Marker for diagnosing diabetic retinopathy

Abstract

The invention discloses a marker for diagnosing diabetic retinopathy. According to the application, the markers CELA3A, CTRC, CELA3B and GLUD1 are determined by researching protein spectra of plasma exosomes of DR patients in different stages and further verification of discovery queue screening and verification queues, and the markers can distinguish the DR patients in different stages, have relatively high diagnosis efficiency, are expected to become markers for monitoring DR progress and provide effective guidance for disease diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to markers for diagnosing diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is the most common microvascular complication caused by diabetes mellitus (DM), which can cause severe visual dysfunction and even blindness. Depending on whether DR proliferates, it can be divided into two categories: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR).

[0003] DR is somewhat hidden in the early stages of the disease. Patients do not feel obvious discomfort in the eyes at the beginning, so it is often not taken seriously. At present, the treatment of DR in the early stage is limited. Only lifestyle intervention and eye hygiene measures cannot control the disease. Once the eyes show obvious discomfort and seek medical treatment, such as blurred vision, visual field loss, and decreased vision, most patients have already reached the middle and late stages of retinal disease, and their vision has been irreversibly damaged. They have missed the best time for treatment and the prognosis is not ideal. Furthermore, the clinical diagnosis of DR is still based on fundus photography and fluorescein angiography performed by ophthalmologists, but these examination equipment are expensive and cannot be popularized in all medical institutions due to differences in culture and medical level in each region. Most importantly, this not only seriously affects the daily life of patients, leading to mobility difficulties, poor quality of life, and even mental problems such as anxiety and depression, but also causes a double blow to the family in terms of manpower and financial resources, resulting in an increase in social burden. It has become a serious public health problem facing my country.

[0004] Therefore, it is of great clinical significance to find economical and affordable biological markers that can predict DR and its progression, so as to enable timely diagnosis and early intervention to prevent and delay the progression of asymptomatic diabetes. Summary of the invention

[0005] To remedy the deficiencies of the prior art, the present invention provides markers for diagnosing diabetic retinopathy.

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

[0007] The first aspect of the present invention provides the use of a reagent for detecting the expression level of a marker in a sample in the preparation of a product for diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression, wherein the marker comprises 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 marker, probes that specifically recognize the marker, or primers that specifically amplify the marker.

[0010] Furthermore, the sample is selected from plasma.

[0011] Furthermore, the plasma is plasma exosomes.

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

[0013] Furthermore, the product includes a chip, a test paper, a test kit or a nucleic acid membrane strip.

[0014] Furthermore, the kit also includes reagents for detecting marker protein or gene expression levels by protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence analysis technology, protein chip method, RT-PCR method, and Southern blotting.

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

[0016] Furthermore, the gene chip comprises a solid phase carrier and a probe fixed on the solid phase carrier, wherein the probe comprises an oligonucleotide probe targeting the marker gene for detecting the transcription level of the marker gene; the protein chip comprises a solid phase carrier and a specific antibody against the marker protein fixed on the solid phase carrier.

[0017] The 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 a protein encoded by it with a substance to be screened; and detecting the expression or activity of the marker gene or the protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the marker gene or the protein encoded by it, the substance to be screened is a candidate drug for treating diabetes / diabetic retinopathy, and the marker comprises any one or more of CELA3A, CTRC, CELA3B and GLUD1.

[0018] The fourth aspect of the present invention provides the use of any one or more markers of CELA3A, CTRC, CELA3B and GLUD1 in constructing a computational model for diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression.

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

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

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

[0022] Further, the system comprises: Acquisition unit: used for acquiring the expression level of the marker in the sample; Processing unit: used for obtaining the result of diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression according to the expression level of the marker.

