WO2023179112A1 - 基于外泌体突触核蛋白的帕金森病早期诊断系统 - Google Patents

基于外泌体突触核蛋白的帕金森病早期诊断系统 Download PDF

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WO2023179112A1
WO2023179112A1 PCT/CN2022/138681 CN2022138681W WO2023179112A1 WO 2023179112 A1 WO2023179112 A1 WO 2023179112A1 CN 2022138681 W CN2022138681 W CN 2022138681W WO 2023179112 A1 WO2023179112 A1 WO 2023179112A1
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synuclein
parkinson
disease
exosomes
content
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French (fr)
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张宝荣
浦佳丽
颜轶群
张燚
温轶
郑冉
胡柳
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浙江大学
浙江养生堂天然药物研究所有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2864—Sleep disorders

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  • the invention relates to the application field of molecular biology technology, and specifically relates to an exosome-based early diagnostic marker for Parkinson's disease and its application.
  • Parkinson's disease is the second most common degenerative disease of the central nervous system in the world. Its core pathological process is the accumulation of misfolded ⁇ -synuclein ( ⁇ -synuclein or ⁇ -Syn for short) in the midbrain. Dopaminergic neuron deposition and neuronal degeneration and loss in the substantia nigra pars compacta cause motor dysfunction.
  • diagnosis of PD is mainly based on clinicians' empirical judgment of clinical symptoms, and most patients have lost more than 50% of midbrain dopamine neurons when PD is diagnosed. Therefore, an accurate and effective early prediction system is particularly necessary to prevent the onset of PD and delay disease progression. However, the insidious onset of PD makes early diagnosis difficult.
  • Exosomes are lipid bilayer membrane vesicles secreted by a variety of cells, with a diameter between 40-100nm. Exosomes contain RNA and abundant proteins and play an important role in the communication of information between cells. Studies have shown that the proteins in exosomes are relatively stable in the blood, and they have great potential as clinical diagnostic markers. They are also a hotspot and breakthrough point in diagnostic research for many diseases.
  • ⁇ -synuclein can spread between cells through exosomes as a carrier during the progression of PD, and can be secreted out of cells. Exosomes secreted by central neurons can enter the blood through the circulatory system and be detected in peripheral blood. This secretory activity begins before the onset of PD motor symptoms. Therefore, the ⁇ -synuclein content in peripheral blood exosomes is a possible early diagnostic marker for PD.
  • Rapid eye movement sleep disorder is widely considered to be a prodromal disease of PD, and there is an 80% chance of transforming into PD with age.
  • RBD Rapid eye movement sleep disorder
  • the current diagnosis of RBD relies on polysomnography, which is time-consuming and labor-intensive and has a high rate of missed diagnosis. It is difficult to distinguish it from other sleep problems clinically.
  • the purpose of the present invention is to provide an early diagnostic marker for Parkinson's disease based on exosomes.
  • the biomarker is ⁇ -synuclein, phosphorylated ⁇ -synuclein and ⁇ -synuclein. oligomers.
  • kits includes total exosome extraction reagent EXOQ (SBI (System Biosciences)), central source exosome isolation reagent (including antibody affinity magnetic beads, L1CAM (L1cell adhesion molecule) antibody and antibody elution reagent) and ⁇ -Synuclein detection reagents (including ⁇ -synuclein antibody, ⁇ -synuclein standard, excitation solution and luminescent solution).
  • EXOQ total exosome extraction reagent EXOQ
  • central source exosome isolation reagent including antibody affinity magnetic beads, L1CAM (L1cell adhesion molecule) antibody and antibody elution reagent
  • ⁇ -Synuclein detection reagents including ⁇ -synuclein antibody, ⁇ -synuclein standard, excitation solution and luminescent solution.
  • This kit should also include reagents and consumables such as sample diluent and cleaning solution used in the experiment.
  • the kit using ⁇ -synuclein as a marker can be used for the diagnosis of Parkinson's disease and the prodromal stage of Parkinson's disease, the diagnosis of rapid eye movement sleep disorder, the prediction of the progression of Parkinson's disease, or other purposes.
  • the present invention separates total exosomes and central-derived exosomes in human plasma and detects the ⁇ -synuclein content in them, making it possible to diagnose PD before clinical symptoms appear.
  • the invention discloses an early diagnosis system for Parkinson's disease based on exosomal synuclein.
  • This system includes three processes: exosome extraction, detection of ⁇ -synuclein in exosomes, and early diagnosis.
  • Exosome extraction includes extracting total exosomes and centrally derived exosomes from blood samples; detection of ⁇ -synuclein in exosomes uses ⁇ -synuclein antibodies, chemiluminescence immunoassay technology, and enzyme-linked Immunosorbent assay (ELISA) and other immune-related detection methods quantitatively calculate the content of ⁇ -synuclein in exosomes; the early diagnosis process is done by comparing the ⁇ -synuclein content in subject samples with that of healthy people.
