Disclosure of Invention
In order to solve the limitation of early diagnosis of the abdominal aortic aneurysm, the application aims to provide a group of extracellular vesicle proteins as biomarkers for diagnosing the abdominal aortic aneurysm and application thereof, and based on the extracellular vesicle protein markers provided by the application, the early diagnosis of the abdominal aortic aneurysm can be realized through determination of circulating extracellular vesicle protein markers, thereby providing an effective tool for diagnosing the abdominal aortic aneurysm.
The invention also provides a screening method of the extracellular vesicle protein marker.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the invention firstly provides a series of extracellular vesicle proteins as biomarkers for diagnosing abdominal aortic aneurysm, wherein the extracellular vesicle protein markers are any one or more than two molecular markers 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).
Preferably, the extracellular vesicle protein marker is a combination of two or more of IL-4, IL-6, MCP-1, neurturin and Oncostatin-M.
Further preferred, the extracellular vesicle protein marker is a combination of IL-4, IL-6, MCP-1, neurturin and Oncostatin-M.
Further preferably, the extracellular vesicles are plasma extracellular vesicles.
Based on one general inventive concept, the present invention also provides the use of the extracellular vesicle protein marker for preparing a product for diagnosing an abdominal aortic aneurysm.
In particular, the product is a pharmaceutical agent, a kit, a microarray or a biochip.
Based on one general inventive concept, the present invention also provides a kit, microarray or biochip for diagnosing an Abdominal Aortic Aneurysm (AAA) including the extracellular vesicle protein marker.
Based on a general inventive concept, the invention also provides a screening method of the extracellular vesicle protein marker, comprising the following steps:
1) Extracting extracellular vesicle proteins from a plasma sample using an extracellular vesicle protein extraction kit;
2) Screening the differential expression extracellular vesicle protein by Olink proteome technology, cluster analysis and biological function enrichment analysis, and carrying out statistical analysis on the obtained data according to the differential expression extracellular vesicle protein among different groups;
3) Verifying the expression level of the screened differentially expressed extracellular vesicle proteins by using a Western Blot test and an enzyme-linked immunosorbent assay (ELISA);
4) And evaluating the diagnostic capability of the potential extracellular vesicle protein markers according to the ROC curve and the area under the ROC curve (AUC), and finally screening out the markers or marker combinations with good accuracy, sensitivity and specificity.
Specifically, in step 1), the method further comprises the steps of detecting the morphology of the extracellular vesicles by a Transmission Electron Microscope (TEM), measuring the particle size distribution and the particle concentration of the Extracellular Vesicles (EVs) by a nanoflow detector, detecting specific extracellular vesicle protein markers by Western Blot, and the like.
Specifically, step 2) mainly comprises a. Total protein extraction and immunological ligation, extracting total protein from the obtained extracellular vesicle PBS suspension of blood plasma, and determining protein concentration by using BCA protein quantitative kit;
The method comprises the steps of immune connection of an oligonucleotide sequence antibody, base complementary connection, namely mixing a protein sample with the oligonucleotide sequence antibody, and incubating, wherein the base complementary connection comprises the steps of respectively connecting two designed oligonucleotide sequences to different antibodies of target proteins, adding DNA polymerase after complementary connection, and extending in a system;
b. In the library preparation stage, firstly adding a specific barcode label for each sample after the extension reaction is finished for subsequent library distinguishing and sample identification, and purifying to obtain a library sample;
in the qPCR detection stage, diluting the purified library sample, and adding the diluted library sample into a qPCR reaction system to finish amplification of the library sample;
After amplification is completed, a qPCR instrument is used for collecting fluorescent signals in real time, and Ct values of each sample are recorded;
c. converting Data generated by qPCR detection into Ct values or count files, processing by NPX Signature software and Olink NPX Signature (NPX Manager) software to obtain an original sample NPX Data required by a biological information analysis flow, and manufacturing an original sample NPX Data distribution box diagram and a sample principal component analysis PCA diagram;
d. After quality control is carried out on the original Data, clean Data expressed by protein NPX is obtained, student inspection analysis or variance analysis is carried out on different experimental variables, and differential protein under the condition of the experimental variables is obtained;
e. And performing biological function enrichment analysis on the differential protein data, wherein the biological function enrichment analysis comprises GO biological process, GO molecular function, KEGG pathway, human gene set, reactome gene set and typical pathway.