[0023] Advantages and beneficial effects of the present invention: This application studied the protein spectrum of plasma exosomes in patients with DR at different stages, and through screening of the discovery cohort and further verification of the validation cohort, identified the markers CELA3A, CTRC, CELA3B and GLUD1. These markers can distinguish patients with DR at different stages and have high diagnostic efficacy. They are expected to become markers for monitoring the progression of DR and provide effective guidance for the diagnosis of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1are characteristic images of plasma exosomes, where 1A is a nanoparticle tracking analysis image, 1B is a protein blot image of exosome markers CD63, HSP70, and Calnexin, and 1C is a transmission electron microscopy image of exosomes; Figure 2 It is the enrichment analysis diagram of DEPs, among which, 2A is the GO function enrichment analysis diagram of DEPs, and 2B is the KEGG enrichment analysis result diagram of DEPs; Figure 3 3A is the expression level graph of hub proteins in the training cohort, among which 3A is the expression level graph of CELA3A, 3B is the expression level graph of CTRC, 3C is the expression level graph of CELA3B, and 3D is the expression level graph of GLUD1; Figure 4 This is the correlation analysis diagram between hub protein and fasting blood glucose and glycosylated hemoglobin; Figure 5 5A is the expression level graph of hub proteins in different clinical groups, among which 5A is the expression level graph of CELA3A, 5B is the expression level graph of CTRC, 5C is the expression level graph of CELA3B, and 5D is the expression level graph of GLUD1; Figure 6 are the expression level graphs of hub proteins in the validation cohort, among which 6A is the expression level graph of CELA3A, 6B is the expression level graph of CTRC, 6C is the expression level graph of CELA3B, and 6D is the expression level graph of GLUD1; Figure 7 are ROC curves of hub proteins in the validation cohort, among which 7A-C are ROC curves of CELA3A, 7D-F are ROC curves of CELA3B, 7G-I are ROC curves of CTRC, and 7J-L are ROC curves of GLUD1; Figure 8 This is a correlation analysis chart between clinical indicators and proteins CELA3A, CELA3B, and CTRC. DETAILED DESCRIPTION

[0025] The definitions of some terms used in this specification are provided below. Unless otherwise defined, 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 belongs.

[0026] The present invention provides the use of a reagent for detecting the expression level of a marker in a sample in the preparation of a product for diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression, wherein the marker comprises any one or more of CELA3A, CTRC, CELA3B and GLUD1.

[0027] The reagents include reagents required for detecting the protein expression level of the marker, probes that specifically recognize the marker, or primers that specifically amplify the marker.

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

[0029] In some embodiments, primer refers to a short nucleic acid molecule, such as a DNA oligonucleotide, which can anneal with a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid chain. The primer can be extended along the target nucleic acid molecule by a polymerase. Therefore, the primer can be used to amplify the target nucleic acid molecule, wherein the sequence of the primer is specific for the target nucleic acid molecule, such as the primer will hybridize with the target nucleic acid molecule under very high stringency hybridization conditions.

[0030] Primer or probe of the present application can use phosphoramidite solid phase support method or other well-known method chemical synthesis.Also can use many means known in the art to modify.The limiting examples of these modifications comprise methylation, capping, displacement and modification between nucleotides with one or more analogs of natural nucleotides, for example, modifying uncharged connector (for example, methyl phosphate, phosphotriester, phosphoramidite, carbamate etc.), or modifying charged connector (for example, phosphorothioate, phosphorodithioate etc.).

[0031] The reagent also includes a detectable label.

[0032] In some embodiments, detectable label refers to a composition that can produce a detectable signal indicating the presence of a target polynucleotide in a determination sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Therefore, labeling is any composition that can be detected by a device or method, including but not limited to spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics, chemical detection devices or any other suitable device. In some embodiments, labeling can be detected visually without the aid of a device. Labeling is used to refer to any chemical group or part with detectable physical properties or can cause chemical groups or parts to show any compound of detectable physical properties, such as an enzyme that catalyzes substrate conversion into a detectable product. Labeling also encompasses compounds that suppress the performance of specific physical properties. Labeling can also be a compound as a member of a pair of binding, and another member thereof 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, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

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

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

[0037] In some embodiments, the chip is also referred to as an array, which refers to a solid support comprising a connected nucleic acid or peptide probe. The array typically comprises a plurality of different nucleic acid or peptide probes connected to the substrate surface according to different known positions. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid phase synthesis methods. The array can comprise a flat surface, or can be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass, or any other suitable substrate. The array can be packaged in a certain manner to allow for diagnosis of a fully functional device or manipulation of other methods.

