Extracellular vesicle proteins as biomarkers for diagnosis of abdominal aortic aneurysm and uses thereof

Abstract

The application belongs to the technical field of molecular diagnosis, and particularly relates to a group of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm and application. The application early diagnoses abdominal aortic aneurysm through determination of circulating extracellular vesicle protein markers, and provides an effective tool for diagnosis of abdominal aortic aneurysm. The extracellular vesicle protein marker is any one or two or more molecular marker combinations of interleukin-4 (IL-4), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), nerve growth factor (Neurturin) and tumor suppressor M (Oncostatin-M). The application uses proteins in extracellular vesicles as specific markers for early diagnosis of abdominal aortic aneurysm, which is a less applied field in current early diagnosis of AAA. The specific proteins in EVs may be directly related to the pathological process of AAA, and are different from conventional serum markers, and have higher specificity and sensitivity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular diagnosis, and specifically relates to a group of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm and application thereof. BACKGROUND

[0002] Abdominal aortic aneurysm (AAA) is the most common type of life-threatening aneurysm. The incidence of AAA in men and women is about 5:1. In some countries, the incidence of people over 60 years old is between 2% and 5%. Epidemiological studies have shown that the incidence of men over 65 years old is 1% to 2%, and 150,000 to 200,000 people die from AAA rupture worldwide each year. The incidence of AAA is closely related to risk factors such as age, gender and smoking. The main cause of AAA is degenerative changes or other non-specific factors in the arterial wall, rather than atherosclerosis. More than 90% of AAA is caused by these degenerative changes. Recent studies have shown that the pathophysiology of AAA involves multiple factors, including inflammatory response, matrix metalloproteinase (MMP) activation, oxidative stress, intraluminal thrombus formation, smooth muscle apoptosis and extracellular matrix (ECM) degradation.

[0003] Currently, ultrasound is a suitable method for screening AAA, and CTA (angiography) is the preferred method for evaluation and treatment planning. However, ultrasound has certain limitations for patients with complex anatomical structures, although CTA and MRA (magnetic resonance angiography) detection results are accurate, but the cost is high, and there is a risk of radiation or contrast agent. In addition, since the imaging diagnosis of AAA is usually applied after the appearance of obvious clinical symptoms, at this time the aorta has already undergone irreversible changes, greatly increasing the risk of rupture and death. Therefore, it is necessary to improve the existing AAA risk assessment model and find new biomarkers to provide valuable diagnostic information.

[0004] The expression or circulating levels of multiple proteins are associated with the occurrence, development and rupture risk of AAA, among which gelatinases MMP-2 and MMP-9 are significantly elevated in AAA patients, especially MMP-9, which is responsible for the degradation of collagen and elastin in the extracellular matrix, leading to weakening of the arterial wall and promoting aneurysm formation, and high expression of matrix metalloproteinases MMPs suggests the degradation process of the arterial wall; previous studies have shown that recombinant human C-reactive protein (CRP) can induce the production of nitric oxide, regulate fibrinolysis and change the expression of inflammatory molecules and complement, and these processes are directly related to the formation of AAA; in addition, serum Cystatin C, as a potent cysteine protease inhibitor, plays a role in the formation of abdominal aortic aneurysm, in which the extracellular matrix, including elastin and collagen, is continuously degraded by proteases, leading to gradual weakening of the structure of the arterial wall. Cystatin C may slow down this process by inhibiting the activity of these proteases, preventing the expansion of aneurysm or slowing down its progression. Early diagnosis of AAA by peripheral proteins has certain potential, but also faces many limitations. Most protein markers have weak correlation with the clinical course of AAA, and few have potential for clinical use.

[0005] Exosomes are small vesicles secreted by cells, with a diameter of about 30-150 nanometers, including proteins, lipids, DNA, mRNA, miRNA and other biological molecules. In recent years, exosomes have been paid attention to as potential biomarkers in the early diagnosis of various diseases. Exosomes are secreted by specific cell types, and they contain specific molecules that reflect the state of these cells. The cell specificity of exosomes enables them to reflect pathological changes in the arterial wall earlier and more accurately, helping to identify early AAA patients, while traditional circulating markers (such as MMPs, CRP, etc.) lack such cell or tissue specificity. Exosomes are surrounded by a double-layered phospholipid membrane, which protects the proteins and nucleic acids inside from degradation by the external environment. This structure makes exosomes relatively stable in body fluids such as blood, urine, and saliva, making them easy to detect by non-invasive methods. This stability helps to reliably detect the protein markers contained in exosomes, reducing false negative results and improving the sensitivity of early screening. In addition, the non-invasive detection of specific exosomes makes them highly feasible for large-scale population screening, especially for monitoring high-risk individuals or early detection of diseases.

[0006] Previous studies have shown that exosome protein markers derived from endothelial cells and platelets also have potential applications in the early diagnosis of coronary heart disease, which can be used as a supplement to traditional myocardial injury markers (such as troponin) and for early diagnosis and damage assessment of myocardial infarction.

[0007] In recent years, the proximity extension assay (PEA) technology of Olink (ultra-micro multi-target protein biomarker discovery platform) is suitable for various disease mechanism research and clinical diagnosis and treatment of personalized medicine due to its high sensitivity, specificity, extremely low sample requirement and other characteristics. Although there has been progress in the diagnosis of circulating extracellular vesicle proteins, there is currently no research on the early diagnosis potential of AAA. Therefore, the expression profile of the circulating extracellular vesicle proteome of AAA patients is detected by using the PEA technology of Olink. By comparison and analysis, the differentially expressed circulating extracellular vesicle proteins are determined. Target gene prediction and functional enrichment analysis are performed by using bioinformatics methods, so as to reveal the potential pathological changes of AAA, find new biomarkers and the biological functions and pathways related to AAA, and determine potential new therapeutic targets, thereby providing technical support for the treatment of AAA. SUMMARY

[0008] In order to solve the limitation of early diagnosis of abdominal aortic aneurysm, the purpose of the present application is to provide a group of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm and application. Based on the extracellular vesicle protein markers provided in the present application, early diagnosis of abdominal aortic aneurysm can be achieved by determining the circulating extracellular vesicle protein markers, thereby providing an effective tool for the diagnosis of abdominal aortic aneurysm.

