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

Abstract

The present invention discloses the use of extracellular vesicles derived from oligodendrocytes carrying FTH1 in differentiating between multiple system atrophy and Parkinson's disease, belonging to the field of biomedical technology. The present invention utilizes fluorescently labeled CNPase antibodies and FTH1 antibodies to accurately detect extracellular vesicles derived from oligodendrocytes carrying FTH1 in plasma. By detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 (FTH1-positive) in plasma, it is possible to accurately distinguish between MSA patients, PD patients, and healthy individuals. This provides a highly effective biomarker for MSA diagnosis, improves the ability to diagnose MSA early and differentiate between MSA and PD, and effectively addresses the complexity and low sensitivity issues faced by traditional diagnostic methods.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to 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 disorder, with the main symptoms being resting tremor, rigidity, bradykinesia, and postural instability. Neuropathologically, it is characterized by the presence of numerous Lewy bodies and filamentous inclusions of α-synuclein (α-syn) in some brain cells, including dopaminergic neurons in the substantia nigra. In addition to PD and dementia with Lewy bodies (DLB), multiple system atrophy (MSA) is the third most common synucleinopathy. It is characterized by the presence of abundant filamentous α-synuclein inclusions in brain cells, particularly oligodendrocytes (Papp-Lantos bodies). Due to the current lack of reliable and readily available biomarkers, differentiating PD from MSA based on clinical evidence is difficult and has a high misdiagnosis rate.

[0003] Existing biomedical technologies for differentiating MSA from PD include the detection of abnormal α-syn protein deposition in several biological fluids and peripheral tissues, including plasma, olfactory and oral epithelial cells, colon specimens, and skin biopsies. α-syn is isolated from peripheral body fluids by immunoprecipitation in exosomes secreted by the central nervous system. Currently, α-syn is detected in plasma exosomes and quantified by electrochemiluminescence enzyme-linked immunosorbent assay (ELISA). Comparing the ratio of α-syn concentrations in putative oligodendrocyte exosomes to putative neuronal exosomes has a diagnostic efficacy of 0.902, a sensitivity of 89.8%, and a specificity of 86.0%, for differentiating PD from MSA.

[0004] While existing technologies have made some progress in biomarker research for neurodegenerative diseases, the following issues and drawbacks remain: high sample size requirements; cumbersome and time-consuming immunocapture and ELISA assays, requiring multiple steps, which can lead to errors between steps and affect the reliability and reproducibility of results; insufficient sensitivity and specificity; and the lack of non-invasive assays, which require invasive procedures such as tissue biopsies to obtain biological samples. Clinical application and promotion have limited patient acceptance. Therefore, there is a need for a diagnostic technology for MSA and PD that offers increased sensitivity and specificity, requires a smaller sample size, and offers a simpler assay process. Summary of the Invention

[0005] The present invention aims to provide the use of extracellular vesicles derived from oligodendrocytes carrying FTH1 in the differentiation of multiple system atrophy and Parkinson's disease, thereby addressing the problems of the prior art described above. The present invention utilizes fluorescently labeled CNPase antibodies and FTH1 antibodies to accurately detect extracellular vesicles derived from oligodendrocytes carrying FTH1 in plasma. By detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 (FTH1-positive) in plasma, it is possible to accurately differentiate between MSA patients, PD patients, and healthy individuals. This provides an efficient diagnostic biomarker for MSA, improves the ability to diagnose MSA early and differentiate between MSA and PD, and effectively addresses the 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 and 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 auxiliary diagnosis of multiple system atrophy.

[0010] The present invention also provides use of a reagent for detecting the concentration of the above-mentioned biomarker in preparing a product for identifying 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, which 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 identifying 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 fluorescently labeled antibodies against CNPase and FTH1;

[0017] (3) mixing the plasma sample with the CNPase fluorescently labeled antibody and incubating them, then adding the FTH1 fluorescently labeled antibody and continuing the incubation to obtain a mixture;

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

[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, thereby providing an efficient MSA diagnostic biomarker. The present invention uses a labeling kit to perform different fluorescent labeling 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 smaller than 500nm. The high-throughput characteristics of the flow cytometer enable a large amount of data to be quickly obtained in a single test, thereby improving the analysis efficiency and the consistency of the data display. The detection process can be completed within 4 hours, maintaining the freshness of the plasma sample and ensuring the timeliness of the test.

[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, effectively solving 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 following briefly introduces the drawings required for use in the embodiments. 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 any 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 are shown.

[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 Receiver operating characteristic (ROC) curves for differentiating MSA and PD patients or HCs from 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative 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 Notes:

[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 a key role in myelin synthesis and participates in this process together with the 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] Plasma sample preparation: Venous blood samples were collected from participants in the morning, fasting, using tubes coated with ethylenediaminetetraacetic acid. Blood samples were centrifuged at 1500 × g for 15 minutes at 4°C, and the supernatant was centrifuged at 12,000 × g for 30 minutes at 4°C. The supernatant plasma was stored at −80°C for use in nanoscale flow cytometry.

[0040] EVs were analyzed using a Cytoflex nanoscale flow cytometer. Fluorophores were conjugated to antibodies using the Zenon IgG labeling kit according to the manufacturer's instructions. Antibodies against CNPase (of oligodendrocyte origin) and FTH1 were labeled using different fluorescent labeling kits. Five microliters of plasma was placed in a flow cytometer tube. 0.1 micrograms of fluorescently conjugated CNPase antibody was then added and incubated for 30 minutes, followed by 0.2 micrograms of fluorescently conjugated FTH1 antibody for 20 minutes. All procedures were performed at room temperature in the dark. After incubation, the mixture was diluted 1:60 with PBS, vortexed, and centrifuged for 10 seconds. Vesicles smaller than 500 nm were quantified using the nanoscale flow cytometer in VSSC-H mode on a Cytoflex S platform (Beckman Coulter, Milano, Italy). The concentration of EVs dually positive for CNPase and FTH1 was calculated based on the flow rate and PBS dilution ratio. All samples were analyzed within 4 hours, and single-batch cell counting and analysis were performed on the same day.

[0041] 2. Data Processing

[0042] Statistical analysis was performed using GraphPad Prism 10 and SPSS 26.0. Differences in the mean concentrations of CNPase- and FTH1-double-positive EVs between MSA and PD or HC groups were assessed using the Kruskal-Wallis test, followed by post hoc comparisons between MSA and PD or HC using the Dunn test. Receiver operating characteristic (ROC) curve analysis was used 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 the figure. Figure 1 The statistical results of EVs concentration are shown in Figure 2 As shown in the results, 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 in the figure, the diagnostic utility of plasma FTH1-positive oligodendrocyte-derived EVs in distinguishing MSA from PD and HC was evaluated by the ROC curve. 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 in the preparation of a product for assisting the diagnosis of multiple system atrophy, 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 healthy subjects.

2. Use of a reagent for detecting the concentration of extracellular vesicles derived from oligodendrocytes carrying FTH1 in the preparation of a product for identifying multiple system atrophy and Parkinson's disease, 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.

3. The use according to claim 1 or 2, characterized in that The product includes a reagent or a kit.

4. Application of FTH1-carrying oligodendrocyte-derived extracellular vesicles in constructing a diagnostic model for auxiliary diagnosis of multiple system atrophy.

5. Application of FTH1-carrying oligodendrocyte-derived extracellular vesicles in constructing a diagnostic model for differentiating multiple system atrophy and Parkinson's disease.

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