Specifically, in step 2) a, the reaction system is reaction buffer (10 mM Tris-HCl, pH 8.0;50mM KCl;5mM MgCl2), base complementary reaction conditions of 25 ℃ for 30 minutes, DNA polymerization reaction conditions of 37 ℃ for 20 minutes, and then heating at 95 ℃ for 5 minutes to terminate the reaction.
Specifically, in step 2) b, the total reaction system volume was 20. Mu.L, including 10. Mu.L of 2X QPCR MASTER Mix, 1. Mu.L of library sample template, 0.4. Mu.L of target primer pair (final concentration 200 nM), and 8.6. Mu.L of sterile ultra-pure water. In qPCR reactions, initial denaturation is first carried out at 95℃for 5min to ensure complete denaturation of the DNA template, followed by 40 cycles including heating at 95℃for 15 seconds and annealing at 60℃for 1 min.
Specifically, the step 3) mainly comprises a, separating and purifying extracellular vesicle proteins by an ultracentrifugation method to obtain a high-purity extracellular vesicle sample, b, using RIPA lysis and extraction buffer to lyse and extract the extracellular vesicle proteins, and performing Western Blot and ELISA detection.
Specifically, the screening criterion of step 4) is that the AUC value is >0.75.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention can find diseases in early or even asymptomatic stages of aneurysm occurrence by detecting specific protein markers in EV. This molecular level detection method is capable of identifying early biological changes associated with lesions without relying on structural changes of blood vessels, thus enabling diagnosis at an earlier stage.
2. EV detection can be accomplished by simple blood sample collection, a non-invasive assay. The liquid biopsy is convenient, can be sampled for a plurality of times, is used for follow-up and monitoring the disease progress, and is particularly suitable for long-term management and periodical screening. Specific protein markers in EVs can reflect pathophysiological processes (such as inflammation, vascular wall degradation, etc.) associated with abdominal aortic aneurysms more accurately.
3. The invention can realize high specificity and high sensitivity diagnosis of the abdominal aortic aneurysm by the joint detection of the multiple markers, and reduce the probability of misdiagnosis and missed diagnosis. By detecting the EV protein marker profile of an individual patient, the pathological condition of each patient can be assessed, providing personalized diagnostic and therapeutic regimens. Such personalized assessment based on molecular markers helps to guide early intervention and optimize therapeutic decisions.
4. According to the invention, through EV protein marker detection, the application range of screening can be enlarged, and the method is not only suitable for high-risk groups, but also can be used for early screening of other potential risk groups. EV detection provides a broader and accurate screening tool. The detection cost is lower, the operation is simple, and the method is suitable for screening of basic medical units or resource limited areas. The liquid biopsy can be used as an effective early diagnostic tool, reduces reliance on high-cost imaging equipment, and optimizes medical resource allocation.
5. The invention can obviously improve the early detection capability and the long-term management effect of the abdominal aortic aneurysm, and fills the defects of the prior diagnosis technology in early discovery and personalized evaluation.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.
Example 1
1. Experimental materials
The study collected 10 AAA patients and 5 healthy normal controls as an internal cohort from a first affiliated hospital at the university of zheng, analyzed proteome expression of circulating Extracellular Vesicles (EVs) of AAA patients using a Olink-platform-based PEA technique, and collected 8 AAA patients and 4 healthy normal controls as separate external cohorts from cardiovascular surgery at a second clinical medical college at the university of ku, shenzhen, and performed external validation of junction proteins carried by Extracellular Vesicles (EVs). Diagnosis of AAA is based on criteria established by the vascular surgery society (SVS) and the European vascular surgery society (ESVS). The diameter of the AAA was measured as recommended by ESVS.
The inclusion criteria for AAA patients included (1) diagnosis according to the ESVS2019 guidelines, (2) physiological ages greater than 55 years old, and (3) signed informed consent, consent for storage and analysis of blood samples.
AAA exclusion criteria included (1) evidence of active infection, (2) chronic liver disease (Child-Pugh score. Gtoreq.B), (3) end stage renal disease (CKD 5 stage or creatinine. Gtoreq.2 mg/dL), (3) chronic inflammatory disease, (5) BMI <20 or >35, (6) significant surgery or illness accepted in the past 30 days, (7) history of organ transplantation with immunosuppressant or steroid drugs, (8) gestation or lactation.