[0009] The present application also provides a screening method of the extracellular vesicle protein markers.

[0010] In order to achieve the above-mentioned purposes, the technical scheme of the present application is realized as follows:

[0011] The present application first provides a series of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm. The extracellular vesicle protein markers are any one or a combination of two or more molecular markers of interleukin-4 (IL-4), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), Neurturin and Oncostatin-M.

[0012] Preferably, the extracellular vesicle protein markers are a combination of two or more of IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M.

[0013] Further preferably, the extracellular vesicle protein markers are a combination of IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M.

[0014] Further preferably, the extracellular vesicle is plasma extracellular vesicle.

[0015] Based on one general inventive concept, the present application also provides use of the extracellular vesicle protein marker in the preparation of a product for diagnosing abdominal aortic aneurysm.

[0016] Specifically, the product is a medicament, a kit, a microarray or a biochip.

[0017] Based on one general inventive concept, the present application also provides a kit, a microarray or a biochip comprising the extracellular vesicle protein marker for diagnosing abdominal aortic aneurysm (AAA).

[0018] Based on one general inventive concept, the present application also provides a screening method of the extracellular vesicle protein marker, comprising the following steps:

[0019] 1) extracting extracellular vesicle proteins from a plasma sample using an extracellular vesicle protein extraction kit;

[0020] 2) screening differentially expressed extracellular vesicle proteins using Olink proteome technology, cluster analysis and biological function enrichment analysis, and statistically analyzing the obtained data according to the differences in differentially expressed extracellular vesicle proteins between different groups;

[0021] 3) verifying the expression levels of the screened differentially expressed extracellular vesicle proteins using Western Blot and enzyme-linked immunosorbent assay (ELISA);

[0022] 4) analyzing the obtained experimental results, drawing a ROC curve for each differentially expressed extracellular vesicle protein, and evaluating the diagnostic ability of potential extracellular vesicle protein markers according to the ROC curve and the area under the ROC curve (AUC), and finally screening out markers or marker combinations with good accuracy, sensitivity and specificity.

[0023] Specifically, in step 1), the method further comprises the steps of detecting the morphology of extracellular vesicles by transmission electron microscopy (TEM), measuring the particle size distribution and particle concentration of extracellular vesicles (EVs) using a nano-flow detector, and detecting specific extracellular vesicle protein markers by Western Blot.

[0024] Specifically, step 2) mainly includes the following operations: a. total protein extraction and immunolinking, extracting total protein from the obtained plasma extracellular vesicle PBS suspension, and determining the protein concentration using a BCA protein quantification kit;

[0025] The immunological connection and base complementary connection of the oligonucleotide sequence antibody, the immunological connection step: the protein sample is mixed with the oligonucleotide sequence antibody and incubated; the base complementary connection: two designed oligonucleotide sequences are used to be connected to different antibodies of the target protein respectively; after the complementary connection, a DNA polymerase is added, and extension is carried out in the system;

[0026] b. In the library preparation stage, first, a specific barcode label is added to each sample after the extension reaction is completed, which is used for subsequent library differentiation and sample identification, and purification, to obtain a library sample;

[0027] In the qPCR detection stage, the purified library sample is diluted and added to the qPCR reaction system to complete the amplification of the library sample;

[0028] After the amplification is completed, the qPCR instrument is used to collect the fluorescence signal in real time, and the Ct value of each sample is recorded; through the data analysis software, the Ct value is converted into the NPX value, to provide standardized protein expression data for subsequent differential expression analysis;

[0029] c. The data generated by the qPCR detection are converted into Ct values or counts files, which are processed by the NPX Signature software and the Olink NPX Signature (NPX Manager) software, to obtain the original sample NPX Data required by the biological information analysis process, and to make the original sample NPX Data box plot and the sample principal component analysis PCA plot;

[0030] d. According to the original sample NPX Data box plot and the sample principal component analysis PCA plot, the sample dimensionality reduction clustering situation is viewed; after the original data are quality controlled, clean data of protein NPX expression are obtained, and through student's test analysis or variance analysis on different experimental variables, differential proteins under the experimental variable condition are obtained;

[0031] e. The differential protein data are further subjected to biological function enrichment analysis, including GO biological process, GO molecular function, KEGG pathway, human gene set, Reactome gene set and typical pathway.

[0032] Specifically, in step 2) a, the reaction system is: reaction buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 5 mM MgCl2); base complementary reaction conditions: incubation at 25℃ for 30 minutes; DNA polymerization reaction conditions: reaction at 37℃ for 20 minutes, followed by heating at 95℃ for 5 minutes to terminate the reaction.

[0033] Specifically, in step 2) b, the total reaction system volume is 20 μL, including 10 μL of 2x qPCR Master Mix, 1 μL of library sample template, 0.4 μL of target primer pair (final concentration of 200 nM), and 8.6 μL of sterile ultrapure water. In the qPCR reaction, first, initial denaturation is performed at 95℃ for 5 minutes to ensure complete denaturation of the DNA template; then 40 cycles of 95℃ heating for 15 seconds and 60℃ annealing and extension for 1 minute are performed.

[0034] Specifically, the main operation of step 3) is a. First, the extracellular vesicle protein is separated and purified by ultracentrifugation method to obtain a high-purity extracellular vesicle sample; b. The extracellular vesicle is lysed and the protein is extracted using RIPA lysis and extraction buffer, and Western Blot and ELISA detection are performed.

[0035] Specifically, the screening criteria of step 4) is that the AUC value is greater than 0.75.

