Extracellular vesicle protein as biomarker for diagnosing abdominal aortic aneurysm and application of extracellular vesicle protein

Abstract

The invention 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 of the extracellular vesicle proteins. The abdominal aortic aneurysm is diagnosed in the early stage by measuring the circulating extracellular vesicle protein marker, and an effective tool is provided for diagnosis of the abdominal aortic aneurysm. The extracellular vesicle protein marker is a molecular marker combination of any one or more than two of an interleukin-4 (IL-4), an interleukin-6 (IL-6), a monocyte chemotactic protein-1 (MCP-1), a nerve growth factor (Neurturin) and a tumor inhibition factor M (Oncostatin-M). The extracellular vesicle protein marker is a molecular marker combination of any one or more than two of the interleukin-4 (IL-4), the interleukin-6 (IL-6), the monocyte chemotactic protein-1 (MCP-1), the nerve growth factor (Neurturin) and the tumor inhibition factor M (Oncostatin-M). Protein in extracellular vesicles is used as a specific marker for early diagnosis of abdominal aortic aneurysm, which is a field which is less applied to early diagnosis of AAA at present. Specific protein in EVs may be directly related to the pathological process of AAA, and different from conventional serum markers, the specific protein has higher specificity and sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and in particular relates to a group of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm and their application. Background Art

[0002] Abdominal aortic aneurysm (AAA) is the most common type of life-threatening aneurysm. The male-female incidence of AAA is about 5:1. In some countries, the incidence in people over 60 years old is between 2% and 5%. Epidemiological studies have shown that the incidence in men over 65 years old is 1% to 2%, and 150,000 to 200,000 people die from AAA rupture each year worldwide. The incidence of AAA is closely related to risk factors such as age, gender and smoking. The main cause of AAA is degenerative changes in the arterial wall or other nonspecific factors, not atherosclerosis. More than 90% of AAA are caused by these degenerative changes. The latest studies have shown that the pathophysiology of AAA involves multiple factors, including inflammatory response, matrix metalloproteinase (MMPs) activation, oxidative stress, intraluminal thrombosis, smooth muscle apoptosis and extracellular matrix (ECM) degradation.

[0003] Currently, ultrasound is a suitable method for screening AAA, while CTA (angiography) is the first choice for evaluation and planning treatment. However, ultrasound has certain limitations for patients with complex anatomical structures. Although CTA and MRA (magnetic resonance angiography) have accurate test results, they are expensive and involve radiation or contrast agent risks. In addition, since the imaging diagnosis of AAA is usually applied only after obvious clinical symptoms appear, the aorta has undergone irreversible changes at this time, 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 them, the levels of gelatinase MMP-2 and MMP-9 are significantly increased in AAA patients, especially MMP-9, which is responsible for degrading collagen and elastin in the extracellular matrix, leading to weakening of the arterial wall and promoting aneurysm formation. The high expression of matrix metalloproteinases (MMPs) indicates 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, which are directly related to the formation of AAA. In addition, serum cystatin C (Cystatin C) is a potent cysteine ​​protease inhibitor. During the formation of abdominal aortic aneurysm, the extracellular matrix (including elastin and collagen) is continuously degraded by proteases, resulting in the 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 aneurysms or slowing their progression. Early diagnosis of AAA through peripheral protein has certain potential, but it also faces many limitations. Most protein markers are weakly correlated with the clinical course of AAA and have little potential for clinical use.

[0005] Extracellular vesicles (exosomes) are small vesicles secreted by cells, with a diameter of about 30-150 nanometers, including a variety of biological molecules such as proteins, lipids, DNA, mRNA, and miRNA. In recent years, extracellular vesicles have attracted attention as potential biomarkers in the early diagnosis of various diseases. Extracellular vesicles are secreted by specific cell types, and they contain specific molecules that reflect the state of these cells. This cell specificity of extracellular vesicles 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 this cell or tissue specificity. Extracellular vesicles are wrapped in a double-layer phospholipid membrane, which protects the proteins and nucleic acids inside them from degradation by the external environment. This structure makes extracellular vesicles relatively stable in body fluids such as blood, urine, and saliva, and easy to detect through non-invasive methods. This stability helps to reliably detect the protein markers they contain, reduce false-negative results, and thus improve the sensitivity of early screening. In addition, its non-invasive detection characteristics also make extracellular vesicle markers highly feasible in large-scale population screening, especially for monitoring high-risk individuals or early detection of diseases.