The study recruited patients diagnosed with AAA and healthy volunteers. Venous blood was collected using whole blood collection EDTA tubes (BD vacuum blood collection tubes) to inhibit clotting. The blood sample was centrifuged at 1500 Xg for 20 minutes at 4℃to remove cells from the blood. The supernatant was taken and centrifuged at 3000×g for 15min at 4 ℃, the supernatant (plasma) was collected, and then the first 2mL of plasma per sample was flash frozen at-80 ℃ to maintain stability and integrity of the plasma biomolecules.
2. Experimental method
2.1 Extraction of extracellular vesicle proteins from plasma samples using ExoQuick extracellular vesicle protein kit (EXOQ A1; systems biosciences, USA) and strict adherence to manufacturer's protocol, the specific steps are:
250. Mu.L of plasma sample was mixed with the rapid extracellular vesicle pellet in 36. Mu. L ExoQuick kit, incubated at 4℃for 30 min, then centrifuged at 1500 Xg for 30 min, after discarding the supernatant, centrifuged at 1500 Xg for 5min, and the resulting extracellular vesicle-nanoscale particles (extracellular vesicles) were resuspended in 100. Mu.L of sterile Phosphate Buffered Saline (PBS) and stored at-80℃for subsequent analysis.
2.2 Detection of the morphology of isolated extracellular vesicles by Transmission Electron Microscopy (TEM), the specific steps are:
2.2.1 detection of isolated extracellular vesicle morphology by Transmission Electron Microscopy (TEM)
The extracellular vesicle solution is prepared by taking a proper amount of extracellular vesicle PBS suspension obtained in the step 2.1, measuring the protein concentration to be 0.5 mug/mu L by using a BCA protein quantitative kit, and diluting to be 10 mu L for detection by a transmission electron microscope. The sample negative dyeing treatment is that 10 mu L of extracellular vesicle solution is dripped on a copper mesh and incubated for 10 minutes at room temperature;
Then, washing the surface of the copper mesh with sterile distilled water, absorbing excessive liquid by using water absorbing paper, dripping 10 mu L of 2% uranyl acetate onto the copper mesh, carrying out negative dyeing for 1 min, drying under an incandescent lamp for 2min after the water absorbing paper absorbs floating liquid, observing the treated copper mesh under a Transmission Electron Microscope (TEM), and recording morphological characteristics (such as diameter range and cup-shaped structure) of extracellular vesicles.
2.2.2 Measurement of particle size distribution and particle concentration of Extracellular Vesicles (EVs) using a nanoflow detector (Flow NanoAnalyzer, nanoFCM inc., xiamen, china), the specific procedure being:
Sample preparation, sampling from the extracellular vesicle PBS suspension in step 2.1, diluting (or adjusting dilution times according to equipment requirements) according to 1:100, particle size and concentration measurement, namely injecting the diluted sample into a nanoflow detector (Flow NanoAnalyzer, nanoFCM Inc., xiamen, china), recording particle size distribution (such as median diameter and particle size range) and particle concentration (such as particle number/mL), and data analysis, namely using software of the detector to analyze data, and outputting the result so as to further evaluate the extracellular vesicle separation effect.
2.2.3BCA protein quantification and sample loading are specifically performed as follows:
Protein concentration determination, namely, strictly operating according to the instruction of a kit, determining the protein concentration of extracellular vesicles by using a BCA protein quantitative kit, ensuring the concentration of extracellular vesicle samples to be in the range of 10-30 mug according to the determination result, preparing SDS (sodium dodecyl sulfate) samples, calculating the loading quantity of the extracellular vesicle samples according to the protein concentration, adding the extracellular vesicle samples into 5 XSDS buffer solution according to a proportion, uniformly mixing by vortex, denaturing in a 95 ℃ water bath for 5 minutes, and then carrying out protein electrophoresis analysis.