[0036] Compared with the prior art, the beneficial effects of the present application are:

[0037] 1. By detecting specific protein markers in EVs, the present application can detect diseases in the early stage of aneurysm occurrence or even in the asymptomatic stage. This molecular level detection method can identify early biological changes related to the lesion, without relying on structural changes of blood vessels, thus achieving diagnosis at an earlier stage.

[0038] 2. EV detection can be completed through simple blood sample collection, which is a non-invasive detection. Liquid biopsy is not only convenient, but also can be sampled multiple times for follow-up and monitoring of disease progression, especially suitable for long-term management and regular screening. Specific protein markers in EVs can more accurately reflect the pathophysiological processes related to abdominal aortic aneurysm (such as inflammation, vascular wall degradation, etc.).

[0039] 3. The present application can achieve high specificity and high sensitivity in the diagnosis of abdominal aortic aneurysm through the joint detection of multiple markers, reducing the probability of misdiagnosis and missed diagnosis. By detecting the EV protein marker spectrum of individual patients, the pathological condition of each patient can be evaluated, and personalized diagnosis and treatment plans can be provided. This individualized evaluation based on molecular markers helps guide early intervention and optimize treatment decisions.

[0040] 4. The present invention can expand the scope of screening through the detection of EV protein markers. It is not only applicable to high-risk groups, but also can be used for early screening of other potential risk groups. EV detection provides a more extensive and accurate screening tool. This test is low-cost and simple to operate, suitable for screening in primary medical units or resource-limited areas. This liquid biopsy can serve as an effective early diagnostic tool, reducing dependence on high-cost imaging equipment and optimizing the allocation of medical resources.

[0041] 5. The present invention can significantly improve the early detection capability and long-term management effect of abdominal aortic aneurysm, filling the deficiencies of existing diagnostic technologies in early detection and personalized evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Figure 2 shows the characterization and internalization of AAA extracellular vesicles (AAA-Exo) and normal human plasma extracellular vesicles (Nor-Exo). Figure A shows the complete workflow for high-throughput sequencing and analysis of AAA extracellular vesicles and normal human plasma extracellular vesicle samples; Figure B shows an electron microscopy image of isolated extracellular vesicles, with the scale bar at 200 nm; Figure C shows the high-sensitivity flow cytometry results for extracellular vesicle nanoparticle analysis; Figure D shows a Western blot showing Tsg101 / Alix / CD9 proteins and EV negative marker protein Calnexin in extracellular vesicles and cell control samples.

[0043] Figure 2 Figure 2 is a plasma protein distribution and analysis diagram, where A is a heat map of all expressed plasma proteins between AAA and the control group; B is a box plot of NPX distribution of all expressed plasma proteins between AAA and the control group, where QC_Warning is marked as a red Warning sample, and Pass is a QC Pass sample, marked as a turquoise Pass; C is a principal component analysis diagram of all expressed plasma proteins;

[0044] Figure 3 Figure 1 is a plasma protein map, where A is a volcano plot of plasma proteins, with red dots representing upregulated proteins, gray dots representing insignificant proteins, and blue dots representing downregulated proteins; B is the number of differentially expressed proteins (DEPs) in the two modules; C is a heat map of plasma proteins, which shows the expression profiles of five proteins under the critical values ​​of absolute logFC>0.5 and p<0.05, where AAA represents the AAA group and Health represents the control normal group; D is a dimensionality reduction analysis of the plasma protein expression profiles of AAA and control samples; E is a Pearson correlation analysis of normal and AAA samples;

[0045] Figure 4Figure 6 is a diagram of the biological significance of DEPs target genes, wherein A is the PPI network between potential biomarkers established by using STRING database, and the edge width represents the comprehensive score; PPI is protein-protein interaction; the top 20 clusters obtained by Metascape protein target gene pathway and process enrichment analysis are colored by p value; B is the enrichment term network of DEPs target genes; terms with a similarity greater than 0.3 are connected by edges, the left figure is colored by enrichment terms, nodes with the same enrichment terms are usually close to each other, and the right figure is colored by p value, and terms containing more genes tend to have more significant p values;

[0046] Figure 5 Figure 8 is a diagram of extrapolation of hub proteins carried by EVs to an independent external cohort, wherein A is a Western blot showing IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M proteins in plasma, Health-Exo = 4, and AAA-Exo = 8; B is a diagram based on Western blot band gray scale analysis, indicating the distribution of expression levels of the five hub proteins between the AAA group and the healthy control group in the external cohort, and the AAA-Exo samples are marked in blue, and the Health-Exo samples are marked in gray; C is a quantitative column chart of expression levels based on Elisa verification, indicating the distribution of expression levels of the five hub proteins between the AAA group and the healthy control group in the external cohort, and the AAA-Exo samples are marked in red, and the Health-Exo samples are marked in gray; D is a ROC curve of core proteins in the external cohort verified by Elisa. DETAILED DESCRIPTION

[0047] The application will be further described in conjunction with the following examples, but the embodiments of the application are not limited thereto. Unless otherwise specified, the reagents, methods and devices used in the application are conventional reagents, methods and devices in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied to the method of the application.

[0048] Example 1

[0049] 1. Experimental materials

[0050] In this study, 10 AAA patients and 5 healthy normal controls were collected from the First Affiliated Hospital of Zhengzhou University as an internal cohort, and the proteome expression of circulating extracellular vesicles (EVs) in AAA patients was analyzed using PEA technology based on the Olink platform; 8 AAA patients and 4 healthy normal controls were collected from the Department of Cardiovascular Surgery, the Second Clinical Medical College of Jinan University (Shenzhen People's Hospital) as an independent external cohort for external verification of hub proteins carried by extracellular vesicles (EVs). The diagnosis of AAA was based on the standards established by the Society for Vascular Surgery (SVS) and the European Society for Vascular Surgery (ESVS). The diameter of AAA was measured according to the method recommended by ESVS.

