Biomarker for aortic dissection diagnosis and application thereof

Abstract

The invention provides application of a detection reagent of an exosome or extracellular envelope derived biomarker in preparation of a composition or a kit for evaluating, diagnosing, screening and / or monitoring an aortic dissection of a subject. The exosome or extracellular envelope derived biomarker provided by the invention is applied to detection, and has high sensitivity and specificity. Compared with the traditional method, the reaction time is greatly shortened. Good clinical application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and in particular relates to biomarkers of aortic dissection and applications thereof. Background Art

[0002] Acute aortic dissection (AAD) is a life-threatening cardiovascular disease with an increased mortality rate of approximately 1%-2% per hour after symptom onset without treatment. Early diagnosis is crucial for the prognosis of AAD. One of the main challenges in diagnosing AAD is distinguishing AAD from other sudden severe chest pain diseases, as these patients present with similar symptoms but require different treatment strategies.

[0003] AAD is a disease associated with the aortic intima. To accelerate the diagnosis of AAD, the search for potential biomarkers has focused on markers associated with vascular smooth muscle (smooth muscle myosin), vascular stroma (cadherin), aortic elastic lamina (soluble elastin fragments), or endothelial cell damage (CD40 ligand), as well as markers associated with blood exposure to non-intimal vascular surfaces (D-dimer). Currently, only D-dimer has a guiding role in the rapid clinical diagnosis of suspected aortic dissection (AD). However, D-dimer has low specificity in patients with pseudoluminal thrombosis, extensive dissection lesions, and small or young patients. Although D-dimer levels are significantly elevated in patients with AAD compared with acute myocardial infarction, D-dimer cannot distinguish patients with AAD from those with pulmonary embolism.

[0004] Therefore, there is an urgent need for a biomarker that can be used to quickly diagnose AAD in clinical practice. Summary of the Invention

[0005] To address at least one of the above issues, the present invention discloses enriching extracellular vesicles from plasma of a cohort of patients with aortic dissection and a cohort of healthy controls, and using mass spectrometry to analyze the proteomic information contained in these extracellular vesicles to identify diagnostic biomarkers for aortic dissection. Using the biomarkers provided by this disclosure, it is possible to assess, diagnose, screen, and / or monitor aortic dissection in subjects with high sensitivity and specificity.

[0006] According to one aspect of the present disclosure, a biomarker detection reagent is provided for use in preparing a kit for evaluating, diagnosing, screening and / or monitoring aortic dissection, wherein the biomarker comprises one or more of the following: human syndecan 1 (SDC1), human EF-hand calcium binding domain 6 (EFCAB6), amine oxidase copper containing 1 (AOC1), regulation of nuclear pre-mRNA domain containing 1A (RPRD1A), FYVE and coiled-coil domain autophagy adaptor 1 (FYCO1), centrosomal protein 295 (CEP295), myotubularin-related protein 11 (MTMR11), solute carrier family 25 member 24 (SC24), and solute carrier family 25 member 24 (SC24). 25 Member 24, SLC25A24), Katanin p60 ATPase-containing subunit A1 (KATNA1), Acyl-CoA Dehydrogenase, Long Chain (ACADL), NCK-associated protein 5-like (NCKAP5L), Kinesin Family Member C1 (KIFC1), Aminoacylase 3 (ACY3), G Protein-Coupled Receptor Kinase 7 (GRK7), RNA Binding Motif Protein 17 (RBM17), Galectin 8 (LGALS8), Ovostatin 1 (Ovostatin 1, OVOS1), Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 3,MICAL3), ATPBinding CassetteSubfamily C Member 9 (ABCC9), Alstrom Syndrome Protein 1 (ALMS1), Ankyrin Repeat Domain 6 (ANKRD6), ADP-Ribosylhydrolase Like 1 (ADPRHL1), Caspase 9 (CASP9), Spectrin Repeat Containing Nuclear Envelope Protein 3 (SYNE3), DEP Domain Containing 7 (DEPDC7), 3-Hydroxyisobutyryl-CoA Hydrolase (HIBCH), Protocadherin gamma subfamily A11 A11 (PCDHGA11), Tripartite Motif Containing 38 (TRIM38), Midkine (MDK), Solute Carrier Family 4 Member 5 (SLC4A5), Kelch repeat and BTB domain containing 7 (KBTBD7), Cell Death-Inducing DFFA-Like Effector A (CIDEA), Serum Amyloid A2 (SAA2), Integrator Complex Subunit 6 Like (INTS6L), Hepatoma-Derived Growth Factor-Like 3 (HDGFL3), Tektin 2 (TEKT2), ​​Endothelial PAS Domain Protein 1 (EPP1) 1, EPAS1), aminomethyltransferase (Aminomethyltransferase,AMT), Growth Arrest Specific 8 (GAS8), DExH-box Helicase 33 (DHX33), Von Willebrand Factor A Domain Containing 3B (VWA3B), Zinc Finger Protein 862 (ZNF862), TATA-Box Binding Protein Associated Factor, RNA Polymerase I Subunit C (TAF1C), Alanyl-tRNA Synthetase 2 (AARS2), Protein Phosphatase 4 Regulatory Subunit 4 (PPP4R4), Mahogunin Ring Finger 1 (MGRN1), Shroom Family Member 2 2, SHROOM2), Exocyst Complex Component 3 Like 4 (EXOC3L4), F-Box And Leucine Rich Repeat Protein 6 (FBXL6), Myosin Heavy Chain 16 (MYH16), KN Motif And Ankyrin Repeat Domains 3 (KANK3) or Serumamyloid A1 (SAA1).

[0007] In some embodiments, the biomarkers include one or more of: SDC1, EFCAB6, AOC1, RPRD1A, FYCO1, CEP295, SLC25A24, KATNA1, ACADL, ACY3, RBM17, LGALS8, ADPRHL1, CASP9, SYNE3, HIBCH, PCDHGA11, MDK, KBTBD7, SAA2, AARS2, PPP4R4, and SAA1.

[0008] In some embodiments, the biomarkers include one or more of the following: SDC1, EFCAB6, AOC1, FYCO1, CEP295, PCDHGA11, MDK, KBTBD7, or SAA1.

[0009] In some embodiments, the aortic dissection includes type A and / or type B aortic dissection. In some embodiments, the aortic dissection includes acute aortic dissection, subacute aortic dissection and / or chronic aortic dissection. In a preferred embodiment, the aortic dissection is type A acute aortic dissection.

[0010] In some embodiments, the detection reagent is used to detect the expression level of the marker in an extracellular vesicle sample from a subject.

[0011] In some embodiments, the assessment, diagnosis, screening and / or monitoring comprises the following steps: (1) detecting the expression level of the biomarker (e.g., protein expression level) in an extracellular vesicle sample from a subject, and (2) determining whether the subject has aortic dissection or is at risk of aortic dissection based on the expression level of the biomarker.

