KR101391506B1 - Marker For Diagnosing Diabetic Retinopathy and Use Thereof - Google Patents
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Abstract
The present invention relates to a composition for diagnosing diabetic retinopathy, a kit for diagnosing diabetic retinopathy and an information providing method for diagnosing diabetic retinopathy using the same, .
Description
The present invention relates to a composition for diagnosing diabetic retinopathy and a kit for diagnosing diabetic retinopathy. The present invention also relates to an analytical method for providing information necessary for diagnosis of diabetic retinopathy.
Diabetes mellitus is a serious disease with various acute and chronic complications resulting from an increase in blood sugar due to abnormality in the production or utilization of insulin. In the United States, 9.6% (over 20 million people) of people aged 20 or older have diabetes and more than 50 million people with pre-diabetes who are at high risk of developing the disease (2005 national diabetes fact sheet). In 2002, $ 132 billion was spent on direct and indirect medical expenses related to diabetes.
According to the National Health and Nutrition Examination Survey conducted by Korea Centers for Disease Control and Prevention in 2005, 9.0% of males over 30 years old and 7.2% of females were diabetic. According to the '2007 Korean Diabetes Research Report' published by the Korean Diabetes Association, the prevalence of diabetes is about 8%, new patients are getting 10% every year, and diabetes related medical expenses are about 20% ). Considering the recent rapid increase in the number of diabetic patients currently estimated at 400 to 5 million, it could reach 10 million in 10-20 years.
As the duration of diabetes mellitus increases, diverse complications of the system are accompanied by cardiovascular disease, diabetic nephropathy, diabetic neuropathy and diabetic retinopathy. Diabetic retinopathy (DR) occurs in more than 60% of diabetic patients within 10 years of diabetes diagnosis and more than 90% in 20 years.
Diabetic retinopathy is a microangiopathy of diabetes characterized by retinal vascular permeability changes, vascular occlusion, ischemia, neovascularization and subsequent fibrovascular proliferation.
Diabetic retinopathy is the largest cause of acquired blindness in the United States, with 12,000 to 24,000 cases of blindness annually in the United States. In the United States, the prevalence of diabetic retinopathy is estimated to be about 40% of diabetic patients and about 8% is reported to be a serious condition that can lead to blindness.
(NPDR) and late proliferative diabetic retinopathy (PDR) according to the progression of diabetic retinopathy (Fig. 1).
Non-proliferative diabetic retinopathy (NPDR) is characterized by retinal capillary obstruction and permeability changes, resulting in retinal hemorrhage, microaneurysm, exudate, and edema. In addition, accompanying edema of the macula (DME, diabetic macular edema) can cause serious visual impairment even in this period.
Proliferative diabetic retinopathy (PDR) is a stage in which neovascularization is proliferated due to ischemia due to extensive vascular occlusion of the retina. This proliferation progresses to vitreous formation in the retina and fibrovascular proliferation occurs, resulting in complications such as vitreous hemorrhage, tractional retinal detachment, and neovascular glaucoma where the retina falls from its original attachment site This is the stage where blindness progresses.
Although there is still a growing number of patients with blindness that continue to progress to blindness despite laser or vitreous surgery, there is a growing need for early detection and progression of diabetic retinas and early treatment of high-risk patients, but the etiology of diabetic retinopathy It is not yet clear, and biomarkers to determine the progression of diabetic retinopathy are also very limited.
To date, studies on diabetic retinopathy have been focused mainly on biochemical and molecular biology studies of individual proteins in the vitreous. In addition, the proteomic study of diabetic retinopathy is a study of the Profiling (Discovery) phase of vitreous protein bodies that identify proteins in the patient's vitreous with 2-DE and mass spectrometry. There is little research on validation or verification of whether these vitelline proteins are expressed in blood or available as clinical biomarkers.
Therefore, it is necessary to anticipate the early diagnosis and progress of diabetic retinopathy by developing new diagnostic markers which are clinically specific and sensitive, as well as antibodies capable of detecting them.
