Markers for diagnosing diabetic retinopathy

Abstract

The application discloses a marker for diagnosing diabetic retinopathy. The application determines the markers CELA3A, CTRC, CELA3B and GLUD1 by studying the protein spectrum of plasma exosomes of patients with DR at different stages, further verifying the markers through finding a screening queue and a verification queue, and the markers can distinguish DR patients at different stages, have high diagnostic efficiency, and are expected to become markers for monitoring the progress of DR, thereby providing effective guidance for the diagnosis of diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to biomarkers for diagnosing diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus (DM), causing severe visual impairment and even blindness. Based on whether it proliferates, DR can be divided into two categories: nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR).

[0003] Retinopathy of prematurity (DR) is often insidious in its early stages, with patients experiencing no obvious eye discomfort initially, leading to its neglect. Furthermore, current treatment options for early-stage DR are limited; lifestyle interventions and eye hygiene alone are insufficient to control the condition. By the time patients seek medical attention for significant eye discomfort, such as blurred vision, visual field defects, or decreased visual acuity, the retinal disease is usually in its middle or late stages, resulting in irreversible vision loss and missed opportunities for optimal treatment, leading to a poor prognosis. Moreover, clinical diagnosis of DR still relies on fundus photography and fluorescein angiography performed by ophthalmologists; however, these diagnostic devices are expensive and limited by regional cultural and healthcare disparities, making them unavailable in all medical institutions.

[0004] Therefore, finding cost-effective biomarkers that can predict DR and its progression is of great clinical significance for timely diagnosis, early intervention, and prevention and delay of the progression of asymptomatic diabetes. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides biomarkers for diagnosing diabetic retinopathy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A first aspect of the invention provides the use of a reagent for detecting the expression level of a biomarker in a sample in the preparation of products for diagnosing diabetes / diabetic retinopathy / diabetic retinopathy progression, said biomarker including any one or more of CELA3A, CTRC, CELA3B, and GLUD1.

[0008] Furthermore, the progression of diabetic retinopathy includes two stages: NPDR and PDR.

[0009] Furthermore, the reagents include reagents required for detecting the protein expression level of the biomarker, probes that specifically recognize the biomarker, or primers that specifically amplify the biomarker.

[0010] Furthermore, the sample was selected from blood plasma.

[0011] Furthermore, the plasma is plasma exosomes.

[0012] A second aspect of the present invention provides a product for diagnosing diabetes / diabetic retinopathy / diabetic retinopathy progression, the product comprising a reagent for detecting the expression level of a biomarker in a sample, the biomarker including any one or more of CELA3A, CTRC, CELA3B and GLUD1.

[0013] Furthermore, the products include chips, test strips, reagent kits, or nucleic acid membrane strips.

[0014] Furthermore, the kit also includes reagents for detecting the expression levels of biomarker proteins or genes by means of Western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry, protein chip method, RT-PCR, and DNA blotting.

[0015] Furthermore, the chip includes a gene chip and a protein chip.

[0016] Furthermore, the gene chip includes a solid-phase carrier and probes immobilized on the solid-phase carrier, the probes including oligonucleotide probes targeting the marker gene for detecting the transcriptional level of the marker gene; the protein chip includes a solid-phase carrier and specific antibodies against the marker protein immobilized on the solid-phase carrier.

[0017] A third aspect of the present invention provides a method for screening candidate drugs for treating diabetes / diabetic retinopathy, the method comprising: treating a culture system expressing or containing a marker gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the marker gene or its encoded protein in the system; wherein, when the substance to be screened inhibits the expression level or activity of the marker gene or its encoded protein, the substance to be screened is a candidate drug for treating diabetes / diabetic retinopathy, the marker including any one or more of CELA3A, CTRC, CELA3B, and GLUD1.

[0018] The fourth aspect of the invention provides the application of any one or more of the biomarkers CELA3A, CTRC, CELA3B, and GLUD1 in constructing computational models for diagnosing diabetes / diabetic retinopathy / diabetic retinopathy progression.

