an agent for measuring mRNA or protein level of one or more genes selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF). to provide.
  • AGP alpha-1 acid glycoprotein
  • PEDF pigment epithelium-derived factor
  • Alpha-1 acid glycoprotein is an acute plasma alpha-globulin glycoprotein that is regulated by two polymorphic genes and, according to the gene ontology classification, is a protein involved in immune response and fibrin lysis. Is AGP (GeneBank Accession No. AAB33887, Uniprot: P02763). However, the association of AGP with diabetic retinopathy is unknown at all. AGP is characterized by an increase in the gene expression level or the expression level of the protein in the diabetic retinopathy subject compared to the normal control (individual non-diabetic retinopathy). The diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.
  • PEDF Porture epithelium-derived factor
  • the genetic information is PEDF (GeneBank Accession No. AAK92491, Uniprot: P36955). However, no association between PEDF and diabetic retinopathy is known at all. PEDF is characterized by an increase in the gene expression level or expression level of the protein in diabetic retinopathy individuals compared to normal controls (individuals not diabetic retinopathy).
  • the diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.
  • the composition is in addition to the agent for measuring the mRNA or protein level of one or more genes selected from AGP and PEDF, APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG ( Diabetes comprising agents for measuring mRNA or protein levels of one or more genes selected from the group consisting of Plasminogen, HRP (Histidine-rich protein), AFM (Afamin), Ceruloplasmin (CP) and Complement factor B (CFB) It may be a composition for diagnosing retinopathy.
  • APOC1 Apolipoprotein C1
  • APOB100 Apolipoprotein B100
  • APOC3 Apolipoprotein C3
  • VTN Vitronectin
  • PLG Diabetes comprising agents for measuring mRNA or protein levels of one or more genes selected from the group consisting of Plasminogen, HRP (Histidine-rich protein), AFM
  • the APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), and APOC3 (Apolipoprotein C3) are all proteins involved in lipid metabolism according to the gene ontology classification, and the genetic information is APOC1 (GeneBank Accession No. AAD02506). , Uniprot: P02654), APOB100 (GeneBank Accession No. AAB04636, Uniprot: P04114), APOC3 (GeneBank Accession No. AAS68230, Uniprot: P02656). However, no association between APOC1, APOB100 and APOC3 with diabetic retinopathy is known at all.
  • VTN Vitronectin
  • PLG Plasminogen
  • HRP Histidine-rich protein
  • AFM (Afamin) is a protein involved in transport according to the gene ontology classification, and the genetic information can be found in GeneBank Accession No. AAI09021, Uniprot: P43652. However, the association of AFM with diabetic retinopathy is unknown at all.
  • CP ceruloplasmin
  • CFB Complement factor B
  • the APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB are all reduced in gene expression level or expression level of the protein in diabetic retinopathy compared to normal control (individual not diabetic retinopathy).
  • the diabetic retinopathy subject is preferably a non-proliferative diabetic retinopathy subject.
  • mRNA expression level measurement refers to measuring the amount of mRNA in the process of confirming the presence and expression of mRNA of the genes for diagnosing diabetic retinopathy in a biological sample in order to diagnose diabetic retinopathy.
  • Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, etc., but are not limited thereto. Since nucleic acid information of genes according to the present invention is known to GeneBank et al., Those skilled in the art can design primer pairs or probes that specifically amplify specific regions of these genes based on the sequences.
  • protein expression level measurement refers to a process of confirming the presence and degree of expression of a protein expressed from a gene for diagnosing diabetic retinopathy in a biological sample to diagnose diabetic retinopathy. Protein expression level measurement is to determine the amount of protein using an antibody that specifically binds to the protein of the gene, preferably refers to measuring the protein expression level itself without using an antibody.
  • the protein expression level measurement or comparative analysis method is protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, Surface Enhanced Laser Desorption / SELDI-TOF Ionization Time of Flight Mass Spectrometry, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase chromatography-mass spectrometry liquid chromatography-Mass Spectrometry (LC-MS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbentassay (ELISA), but are not limited thereto.