2.2.4Western Blot detection of extracellular vesicle protein markers
And transferring the protein after electrophoresis, namely taking down the separation gel after electrophoresis, and electrically transferring the protein in the target area in the gel to the PVDF film. Extracellular vesicle-positive protein markers include Tsg101 (# ab125011, abcam, U.S.), alix (# ab186429, abcam, U.S.), CD9 (# ab263019, abcam, U.S.), extracellular vesicle-negative markers Calnexin (# 10427-2-AP, proteintech, U.S.), membrane blocking, blocking of membranes in 3% BSA blocking solution for 1 hour at room temperature to reduce non-specific binding, primary antibody incubation: primary antibody diluted to recommended concentration (typically 1:1000), incubation overnight at 4 ℃, antibodies employed are Tsg101 antibodies (# ab125011, abcam), alix antibodies (# ab186429, abcam), CD9 antibodies (# ab263019, abcam), calnexin antibodies (# 10427-2-AP, proteintech), primary antibody dilutions typically using TBST (TBS+0.1% Tween-20) or PBS buffer.
Secondary antibody incubation, namely, using HRP (horseradish peroxidase) labeled anti-rabbit secondary antibody, diluting to 1:5000 or manufacturer recommended concentration, and adopting anti-rabbit secondary antibody which is suitable for Tsg101, alix and Calnexin (such as the secondary antibody provided by Proteintech). After incubation for 1 hour at room temperature, the membrane was washed 3 times with TBST for 10 minutes each, developed and fixed by immersing the membrane in ECL developer (enhanced chemiluminescent kit such as Thermo #34095 is recommended), recording chemiluminescent signals using an imager such as Bio-Rad Chemiedoc or other brands, and confirming target protein expression by comparison to protein molecular weight standards based on the size and molecular weight of the target protein band.
Supplementary explanation:
Primary and secondary antibody selection:
The primary antibody is used for specifically recognizing target proteins (Tsg 101, alix, CD9, calnexin), the secondary antibody is paired with a primary antibody source animal species (such as anti-rabbit secondary antibody paired with rabbit source primary antibody), and the secondary antibody marks HRP for detecting signals by ECL development.
Blocking and membrane wash notes:
the blocking solution is usually low-fat milk powder (5%) or BSA (3%).
The TBST concentration for washing is typically 0.1% -0.2% Tween-20, ensuring removal of unbound antibody.
Developing system:
High sensitivity ECL imaging reagents are suggested to ensure a clear signal of the target protein band.
The exposure time is adjustable to ensure that the signal is not oversaturated.
2.3 Obtaining differential expression extracellular vesicle protein by utilizing Olink proteome technology, cluster analysis, biological function enrichment analysis and the like, wherein the specific steps are as follows:
olink proteome technology is a combination innovative protein detection and quantification technology based on Proximity Extension Assay (PEA) a protein and antibody immunoreaction technology and oligonucleotide amplification technology.
The Olink protein Biomarker detection is divided into a Target series (48, 96) and an Explorer series (384, 3072), wherein the Target is detected by adopting a qPCR mode, specifically, a prepared protein sample is diluted according to different Target panel protein concentrations, then is incubated with an antibody connected with an oligonucleotide sequence together, two oligonucleotide sequences connected with the protein are subjected to base complementary pairing, and under the action of DNA polymerase, the paired oligonucleotide sequences are extended in a reaction system and are subjected to PCR detection after pre-amplification. Among them, olink Target-96 series of targeted protein biomarker sets (Ding Z,Wang N,Ji N,et al.Proteomics technologies for cancer liquid biopsies[J].Molecular Cancer,2022,21(1):1-11). that can be used to analyze specific proteins associated with specific diseases or biological functions can gain new insight into disease processes, improve disease detection, and facilitate a better understanding of biology than previous protein immunization techniques.
2.3.1 Total protein extraction and immunological ligation
Total extracellular vesicle 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 ℃, extracellular vesicle protein extraction reagent: RIPA lysis buffer (Thermo Scientific, # 89900), 1 Xphosphatase inhibitor and PMSF were added, protein concentration assay: BCA protein quantification kit (Thermo Scientific, # 23225).
The extraction step comprises adding equal volume of RIPA lysate into extracellular vesicle suspension, mixing, incubating in ice bath for 30 min, centrifuging at 4deg.C at 13,000Xg for 10min, collecting supernatant, measuring protein concentration by BCA method, diluting and preserving for subsequent detection.
Immune ligation of oligonucleotide sequence antibodies:
antibody and sequence design oligonucleotide sequence antibodies were purchased from Olink Proteomics company and their sequences were optimally designed for the target protein (see literature for specific sequences, optimal designs and methods of acquisition) (Ding,Zhiyong,et al."Proteomics technologies for cancer liquid biopsies."Molecular Cancer 21.1(2022):53.)).
