A CD5 biomarker for the diagnosis of diabetic foot and its application

Abstract

The present invention discloses a CD5 biomarker for diagnosing diabetic foot and its application, belonging to the field of medical diagnosis technology. The present invention provides a highly sensitive and specific method for diagnosing diabetic foot. Through Olink Proteomics technology, the expression level of CD5 in diabetic foot patients can be accurately detected, providing a potential large-scale screening indicator for clinicians, improving the accuracy of early diagnosis, and simplifying the operation process.

Description

Technical Field

[0001] The present invention relates to the technical field of medical diagnosis, and in particular to a CD5 biomarker for diagnosing diabetic foot and an application thereof. Background Art

[0002] Diabetic foot is a common and serious complication of diabetes, characterized by lower extremity ulcers and infections. Traditional diagnostic methods rely mainly on clinical symptoms and imaging examinations, but these methods have low sensitivity for early diagnosis and accurate assessment of disease progression. In recent years, studies have found that inflammation plays an important role in the occurrence and development of diabetic foot, but the specific mechanism is not yet fully understood.

[0003] The existing technologies for diagnosing diabetic foot often have low sensitivity and specificity, and cannot accurately identify early diabetic foot. In addition, the operation is complicated and time-consuming, making it difficult to apply to large-scale clinical screening and unable to achieve high-throughput detection, which limits the possibility of combined application of multiple markers. Summary of the invention

[0004] The purpose of the present invention is to provide a CD5 biomarker for the diagnosis of diabetic foot and its application to solve the problems existing in the above-mentioned prior art. The present invention aims to solve the problems of low sensitivity and specificity, complex operation, and inability to achieve high-throughput detection in the prior art, and provides a new CD5 biomarker for the diagnosis of diabetic foot and its application method, aiming to improve the accuracy of early diagnosis and provide a large-scale screening indicator for clinical practice.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the use of CD5 as a biomarker in the preparation of a product for diagnosing or monitoring diabetic foot.

[0007] The second technical solution of the present invention is a product for diagnosing or monitoring diabetic foot, with CD5 as the detection target.

[0008] Based on the above technical solution, the present invention has the following technical effects:

[0009] The present invention provides a highly sensitive and specific method for diagnosing diabetic foot. Through Olink Proteomics technology, the expression level of CD5 in diabetic foot patients can be accurately detected, providing a potential large-scale screening indicator for the clinic, improving the accuracy of early diagnosis, and simplifying the operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 This is a box plot comparing CD5 between the diabetic foot group and the diabetes group;

[0012] Figure 2 The volcano plot is a comparison between the diabetic foot group and the diabetes group;

[0013] Figure 3 This is the CD5 ROC analysis chart;

[0014] Figure 4 This is the result diagram of GO enrichment analysis of differential factors;

[0015] Figure 5 This is the result diagram of KEGG enrichment analysis of differential factors. DETAILED DESCRIPTION

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0018] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0019] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0022] The embodiments of the present invention provide the use of CD5 as a biomarker in the preparation of a product for diagnosing or monitoring diabetic foot.

[0023] In the prior art, CD5 is mainly used for auxiliary diagnosis of mature B-cell tumors. For example, in the patent named "Auxiliary diagnosis prediction method, device, equipment and medium for mature B-cell tumors", CD5 is used for tumor detection, and its detection methods include flow cytometry and related gene mutation detection. However, these methods have the following shortcomings compared with olink technology: flow cytometry is complicated to operate, sample processing steps are cumbersome, and time cost is high. The throughput is limited, and only a limited number of samples can be analyzed each time, which is not suitable for large-scale screening. Gene mutation detection: The operation is complicated and time-consuming, and multiple markers cannot be detected at the same time, and the diagnostic efficiency is low.

[0024] In some specific embodiments, the PEA technique is used to quantify the expression level of CD5.