  • MALDI-TOF Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry
  • diabetic retinopathy refers to a diabetic complication in which peripheral circulatory disorder occurs due to diabetes, which causes disorders in the microcirculation of the retina, resulting in decreased vision.
  • the diabetic retinopathy according to the invention is non-proliferative diabetic retinopathy.
  • the agent measuring the mRNA level is specific for at least one gene selected from AGP and PEDF and additionally at least one gene selected from APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB. Primer pairs, probes or antisense nucleotides that bind to each other.
  • primer pair includes primer pairs of all combinations of forward and reverse primers that recognize a target gene sequence, but preferably, a primer pair that provides an assay with specificity and sensitivity. to be. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample so that only the target gene sequence containing the complementary primer binding site is amplified and does not cause nonspecific amplification. .
  • the term "probe” refers to a substance that can specifically bind to a target substance to be detected in a sample, and through the binding, a substance that can specifically confirm the presence of the target substance in the sample. it means.
  • the type of probe is a material commonly used in the art, but is not limited.
  • the probe may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA, or a DNA. It is PNA.
  • the probes include those derived from or similar to organisms or produced ex vivo as biomaterials, for example enzymes, proteins, antibodies, microorganisms, flora and fauna, organ cells, neurons, DNA, and RNA.
  • DNA may include cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.
  • antisense refers to a nucleotide in which an antisense oligomer hybridizes with a target sequence in RNA by Watson-Crick base pairing, allowing formation of mRNA and RNA: oligomeric heterodimers typically within the target sequence.
  • the agent measuring the protein level is specific for at least one protein selected from AGP and PEDF and additionally at least one protein selected from APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP and CFB. It may include an antibody that binds to.
  • an antibody refers to a specific protein molecule directed against an antigenic site.
  • an antibody means an antibody that specifically binds to at least one protein selected from AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB.
  • Antibodies of the invention include all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Generating antibodies can be readily prepared using techniques well known in the art.
  • the antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains.
  • the functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, Fv, etc.
  • the present invention provides a kit for diagnosing diabetic retinopathy comprising the composition for diagnosing diabetic retinopathy.
  • the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.
  • MRM multiple reaction monitoring
  • the diabetic retinopathy diagnostic kit may further include one or more other component compositions, solutions, or devices suitable for analytical methods.
  • the diagnostic kit may be a diagnostic kit comprising essential elements necessary to perform reverse transcriptase.
  • the reverse transcription polymerase kit contains each primer pair specific for the marker gene.
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific for the nucleic acid sequence of the control gene.
  • reverse transcriptase kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC - May include DEPC-water, sterile water, and the like.
  • reaction buffers pH and magnesium concentrations vary
  • enzymes such as deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase
  • DNAse DNAse
  • RNAse inhibitor DEPC - May include DEPC-water, sterile water, and the like.
  • the diagnostic kit may be a diagnostic kit comprising an essential element necessary to perform a DNA chip.
  • the DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, an enzyme, or the like for preparing a fluorescent probe.
  • the substrate may also comprise cDNA or oligonucleotide corresponding to the control gene or fragment thereof.
  • the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing an ELISA.
  • ELISA kits include antibodies specific for the protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies.
  • the ELISA kit can also include antibodies specific for the control protein.
  • Other ELISA kits can bind reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with the antibody) and substrates or antibodies thereof. Other materials and the like.
  • the present invention provides a method for providing information for diagnosing diabetic retinopathy using the diabetic retinopathy diagnostic composition or the diabetic retinopathy diagnostic kit.
  • the information providing method is an expression level or protein expression level of one or more genes selected from alpha-1 acid glycoprotein (AGP) and pigment epithelium-derived factor (PEDF) from biological samples isolated from patients with suspected diabetic retinopathy. Measuring; And it may be a method of providing information for diagnosing diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a normal control sample.
  • AGP alpha-1 acid glycoprotein
  • PEDF pigment epithelium-derived factor
  • biological sample means a tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid or urine, or the like, in which the gene expression level or protein expression level is different due to the development of diabetic retinopathy. However, it is not limited thereto.
