Application of ferritin-carrying neuron-derived extracellular vesicles in auxiliary diagnosis of Parkinson's disease

Abstract

The application discloses application of ferritin-carrying neuron-derived extracellular vesicles in auxiliary diagnosis of Parkinson's disease and belongs to the field of biomedical technology. The application realizes effective identification of specific ferritin-carrying neuron-derived extracellular vesicles in a flow cytometer by fluorescence labeling of L1 cell adhesion molecule antibodies and ferritin heavy chain antibodies, and can accurately distinguish PD patients, MSA patients and healthy people by analyzing the concentration of the ferritin-carrying neuron-derived extracellular vesicles in blood plasma, thereby providing an efficient PD diagnosis biomarker and greatly improving the ability of early diagnosis of PD and identification of MSA and PD, and effectively solving the problems of complexity and low sensitivity faced by traditional diagnosis methods.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of neuronal extracellular vesicles carrying ferritin in the auxiliary diagnosis of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common degenerative disease of the central nervous system. Currently, its diagnosis mainly relies on medical history and clinical examination. However, because the clinical manifestations of PD often overlap with those of other atypical Parkinsonian syndromes (such as multiple system atrophy, MSA), the misdiagnosis rate is high. Therefore, there is an urgent need to find objective biomarkers to assist in the diagnosis and differential diagnosis of PD.

[0003] Abnormal iron metabolism is one of the important pathogenic mechanisms of neurodegenerative diseases (PD). Ferritin, as the main storage form of iron in the body, plays a crucial role in maintaining iron homeostasis. Autopsy studies of PD patients have shown a reduction in ferritin in the substantia nigra region, suggesting abnormal ferritin levels in the body. Extracellular vesicles (EVs) mediate the extracellular release of ferritin; therefore, detecting the number of neuronal-derived ferritin granules in plasma holds promise as a potential diagnostic biomarker for PD, aiding in accurate diagnosis. Although current technologies have made some progress in the research of biomarkers for neurodegenerative diseases, several significant problems and limitations remain: high sample volume requirements, with current technologies typically requiring 500 μl or more of plasma samples; complex detection processes, with existing immune capture and ELISA procedures being cumbersome and time-consuming, requiring multiple operations, which may lead to errors between different steps, thus affecting the reliability and reproducibility of the results; insufficient sensitivity and specificity; and a lack of non-invasive detection methods, as existing biological samples often require invasive procedures such as tissue biopsy, limiting their widespread application in routine medical care. Therefore, it is necessary to provide a novel PD diagnostic biomarker and detection method that is convenient to use. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ferritin-carrying neuronal extracellular vesicles in the auxiliary diagnosis of Parkinson's disease, thereby addressing the problems existing in the prior art. This invention utilizes fluorescent labeling of L1 cell adhesion molecule antibodies and ferritin heavy chain antibodies to enable the effective identification of specific ferritin-carrying neuronal extracellular vesicles in flow cytometry. By analyzing the concentration of ferritin-carrying neuronal extracellular vesicles in plasma, it is possible to accurately distinguish between PD patients, MSA patients, and healthy individuals, providing a highly efficient PD diagnostic biomarker and significantly improving the ability to diagnose early PD and differentiate between MSA and PD. This effectively solves the problems of complexity and low sensitivity faced by traditional diagnostic methods.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a biomarker for assisting in the diagnosis of Parkinson's disease, wherein the biomarker is a neuronal extracellular vesicle carrying ferritin;

[0007] The concentration of neuronal extracellular vesicles carrying ferritin in the plasma of Parkinson's disease patients is lower than that in patients with multiple system atrophy and healthy individuals.

[0008] The present invention also provides the use of products for detecting the concentration of the above-mentioned biomarkers in the preparation of products for diagnosing Parkinson's disease.

[0009] The present invention also provides a product for assisting in the diagnosis of Parkinson's disease, the product comprising a reagent for detecting the concentration of extracellular vesicles of neuronal origin carrying ferritin; the reagent is a reagent for detection by nanoscale flow cytometry.