  • ELISA enzyme-linked Immunosorbent assay
  • ⁇ -synuclein For comparison, if the level of ⁇ -synuclein is increased, it indicates that the patient is in the prodromal stage of Parkinson's disease.
  • the ⁇ -synuclein content in total plasma exosomes was slightly increased in RBD patients and significantly increased in PD patients.
  • Plasma centrally derived exosomes The content of ⁇ -synuclein in the body is significantly increased in both RBD and PD, which has early diagnostic value.
  • the ⁇ -synuclein content in total plasma exosomes increases with disease progression and has predictive value for disease progression.
  • the present invention provides an early diagnosis system for Parkinson's disease based on exosomal synuclein, which is characterized by detecting the ⁇ -synuclein content in blood-derived exosomes in the sample.
  • the detection reagents used include total exosome extraction reagents, central-derived exosome isolation reagents and alpha-synuclein content detection reagents.
  • the test sample includes the subject's serum or plasma.
  • the detection includes extracting exosomes, and the extracted exosomes include total exosomes derived from peripheral blood and exosomes derived from central nervous system.
  • detecting includes detecting alpha-synuclein, phosphorylated alpha-synuclein, and alpha-synuclein oligomers.
  • the detection method used for detection includes chemiluminescence immunoassay technology, enzyme-linked immunosorbent assay technology (ELISA) and other immune-related detection methods.
  • chemiluminescence immunoassay technology enzyme-linked immunosorbent assay technology (ELISA) and other immune-related detection methods.
  • ELISA enzyme-linked immunosorbent assay technology
  • the detection reagent used for the detection includes an alpha-synuclein antibody, including monomeric, oligomeric, and phosphorylated antibodies.
  • the system of the present invention is used for early diagnosis of Parkinson's disease and/or prodromal diagnosis of Parkinson's disease, optionally, for identifying patients with rapid eye movement sleep disorder and/or people at high risk for Parkinson's disease. .
  • the present invention provides use of a reagent for detecting ⁇ -synuclein content in exosomes in preparing a system for diagnosing early Parkinson's disease.
  • the diagnosis includes comparing the amount of alpha-synuclein in a sample of the subject to a healthy population, and if the amount of alpha-synuclein is elevated, it indicates that the subject is in prodromal form of Parkinson's disease. period stage.
  • the present invention provides the use of a reagent for detecting ⁇ -synuclein content in exosomes in preparing a system for identifying patients with rapid eye movement sleep disorder and/or people at high risk of Parkinson's disease.
  • reagents for detecting ⁇ -synuclein content in exosomes include total exosome extraction reagents, central-derived exosome isolation reagents, and ⁇ -synuclein content detection reagents.
  • the invention provides a biomarker, wherein the biomarker is selected from the group consisting of alpha-synuclein, phosphorylated alpha-synuclein and alpha-synuclein oligomers. , and the biomarkers are used for early diagnosis of Parkinson's disease based on exosomes.
  • the present invention provides the use of a biomarker in preparing a kit for early diagnosis of Parkinson's disease, wherein the biomarker is selected from the group consisting of ⁇ -synuclein, phosphorylated ⁇ -synuclein and ⁇ -Group of synuclein oligomers.
  • the present invention provides the use of a biomarker in preparing a diagnostic kit for the prodromal stage of Parkinson's disease, wherein the biomarker is selected from the group consisting of alpha-synuclein, phosphorylated alpha-synuclein and Groups of ⁇ -synuclein oligomers.
  • the present invention provides the use of a biomarker in preparing a rapid eye movement sleep disorder diagnostic kit, wherein the biomarker is selected from the group consisting of alpha-synuclein, phosphorylated alpha-synuclein and Groups of ⁇ -synuclein oligomers.
  • the present invention provides a method for early diagnosis of Parkinson's disease, which method includes the following steps:
  • the collected blood samples are plasma or serum;
  • the present invention provides a method for monitoring disease progression in a subject, the method comprising the following steps:
  • the collected blood samples are plasma or serum;
  • the present invention achieves the purpose of non-invasive, efficient and early diagnosis of Parkinson's disease through blood-derived markers. It is of great significance for the early diagnosis of Parkinson's disease, can improve the accuracy of diagnosis, and has certain significance for early clinical intervention and reduction of later disability. auxiliary guidance significance.
  • the present invention has the following advantages:
  • the present invention uses the exosomal protein ⁇ -synuclein as a marker for early diagnosis of PD. It has high sensitivity (0.750) and high specificity (0.688), and the area under the ROC curve is 0.741. It is an excellent marker for early diagnosis and/or PD. Or a completely new approach to treatment.
  • the present invention focuses on the diagnosis of PD in the prodromal stage and finds that centrally derived exosome ⁇ -synuclein increases long before the onset of PD.
