Application of astrocyte-derived extracellular vesicles carrying Tau in auxiliary diagnosis of progressive suprakaryotic paralysis

Abstract

The invention discloses application of astrocyte-derived extracellular vesicles carrying Tau in auxiliary diagnosis of progressive suprakaryotic paralysis, and belongs to the technical field of biomedicine. The specific Tau protein antibody and the anti-glutamic acid transporter 1 antibody are labeled by fluorescence, astrocyte-derived extracellular vesicles carrying Tau in the plasma are accurately detected, PSP patients, PD patients and healthy people can be accurately distinguished by analyzing the concentration of astrocyte-derived EVs carrying Tau in the plasma, and the kit has the advantages of high sensitivity, high sensitivity, high sensitivity and the like. An efficient PSP diagnosis biomarker and a diagnosis model are provided, and a new experience method and technical support are provided for PSP diagnosis and identification.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of astrocyte-derived extracellular vesicles carrying Tau in assisting diagnosis of progressive supranuclear palsy. Background Art

[0002] Progressive supranuclear palsy (PSP) is a common atypical Parkinson's syndrome, and its pathological characteristics are abnormal aggregation of Tau protein in neurons, oligodendrocytes and astrocytes. Since Parkinson's disease (PD) and other atypical Parkinson's syndromes have multiple clinical subtypes and symptoms often overlap, the diagnosis process is very complicated, resulting in a high misdiagnosis rate. Therefore, reliable biomarkers are urgently needed to facilitate early diagnosis and effectively distinguish PSP from other neurodegenerative diseases.

[0003] In existing studies, abnormal Tau protein deposition has been detected in a variety of biological fluids and peripheral tissues, including plasma, olfactory epithelial cells, oral epithelial cells, colon samples, and skin biopsy tissues. Compared with tissue biopsy and other methods of obtaining, plasma, as a routinely available clinical sample, has significant advantages.

[0004] Current research extracts and analyzes disease-related extracellular vesicles (EVs) from plasma through methods such as immunocapture and enzyme-linked immunosorbent assay (ELISA). However, these methods usually require a large volume of serum samples (usually 500 μL) and the detection process is complicated. The existing immunocapture and ELISA steps are cumbersome and time-consuming, requiring multiple operations, which may lead to errors between different steps, thereby affecting the reliability and repeatability of the results; insufficient sensitivity and specificity; lack of non-invasive detection methods, existing biological samples often need to be obtained through invasive operations such as tissue biopsy, limiting their widespread application in routine medical care. Summary of the invention

[0005] The purpose of the present invention is to provide an application of astrocyte-derived extracellular vesicles carrying Tau in the auxiliary diagnosis of progressive supranuclear palsy to solve the problems existing in the above-mentioned prior art. The present invention uses fluorescently labeled specific Tau protein antibodies and anti-glutamate transporter 1 antibodies to accurately detect astrocyte-derived extracellular vesicles carrying Tau in plasma. By analyzing the concentration of astrocyte-derived EVs carrying Tau in plasma, PSP patients and PD patients and healthy people can be accurately distinguished, providing an efficient PSP diagnostic biomarker and diagnostic model, and providing new empirical methods and technical support for the diagnosis and identification of PSP.

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

[0007] The present invention provides a biomarker for assisting the diagnosis of progressive supranuclear palsy, wherein the biomarker is an astrocyte-derived extracellular vesicle carrying Tau protein;

[0008] The Tau protein includes Tau or pTau181.

[0009] Furthermore, the higher the concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma, the higher the risk of progressive supranuclear palsy.

[0010] The present invention also provides use of a product for detecting the concentration of the above biomarker in preparing a product for diagnosing progressive supranuclear palsy.

[0011] The present invention also provides a biomarker for distinguishing progressive supranuclear palsy and Parkinson's disease, wherein the biomarker is an astrocyte-derived extracellular vesicle carrying Tau protein; the Tau protein includes Tau or pTau181.

[0012] Furthermore, the concentration of astrocyte-derived extracellular vesicles carrying Tau protein in the plasma of patients with progressive supranuclear palsy is higher than that in patients with Parkinson's disease.