  • the AGP and PEDF has a characteristic that the expression level of the gene or protein expression level is increased compared to the normal control, and when the level is increased can be diagnosed with diabetic retinopathy and provide information.
  • the diabetic retinopathy is non-proliferative diabetic retinopathy.
  • the measuring and comparing step is APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM ( Afamin), Ceruloplasmin (CP) and Complement factor B (CFB) may further comprise the step of measuring and comparing the expression level of the protein or the expression level of the protein selected from the group consisting of.
  • the APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB are characterized by a decrease in the expression level of the gene and the expression level of the protein compared to the normal control, diabetic retinopathy Can diagnose and provide information. At this time, the diabetic retinopathy is non-proliferative diabetic retinopathy.
  • comparing the expression level of the gene of AGP or PEDF or the expression level of the protein with the expression level of the gene of the individual sample of proliferative diabetic retinopathy or the expression level of the protein determining that the increase is non-proliferative diabetic retinopathy It may further include.
  • AGP and PEDF of the present invention increase the gene or protein expression level in non-proliferative diabetic retinopathy, when it is increased compared to the gene or protein expression level of proliferative diabetic retinopathy (PDR) individuals, It can be determined as dietary diabetic retinopathy (NPDR).
  • PDR proliferative diabetic retinopathy
  • At least one selected from the group consisting of APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB as a complex marker decreases the gene or protein expression level in non-proliferative diabetic retinopathy, thereby proliferating diabetic retina.
  • a decrease in comparison with the level of gene or protein expression in an individual can be determined as non-proliferative diabetic retinopathy (NPDR).
  • the expression level of the gene of the present invention can measure or compare the mRNA expression level.
  • the mRNA expression level measurement or comparison may include, but is not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting or DNA chip.
  • the protein expression level of the present invention can be measured and compared using an antibody that specifically binds to the protein.
  • the antibody and the protein of interest in the biological sample form an antigen-antibody complex, and a method of detecting the antibody is used.
  • the term “antigen-antibody complex” means a combination of a protein antigen and an antibody that recognizes it to identify the presence or absence of the gene of interest in a biological sample.
  • the detection of the antigen-antibody complex can be detected using methods as known in the art, such as spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbing, chemical and other methods.
  • the protein expression level measurement and comparison in the present invention is characterized by measuring and comparing the protein expression level itself without using an antibody.
  • the protein expression level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry (MALDI-TOF) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid phase Liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), western blot, and enzyme linked immunosorbentassay (ELISA). .
  • MALDI-TOF Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry
  • SELDI-TOF Surface Enhanced
  • the LC-MRM method was used to measure and compare the protein expression levels of AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP or CFB itself.
  • the protein in the biological sample was passed through the LC analysis column with a concentration gradient of 5% to 85% for 30 minutes with a solution of 5% distilled water, 95% acetonitrile, and 0.1% formic acid based on volume%. Since the resolution of a specific material may vary depending on the solution mixing ratio, a concentration gradient was performed, and the above range was selected for an optimal range for separating various proteins at the same time.
  • SIM Multiple reaction monitoring
  • MRM Multiple reaction monitoring
  • MRM Multiple reaction monitoring
  • MRM Multiple reaction monitoring
  • MRM Multiple reaction monitoring
  • MRM selects one ion from one broken ion one more time to source another MS in series. It is a method of using ions obtained from the collision after passing through it once more.
  • SIM there is a problem that the selected quantitative ion may interfere with the quantification when the selected quantitative ion is an ion that is also detected in plasma.
  • MRM even if ions with the same mass are broken once more, the molecular structure is different and tends to be differentiated. Can be obtained.
  • MRM mode in mass spectrometry, it was possible to simultaneously analyze the desired materials with better analytical sensitivity.
  • the present invention is to measure the expression level or expression level of one or more genes selected from the group consisting of Alpha-1 acid glycoprotein (AGP) and Pigment epithelium-derived factor (PEDF) from the biological sample ; And it provides a method for diagnosing diabetic retinopathy comprising comparing the expression level of the gene or the expression level of the protein with a normal control sample.