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

[0007] In recent years, the proximity extension assay (PEA) technology of Olink (ultra-trace multi-targeted protein biomarker discovery platform) has been used for a variety of disease mechanism studies and clinical diagnosis and treatment of personalized medicine due to its high sensitivity, specificity, and extremely low sample requirements. Although there has been progress in the diagnosis of circulating extracellular vesicle proteins, there is currently no research on the potential for early diagnosis of AAA. Therefore, the present invention uses Olink's PEA technology to detect the expression profile of the circulating extracellular vesicle proteome of AAA patients. Through comparative analysis, differentially expressed circulating extracellular vesicle proteins were identified. Bioinformatics methods are used to predict target genes and perform functional enrichment analysis in order to reveal the potential pathological changes of AAA, aiming to find new biomarkers and their biological functions and pathways related to AAA, and to identify potential new therapeutic targets to provide technical support for the treatment of AAA. Summary of the invention

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

[0009] The present invention also provides a method for screening the extracellular vesicle protein marker.

[0010] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0011] The present invention first provides a series of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm, wherein the extracellular vesicle protein markers are any one or a combination of two or more molecular markers selected from interleukin-4 (IL-4), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor (Neurturin) and tumor suppressor factor M (Oncostatin-M).

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

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

[0014] Further preferably, the extracellular vesicles are plasma extracellular vesicles.

[0015] Based on a general inventive concept, the present invention also provides the 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 a general inventive concept, the present invention also provides a kit, a microarray or a biochip for diagnosing abdominal aortic aneurysm (AAA), including the extracellular vesicle protein marker.

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

[0019] 1) Extracting extracellular vesicle proteins from plasma samples using an extracellular vesicle protein extraction kit;

[0020] 2) Using Olink proteomics technology, cluster analysis and biological function enrichment analysis, differentially expressed extracellular vesicle proteins were screened; the obtained data were statistically analyzed based on the differences in differentially expressed extracellular vesicle proteins between different groups;

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

[0022] 4) Analyze the experimental results obtained and draw a ROC curve for each differentially expressed extracellular vesicle protein; evaluate the diagnostic ability of potential extracellular vesicle protein markers based on the ROC curve and the area under the ROC curve (AUC), and finally screen out markers or marker combinations with good accuracy, sensitivity and specificity.

[0023] Specifically, step 1) also includes 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) by nanoflow cytometry, and detecting specific extracellular vesicle protein markers by Western Blot.

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

[0025] Immunoligation and base complementary ligation of oligonucleotide sequence antibodies. Immunoligation steps: protein sample and oligonucleotide sequence antibody are mixed and incubated; base complementary ligation: two designed oligonucleotide sequences are respectively connected to different antibodies of the target protein; after complementary ligation, DNA polymerase is added to extend in the system;

[0026] b. In the library preparation stage, first, a specific barcode tag is added to each sample after the extension reaction is completed for subsequent library differentiation and sample identification, and then purified 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 amplification, the fluorescence signal was collected in real time using a qPCR instrument, and the Ct value of each sample was recorded. The Ct value was converted into NPX value using data analysis software to provide standardized protein expression data for subsequent differential expression analysis.

[0029] c. Convert the data generated by qPCR detection into Ct value or counts file, and after processing by NPX Signature software and Olink NPX Signature (NPX Manager) software, obtain the original sample NPX Data required for the bioinformatics analysis process, and make the original sample NPX Data distribution box plot and sample principal component analysis PCA diagram;

[0030] d. Check the sample dimensionality reduction clustering according to the original sample NPX Data distribution box plot and the sample principal component analysis PCA plot; after quality control of the original data, obtain the clean data of protein NPX expression, and perform Student's test analysis or variance analysis on different experimental variables to obtain the differential proteins under the experimental variable conditions;

[0031] e. Perform biological function enrichment analysis on the differential protein data, 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 (10mM Tris-HCl, pH 8.0; 50mM KCl; 5mM MgCl2); base complementation 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.