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

[0039] In some embodiments, the gene chip includes a solid phase carrier and a probe fixed on the solid phase carrier, wherein the probe includes an oligonucleotide probe for the marker gene for detecting the transcription level of the marker gene; the protein chip includes a solid phase carrier and a specific antibody for the marker protein fixed on the solid phase carrier. The gene chip can be used to detect the expression levels of multiple genes including the above-mentioned markers (for example, multiple genes related to diabetes / diabetic retinopathy). The protein chip can be used to detect the expression levels of multiple proteins including the above-mentioned marker proteins (for example, multiple proteins related to diabetes / diabetic retinopathy). By simultaneously detecting multiple markers related to diabetes / diabetic retinopathy, the accuracy of diagnosing diabetes / diabetic retinopathy can be greatly improved.

[0040] In some embodiments, the components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit usually include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the present invention will also usually include a container for accommodating reactants, sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which the required vial can be retained.

[0041] The solid support of the kit can be, for example, plastic, silicon, 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 a probe fixed on the substrate; the substrate can be any substrate suitable for fixing the probe, including but not limited to nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica gel wafer, micro-magnetic beads.

[0043] In some embodiments, the sample includes, but is not limited to, bone marrow, peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, breast milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder washing fluid, and bronchoalveolar lavage fluid.

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

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

[0046] The present invention provides the use of any one or more markers of CELA3A, CTRC, CELA3B and GLUD1 in constructing a system / device for diagnosing diabetes / diabetic retinopathy / diagnosing the progression of diabetic retinopathy.

[0047] The system comprises: Acquisition unit: used for acquiring the expression level of the marker in the sample; Processing unit: used for obtaining the result of diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression according to the expression level of the marker.

[0048] In some embodiments, the processing unit (processor) may include one or more microprocessors or digital processors. The processor may call program codes stored in a memory to execute related functions. The processor is also called a central processing unit (CPU), which may be a very large scale integrated circuit, a computing core (Core) and a control core (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 protein expression detection, polysome qPCR, enzyme-linked immunosorbent assay, or transcriptome sequencing.

[0050] In some embodiments, the system / device may further include a result display unit, and the result display unit is used to display the conclusion obtained by the processing unit.

[0051] In some implementations, the result display unit displays the result by screen display, voice broadcast or printing.

[0052] In some embodiments, the implementation of the system may include performing or completing the selected tasks manually, automatically, or in combination thereof. Moreover, actual instruments and devices according to embodiments of the system of the present invention may implement multiple selected tasks using an operating system by hardware, by software, or by firmware, or by a combination thereof.

[0053] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0054] Example

[0055] 1. Materials and Methods Participants and sample collection In the study of this application, the discovery cohort included 6 controls (HC), 6 patients with type 2 diabetes (DM) without clinically obvious retinopathy, 6 patients with NPDR (NPDR), and 5 patients with PDR (PDR). The validation cohort included 26 controls (HC), 30 patients with DM (DM), 26 patients with NPDR (NPDR), and 25 patients with PDR (PDR). The age of the subjects ranged from 40 to 80 years old. Glycated hemoglobin (HbA1c) levels were 7.0%-10.5%. Patients with NPDR and PDR were diagnosed by fundus fluorescein angiography. For patients with inconsistent disease progression in both eyes, the eye with the more severe disease 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, systemic persistent infection, cerebral hemorrhage and / or cerebral infarction within 90 days, and other severe systemic diseases were excluded. The design of this study conforms to 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 subjects. Peripheral blood samples were collected from the above two groups. Plasma was extracted by centrifugation at 1,500 g for 20 minutes at 4°C and stored at -80°C.

[0056] Extraction of plasma exosomes Plasma samples were thawed, centrifuged at 2,000 g for 10 min at 4°C, and the supernatant was transferred to a new tube. The supernatant was then centrifuged at 10,000 g for 30 min at 4°C to remove large vesicles and transferred to a new tube. Exosomes were extracted using qEVoriginal Size Exclusion Columns (Cat. No.: SP1, IZON Science, New Zealand). The qEV column was washed three times with 10 mL 1× PBS (Cat. No.: 20012-043, Gibco, USA), 10 mL 0.5 M NaOH (Cat. No.: 1310-73-2, Millipore, Germany), and 20 mL 1× PBS, respectively, and then the supernatant was added. When the supernatant finally entered the frit at the top of the column, 2.5 mL 1× PBS was added. Subsequently, 1.5 mL of liquid was collected after 3 mL of empty volume flowed out and exosomes were further enriched using magnetic beads.