Under these circumstances, the present inventors have made intensive efforts to develop markers useful for the early diagnosis of diabetic retinopathy. As a result, they have found that diabetic retinopathy specific proteins are increased and decreased in diabetic retinopathy patients using LC-MS / The present inventors have completed the present invention.
It is an object of the present invention to provide a marker for diagnosing diabetic retinopathy which is selected from one or more of AGP and PEDF, which are new diagnostic markers for diabetic retinopathy which can effectively diagnose the early diagnosis and progress of diabetic retinopathy.
It is still another object of the present invention to provide a composition for diagnosing diabetic retinopathy comprising a mRNA of one or more genes selected from the markers or an agent for measuring the level of the mRNA.
It is still another object of the present invention to provide a kit for diagnosing diabetic retinopathy comprising the composition.
It is still another object of the present invention to provide a method for providing information necessary for diagnosis of diabetic retinopathy using the composition or kit for diagnosing diabetic retinopathy.
In one aspect of the present invention, the present invention provides a marker for diagnosing at least one type of diabetic retinopathy selected from AGP and PEDF.
The term "diagnosis" as used herein means to identify the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of diabetic retinopathy. Preferably, the presence of non-proliferative diabetic retinopathy, which is an early stage of diabetic retinopathy, is checked.
As used herein, the term "diagnostic marker" refers to a significant increase in gene expression level or protein expression level in non-proliferative diabetic retinopathy subjects compared to a normal control (non-diabetic retinopathy) or proliferative diabetic retinopathy subjects (Such as mRNA), lipids, glycolipids, glycoproteins, saccharides (monosaccharides, disaccharides, oligosaccharides, etc.), and the like that exhibit reduced activity.
For the purposes of the present invention, the marker for diagnosing diabetic retinopathy may be AGP or PEDF. More preferably APOP1 (Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Plasminogen), Histidine-rich protein, AFM (Afamin), CP (Ceruloplasmin) And complementary factor B (CFB).
Diabetic retinopathy is divided into early nonproliferative diabetic retinopathy (NPDR) and later proliferative diabetic retinopathy (PDR) according to progression. Non-proliferative diabetic retinopathy is characterized by non-vascular development, proliferative diabetic retinopathy is different in its mechanism such as development of blood vessels, non-proliferative diabetic retinopathy is not necessarily progressive to proliferative diabetic retinopathy Thus, markers known as diagnostic markers for proliferative diabetic retinopathy can not necessarily be used as diagnostic markers for non-proliferative diabetic retinopathy.
The present inventors have proved that AGP or PEDF can be used as a marker for the diagnosis of diabetic retinopathy by the following proof.
In one embodiment of the present invention, plasma samples of individuals with MH (macular hole), PDR (proliferative diabetic retinopathy), and NPDR (non-proliferative diabetic retinopathy) were analyzed to identify biomarkers over- or under- expressed in diabetic retinopathy . Specifically, PDR and MH samples were analyzed to identify PDR-specific candidate markers. Based on this, 11 kinds of NPDR-specific markers (AGP, PEDF, APOC1, APOB100, APOC3, VTN, PLG, HRP, CFB) was finally excavated.
In another embodiment of the present invention, plasma samples of normal controls (non-diabetic retinopathy) and NPDR individuals were analyzed to establish the validity of the above-identified 11 NPDR-specific markers.
In one aspect, the present invention provides a composition for diagnosing diabetic retinopathy comprising an agent for measuring the mRNA of at least one gene selected from AGP (Alpha-1 acid glycoprotein) and PEDF (Pigment epithelium-derived factor) to provide.
AGP (Alpha-1 acid glycoprotein) is an acute-phase plasma alpha-globulin glycoprotein that is regulated by two polymorphic genes and is involved in immune response and fibrinolysis according to the gene ontology classification. Is AGP (GeneBank Accession No. AAB33887, Uniprot: P02763). However, the association of AGP with diabetic retinopathy was not known at all. AGP is characterized by an increase in the level of gene expression or expression of the protein in diabetic retinopathy compared to the normal control (non-diabetic retinopathy). Wherein the diabetic retinopathy patient is preferably a non-proliferative diabetic retinopathy patient.