[0010] Furthermore, the method for detecting the concentration of neuronal-derived extracellular vesicles carrying ferritin is as follows:

[0011] (1) Collect plasma samples;

[0012] (2) Preparation of fluorescently labeled antibodies against L1 cell adhesion molecules and ferritin heavy chain;

[0013] (3) After mixing and incubating the plasma sample with the fluorescently labeled antibody of L1 cell adhesion molecule, add the fluorescently labeled antibody of antiferritin heavy chain and continue incubation to obtain a mixture;

[0014] (4) The concentration of neuronal extracellular vesicles carrying ferritin in the mixture was detected by nanoscale flow cytometry.

[0015] This invention also provides the application of neuronal extracellular vesicles carrying ferritin in constructing a multivariate logistic regression model to assist in the diagnosis of Parkinson's disease.

[0016] This invention also provides the application of neuronal extracellular vesicles carrying ferritin in constructing a multivariate logistic regression model to differentiate between multiple system atrophy and Parkinson's disease.

[0017] The present invention also provides a method for constructing a multivariate logistic regression model to assist in the diagnosis of Parkinson's disease. The multivariate logistic regression model is constructed using the concentration of extracellular vesicles carrying ferritin in plasma, the subject's gender, and age as input variables.

[0018] The probability logit of the multivariate logistic regression model is 9.047 - 6.587 × 10⁻⁶. -6× Concentration of extracellular vesicles from neurons carrying ferritin + 0.600 × sex + 0.046 × age; where the value is 1 for males and 0 for females.

[0019] When the probability is greater than 0.65, the subject is diagnosed with Parkinson's disease; otherwise, they are considered healthy.

[0020] The present invention also provides a multivariate logistic regression model constructed by the above construction method.

[0021] The present invention also provides a method for constructing a multivariate logistic regression model to distinguish between multiple system atrophy and Parkinson's disease. The multivariate logistic regression model is constructed using the concentration of extracellular vesicles of neurons carrying ferritin in plasma, the subject's gender, and age as input variables.

[0022] The probability logit of the multivariate logistic regression model is 8.347 - 6.330 × 10⁻⁶. -6 × Concentration of extracellular vesicles from neurons carrying ferritin + 0.199 × sex + 0.053 × age; where the value is 1 for males and 0 for females.

[0023] When the probability is greater than 0.71, the subject is diagnosed with Parkinson's disease; otherwise, it is diagnosed with multiple system atrophy.

[0024] The present invention also provides a multivariate logistic regression model constructed by the above construction method.

[0025] The present invention discloses the following technical effects:

[0026] This invention provides a highly efficient biomarker for PD diagnosis by accurately differentiating between PD patients, MSA patients, and healthy individuals by detecting the concentration of neuronal extracellular vesicles carrying ferritin in plasma. The invention uses a labeling kit to fluorescently label L1 cell adhesion molecule-antibody and ferritin heavy chain antibody, enabling the effective identification of specific neuronal extracellular vesicles carrying ferritin in flow cytometry. The use of a combination of multiple fluorescently labeled antibodies enhances detection capabilities. Quantitative analysis of vesicles with a diameter less than 500 nm is performed using a Cytoflex S nanoscale flow cytometer. The high-throughput characteristics of flow cytometry allow for the rapid acquisition of large amounts of data in a single test, thereby improving analytical efficiency and result consistency. Furthermore, the detection process is time-efficient, maintaining the freshness of plasma samples and ensuring timely detection.

[0027] This invention, by combining fluorescent labeling of specific antibodies with an efficient sample processing procedure, greatly improves the ability to diagnose PD early and differentiate between MSA and PD, effectively solving the problems of complexity and low sensitivity faced by traditional diagnostic methods. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The results of detecting neuronal-derived EVs labeled with L1 cell adhesion molecule in plasma in Example 1 are shown below; where A is a TEM image of EVs with a scale bar of 100 nm; B is the Western blot analysis result of EVs; and C is the concentration and particle size distribution result of EVs.

[0030] Figure 2 The concentration of neuronal-derived extracellular vesicles carrying ferritin in the plasma of PD, MSA, and HCs in Example 1;

[0031] Figure 3 The concentration of neuronal extracellular vesicles carrying ferritin in the plasma of PD, MSA, and HCs in Example 2;

[0032] Figure 4 The ROC curve for distinguishing PD and MSA is shown for extracellular vesicles of neurons carrying ferritin in plasma in Example 2.