[0019] Furthermore, the progression of diabetic retinopathy includes two stages: NPDR and PDR.

[0020] The fifth aspect of the invention provides the use of any one or more of the biomarkers CELA3A, CTRC, CELA3B, and GLUD1 in constructing a system / device for diagnosing diabetes / diagnosing diabetic retinopathy / diagnosing the progression of diabetic retinopathy.

[0021] Furthermore, the progression of diabetic retinopathy includes two stages: NPDR and PDR.

[0022] Furthermore, the system includes: an acquisition unit for acquiring the expression level of the biomarker in the sample; and a processing unit for obtaining the results of diagnosing diabetes / diagnosing diabetic retinopathy / diagnosing the progression of diabetic retinopathy based on the expression level of the biomarker.

[0023] Advantages and benefits of the present invention: This application, through the study of the protein profile of plasma exosomes of DR patients at different stages, and through further validation by discovery cohort screening and validation cohort, identified biomarkers CELA3A, CTRC, CELA3B and GLUD1. These biomarkers can distinguish DR patients at different stages, have high diagnostic efficacy, and are expected to become biomarkers for monitoring DR progression, providing effective guidance for the diagnosis of the disease. Attached Figure Description

[0024] Figure 1 These are characteristic images of plasma exosomes. Among them, 1A is a nanoparticle tracking analysis image, 1B is a Western blot image of exosome markers CD63, HSP70 and Calnexin, and 1C is a transmission electron microscope image of exosomes. Figure 2 These are enrichment analysis plots of DEPs, where 2A is the GO function enrichment analysis plot of DEPs and 2B is the KEGG enrichment analysis result plot of DEPs. Figure 3 These are the expression levels of pivot proteins in the training cohort. 3A is the expression level of CELA3A, 3B is the expression level of CTRC, 3C is the expression level of CELA3B, and 3D is the expression level of GLUD1. Figure 4 This is a correlation analysis diagram of pivot proteins with fasting blood glucose and glycated hemoglobin; Figure 5These are graphs showing the expression levels of pivot proteins in different clinical groups. 5A represents the expression level of CELA3A, 5B represents the expression level of CTRC, 5C represents the expression level of CELA3B, and 5D represents the expression level of GLUD1. Figure 6 These are expression level maps of pivot proteins in the validation cohort, where 6A is the expression level map of CELA3A, 6B is the expression level map of CELA3B, 6C is the expression level map of CTRC, and 6D is the expression level map of GLUD1. Figure 7 These are the ROC curves of the pivot proteins in the validation cohort. Among them, 7A-C is the ROC curve of CELA3A, 7D-F is the ROC curve of CELA3B, 7G-I is the ROC curve of CTRC, and 7J-L is the ROC curve of GLUD1. Figure 8 This is a correlation analysis chart between clinical indicators and proteins CELA3A, CELA3B, and CTRC. Detailed Implementation

[0025] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] This invention provides the application of reagents for detecting the expression level of biomarkers in samples in the preparation of products for diagnosing diabetes / diabetic retinopathy / diabetic retinopathy progression, wherein the biomarkers include any one or more of CELA3A, CTRC, CELA3B and GLUD1.

[0027] The reagents include reagents required to detect the protein expression level of the biomarker, probes that specifically recognize the biomarker, or primers that specifically amplify the biomarker.

[0028] In some embodiments, the probe that specifically identifies the marker can be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives. The length of the probe is not limited; any length is acceptable as long as specific hybridization and binding to the target nucleotide sequence are achieved. The probe length can be as short as 25, 20, 15, 13, or 10 bases. Similarly, the probe length can be as long as 60, 80, 100, 150, 300 bases or longer, even encompassing the entire gene.

[0029] In some implementations, primers are short nucleic acid molecules, such as DNA oligonucleotides, that can form a hybrid between the primer and the target nucleic acid strand through nucleic acid hybridization and annealing with a complementary target nucleic acid molecule. The primer can be extended along the target nucleic acid molecule using polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, where the primer sequence is specific to the target nucleic acid molecule; for example, the primer will hybridize with the target nucleic acid molecule under very high-tightness hybridization conditions.