Pigment epithelium-derived factor (PEDF) is a multifunctional secretory protein with anti-angiogenic, anti-tuberculinic and neurotrophic functions and is classified as a retinoblastoma and preactivated B-lymphocytes according to the gene ontology classification B-lymphocytes). The gene information is PEDF (GeneBank Accession No. AAK92491, Uniprot: P36955). However, the association of PEDF with diabetic retinopathy was not known at all. PEDF is characterized by an increase in gene expression level or expression level of the protein in diabetic retinopathy subjects compared to a normal control group (non-diabetic retinopathy). Wherein the diabetic retinopathy patient is preferably a non-proliferative diabetic retinopathy patient.
(Apolipoprotein C1), APOB100 (Apolipoprotein B100), APOC3 (Apolipoprotein C3), VTN (Vitronectin), PLG (Phosphatidylcholine), and the like, in addition to the mRNA of one or more genes selected from AGP and PEDF, A diabetic agent comprising an agent for measuring the mRNA of the at least one gene selected from the group consisting of Plasminogen, HRP (Histidine-rich protein), AFM (Afamin), CP (Ceruloplasmin) and CFB May be a composition for diagnosing retinopathy.
The VTN (Vitronectin), PLG (Plasminogen) and HRP (Histidine-rich protein) of the APOL1 (apolipoprotein C1), APOB100 (Apolipoprotein B100) and APOC3 (Apolipoprotein C3) are proteins involved in lipid metabolism. And fibrinolysis. In addition, AFM (Afamin) and CP (Ceruloplasmin) are proteins involved in transport. In addition, the complement factor B (CFB) is a protein involved in retinoblastoma and preactivated B-lymphocytes. Each gene information includes APOC1 (GeneBank Accession No. AAD02506, Uniprot: P02654), APOB100 (GeneBank Accession No. AAB04636, Uniprot: P04114), APOC3 (GeneBank Accession No. AAS68230, Uniprot: P02656), VTN CPA (GeneBank Accession No. AAA60113, Uniprot: P00747), HRP (GeneBank Accession No. AAI50592, Uniprot: P04196), AFM (GeneBank Accession No. AAI09021, Uniprot: P43652) No. AAF02483, Uniprot: P00450), and CFB (GeneBank Accession No. AAA16820, Uniprot: P00751). However, the association of each gene with diabetic retinopathy was not known at all. The expression level of the gene or the expression level of the protein is decreased in diabetic retinopathy compared to the normal control group (non-diabetic retinopathy) by the APOC1, APOB100, APOC3, PLG, HRP, AFM, CP and CFB I have. Wherein the diabetic retinopathy patient is preferably a non-proliferative diabetic retinopathy patient.
As used herein, the term "measurement of mRNA expression level" is used to determine the presence and expression level of mRNAs of diabetic retinopathy diagnosis genes in a biological sample to diagnose diabetic retinopathy. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.
The agent for measuring the mRNA expression level of the gene is preferably a primer pair or a probe. Since the nucleic acid information of the genes is known by GeneBank et al., A person skilled in the art can use a primer or a probe specifically amplifying a specific region of these genes Can be designed.
The term " measurement of protein expression level "used in the present invention is a process for confirming the presence and expression level of a protein expressed from a gene for diabetic retinopathy in a biological sample to diagnose diabetic retinopathy. The amount of the protein can be confirmed by using an antibody that specifically binds to the protein of the gene. Preferably, the protein expression level itself is measured without using the antibody.
As a method for measuring or comparing the protein expression level, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI- of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis analysis, liquid chromatography (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assay (ELISA).
As used herein, the term "diabetic retinopathy" refers to a complication in which peripheral circulatory disturbance is caused by diabetes, in which the microcirculation of the retina is disturbed and the visual acuity is reduced, preferably non-proliferative diabetic retinopathy .