[0033] Figure 5 The ROC curve for distinguishing PD and HC is shown for extracellular vesicles of neurons carrying ferritin in plasma in Example 2.

[0034] Figure 6 The ROC curve for the multivariate logistic regression model used to distinguish between PD and MSA in Example 2;

[0035] Figure 7 The ROC curve used in Example 2 for the multivariate logistic regression model to distinguish between PD and HC. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] Previous research by the inventors' research group revealed significant differences in the concentration of ferritin-carrying neuronal-derived extracellular vesicles among Parkinson's disease patients, multiple system atrophy patients, and healthy individuals. Based on this, this invention aims to investigate the diagnostic efficacy of ferritin-carrying neuronal-derived extracellular vesicles in the auxiliary diagnosis of Parkinson's disease.

[0042] Example 1

[0043] I. Experimental Materials

[0044] The included sample consisted of 10 patients with Parkinson's disease (PD), 10 patients with multiple system atrophy (MSA), and 10 healthy individuals (HC). Informed consent was obtained from all participants' families (legal representatives), and the samples were reviewed by the ethics committee.

[0045] Plasma sample preparation: Venous blood samples were collected from fasting participants in the morning using test tubes coated with EDTA. The blood samples were then centrifuged at 1500×g for 15 minutes (4°C), and the supernatant was centrifuged at 12,000×g for 10 minutes (4°C). The supernatant plasma was stored at -80°C for nanoscale flow cytometry and nanoparticle tracking analysis (NTA).

[0046] Neuronal-derived EVs: EVs labeled with the anti-L1 cell adhesion molecule were isolated from ultracentrifuged plasma EVs using an immunocapture protocol. 4 ml of plasma was added to a 4.2 ml ultracentrifuge tube, accurately weighed, and balanced with PBS. The balanced ultracentrifuge tube was placed in an SW 60ti horizontal rotor and centrifuged at 180,000 × g, 4°C, for 120 minutes. After ultracentrifugation, the tube was removed, and all supernatant was aspirated with a 1000 μl pipette, taking care not to touch the bottom of the tube. EVs labeled with the anti-L1 cell adhesion molecule were obtained.

[0047] Different solvents were selected for resuspending the neuronal-derived EVs according to the purpose of enriching them. When enriching neuronal-derived EVs for Western blot validation, RIPA was chosen to resuspend the lysed proteins. When enriching neuronal-derived EVs for transmission electron microscopy to observe EV morphology or for determining particle size using nanoparticle tracking analysis, 70 μl of 0.1 M glycine (pH=3) was used for resuspending, and the mixture was vortexed for 15 min to elute the immunocaptured neuronal-derived EVs. The suspension was then centrifuged at 100×g for 2 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube using a clean pipette tip. 5 μl of 1 M tris (pH=7) was added to balance the pH of the EV suspension. The resuspended EVs were then transferred to a 1.5 ml centrifuge tube. 10 μg of L1 celladhesion molecule antibody and 5 μl of 100× protease inhibitor were added to the 1.5 ml centrifuge tube, mixed well, and incubated overnight at 4°C on a rotary shaker. Cut off the tip of a 200 μl pipette and transfer 25 μl of Protein A / G agarose beads (sc-2003, Santa Cruz Biotechnology, Dallas, United States) into a 1.5 ml microcentrifuge tube. Rinse the agarose beads three times with 500 μl of 0.1% BSA, centrifuging at 100×g for 2 min each time, and aspirate the supernatant with a pipette. Add the Protein A / G agarose beads to the overnight incubated sample-antibody suspension, mix well, and place on a rotary shaker at 4°C for 3 hours. Centrifuge the suspension at 100×g for 2 min and aspirate the supernatant with a pipette. When enriching neuronal-derived EVs for immunoblotting verification, add 50 μl of 1× loading buffer, vortex for 15 min, centrifuge at 12000g×g for 2 min, and collect the supernatant with a pipette.

[0048] II. Experimental Methods

[0049] Transmission electron microscopy (TEM) analysis: 5 μl of L1 cell adhesion molecule-labeled EVs were placed on a copper grid coated with a carbon support film. After incubation for 60 seconds, excess sample was removed with filter paper. The copper grid was then stained with 2% uranium acetate solution for 60 seconds. Excess uranium acetate solution was removed with filter paper, and the sample was allowed to air dry. Electron microscopy images were acquired using a Hitachi H-7650 at 80 kV.