  • the diagnosis of PD can be advanced to the RBD stage. It provides a good basis for early diagnosis and treatment, can be used as an early screening and progression prediction indicator for PD, and can be well applied in clinical practice.
  • the present invention establishes a method for extracting total exosomes and centrally derived exosomes from the plasma of PD patients, RBD patients and healthy people and detecting the content of ⁇ -synuclein in them, and provides a biomarker for early PD (RBD) diagnosis. Objects and their applications.
  • the present invention mainly enriches the total exosomes and centrally derived exosomes in the blood, detects the content of ⁇ -synuclein in them through chemiluminescence immunoassay technology, determines the amount of the detected analyte through the test group, and The detection group is used clinically to provide doctors with an effective auxiliary diagnostic reference.
  • the biomarker is ⁇ -synuclein, including phosphorylated ⁇ -synuclein and ⁇ -synuclein oligomers.
  • the blood sample used is serum or plasma.
  • the present invention provides an application of early PD diagnostic biomarkers based on blood exosomes.
  • the detection method of the biomarkers is chemiluminescence immunoassay technology, enzyme-linked immunosorbent assay technology (ELISA), and other immune-related detection methods. method.
  • the PD early diagnosis kit used mainly includes exosome extraction, isolation of centrally derived exosomes, and alpha-synuclein detection related reagents.
  • the PD early diagnosis kit contains an exosome extraction reagent, which is EXOQ from SBI Company.
  • the PD early diagnosis kit contains a center-derived exosome isolation reagent, which is an L1CAM antibody and magnetic beads (Dynabeads, Invitrogen).
  • the PD early diagnosis kit contains an ⁇ -synuclein detection reagent, which is an ⁇ -synuclein antibody labeled with biotin and acridinium ester and a chemiluminescent excitation solution.
  • Figure 1 shows the identification results of exosomes, (a) Western blot results of plasma exosome markers Alix and Flotillin; (b) Western blot results of Alix and L1CAM captured by anti-L1CAM or normal mouse IgG; (c) Exosome concentration and particle size detection; (d) Electron microscope observation of exosome morphology.
  • Figure 2 shows the ROC plot of ⁇ -synuclein content and diagnostic effect in total plasma exosomes and centrally derived exosomes.
  • Example 1 Preparation of total exosomes and centrally derived exosomes.
  • This example involves the preparation of total exosomes and centrally derived exosomes.
  • EXOQ reagent (EXOQ20A-1, SBI)
  • EXOQ20A-1 500 ⁇ l of diluted plasma and add 63 ⁇ l of EXOQ reagent, mix gently and let stand.
  • Figure 1 shows the identification results of exosomes of the present invention, wherein (a) Western blotting results of plasma exosome markers Alix and Flotillin; (b) Alix is captured by anti-L1CAM or normal mouse IgG. Western blot results of L1CAM and L1CAM; (c) Exosome concentration and particle size detection; (d) Electron microscope observation of exosome morphology.
  • the kit involved in the present invention contains: plasma total exosome extraction reagents: EXOQ (SBI), DPBS, RIPA lysis solution.
  • Extraction reagents for plasma center-derived exosomes L1CAM antibody, DPBS, antibody affinity magnetic beads.
  • Alpha-synuclein detection reagent labeled alpha-synuclein antibody, alpha-synuclein protein standard, IgG affinity magnetic beads, luminescence pre-excitation solution and excitation solution, magnetic bead cleaning solution.
  • the kit should also contain common consumables used during the procedure.
  • the present invention provides a kit consisting of the following reagents:
  • EXOQ SBI
  • DPBS E607009, Sangon Biotech
  • RIPA lysis buffer FD009, FD Biotech
  • protease inhibitor cocktail FD1001, FD Biotech
  • L1CAM antibody Human CAM antibody
  • IgG affinity magnetic beads Dynabeads, Invitrogen
  • DPBS E607009, Sangon Biotech
  • biotin-labeled ⁇ -synuclein antibody acridinium ester-labeled ⁇ -synuclein antibody
  • ⁇ -synuclein protein standard 12093 -HNAE, sino biological
  • BSA A600332, Sangon Biotech
  • pre-excitation solution main component is 0.2% hydrogen peroxide
  • excitation solution main component is 0.35M sodium hydroxide
  • magnetic bead cleaning buffer Tween- 20(A100777, Sangon Biotech).
  • the present invention provides help for clinical diagnosis and early screening of PD by isolating plasma exosomes and plasma center-derived exosomes from PD patients, RBD patients and age-matched healthy controls, and detecting ⁇ -synuclein content in them. . Specifically, it includes the following steps:
  • the collected subjects met the following conditions: (1) clinically confirmed new cases of PD and RBD; (2) excluding family history, brain trauma history, brain tumor history and other serious systemic diseases; (3) controls For healthy people matching the age of the patient.