Preferably, the agent for measuring the level of mRNA is one or more genes selected from AGP and PEDF, and additionally one or more genes selected from among APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, , A probe, or an antisense nucleotide.
As used herein, the term "primer pair" is a primer pair that contains all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, but preferably provides specific and sensitive assay results . The nucleic acid sequence of the primer is inconsistent with the non-target sequence present in the sample and can be given high specificity when it is a primer that amplifies only the target gene sequence containing a complementary primer binding site and does not induce nonspecific amplification .
As used herein, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in the sample through the binding do. The probe molecule may be a peptide nucleic acid (PNA), a peptide, a polypeptide, a protein, an RNA, or a DNA, and may be a peptide nucleic acid It is preferably PNA. More specifically, the probe may be a biomolecule derived from or derived from an organism, or prepared in vitro, such as an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and an organ, a nerve cell, DNA, and RNA DNA includes cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.
The term "antisense" as used in the present invention refers to an antisense oligomer that hybridizes with a target sequence in an RNA by Watson-Crick base pairing to allow the formation of an RNA: oligomer heterodimer, typically in the target sequence, Refers to an oligomer having a sequence of bases and a backbone between subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence.
Preferably the agent for measuring the protein level is one or more proteins selected from AGP and PEDF and additionally one or more proteins selected from APOCl, APOBlOO, APOC3, VTN, PLG, HRP, AFM, CP and CFB Binding < / RTI >
The term "antibody" as used herein refers to a specific protein molecule directed against an antigenic site. For the purpose of the present invention, the antibody refers to 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, monoclonal antibodies, and recombinant antibodies. The production of the antibody can be easily carried out 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 with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.
In another aspect, the present invention provides a diagnostic kit for diabetic retinopathy comprising the composition for diagnosing diabetic retinopathy. Preferably, 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.
In addition, preferably, the diabetic retinopathy diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.
Preferably, the diagnostic kit comprises a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each of the above genes, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits may be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC DEPC-water, sterile water, and the like.
Preferably a diagnostic kit comprising essential elements necessary for carrying out a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.
Also preferably, it may be a diagnostic kit characterized by comprising essential elements necessary for performing ELISA. The ELISA kit comprises an antibody specific for the protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.
In another aspect, the present invention provides a method for diagnosing diabetic retinopathy using the composition for diagnosing diabetic retinopathy or the kit for diagnosing diabetic retinopathy.
Preferably, the information providing method is a method for detecting an expression level or protein expression level of at least one gene selected from AGP (Alpha-1 acid glycoprotein) and PEDF (Pigment epithelium-derived factor) from a biological sample isolated from a patient suspected of diabetic retinopathy ; And
And comparing the expression level of the gene or the expression level of the protein with that of a normal control sample, may be an information providing method for diagnosing diabetic retinopathy.
The term "biological sample " used in the present invention includes samples such as tissues, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid or urine which have different levels of gene expression or protein expression by the onset of diabetic retinopathy , But is not limited thereto.
Since the expression level of the gene or the expression level of the protein is elevated in comparison with the normal control, AGP and PEDF can be diagnosed as diabetic retinopathy and provide information by increasing the level. Preferably, the diabetic retinopathy is non-proliferative diabetic retinopathy.
Preferably, the step of measuring and comparing comprises the steps of: measuring the concentration of Apolipoprotein C1, APOB100, Apolipoprotein C3, Apolipoprotein C3, VTN, Plasminogen, Histidine-rich protein, AFM, Afamin), CP (Ceruloplasmin) and CFB (Complement factor B), or the level of expression of the protein.
Since the levels of expression and protein expression levels of the APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB genes are decreased as compared with the normal control group, Diagnosis can be made and information can be provided. Preferably, the diabetic retinopathy is non-proliferative diabetic retinopathy.
Preferably, the expression level of the AGP or PEDF gene or the expression level of the protein is compared with the expression level of the gene of the proliferative diabetic retinopathy subject sample or the expression level of the protein, and when the increase is determined to be non-proliferative diabetic retinopathy May be further included.