[0050] Western blot analysis: 15 μg of total protein in each sample was separated using a 4-20% Bis-Tris gel (M42010C, Genscript) and then electrotransferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the sample was incubated overnight at 4°C with anti-Anti-L1 cell adhesion molecule antibody and CD63 polyclonal antibody.

[0051] Nanoparticle tracking analysis: L1 cell adhesion molecule-labeled EVs were diluted 1:30 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.

[0052] Cytoflex nanoscale flow cytometry analysis of EVs: First, prepare the first labeling solution, using Zenon... TM Solution A (Zenon) in the Alexa Fluor 488 mouse IgG1 labeling kit TM Mix 5 μl of mouse IgG labeling reagent with 1 μg of L1 cell adhesion molecule-antibody (ab80832), and react at room temperature in the dark for 15 min. Then add 5 μl of solution B (Zenon) from the kit. TM After thoroughly mixing with the blocking reagent, react at room temperature in the dark for 5 minutes. Then prepare the second labeling solution using Zenon... TM Pacific Blue TM Solution A in the mouse IgG2a labeling kit (Zenon) TM Pacific Blue TM Mix 5 μl of the labeling reagent with 1 μg of monoclonal ferritin heavy chain antibody (sc-376594), mix thoroughly, and react at room temperature in the dark for 15 min. Then add 5 μl of solution B (Zenon) from the kit. TMThe first labeling solution was mixed thoroughly and reacted at room temperature in the dark for 5 minutes. Plasma samples were collected from the PD, MSA, and HC groups. 5 μl of plasma was taken from each sample, and 2 μl of the first labeling solution was added. After thorough mixing, the mixture was reacted at room temperature for 30 minutes. Then, 2 μl of the second labeling solution was added, and the mixture was reacted thoroughly at room temperature in the dark for 30 minutes. Finally, 200 μl of phosphate buffered saline solution was added and thoroughly mixed with the sample before testing. The number of labeled ferritin-carrying neuronal extracellular vesicles was detected using a Violet Side Scatter High (VSSC-H) flow cytometer at a medium flow rate of 30 μl / min. Three detection channels were set up to detect the labeling signals of the first and second labels, and to detect the labels of both labels simultaneously. The detection time for each sample was set to 30 seconds.

[0053] III. Data Processing

[0054] Statistical analysis was performed using GraphPad Prism 10. The Kruskal-Wallis test was used to assess intergroup differences in the mean concentration of ferritin-carrying neuronal EVs, followed by the Dunn test for post-hoc comparisons of PD, MSA, and HC. All tests were two-tailed, and statistical significance was defined as P < 0.05.

[0055] IV. Results and Analysis

[0056] 1. Results of transmission electron microscopy (TEM) analysis

[0057] like Figure 1 As shown in Figure A, the diameter of neuronal-derived EVs labeled with L1 cell adhesion molecule is approximately 100 nm.

[0058] 2. Western blot analysis results

[0059] like Figure 1 As shown in B, neuronal-derived EVs labeled with L1 cell adhesion molecule contain both L1CAM protein and the EV marker CD63 protein.

[0060] 3. Results of nanoparticle tracking analysis

[0061] like Figure 1 As shown in Figure C, the particle size of L1 cell adhesion molecule-labeled neuronal-derived EVs in plasma is mainly distributed around 100 nm, which is consistent with the particle size range of exosomes; the concentration of EVs with this particle size can reach 2.5 × 10⁻⁶. 8 / ml.

[0062] 4. Results of nanoscale flow cytometry analysis

[0063] like Figure 2 As shown, ferritin-carrying neuronal-derived EVs can effectively differentiate between PD, MSA, and HC. Among the 10 PD, 10 MSA, and 10 HC patients included, the concentration of ferritin-containing neuronal-derived EVs in the plasma of PD patients was significantly lower than that of MSA and HC patients (PD vs. HC: P<0.001; PD vs. MSA: P<0.01). Figure 2 This result indicates that neuronal-derived EVs carrying ferritin have the potential to aid in the diagnosis of PD.