[0025] In some specific embodiments, the method for diagnosing or monitoring diabetic foot is: quantitatively detecting CD5 in the test sample and comparing it with the control sample or the reference sample. If the CD5 level in the test sample is significantly increased, it indicates that the patient suffers from diabetic foot.

[0026] In some specific embodiments, the product includes a reagent, a kit, a chip, a test paper or a high-throughput sequencing platform.

[0027] The embodiment of the present invention also provides a product for diagnosing or monitoring diabetic foot, with CD5 as the detection target.

[0028] In some specific embodiments, the product includes a reagent, a kit, a chip, a test paper or a high-throughput sequencing platform.

[0029] Example 1

[0030] The present invention adopts Olink Proteomics technology and detects the expression level of CD-5 in the plasma of diabetic patients and diabetic foot patients through the Proximity Extension Assay (PEA) platform.

[0031] The specific steps are as follows:

[0032] 1. Research subjects The present invention studied and analyzed plasma samples from 30 patients with diabetes, diabetic foot patients and healthy people. Plasma samples were collected from the healthy control group, diabetes group and diabetic foot group, respectively, and the sample collection time was in the morning fasting state.

[0033] Sample source: Patients who visited Changsha Central Hospital from October 1, 2023 to February 31, 2024.

[0034] Inclusion criteria: (1) Patients with confirmed type 2 diabetes: Patients must be diagnosed with type 2 diabetes by any one or more of the following methods: ①. Fasting blood glucose level ≥ 126 mg / dL (7.0 mmol / L) ②. Random blood glucose level ≥ 200 mg / dL (11.1 mmol / L) ③. Oral glucose tolerance test (OGTT) 2-hour blood glucose level ≥ 200 mg / dL (11.1 mmol / L) ④. Glycated hemoglobin (HbA1c) level ≥ 6.5%. The diagnosis must be based on the diagnostic criteria of the World Health Organization (WHO) or the American Diabetes Association (ADA). (2) Patients with diabetic foot ulcers (DFU): Patients have one or more open skin lesions on their feet, and the wounds must meet the following conditions: ①. The ulcer is related to diabetes and is not caused by other reasons (such as trauma, burns, infection, etc.). ②. The ulcer lasts ≥ 4 weeks and there must be a clear clinical diagnosis record. ③. The ulcer grade is 1 to 4 according to the Wagner grading system. (3) Informed consent and willingness to participate in the study: ①. Patients need to understand the purpose, methods, potential risks and benefits of this study.

[0035] Exclusion criteria: (1) Presence of systemic infectious diseases: The patient currently has any form of systemic infectious disease, such as active tuberculosis, HIV / AIDS, active hepatitis, etc. (2) Other types of chronic wounds or ulcers: ① The patient suffers from other types of chronic wounds or ulcers not related to diabetes, such as venous ulcers, pressure ulcers, etc. ② The patient's ulcer is related to vascular diseases (such as arteriosclerotic occlusive disease), excluding diabetic microvascular complications. (3) Immunosuppression or anticoagulant therapy: ① The patient is currently receiving any one or more of the following treatments: immunosuppressive therapy, such as glucocorticoids, immunosuppressants, etc. Anticoagulant therapy, such as warfarin, heparin, direct oral anticoagulants (DOACs), etc. ② The patient has a clear history of immune system diseases (such as systemic lupus erythematosus, rheumatoid arthritis, etc.) or blood coagulation disorders (such as hemophilia).

[0036] 2. Sample collection Fasting plasma samples were collected from the healthy control group, diabetic group and diabetic foot group:

[0037] (1) Collect whole blood samples into EDTA anticoagulant tubes (purple caps);

[0038] (2) Gently invert the tube 5-10 times and then centrifuge it at 1500-2000 g for 10 min at 4°C.

[0039] (3) The upper layer of plasma was transferred to a pre-cooled -192°C ultra-low temperature screw-cap cryotube, quickly frozen with liquid nitrogen, stored at -80°C for a long time (to avoid repeated freezing and thawing), and transported on dry ice.

