Application of oligodendrocyte-derived extracellular vesicles carrying FTH1 in identification of multi-system atrophy and Parkinson's disease

Abstract

The invention discloses application of FTH1-carrying oligodendrocyte-derived extracellular vesicles in identification of multi-system atrophy and Parkinson's disease, and belongs to the technical field of biomedicine. According to the present invention, the fluorescence labeling CNPase antibody and the FTH1 antibody are adopted to accurately detect the oligodendroglia-derived extracellular vesicles carrying FTH1 in the plasma, and the concentration of the oligodendroglia-derived extracellular vesicles carrying FTH1 (FTH1 positive) in the plasma is detected so as to accurately distinguish MSA patients, PD patients and healthy people, such that the efficient MSA diagnosis biomarker is provided, and the application prospect is broad. The MSA early diagnosis capability and the MSA and PD identification capability are improved, and the problems of complexity and low sensitivity of a traditional diagnosis method are effectively solved.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to the application of oligodendrocyte-derived extracellular vesicles carrying FTH1 in identifying multiple system atrophy and Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, with the main symptoms being resting tremor, rigidity, bradykinesia, and postural instability. Neuropathologically, it is characterized by the presence of numerous alpha-synuclein (α-syn) filamentous inclusions in some brain cells, including dopaminergic neurons in the substantia nigra, and Lewy bodies. In addition to PD and dementia with Lewy bodies (DLB), multiple system atrophy (MSA) is the third major synucleinopathy. It is characterized by the presence of abundant filamentous α-synuclein inclusions in brain cells, especially oligodendrocytes (Papp-Lantos bodies). Due to the lack of reliable and easily accessible biomarkers at this stage, it is difficult to differentiate between PD and MSA based on clinical evidence, and the misdiagnosis rate is high.

[0003] Existing biomedical technologies for distinguishing MSA from PD include: abnormal α-syn protein deposition has been detected in some biological fluids and peripheral tissues (including plasma, olfactory and oral epithelial cells, colon specimens, and skin biopsies). α-syn in exosomes secreted by the central nervous system is isolated from peripheral body fluids by immunoprecipitation. Currently, α-syn in plasma exosomes is detected and quantitatively analyzed by electrochemiluminescence enzyme-linked immunosorbent assay (ELISA). By comparing the α-syn concentration ratio of putative oligodendrocyte exosomes with that of putative neuronal exosomes, the diagnostic efficacy AUC for distinguishing PD from MSA is 0.902, the sensitivity is 89.8%, and the specificity is 86.0%.

[0004] Although existing technologies have made some progress in the study of biomarkers for neurodegenerative diseases, the following problems and defects still exist: high sample volume requirements; existing immunocapture and ELISA detection steps are cumbersome and time-consuming, requiring multiple operations, which may lead to errors between steps, thus affecting the reliability and repeatability of the results; insufficient sensitivity and specificity; existing biological samples need to be obtained through invasive operations such as tissue biopsy, lack of non-invasive detection methods, and low acceptance of clinical application and promotion by patients. Therefore, it is necessary to provide an MSA and PD auxiliary diagnosis technology with higher sensitivity and specificity, less sample volume required, and simpler detection process. Summary of the invention

[0005] The purpose of the present invention is to provide an application of oligodendrocyte-derived extracellular vesicles carrying FTH1 in distinguishing multiple system atrophy and Parkinson's disease, so as to solve the problems existing in the above-mentioned prior art. The present invention uses fluorescently labeled CNPase antibodies and FTH1 antibodies to accurately detect oligodendrocyte-derived extracellular vesicles carrying FTH1 in plasma. By detecting the concentration of oligodendrocyte-derived extracellular vesicles carrying FTH1 (FTH1 positive) in plasma, it is possible to accurately distinguish between MSA patients, PD patients and healthy people, and provide an efficient MSA diagnostic biomarker, which improves the ability of early diagnosis of MSA and differentiation of MSA and PD, and effectively solves the problems of complexity and low sensitivity faced by traditional diagnostic methods.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a biomarker for assisting in the diagnosis of multiple system atrophy or distinguishing multiple system atrophy from Parkinson's disease. The biomarker is an extracellular vesicle derived from oligodendrocytes carrying FTH1.

[0008] Furthermore, the concentration of the oligodendrocyte-derived extracellular vesicles carrying FTH1 in the plasma of patients with multiple system atrophy is lower than that in patients with Parkinson's disease and healthy subjects.

[0009] The present invention also provides the use of a reagent for detecting the concentration of the above-mentioned biomarker in the preparation of a product for assisting in the diagnosis of multiple system atrophy.