[0051] The inclusion criteria for AAA patients include: (1) diagnosed according to the ESVS 2019 guidelines; (2) physiological age greater than 55 years old; (3) signed informed consent, agreed to store and analyze blood samples.

[0052] The exclusion criteria for AAA include: (1) evidence of active infection; (2) chronic liver disease (Child-Pugh score≥B); (3) end-stage renal disease (CKD stage 5 or creatinine≥2mg / dL); (3) chronic inflammatory disease; (5) BMI<20 or>35; (6) received major surgery or suffered from illness within the past 30 days; (7) use of immunosuppressants or steroids; (8) history of organ transplantation; (9) pregnancy or lactation.

[0053] This study recruited patients diagnosed with AAA and healthy volunteers. Venous blood was collected using whole blood collection EDTA tubes (BD Vacutainer) to inhibit blood clotting. The blood sample was centrifuged at 1500xg for 20 minutes at 4°C to remove cells from the blood. The supernatant was centrifuged at 3000xg for 15 minutes at 4°C to collect the supernatant (plasma), and then the first 2mL of plasma from each sample was quickly frozen at -80°C to maintain the stability and integrity of the plasma biomolecules.

[0054] 2. Experimental methods

[0055] 2.1 Extracellular vesicle proteins were extracted from plasma samples using the ExoQuick Extracellular Vesicle Protein Kit (EXOQ5A1; System Biosciences, USA) and strictly following the manufacturer's protocol, and the specific steps were as follows:

[0056] Mix 250 μL of plasma sample with 36 μL of ExoQuick kit fast extracellular vesicle precipitation solution, incubate at 4°C for 30 minutes; then centrifuge at 1500 x g for 30 minutes; after discarding the supernatant, centrifuge again at 1500 x g for 5 minutes, then resuspend the obtained extracellular vesicle nanoscale particles (extracellular vesicles) in 100 μL of sterile phosphate buffered saline (PBS) and store at -80°C for subsequent analysis.

[0057] 2.2 Detect the morphology of isolated extracellular vesicles by transmission electron microscopy (TEM), the specific steps are as follows:

[0058] 2.2.1 Transmission electron microscopy (TEM) detects the morphology of isolated extracellular vesicles

[0059] Prepare the extracellular vesicle solution: take an appropriate amount of extracellular vesicle PBS suspension obtained from step 2.1, determine the protein concentration to be 0.5 μg / μL using the BCA protein quantification kit, and dilute to 10 μL for transmission electron microscopy detection. Sample negative staining treatment: take 10 μL of extracellular vesicle solution and drop it on a copper mesh, incubate at room temperature for 10 minutes;

[0060] Then, wash the surface of the copper mesh with sterile distilled water, and absorb the excess liquid with a water-absorbing paper; drop 10 μL of 2% uranyl acetate onto the copper mesh, and negatively stain for 1 minute; after absorbing the supernatant with a water-absorbing paper, dry it under an incandescent lamp for 2 minutes; transmission electron microscopy observation: place the treated copper mesh under a transmission electron microscope (TEM) for observation; record the morphological characteristics of extracellular vesicles (such as diameter range and cup-shaped structure).

[0061] 2.2.2 Measure the particle size distribution and particle concentration of extracellular vesicles (EVs) using a nano-flow cytometer (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China), the specific operation is as follows:

[0062] Sample preparation: take samples from the extracellular vesicle PBS suspension of step 2.1, dilute by 1:100 (or adjust the dilution multiple according to the requirements of the equipment); particle size and concentration determination: inject the diluted sample into the nano-flow cytometer (Flow NanoAnalyzer, NanoFCM Inc., Xiamen, China); record the particle size distribution (such as median diameter, particle size range) and particle concentration (such as particle number / mL); data analysis: analyze the data using the software provided with the detector, and output the results for further evaluation of the separation effect of extracellular vesicles.

[0063] 2.2.3 BCA protein quantification and sample loading, the specific operation is as follows:

[0064] Protein concentration determination: BCA protein quantification kit was used to determine the protein concentration of extracellular vesicles according to the kit instructions; according to the determination results, the concentration of extracellular vesicle sample was ensured to be in the range of 10-30 μg; SDS sample preparation: according to the protein concentration, the loading amount of extracellular vesicle sample was calculated; the extracellular vesicle sample was added into 5x SDS buffer in proportion and vortexed uniformly; denatured at 95℃ water bath for 5 minutes, and then used for protein electrophoresis analysis.

[0065] 2.2.4 Western Blot detection of extracellular vesicle protein markers

[0066] Protein transfer after electrophoresis: after electrophoresis, the separation gel was removed, and the target area protein in the gel was transferred to the PVDF membrane. The positive protein markers of extracellular vesicles include: Tsg101 (#ab125011, Abeam, USA), Alix (#ab186429, Abeam, USA), CD9 (#ab263019, Abeam, USA), and the negative marker of extracellular vesicles is Calnexin (#10427-2-AP, Proteintech, USA); membrane blocking: blocking the membrane in 3% BSA blocking solution for 1 hour at room temperature to reduce non-specific binding; primary antibody incubation: the primary antibody was diluted to the recommended concentration (usually 1:1000) and incubated at 4℃ overnight; the antibodies used were: Tsg101 antibody (#ab125011, Abeam), Alix antibody (#ab186429, Abeam), CD9 antibody (#ab263019, Abeam), and Calnexin antibody (#10427-2-AP, Proteintech); the diluent of the primary antibody usually uses TBST (TBS + 0.1% Tween-20) or PBS buffer.