[0012] In some embodiments, before step (1), the method further comprises the step of isolating and enriching extracellular vesicles from the subject.

[0013] In some embodiments, the separation and enrichment of extracellular vesicles from the subject comprises one or more of the following: ultracentrifugation, size exclusion, field flow separation, microfluidics, ultrafiltration, and polymer precipitation.

[0014] In some embodiments, the separation and enrichment of extracellular vesicles from a subject is achieved by using ultracentrifugation and / or asymmetric field flow. In some embodiments, the asymmetric field flow can be combined with a multi-angle light scattering system to sort extracellular vesicles.

[0015] In some embodiments, the detection in step (1) includes, but is not limited to, mass spectrometry, chromatography, immunoassay, PCR, and transcriptome sequencing.

[0016] In some embodiments, the detection in step (1) comprises one or more of the following: time-of-flight mass spectrometry (TOF-MS), orbitrap mass spectrometry (Orbitrap MS), quadrupole mass spectrometry (QMS), ion trap mass spectrometry (ITMS), magnetic sector mass spectrometry, FT-ICR mass spectrometry, ion mobility spectrometry-mass spectrometry (IMS-MS), liquid chromatography, solid phase chromatography, enzyme-linked immunosorbent assay, protein blotting, dot blot or immunostaining, lateral flow assay, RT-qPCR, and transcriptome sequencing.

[0017] In some embodiments, step (2) comprises comparing the level of the biomarker from the subject with a threshold value, wherein a level above or equal to the threshold value indicates a higher risk of aortic dissection; a level below the threshold value indicates a lower risk of aortic dissection.

[0018] Those skilled in the art will appreciate that the threshold value can be determined using conventional methods known to those skilled in the art, for example, based on ROC curves, Youden index, sensitivity, specificity, and percentiles of healthy control populations.

[0019] According to yet another aspect of the present disclosure, there is provided a device for assessing, diagnosing, screening, and / or monitoring the risk of aortic dissection in a subject, the device comprising:

[0020] a biomarker detection module for determining the presence and / or level of said biomarker from a subject; and

[0021] The aortic dissection risk judgment module judges the risk of aortic dissection in a subject based on the presence and / or level of the biomarker.

[0022] According to another aspect of the present disclosure, a biomarker for diagnosing aortic dissection is provided, wherein the biomarker comprises one or more of the following: human syndecan 1 (SDC1), human EF-hand calcium binding domain 6 (EFCAB6), amine oxidase copper containing 1 (AOC1), nuclear pre-mRNA domain regulatory protein 1A (RPRD1A), FYVE and coiled-coil domain autophagy adaptor 1 (FYCO1), centrosomal protein 295 (CEP295), myotubularin-related protein 11 (MTMR11), solute carrier family 25 member 24 (SCF24), and solute carrier family 25 member 25. 24, SLC25A24), Katanin p60 ATPase-containing subunit A1 (KATNA1), Acyl-CoA Dehydrogenase, Long Chain (ACADL), NCK-associated protein 5-like (NCKAP5L), Kinesin Family Member C1 (KIFC1), Aminoacylase 3 (ACY3), G Protein-Coupled Receptor Kinase 7 (GRK7), RNA Binding Motif Protein 17 (RBM17), Galectin 8 (LGALS8), Ovostatin 1 (Ovostatin 1, OVOS1), Microtubule-associated monooxygenase, Calponin and LIM Domain Containing 3,MICAL3), ATP Binding Cassette Subfamily C Member 9 (ABCC9), Alstrom Syndrome Protein 1 (ALMS1), Ankyrin Repeat Domain 6 (ANKRD6), ADP-Ribosylhydrolase Like 1 (ADPRHL1), Caspase 9 (CASP9), Spectrin Repeat Containing Nuclear Envelope Protein 3 (SYNE3), DEP Domain Containing 7 (DEPDC7), 3-Hydroxyisobutyryl-CoA Hydrolase (HIBCH), Protocadherin γ subfamily A11 A11 (PCDHGA11), Tripartite Motif Containing 38 (TRIM38), Midkine (MDK), Solute Carrier Family 4 Member 5 (SLC4A5), Kelch repeat and BTB domain containing 7 (KBTBD7), Cell Death-Inducing DFFA-Like Effector A (CIDEA), Serum Amyloid A2 (SAA2), Integrator Complex Subunit 6 Like (INTS6L), Hepatoma-Derived Growth Factor-Like 3 (HDGFL3), Tektin 2 (TEKT2), ​​Endothelial PAS Domain Protein 1 (EPP1), E237-114 (EGFR), E237-115 (EGFR), E237-116 (EGFR), E237-117 (EGFR), E237-118 (EGFR), E237-119 (EGFR), E237-119 (EGFR), E237-119 (EGFR), E237-118 (EGFR), E237-119 (EGFR), E237-119 (EGFR), E237-119 (EGFR), E237-119 (EGFR), 1, EPAS1), aminomethyltransferase (Aminomethyltransferase,AMT), Growth Arrest Specific 8 (GAS8), DExH-box Helicase 33 (DHX33), Von Willebrand Factor A Domain Containing 3B (VWA3B), Zinc Finger Protein 862 (ZNF862), TATA-Box Binding Protein Associated Factor, RNA Polymerase I Subunit C (TAF1C), Alanyl-tRNA Synthetase 2 (AARS2), Protein Phosphatase 4 Regulatory Subunit 4 (PPP4R4), Mahogunin Ring Finger 1 (MGRN1), Shroom family member 2 (Shroom Family Member 2, SHROOM2), Exocyst Complex Component 3 Like 4, EXOC3L4, F-Box And Leucine Rich Repeat Protein 6, FBXL6, Myosin Heavy Chain 16, MYH16, KN Motif And AnkyrinRepeat Domains 3, KANK3, or Serum amyloid A1 (SAA1).

[0023] In some embodiments, the biomarkers include one or more of: SDC1, EFCAB6, AOC1, RPRD1A, FYCO1, CEP295, SLC25A24, KATNA1, ACADL, ACY3, RBM17, LGALS8, ADPRHL1, CASP9, SYNE3, HIBCH, PCDHGA11, MDK, KBTBD7, SAA2, AARS2, PPP4R4, and SAA1.

[0024] In some embodiments, the biomarkers include one or more of the following: SDC1, EFCAB6, AOC1, FYCO1, CEP295, PCDHGA11, MDK, KBTBD7, or SAA1.

[0025] In some embodiments, the aortic dissection comprises type A and / or type B aortic dissection. In some embodiments, the aortic dissection comprises acute aortic dissection, subacute aortic dissection, and / or chronic aortic dissection.

[0026] In some embodiments, the biomarker is differentially expressed in a sample from a subject with aortic dissection compared to a healthy subject.

[0027] In some embodiments, the biomarker is significantly differentially expressed in samples from subjects with aortic dissection compared to healthy subjects.