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

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

[0035] Specifically, the screening criterion of step 4) is that the AUC value is >0.75.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention can detect the disease in the early stage of aneurysm development or even in the asymptomatic stage by detecting specific protein markers in EVs. This molecular-level detection method can identify early biological changes associated with lesions without relying on structural changes in 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 test. Liquid biopsy is not only convenient, but also can be sampled multiple times for follow-up and monitoring of disease progression, which is particularly suitable for long-term management and regular screening. Specific protein markers in EVs can more accurately reflect the pathophysiological processes associated with abdominal aortic aneurysm (such as inflammation, vascular wall degradation, etc.).

[0039] 3. The present invention can achieve high specificity and high sensitivity diagnosis of abdominal aortic aneurysm through the combined 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 assessment based on molecular markers helps guide early intervention and optimize treatment decisions.

[0040] 4. The present invention can expand the scope of application of screening through EV protein marker detection. It is not only suitable for high-risk groups, but also for early screening of other potential risk groups. EV detection provides a broader and more accurate screening tool. This test is low in cost and simple to operate, and is suitable for screening in primary medical units or resource-limited areas. This liquid biopsy can be used 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 Characterization and internalization diagram of AAA extracellular vesicles (AAA-Exo) and normal human plasma extracellular vesicles (Nor-Exo), where A is the complete workflow for high-throughput sequencing and analysis of AAA extracellular vesicles and normal human plasma extracellular vesicle samples; B is an electron microscope image of isolated extracellular vesicles, with a scale of 200 nanometers; C is a high-sensitivity flow cytometry result for extracellular vesicle nanoparticle analysis; D is 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 1 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 diagram 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 cyan 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 map of plasma proteins, red dots represent up-regulated proteins, gray dots represent insignificant proteins, and blue dots represent down-regulated 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 samples and AAA samples;

[0045] Figure 4Figure 1 is a biological significance diagram of target genes of plasma differentially expressed proteins (DEPs), where A is a PPI network between potential biomarkers established using the 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-values; B is an enrichment term network of DEPs target genes; terms with similarity greater than 0.3 are connected by edges, the left figure is colored by enrichment terms, and nodes with the same enrichment terms are usually close to each other, and the right figure is colored by p-values, and terms containing more genes tend to have more significant p-values;

[0046] Figure 5 The figure is the extrapolation of hub proteins carried by EV to an independent external cohort, where A is a Western blot showing IL-4, IL-6, MCP-1, Neurturin and Oncostatin-M proteins in plasma, Health-Exo=4 subjects, AAA-Exo=8 subjects; B is a grayscale analysis of Western blot bands, indicating the distribution of expression levels of five hub proteins between the AAA group and the healthy control group in the external cohort, AAA-Exo is marked as blue samples, and Health-Exo is marked as gray samples; C is a quantitative bar graph based on Elisa verification, indicating the distribution of expression levels of five hub proteins between the AAA group and the healthy control group in the external cohort, AAA-Exo is marked as red samples, and Health-Exo is marked as gray samples; D is the ROC curve of the core protein of the external cohort verified by Elisa. DETAILED DESCRIPTION

[0047] The present invention is described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise defined, all professional and scientific terms used herein are identical in meaning to those skilled in the art. In addition, any method and material similar to or equivalent to the recorded content can all be applied to the inventive method.

[0048] Example 1

[0049] 1. Experimental Materials

[0050] In this study, 10 AAA patients and 5 healthy controls were collected from the First Affiliated Hospital of Zhengzhou University as an internal cohort, and the proteomic 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 controls were collected from the Department of Cardiovascular Surgery of the Second Clinical Medical College of Jinan University (Shenzhen People's Hospital) as an independent external cohort for external validation of hub proteins carried by extracellular vesicles (EVs). The diagnosis of AAA was based on the criteria established by the Society of Vascular Surgery (SVS) and the European Society of Vascular Surgery (ESVS). The diameter of AAA was measured according to the method recommended by ESVS.