[0013] The present invention also provides the use of a product for detecting the concentration of the above biomarker in the preparation of a product for identifying progressive supranuclear palsy and Parkinson's disease.

[0014] The present invention also provides a diagnostic model for progressive supranuclear palsy, wherein the input variable of the diagnostic model is the concentration of astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181;

[0015] The higher the concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma, the higher the risk of progressive supranuclear palsy.

[0016] The present invention also provides a diagnostic model for distinguishing progressive supranuclear palsy from Parkinson's disease, wherein the input variable of the diagnostic model is the concentration of astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181;

[0017] The concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in the plasma of patients with progressive supranuclear palsy is higher than that in patients with Parkinson's disease.

[0018] Furthermore, the method for determining the concentration of astrocyte-derived extracellular vesicles carrying Tau protein is:

[0019] (1) Collect plasma samples;

[0020] (2) preparing fluorescently labeled antibodies against glutamate transporter 1 and Tau;

[0021] (3) after the plasma sample and the anti-glutamate transporter 1 fluorescently labeled antibody are mixed and incubated, the Tau fluorescently labeled antibody is added and incubated for a further period of time to obtain a mixture;

[0022] (4) Using a nanoscale flow cytometer to detect the concentration of astrocyte-derived extracellular vesicles carrying Tau protein in the mixture.

[0023] The present invention discloses the following technical effects:

[0024] The present invention utilizes fluorescently labeled specific Tau protein antibodies and anti-glutamate transporter 1 (Anti-Glutamate Transporter 1) antibodies to accurately detect astrocyte-derived extracellular vesicles (EVs) carrying Tau in plasma. By analyzing the concentration of astrocyte-derived EVs carrying Tau in plasma, PSP patients, PD patients and healthy people can be accurately distinguished, providing an efficient PSP diagnostic biomarker and diagnostic model.

[0025] In the process of constructing the diagnostic model, the present invention significantly enhances the detection ability of various Tau protein forms and specific cells through the combination of different fluorescently labeled antibodies, and provides detection sensitivity and specificity; the flow cytometer is used to quantitatively analyze vesicles with a diameter of less than 500nm, and 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 consistency of the results; the entire detection process can be completed within 4 hours, maintaining the freshness of the sample and ensuring the timeliness of the detection. The present invention greatly improves the ability of early diagnosis of PSP by combining the fluorescent labeling of specific antibodies and an efficient sample processing process, and effectively solves the complexity and low sensitivity problems faced by traditional diagnostic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1are the characteristic analysis results of EVs; wherein, A is the TEM image of EVs; B is the Western blot analysis image of EVs obtained by ultracentrifugation; C is the distribution map of nanoparticles in EVs of the PSP group; D is the concentration comparison of GLT-1-positive EVs in plasma (plasma GLT-1), GLT-1-positive EVs in ultracentrifuged plasma supernatant (UC supernatant GLT-1), and IgG-positive EVs in plasma (plasma IgG) in the PSP group;

[0028] Figure 2 is the concentration of astrocyte-derived EVs carrying pTau181 in the plasma of PSPs, PDs, and HCs;

[0029] Figure 3 ROC curve of astrocyte-derived EVs carrying pTau181 in plasma for distinguishing PSP from PD;

[0030] Figure 4 ROC curve of astrocyte-derived EVs carrying pTau181 in plasma for distinguishing PSP from HC. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. Experimental Materials

[0038] Included samples: 105 patients with progressive supranuclear palsy (PSP), 80 patients with Parkinson's disease (PD), and 80 healthy controls (HC).

[0039] Plasma sample preparation: 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 nanoscale flow cytometry and nanoparticle tracking analysis (NTA).

[0040] Isolation of astrocyte-derived EVs: According to the manufacturer's instructions, 10 μg of Anti-Glutamate Transporter 1 (Anti-GLT-1) antibody was coated on 1 mg of M-270 epoxy beads using the Dynabeads Antibody Coupling Kit (14311D, Invitrogen). Anti-Glutamate Transporter 1-labeled EVs were isolated from ultrapure plasma EVs using immunocapture technology.