[0030] The primers or probes of this application can be chemically synthesized using phosphorimide solid-phase support or other well-known methods. They can also be modified using many techniques known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more analogs of natural nucleotides, and modifications between nucleotides, such as modifying uncharged linkers (e.g., methyl phosphate, triphosphate, phosphorimide, carbamate, etc.) or modified charged linkers (e.g., thiophosphate, dithiophosphate, etc.).

[0031] The reagent also includes a detectable marker.

[0032] In some embodiments, a detectable label refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent components, magnetic particles, and bioluminescent components. Therefore, a label is any composition detectable by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrochemical, optical, chemical detection devices, or any other suitable device. In some embodiments, a label can be visually detected without the aid of a device. The term "label" is used to refer to any chemical group or portion having a detectable physical property, or any compound capable of causing a chemical group or portion to exhibit a detectable physical property, such as an enzyme that catalyzes the conversion of a substrate into a detectable product. Labels also encompass compounds that inhibit the expression of a particular physical property. A label can also be a compound that is a member of a binding pair, the other member of which has a detectable physical property.

[0033] Among them, radioactive isotopes include but are not limited to 3 H, 14 C 35 S, 125 I, 131 I.

[0034] Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase.

[0035] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0036] The products include chips, test strips, reagent kits, or nucleic acid membrane strips.

[0037] In some implementations, a chip, also referred to as an array, refers to a solid support containing linked nucleic acid or peptide probes. Arrays typically contain a variety of different nucleic acid or peptide probes attached to a substrate surface at different known locations. These arrays, also known as “microarrays,” can typically be produced using mechanosynthesis or photoguided synthesis, which combines photolithography and solid-phase synthesis methods. Arrays can comprise flat surfaces or can be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. Arrays can be packaged in a manner that allows for diagnostic or other manipulation of a fully functional device.

[0038] The chips include gene chips and protein chips.

[0039] In some embodiments, the gene chip includes a solid-phase carrier and probes immobilized on the solid-phase carrier, the probes including oligonucleotide probes targeting the marker gene for detecting the transcriptional level of the marker gene; the protein chip includes a solid-phase carrier and a specific antibody against the marker protein immobilized on the solid-phase carrier. The gene chip can be used to detect the expression levels of multiple genes, including the aforementioned markers (e.g., multiple genes associated with diabetes / diabetic retinopathy). The protein chip can be used to detect the expression levels of multiple proteins, including the aforementioned marker proteins (e.g., multiple proteins associated with diabetes / diabetic retinopathy). By simultaneously detecting multiple markers associated with diabetes / diabetic retinopathy, the accuracy of diagnosing diabetes / diabetic retinopathy can be significantly improved.

[0040] In some embodiments, the components of the kit may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of the present invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0041] The solid support of the kit may be, for example, plastic, silicon wafer, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide.

[0042] In some embodiments, the nucleic acid membrane strip includes a substrate and probes fixed on the substrate; the substrate can be any substrate suitable for fixing the probes, including but not limited to nylon membranes, nitrocellulose membranes, polypropylene membranes, glass slides, silicone wafers, and micro-magnetic beads.

[0043] In some embodiments, the samples include, but are not limited to, bone marrow, peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, breast milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytokine, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid.

[0044] In a preferred embodiment, the sample is selected from blood plasma.

[0045] In a specific implementation, the plasma is plasma exosomes.

[0046] This invention provides the use of any one or more of the biomarkers CELA3A, CTRC, CELA3B, and GLUD1 in constructing systems / devices for diagnosing diabetes / diabetic retinopathy / diabetic retinopathy progression.

[0047] The system includes: an acquisition unit for acquiring the expression level of the biomarker in a sample; and a processing unit for obtaining the results of diagnosing diabetes / diabetic retinopathy / diabetic retinopathy progression based on the expression level of the biomarker.

