A diagnostic kit for unstable carotid atherosclerotic plaque

Abstract

The present invention is applicable to the field of biomedical technology and provides a diagnostic kit for unstable carotid atherosclerotic plaques. The present invention aims to collect urine samples from patients with carotid atherosclerotic plaques, perform proteomic mass spectrometry sequencing, and conduct comprehensive analysis in combination with transcriptomic and proteomic data of tissue samples. In this process, we successfully discovered two reliable diagnostic biomarkers: ADAMDEC1 and IQGAP2. These two biomarkers can not only be used to prepare diagnostic kits to achieve accurate diagnosis of unstable carotid atherosclerotic plaques, but also provide strong support for the judgment of disease prognosis. In addition, the discovery of the present invention has also opened up a new method for the diagnosis of carotid plaque stability and provided a more reliable basis for clinical prognosis evaluation and the identification of new therapeutic targets.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a diagnostic kit for unstable carotid atherosclerotic plaques. Background Art

[0002] Carotid atherosclerotic plaques refer to the formation of fibrofatty lesions in the carotid artery wall. Carotid atherosclerotic plaques may lead to numerous conditions, including cerebral ischemia and ischemic stroke. Ischemic stroke is associated with extremely high mortality and poor prognosis, with approximately 20% of ischemic strokes caused by large vessel atherosclerosis. Carotid atherosclerotic plaques can be categorized as stable or unstable based on their morphology. Unstable plaques, characterized by inflammation, intraplaque hemorrhage, and high lipid content, are more susceptible to rupture, potentially leading to serious cerebrovascular disease. Therefore, early assessment of plaque stability is crucial for clinical decision-making and patient prognosis assessment.

[0003] Although ultrasound and imaging techniques are currently available for assessing carotid atherosclerotic plaques, ultrasound examinations suffer from poor accuracy and MRI examinations are expensive. The search for new, more accurate biomarkers remains a research hotspot and a challenge. Therefore, omics sequencing technologies, such as transcriptomics, proteomics, and metabolomics, based on histological and pathological classification of clinical samples, have been used to identify biomarkers that can accurately identify the stability of carotid atherosclerotic plaques in patients. However, most of these studies are single-omics studies and have not yet been widely applied in clinical practice. Current proteomic research on identifying carotid atherosclerotic plaque stability primarily focuses on sequencing plaque tissue samples; no research has examined urine samples. Only one study measured the urine proteome of 35 patients with asymptomatic carotid stenosis and identified novel potential urine biomarkers for noninvasive early screening and risk stratification of carotid stenosis. However, these biomarkers do not directly reflect the nature of the plaque and fail to address further questions. To address this issue, the present invention proposes a diagnostic kit for unstable carotid atherosclerotic plaques. Summary of the Invention

[0004] The purpose of the present invention is to provide a diagnostic kit for unstable carotid atherosclerotic plaques, aiming to solve the problems raised in the above background technology.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A use of a biomarker in preparing a diagnostic product for unstable carotid atherosclerotic plaque, wherein the biomarker is selected from one or both of ADAMDEC1 and IQGAP2.

[0007] A reagent for detecting biomarker levels in the preparation of a diagnostic product for unstable carotid atherosclerotic plaques, wherein the biomarker is selected from one or both of ADAMDEC1 and IQGAP2.

[0008] Further, the biomarker level is the protein level of the biomarker.

[0009] Furthermore, the reagent is used to measure the level of biomarkers in biological samples by ELISA.

[0010] Furthermore, the expression level of the biomarker is significantly increased in unstable carotid atherosclerotic plaques.

[0011] A diagnostic kit for unstable carotid atherosclerotic plaques comprises one or both of a reagent for detecting ADAMDEC1 levels and a reagent for detecting IQGAP2 levels.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention has discovered two reliable diagnostic biomarkers: ADAMDEC1 and IQGAP2. These two biomarkers can not only be used to develop diagnostic kits for the accurate diagnosis of unstable carotid atherosclerotic plaques, but also provide strong support for disease prognosis. Furthermore, this discovery has opened up new methods for diagnosing carotid plaque stability and provided a more reliable basis for clinical prognosis assessment and the identification of new therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The proteomic data of 179 urine samples were analyzed for differential expression and 230 differentially expressed proteins were obtained.

[0015] Figure 2 Figure 2 shows the differential expression of ADAMDEC1 and IQGAP2 in transcriptomics and proteomics of carotid atherosclerotic plaques. ** indicates P < 0.01, *** indicates P < 0.001.

[0016] Figure 3 2 is the ROC curve of ADAMDEC1 and IQGAP2 in Example 3.

[0017] Figure 4 The expression levels of ADAMDEC1 and IQGAP2 molecules were increased in unstable carotid atherosclerotic plaques, and the differences were statistically significant; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0018] Figure 52 is the ROC curve of ADAMDEC1 and IQGAP2 in Example 4. DETAILED DESCRIPTION

[0019] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0020] The present invention involves collecting urine samples from people with carotid atherosclerotic plaques for proteomic mass spectrometry sequencing, and combining them with transcriptomic and proteomic data of tissue samples for comprehensive analysis to find reliable diagnostic biomarkers.