[0067] Secondary antibody incubation: HRP (horseradish peroxidase) labeled anti-rabbit secondary antibody was used, which was diluted to 1:5000 or the recommended concentration by the manufacturer; the anti-rabbit secondary antibody used was suitable for Tsg101, Alix and Calnexin (such as the secondary antibody provided by Proteintech). After incubation at room temperature for 1 hour, the membrane was washed with TBST for 3 times, 10 minutes each time; development and fixation: the membrane was immersed in ECL developing solution (enhanced chemiluminescence kit was recommended, such as Thermo #34095); the chemiluminescence signal was recorded using an imager (such as Bio-Rad ChemiDoc or other brands); according to the size and molecular weight of the target protein band, the expression of the target protein was confirmed by comparison with the protein molecular weight standard.

[0068] Supplementary notes:

[0069] Selection of primary and secondary antibodies:

[0070] Primary antibody for specific recognition of target protein (Tsg101, Alix, CD9, Calnexin); secondary antibody paired with primary antibody animal species (such as anti-rabbit secondary antibody paired with rabbit-derived primary antibody); secondary antibody labeled with HRP for signal detection by ECL development.

[0071] Blocking and washing film considerations:

[0072] The blocking solution is usually low-fat milk powder (5%) or BSA (3%).

[0073] The concentration of TBST used for cleaning is generally 0.1%-0.2% Tween-20, which ensures the removal of unbound antibodies.

[0074] Development system:

[0075] It is recommended to use high-sensitivity ECL development reagent to ensure clear target protein band signal.

[0076] Exposure time can be adjusted to ensure that the signal is not oversaturated.

[0077] 2.3 Obtain differentially expressed extracellular vesicle proteins using Olink proteomic technology, cluster analysis, and biological function enrichment analysis, etc. The specific steps are as follows:

[0078] Olink proteomic technology is a combined innovative protein detection and quantification technology based on Proximity Extension Assay (PEA), a protein and antibody immune reaction technology, and oligonucleotide amplification technology.

[0079] Olink protein biomarker detection is divided into Target series (48, 96) and Explore series (384, 3072), among which Target uses qPCR for detection, specifically: the prepared protein sample is diluted according to the different target panel protein concentration, then incubated with the antibody connected with the oligonucleotide sequence, and the two oligonucleotide sequences connected to the protein are paired by base complementation, then under the action of DNA polymerase, the paired oligonucleotide sequences are extended in the reaction system, and after pre-amplification, PCR detection is performed. Among them, Olink Target 96 series can be used to analyze the target protein biomarker group of specific proteins related to specific diseases or biological functions (Ding Z, Wang N, Ji N, et al. Proteomics technologies for cancer liquid biopsies [J]. Molecular Cancer, 2022, 21(1): 1-11). Compared with previous protein immunization techniques, this technique can provide new insights into the disease process, improve disease detection, and help better understand biology.

[0080] 2.3.1 Total protein extraction and immunoconnection

[0081] Extracellular vesicle total protein extraction: total protein was extracted from the plasma extracellular vesicle PBS suspension obtained in step 2.1, ensuring that the extracellular vesicle sample remained stable when stored at -80°C; extracellular vesicle protein extraction reagent: RIPA lysis buffer (Thermo Scientific, #89900) with 1× phosphatase inhibitor and PMSF; protein concentration determination: BCA protein quantification kit (Thermo Scientific, #23225).

[0082] Extraction steps: equal volume of RIPA lysis buffer was added to the extracellular vesicle suspension, mixed thoroughly and incubated in ice bath for 30 minutes; centrifuged at 13,000 x g for 10 minutes at 4°C, and the supernatant was collected; the protein concentration was determined by BCA method, diluted and saved for subsequent detection.

[0083] Oligonucleotide sequence antibody immunoconnection:

[0084] Antibody and sequence design: The oligonucleotide sequence antibody was purchased from Olink Proteomics Company, and its sequence was designed and optimized for the target protein (for specific sequence, optimization design and acquisition method, see literature (Ding, Zhiyong, et al. "Proteomics technologies for cancer liquid biopsies." Molecular Cancer 21.1 (2022): 53.)).

[0085] Immune connection step: The protein sample was mixed with the oligonucleotide sequence antibody at the recommended concentration and incubated on ice for 30 minutes; base complementary connection: two oligonucleotide sequences designed specifically were connected to different antibodies of the target protein; after complementary connection, DNA polymerase (Thermo Scientific, #EP0502) was added to extend in the system; reaction system: reaction buffer (10 mM Tris-HCl, pH 8.0; 50 mM KCl; 5 mM MgCl2); base complementary reaction conditions: incubation at 25°C for 30 minutes; DNA polymerization reaction conditions: reaction at 37°C for 20 minutes, followed by heating at 95°C for 5 minutes to terminate the reaction.

[0086] 2.3.2 Sample library preparation and qPCR detection

[0087] In the library preparation stage, first, a specific barcode label is added to each sample after the extension reaction, which is used for subsequent library differentiation and sample identification; the equipment required for library preparation includes an Applied Biosystems QuantStudio 5 qPCR instrument, and barcode primers and library preparation reagents purchased from Olink Proteomics Company. In specific operation, the primer with barcode label is mixed with the product of extension reaction, and then secondary amplification is carried out to improve specificity and signal strength; the product after amplification is purified by magnetic beads (such as using AMPure XP magnetic beads, Beckman Coulter) to obtain high-purity library sample, which provides high-quality template for subsequent qPCR detection.

[0088] In the qPCR detection stage, the purified library sample is added to the qPCR reaction system according to the appropriate dilution ratio, and the total reaction system volume is 20 μL, including 10 μL of 2×qPCR Master Mix (such as Thermo Fisher, #4369510), 10 μL of nuclease-free water, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, and 1 μL of 10 μM probe. The specific primer and probe sequences are shown in Table 1.

[0089] A25742), 1 μΐ of library sample template, 0.4 μΐ of target primer pair (final concentration of 200 nM), and 8.6 μΐ of sterile ultrapure water. In the qPCR reaction, first, initial denaturation was performed at 95 °C for 5 minutes to ensure complete denaturation of the DNA template; then 40 cycles were performed, including heating at 95 °C for 15 seconds and annealing and extension at 60 °C for 1 minute to complete the amplification of the library.