[0028] In some embodiments, the expression level of the biomarker is significantly increased or significantly decreased in a sample from a subject with aortic dissection compared to a healthy subject.

[0029] In some embodiments, the biomarker is differentially expressed in samples from subjects with aortic dissection compared to subjects with myocardial infarction.

[0030] In some embodiments, the biomarker is differentially expressed in samples from subjects with aortic dissection compared to subjects with myocardial infarction.

[0031] In some embodiments, the biomarker is expressed at a significantly increased or decreased level in a sample from a subject with aortic dissection compared to a subject with myocardial infarction.

[0032] In some embodiments, the biomarker is derived from an extracellular vesicle of the subject.

[0033] In some embodiments, the extracellular vesicles are selected from one or more of exosomes, migrasomes, microvesicles, apoptotic bodies, and inducible extracellular vesicles. In some embodiments, the biomarkers are derived from exosomes.

[0034] In some embodiments, the extracellular vesicles are derived from bodily fluids.

[0035] In some embodiments, the extracellular vesicles can be isolated and enriched by one or more of the following methods: ultracentrifugation, size exclusion, field flow fractionation, microfluidics, ultrafiltration, and polymer precipitation.

[0036] In some embodiments, the body fluid comprises one or more of the following: whole blood, serum, plasma, urine, alveolar lavage fluid, cerebrospinal fluid, serous fluid, pleural effusion, peritoneal lavage fluid, peritoneal fluid, and processed products thereof.

[0037] According to another aspect of the present disclosure, a kit for evaluating, diagnosing, screening and / or monitoring aortic dissection is provided, wherein the kit comprises a detection reagent for the above-mentioned biomarker.

[0038] In some embodiments, the kit includes a standard.

[0039] In some embodiments, the standard preferably includes a negative standard and / or a positive standard. The biomarkers provided by the present disclosure can be used to evaluate, diagnose, screen and / or monitor aortic dissection in subjects with high sensitivity and specificity. Compared with traditional methods, the advantages of the biomarkers provided by the present disclosure over traditional aortic CTA are that they are easy to promote and inexpensive. In addition, the biomarkers provided by the present disclosure and the methods for evaluating, diagnosing, screening and / or monitoring aortic dissection in subjects significantly reduce the impact of high-abundance proteins in body fluids, greatly reducing the false negative rate of screening results. It has unique advantages in detecting low-abundance protein markers in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The experimental flow chart for screening diagnostic markers for aortic dissection is shown.

[0041] Figure 2 The electron microscopy observation images and nanoparticle tracking analysis (NTA) results of the aortic dissection group and the healthy control group of the experimental cohort are shown.

[0042] Figure 3 The diagram shows the differential expression of proteins in the plasma exosome proteome of individuals in the aortic dissection group and the healthy control group in the experimental cohort. The horizontal axis is log2 (Fold Change), where Fold Change = centered intensity of the AAD group / centered intensity of the Ctrl group; the vertical axis is -log 10 (P value).

[0043] Figure 4 Flowchart showing the validation cohort for diagnostic markers of aortic dissection.

[0044] Figure 5 Electron microscopy observations and nanoparticle tracking analysis (NTA) results of the validation cohort of aortic dissection patients and healthy controls are shown.

[0045] Figure 6Electron microscopy observations and nanoparticle tracking analysis (NTA) results of patients with acute myocardial infarction in the validation cohort are shown.

[0046] Figure 7 A schematic diagram showing the differential expression of AOC1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0047] Figure 8 A schematic diagram shows the differential expression of SDC1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0048] Figure 9 A schematic diagram shows the differential expression of MDK in plasma exosomal proteins in the validation cohort aortic dissection group and the healthy control group. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0049] Figure 10 A schematic diagram shows the differential expression of EFCAB6 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0050] Figure 11 A schematic diagram shows the differential expression of RPRD1A in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0051] Figure 12 Figure 2 shows the differential expression of SAA1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0052] Figure 13 The figure shows the differential expression of SAA2 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0053] Figure 14Figure showing the plasma exosome protein ABCC9 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0054] Figure 15 Figure showing the plasma exosome protein TRIM38 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0055] Figure 16 Figure showing the plasma exosome protein KANK3 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0056] Figure 17 Figure showing the plasma exosome protein MICAL3 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0057] Figure 18 Figure showing the plasma exosome protein FYCO1 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0058] Figure 19 Figure showing the plasma exosome protein CEP295 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0059] Figure 20 Figure showing the plasma exosome protein MTMR11 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0060] Figure 21 Figure showing the plasma exosome protein SLC25A24 differential protein diagram of individuals in the validation cohort of aortic dissection group and healthy control group. Among them, Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute type A aortic dissection group.

[0061] Figure 22A schematic diagram shows the differential expression of KATNA1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0062] Figure 23 A schematic diagram showing the differential expression of ACADL in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0063] Figure 24 A schematic diagram shows the differential expression of NCKAP5L in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0064] Figure 25 A schematic diagram showing differential expression of ACY3 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0065] Figure 26 A schematic diagram shows the differential expression of KIFC1 in plasma exosomal proteins between individuals in the validation cohort, including the aortic dissection group and the healthy control group. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0066] Figure 27 A schematic diagram shows the differential expression of GRK7 in plasma exosomal proteins between individuals in the validation cohort, including the aortic dissection group and the healthy control group. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0067] Figure 28 A schematic diagram shows the differential expression of RBM17 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0068] Figure 29 A schematic diagram shows the differential expression of LGALS8 in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0069] Figure 30A schematic diagram shows the differential expression of OVOS1 in plasma exosomal proteins between the aortic dissection group and the healthy control group in the validation cohort. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0070] Figure 31 A schematic diagram shows the differential expression of ALMS1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0071] Figure 32 A schematic diagram showing differential expression of ANKRD6 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0072] Figure 33 A schematic diagram shows the differential expression of ADPRHL1 in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0073] Figure 34 A schematic diagram shows the differential expression of CASP9 in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0074] Figure 35 A schematic diagram shows the differential expression of SYNE3 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0075] Figure 36 A schematic diagram shows the differential expression of DEPDC7 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0076] Figure 37 A schematic diagram of HIBCH differentially expressed exosomal proteins in the aortic dissection group and the healthy control group in the validation cohort is shown. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0077] Figure 38A schematic diagram shows the differential expression of PCDHGA11 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0078] Figure 39 The figure shows the differential expression of SLC4A5 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0079] Figure 40 A schematic diagram shows the differential expression of KBTBD7 in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0080] Figure 41 A schematic diagram shows the differential expression of CIDEA in plasma exosomal proteins in the validation cohort aortic dissection group and healthy control group. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0081] Figure 42 A schematic diagram shows the differential expression of INTS6L in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0082] Figure 43 A schematic diagram shows the differential expression of HDGFL3 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0083] Figure 44 A schematic diagram shows the differential expression of TEKT2 in plasma exosomal proteins between individuals in the validation cohort, including the aortic dissection group and the healthy control group. Ctrl represents the healthy control group, AAD represents the acute myocardial infarction group, and AMI represents the acute type A aortic dissection group.