[0048] In some implementations, the processing unit (processor) may include one or more microprocessors or digital processors. The processor can call program code stored in memory to perform related functions. The processor, also known as a central processing unit (CPU), can be a very large-scale integrated circuit and serves as both the computing core and the control unit.

[0049] In some embodiments, the system / device may further include a detection unit, which may be used to perform one or more of the following: protein expression level detection, polysome qPCR, enzyme-linked immunosorbent assay (ELISA), or transcriptome sequencing.

[0050] In some embodiments, the system / device may further include a result display unit for displaying the conclusions reached by the processing unit.

[0051] In some implementations, the result display unit displays the results via screen display, sound broadcast, or printing.

[0052] In some implementations, the system may be implemented manually, automatically, or in combination thereof to perform or complete the selected tasks. Furthermore, the actual instruments and equipment according to embodiments of the system of the present invention may implement multiple selected tasks via hardware, software, firmware, or a combination thereof using an operating system.

[0053] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0054] Example 1: Materials and Methods Participants and Sample Collection In this study, the cohort included 6 controls (HC), 6 patients with type 2 diabetes (DM) without clinically obvious retinopathy, 6 patients with non-retinopathy retinopathy (NPDR), and 5 patients with prolapsed retinopathy (PDR). The validation cohort included 26 controls (HC), 30 patients with DM, 26 patients with NPDR, and 25 patients with PDR. The participants ranged in age from 40 to 80 years. Glycated hemoglobin (HbA1c) levels ranged from 7.0% to 10.5%. NPDR and PDR were diagnosed by fundus fluorescein angiography. For patients with inconsistent disease progression in both eyes, the more severely affected eye was in the DR stage. Patients with intraocular pressure >21 mmHg, glaucoma, retinal vascular disease, uveitis, optic nerve disease, hereditary eye disease, high myopia (>-6.0D) and other eye diseases, as well as infectious diseases (AIDS, syphilis, hepatitis B, etc.), autoimmune diseases, malignant tumors, persistent systemic infections, cerebral hemorrhage and / or cerebral infarction within 90 days, and other serious systemic diseases were excluded. This study design complied with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Xuanwu Hospital, Capital Medical University (2023-046). Written consent was obtained from all participants. Peripheral blood samples were collected from both groups. Plasma was extracted by centrifugation at 1,500 g for 20 minutes at 4°C and stored at -80°C.

[0055] Plasma exosomes were extracted from thawed plasma samples and centrifuged at 2,000 g for 10 minutes at 4°C. The supernatant was then transferred to a new tube. The supernatant was then centrifuged at 10,000 g for 30 minutes at 4°C to remove large vesicles and transferred to a new tube. Exosomes were extracted using qEV original size exclusion columns (catalog number: SP1, IZON Science, New Zealand). The qEV column was washed three times with 10 mL of 1×PBS (catalog number: 20012-043, Gibco, USA), 10 mL of 0.5 M NaOH (catalog number: 1310-73-2, Millipore, Germany), and 20 mL of 1×PBS, respectively, and then the supernatant was added. When the supernatant finally reached the sieve plate at the top of the column, 2.5 mL of 1×PBS was added. Subsequently, after 3 mL of empty volume eluent, 1.5 mL of liquid was collected and the exosomes were further enriched using magnetic beads.

[0056] Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to resuspend and mix exosomes in 100 μL PBS. Then, 5 μL of the exosome solution was diluted 1,000-fold with PBS and injected into a Nanosight NS300 nanoparticle tracking analyzer (Malvern Panalytical, UK). The particle size and concentration of the exosomes were measured according to standard procedures. A JEM-1400 TEM (JEOL, Akishima, Japan) was used to observe the morphology of the exosomes. In simpler terms, 4 μL of the exosome solution was loaded onto a 400 copper grid and incubated at room temperature (RT) for 1 minute, then excess liquid was blotted away with filter paper. Then, 7 μL of uranyl acetate dihydrate (China Kedo Co., Ltd., catalog number: CD106833) was added for 1 minute, and excess liquid was discarded. The exosomes were dried at RT and their morphology was observed using TEM.