[0090] After the amplification was completed, the fluorescence signal was collected in real time using the software (QuantStudio Design & Analysis Software) provided with the qPCR instrument, and the Ct value of each sample was recorded; the Ct value was converted into NPX value (Olink Normalized Protein eXpression) by the data analysis software, to provide standardized protein expression data for subsequent differential expression analysis.

[0091] 2.3.3 The bml file or bcl file generated by qPCR detection was converted into Ct value or counts file, to obtain the data input file required by NPX Manager software, then the qPCR quantitative data was pre-processed by NPX Signature software, and then imported into Olink NPX Signature (NPX Manager) software to make sample type annotation, protein NPX calculation, data quality control, data export and save analysis certificate (Certificate of Analysis, CoA), so as to obtain the input file required by the biological information analysis process: NPX Data, and make original sample NPX Data boxplot and sample principal component analysis PCA graph.

[0092] 2.3.4 According to the original sample NPX Data boxplot and sample principal component analysis PCA graph, the sample dimensionality reduction clustering situation was observed, whether there were outlier samples, so as to decide whether to exclude the outlier samples in subsequent analysis; then a protein expression clustering heat map of all proteins was made, to facilitate a preliminary understanding of the overall protein expression pattern in the present application, and to develop abnormal protein expression.

[0093] 2.3.5 After the previous original data quality control, clean data of protein NPX expression (Clean Data) was obtained, then protein expression clustering heat map analysis, protein correlation coefficient heat map analysis could be carried out for each panel, and expression boxplot analysis could also be carried out for the target protein.

[0094] Based on the clean data of protein NPX expression, the present application performs Student's test analysis or variance analysis on different experimental variables to obtain differential proteins under the condition of experimental variables, and performs visualization analysis on differential protein data, including drawing differential protein volcano plot, Panel differential protein clustering heat map, etc. The present application also performs Pearson correlation analysis and UMAP analysis to evaluate the similarities and differences of extracellular vesicle protein expression of AAA patients and healthy controls.

[0095] 2.3.6 The present application also performs biological function enrichment analysis on differential protein data. All identified AAA extracellular vesicle proteins are annotated using the STRING database, and protein-protein interaction (PPI) analysis is performed. After preliminary analysis, the present application performs comprehensive atlas and process enrichment analysis on the predicted targets of AAA extracellular vesicle proteins, which integrates multiple ontology-derived data, including GO biological process, GO molecular function, KEGG pathway, human gene set, Reactome gene set and canonical pathway.

[0096] At the same time, the biological functions of the Panel proteins corresponding to Gene Ontology (http: / / www.geneontology.org / ), KEGG Pathway (http: / / www.genome.jp / kegg / ), Reactome Pathway (http: / / metascape.org) and DOSE are explored.

[0097] Finally, all proteins analyzed by Olink technology are annotated by protein-related biological information database, subcellular localization analysis and protein interaction network analysis.

[0098] 2.4 The expression levels of five differentially expressed proteins (Oncostatin-M, Interleukin-4, Interleukin-6, Neurturin and MCP-1) in the external cohort are further verified by Western Blot and enzyme-linked immunosorbent assay (ELISA), and the specific steps are as follows:

[0099] 2.4.1 In this step, the extracellular vesicle proteins are first separated and purified by ultracentrifugation method; the specific operation is as follows: the sample is centrifuged at 10,000xg at 4℃ for 20 minutes to remove cell debris and other impurities; then the supernatant is transferred to an ultracentrifuge tube, and the extracellular vesicles are precipitated by centrifugation at 100,000xg at 4℃ for 90 minutes; after collecting the obtained precipitate, the extracellular vesicle particles are resuspended with sterile PBS, and centrifuged again at 100,000xg for 90 minutes to obtain high-purity extracellular vesicle samples.

[0100] Next, the extracellular vesicles were lysed and protein extracted using RIPA lysis and extraction buffer (Thermo Scientific, #89900); phosphatase inhibitors and PMSF (phenylmethylsulfonyl fluoride) were added in the RIPA buffer at a volume ratio of 100:1 to protect protein activity; after the lysate was incubated on ice for 30 minutes, it was centrifuged at 13,000 x g at 4°C for 10 minutes, and the supernatant was collected as a protein sample and the protein concentration was determined using a BCA protein quantification kit; the resulting protein sample was used for Western Blot and ELISA detection.

[0101] In the Western Blot detection, the following antibodies were used to detect the target extracellular vesicle proteins: IL-4 antibody (Proteintech, #66142), IL-6 antibody (Proteintech, #21865), MCP-1 antibody (Proteintech, #26161), Neurturin antibody (Proteintech, #19709), and OSM antibody (Proteintech, #27792). After loading the target protein at a concentration of 10-30 μg, the proteins were separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and sequentially added with primary and secondary enzyme-labeled antibodies, and finally developed using a chemiluminescence reagent to observe the expression level of the specific protein.

[0102] Meanwhile, to further verify the concentration of the target protein, an ELISA kit was used to detect the extracellular vesicle protein sample. The kits used included: human IL-4 ELISA kit (Multi Sciences, EK104 / 2-96), human IL-6 ELISA kit (Multi Sciences, EK106-96), human MCP-1 ELISA kit (Multi Sciences, EK187-96), human Neurturin ELISA kit (YOBIBIO, U96-1476E), and human OSM ELISA kit (YOBIBIO, U96-1578E). During the detection process, the manufacturer's instructions were strictly followed; after loading, incubation and washing, the enzyme-labeled secondary antibody was added, followed by color development and termination of the reaction, and then the absorbance value was measured at 450 nm wavelength using an enzyme-labeled instrument, and the concentration of each target protein was calculated to evaluate its expression level.