The immunological ligation step comprises mixing protein sample with oligonucleotide sequence antibody according to the recommended concentration, incubating on ice for 30min, base complementation ligation comprising respectively ligating two oligonucleotide sequences designed specifically to different antibodies of target protein, complementation ligation followed by adding DNA polymerase (Thermo Scientific, #EP 0502), extending in the system, reaction system comprising reaction buffer (10 mM Tris-HCl, pH 8.0;50mM KCl;5mM MgCl2), base complementation reaction conditions comprising incubation at 25deg.C for 30min, DNA polymerization reaction conditions comprising reaction at 37deg.C for 20 min, and subsequent heating at 95deg.C for 5 min to terminate the reaction.
2.3.2 Sample library preparation and qPCR detection
In the library preparation phase, a specific barcode tag is added to each sample for subsequent library discrimination and sample identification after the extension reaction is completed, and the equipment required for library preparation comprises Applied Biosystems QuantStudio qPCR instrument, and bar code primers and library preparation reagents purchased from Olink Proteomics company. In a specific operation, the primer with the barcode label is fully mixed with the product of the extension reaction, and then secondary amplification is carried out to improve the specificity and the signal intensity, and the amplified product is purified by magnetic beads (such as using AMPure XP magnetic beads, beckman Coulter) to obtain a library sample with high purity, so that a high-quality template is provided for subsequent qPCR detection.
In the qPCR detection stage, the purified library sample is added to a qPCR reaction system at a suitable dilution, the total reaction system volume is 20. Mu.L, including 10. Mu.L of 2X QPCR MASTER Mix (e.g., thermo Fisher,
#A25742), 1. Mu.L of library sample template, 0.4. Mu.L of target primer pair (final concentration 200 nM), and 8.6. Mu.L of sterile ultra-pure water. In the qPCR reaction, initial denaturation was first performed at 95℃for 5min to ensure complete denaturation of the DNA template, followed by 40 cycles including heating at 95℃for 15 seconds and annealing at 60℃for 1 min to complete amplification of the library.
After amplification is completed, fluorescence signals are acquired in real time by using software (QuantStudio Design & Analysis Software) of a qPCR instrument, ct values of each sample are recorded, and the Ct values are converted into NPX values (Olink Normalized Protein eXpression) by data analysis software, so that standardized protein expression data are provided for subsequent differential expression analysis.
2.3.3 Converting bml files or bcl files generated by qPCR detection into Ct values or counts files to obtain Data input files required by NPX Manager software, preprocessing qPCR quantitative Data by NPX Manager software, importing Olink NPX Signature (NPX Manager) software, performing sample type annotation, protein NPX calculation, data quality control, data deriving and analysis certificate (CERTIFICATE OF ANALYSIS, coA) to obtain input files required by biological information analysis flow, namely NPX Data, and manufacturing an original sample NPX Data distribution box diagram and a sample principal component analysis PCA diagram.
2.3.4 Analyzing the PCA diagram according to the NPX Data distribution box diagram of the original sample and the principal components of the sample, checking whether the sample has outlier samples or not according to the dimension-reducing clustering condition of the sample so as to facilitate the subsequent analysis to determine whether the outlier samples are removed or not, and then making all protein expression clustering heat diagrams so as to facilitate the preliminary understanding of the whole protein expression mode and develop the protein with abnormal expression.
2.3.5 After the quality control of the original data, clean data (CLEANDATA) expressed by protein NPX can be obtained, then protein expression cluster heat map analysis and protein correlation coefficient heat map analysis can be carried out aiming at each Panel, and expression box diagram analysis can be carried out on target proteins;
based on clean data expressed by protein NPX, the invention carries out student inspection analysis or variance analysis on different experimental variables to obtain differential protein under the condition of the experimental variables, carries out visual analysis on the differential protein data, comprises drawing a differential protein volcanic diagram, a Panel differential protein cluster heat diagram and the like, and carries out pearson correlation analysis and UMAP analysis to evaluate the difference of extracellular vesicle protein expression of AAA patients and healthy control groups.
2.3.6 The invention also performs a biofunction enrichment analysis on the differential protein data. All identified AAA extracellular vesicle proteins were annotated with the STRING database and protein-protein interactions (PPI) analysis was performed. After the preliminary analysis, the invention carries out comprehensive map and process enrichment analysis on the predicted target point of the AAA extracellular vesicle protein, and the analysis integrates a plurality of data of ontology sources, including GO biological process, GO molecular function, KEGG pathway, human gene set, reactome gene set and typical pathway.