[0084] Figure 45 Figure 2 shows the differential expression of EPAS1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0085] Figure 46Figure 8 shows a schematic diagram of the differential proteins of plasma exosome protein AMT in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0086] Figure 47 Figure 9 shows a schematic diagram of the differential proteins of plasma exosome protein GAS8 in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0087] Figure 48 Figure 10 shows a schematic diagram of the differential proteins of plasma exosome protein DHX33 in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0088] Figure 49 Figure 11 shows a schematic diagram of the differential proteins of plasma exosome protein VWA3B in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0089] Figure 50 Figure 12 shows a schematic diagram of the differential proteins of plasma exosome protein ZNF862 in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0090] Figure 51 Figure 13 shows a schematic diagram of the differential proteins of plasma exosome protein TAF1C in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0091] Figure 52 Figure 14 shows a schematic diagram of the differential proteins of plasma exosome protein AARS2 in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0092] Figure 53 Figure 15 shows a schematic diagram of the differential proteins of plasma exosome protein PPP4R4 in the validation cohort of the aortic dissection group and the healthy control group. Ctrl is the healthy control group, AAD is the acute myocardial infarction group, and AMI is the acute aortic dissection group.

[0093] Figure 54A schematic diagram shows the differential expression of MGRN1 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0094] Figure 55 A schematic diagram shows the differential expression of SHROOM2 in plasma exosomes between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0095] Figure 56 The figure shows the differential expression of plasma exosomal protein EXOC3L4 between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0096] Figure 57 A schematic diagram shows the differential expression of FBXL6 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0097] Figure 58 A schematic diagram shows the differential expression of MYH16 in plasma exosomal proteins between individuals in the validation cohort (control group, healthy control group, AAD group, and AMI group).

[0098] Figure 59 The ROC curve of SDC1, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0099] Figure 60 The ROC curve of AOC1, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0100] Figure 61 The ROC curve of MDK, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0101] Figure 62 The ROC curve of EFCAB6, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0102] Figure 63 Shown is the ROC curve of RPRD1A, a plasma diagnostic marker for aortic dissection in the validation cohort.

[0103] Figure 64ROC curve for plasma diagnostic marker SAA1 for aortic dissection in the validation cohort population is shown.

[0104] Figure 65 ROC curve for plasma diagnostic marker SAA2 for aortic dissection in the validation cohort population is shown.

[0105] Figure 66 ROC curve for plasma diagnostic marker ABCC9 for aortic dissection in the validation cohort population is shown.

[0106] Figure 67 ROC curve for plasma diagnostic marker TRIM38 for aortic dissection in the validation cohort population is shown.

[0107] Figure 68 ROC curve for plasma diagnostic marker KANK3 for aortic dissection in the validation cohort population is shown.

[0108] Figure 69 ROC curve for plasma diagnostic marker MICAL3 for aortic dissection in the validation cohort population is shown.

[0109] Figure 70 ROC curve for plasma diagnostic marker FYCOl for aortic dissection in the validation cohort population is shown.

[0110] Figure 71 ROC curve for plasma diagnostic marker CEP295 for aortic dissection in the validation cohort population is shown.

[0111] Figure 72 ROC curve for plasma diagnostic marker MTMR11 for aortic dissection in the validation cohort population is shown.

[0112] Figure 73 ROC curve for plasma diagnostic marker SLC25A24 for aortic dissection in the validation cohort population is shown.

[0113] Figure 74 ROC curve for plasma diagnostic marker KATNA1 for aortic dissection in the validation cohort population is shown.

[0114] Figure 75 ROC curve for plasma diagnostic marker ACADL for aortic dissection in the validation cohort population is shown.

[0115] Figure 76 ROC curve for plasma diagnostic marker NCKAP5L for aortic dissection in the validation cohort population is shown.

[0116] Figure 77 ROC curve for plasma diagnostic marker KIFCl for aortic dissection in the validation cohort population is shown.

[0117] Figure 78 The ROC curve of ACY3, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0118] Figure 79 The ROC curve of GRK7, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0119] Figure 80 The ROC curve of RBM17, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0120] Figure 81 The ROC curve of LGALS8, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0121] Figure 82 The ROC curve of OVOS1, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0122] Figure 83 The ROC curve of ALMS1, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0123] Figure 84 The ROC curve of ANKRD6, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0124] Figure 85 The ROC curve of ADPRHL1, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0125] Figure 86 The ROC curve of CASP9, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0126] Figure 87 The ROC curve of SYNE3, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0127] Figure 88 The ROC curve of DEPDC7, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0128] Figure 89 The ROC curve of HIBCH, a plasma diagnostic marker for aortic dissection, in the validation cohort is shown.

[0129] Figure 90 The ROC curve of PCDHGA11, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0130] Figure 91An ROC curve is shown for the plasma diagnostic marker SLC4A5 for aortic dissection in the validation cohort.

[0131] Figure 92 An ROC curve is shown for the plasma diagnostic marker KBTBD7 for aortic dissection in the validation cohort.

[0132] Figure 93 An ROC curve is shown for the plasma diagnostic marker CIDEA for aortic dissection in the validation cohort.

[0133] Figure 94 An ROC curve is shown for the plasma diagnostic marker INTS6L for aortic dissection in the validation cohort.

[0134] Figure 95 An ROC curve is shown for the plasma diagnostic marker HDGFL3 for aortic dissection in the validation cohort.

[0135] Figure 96 An ROC curve is shown for the plasma diagnostic marker TEKT2 for aortic dissection in the validation cohort.

[0136] Figure 97 An ROC curve is shown for the plasma diagnostic marker EPAS1 for aortic dissection in the validation cohort.

[0137] Figure 98 An ROC curve is shown for the plasma diagnostic marker AMT for aortic dissection in the validation cohort.

[0138] Figure 99 An ROC curve is shown for the plasma diagnostic marker GAS8 for aortic dissection in the validation cohort.

[0139] Figure 100 An ROC curve is shown for the plasma diagnostic marker DHX33 for aortic dissection in the validation cohort.

[0140] Figure 101 An ROC curve is shown for the plasma diagnostic marker VWA3B for aortic dissection in the validation cohort.

[0141] Figure 102 An ROC curve is shown for the plasma diagnostic marker ZNF862 for aortic dissection in the validation cohort.

[0142] Figure 103 An ROC curve is shown for the plasma diagnostic marker TAF1C for aortic dissection in the validation cohort.

[0143] Figure 104 An ROC curve is shown for the plasma diagnostic marker AARS2 for aortic dissection in the validation cohort.