[0064] Example 2

[0065] To validate the diagnostic utility of ferritin-carrying neuronal EVs in diagnosing Parkinson's disease (PD) and differentiating PD from MSA and PD from HC, this study included an independent validation cohort comprising 90 PD patients, 39 MSA patients, and 50 healthy individuals (HC). The plasma concentration of ferritin-carrying neuronal EVs in the included samples was measured according to the method described in Example 1. The Kruskal-Wallis test was used to assess inter-group differences in the mean concentration of ferritin-carrying neuronal EVs, and the Dunn test was used for post-hoc comparisons between PD, MSA, and HC. Finally, receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of this marker in differentiating PD, MSA, or HC. All tests were two-tailed, and statistical significance was defined as P < 0.05.

[0066] The results are as follows Figure 3 As shown, among the 90 PD patients, 39 MSA patients, and 50 HC patients included, the concentration of ferritin-containing neuronal EVs in the plasma of PD patients was significantly lower than that of MSA and HC patients (PD vs. HC: P<0.001; PD vs. MSA: P<0.001). Figure 3 ).

[0067] ROC analysis assessed the diagnostic utility of ferritin-carrying neuronal-derived EVs in distinguishing between PD and MSA, with an area under the curve (AUC) of 0.78 (95% CI 0.70 to 0.86). Figure 4 When distinguishing between PD and HC, the AUC value was 0.81 (95% CI 0.73 to 0.89). Figure 5 ).

[0068] The above results indicate that assessing the number or concentration of ferritin-carrying neuronal EVs in plasma can effectively differentiate between PD patients and MSA patients, as well as between PD patients and the HC population, thus achieving an auxiliary diagnostic effect for PD. Based on these results, a multivariate model was established using binary logistic regression, which included ferritin-carrying neuronal EVs, gender, and age, as shown in Tables 1 and 2. The multivariate logistic regression model for differentiating between PD and MSA is: logit(probability) = 8.347 - 6.330 × 10⁻¹⁰ -6 ×External vesicle concentration + 0.199 × gender + 0.053 × age, when this probability > 0.71, the subject is considered to be diagnosed with PD; the multivariate logistic regression model for distinguishing between PD and HC is: logit(probability) = 9.047 - 6.587 × 10 -6 ×External vesicle concentration + 0.600 × gender + 0.046 × age; when this probability > 0.65, the subject is considered to be diagnosed with PD. ROC analysis evaluated the diagnostic utility of the above multivariate logistic regression model in distinguishing between PD and MSA, with an area under the curve (AUC) of 0.818 (95% CI 0.742 to 0.889). Figure 6 When distinguishing between PD and HC, the AUC value was 0.848 (95% CI 0.781 to 0.905). Figure 7 ).

[0069] Table 1 shows the equation variables used to distinguish between PD and MSA in the multivariate logistic regression model.

[0070]

[0071] Table 2 shows the equation variables used to distinguish between PD and HC in the multivariate logistic regression model.

[0072]

[0073]

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of products for detecting biomarker concentrations in the preparation of products for diagnosing Parkinson's disease, characterized in that, The biomarker is a neuronal extracellular vesicle carrying ferritin; The concentration of neuronal extracellular vesicles carrying ferritin in the plasma of Parkinson's disease patients is lower than that in patients with multiple system atrophy and healthy individuals.

2. The application of neuronal extracellular vesicles carrying ferritin in constructing a multivariate logistic regression model to differentiate between multiple system atrophy and Parkinson's disease, characterized in that... The multivariate logistic regression model was constructed using the concentration of extracellular vesicles carrying ferritin in plasma, the subject's gender, and age as input variables.

3. A method for constructing a multivariate logistic regression model to differentiate between multiple system atrophy and Parkinson's disease, characterized in that, The multivariate logistic regression model was constructed using the concentration of extracellular vesicles carrying ferritin in plasma, the subject's gender, and age as input variables. The probability logit of the multivariate logistic regression model is 8.347 - 6.330 × 10⁻⁶. -6 × Concentration of extracellular vesicles from neurons carrying ferritin + 0.199 × sex + 0.053 × age; where the value is 1 for males and 0 for females. When the probability is greater than 0.71, the subject is diagnosed with Parkinson's disease; otherwise, it is diagnosed with multiple system atrophy.

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