  • AGP Alpha-1 acid glycoprotein
  • PEDF Pigment epithelium-derived factor
  • the measuring and comparing step are APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), HRP (Histidine-rich protein), AFM (Afamin),
  • the method may further include measuring and comparing the expression level of one or more genes selected from the group consisting of Ceruloplasmin (CP) and Complement factor B (CFB) or expression levels of proteins.
  • CP Ceruloplasmin
  • CFB Complement factor B
  • the present invention provides Alpha-1 acid glycoprotein (AGP), Pigment epithelium-derived factor (PEDF), APOC1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), Hisidine-rich protein (HRP), AFM (Afamin), Ceruloplasmin (CP), and Complement factor B (CFB) are provided for use as a marker for diagnosing diabetic retinopathy.
  • AGP Alpha-1 acid glycoprotein
  • PEDF Pigment epithelium-derived factor
  • APOC1 Apolipoprotein C1
  • APOB100 Polipoprotein B100
  • APOC3 Apolipoprotein C3
  • VTN Vitronectin
  • PLG Plasminogen
  • HRP Hisidine-rich protein
  • AFM Afamin
  • Ceruloplasmin CP
  • CFB Complement factor B
  • MRM analysis of vitreous proteins in patients with proliferative diabetic retinopathy (PDR) and macular hole (MH) revealed MDR-specific candidate markers, which differ in expression.
  • NPDR non-proliferative diabetic retinopathy
  • Table 1 SEQ ID NO: Protein name Expression Gene Accession number Uniprot One Alpha-1 acid glycoprotein (AGP) Increased expression AAB33887 P02763 2 Pigment epithelium-derived factor (PEDF) Increased expression AAK92491 P36955 3 APOC1 (Apolipoprotein C1) Reduced expression AAD02506 P02654 4 APOB100 (Apolipoprotein B100) Reduced expression AAB04636 P04114 5 Apolipoprotein C3 (APOC3) Reduced expression AAS68230 P02656 6 Vitronectin (VTN) Reduced expression AAH05046 P04004 7 PLG (Plasminogen) Reduced expression AAA60113 P00747 8 Histidine-rich protein (HRP) Reduced expression AAI50592 P04196 9 AFM (Afamin) Reduced expression AAI09021 P43652 10 Ceruloplasmin (CP) Reduced expression AAF02483 P00450 11 Complement factor B (CFB) Reduced
  • Plasma samples from 45 patients with non-proliferative diabetic retinopathy (NPDR) early in diabetic retinopathy (NPDR) were obtained for LC-MS / MS test samples.
  • Clinical characteristics of the 45 patients with non-proliferative diabetic retinopathy (NPDR) and control patients are shown in Table 2 below. The three stages were mild, moderate and severe according to the progression of non-proliferative diabetic retinopathy.
  • NoDR Diabetic but no diabetic retinopathy
  • MI NPDR Mild NPDR
  • MO NPDR Moderate NPDR
  • SV NPDR Severe NPDR
  • Plasma samples were quantified using Bradford, of which 200 ⁇ g of plasma was taken, denatured into urea, reduced and alkylated with DTT and iodoacetic acid.
  • trypsin was treated at a ratio of 50: 1 (protein: trypsin, w / w) to make denatured proteins into peptides, and the peptides were desalted using C18 ZipTip and lyophilized. This was dissolved in solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid) and spiked with 100 fmol of beta-galactosidase peptide, an internal standard, and analyzed by MRM. .
  • MS / MS analysis was performed on the proteins selected in the excavation study. Based on this, a representative peptide for each protein was selected (Q1 transition), and the highest intensity ion (Q3) was selected among fragmentation ions generated by electrically breaking the peptide. Two peptides per protein were selected and two fragmentation ions per peptide were selected to determine Q1 / Q3 as four transitions for one protein. Transitions were selected using the MRM-Initiated Detection and Sequencing (MIDAS) workflow program (MRMPliot, version 2.0, Appliedbiosystems, USA) for some transitions that were difficult to select due to low peaks. For transitions not captured by the MIDAS workflow program, the peptides were selected by selecting peptides with high observed numbers using the Peptide Atlas database.