[0010] The present invention also provides the use of a reagent for detecting the concentration of the above biomarker in the preparation of a product for distinguishing multiple system atrophy and Parkinson's disease.

[0011] Optionally, the product comprises a reagent or a kit.

[0012] The present invention also provides a product for assisting in the diagnosis of multiple system atrophy, wherein the product comprises a reagent for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1; the reagent is a reagent for nanoscale flow cytometry detection.

[0013] The present invention also provides a product for distinguishing multiple system atrophy and Parkinson's disease, wherein the product comprises a reagent for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1; the reagent is a reagent for nanoscale flow cytometry detection.

[0014] Furthermore, the method for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 comprises the following steps:

[0015] (1) Collect plasma samples;

[0016] (2) preparing CNPase fluorescently labeled antibodies and FTH1 fluorescently labeled antibodies;

[0017] (3) after the plasma sample and the CNPase fluorescently labeled antibody are mixed and incubated, the FTH1 fluorescently labeled antibody is added and incubated for a further period of time to obtain a mixture;

[0018] (4) Using a nanoscale flow cytometer to detect the concentration of oligodendrocyte-derived extracellular vesicles carrying CNPase and FTH1 in the mixture.

[0019] The present invention also provides the use of the above-mentioned biomarkers in constructing a diagnostic model for auxiliary diagnosis of multiple system atrophy.

[0020] The present invention also provides the use of the above biomarkers in constructing a diagnostic model for distinguishing multiple system atrophy and Parkinson's disease.

[0021] The present invention discloses the following technical effects:

[0022] The present invention can accurately distinguish between MSA patients, PD patients and healthy people by detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 (FTH1 positive) in plasma, and provides an efficient MSA diagnostic biomarker. The present invention uses a labeling kit to carry out different fluorescent labels on CNPase antibodies and FTH1 antibodies, so that specific FTH1-positive oligodendrocyte-derived extracellular vesicles can be effectively identified in a flow cytometer. By using a combination of multiple fluorescently labeled antibodies, the detection capability is improved. The Cytoflex S nanoscale flow cytometer is used to quantitatively detect extracellular vesicles less than 500nm. The high-throughput characteristics of the flow cytometer enable a large amount of data to be quickly obtained in one detection, thereby improving the analysis efficiency and consistency of data display. The detection process can be completed within 4 hours, maintaining the freshness of the plasma sample and ensuring the timeliness of the detection.

[0023] The present invention greatly improves the ability of early diagnosis of MSA and differentiation of MSA and PD by combining fluorescent labeling of specific antibodies with an efficient sample processing process, and effectively solves the problems of complexity and low sensitivity faced by traditional diagnostic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1The results of flow cytometry analysis of FTH1-positive oligodendrocyte-derived EVs in the plasma of HC, PD, and MSA patients.

[0026] Figure 2 Statistical analysis results of the concentration of FTH1-positive oligodendrocyte-derived EVs in MSA, PD and HC plasma by flow cytometry (***: p < 0.001);

[0027] Figure 3 ROC curve for distinguishing MSA and PD patients or HCs for FTH1-positive oligodendrocyte-derived EVs. DETAILED DESCRIPTION

[0028] 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.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment 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. The intermediate value in any stated value or stated range, and each smaller range between 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.

[0030] 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.

[0031] 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 invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Terminology Note:

[0034] CNPase (cyclic nucleotide-3' phosphohydrolase) is an enzyme involved in myelin synthesis, specifically 2',3'-cyclic nucleotide-3'-phosphohydrolase, also known as phosphodiesterase or cyclic nucleotide phosphodiesterase. CNPase plays an important role in the synthesis of myelin and participates in this process together with protein lipoprotein (PLP).

[0035] FTH1 (Ferritin Heavy Chain 1) refers to ferritin heavy chain 1, which is a spherical molecule composed of 24 different subunits and is mainly involved in the storage and release of iron ions.

[0036] Example 1

[0037] 1. Materials and Methods

[0038] Included samples: 49 patients with multiple system atrophy (MSA), 46 patients with Parkinson's disease (PD) and 48 healthy controls (HC).

[0039] Preparation of plasma samples: Venous blood samples were collected from fasting participants in the morning using tubes coated with ethylenediaminetetraacetic acid. The blood samples were then centrifuged at 1500×g for 15 min (4°C), and the supernatant was centrifuged at 12,000×g for 30 min (4°C). The supernatant plasma was stored at -80°C for nano-flow cytometry.