  • the area under the ROC curve for ⁇ -synuclein in plasma center-derived exosomes for diagnosing PD is 0.761.
  • the ⁇ -synuclein content in total plasma exosomes increases with disease progression and has predictive value for disease progression (see Figure 2a).
  • the present invention provides a method for early diagnosis of PD, which method includes the following steps: (a) collecting blood samples from subjects, and the collected blood samples are plasma and serum. (b) Exosomes are extracted from collected blood samples. (c) Isolating centrally derived exosomes from collected blood samples. (d) Detect the ⁇ -synuclein content in the exosomes in steps (b) and (c) above, and calculate the ⁇ -synuclein protein expression value. (e) Compare the obtained protein expression value with the amount of the selected analyte in healthy subjects. If the expression of ⁇ -synuclein is increased, it indicates that the subject has RBD and is at high risk of PD. .
  • the collected subjects met the following conditions: (1) clinically confirmed new RBD cases; (2) excluding family history, brain trauma history, brain tumor history and other serious systemic diseases; (3) controls with Patients were age-matched to healthy individuals.
  • the present invention also provides a method for monitoring disease progression, which method includes the following steps: (a) collecting blood samples from subjects at different time points, and the collected blood samples are plasma and serum. (b) Exosomes are extracted from collected blood samples. (c) Isolating centrally derived exosomes from collected blood samples. (d) Detect the ⁇ -synuclein content in the exosomes in steps (b) and (c) above, and calculate the ⁇ -synuclein expression value.
  • the collected subjects met the following conditions: (1) clinically confirmed new PD cases; (2) excluding family history, brain trauma history, brain tumor history and other serious systemic diseases; (3) controls with Patients were age-matched to healthy individuals.

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Abstract