[0041] 2. Experimental Methods

[0042] Transmission electron microscopy (TEM) analysis: 5 μL Anti-Glutamate Transporter 1 labeled EVs were placed on a copper grid coated with a carbon support film. After incubation for 60 seconds, the excess sample was removed with filter paper. The copper grid was then stained with 2% uranyl acetate solution for 60 seconds. After removing the excess uranyl acetate solution with filter paper, the sample was allowed to air dry. Electron microscopy images were taken using a Tecnai F20 transmission electron microscope at 200 kV.

[0043] Western blot analysis: The total protein concentration of EVs obtained by ultracentrifugation was assessed using the BCA method. 15 μg of total protein contained in the sample was separated using 4-20% Bis-Tris gel (M42010C, Genscript) and then electrotransferred to a nitrocellulose membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with Anti-Glutamate Transporter 1 antibody, AliX polyclonal antibody, TSG 101 polyclonal antibody, Actin polyclonal antibody, and CD9 monoclonal antibody.

[0044] Nanoparticle Tracking Analysis: 2 μL plasma samples from PSP patients were diluted 1:500 in PBS (pH 7.4). These diluted samples were then analyzed using the ZetaView platform (Particle Metrix) to assess the distribution and concentration of nanoparticles.

[0045] Analysis of EVs by Cytoflex nanoscale flow cytometer: The antibodies were conjugated with fluorophores using the Zenon IgG labeling kit according to the instructions of the kit. Anti-Glutamate Transporter 1 antibody (labeling astrocytes) and pTau181 antibody were labeled with different fluorescent labeling kits. 5 μL of plasma was placed in a flow cytometer tube. Then 0.1 μg of fluorescently conjugated Anti-Glutamate Transporter 1 antibody was added and incubated for 30 minutes, followed by 0.2 μg of fluorescently conjugated pTau181 antibody incubated for 20 minutes. All processes were performed at room temperature and protected from light. After incubation, the mixture was diluted 1:60 with PBS, vortexed and centrifuged for 10 seconds. The vesicles below 500 nm were quantitatively detected using a nanoscale flow cytometer on the Cytoflex S platform (Beckman Coulter, Milano, Italy) in VSSC-H mode. The concentration of EVs carrying pTau181 protein and Anti-Glutamate Transporter 1 was calculated according to the flow rate and PBS dilution ratio. All samples were tested within 4 hours and a single batch of cell counting analysis was performed on the same day.

[0046] 3. Data Processing

[0047] Statistical analysis was performed using GraphPad Prism 10 and SPSS26.0. The Kruskal-Wallis test was used to assess the intergroup differences in the mean concentration of astrocyte-derived EVs carrying Tau, followed by Dunn's test for post hoc comparisons between PSP and PD, and PSP and HC. Receiver operating characteristic (ROC) curve analysis was used to assess the sensitivity and specificity of these markers in distinguishing PSP from HCs or PD. Spearman correlation analysis was used to assess the correlation between clinical indicators and the concentration of astrocyte-derived EVs carrying Tau in the PSP group, and Bonferroni correction was used to adjust for multiple comparisons. All tests were two-tailed, and statistical significance was set at P < 0.05.

[0048] 4. Results and Analysis

[0049] 1. TEM analysis results:

[0050] TEM analysis showed that the diameter of astrocyte-derived EVs was approximately 100 nm ( Figure 1 A).

[0051] 2. Western blot analysis results:

[0052] Western blot analysis showed that GLT-1 was enriched together with the general EV markers Alix, TSG 101, Actin, and CD9 in the reference EVs sample obtained by ultracentrifugation ( Figure 1 B).

[0053] 3. Nanoparticle tracking analysis results:

[0054] Nanoparticle tracking analysis showed that GLT-1-positive EVs in PSP plasma showed a broad peak centered at 100 nm ( Figure 1 C). The concentrations of GLT-1-positive EVs in PSP plasma, GLT-1-positive EVs in the plasma supernatant after ultracentrifugation, and IgG-positive EVs in PSP plasma (for control) are shown in Figure 2. Figure 1 As shown in D.