[0057] Western blotting was used to detect exosome-specific markers CD63 and HSP70, and the negative control Calnexin. Plasma exosomes were lysed at RT with 100 μL of a cocktail of 7M urea and protease inhibitors containing 2% SDS (catalog number: 78428, Thermo Scientific, USA) for 30 min, followed by centrifugation at 12,000 rpm for 20 min at 4 °C. The supernatant was carefully transferred to a new EP tube. The protein concentration in the supernatant was determined using the Bicinchonininc acid (BCA, BCA Protein Quantification Kit [catalog number: 23227, Thermo Scientific, USA]) method.

[0058] 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared using the 10% SDS-PAGE Gel FastPreparation Kit (Catalog No.: WB2102, Biotides, China). 10 μL of protein sample was added and electrophoresed using a PowerPac™ HC power supply (Bio-Rad, USA). The protein sample was then transferred to a polyvinylidene fluoride (PVDF) membrane (Catalog No.: 1620177, Bio-Rad, USA) using a rapid multichannel semi-dry transfer instrument (ACEBiotechnology, China). After blocking with 5% skim milk, the PVDF membrane was incubated with primary antibody (CD63 (E1W3T) rabbit mAb [1:1000, Cat. No.: 52090, Cell Signaling Technology, USA] and HSP70 antibody [1:1000, Catalog No.: 4872, Cell Signaling Technology, USA]). The membrane was washed with 0.1% ethyl acetate solution at RT. It was then rinsed with 0.1% triple-buffered saline containing Tween-20 and incubated with secondary antibody (anti-rabbit IgG, horseradish peroxidase (HRP) conjugated antibody [1:10000, Catalog No.: 7074, Cell Signaling Technology, USA]) at RT for 1 hour. Chemiluminescence was performed using the Omni-ECL™ Femto Light chemiluminescence kit (Catalog No.: SQ201, Epizyme, China) and Touch ELISA was used. Imager™ electronic compression imaging system (eBolt, China) acquires images.

[0059] LC-MS analysis for the detection of plasma exosomal proteins was performed using a timsTOF Pro2 mass spectrometer (Bruker, Germany) and a Bruker NanoElute (Bruker, Germany) for 4D label-free quantitative proteomics analysis. Proteins were extracted using EasyPept Ex (catalog number: OSFP0001, EasyPept, Shanghai, China) and their concentrations were determined using the BCA method. A total of 30 μg of exosomal protein from each sample was digested overnight. Peptides were then concentrated and desalted using a Monospin desalting column before chromatographic separation using a NanoElute nanofluidic chromatography system. The separated peptides were ionized using a CaptiveSpray ion source and then detected by a timsTOF Pro2 tandem mass spectrometer in DIA (data-dependent acquisition) mode.

[0060] Protein identification and quantification: Raw mass spectrometry data were processed using PaSER 3.0 software for identification and quantification. Proteins were identified and matched in the human SwissProt and Exocarta (http: / / www.exocarta.org / ) databases. The intensity of each protein peak was normalized to obtain NORM_INTENSITY, which was then used for quantification.

[0061] Differentially expressed proteins (DEPs) were identified and enriched by calculating the mean expression level of each protein in the four groups. The reduced change in expression (FC) was then determined based on the mean expression levels of each protein in the DM and HC groups, the NPDR and DM groups, and the PDR and NPDR groups. Proteins with FC > 1.2 were then selected from the three groups. Finally, the Kruskal-Wallis test was used to identify DEPs with p-values ​​less than 0.05. Functional enrichment analysis of DEPs was performed using DAVID (https: / / david.ncifcrf.gov / ). A p-value less than 0.05 was considered statistically significant. The STRING database (https: / / cn.string-db.org / ) was used to construct the PPI network of the DEPs.

[0062] Central protein identification and clinical relevance analysis were performed using the Wilcox test to identify hub proteins from DEPs, and the results were displayed using a block diagram. The clinical characteristics of the four patient groups were explored using the "compareGroups" software package. The relationship between clinical characteristics and hub proteins was investigated using Spearman analysis. The expression levels of hub proteins in different clinical groups were compared using the Wilcox test.