[0103] 2.5 Analysis of the results of the foregoing experiments

[0104] Specifically, all statistical tests in the foregoing experimental methods are two-tailed tests; P values less than 0.05 and false discovery rates (FDR) less than 0.05 are considered statistically significant; the descriptive statistics of normally distributed continuous variables are reported in the form of mean ± standard deviation; the comparison of continuous variables uses the corresponding Wilcoxon rank-sum test or Student's t-test; categorical variables are analyzed using the chi-square test or Fisher's exact test; all data processing, statistical analysis and chart drawing are performed using R software (version 4.4.3).

[0105] 3. Experimental results

[0106] 3.1 Identification of the isolated plasma extracellular vesicles

[0107] The overall process of the method of the present application is shown in Figure 1 A, which is used for the isolation and identification of plasma extracellular vesicles; the characterization of plasma extracellular vesicles is based on their diameter, morphology and surface protein expression. First, the isolated plasma extracellular vesicles were observed by transmission electron microscopy (TEM) ( Figure 1 B), and the isolated plasma extracellular vesicle particles showed a typical cup-shaped structure, which is a typical morphological marker of extracellular vesicles, further proving the presence of extracellular vesicle nanoparticles in the sample. In addition, the sample was analyzed for particle size distribution using a high-sensitivity flow nanometer analyzer (Flow NanoAnalyzer) ( Figure 1 C), and the results showed that the median diameter of AAA extracellular vesicles (AAA-Exo) was 85.18 nanometers, which is consistent with the reported range of extracellular vesicle particle size (30-150 nanometers), further confirming the presence and particle size characteristics of extracellular vesicles.

[0108] The present application also detected the surface proteins of extracellular vesicles by Western blotting ( Figure 1 D). The results showed that the extracellular vesicle positive markers Tsg101, Alix and CD9 were clearly expressed in the sample, while the extracellular vesicle negative marker Calnexin was not detected. This indicates that the extracted sample contains high-purity extracellular vesicles and substantially excludes contamination by cell debris and other non-extracellular vesicle components. Combined with the results of TEM observation, flow nanometer analysis and Western blot detection, the sample isolated by the method of the present application is successfully identified as plasma extracellular vesicles; these extracellular vesicles will be used for the subsequent screening and analysis of protein biomarkers.

[0109] 3.2 Detection of protein biomarkers of AAA patient plasma extracellular vesicles using Olink proteome technology

[0110] The present invention analyzed 92 protein biomarkers in plasma extracellular vesicles of AAA patients and healthy controls. Figure 2 As shown. Through the heat map ( Figure 2 A), the distribution pattern of extracellular vesicle proteins in different samples can be visually observed. The results showed that there were significant differences in protein expression between the AAA group and the healthy control group, and some proteins showed unique expression patterns in the two groups. Further box plot analysis ( Figure 2 B) shows that the NPX (normalized protein expression) levels between the AAA group and the healthy control group showed significant differences in multiple proteins, revealing the protein expression characteristics between the two groups. Principal component analysis (PCA) ( Figure 2 C) Results show that the samples can be clearly separated in principal component space, indicating that the differences in extracellular vesicle protein expression between AAA patients and healthy controls are significant and consistent. These results support the use of extracellular vesicle proteins as potential biomarkers to distinguish AAA patients from healthy individuals.

[0111] The judgment criteria used in the significance analysis was P < 0.05, based on the results of t-test or analysis of variance for differential expression analysis. Combined with the calculation of this threshold, the statistical significance of the expression levels of extracellular vesicle proteins between the two groups was further verified. Validation by external cohorts ( Figure 3 A, B, C), it was found that there were five protein biomarkers in the extracellular vesicles of AAA patients and healthy controls that showed significant expression differences, namely Oncostatin-M, Interleukin-4, Interleukin-6, Neurturin and MCP-1 (for the five protein biomarkers, please refer to https: / / www.genecards.org / ). Among them, the expression of Oncostatin-M, Neurturin and MCP-1 in the extracellular vesicles of AAA patients was significantly upregulated, while the expression of Interleukin-4 and Interleukin-6 was significantly downregulated.

[0112] The purpose of this invention, in terms of the external and internal cohorts, is to ensure the stability and broad applicability of the extracellular vesicle protein markers screened. The internal cohort is used to initially identify potential differentially expressed proteins, while the external cohort serves as an independent validation group to further evaluate the performance of these markers in different populations, thereby improving the reliability and scalability of marker screening. Through this dual-cohort design, the present invention ensures that the screened extracellular vesicle proteins show consistent and significant differences across different cohorts, providing a solid experimental basis for their use as potential diagnostic markers for AAA.

[0113] In addition, Pearson correlation analysis and UMAP analysis were also performed to assess the similarities and differences of extracellular vesicle protein expression between AAA patients and healthy controls Figure 3 D, E). These results showed that there were significant differences in plasma extracellular vesicle protein expression between AAA patients and healthy people.

[0114] 3.3 Comprehensive functional enrichment of AAA extracellular vesicle protein set target proteins

[0115] In the aforementioned experimental methods of the present application, protein-protein interaction (PPI) analysis was performed to generate a PPI interaction network labeled with UniProt ID and gene symbol. After removing isolated target genes lacking interaction and applying a confidence threshold of 900, the gene interaction network was visualized using Cytoscape Figure 4 A). The top 20 enrichment terms of the key clusters in this analysis are shown in the figure. To further clarify the relationships between these top terms, the present application constructed a functional annotation network, as shown in Figure 4 B. This network can provide a deeper understanding of the potential interrelated pathways and processes affected by AAA exon-derived proteins, thereby deepening the understanding of their roles in the molecular mechanisms of AAA.