[0040] The sample groups are shown in Table 1.

[0041] Table 1

[0042]

[0043] 3. Protein content was processed by log2 with the standardized protein expression value to obtain the standardized protein expression value (NPX). The specific information of 92 immune response-related proteins in the diabetic group and the diabetic foot group is shown in Table 2.

[0044] Table 2

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[0053] 4. Data analysis Differential expression analysis (DEPs) was performed using the R software package, and the diagnostic efficacy of CD-5 was evaluated by principal component analysis (PCA) and receiver operating characteristic (ROC) curve. A logistic regression model was constructed to verify the efficacy of CD-5 in the diagnosis of diabetic foot in combination with five markers. The specific analysis methods are as follows:

[0054] (1) The R software package was used to perform differential expression analysis and screen out proteins with significant differences.

[0055] (2) The clustering of samples was analyzed by PCA to reveal the expression differences between different groups.

[0056] (3) The ROC curve was used to evaluate the diagnostic performance of CD-5 and the area under the curve (AUC) was calculated.

[0057] Specificity and sensitivity calculation (ROC curve was drawn using the OmicStudio tool on the website https: / / www.omicstudio.cn / tool / 58, and the corresponding specificity and sensitivity were obtained):

[0058] Threshold determination: Determine the optimal diagnostic threshold based on the Youden index (by calculating the Youden index of each point on the ROC curve, and selecting the CD-5 value corresponding to the maximum value as the threshold):

[0059] YI (Youden Index) = Sensitivity + Specificity - 1;

[0060] Threshold: 2.71.

[0061] Sensitivity and specificity calculation: Under the optimal threshold, calculate the number of true and false positives and true and false negatives, and then calculate the sensitivity and specificity:

[0062] True Positives: 8;

[0063] False Negatives: 2;

[0064] True Negatives: 10;

[0065] False Positives: 0;

[0066]

[0067]

[0068] 5. Experimental results The experimental results show that the expression level of CD-5 in patients with diabetic foot is significantly higher than that in the healthy control group and the diabetic group, and has a high diagnostic efficacy. The specific results are as follows:

[0069] (1) Comparison box plot of NPX values ​​of CD-5 ( Figure 1 ) showed the expression difference between diabetic foot group and diabetes group.

[0070] (2) Volcano plot of differential expression of CD-5 ( Figure 2 ) showed that its expression was significantly upregulated in the diabetic foot group.

[0071] (3) ROC curve analysis Figure 3 )The results showed that CD-5 had a high diagnostic accuracy, with an AUC value of 0.89.

[0072] (4) Functional enrichment analysis (GO) Figure 4 ) showed that the differential factors were mainly enriched in "outside the cell membrane", "inflammatory response", and "extracellular space and cytokine-mediated signaling pathways".

[0073] (5) Pathway enrichment analysis (KEGG) Figure 5 ) showed that the differential factors were mainly enriched in "cytokine-cytokine receptor interaction", "malaria" and "rheumatoid arthritis".

[0074] In summary, the present invention provides a highly sensitive and specific method for diagnosing diabetic foot. Through Olink Proteomics technology, the expression level of CD5 in diabetic foot patients can be accurately detected, providing a potential large-scale screening indicator for the clinic, improving the accuracy of early diagnosis, and simplifying the operation process.

[0075] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of CD5 as a biomarker in the preparation of products for diagnosing or monitoring diabetic foot.

2. The use according to claim 1, characterized in that: The expression level of CD5 was quantitatively detected using PEA technology.

3. The use according to claim 1, characterized in that: The method for diagnosing or monitoring diabetic foot is: quantitatively detecting CD5 in the sample to be tested, and comparing it with a control sample or a reference sample. If the CD5 level in the sample to be tested is significantly increased, it indicates that the patient suffers from diabetic foot.

4. The use according to claim 1, characterized in that: The products include reagents, kits, chips, test strips or high-throughput sequencing platforms.

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