Since AGP and PEDF of the present invention increase the expression level of the gene or protein in non-proliferative diabetic retinopathy, when compared with the gene or protein expression level of the proliferative diabetic retinopathy (PDR), the expression level of NPDR . ≪ / RTI >
In addition, at least one of the complex markers selected from the group consisting of APOC1, APOB100, APOC3, VTN, PLG, HRP, AFM, CP, and CFB is decreased in non-proliferative diabetic retinopathy, When compared with the gene or protein expression level of the retinopathy (PDR) individual, it can be judged to be NPDR during the progression stage of diabetic retinopathy.
Preferably, the expression level of the gene of the invention is preferably capable of measuring and comparing mRNA expression levels.
The measurement or comparison of the mRNA expression level may be performed using a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, northern blotting or a DNA chip. It is not. Through these measurement methods, mRNA expression level in normal control group and mRNA expression level in diabetic retinopathy patients can be confirmed, and the degree of diabetic retinopathy can be diagnosed or predicted by comparing the amount of these mRNA expression levels.
Preferably, 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 in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting the antibody-antibody complex is used.
As used herein, the term "antigen-antibody complex" refers to a combination of a protein antigen and an antibody recognizing the presence or absence of the gene in the biological sample. The detection of the antigen-antibody complex can be detected using methods such as those known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods.
More preferably, in the present invention, the measurement and comparison of the protein expression level is characterized by measuring and comparing the protein expression level itself without using the antibody.
For the purpose of the present invention, protein level analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheronian immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, But are not limited to, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA).
In a specific embodiment of the invention, the LC-MRM method is used to measure and compare the protein expression levels of AGP, PEDF, APOCl, APOBlOO, APOC3, VTN, PLG, HRP, AFM, CP or CFB per se.
Specifically, the protein in the biological sample is passed through an LC analysis column with a gradient of 5% to 85% by volume in a solution of 5% distilled water, 95% acetonitrile, 0.1% formic acid for 30 minutes. Concentration gradients were performed because the resolution for a specific substance may vary depending on the solution mixing ratio, and thus the range is an optimal range for simultaneously separating various proteins.
Mass spectrometry is performed by MS / MS mode (Multiple reaction monitoring). While SIM (Selected Ion Monitoring) is a method of using ions generated by collision with the source part of the mass spectrometer, MRM once again selects a specific ion among the broken ions to connect another consecutively connected MS source And then collide with another once more, and then use ions obtained from the collision. More specifically, there is a problem in the SIM that disturbance may occur when the selected quantitative ions are ions that are also detected in plasma. On the other hand, in the case of using MRM, when ions having the same mass are further broken, the molecular structure is different and differentiated. Therefore, when the ion is used as a quantitative ion, disturbing peaks in the background are removed, Can be obtained. Thus, by using the MRM mode during mass spectrometry, desired materials can be simultaneously analyzed at better analytical sensitivity.
Through the above analysis methods, it is possible to compare the protein expression level in the normal control group with the protein expression level in the diabetic retinopathy patient, judge whether the expression level of the marker gene for diabetic retinopathy is significantly increased or not, It can diagnose the onset.
As described above, the present invention provides a marker capable of diagnosing diabetic retinopathy, so that by measuring and comparing the expression level of a gene whose expression is increased or decreased in diabetic retinopathy patients or its protein, Diagnosis and disease severity can be significantly predicted or identified. In addition, the marker of the present invention enables non-invasive diagnosis and enables early diagnosis of simple and effective diabetic retinopathy by blood, urine test or the like.
FIG. 1 is an eye photograph of a patient with non-proliferative diabetic retinopathy and proliferative diabetic retinopathy,
2 is a flowchart schematically showing the procedure of the first to sixth embodiments,
3 is a graph showing an ROC curve and an interactive plot of AGP according to Example 6. FIG.