[0051] The inclusion criteria for AAA patients included: (1) diagnosed according to the ESVS 2019 guidelines; (2) physiological age greater than 55 years; and (3) signing an informed consent form and agreeing to the storage and analysis of blood samples.

[0052] AAA exclusion criteria included: (1) evidence of active infection; (2) chronic liver disease (Child-Pugh score ≥ B); (3) end-stage renal disease (CKD stage 5 or creatinine ≥ 2 mg / dL); (4) chronic inflammatory disease; (5) BMI < 20 or > 35; (6) major surgery or illness in the past 30 days; (7) use of immunosuppressants or steroid drugs; (8) history of organ transplantation; and (9) pregnancy or lactation.

[0053] Patients diagnosed with AAA and healthy volunteers were recruited for this study. Venous blood was collected using whole blood collection EDTA tubes (BD Vacuum Blood Collection Tubes) to inhibit coagulation. Blood samples were centrifuged at 1500×g for 20 minutes at 4°C to remove cells from the blood. The supernatant was taken and centrifuged at 3000×g for 15 minutes at 4°C to collect the supernatant (plasma). Subsequently, the first 2 mL of plasma from each sample was quickly frozen at -80°C to maintain the stability and integrity of 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; Systems Biosciences, USA) and strictly followed the manufacturer's protocol. The specific steps were as follows:

[0056] 250 μL of plasma sample was mixed with 36 μL of rapid extracellular vesicle precipitation solution in the ExoQuick kit and incubated at 4°C for 30 minutes; then centrifuged at 1500×g for 30 minutes; after discarding the supernatant, centrifuged again at 1500×g for 5 minutes, and then the resulting extracellular vesicle nanoparticles (extracellular vesicles) were resuspended in 100 μL of sterile phosphate-buffered saline (PBS) and stored at -80°C for subsequent analysis.

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

[0058] 2.2.1 Transmission electron microscopy (TEM) detection of isolated extracellular vesicles

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

[0060] Then, the surface of the copper mesh was cleaned with sterile distilled water, and excess liquid was absorbed with absorbent paper; 10 μL of 2% uranyl acetate was added to the copper mesh and negatively stained for 1 minute; after the floating liquid was absorbed with absorbent paper, it was dried under an incandescent lamp for 2 minutes; transmission electron microscopy observation: the treated copper mesh was placed under a transmission electron microscope (TEM) for observation; the morphological characteristics of the extracellular vesicles (such as diameter range and cup-shaped structure) were recorded.

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

[0062] Sample preparation: Take a sample from the extracellular vesicle PBS suspension in step 2.1 and dilute it at 1:100 (or adjust the dilution factor according to the equipment requirements); Particle size and concentration determination: Inject the diluted sample into a nanoflow detector (FlowNanoAnalyzer, NanoFCM Inc., Xiamen, China); Record the particle size distribution (such as median diameter, particle size range) and particle concentration (such as number of particles / mL); Data analysis: Use the software provided by the detector to analyze the data and output the results for further evaluation of the extracellular vesicle separation effect.

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

[0064] Protein concentration determination: Strictly follow the instructions of the kit and use the BCA protein quantification kit to determine the extracellular vesicle protein concentration; according to the determination results, ensure that the concentration of the extracellular vesicle sample is in the range of 10-30μg; SDS sample preparation: calculate the amount of extracellular vesicle sample according to the protein concentration; add 5×SDS buffer to the extracellular vesicle sample in proportion and vortex to mix; denature in a 95℃ water bath for 5 minutes, and then use for protein electrophoresis analysis.

[0065] 2.2.4 Western Blot Detection of Extracellular Vesicle Protein Markers

[0066] Protein transfer after electrophoresis: After electrophoresis, remove the separation gel and transfer the target area protein in the gel to the PVDF membrane. Extracellular vesicle positive protein markers include: Tsg101 (#ab125011, Abcam, USA), Alix (#ab186429, Abcam, USA), CD9 (#ab263019, Abcam, USA), and extracellular vesicle negative markers are: Calnexin (#10427-2-AP, Proteintech, USA); Membrane blocking: Block the membrane in 3% BSA blocking solution at room temperature for 1 hour to reduce nonspecific binding; Primary antibody incubation: Primary antibody dilute The primary antibody was diluted to the recommended concentration (usually 1:1000) and incubated overnight at 4°C. The antibodies used were: Tsg101 antibody (#ab125011, Abcam), Alix antibody (#ab186429, Abcam), CD9 antibody (#ab263019, Abcam), and Calnexin antibody (#10427-2-AP, Proteintech). TBST (TBS+0.1% Tween-20) or PBS buffer was usually used as the dilution buffer for the primary antibody.