Meanwhile, the biological functional 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 protein is explored.
Finally, annotation of a protein-related bioinformatic database, subcellular localization analysis and protein interaction network analysis are performed on all proteins analyzed by Olink technology.
2.4 Further validation of the expression levels of five differentially expressed proteins (Oncostatin-M, interleukin-4, interleukin-6, neurturin, and MCP-1) in the external cohort using Western Blot and enzyme-Linked immunosorbent assay (ELISA), the specific steps are:
2.4.1 in this step, the extracellular vesicle proteins were first isolated and purified by ultracentrifugation, specifically by centrifuging the sample at 10,000Xg for 20 minutes at 4℃to remove cell debris and other impurities, then transferring the supernatant into an ultracentrifuge tube, centrifuging at 100,000Xg for 90 minutes at 4℃to precipitate extracellular vesicles, collecting the resulting precipitate, re-suspending extracellular vesicle particles with sterile PBS, and centrifuging again at 100,000Xg for 90 minutes to finally obtain a high purity extracellular vesicle sample.
Extracellular vesicles were then lysed and protein extracted using RIPA lysis and extraction buffer (Thermo Scientific, # 89900), phosphatase inhibitor and PMSF (phenolmethanesulfonyl fluoride) were added to RIPA buffer at a volume ratio of 100:1 to preserve protein activity, the lysate was incubated on ice for 30min, centrifuged at 13,000Xg for 10 min at 4 ℃, the supernatant was collected as protein sample and protein concentration was determined using BCA protein quantification kit, and the resulting protein samples were used for Western Blot and ELISA detection.
In Western Blot detection, target extracellular vesicle proteins were detected using 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 according to the target protein concentration (10-30 mug), carrying out SDS-PAGE electrophoresis to separate the protein, transferring the protein to a PVDF membrane, sequentially adding a primary antibody and an enzyme-labeled secondary antibody, and finally using a chemiluminescent reagent for developing and observing the expression level of the specific protein.
Meanwhile, to further verify the concentration of the target protein, an extracellular vesicle protein sample was detected using an ELISA kit. The kits used include human IL-4ELISA kit (Multi Sciences, EK 104/2-96), human IL-6ELISA kit (Multi Sciences, EK 106-96), human MCP-1ELISA kit (Multi Sciences, EK 187-96), human Neurturin ELISA kit (YOBIBIO, U96-1476E) and human OSM ELISA kit (YOBIBIO, U96-1578E). In the detection process, the operation is strictly according to the specification of the manufacturer, incubation and washing are carried out after sample addition, enzyme-labeled secondary antibodies are added, after the reaction is developed and terminated, the absorbance value is measured by using an enzyme-labeled instrument at the wavelength of 450nm, and the concentration of each target protein is calculated to evaluate the expression level.
2.5 Analysis of the results of the foregoing experiments
Specifically, all statistical tests in the experimental method are double-tail tests, P values smaller than 0.05 and False Discovery Rate (FDR) smaller than 0.05 are regarded as having statistical significance, descriptive statistics of normal distribution continuous variables are reported in the form of mean value +/-standard deviation, comparison of continuous variables adopts corresponding Wilcoxon rank sum test or student t test, classification variables are analyzed by chi-square test or Fisher exact test, and all data processing, statistical analysis and chart drawing are performed by using R software (version 4.4.3).
3. Experimental results
3.1 Identification of isolated plasma extracellular vesicles
The overall flow of the method of the invention is shown in FIG. 1A for the isolation and identification of plasma extracellular vesicles, the characterization of which is based on their diameter, morphology and surface protein expression. First, the isolated plasma extracellular vesicle particles exhibited a typical cuplike structure as observed by Transmission Electron Microscopy (TEM) (fig. 1B), which is characterized by the typical morphological signature of extracellular vesicles, further demonstrating the presence of extracellular vesicle nanoparticles in the sample. Further, the sample was subjected to particle size distribution analysis (FIG. 1C) using a high-sensitivity flow nanoanalyzer (Flow NanoAnalyzer), and the result showed that the AAA extracellular vesicles (AAA-Exo) had a median diameter of 85.18 nm, which was consistent with the reported extracellular vesicles particle size range (30-150 nm), and further confirmed the presence and particle size characteristics of the extracellular vesicles.