4 is a graph showing an ROC curve and an interactive plot of the PEDF according to Example 6,
5 is a graph showing ROC curves and interactive plots of APOC1 according to Example 6,
6 is a graph showing an ROC curve and an interactive plot of the
7 is a graph showing an ROC curve and an interactive plot of APOC3 according to Example 6. FIG.
8 is a graph showing an ROC curve and an interactive plot of VTN according to the sixth embodiment,
9 is a graph showing an ROC curve and an interactive plot of the PLG according to the sixth embodiment,
10 is a graph showing an ROC curve and an interactive plot of HRP according to Example 6. FIG.
11 is a graph showing an ROC curve and an interactive plot of the AFM according to the sixth embodiment,
12 is a graph showing an ROC curve and an interactive plot of the CP according to the sixth embodiment,
13 is a graph showing an ROC curve and an interactive plot of the CFB according to the sixth embodiment.
Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.
Example One: Diabetic retinopathy Overexpression or Low expression Protein discovery
First, PDR - specific candidate markers were identified by MRM analysis of vitreous protein in patients with proliferative diabetic retinopathy (PDR) and macular hole (MH) patients.
Protein expression in vitreous and corresponding plasma samples of patients with secondary proliferative diabetic retinopathy (PDR), macular hole (MH) and non-proliferative diabetic retinopathy (NPDR) patients based on the degree of PDR protein expression obtained by the above procedure And MRM. As a result, genes showing differences in expression compared to patients with macular hole (MH) and diabetic retinopathy (PDR) were found (Table 1). Two overexpression and two low overexpression, respectively, were able to finally select proteins that were specifically expressed in non - proliferative diabetic retinopathy (NPDR).
Example 2: Patient Selection and Plasma Collection
A sample for LC-MS / MS test was obtained from 45 plasma samples from non-diabetic retinopathy patients (NPDR) early in diabetic retinopathy and from control plasma samples (diabetic but not diabetic retinopathy patients, NoDR). The clinical characteristics of the non-proliferative diabetic retinopathy patients (NPDR) and the control patients are shown in Table 2 below. The patients were divided into mild, moderate and severe groups according to progression of non - proliferative diabetic retinopathy.
(Female / male)
Elapsed time (years)
(占 퐂 / 占 퐇)
(NoDR: No diabetic retinopathy, MI NPDR: Mild NPDR, MO NPDR: Moderate NPDR, SV NPDR: Severe NPDR)
Example 3: Pretreatment of plasma sample
Plasma samples were quantitated using Bradford, 200 μg of plasma was taken down to Urea and reduced and alkylated with DTT and iodoacetic acid. Trypsin was treated with trypsin at a ratio of 50: 1 (protein: trypsin, w / w) to make a denatured protein. The peptide was desalted and freeze-dried using C18 ZipTip. The resulting solution was dissolved in solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid), and 100 fmol of an internal standard beta-galactosidase peptide was spiked and analyzed by MRM .
Example 4: Transition Selection of
MS / MS analysis was performed on the proteins selected in the excavation studies to select the transitions of the proteins. Based on this, representative peptides for each protein were selected (Q1 transition) and the most intense fragmentation ions (Q3) were generated by electrically breaking the peptide. Two peptides were selected per protein and two fragmentation ions were selected for each peptide. Q1 / Q3 was determined as four transitions for one protein. Transitions were selected using the MIDAS (MRM-Initiated Detection and Sequencing) workflow program (MRMPliot, version 2.0, Applied Biosystems, USA) for some of the peaks that were difficult to select experimentally. The transitions not captured by the MIDAS workflow program were selected using the Peptide Atlas database to select peptides with high observed numbers.
Example 5: LC And MRM
LC was a MDLC nanoflow Tempo LC from MDS. For the separation of peptides, C18 resin having a diameter of 3 탆 and a pore size of 200 Å was directly filled using a fused sillica capillary column having a length of 15 cm and an inner diameter of 100 탆. 1.0 μl of the peptide sample was injected directly into the analitical column without passing through the trap column, and the flow rate was 400 nl / min. The column was equilibrated with solution A (95% distilled water, 5% acetonitrile, 0.1% formic acid) for 10 minutes and then 5% to 85% with solution B (5% distilled water, 95% acetonitrile, 0.1% formic acid) The peptide was eluted through a concentration gradient of 85% for 5 minutes.