[0067] Secondary antibody incubation: Use HRP (horseradish peroxidase) labeled anti-rabbit secondary antibody, diluted to 1:5000 or the concentration recommended by the manufacturer; the anti-rabbit secondary antibody used is: suitable for Tsg101, Alix and Calnexin (such as the secondary antibody provided by Proteintech). After incubation at room temperature for 1 hour, wash the membrane 3 times with TBST, 10 minutes each time; development and fixation: immerse the membrane in ECL developer (recommended to use enhanced chemiluminescence kit, such as Thermo #34095); use an imager (such as Bio-Rad ChemiDoc or other brands) to record the chemiluminescent signal; according to the size and molecular weight of the target protein band, confirm the expression of the target protein by comparing it with the protein molecular weight standard.

[0068] Additional notes:

[0069] Primary and secondary antibody selection:

[0070] The primary antibody is used to specifically recognize the target protein (Tsg101, Alix, CD9, Calnexin); the secondary antibody is paired with the animal species from which the primary antibody was derived (e.g., an anti-rabbit secondary antibody is paired with a rabbit primary antibody); the secondary antibody is labeled with HRP to facilitate the detection of the signal by ECL development.

[0071] Notes on sealing and membrane washing:

[0072] Low-fat milk powder (5%) or BSA (3%) is often used as the blocking solution.

[0073] The concentration of TBST used for washing is generally 0.1%-0.2% Tween-20 to ensure the removal of unbound antibodies.

[0074] Development system:

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

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

[0077] 2.3 Using Olink proteomics technology, cluster analysis and biological function enrichment analysis to obtain differentially expressed extracellular vesicle proteins, the specific steps are as follows:

[0078] Olink proteomics technology is an innovative protein detection and quantification technology based on the combination of Proximity Extension Assay (PEA), a protein and antibody immune response 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 different target panel protein concentrations, and then incubated with antibodies connected to oligonucleotide sequences. After the two oligonucleotide sequences connected to the protein are base-paired, the paired oligonucleotide sequences are extended in the reaction system under the action of DNA polymerase, and PCR detection is performed after pre-amplification. Among them, the Olink Target 96 series can be used to analyze targeted protein biomarker groups of specific proteins associated with 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 immunoassay technologies, this technology can gain new insights into disease processes, improve disease detection, and help to better understand biology.

[0080] 2.3.1 Total protein extraction and immunoligation

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

[0082] Extraction steps: Add an equal volume of RIPA lysis buffer to the extracellular vesicle suspension, mix thoroughly and incubate in an ice bath for 30 minutes; centrifuge at 13,000×g for 10 minutes at 4°C and collect the supernatant; determine the protein concentration using the BCA method, dilute and save for subsequent detection.

[0083] Immunoligation of Oligonucleotide Sequences to Antibodies:

[0084] Antibody and sequence design: Oligonucleotide sequence antibodies were purchased from Olink Proteomics, and their sequences were optimized for the target protein (for specific sequences, optimization designs, and acquisition methods, see the literature (Ding, Zhiyong, et al. "Proteomics technologies for cancer liquid biopsies." Molecular Cancer 21.1(2022):53.)).

[0085] Immunoligation steps: protein samples were mixed with oligonucleotide sequence antibodies at the recommended concentration and incubated on ice for 30 minutes; base complementary ligation: two specifically designed oligonucleotide sequences were used to connect to different antibodies of the target protein respectively; DNA polymerase (Thermo Scientific, #EP0502) was added after complementary ligation and extended in the system; reaction system: reaction buffer (10mM Tris-HCl, pH 8.0; 50mM KCl; 5mM 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.