  • MIDAS MRM-Initiated Detection and Sequencing
  • LC used MDLC nanoflow TempoLC of MDS.
  • C18 resin with a diameter of 3 ⁇ m and a pore size of 200 mm was directly filled using a fused sillica capillary column of 15 cm in length and 100 ⁇ m in diameter.
  • Peptide samples were injected by direct injection method, 1.0 ⁇ l was injected directly into the analitical column without passing through the trap column, and the flow rate was used as 400nl / min.
  • Mass spectrometry was monitored in MRM mode for transitions to selected proteins using 4000 QTrap instrument, Applied Biosystems' hybrid triple quadrupole / linear ion trap. Ion voltage was used at 2000 Volt and resolution at Quadruple 1 (Q1) and Quadruple 3 (Q3) was set in units. The dwell time for the transition was set to 20 milliseconds so that the total cycle time would be 2.5 seconds. Neubulizing gas was used in 5 units and heater temperature was set at 150 °C.
  • beta-galactosidase peptide (beta-galactosidase peptide, Transition 542.3 / 636.3) spiked in each sample was also monitored at the same time.
  • MS run time was run for 60 minutes in time synchronization with LC.
  • MS and LC were run using Analyst 2.1.2.
  • beta-galatosidase peptide (Transition 542.3 / 636.3) was used to quantify MRM at 8 concentration points of blank, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, and 100.0 fmol. Standard curve was determined.
  • the individual MRM results were generated by extracting ion ion chromatography (XIC) of the MRM transitions using MultiQuant (AppliedBiosystems, ver1.0), and the peak area of each transition was calculated and plotted again over time. . Normalize the area of each XIC peak to the XIC peak area of the beta-galatosidase peptide (Transition 542.3 / 636.3), an internal standard, and use this value to perform relative quantitative analysis for each protein.
  • ROC Receiveiver Operating Characteristic Curve and Interactive plot were prepared using MedCalc (MedCalc Software, Belgium, vesion 11.3.3) and ANOVA (Analysis of variance) statistical analysis was performed. Sigma Plot (Systat Software Inc, USA, version 10.1) was used for some plotting and t-test analysis.
  • FIGS. 3 and 4 show that ROG curves and interactive plots of AGP and PEDF, respectively, are increased in expression compared to NoDR samples, and thus can be used as diagnostic markers specific to non-proliferative diabetic retinopathy.
  • APOC1 Apolipoprotein C1
  • APOB100 Apolipoprotein B100
  • APOC3 Apolipoprotein C3
  • VTN Vitronectin
  • PLG Plasminogen
  • HRP Histidine-rich protein
  • AFM Afamin
  • ROC curves and interactive plots of CP Ceruloplasmin
  • CFB Complement factor B

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Abstract

La présente invention concerne une composition et une trousse comprenant un agent pour la mesure d'un niveau d'un gène ou d'une protéine qui peuvent être utilisés pour diagnostiquer et déterminer la progression d'un patient atteint d'une rétinopathie diabétique, pour le diagnostic d'une rétinopathie diabétique, un procédé pour fournir des informations pour le diagnostic d'une rétinopathie diabétique, et un procédé de diagnostic d'une rétinopathie diabétique à l'aide de la composition et de la trousse.
PCT/KR2012/005626 2011-07-14 2012-07-13 Marqueur pour le diagnostic d'une rétinopathie diabétique Ceased WO2013009146A2 (fr)

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WO2020179953A1 (fr) * 2019-03-07 2020-09-10 (주)레티마크 Marqueur composite radioactif pour le diagnostic d'un mélanome malin et utilisation de ce dernier
WO2020189821A1 (fr) * 2019-03-20 2020-09-24 (주)레티마크 Marqueur sanguin pour diagnostiquer les principales maladies responsables de cécité, et procédé de diagnostic les utilisant
WO2021150011A1 (fr) * 2020-01-22 2021-07-29 계명대학교 산학협력단 Biomarqueur pour le diagnostic précoce du diabète ou des complications diabétiques, et utilisation associée
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