The invention also detects the surface protein of the extracellular vesicles by a Western blot Western blotting method (figure 1D). The results showed that the extracellular vesicle positive markers Tsg101, alix and CD9 were all clearly expressed in the samples, whereas the extracellular vesicle negative marker Calnexin was not detected. This indicates that the extracted sample contains high purity extracellular vesicles and is substantially free of contamination by cell debris and other non-extracellular vesicle components. By combining TEM observation, flow nano analysis and Western blot detection results, the samples separated by the method are successfully identified as plasma extracellular vesicles, and the extracellular vesicles are used for screening and analyzing protein biomarkers in subsequent experiments.
3.2 Detection of plasma extracellular vesicle protein biomarkers of AAA patients Using Olink proteome technology
The 92 protein biomarkers in plasma extracellular vesicles of AAA patients and healthy controls were analyzed by the present invention and the results are shown in FIG. 2. By means of the thermal map (fig. 2A), the distribution pattern of extracellular vesicle proteins in different samples can be intuitively observed. The results show that there is a clear difference in protein expression between the AAA group and the healthy control group, and that part of the proteins exhibit unique expression patterns in both groups. Further block diagram analysis (fig. 2B) showed that NPX (normalized protein expression) levels between AAA group and healthy control group exhibited significant differences over various proteins, revealing protein expression characteristics between the two groups. The Principal Component Analysis (PCA) (fig. 2C) results showed that the samples were clearly separable in the principal component space, indicating that AAA patients and healthy controls were significantly and consistently different in extracellular vesicle protein expression profile. Together, these results support that extracellular vesicle proteins can be used as potential biomarkers for distinguishing AAA patients from healthy individuals.
The decision criteria used in the significance analysis were P <0.05, based on the t-test or analysis of variance results of the differential expression analysis. In conjunction with this threshold calculation, the statistical significance of extracellular vesicle protein expression levels between the two groups was further verified. Through the verification of the external cohort (FIG. 3A, B, C), five protein biomarkers in the extracellular vesicles of AAA patients and healthy controls were found to exhibit significant expression differences, oncostatin-M, interleukin-4, interleukin-6, neurturin, and MCP-1 (five protein biomarkers see https:// www.genecards.org /), respectively, with significantly up-regulated expression of Oncostatin-M, neurturin and MCP-1 in the extracellular vesicles of AAA patients and significantly down-regulated expression of interleukin-4 and interleukin-6.
With regard to the arrangement of the external and internal queues, the object of the present invention is to ensure that the screened extracellular vesicle protein markers have stability and broad applicability. The internal queue is used for initially finding potential differential proteins, while the external queue is used as an independent verification group for further evaluating the performances of the markers in different populations, thereby improving the reliability and popularization of marker screening. Through the design of the double queues, the invention ensures that the screened extracellular vesicle proteins show consistent and obvious differences in different queues, and provides a firm experimental basis for the screened extracellular vesicle proteins serving as potential diagnosis markers of AAA.
In addition, pearson-related analysis and UMAP analysis were performed to evaluate differences in extracellular vesicle protein expression in AAA patients and healthy control groups (fig. 3D, E). These results show that there is a significant difference in plasma extracellular vesicle protein expression between AAA patients and healthy people.
3.3 Comprehensive functional enrichment of extracellular vesicle proteomes target proteins
In the experimental method of the present invention described above, a PPI interaction network labeled with UniProt ID and gene symbol was generated by protein-protein interaction (PPI) analysis. After rejecting isolated target genes lacking interactions and applying confidence threshold 900, the gene interaction network is visualized using Cytoscape (fig. 4A). The top 20 enrichment terms of the key clusters in this analysis are shown. To further clarify the relationship between these top-level terms, the present invention constructs a functional annotation network, as shown in FIG. 4B. The network allows a deeper understanding of the potential interrelated pathways and processes of AAA-exon derived protein effects, thereby enhancing an understanding of their role in AAA molecular mechanisms.
In the profile and process enrichment analysis of predicted targets for AAA extracellular vesicle proteins, a number of data from bulk sources are integrated, including GO biological processes, GO molecular functions, KEGG pathways, human gene sets, reactome gene sets, and canonical pathways. As a result, several markedly enriched terms, in particular "cell response to cytokine stimulation", were found "
(GO: 0071345), "inflammatory response" (GO: 0006954), chemokine signaling pathway (hsa 04062), activin-1 pathway (M167), and Glucocorticoid Receptor (GR) regulatory pathway (M115). According to previous studies, it was found that the inflammatory response and the production and activation of various proteases play a vital role in promoting the formation and development of aortic aneurysms.