[0086] 2.3.2 Sample library preparation and qPCR detection

[0087] In the library preparation stage, firstly, 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; the equipment required for library preparation includes Applied Biosystems QuantStudio 5qPCR instrument, as well as barcode primers and library preparation reagents purchased from Olink Proteomics. In the specific operation, the primers with barcode labels are fully mixed with the products of the extension reaction, and then secondary amplification is performed to improve specificity and signal strength; the amplified products are purified by magnetic beads (such as using AMPure XP magnetic beads, BeckmanCoulter) to obtain high-purity library samples, providing high-quality templates for subsequent qPCR detection.

[0088] In the qPCR detection stage, the purified library samples were added to the qPCR reaction system according to the appropriate dilution factor. The total reaction volume was 20 μL, including 10 μL of 2×qPCR Master Mix (such as Thermo Fisher,

[0089] #A25742), 1 μL of library sample template, 0.4 μL of target primer pair (final concentration 200 nM), and 8.6 μL of sterile ultrapure water. In the qPCR reaction, the initial denaturation was first performed at 95°C for 5 minutes to ensure complete denaturation of the DNA template; followed by 40 cycles of 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 amplification, the software provided by the qPCR instrument (QuantStudio Design & Analysis Software) was used to collect the fluorescence signal in real time and record the Ct value of each sample. The Ct value was converted into NPX value (Olink Normalized Protein eXpression) by data analysis software to provide standardized protein expression data for subsequent differential expression analysis.

[0091] 2.3.3 Convert the bml file or bcl file generated by qPCR detection into a Ct value or counts file to obtain the data input file required by the NPX Manager software. Then pre-process the qPCR quantitative data with the NPX Signature software and import it into the Olink NPX Signature (NPX Manager) software to perform sample type annotation, protein NPX calculation, data quality control, data export and save the Certificate of Analysis (CoA), thereby obtaining the input file required for the bioinformatics analysis process: NPX Data, and make the original sample NPX Data distribution box plot and sample principal component analysis PCA plot.

[0092] 2.3.4 Based on the original sample NPX Data distribution box plot and the sample principal component analysis PCA diagram, check the sample dimensionality reduction clustering to see if there are any outlier samples, so that subsequent analysis can determine whether to remove the outlier samples; then make a cluster heat map of all protein expression to facilitate a preliminary understanding of the overall protein expression pattern in this application, and to develop abnormally expressed proteins.

[0093] 2.3.5 After the previous raw data quality control, the clean data (CleanData) of protein NPX expression will be obtained. Then, protein expression clustering heat map analysis and protein correlation coefficient heat map analysis can be performed for each panel, and expression box plot analysis of the target protein can also be performed;

[0094] Based on the clean data of protein NPX expression, the present invention performs Student's test analysis or variance analysis on different experimental variables to obtain differential proteins under the experimental variable conditions, and performs visualization analysis of the differential protein data, including drawing differential protein volcano diagrams, Panel differential protein clustering heat maps, etc.; the present invention also performs Pearson correlation analysis and UMAP analysis to evaluate the similarities and differences in the expression of extracellular vesicle proteins between AAA patients and healthy control groups.

[0095] 2.3.6 The present invention also performs biological function enrichment analysis on differential protein data. All identified AAA extracellular vesicle proteins were annotated using the STRING database, and protein-protein interaction (PPI) analysis was performed. After preliminary analysis, the present invention performed a comprehensive map and process enrichment analysis on the predicted targets of AAA extracellular vesicle proteins. This analysis integrates data from multiple ontology sources, including GO biological processes, GO molecular functions, KEGG pathways, human gene sets, Reactome gene sets, and typical pathways.

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

[0097] Finally, all proteins analyzed by Olink technology were annotated with protein-related bioinformatics databases, subjected to subcellular localization analysis, and protein interaction network analysis.

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

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

[0100] Then, RIPA lysis and extraction buffer (Thermo Scientific, #89900) was used to lyse the extracellular vesicles and extract proteins. Phosphatase inhibitors and PMSF (phenol methylsulfonyl fluoride) were added to 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×g at 4°C for 10 minutes, and the supernatant was collected as a protein sample. The protein concentration was determined using a BCA protein quantification kit. The obtained protein samples were used for Western Blot and ELISA detection.