[0063] The expression level of the pivot protein was verified by enzyme-linked immunosorbent assay (ELISA). The expression level of the pivot protein was determined using ELISA. The following procedures were performed according to the ELISA kit instructions for CELA3A (HM11975, Bioswamp, Wuhan), CELA3B (JOTEK6849Hu, Jotbody, Shenzhen), GLUD1 (HM13755, Bioswamp, Wuhan), and CTRC (SYP-H0971, UpingBio, Hangzhou): Standard samples of different concentrations were prepared using a serial dilution method. Monoclonal antibody-coated microplates were divided into standard wells, blank wells, and sample wells. 50 μL of standard was added to each standard well. 40 μL of the test sample and 10 μL of biotinylated antibody were added to each sample well. 50 μL of HRP conjugate reagent was added to each well (except for the blank wells), gently mixed, sealed, and incubated at 37°C for 1 hour. The plates were washed 5 times with buffer. Then, 50 μL of colorimetric reagent solutions A and B were added to each well, gently shaken to mix, and incubated in the dark for 10 minutes. Subsequently, 50 μL of stop solution was added to each well; the color changed from blue to yellow, indicating that the reaction had stopped. After 15 minutes, the absorbance of each well was measured using a microplate reader, and the concentration of the target analyte in the sample was calculated according to the standard curve. One-way ANOVA was used to verify the significance of differences between groups.

[0064] ROC analysis of pivot proteins and correlation analysis between pivot proteins and clinical indicators were performed using GraphPadPrism 9 to plot ROC curves for CELA3A, CELA3B, CTRC, and GLUD1 proteins, and the area under the curve (AUC) was calculated to represent their performance in predicting disease occurrence. Pearson correlation analysis was used to analyze the correlation between pivot protein expression levels and clinical indicators.

[0065] 2. Experimental Results: Characteristics of Plasma Exosomes. The size of plasma exosomes ranged from 30 nm to 200 nm, with a peak size of 100 nm and a concentration of 10¹¹ per mL. Figure 1 A). Western blotting results showed that the extracted exosomes were rich in the transmembrane protein CD63 and the solute protein HSP70 (positive markers of exosomes), while the intracellular protein Calnexin (a negative marker of exosomes) was absent. Figure 1 B). TEM analysis revealed cup-shaped spherical particles with a diameter of approximately 100 nm, consistent with the morphological characteristics of exosomes. Figure 1 C). The above results indicate that plasma exosomes were successfully isolated and structurally intact, and can be used for further analysis.

[0066] A total of 12,021 peptides and 1,830 proteins were identified using the label-free DEP4D technique. Among these, 32 DEPs were obtained from the DM and HC groups, the NPDR and DM groups, and the PDR and NPDR groups, based on FC > 1.2 and P < 0.05 (Table 1). Gene ontology (GO) analysis showed that these proteins are mainly located in synapses and mitochondria, participating in synaptic transmission, such as chemical synaptic transmission, neuromuscular process control homeostasis, positive regulation of insulin secretion, neurotransmitter uptake, neurotransmitter secretion regulation, and synaptic vesicle extravasation. Figure 2 A). Kyoto Genome Encyclopedia (KEGG) analysis indicates that DEPs are primarily associated with neurodegeneration, diabetic cardiomyopathy, pancreatic secretion, and synaptic vesicle circulation. Figure 2 B).

[0067] Table 1 Differentially expressed proteins

[0068]

[0069] Four pivot proteins (CELA3A, CELA3B, CTRC, and GLUD1) were identified using Wilcox. Significant increases in the expression levels of CELA3A and CTRC were found in the HC and DM groups, the DM and NPDR groups, and the NPDR and PDR groups. Figure 3 AB). Meanwhile, the expression levels of CELA3B and GLUD1 were significantly upregulated in the DM group and NPDR group, and in the NPDR group and PDR group. Figure 3 Correlation analysis showed that HbA1c was significantly positively correlated with CELA3A and CTRC, and fasting blood glucose was significantly correlated with CELA3A (CD). Figure 4 Furthermore, the researchers investigated the expression levels of pivotal proteins in different clinical groups. The expression levels of CELA3A, CELA3B, CTRC, and GLUD1 were significantly higher in the DME and retinal hemorrhage groups. Figure 5 ).