[0055] 4. Nanoscale flow cytometer analysis results:

[0056] like Figure 2As shown, astrocyte-derived EVs carrying pTau181 can effectively distinguish PSP from PD and HC: among the 105 PSP patients, 80 PD patients and 80 HC included, the concentration of astrocyte-derived EVs containing pTau181 in the plasma of PSP patients was significantly higher than that of HC and PD patients (PSP vs HCs: PpTau181 < 0.0001; PSP vs PD: PpTau181 < 0.0001; Figure 2 ).

[0057] ROC analysis evaluated the diagnostic utility of astrocyte-derived EVs carrying pTau181 in plasma in differentiating PSP from PD and HC. In differentiating PSP from PD, the area under the curve (AUC) value was: 0.815 (95% CI 0.752-0.877) ( Figure 3 ). When distinguishing PSP from HC, the AUC value was 0.917 (95% CI 0.876-0.958) ( Figure 4 When the concentration of astrocyte-derived EVs carrying pTau181 in plasma is less than 1071500 / mL, the patient is judged to have a low risk of Parkinson's disease or progressive supranuclear palsy; when the concentration of astrocyte-derived EVs carrying pTau181 in plasma is in the range of 1071500-1600500 / mL, the patient is judged to have a high risk of Parkinson's disease and a low risk of progressive supranuclear palsy; when the concentration of astrocyte-derived EVs carrying pTau181 in plasma is greater than 1600500 / mL, the patient is judged to have a high risk of progressive supranuclear palsy.

[0058] The above results indicate that by evaluating the number or concentration of astrocyte-derived EVs carrying pTau181 in plasma, PSP patients and PD patients, as well as PSP patients and HC populations, can be effectively distinguished, thereby achieving the effect of auxiliary diagnosis of PSP.

[0059] 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 the diagnosis of progressive supranuclear palsy, characterized in that: The biomarker is an astrocyte-derived extracellular vesicle carrying Tau protein; The Tau protein includes Tau or pTau181.

2. The biomarker according to claim 1, characterized in that The higher the concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma, the higher the risk of progressive supranuclear palsy.

3. Use of a product for detecting the concentration of the biomarker according to claim 1 or 2 in the preparation of a product for diagnosing progressive supranuclear palsy.

4. A biomarker for distinguishing progressive supranuclear palsy from Parkinson's disease, characterized in that: The biomarker is an astrocyte-derived extracellular vesicle carrying Tau protein; the Tau protein includes Tau or pTau181.

5. The biomarker according to claim 4, characterized in that The concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in the plasma of patients with progressive supranuclear palsy is higher than that in patients with Parkinson's disease.

6. Use of a product for detecting the concentration of the biomarker according to claim 4 or 5 in the preparation of a product for distinguishing progressive supranuclear palsy and Parkinson's disease.

7. A diagnostic model for progressive supranuclear palsy, characterized in that: The input variable of the diagnostic model is the concentration of astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181; The higher the concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in plasma, the higher the risk of progressive supranuclear palsy.

8. A diagnostic model for distinguishing progressive supranuclear palsy from Parkinson's disease, characterized in that: The input variable of the diagnostic model is the concentration of astrocyte-derived extracellular vesicles carrying Tau protein; the Tau protein includes Tau or pTau181; The concentration of the astrocyte-derived extracellular vesicles carrying Tau protein in the plasma of patients with progressive supranuclear palsy is higher than that in patients with Parkinson's disease.

9. The diagnostic model according to claim 7 or the diagnostic model according to claim 8, characterized in that: The method for determining the concentration of astrocyte-derived extracellular vesicles carrying Tau protein is: (1) Collect plasma samples; (2) preparing fluorescently labeled antibodies against glutamate transporter 1 and Tau; (3) after the plasma sample and the anti-glutamate transporter 1 fluorescently labeled antibody are mixed and incubated, the Tau 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 astrocyte-derived extracellular vesicles carrying Tau protein in the mixture.

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