[0116] In the map and process enrichment analysis of the predicted targets of AAA extracellular vesicle proteins, multiple ontology-derived data were integrated, including GO biological processes, GO molecular functions, KEGG pathways, human gene sets, Reactome gene sets, and canonical pathways. Several significantly enriched terms were found, especially "cellular response to cytokine stimulus"

[0117] (GO:0071345), "inflammatory response" (GO:0006954), chemokine signaling pathway (hsa04062), activation protein-1 pathway (M167), and glucocorticoid receptor (GR) regulatory pathway (M115). According to previous studies, inflammatory response and the production and activation of various proteases play a crucial role in promoting the formation and development of aortic aneurysm.

[0118] 3.4 Western blot and ELISA verification of differentially expressed proteins in AAA circulating extracellular vesicles

[0119] Five differentially expressed proteins were identified as hub proteins in the circulating extracellular vesicles of AAA patients and healthy controls, including IL-4, IL-6, MCP-1, Neurturin, and Oncostatin-M. The criteria for screening these proteins as hub proteins were mainly based on the following quantitative indicators: (1) meeting the significance threshold (P < 0.05) in differential expression analysis; (2) having high connectivity or key positions in the protein-protein interaction (PPI) network; and (3) significantly participating in AAA-related pathological processes (such as inflammatory response, cytokine signaling pathway, etc.) in biological function enrichment analysis. Based on these quantitative criteria, the key roles of these proteins were further verified by experiments.

[0120] The present invention uses Western Blot and ELISA to verify the expression of hub protein in an external validation cohort (8 patients and 4 healthy controls). In the Western Blot experiment, Figure 5 A shows the bands of these five proteins and the grayscale analysis results of the bands ( Figure 5 B) shows that the expression of MCP-1, Neurturin, and Oncostatin-M was significantly increased in AAA patients compared with healthy controls (P<0.05), while IL-4 and IL-6 did not show statistically significant differences. Subsequently, the expression levels of these proteins were further verified by ELISA.

[0121] ELISA results showed that ( Figure 5 C), the levels of IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M in the circulating extracellular vesicles of AAA patients were significantly higher than those in the healthy control group (P<0.05). Figure 5 The receiver operating characteristic (ROC) curves in Figure D further evaluated the potential of these proteins as diagnostic biomarkers. The AUC values ​​were 0.760 (95% CI: 0.486-1.000 for IL-4), 0.840 (95% CI: 0.630-1.000 for IL-6), 0.800 (95% CI: 0.564-1.000 for MCP-1), 0.840 (95% CI: 0.617-1.000 for neurturin), and 0.900 (95% CI: 0.731-1.000 for oncostatin-M), respectively. These results indicate that oncostatin-M and neurturin, in particular, possess high AUC values, demonstrating strong diagnostic ability and can significantly differentiate AAA patients from healthy controls.

[0122] Compared with the prior art, the present invention has the following advantages:

[0123] 1. Novel biomarker screening: The present invention utilizes proteins in extracellular vesicles as specific markers for early diagnosis of abdominal aortic aneurysm, which is a less explored area in the early diagnosis of AAA. Specific proteins in EVs can be directly related to the pathological processes of AAA, such as vascular wall remodeling, inflammation, oxidative stress, etc., which are more specific and sensitive than conventional serum markers.

[0124] 2. Innovative detection method: The present invention proposes to achieve early diagnosis through specific protein markers in circulating EVs. This method is earlier and more sensitive than imaging diagnosis, and can be obtained through non-invasive sampling such as blood or urine. Compared with traditional imaging relying on the size detection of aneurysm, EV markers can provide early warning when aneurysm has not yet significantly increased.

[0125] 3. Personalized diagnostic model: Through high-throughput detection technology, protein markers highly related to abdominal aortic aneurysm are screened out, and through the establishment of a 5-key biomarker diagnostic model and diagnostic method that can distinguish AAA patients from healthy individuals, it not only helps diagnosis, but also provides information on disease progression, enabling early intervention and individualized treatment.

[0126] 4. Non-invasive and repeatable liquid biopsy: Traditional imaging may require frequent monitoring of aneurysm growth, while non-invasive detection of EV protein markers not only reduces the burden on patients, but also provides a means for regular assessment of AAA development, improving long-term management effectiveness.

[0127] 5. Specific protein marker combination: The present invention protects a specific combination of protein markers, especially proteins that are significantly increased or decreased in EVs circulating in patients with abdominal aortic aneurysm. The screening and verification process of these proteins is one of the core innovation points, and the innovation points of the present invention include the screening of such protein markers and their application in diagnosis.

[0128] 6. Diagnostic method and process: The detection method of the present invention, including the specific technical process of sample collection, EV separation, protein detection (such as ELISA, mass spectrometry, immunoblotting, etc.) and data analysis, is one of the key points of the present invention to be protected.

[0129] 7. Multi-marker algorithm model: Through the combined detection of multiple protein markers, a mathematical model or algorithm (such as machine learning, statistical model, etc.) can be established to achieve early diagnosis of AAA patients. At the same time, the application of the method of the present invention in diagnosis is also a core invention point of the present invention.

Claims

1. Use of an extracellular vesicle protein marker in the manufacture of a product for the diagnosis of abdominal aortic aneurysm, characterized in that, The product is a kit, which comprises a reagent or a biochip, and the biochip comprises a microarray biochip; The extracellular vesicle protein marker is a combination of five molecular markers of interleukin-4, interleukin-6, monocyte chemotactic protein-1, nerve growth factor and tumor suppressor M; The extracellular vesicle is a plasma extracellular vesicle.

2. A kit for diagnosing abdominal aortic aneurysm comprising an extracellular vesicle protein marker, characterized in that, The kit comprises a reagent or a biochip, and the biochip comprises a microarray biochip; the extracellular vesicle protein marker is a combination of five molecular markers of interleukin-4, interleukin-6, monocyte chemotactic protein-1, nerve growth factor and tumor suppressor M; The extracellular vesicle is a plasma extracellular vesicle.

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