[0144] Figure 105 The ROC curve of PPP4R4, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0145] Figure 106 The ROC curve of MGRN1, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0146] Figure 107 The ROC curve of SHROOM2, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0147] Figure 108 Shown is the ROC curve of the plasma diagnostic marker EXOC3L4 for aortic dissection in the validation cohort.

[0148] Figure 109 The ROC curve of FBXL6, a plasma diagnostic marker for aortic dissection in the validation cohort is shown.

[0149] Figure 110 The ROC curve of MYH16, a plasma diagnostic marker for aortic dissection in the validation cohort is shown. DETAILED DESCRIPTION

[0150] This study used plasma from a cohort of patients with a clinically confirmed acute aortic dissection and a healthy control cohort to enrich exosomes using ultracentrifugation combined with asymmetric field flow (AF4). Bioinformatics analysis of the proteomic information contained in the exosomes was performed using mass spectrometry to screen and identify diagnostic markers for aortic dissection. The specificity and sensitivity of the selected markers were verified using a validation cohort.

[0151] The present invention discloses a strategy of enriching exosomes by ultracentrifugation and merging with AF4, which significantly reduces the impact of non-exosomal vesicles on the detection results.

[0152] definition

[0153] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0154] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0155] As used herein, the expression "about" is understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If the use of the term is not understood by one of ordinary skill in the art based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0156] As used herein, the term "extracellular vesicles" is a general term for cystic structures released outside cells. Their density, shape, and size are primarily determined by their contents, including proteins, lipids, enzymes, and ions. Extracellular vesicles contain various transmembrane proteins, surface membrane proteins, lipid-anchored membrane proteins, and soluble proteins within the vesicles. Examples of extracellular vesicles include, but are not limited to, exosomes, migrasomes, microvesicles, apoptotic bodies, and inducible extracellular vesicles. Extracellular vesicles participate in various life processes, including the transmission of substances and information between cells and the development and progression of diseases.

[0157] The term "exosome" used in this article is a type of extracellular vesicle, which is an extracellular vesicle surrounded by a phospholipid bilayer and encapsulates a variety of biological molecules (including nucleic acids, proteins, and lipids). Exosomes are released by the inward indentation of endosomes to form multivesicular bodies (MVBs), which are then fused with the cell membrane and released outside the cell through exocytosis. Exosomes can not only transmit mRNA and miRNA laterally, but also have antigen presentation, immune activation, and immunosuppression activities, and can express markers such as Alix, CD63, CD81, CD9, LAMP1, and TSG101.

[0158] The term "aortic dissection (AD)" as used herein refers to a serious medical condition in which the inner layer of the aorta is torn. Aortic dissection is a tear in the aorta's intima and media due to various reasons. The aortic intima and media separate, blood flows in, and the aortic lumen is divided into a true lumen and a false lumen. AD can be divided into two types, A and B. Type A is when the dissection involves the ascending aorta; type B is when the dissection only involves the descending thoracic aorta and its distal end. The AD staging method is: the onset time is ≤14 days as the acute stage, the onset time is 15 to 90 days as the subacute stage, and the onset time is >90 days as the chronic stage. Currently, the recognized acute stage AD is the onset time of less than 2 weeks.

[0159] The term "myocardial infarction (MI)" as used herein refers to acute myocardial injury, in which the serum cardiac troponin (cTn) value is increased and / or decreased, and at least once exceeds the 99% upper reference value (URL). Clinical evidence of myocardial infarction includes: (1) symptoms of acute myocardial ischemia; (2) new ischemic electrocardiographic changes; (3) the occurrence of pathological Q waves; (4) imaging evidence of new loss of viable myocardium or new segmental wall motion abnormalities; (5) coronary artery thrombosis confirmed by coronary angiography, intracoronary imaging or autopsy. Myocardial infarction is usually divided into five types. Type 1: Acute rupture or erosion of coronary atherosclerotic plaques, platelet activation, and subsequent coronary artery thrombotic obstruction, causing myocardial ischemia, damage or necrosis. Myocardial damage and at least one clinical evidence of myocardial ischemia are required. Type 2: Not related to acute rupture or erosion of coronary atherosclerotic plaques or thrombosis, but caused by an imbalance between myocardial oxygen supply and demand. Type 3: Refers to cardiac death accompanied by symptoms of myocardial ischemia and new ischemic electrocardiographic changes or ventricular fibrillation (VF), but death occurs before a blood sample for biomarkers is obtained or before a clear increase in cardiac biomarkers is confirmed, and autopsy confirms myocardial infarction. Type 4: Includes myocardial infarction related to percutaneous coronary intervention (PCI) (type 4a), myocardial infarction related to coronary stent or support thrombosis (type 4b), and myocardial infarction related to restenosis (type 4c). Type 5: Myocardial infarction related to coronary artery bypass grafting (CABG). Myocardial infarction that occurs again within 28 days of the first myocardial infarction is called re-infarction, and recurrent myocardial infarction after 28 days is called recurrent myocardial infarction.

[0160] The term "field flow" as used in this article refers to a technology that uses the interaction of an external field (such as a flow field, temperature field, electric field, etc.) with sample molecules or particles to cause components of different sizes or properties to produce different migration speeds in the separation channel, thereby achieving separation. Asymmetric field flow (AF4) is a type of flow field flow, which is characterized by the asymmetric geometric structure of the separation channel. The upper wall of the channel is a porous membrane, and the lower wall is a solid surface. The flow field is applied through the upper wall to form a field flow perpendicular to the flow direction, so that the sample molecules or particles are separated according to size. This technology has the advantage of high-resolution separation of nanoparticles in a large size range and can be used to separate different extracellular vesicle subpopulations.

[0161] The term "mass spectrometry (MS)" as used herein refers to a technique for identifying and / or quantifying molecules in a sample. MS includes ionizing the molecules in the sample to form charged molecules; separating the charged molecules according to their mass-to-charge ratio; and detecting the charged molecules. MS allows for qualitative and quantitative detection of molecules in a sample. The molecules can be ionized and detected by any suitable method known to those skilled in the art. In some exemplary embodiments, the mass spectrometry includes time-of-flight mass spectrometry (TOF-MS), orbital trap mass spectrometry (Orbitrap MS), quadrupole mass spectrometry (QMS), ion trap mass spectrometry (ITMS), magnetic sector mass spectrometry, FT-ICR mass spectrometry, ion mobility spectrometry-mass spectrometry (IMS-MS), etc.

[0162] As used herein, the term "biomarker" or "marker" refers to a biochemical marker that can indicate changes or potential changes in the structure or function of a system, organ, tissue, cell, or subcellular structure. Biomarkers have a wide range of applications. Biomarkers can be used to diagnose and stage diseases, or to evaluate the safety and efficacy of new drugs or therapies in a target population.

[0163] As used herein, the term "subject" refers to any animal, mammal, or human. The subject has, may have, or is suspected of having one or more diseases. In some embodiments, the subject is a human.