[0101] In 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 sample according to the target protein concentration (10-30μg), SDS-PAGE electrophoresis was performed to separate the proteins, and the membrane was transferred to PVDF membrane, and primary antibody and enzyme-labeled secondary antibody were added in turn. Finally, chemiluminescent reagent was used to develop and observe the expression level of specific proteins.

[0102] At the same time, to further verify the concentration of the target protein, ELISA kits were used to detect extracellular vesicle protein samples. 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 adding the sample, incubation and washing, enzyme-labeled secondary antibody was then added, and after color development and termination of the reaction, the absorbance was measured using an enzyme reader at a wavelength of 450nm, and the concentration of each target protein was calculated to evaluate its expression level.

[0103] 2.5 Analysis of the above experimental results

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

[0105] 3. Experimental results

[0106] 3.1 Identification of isolated plasma extracellular vesicles

[0107] The overall process of the method of the present invention is as follows Figure 1 As shown in A, it 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, transmission electron microscopy (TEM) observation ( Figure 1 B), the isolated plasma extracellular vesicle particles showed a typical cup-shaped structure, which is a typical morphological feature of extracellular vesicles, further proving the presence of extracellular vesicle nanoparticles in the sample. In addition, the particle size distribution of the sample was analyzed using a highly sensitive flow nanoanalyzer (Flow NanoAnalyzer). Figure 1 C), the results showed that the median diameter of AAA extracellular vesicles (AAA-Exo) was 85.18 nm, which was consistent with the reported extracellular vesicle size range (30-150 nm), further confirming the existence and size characteristics of extracellular vesicles.

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

[0109] 3.2 Detection of extracellular vesicle protein biomarkers in plasma of AAA patients using Olink proteomics 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) The results show that the samples can be clearly separated in the principal component space, indicating that the differences in the expression characteristics of extracellular vesicle proteins between AAA patients and healthy controls are significant and consistent. These results together support that extracellular vesicle proteins can be used as potential biomarkers to distinguish AAA patients from healthy individuals.

[0111] The judgment criteria used in the significance analysis were P < 0.05, based on the results of the 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 (see https: / / www.genecards.org / for the five protein biomarkers). 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] Regarding the setting of external cohorts and internal cohorts, the purpose of the present invention is to ensure that the extracellular vesicle protein markers screened are stable and widely applicable. The internal cohort is used to preliminarily discover potential differential proteins, while the external cohort is used as an independent validation group to further evaluate the performance of these markers in different populations, thereby improving the reliability and generalizability of marker screening. Through this dual-cohort design, the present invention ensures that the screened extracellular vesicle proteins show consistent and significant differences in different cohorts, providing a solid experimental basis for them as potential diagnostic markers for AAA.

[0113] In addition, the present invention also performed Pearson correlation analysis and UMAP analysis to evaluate the similarities and differences in the expression of extracellular vesicle proteins between AAA patients and healthy controls ( Figure 3 D, E). These results show that there are significant differences in the expression of plasma extracellular vesicle proteins between AAA patients and healthy subjects.

[0114] 3.3 Comprehensive functional enrichment of target proteins in the AAA extracellular vesicle proteome

[0115] In the aforementioned experimental method of the present invention, a PPI interaction network labeled with UniProt ID and gene symbol was generated by protein-protein interaction (PPI) analysis. After eliminating isolated target genes lacking interaction and applying a confidence threshold of 900, the gene interaction network was visualized using Cytoscape ( Figure 4 A). The figure shows the top 20 enriched terms of the key clusters in this analysis. To further clarify the relationship between these top terms, the present invention constructed a functional annotation network, such as Figure 4 B. This network enables a deeper understanding of the potential interconnected pathways and processes affected by AAA exosome-derived proteins, thereby deepening the understanding of their roles in the molecular mechanisms of AAA.

[0116] In the profile and process enrichment analysis of predicted targets of AAA extracellular vesicle proteins, data from multiple ontology sources 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 stimulation"

[0117] (GO:0071345), "inflammatory response" (GO:0006954), chemokine signaling pathway (hsa04062), activator 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 vital role in promoting the formation and development of aortic aneurysms.