[0070] ELISA results from an independent validation cohort (n=107) of CELA3A, CELA3B, GLUD1, and CTRC expression levels showed that the overall trend of CELA3A, CELA3B, and CTRC protein expression was consistent with the proteomics findings. Figure 6 The expression levels of CELA3A, CELA3B, and CTRC proteins differed significantly between the HC and PDR groups, and between the DM and PDR groups. Furthermore, CELA3B and CTRC also showed significant differences between the HC and NPDR groups, and between the DM and NPDR groups. This suggests that these three proteins may be involved in the progression from DM to DR.

[0071] ROC curve analysis of the validation cohort: To further investigate the diagnostic value of the three proteins, ROC curves were plotted. The results are as follows... Figure 7 As shown, the AUC values ​​of CELA3A in the HC group and DM group, and in the DM group and NPDR group were 0.7237 and 0.8885, respectively. The AUC values ​​of CELA3B in the DM group and NPDR group, and in the NPDR group and PDR group were 0.6077 and 0.6346, respectively. The AUC value of CTRC in the DM group and NPDR group was 0.7912. The AUC values ​​of GLUD1 in the DM group and NPDR group, and in the NPDR group and PDR group were 0.6564 and 0.6892, respectively. These results indicate that CELA3A, CELA3B, CTRC, and GLUD1 can serve as effective biomarkers for distinguishing different stages of DR.

[0072] Correlation analysis between clinical indicators and proteins CELA3A, CELA3B, and CTRC revealed statistically significant differences in duration of diabetic renal syndrome (DR), fasting plasma glucose (FPG), HbA1c, diastolic blood pressure (DBP), triglycerides (TG), and high-density lipoprotein (HDL). Further analysis of the correlation between these three pivotal proteins and clinical indicators... Figure 8 The results showed that CELA3A was significantly positively correlated with DR duration, TG, and aspartate aminotransferase (AST), and significantly negatively correlated with potassium (K). CELA3B was significantly positively correlated with total cholesterol (TG) and calcium (Ca), and significantly negatively correlated with high-density lipoprotein (HDL). CTRC was significantly positively correlated with DR duration and TG, and significantly negatively correlated with HDL.

[0073] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of reagents for detecting the expression level of biomarkers in samples in the preparation of products for diagnosing diabetic retinopathy / diagnosing the progression of diabetic retinopathy, wherein the biomarkers include any one or more of CELA3A, CTRC, CELA3B and GLUD1; The progression of diabetic retinopathy includes two stages: NPDR and PDR.

2. The application according to claim 1, characterized in that, The reagents include reagents required to detect the protein expression level of the biomarker, probes that specifically recognize the biomarker, or primers that specifically amplify the biomarker.

3. The application according to claim 1, characterized in that, The sample was selected from blood plasma.

4. The application according to claim 3, characterized in that, The plasma is plasma exosomes.

5. The application according to claim 1, characterized in that, The products include chips, test strips, and reagent kits.

6. The application according to claim 5, characterized in that, The kit also includes reagents for detecting biomarker protein or gene expression levels by means of Western blotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry, protein chip method, RT-PCR, and DNA blotting.

7. The application according to claim 6, characterized in that, The enzyme-linked immunosorbent assay (ELISA) includes a sandwich assay.

8. The application according to claim 5, characterized in that, The chips include gene chips and protein chips.

9. The application according to claim 8, characterized in that, The gene chip includes a solid-phase carrier and probes immobilized on the solid-phase carrier, the probes including oligonucleotide probes targeting the marker gene for detecting the transcriptional level of the marker gene; the protein chip includes a solid-phase carrier and specific antibodies against the marker protein immobilized on the solid-phase carrier.

Images