[0164] The term "False Discovery Rate" (FDR) used in this article is a necessary parameter used in general expression profile analysis, which is the expected value of the ratio of the number of false rejections (rejection of true (null) hypotheses) to the number of all rejected null hypotheses.

[0165] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the examples. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. The actual protection scope of the present invention is set forth in the claims. In the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following examples, without mentioning the source, are all commercially available on the market or obtained by conventional methods known to those skilled in the art.

[0166] Example

[0167] Materials and methods

[0168] Study and validation cohort plasma samples

[0169] Blood samples were collected with ethical approval from the People's Hospital of Xinjiang Uygur Autonomous Region (No. KY2023042008), and informed consent was obtained from all blood donors.

[0170] The study cohort included 50 healthy blood donors (aged 34-85 years, 42 males and 8 females) and 50 patients with acute type A aortic dissection (aged 29-69 years, 40 males and 10 females), whose blood was collected on a fasting basis at the People's Hospital of Xinjiang Uygur Autonomous Region.

[0171] The validation cohort included 47 healthy blood donors (aged 35-77 years, 20 males and 27 females), 54 patients with acute type A aortic dissection (aged 42-71 years, 46 males and 8 females), and 106 patients with type 1 acute myocardial infarction (aged 31-85 years, 87 males and 19 females). Fasting blood was collected at the People's Hospital of Xinjiang Uygur Autonomous Region.

[0172] Blood samples from patients with acute type A aortic dissection and acute myocardial infarction in both the study and validation cohorts were collected in the ward before surgery. Peripheral blood was collected using a 21G butterfly needle (10 mL / person) into BD Vacutainer tubes (Cat. #367863, BD Biosciences, USA, EDTA anticoagulant). Blood samples were stored at 4°C and centrifuged at 3000 × g for 10 minutes at room temperature within 2 hours of collection to obtain plasma, which was aliquoted into 0.5 mL cryovials (Cat. No. BP-15405, Biosidis (Hangzhou) Technology Co., Ltd.) and stored at −80°C.

[0173] Enrichment of plasma exosomes by ultracentrifugation combined with asymmetric field flow

[0174] This study primarily used an Agilent quaternary pump and autosampler (Agilent, USA) coupled with an asymmetric field-flow separation and multi-angle light scattering system (AF4-MALS, Wyatt Technology, USA) to enrich plasma exosomes. The field-flow system was assembled using a 10 kDa regenerated cellulose membrane (RC10 membrane) within a 152 mm separation channel. 400 μL of plasma was diluted to 1.25 mL with 1× PBS and loaded into a TLS-55 rotor. The sample was centrifuged at 150,000 × g for 1.5 h at 4°C (OptimaMAX-XP, Beckman Coulter, USA) to obtain a mixed system containing exosomes. The mixed system was thoroughly resuspended in 100 μL of PBS and injected into the inlet channel of the AF4-MALS. The sonicated PBS was used as the equilibration and elution buffer for injection into the AF4-MALS asymmetric field-flow separation system. Detailed flow rates and parameters of the field-flow system used in this study are shown in Table 1. ASTRA software (Wyatt Technology, USA) was used to record and process data from the UV+RI+MALS pipeline. Four major peaks, P1, P2, P3, and P4, were observed. Fractions corresponding to each peak were transferred to a 100 kDa ultrafiltration tube (Merck Millipore) and centrifuged at 3000 × g at 4°C to a 100 μL volume for subsequent analysis.

[0175] Table 1: AF4-MALS field flow parameters

[0176]

[0177] NTA characterization of enriched plasma exosomes

[0178] Nanoparticle tracking analysis (NTA) was performed using a Nanosight NS300 with a green laser (Malvern Panalytical, UK) to examine the size distribution and particle density of exosomes. Briefly, enriched exosome samples were exchanged into PBS buffer and measured at 23.5°C. An sCMOS camera with a camera level of 13 was used, capturing a total of 749 frames. All other settings were set to automatic. Image data were analyzed using NanoSight NTA software (version 3.4). Three replicates were performed for each sample.

[0179] TEM characterization of enriched plasma exosomes

[0180] Exosome staining was performed using a transmission electron microscope (TEM). 3 μl of the liquid sample was applied to a precleaned carbon-coated circular-hole copper grid (Cat. #BZ110223b, Beijing Zhongjing Keyi Technology Co., Ltd.) and incubated at room temperature for 1 minute. The liquid was then removed with filter paper (Cat. #1004110, Whatman) and the grid was stained with 2% uranyl acetate (Cat. #22451, Electron Microscopy Sciences) for 1 minute. The uranyl acetate was removed with filter paper, and the grid was allowed to dry completely before imaging. Images were taken using a Tecnai Spirit electron microscope (FEI, Hillsboro, OR, USA) operating at 120 kV and a magnification of 36,000×.

[0181] Identification of plasma exosome proteins by mass spectrometry

[0182] The collected plasma exosome samples were dried using a refrigerated centrifugal concentrator (CV600, Beijing Jim Technology Co., Ltd.), lysed using urea lysis buffer (8 M urea, 50 mM ammonium bicarbonate), and sonicated (Weimi, Shanghai, China). Protein concentration was then determined using a BCA assay kit (Thermo Fisher Scientific). Samples were then treated with 10 mM dithiothreitol (reduction at room temperature for 30 minutes) and 20 mM iodoacetamide (alkylation at room temperature for 30 minutes in the dark). The protein mixture was then digested with endoproteinase Lys-C (enzyme / protein ratio 1:100, w / w) at 37°C for 4 hours and then with trypsin (enzyme / protein ratio 1:50, w / w) at 37°C for 12 hours. The resulting peptides were quenched with 10% trifluoroacetic acid to a final concentration of 1% and desalted using a custom-made C18 StageTip column. The resulting peptides were used for mass spectrometry analysis.

[0183] All nano-LC-MS / MS experiments were performed on an Orbitrap Exploris 480 (Thermo Scientific) equipped with an Easyn-LC 1200 high-performance liquid chromatography system (Thermo Scientific). Peptides were loaded onto a 100 μm id × 2 cm quartz trap column packed with reversed-phase silica (resil-pur C18 AQ, 5 μm, Dr. Maisch GmbH) and then separated on a 75 μm id × 25 cm C18 column packed with reversed-phase silica (resil-pur C18 AQ, 1.9 μm, Dr. Maisch GmbH). Bound peptides were eluted with a 103-minute linear gradient. The eluents consisted of solvent A and solvent B. Solvent A consisted of 0.1% formic acid in water, and solvent B consisted of 80% acetonitrile and 0.1% formic acid. The elution gradient was 4-11% B in 4 min; 11-21% B in 28 min; 21-30% B in 29 min; 30-42% B in 27 min; 42-99% B in 5 min; and 99% B in 10 min at a flow rate of 300 nl / min. Mass spectral data were acquired at a high resolution of 120,000 (m / z 200) over a mass range of 400-1210 m / z. The target value was 4.00E+05, and the maximum injection time was 50 ms. Following a full scan, there were 40 windows with an isolation width of 16 m / z for fragmentation with an ion routing multipole, and the HCD normalized collision energy was 30%. MS / MS spectra were acquired at a resolution of 30,000 over a mass range of 200-2000 m / z. The target value was 4.00E+05, and the maximum injection time was 50 ms. The nanoelectrospray ion source was set up with a spray voltage of 2.0 kV, no sheath gas flow, and a heated capillary temperature of 320 °C.