一种基于外泌体突触核蛋白的帕金森病早期诊断系统。在对患者的实验中，发现与健康对照组比较，血浆总外泌体中的α-突触核蛋白含量在RBD患者中轻度升高，在PD患者中显著升高，血浆中枢来源外泌体中α-突触核蛋白含量在RBD和PD中均显著升高，具有早期诊断价值。血浆总外泌体中的α-突触核蛋白含量随疾病进展而升高，具有预测疾病进展价值。通过血液来源的标记物达到了无创、高效、早期诊断帕金森病的目的，对于早期诊断帕金森病具有重要意义，可以提高诊断的准确性，对临床早期干预、减少后期残疾，具有一定的辅助指导意义。

Description

基于外泌体突触核蛋白的帕金森病早期诊断系统 发明领域
本发明涉及分子生物学技术的应用领域，具体涉及一种基于外泌体的帕金森病早期诊断标志物及其应用。
发明背景
帕金森病(Parkinson’s Disease,PD)是全球第二大常见中枢神经系统退行性疾病，其核心病理过程是错误折叠的α-突触核蛋白(α-synuclein或简称为α-Syn)在中脑黑质致密部多巴胺能神经元沉积和神经元变性丢失造成运动功能障碍。目前PD的诊断主要基于临床医生对于临床症状的经验性判断，而大多数患者在诊断PD时中脑多巴胺神经元就已损失50％以上。因此，准确有效的早期预测系统对于预防PD起病、延缓疾病进展尤为必要。然而，PD的隐匿性发作使早期诊断变得困难。
外泌体是一种由多种细胞分泌的脂质双层膜囊泡，直径在40-100nm之间。在外泌体中包含RNA和丰富的蛋白质，在细胞间信息交流过程中发挥着重要作用。研究表明外泌体中的蛋白在血液中比较稳定，其作为临床诊断标志物的潜力很大，也是众多疾病诊断研究的热点和突破口。
近年来研究发现，α-突触核蛋白在PD进展过程中可通过外泌体作为载体在细胞间传播，并可向细胞外分泌。中枢神经元分泌的外泌体可通过循环系统进入血液并在外周血中检测。这种分泌的活动在PD运动症状出现之前就开始出现了。因此外周血外泌体α-突触核蛋白含量是可能的PD早期诊断标志物。
快动眼睡眠障碍(rapid eye movement sleep behavior disorder，RBD)被广泛认为是PD的前驱期疾病，随着年龄的增长有80％的可能转变为PD。但是目前RBD的诊断有赖于多导睡眠监测，费时费力且漏诊率高，在临床上很难将其与其他睡眠问题区分开。
发明内容
本发明的目的在于提供一种基于外泌体的帕金森病早期诊断标志物，所述的生物标记物为α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体。
本发明的另一个目的是提供所述生物标记物在制备PD早期诊断试剂盒中的应 用。所述试剂盒包括总外泌体提取试剂EXOQ(SBI(System Biosciences))，中枢来源外泌体分离试剂(包括抗体亲和磁珠，L1CAM(L1cell adhesion molecule)抗体和抗体洗脱试剂)和α-突触核蛋白检测试剂(包括α-突触核蛋白抗体，α-突触核蛋白标准品，激发液和发光液)。本试剂盒还应包括实验中用到的样品稀释液和清洗液等试剂和耗材。
以α-突触核蛋白为标志物的试剂盒可用于帕金森病及帕金森病前驱期的诊断、快动眼睡眠障碍的诊断、帕金森病疾病进展预测等用途，或其他用途。
本发明分离了人血浆中的总外泌体和中枢来源外泌体并检测其中α-突触核蛋白含量，使PD出现临床症状前对其做出诊断成为可能。
本发明公开了一种基于外泌体突触核蛋白的帕金森病早期诊断系统。本系统包括外泌体提取、外泌体中α-突触核蛋白(α-synuclein)的检测和早期诊断三个过程。外泌体提取包括从血液样品中提取总外泌体和中枢来源外泌体；外泌体中α-突触核蛋白检测使用α-突触核蛋白抗体、应用化学发光免疫分析技术、酶联免疫吸附测定技术(ELISA)及其他免疫相关的检测方法定量计算外泌体中α-突触核蛋白的含量；早期诊断过程通过将受试者样品中的α-突触核蛋白含量与健康人群作比较，若α-突触核蛋白含量升高，则表明处于帕金森病前驱期阶段。在对患者的实验中，发现与健康对照组比较，血浆总外泌体中的α-突触核蛋白含量在RBD患者中轻度升高，在PD患者中显著升高，血浆中枢来源外泌体中α-突触核蛋白含量在RBD和PD中均显著升高，具有早期诊断价值。血浆总外泌体中的α-突触核蛋白含量随疾病进展而升高，具有预测疾病进展价值。
因此，在一方面，本发明提供一种基于外泌体突触核蛋白的帕金森病早期诊断系统，其特征在于，通过检测样品中血液来源外泌体中α-突触核蛋白含量，实现帕金森病的早期诊断，所使用的检测试剂包括总外泌体提取试剂，中枢来源外泌体分离试剂和α-突触核蛋白含量检测试剂。
在一个实施方案中，检测样品包括受试者的血清或血浆。
在另一个实施方案中，检测包括提取外泌体，所提取的外泌体包括外周血来源的总外泌体和中枢来源外泌体。
在又一个实施方案中，检测包括检测α-突触核蛋白、磷酸化α-突触核蛋白和α-突触核蛋白寡聚体。
在另一个实施方案中，检测所使用的检测方法包括化学发光免疫分析技术、酶联 免疫吸附测定技术(ELISA)以及其他免疫相关的检测方法。
在又一个实施方案中，检测所使用的检测试剂包含α-突触核蛋白抗体，所述α-突触核蛋白包括单体、寡聚体和磷酸化抗体。
在另一个实施方案中，本发明的系统用于帕金森病早期诊断和/或帕金森病前驱期诊断，任选地，用于鉴别快动眼睡眠障碍患者和/或帕金森病高风险人群。