[0021] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0022] Example 1: Collect urine samples for proteomic mass spectrometry analysis;

[0023] 1.1 Experimental methods;

[0024] Urine samples for proteomic analysis were obtained from consecutive patients with carotid artery stenosis who underwent carotid endarterectomy (CEA) surgery at the First Bethune Hospital of Jilin University between January 2020 and August 2022. Early morning midstream urine samples were collected from patients and immediately centrifuged to remove cellular components and debris, and the supernatant was stored at -80°C for subsequent analysis. Carotid artery specimens obtained from patients with carotid artery stenosis after surgery were histologically analyzed and classified according to American Heart Association (AHA) criteria.

[0025] Each sample component was analyzed using the same liquid chromatography-tandem mass spectrometry (LC-MS / MS) parameters. Mass spectrometry data were searched against the SwissProt human proteomics database (www.uniprot.org, containing 20,121 sequences) using Mascot software (Matrix Science, Inc., version 2.5.01). Results were filtered using a Q-value cutoff of 0.01 (corresponding to an FDR of 1%) to exclude outliers. Protein intensities were calculated by summing the intensities of the individual peptides. Proteins exceeding 80% of the samples in each group were retained for further examination.

[0026] 1.2 Experimental results;

[0027] A total of 3828 and 4109 proteins were detected in the stable and unstable carotid atherosclerotic plaque groups, respectively, with an average of 2089 and 2403 proteins detected, respectively. 230 differentially expressed proteins were identified by proteomic analysis of 179 urine samples ( Figure 1 ).

[0028] Example 2: Comprehensive analysis of multi-omics data;

[0029] 2.1 Experimental method;

[0030] The transcriptomic data of carotid atherosclerotic plaques was sourced from the GEO database (http: / / www.ncbi.nlm.nih.gov / geo / ). The tissue proteomic data of this disease was sourced from the main text or supplementary materials of literature retrieved from PubMed, Web of Science, Embase, etc.

[0031] 2.2 Experimental results;

[0032] In the transcriptomics and proteomics of carotid atherosclerotic plaques, both ADAMDEC1 and IQGAP2 showed differential expression (see Figure 2 ).

[0033] Example 3: Diagnostic efficacy of ADAMDEC1 and IQGAP2;

[0034] 3.1 Experimental method;

[0035] The diagnostic efficacy of ADAMDEC1 and IQGAP2 was demonstrated using ROC curve analysis.

[0036] 3.2 Experimental results;

[0037] When evaluating diagnostic efficacy, the AUC value (area under the curve) is usually used for measurement. According to the conventional judgment criteria, 0.5 < AUC < 0.7 indicates a certain classification ability but average effect; 0.7 < AUC < 0.9 indicates a good classification ability. The experimental results showed that the AUC value of ADAMDEC1 was 0.841 and the AUC value of IQGAP2 was 0.881 (see Figure 3 ), indicating that both have good diagnostic value.

[0038] Example 4: Detection of ADAMDEC1 and IQGAP molecules in clinical patients;

[0039] 4.1 Experimental method;

[0040] Urine specimens of 93 patients (62 unstable carotid atherosclerotic plaques and 31 stable carotid atherosclerotic plaques) were collected from the First Bethune Hospital of Jilin University, and protein verification was performed using ELISA experiments.

[0041] Protein in urine was measured using commercially available ELISA kits according to the manufacturer's instructions (Jiangsu Meimian Industrial Co., Ltd.). The product numbers are Human ADAMDEC1 Protein (ADAMDEC1) ELISA Kit (MM-63977H1) and Human IQ Motif-Containing GTPase Activating Protein 2 (IQGAP2) ELISA Kit (MM-63972H1).

[0042] 4.2 Experimental Results

[0043] The experimental results showed that the expression levels of ADAMDEC1 and IQGAP2 molecules were significantly increased in unstable carotid atherosclerotic plaques (see Figure 4 ).

[0044] To further verify the diagnostic capabilities of these two molecules, we calculated their AUC values. The results showed that the AUC value of ADAMDEC1 was 0.878 and the AUC value of IQGAP2 was 0.79 (see Figure 5 ), which indicates that both have good classification ability in clinical detection.

[0045] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Use of a biomarker in the preparation of a diagnostic product for unstable carotid atherosclerotic plaque, characterized in that: The biomarker is selected from one or both of ADAMDEC1 and IQGAP2.

2. Use of a reagent for detecting biomarker levels in the preparation of a diagnostic product for unstable carotid atherosclerotic plaques, characterized in that: The biomarker is selected from one or both of ADAMDEC1 and IQGAP2.

3. The use according to claim 2, characterized in that The biomarker level is the protein level of the biomarker.

4. The use according to claim 3, characterized in that The reagents are used to measure the levels of biomarkers in biological samples by ELISA.

5. The use according to claim 4, characterized in that The expression level of the biomarker is significantly increased in unstable carotid atherosclerotic plaques.

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