[0184] Raw DIA data from the Orbitrap Exploris 480 were analyzed using Spectronaut (Biognosys, version 14). Protein identification and quantification were performed using the “Direct-DIA” mode. The UniProt human proteome database was used for the search. The most important search parameters were set to default settings: trypsin was selected as the enzyme, and two missed cleavages were allowed; the mass tolerance for MS1 and MS2 was set to a correction factor of 1; cysteine ​​carbamidomethylation was specified as a fixed modification variable; and oxidation and acetylation of the protein N-terminal amino acid were selected as variable modifications. An FDR < 1% was used for peptide spectrum matching (PSM), peptide, and protein identification. The data were filtered by Qvalue, and “Global Normalization” was set to “Median” with cross-run normalization enabled to obtain the protein expression signal intensity (centered intensity).

[0185] Statistical analysis methods

[0186] Bioinformatics analysis was performed using scripts based on the OpenSource R (version 4.4.1) toolkit. Key software packages included limma (Matthew E. Ritchie, et al., limma powers differential expression analyses for RNA-sequencing and microarray studies, Nucleic Acids Research, Volume 43, Issue 7, 20 April 2015, e47) for quality control and differential analysis of proteomic data. A multi-sample one-way ANOVA test was used to compare differential expression of plasma exosome proteomes between healthy controls and patients with AAD, generating a volcano plot.

[0187] Multi-group sample one-way ANOVA test was used to compare the Log2 values ​​of the protein abundance (centered intensity) of each biomarker between healthy controls and AAD diseases, or between different diseases.

[0188] Diagnostic performance of each marker in differentiating AAD from other diseases was assessed using receiver operating characteristic (ROC) analysis. Area under the ROC curve and Youden index, sensitivity, specificity were calculated. Non-parametric ROC analysis was performed using the Log2 values of each biomarker protein abundance centered intensity as a continuous variable, respectively, and D-dimer, troponin t (C-TnT), creatine kinase isoenzyme MB (CK-MB). Sensitivity and specificity ROC curve analysis and plotting of each protein marker were performed using Graphpad Prism 9.5. All P values are two-tailed and adjusted for multiple testing. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001. Two-sided P values and 95% confidence intervals were used.

[0189] Example 1, Screening of plasma exosome diagnostic markers for aortic dissection

[0190] According to the procedure shown in Figure 1 , the study cohort was used to screen plasma exosome diagnostic markers for aortic dissection.

[0191] Plasma exosomes were combined by ultracentrifugation. Four main peaks, P1, P2, P3, P4 were observed. By characterizing the components corresponding to P1, P2, P3, P4 peaks by NTA and TEM, it was found that exosomes mainly exist in the components corresponding to P4 peak.

[0192] Figure 2 Exemplary electron microscope observation and NTA detection results of P4 peak components of plasma exosome-enriched exosomes from healthy people and acute type A aortic dissection patients are shown. As shown in Figure 2 , the results found that the size of the vesicles in P4 peak was mainly concentrated in 50-200 nm, and it was clear that the P4 peak enrichment product was exosomes.

[0193] For P4 components, exosome proteomic difference analysis was performed on the two groups of people according to mass spectrometry identification method and statistical analysis method, and the results are shown in Figure 3 .

[0194] Figure 3The results showed that compared with the healthy control group, there were proteins with significantly down-regulated, up-regulated, and no significant changes in expression in patients with acute type A aortic dissection (AAD). The P values ​​of the differences were ranked by the empirical Bayesian moderated t-test based on the log2 values ​​of the centered intensity of the AAD group and the centered intensity of the Ctrl group. 51 proteins with a P value less than 0.05 and a Fold Change (the ratio of the centered intensity of the AAD group to the centered intensity of the Ctrl group) greater than 2 were selected as plasma exosome diagnostic markers: SDC1, EFCAB6, AOC1, RPRD1A, FYCO1, CEP295, MTMR11, SLC25A24, KATNA1, ACADL, NCKAP5L, KIFC1, ACY3, GRK7, RBM17, LGALS8, OVOS1, MICAL3, ABCC9, ALMS1, ANKRD6, ADPRHL1, and CA SP9, SYNE3, DEPDC7, HIBCH, PCDHGA11, TRIM38, MDK, SLC4A5, KBTBD7, CIDEA, SAA2, INTS6L, HDGFL3, TEKT2, EP AS1, AMT, GAS8, DHX33, VWA3B, ZNF862, TAF1C, AARS2, PPP4R4, MGRN1, SHROOM2, EXOC3L4, FBXL6, MYH16, KANK3.

[0195] Example 2: Validation of plasma exosome diagnostic markers for aortic dissection

[0196] This embodiment adopts the verification queue, according to Figure 4 The process shown verifies the accuracy and sensitivity of the 51 proteins screened in Example 1 in the diagnosis of AAD.

[0197] By ultracentrifugation combined with asymmetric field flow, plasma exosomes from 47 healthy subjects, 54 patients with acute type A aortic dissection, and 106 patients with acute myocardial infarction in the validation cohort were enriched, and the P4 peak component was collected for subsequent research.

[0198] Figure 5 and Figure 6 The electron microscopy images and NTA detection results of the P4 peak component enriched in exosomes from the plasma of healthy people and patients with acute type A aortic dissection are shown as examples. Figure 5-6As shown, the results showed that vesicles (exosomes) of 50-200 nm were also mainly concentrated in the P4 peak enrichment product, so the P4 peak enrichment product was used for subsequent detection.

[0199] Using mass spectrometry, we performed exosome proteomic analysis on the components corresponding to the P4 peak in healthy individuals, patients with acute type A aortic dissection (AAD), and patients with acute myocardial infarction (AMI). We obtained the centered intensity of the expression signals of the 51 proteins selected in Example 1 and performed statistical analysis. In addition, we detected the acute phase-related protein SAA1 located in the exosomes as a positive control. The results are shown in Figure 2. Figure 7-58 shown.

[0200] In order to further verify the sensitivity and specificity of the protein markers screened in the present disclosure, the ROC curve was used to analyze the diagnostic performance of each marker in distinguishing AAD from other diseases.