[0057] Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) Exosomes were resuspended in 100 μL PBS and mixed. Then, 5 μL of the exosome solution was diluted 1,000-fold with PBS and injected into the Nanosight NS300 nanoparticle tracking analyzer (Malvern Panalytical, UK). The particle size and concentration of exosomes were measured according to the standard protocol. The JEM-1400 TEM (JEOL, Akishima, Japan) was used to observe the morphology of exosomes. Briefly, 4 μL of the exosome solution was loaded onto a 400 copper grid and incubated at room temperature (RT) for 1 min, and then the excess liquid was removed with filter paper. Then 7 μL of uranyl acetate dihydrate (Kodo, China, Cat. No.: CD106833) was added for 1 min, and the excess liquid was discarded. The morphology of the exosomes was observed by TEM after drying at RT.

[0058] Western blotting Western blotting was used to detect exosome surface specific markers CD63, HSP70 and negative control Calnexin. Plasma exosomes were lysed with 100 μL of 7M urea and protease inhibitor cocktail (Cat. No. 78428, Thermo Scientific, USA) containing 2% SDS at RT for 30 minutes, and then centrifuged at 12,000 rpm for 20 minutes at 4 °C. The supernatant was carefully transferred to a new EP tube. The protein concentration of the supernatant was determined using the Bicinchonininc acid (BCA, BCA Protein Assay Kit [Cat. No. 23227, Thermo Scientific, USA]) method.

[0059] 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was prepared using 10% SDS-PAGE Gel FastPreparation Kit (Cat. No.: WB2102, Biotides, China). 10 μL of protein sample was added and electrophoresed using PowerPac™ HC power source (Bio-Rad, USA). Protein samples were transferred to polyvinylidene fluoride (PVDF) membrane (Cat. No.: 1620177, Bio-Rad, USA) using a fast multi-channel semi-dry transfer instrument (ACE Biotechnology, China). After blocking with 5% skim milk, the PVDF membrane was incubated with primary antibodies (CD63 (E1W3T) rabbit mAb [1:1000, Cat.No.:52090, Cell Signaling Technology, USA], HSP70 antibody [1:1000, Cat.No.: 4872, Cell Signaling Technology, USA]). The membrane was washed with 0.1% ethyl acetate solution at RT and then washed with 0.1% ethyl acetate solution. The membrane was rinsed with 0.1% Tris-buffered saline containing Tween-20 and incubated with secondary antibodies (anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody [1:10000 Cat.No.:7074, Cell Signaling Technology, USA]) at RT for 1 hour. Chemiluminescence was performed using the Omni-ECL™ Femto Light Chemiluminescence Kit (Cat.No.: SQ201, Epizyme, China) and the Touch Images were acquired using an Imager™ electronic compression imager (eBolt, China).

[0060] LC-MS analysis for detection of plasma exosomal proteins 4D label-free quantitative proteomics analysis was performed using a timsTOF Pro2 mass spectrometer (Bruker, Germany) and a Brucker NanoElute (Bruker, Germany). Proteins were extracted using EasyPept Ex (Cat. No.: OSFP0001, EasyPept, Shanghai, China), and their concentrations were determined using the BCA method. A total of 30 μg of exosome proteins in each sample were digested overnight. The peptides were then concentrated and desalted using a Monospin desalting column and then chromatographically separated using a nanoflow liquid chromatography system NanoElute. The separated peptides were ionized by a CaptiveSpray ion source and detected in the DIA (data-dependent acquisition) mode on a tandem mass spectrometer timsTOF Pro2.

[0061] Protein identification and quantification The raw mass spectrometry data were processed by PaSER 3.0 software for identification and quantitative analysis. 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, and then quantitative analysis was performed.

[0062] Identification and enrichment analysis of differentially expressed proteins (DEPs) The mean expression level of each protein in the four groups was calculated, and then the fold change (FC) was determined based on the mean value of each protein in the DM group versus the HC group, the NPDR group versus the DM group, and the PDR group versus the NPDR group. Subsequently, proteins with FC>1.2 in the three groups were selected. Finally, the Kruskal-Wallis test was used to identify DEPs with a p-value 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 statistically significant. The STRING database (https: / / cn.string-db.org / ) was used to construct the PPI network of DEPs.