Mass spectrometry was performed in MRM mode for the transition of selected proteins using a hybrid triple quadrupole / linear ion trap of Applied Biosystems, 4000 QTrap instrument. The Ion voltage was 2000 volts and the resolution in Quadruple 1 (Q1) and Quadruple 3 (Q3) was set to unit. The dwell time for the transition was set to 20 milliseconds and the total cycle time was set to 2.5 seconds. Neubulizing gas was used in 5 units and the heater temperature was set at 150 ℃. 50 fmole beta-galactosidase peptide (Transition 542.3 / 636.3) spiking in each sample was also monitored simultaneously to demonstrate batch-to-batch variation. The MS run time was run for 60 minutes with LC and time synchronization, and MS and LC were run using Analyst 2.1.2.
Example 6: Data Analysis
For relative quantitation, MRM was quantified at eight concentrations of blank, 0.5, 1.0, 5.0, 10.0, 25.0, 50.0 and 100.0 fmol using beta-galactosidase peptide (Transition 542.3 / 636.3) Standard curve was determined. The MRM results of each individual were generated by ion chromatography (XIC, Extraction Chromatography) of corresponding MRM transition using MultiQuant (Applied Biosystems, ver1.0), and the peak area of each transition was calculated and plotted again over time . The area of each XIC peak was normalized to the XIC-formed peak area of the internal standard beta-galactosidase peptide (Transition 542.3 / 636.3), and relative quantitative analysis was performed on each protein Respectively. For statistical analysis, ROC (Receiver 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.
As a result of the above analysis, eleven proteins showing significant differences among the expressed proteins were identified. The ROC curve and the interactive plot of each protein are shown in FIGS. 3 to 13.
As a result, FIG. 3 and FIG. 4 show that ROC curve and interactive plot of AGP and PEDF, respectively, are increased in expression compared to NoDR samples and can be used as diagnostic markers specific for non-proliferative diabetic retinopathy. 5 to 13 are graphs showing the results of immunohistochemical staining for apolipoprotein C1, APOB100 (apolipoprotein B100), APOC3 (apolipoprotein C3), VTN (vitronectin), PLG (plasminogen), HRP (histidine- The ROC curve and the interactive plot of CP (Ceruloplasmin) and CFB (Complement Factor B) decreased in expression compared to NoDR samples, respectively, indicating that it could be used as a diagnostic marker specific for the additional non-proliferative diabetic retinopathy together with AGP and PEDF Respectively.
Claims (14)
And comparing the expression level of the gene or the expression level of the protein with a normal control sample.
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| WO2020179953A1 (en) * | 2019-03-07 | 2020-09-10 | (주)레티마크 | Composite marker for diagnosis of diabetic retinopathy and use thereof |
| WO2020189821A1 (en) * | 2019-03-20 | 2020-09-24 | (주)레티마크 | Blood marker for diagnosing major blindness-causing eye diseases, and diagnostic method using same |
| KR20210095071A (en) * | 2020-01-22 | 2021-07-30 | 계명대학교 산학협력단 | Biomarker for early diagnosing diabetes or diagnosing diabetic complications and use thereof |
| RU2765437C1 (en) * | 2021-08-17 | 2022-01-31 | Федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр глазных болезней имени Гельмгольца" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ ГБ им. Гельмгольца" Минздрава России) | Method for determining gene expression level, coding pedf, in oryctolagus cuniculus rabbit eye tissues and a kit for its determining |
| KR102480069B1 (en) * | 2022-09-08 | 2022-12-26 | 줄리아 연구소 주식회사 | Composition for diagnosing diabetic retinopathy using biomarkers, method for providing information for diagnosing diabetic retinopathy, and screening method for substances for treatment of diabetic retinopathy |
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