[0201] The results were compared with the existing AAD markers D-dimer, troponin-t, and creatine kinase isoenzyme MB. The samples were from the validation cohort of 47 healthy individuals, 54 patients with acute type A aortic dissection, and 106 patients with type 1 acute myocardial infarction.

[0202] D-dimer was detected using a D-dimer assay kit (immunoturbidimetric method, SIEMENS) according to the kit instructions. COAG 360 (SIEMENS) was used to measure the concentration at 540 nm, and the concentration unit of the test result was mg / L.

[0203] Troponin-t was detected using a troponin-t chemiluminescence immunoassay kit (Genentech) according to the kit instructions. Getein 1600 (Genentech) was used for detection, and the test results are expressed in mmol / L.

[0204] Creatine kinase isoenzyme MB was measured using a Creatine Kinase Isoenzyme MB Assay Kit (immunosuppression method, Hitachi LST) according to the kit instructions. The assay was performed using LST 008AS (Hitachi), and the concentration unit of the assay result is mmol / L.

[0205] The results are shown in Figure 59-110 middle.

[0206] The sensitivity and specificity of the plasma exosome diagnostic markers screened in Example 8 for identifying aortic dissection were verified using ROC curves when the Youden index was highest. The results are shown in Tables 2 and 3.

[0207] Table 2: Sensitivity and specificity of each marker compared to the negative control validation cohort

[0208]

[0209]

[0210] Note: Sensitivity (%) and specificity (%) are the values ​​when the Youden Index of the protein is the highest.

[0211] As shown in Table 2, the plasma exosome protein markers screened in Example 1 were used to determine the risk of aortic dissection, and they were able to distinguish between patients with aortic dissection and healthy controls.

[0212] Table 3: Sensitivity and specificity of each marker relative to the acute myocardial infarction validation cohort

[0213]

[0214]

[0215] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of a biomarker detection reagent in the preparation of a kit for evaluating, diagnosing, screening and / or monitoring aortic dissection, wherein the biomarker comprises one or more of the following: SDC1, EFCAB6, AOC1, RPRD1A, FYCO1, CEP295, MTMR11, SLC25A24, KATNA1, ACADL, NCKAP5L, KIFC1, ACY3, GRK7, RBM17, LGALS8, OVOS1, MICAL3, ABCC9, ALMS1, ANKRD6, ADPRHL1, CASP9, SYNE3, DEPDC7, HIBCH, PCDHGA11, TRIM38, MDK, SLC4A5, KBTBD7, CIDEA, SAA2, INTS6L, HDGFL3, TEKT2, EPAS1, AMT, GAS8, DHX33, VWA3B, ZNF862, TAF1C, AARS2, PPP4R4, MGRN1, SHROOM2, EXOC3L4, FBXL6, MYH16, KANK3 and SAA1.

2. The use according to claim 1, characterized in that The biomarkers include one or more of the following: SDC1, EFCAB6, AOC1, RPRD1A, FYCO1, CEP295, SLC25A24, KATNA1, ACADL, ACY3, RBM17, LGALS8, ADPRHL1, CASP9, SYNE3, HIBCH, PCDHGA11, MDK, KBTBD7, SAA2, AARS2, PPP4R4, and SAA1.

3. The use according to claim 1, characterized in that The biomarkers include one or more of the following: SDC1, EFCAB6, AOC1, FYCO1, CEP295, PCDHGA11, MDK, KBTBD7 and SAA1.

4. The use according to claim 1, characterized in that The detection reagent is used to detect the expression level of the marker in an extracellular vesicle sample from a subject; Preferably, the extracellular vesicles are selected from one or more of exosomes, migrasomes, microvesicles, apoptotic bodies, and inducible extracellular vesicles, more preferably exosomes; Preferably, the exosomes are derived from body fluids; Preferably, the body fluid comprises one or more of the following: whole blood, serum, plasma, urine, alveolar lavage fluid, cerebrospinal fluid, serous fluid, pleural effusion, peritoneal lavage fluid, peritoneal fluid, and processed products thereof.

5. The use according to claim 1, characterized in that The aortic dissection includes type A and type B aortic dissection, or includes acute aortic dissection, subacute aortic dissection and chronic aortic dissection.

6. The use according to claim 1, characterized in that The aortic dissection is type A acute aortic dissection.

7. The use according to claim 1, characterized in that The evaluation, diagnosis, screening and / or monitoring may include the following steps: (1) detecting the presence and / or level of said biomarker from a subject, and (2) Determining whether the subject has a risk of aortic dissection based on the presence and / or level of the biomarker.

8. The use according to claim 7, characterized in that Prior to step (1), the method further comprises the step of isolating and enriching the extracellular vesicles of the subject; Preferably, the method for isolating and enriching the extracellular vesicles of the subject comprises one or more of the following: ultracentrifugation, size exclusion, field flow separation, microfluidics, ultrafiltration and polymer precipitation; Preferably, the separation and enrichment of extracellular vesicles from the subject is achieved by using ultracentrifugation and / or asymmetric field flow; Preferably, the asymmetric field flow is combined with a multi-angle light scattering system to sort extracellular vesicles.

9. The use according to claim 7, characterized in that The detection in step (1) comprises one or more of the following: mass spectrometry, chromatography, immunoassay, PCR, transcriptome sequencing; Preferably, the detection in step (1) comprises one or more of the following: time-of-flight mass spectrometry, orbitrap mass spectrometry, quadrupole mass spectrometry, ion trap mass spectrometry, magnetic sector mass spectrometry, FT-ICR mass spectrometry, ion mobility spectrometry-mass spectrometry, liquid chromatography, solid phase chromatography, enzyme-linked immunosorbent assay, protein blotting, dot blot or immunostaining, lateral flow assay, RT-qPCR and transcriptome sequencing.

10. A device for assessing, diagnosing, screening, and / or monitoring a subject's risk of aortic dissection using a biomarker, the device comprising: a biomarker detection module for determining the presence and / or level of said biomarker from a subject; and an aortic dissection risk assessment module, for assessing the risk of aortic dissection in a subject based on the presence and / or level of the biomarker; The biomarkers include one or more of the following: SDC1, EFCAB6, AOC1, RPRD1A, FYCO1, CEP295, MTMR11, SLC25A24, KATNA1, ACADL, NCKAP5L, KIFC1, ACY3, GRK7, RBM17, LGALS8, OVOS1, MICAL3, ABCC9, ALMS1, ANKRD6, ADPRHL1, CASP9, SYNE3, D EPDC7, HIBCH, PCDHGA11, TRIM38, MDK, SLC4A5, KBTBD7, CIDEA, SAA2, INTS6L, HDGFL3, TEKT2, EPAS1, AMT, GAS8, DHX33, VWA3B, ZNF862, TAF1C, AARS2, PPP4R4, MGRN1, SHROOM2, EXOC3L4, FBXL6, MYH16, KANK3 and SAA1.

Images