[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; (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 experimentally.

[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 results of band grayscale analysis ( Figure 5 B) showed that the expression of MCP-1, Neurturin and Oncostatin-M in AAA patients was significantly increased compared with the healthy control group (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 ROC curves in D further evaluated the potential of these proteins as diagnostic markers. The AUC values ​​were 0.760 (95% CI: 0.486-1.000, IL-4), 0.840 (95% CI: 0.630-1.000, IL-6), 0.800 (95% CI: 0.564-1.000, MCP-1), 0.840 (95% CI: 0.617-1.000, Neurturin), and 0.900 (95% CI: 0.731-1.000, Oncostatin-M). These results show that Oncostatin-M and Neurturin, in particular, have high AUC values, showing extremely strong diagnostic ability and can significantly distinguish AAA patients from healthy people.

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

[0123] 1. Novel biomarker screening: This invention uses proteins in extracellular vesicles as specific markers for early diagnosis of abdominal aortic aneurysm, which is currently less used in the early diagnosis of AAA. Specific proteins in EVs may be directly related to the pathological process of AAA (such as vascular wall remodeling, inflammation, oxidative stress, etc.), which is different from conventional serum markers and has higher specificity and sensitivity.

[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 that relies on aneurysm size detection, EV markers can provide early warning before the aneurysm has significantly increased.

[0125] 3. Personalized diagnostic model: Through high-throughput detection technology, protein markers that are highly correlated with abdominal aortic aneurysm are screened out. By establishing 5 key biomarker diagnostic models and diagnostic methods that can distinguish AAA patients from healthy individuals, it not only helps with diagnosis, but also provides information on disease progression, achieving early intervention and personalized 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 to regularly evaluate the development of AAA and improve long-term management effects.

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

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

[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 the core invention of the present invention.

Claims

1. Extracellular vesicle protein as a biomarker for diagnosing abdominal aortic aneurysm, characterized in that: The extracellular vesicle protein marker is any one or a combination of two or more molecular markers selected from interleukin-4, interleukin-6, monocyte chemoattractant protein-1, nerve growth factor and tumor suppressor factor M.

2. The biomarker according to claim 1, characterized in that The extracellular vesicle protein marker is a combination of two or more of interleukin-4, interleukin-6, monocyte chemoattractant protein-1, nerve growth factor and tumor suppressor factor M.

3. The biomarker according to claim 1, characterized in that The extracellular vesicles are plasma extracellular vesicles.

4. Use of the extracellular vesicle protein marker according to any one of claims 1 to 3 in the preparation of a product for diagnosing abdominal aortic aneurysm, characterized in that: The product is a medicament, a kit, a microarray or a biochip.

5. A kit, microarray or biochip for diagnosing abdominal aortic aneurysm, comprising the extracellular vesicle protein marker according to any one of claims 1 to 3.

6. A method for screening the extracellular vesicle protein marker according to any one of claims 1 to 3, comprising the following steps: 1) Extracellular vesicle proteins were extracted from plasma samples using an extracellular vesicle protein extraction kit; 2) Using Olink proteomics technology, cluster analysis and biological function enrichment analysis, differentially expressed extracellular vesicle proteins were screened; the obtained data were statistically analyzed based on the differences in differentially expressed extracellular vesicle proteins between different groups; 3) Use Western Blot and ELISA to verify the expression levels of the differentially expressed extracellular vesicle proteins screened; 4) Analyze the experimental results obtained and draw ROC curves for each differentially expressed extracellular vesicle protein; evaluate the diagnostic ability of potential extracellular vesicle protein markers based on the ROC curves and the area under the ROC curves, and finally screen out markers or marker combinations with good accuracy, sensitivity and specificity.

7. The method according to claim 6, characterized in that Step 1) also includes detecting the morphology of extracellular vesicles by transmission electron microscopy, measuring the particle size distribution and particle concentration of extracellular vesicles by nanoflow cytometry, and detecting specific extracellular vesicle protein markers by Western Blot.

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