在另一方面，本发明提供检测外泌体中α-突触核蛋白含量的试剂在制备用于诊断早期帕金森病的系统中的用途。
在一个实施方案中，诊断包括通过将受试者样品中的α-突触核蛋白含量与健康人群作比较，若α-突触核蛋白含量升高，则表明受试者处于帕金森病前驱期阶段。
在又一方面，本发明提供检测外泌体中α-突触核蛋白含量的试剂在制备用于鉴别快动眼睡眠障碍患者和/或帕金森病高风险人群的系统中的用途。
在一个实施方案中，检测外泌体中α-突触核蛋白含量的试剂包括总外泌体提取试剂、中枢来源外泌体分离试剂和α-突触核蛋白含量检测试剂。
在另一方面，本发明提供一种生物标记物，其中所述生物标记物选自包括α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组，并且所述生物标记物用于基于外泌体早期诊断帕金森病。
在又一方面，本发明提供生物标记物在制备帕金森病早期诊断试剂盒中的用途，其中所述生物标记物选自包括α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组。
在另一方面，本发明提供生物标记物在制备帕金森病前驱期诊断试剂盒中的用途，其中所述生物标记物选自包括α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组。
在又一方面，本发明提供生物标记物在制备快动眼睡眠障碍诊断试剂盒中的用途，其中所述生物标记物选自包括α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组。
在另一方面，本发明提供生物标记物在制备帕金森病疾病进展预测试剂盒中的用途，其中所述生物标记物选自包括α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组。
在又一方面，本发明提供一种早期诊断帕金森病的方法，该方法包括以下步骤：
(a)收集受试者血液样品，所述受试者被怀疑患有快动眼睡眠障碍，任选地，所 收集的血液样品为血浆、血清；
(b)从所收集的血液样品中提取外泌体；
(c)从所收集的血液样品中分离中枢来源外泌体；
(d)检测(b)和(c)步骤中外泌体中α-突触核蛋白含量，计算α-突触核蛋白的蛋白表达值；
(e)将所得到的蛋白表达值与健康受试者的α-突触核蛋白的蛋白表达值比较，如果α-突触核蛋白表达升高，则表明受试者患有快动眼睡眠障碍，并存在帕金森病高风险。
在另一方面，本发明提供一种监测受试者疾病进展的方法，该方法包括以下步骤：
(a)在不同时间点收集受试者血液样品，所述受试者患有快动眼睡眠障碍，任选地，所收集的血液样品为血浆、血清；
(b)从所收集的血液样品中提取外泌体；
(c)从所收集的血液样品中分离中枢来源外泌体；
(d)检测(b)和(c)步骤中外泌体中α-突触核蛋白含量，计算α-突触核蛋白蛋白表达值；
(e)如果在稍后时间点中收集的样品中α-突触核蛋白含量较稍早时间点收集的样品中升高，则表明疾病由快动眼睡眠障碍向帕金森病进展。
本发明通过血液来源的标记物达到了无创、高效、早期诊断帕金森病的目的，对于早期诊断帕金森病具有重要意义，可以提高诊断的准确性，对临床早期干预、减少后期残疾，具有一定的辅助指导意义。
本发明与现有技术比较，具有如下优势：
1、本发明将外泌体蛋白α-突触核蛋白作为PD早期诊断的标志物，具有高灵敏度(0.750)和高特异性(0.688)，ROC曲线下面积0.741，为PD的早期诊断和/或治疗提供一条全新的途径。
2、本发明重点关注了PD在前驱期的诊断，发现中枢来源外泌体α-突触核蛋白在PD发病前很早一段时间就出现升高，可以将PD的诊断提前到RBD时期，为早期诊断和治疗提供良好依据，可以作为PD早期筛查、进展预测指标，能够很好的应用于临床上。
本发明建立了提取PD患者、RBD患者和健康人血浆中总外泌体及中枢来源外泌体并检测其中α-突触核蛋白的含量的方法，提供一种早期PD(RBD)诊断生物标志 物及其应用。
本发明主要通过富集血液中的总外泌体和中枢来源外泌体，通过化学发光免疫分析技术检测其中α-突触核蛋白的含量，通过测试组确定被检分析物量值，并在检测组进行临床应用，从而为医生提供有效的辅助诊断参考。
所述的生物标记物为α-突触核蛋白，包括磷酸化α-突触核蛋白及α-突触核蛋白寡聚体。优选的，使用的血液样品为血清或血浆。本发明提供了一种基于血液外泌体的早期PD诊断生物标记物的应用，生物标记物的检测方法为化学发光免疫分析技术，酶联免疫吸附测定技术(ELISA)，及其他免疫相关的检测方法。所用PD早期诊断试剂盒主要包括外泌体提取，中枢来源外泌体分离，α-突触核蛋白检测相关试剂。其中，所述的PD早期诊断试剂盒中含有外泌体提取试剂，该试剂为SBI公司的EXOQ。所述的PD早期诊断试剂盒中含有中枢来源外泌体分离试剂，该试剂为L1CAM抗体及磁珠(Dynabeads，Invitrogen)。所述的PD早期诊断试剂盒中含有α-突触核蛋白检测试剂，试剂为使用生物素和吖啶酯标记的α-突触核蛋白抗体及化学发光激发液。