[0040] Analysis of EVs using Cytoflex nano-flow cytometer: Antibodies were conjugated to fluorophores using the Zenon IgG labeling kit according to the manufacturer’s instructions, and anti-CNPase antibodies (labeling oligodendrocytes) and anti-FTH1 antibodies were labeled using different fluorescent labeling kits. 5 μL of plasma was placed in a flow cytometer tube. Then 0.1 μg of fluorescently conjugated CNPase antibody was added and incubated for 30 min, followed by 0.2 μg of fluorescently conjugated FTH1 antibody for 20 min, all at room temperature and in the dark. After incubation, the mixture was diluted 1:60 with PBS, vortexed, and centrifuged for 10 seconds. Vesicles below 500 nm were quantified using a nano-flow cytometer on a Cytoflex S platform (Beckman Coulter, Milano, Italy) in VSSC-H mode. The concentration of EVs carrying dual positivity for CNPase and FTH1 was calculated based on the flow rate and PBS dilution ratio. All samples were analyzed within 4 h and analyzed in a single batch of cells on the same day.

[0041] 2. Data Processing

[0042] Statistical analysis was performed using GraphPad Prism 10 and SPSS 26.0. The Kruskal-Wallis test was used to assess the differences between the MSA and PD or HC groups in the mean concentrations of CNPase and FTH1 double-positive EVs, followed by Dunn's test for post hoc comparisons between MSA and PD or HC. Receiver operating characteristic (ROC) curve analysis was performed to assess the sensitivity and specificity of these markers in distinguishing MSA from PD or HC.

[0043] 3. Results and Analysis

[0044] The results of flow cytometry detection of FTH1-positive oligodendrocyte-derived EVs are shown in Figure 2. Figure 1 The statistical results of EVs concentration are shown in Figure 2 As shown in the figure, the concentration of FTH1-positive oligodendrocyte-derived EVs in the plasma of MSA patients was significantly lower than that in HC and PD patients (MSA vs. HCs: P < 0.001; MSA vs. PD: P < 0.001; Figure 2 ). The ROC curve analysis results are as follows Figure 3 As shown, the diagnostic utility of plasma FTH1-positive oligodendrocyte-derived EVs in distinguishing MSA from PD and HC was evaluated by the ROC curve, and the area under the curve (AUC) values ​​were as follows: the sensitivity for distinguishing MSA from HC was 69.35%, the specificity was 91.67%, and the AUC was 0.857; the sensitivity for distinguishing MSA from PD was 65.31%, the specificity was 84.78%, and the AUC was 0.771.

[0045] When the concentration of FTH1-positive oligodendrocyte-derived EVs in plasma is less than 8456923 / mL, the patient is judged to have a high risk of MSA; when distinguishing MSA patients from PD patients, when the concentration of FTH1-positive oligodendrocyte-derived EVs in plasma is less than 8341733 / mL, the patient is judged to have a high risk of MSA and a low risk of PD.

[0046] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biomarker for assisting in the diagnosis of multiple system atrophy or distinguishing multiple system atrophy from Parkinson's disease, characterized in that: The biomarker is extracellular vesicles derived from oligodendrocytes carrying FTH1.

2. The biomarker according to claim 1, characterized in that The concentration of the oligodendrocyte-derived extracellular vesicles carrying FTH1 in the plasma of patients with multiple system atrophy is lower than that in patients with Parkinson's disease and healthy subjects.

3. Use of a reagent for detecting the concentration of the biomarker according to claim 1 or 2 in the preparation of a product for assisting the diagnosis of multiple system atrophy.

4. Use of a reagent for detecting the concentration of the biomarker according to claim 1 or 2 in the preparation of a product for distinguishing multiple system atrophy and Parkinson's disease.

5. The use according to claim 3 or 4, characterized in that: The product includes a reagent or a kit.

6. A product for assisting diagnosis of multiple system atrophy, characterized in that: The product contains a reagent for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1; the reagent is a reagent for nanoscale flow cytometry detection.

7. A product for distinguishing multiple system atrophy and Parkinson's disease, characterized in that: The product contains a reagent for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1; the reagent is a reagent for nanoscale flow cytometry detection.

8. The product according to claim 6 or 7, characterized in that The method for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 comprises the following steps: (1) Collect plasma samples; (2) preparing CNPase fluorescently labeled antibodies and FTH1 fluorescently labeled antibodies; (3) after the plasma sample and the CNPase fluorescently labeled antibody are mixed and incubated, the FTH1 fluorescently labeled antibody is added and incubated for a further period of time to obtain a mixture; (4) Using a nanoscale flow cytometer to detect the concentration of oligodendrocyte-derived extracellular vesicles carrying CNPase and FTH1 in the mixture.

9. Use of the biomarker according to claim 1 or 2 in constructing a diagnostic model for assisting diagnosis of multiple system atrophy.

10. Use of the biomarker according to claim 1 or 2 in constructing a diagnostic model for distinguishing multiple system atrophy and Parkinson's disease.

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