3.4 Western blot Western ELISA verification of differentially expressed proteins in extracellular vesicles of AAA circulation
Five differentially expressed proteins were identified as junction proteins, including IL-4, IL-6, MCP-1, neurturin and Oncostatin-M, in circulating extracellular vesicles from AAA patients and healthy controls. The criteria for screening these proteins as junction proteins are based mainly on quantitative indicators that (1) meet a significance threshold (P < 0.05) in differential expression assays, (2) have a higher connectivity or key position in the protein-protein interaction (PPI) network, and (3) are significantly involved in AAA-related pathological processes (such as inflammatory responses, cytokine signaling pathways, etc.) in biofunction enrichment assays. Based on these quantification criteria, the key role of these proteins was further verified experimentally.
The invention adopts Western Blot and ELISA to verify the expression of the junction protein in an external verification queue (8 patients and 4 healthy controls). In Western Blot experiments, FIG. 5A shows the bands of these five proteins, and the band gray scale analysis results (FIG. 5B) indicate that MCP-1, neurturin and Oncostatin-M expression was significantly elevated (P < 0.05) in AAA patients compared to healthy controls, whereas IL-4 and IL-6 showed no statistically significant differences. Subsequently, the expression levels of these proteins were further verified by ELISA.
ELISA results showed (FIG. 5C) that the levels of IL-4, IL-6, MCP-1, neurturin and Oncostatin-M in circulating extracellular vesicles were significantly higher in AAA patients than in healthy control (P < 0.05). Furthermore, the ROC curve in fig. 5D further evaluates the potential of these proteins as diagnostic markers. 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, respectively (95% CI:0.731-1.000, oncostatin-M). These results indicate that, in particular, oncostatin-M and Neurturin have high AUC values, show extremely strong diagnostic capabilities, and can significantly distinguish AAA patients from healthy people.
Compared with the prior art, the invention has the following advantages:
1. Novel biomarker screening the invention utilizes the protein in the extracellular vesicles as a specific marker for early diagnosis of abdominal aortic aneurysm, which is the field of less application in the early diagnosis of AAA at present. The specific proteins in EVs may be directly related to pathological processes of AAA (e.g., vascular wall remodeling, inflammation, oxidative stress, etc.), with higher specificity and sensitivity, as opposed to conventional serum markers.
2. The invention provides an innovative detection method for realizing early diagnosis by specific protein markers in a circulating EV. This method is earlier, more sensitive than imaging diagnosis and can be obtained by non-invasive sampling such as blood or urine. EV markers can provide early warning when the aneurysm has not significantly increased, as compared to traditional imaging, which relies on size detection of the aneurysm.
3. And the personalized diagnosis model screens out protein markers highly related to the abdominal aortic aneurysm by a high-throughput detection technology, and establishes 5 key biomarker diagnosis models and diagnosis methods capable of distinguishing AAA patients from healthy individuals, thereby not only being beneficial to diagnosis, but also providing information of disease progress and realizing early intervention and personalized treatment.
4. Traditional imaging may require frequent monitoring of aneurysm growth, while non-invasive detection of EV protein markers not only reduces patient burden, but also provides a means to evaluate AAA development periodically, improving long-term management.
5. Specific protein marker combinations the present invention protects proteins that are significantly elevated or reduced in specific protein marker combinations, especially in EV in the circulation of patients with abdominal aortic aneurysm. The screening and verification process of the proteins is one of the core innovation points, and the innovation point of the invention comprises the screening of the protein markers and the application of the protein markers in diagnosis.
6. Diagnostic methods and procedures the detection methods of the present invention, including specific technical procedures for sample collection, EV separation, protein detection (e.g., ELISA, mass spectrometry, immunoblotting, etc.), and data analysis, are one of the important points to be protected by the present invention.
7. Multiple marker algorithm model through the combined detection of multiple protein markers, and can establish mathematical models or algorithms (such as machine learning, statistical models and the like) to realize early diagnosis of AAA patients. At the same time, the application of the method in diagnosis is also the core invention point of the invention.