以上所述仅是本发明的优选实施方式。应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和补充，这些改进和补充也应视为在本发明的保护范围内。
附图说明
图1显示外泌体的鉴定结果，(a)血浆外泌体标记物Alix、Flotillin的蛋白质印迹结果；(b)通过抗L1CAM捕获、或正常小鼠IgG捕获，Alix和L1CAM的蛋白质印迹结果；(c)外泌体浓度粒径检测；(d)电镜观察外泌体形态。
图2显示血浆总外泌体和中枢来源外泌体中α-突触核蛋白含量和诊断效果的ROC图。(a)血浆总外泌体α-突触核蛋白含量在PD、RBD和健康对照中的比较。(b)血浆中枢来源外泌体α-突触核蛋白含量在PD、RBD和健康对照中的比较。(c)血浆中枢来源外泌体中α-突触核蛋白诊断PD的ROC图。(d)血浆中枢来源外泌体诊断RBD的ROC图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这 些等价形式同样落于本申请所附权利要求书所限定的范围。需要说明的是，以下实施例中所提供的图示仅以示意方式说明本发明的基本构想，应当理解的是，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例
实施例1——总外泌体及中枢来源外泌体的制备。
本实施例涉及制备总外泌体和中枢来源外泌体。
1.1血浆总外泌体的提取
1.1.1被试者血浆使用DPBS(E607009，Sangon Biotech)1:1稀释，混合均匀。
1.1.2按照EXOQ试剂(EXOQ20A-1，SBI)说明书，取500μl稀释后的血浆加入63μl EXOQ试剂，轻柔混合并静置。
1.1.3离心得到外泌体沉淀，使用200μl DPBS或200μl RIPA裂解液(FD009,弗德生物)吹打重悬，得到待检测样品。
1.2血浆中枢来源外泌体的提取
1.2.1取被试者血浆900μl加入300μl DPBS稀释。
1.2.2将L1CAM抗体(Huabio ET1703-51)与稀释后的血浆充分混合，加入0.25mg IgG亲和磁珠(Dynabeads，Invitrogen)4℃过夜。
1.2.3使用RIPA或DPBS，100℃加热10分钟将外泌体及抗体洗脱，得到待检测样品。
1.3外泌体鉴定。
1.3.1提取出的外泌体使用2％组织固定液固定，经醋酸铀染色后使用透射电镜在100kV下观察外泌体形态。
1.3.2将提取出的外泌体使用RIPA裂解，经蛋白质电泳鉴定外泌体表面标记物Alix和Flotillin以及中枢来源外泌体标记物L1CAM。
1.3.3提取出的外泌体使用Zetaview纳米颗粒跟踪分析仪进行浓度径粒检测。
图1显示的是本发明的外泌体的鉴定结果，其中，(a)血浆外泌体标记物Alix、Flotillin的蛋白质印迹结果；(b)通过抗L1CAM捕获、或正常小鼠IgG捕获，Alix和L1CAM的蛋白质印迹结果；(c)外泌体浓度粒径检测；(d)电镜观察外泌体形态。
实施例2——试剂盒组成
本发明涉及的试剂盒包含：血浆总外泌体提取试剂：EXOQ(SBI)，DPBS，RIPA裂解液。血浆中枢来源外泌体提取试剂：L1CAM抗体，DPBS，抗体亲和磁珠。α-突触核蛋白检测试剂：带标签的α-突触核蛋白抗体，α-突触核蛋白蛋白标准品，IgG亲和磁珠，发光预激发液和激发液，磁珠清洗液。试剂盒还应包含操作过程中使用的常规耗材。
示例性地，本发明提供由以下试剂组成的试剂盒：
EXOQ(SBI)，DPBS(E607009，Sangon Biotech)，RIPA裂解液(FD009,弗德生物)，蛋白酶抑制剂混合物(FD1001，弗德生物)，L1CAM抗体(Huabio ET1703-51)，IgG亲和磁珠(Dynabeads，Invitrogen)，DPBS(E607009，Sangon Biotech)，生物素标记的α-突触核蛋白抗体，吖啶酯标记的α-突触核蛋白抗体，α-突触核蛋白蛋白标准品(12093-HNAE，sino biological)，BSA(A600332，Sangon Biotech)，预激发液(主要成分为0.2％过氧化氢)，激发液(主要成分为0.35M氢氧化钠)，磁珠清洗缓冲液，Tween-20(A100777，Sangon Biotech)。
实施例3——试剂盒应用实验
本发明通过分离PD患者、RBD患者及与其年龄匹配的健康对照的血浆外泌体和血浆中枢来源外泌体，检测其中α-突触核蛋白含量，为PD的临床诊断和早期筛查提供帮助。具体包括以下步骤：
3.1临床样本收集
收集自2019年起至今于浙江大学附属第二医院以标准操作程序(SOP)诊断的PD患者54例，RBD患者149例，并收集健康对照55例，并收集整理相应的病例资料(包括年龄、发病时间、运动症状评分、非运动症状评分等)。后因样本质量问题排除至PD患者44人，RBD患者101人，健康对照48人。
收集的受试者符合以下几个条件：(1)临床确诊的新发PD和RBD病例；(2)排除家族史、脑外伤史、脑肿瘤史和其他严重的系统性疾病；(3)对照为与患者年龄匹配的健康人。