[0063] Identification and clinical relevance of centrin The Wilcox test was used to identify hub proteins from DEPs, and the results were displayed using box plots. The clinical characteristics of the four groups of patients were explored using the "compareGroups" software package. The relationship between clinical characteristics and hub proteins was investigated by Spearman analysis. The Wilcox test was used to compare the expression levels of hub proteins in different clinical groups.

[0064] Validation of hub protein expression levels by ELISA The expression levels of hub proteins were determined by enzyme-linked immunosorbent assay (ELISA). The ELISA kit instructions for CELA3A (HM11975, Bioswamp, Wuhan), CELA3B (JOTEK6849Hu, Jotbody, Shenzhen), GLUD1 (HM13755, Bioswamp, Wuhan), and CTRC (SYP-H0971, UpingBio, Hangzhou) were followed as follows: Standard samples of different concentrations were prepared by gradient dilution. The monoclonal antibody-coated microplate was divided into standard wells, blank wells, and sample wells. 50 μL of standard was added to the standard wells. 40 μL of detection sample and 10 μL of biotinylated antibody were added to the sample wells. 50 μL of HRP conjugate reagent was added to each well (except the blank well), gently shaken, sealed, and incubated at 37°C for 1 hour. Washed 5 times with buffer. Then 50 μL of colorimetric 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, and the color changed from blue to yellow, indicating that the reaction 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 based on the standard curve. One-way ANOVA was used to verify the significance of the differences between the groups.

[0065] ROC analysis of hub proteins and correlation analysis between hub proteins and clinical indicators ROC curves of CELA3A, CELA3B, CTRC, and GLUD1 proteins were plotted using GraphPad Prism 9, and the area under the curve (AUC) was calculated to represent their performance in predicting the occurrence of the disease. Pearson correlation analysis was performed on the correlation between the expression levels of hub proteins and clinical indicators.

[0066] 2. Experimental results Characteristics of plasma exosomes The size of plasma exosomes ranges from 30 nm to 200 nm, with a peak size of 100 nm and a concentration of 1011 cells / mL ( Figure 1 A). Western results showed that the extracted exosomes were rich in transmembrane protein CD63 and solute protein HSP70 (positive markers of exosomes), while the intracellular protein Calnexin (negative marker of exosomes) was absent ( Figure 1 B). TEM analysis showed that cup-shaped round particles with a diameter of about 100 nm were observed, which is 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.

[0067] Determine DEP A total of 12,021 peptides and 1,830 proteins were identified by 4D label-free technology. Among them, 32 DEPs were obtained based on FC>1.2 and P value<0.05 in the DM group and HC group, NPDR group and DM group, and PDR group and NPDR group (Table 1). Gene ontology (GO) analysis showed that these proteins were mainly located in synapses and mitochondria and participated in synaptic transmission, such as chemical synaptic transmission, control balance of neuromuscular processes, positive regulation of insulin secretion, neurotransmitter uptake, regulation of neurotransmitter secretion, and synaptic vesicle extravasation ( Figure 2 A). Kyoto Encyclopedia of Genomes (KEGG) analysis showed that DEPs are mainly associated with neurodegeneration, diabetic cardiomyopathy, pancreatic secretion, and synaptic vesicle recycling ( Figure 2 B).

[0068] Table 1 Differentially expressed proteins

[0069]

[0070] Identification and clinical relevance of hub proteins A total of four hub proteins (CELA3A, CELA3B, CTRC, and GLUD1) were identified using Wilcox analysis. It was found that the expression levels of CELA3A and CTRC were significantly increased in the HC group and DM group, the DM group and NPDR group, and the NPDR group and PDR group ( 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 CD). Correlation analysis showed that HbA1c was significantly positively correlated with CELA3A and CTRC, and fasting blood glucose was significantly correlated with CELA3A ( Figure 4 ). In addition, the researchers also explored the expression levels of hub proteins in different clinical groups. The expression levels of CELA3A, CELA3B, CTRC, and GLUD1 were significantly higher in the DME group and fundus hemorrhage group ( Figure 5 ).

[0071] Verification of the expression levels of CELA3A, CELA3B, GLUD1, and CTRC The ELISA results of the independent validation cohort (n=107) showed that the overall trend of CELA3A, CELA3B, and CTRC protein expression was consistent with the proteomics results ( Figure 6). The expression levels of CELA3A, CELA3B, and CTRC proteins were significantly different between the HC and PDR groups, and between the DM and PDR groups. In addition, CELA3B and CTRC were also significantly different 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.

[0072] ROC curve analysis of the validation cohort In order to further study the diagnostic value of the three proteins, ROC curves were drawn. Figure 7 As shown in the figure, the AUC values ​​of CELA3A in the HC group and DM group, and 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 the NPDR group and PDR group were 0.6077 and 0.6346, respectively. The AUC values ​​of CTRC in the DM group and NPDR group were 0.7912. The AUC values ​​of GLUD1 in the DM group and NPDR group, and the NPDR group and PDR group were 0.6564 and 0.6892, respectively. The above results show that CELA3A, CELA3B, CTRC and GLUD1 can be used as effective biomarkers to distinguish different stages of DR.

[0073] Correlation analysis between clinical indicators and proteins CELA3A, CELA3B, and CTRC There were statistically significant differences in DR duration, fasting plasma glucose (FPG), HbA1c, diastolic blood pressure (DBP), triglycerides (TG), and high-density lipoprotein (HDL). The correlation between the three hub proteins and clinical indicators was further analyzed ( 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.

[0074] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting the expression level of a marker in a sample in the preparation of a product for diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression, wherein the marker comprises any one or more of CELA3A, CTRC, CELA3B and GLUD1; Preferably, the progression of diabetic retinopathy includes two stages: NPDR and PDR.

2. The use according to claim 1, characterized in that: The reagents include reagents required for detecting the protein expression level of the marker, probes that specifically recognize the marker, or primers that specifically amplify the marker.

3. The use according to claim 1, characterized in that: The sample is selected from plasma; Preferably, the plasma is plasma exosomes.

4. A product for diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression, characterized in that: The product includes reagents for detecting the expression level of markers in a sample, and the markers include any one or more of CELA3A, CTRC, CELA3B and GLUD1.

5. The product according to claim 4, characterized in that The products include chips, test strips, test kits or nucleic acid membrane strips.

6. The product according to claim 5, characterized in that The kit also includes reagents for detecting marker protein or gene expression levels by protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence analysis technology, protein chip method, RT-PCR method, and Southern blotting.

7. The product according to claim 5, characterized in that The chips include gene chips and protein chips; Preferably, the gene chip comprises a solid phase carrier and a probe fixed on the solid phase carrier, wherein the probe comprises an oligonucleotide probe targeting the marker gene for detecting the transcription level of the marker gene; the protein chip comprises a solid phase carrier and a specific antibody against the marker protein fixed on the solid phase carrier.

8. A method for screening candidate drugs for treating diabetes / diabetic retinopathy, characterized in that: The method comprises: treating a culture system expressing or containing a marker gene or a protein encoded by the marker gene with a substance to be screened; and detecting the expression or activity of the marker gene or the protein encoded by the marker gene in the system; wherein, when the substance to be screened inhibits the expression level or activity of the marker gene or the protein encoded by the marker gene, the substance to be screened is a candidate drug for treating diabetes / diabetic retinopathy, and the marker comprises any one or more of CELA3A, CTRC, CELA3B and GLUD1.

9. Application of any one or more markers of CELA3A, CTRC, CELA3B and GLUD1 in constructing a computational model for diagnosing diabetes / diabetic retinopathy / diagnosing the progression of diabetic retinopathy; Preferably, the progression of diabetic retinopathy includes two stages: NPDR and PDR.

10. Use of any one or more markers of CELA3A, CTRC, CELA3B and GLUD1 in constructing a system or device for diagnosing diabetes / diabetic retinopathy / diabetes retinopathy progression; Preferably, the progression of diabetic retinopathy includes two stages: NPDR and PDR; Preferably, the system comprises: Acquisition unit: used for acquiring the expression level of the marker in the sample; Processing unit: used for obtaining the result of diagnosing diabetes / diabetes retinopathy / diabetes retinopathy progression according to the expression level of the marker.

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