3.2外泌体蛋白提取
3.2.1使用EXOQ(SBI)试剂提取被试者血浆总外泌体
3.2.2使用L1CAM抗体利用免疫复合物方法收集血浆中枢来源外泌体
3.2.3使用RIPA裂解液提取外泌体总蛋白
3.3通过化学发光免疫分析技术检测外泌体α-突触核蛋白含量。
3.3.1将α-突触核蛋白按照32ng/ml、16ng/ml、8ng/ml、4ng/ml、2ng/ml、1ng/ml、0.5ng/ml、0.25ng/ml、0.125ng/ml的梯度配制标准溶液。
3.3.2将蛋白标准品和待测样品分别与带生物素标签的α-突触核蛋白抗体、带吖啶酯标签的α-突触核蛋白抗体和IgG亲和磁珠共同孵育。
3.3.3将磁珠上的蛋白洗脱，分别向洗脱液中加入预激发液和激发液，检测样品的发光值。
3.3.4根据蛋白标准品发光值绘制标准曲线，并根据标准曲线公式和待测样品发光值计算待测样品浓度。
3.4数据分析：所有统计检验均使用GraphPad Prism软件8(圣地亚哥，CA)进行。数据以平均值±SEMs表示。P<0.05为差异有统计学意义。
3.5结果分析
如图2a所示，与健康对照组比较，血浆总外泌体中的α-突触核蛋白含量在RBD患者中轻度升高，在PD患者中显著升高。如图2c蓝色曲线所示，血浆总外泌体中的α-突触核蛋白诊断PD具有高灵敏度(0.750)和高特异性(0.688)，ROC曲线下面积0.741。如图2b所示，与健康对照组比较，血浆中枢来源外泌体中α-突触核蛋白含量在RBD和PD中均显著升高，具有早期诊断价值。如图2c绿色曲线所示，血浆中枢来源外泌体中α-突触核蛋白诊断PD的ROC曲线下面积为0.761。血浆总外泌体中的α-突触核蛋白含量随疾病进展而升高，具有预测疾病进展价值(参见图2a)。
实施例4——PD早期诊断
本发明提供PD早期诊断的方法，该方法包括以下步骤：(a)收集受试者血液样品，所收集的血液样品为血浆、血清。(b)从所收集的血液样品中提取外泌体。(c)从所收集的血液样品中分离中枢来源外泌体。(d)检测上述(b)、(c)步骤中外泌体中α-突触核蛋白含量，计算α-突触核蛋白蛋白表达值。(e)将所得到的蛋白表达值与健康受试者选定被检分析物量值比较，如α-突触核蛋白表达升高，则表明受试者患有RBD，并存在PD高风险。
4.1实验样本收集
收集自2019年起至今筛查到的RBD患者149例，并收集健康对照55例，并收 集整理相应的病例资料(包括年龄、发病时间、运动症状评分、非运动症状评分等)。后因样本质量问题排除至RBD患者101人，健康对照48人。
收集的受试者符合以下几个条件：(1)临床确诊的新发RBD病例；(2)排除家族史、脑外伤史、脑肿瘤史和其他严重的系统性疾病；(3)对照为与患者年龄匹配的健康人。
4.2提取中枢来源外泌体蛋白
4.2.1使用L1CAM抗体利用免疫复合物方法收集血浆中枢来源外泌体
4.2.2使用RIPA裂解液提取外泌体总蛋白
4.3通过化学发光免疫分析技术检测外泌体α-突触核蛋白含量。
4.4数据分析：所有统计检验均使用GraphPad Prism软件8(圣地亚哥，CA)进行。数据以平均值±SEMs表示。P<0.05为差异有统计学意义。
4.5结果分析
如图2b所示，与健康对照相比，RBD患者中枢来源外泌体中α-突触核蛋白含量明显升高。如图2d所示，中枢来源外泌体中α-突触核蛋白诊断RBD的ROC曲线下面积为0.727。以上数据说明中枢来源外泌体中α-突触核蛋白含量可以在PD前驱期做出诊断。
实施例5——监测疾病进展
本发明还提供监测疾病进展的方法，该方法包括以下步骤：(a)在不同时间点收集受试者血液样品，所收集的血液样品为血浆、血清。(b)从所收集的血液样品中提取外泌体。(c)从所收集的血液样品中分离中枢来源外泌体。(d)检测上述(b)、(c)步骤中外泌体中α-突触核蛋白含量，计算α-突触核蛋白表达值。(e)检测在稍后时间点中收集的样品中α-突触核蛋白含量，若在稍后时间点中收集的样品中α-突触核蛋白含量相比在较稍早时间点收集的样品中升高，则表明受试者的疾病由RBD向PD进展。
5.1实验样本收集
收集自2019年起至今于浙江大学附属第二医院以标准操作程序(SOP)诊断的PD患者54例，并收集健康对照55例，并收集整理相应的病例资料(包括年龄、发病时间、运动症状评分、非运动症状评分等)。后因样本质量问题排除至PD患者44人，健康对照48人。
收集的受试者符合以下几个条件：(1)临床确诊的新发PD病例；(2)排除家族史、 脑外伤史、脑肿瘤史和其他严重的系统性疾病；(3)对照为与患者年龄匹配的健康人。
4.2提取血浆总外泌体蛋白
4.2.1使用EXOQ试剂盒提取血浆外泌体
4.2.2使用RIPA裂解液提取外泌体总蛋白
4.3通过化学发光免疫分析技术检测外泌体α-突触核蛋白含量。
4.4数据分析：所有统计检验均使用GraphPad Prism软件8(圣地亚哥，CA)进行。数据以平均值±SEMs表示。P<0.05为差异有统计学意义。
4.5结果分析
结果发现α-突触核蛋白含量与帕金森病统一评定量表UPDRS III评分成正相关(p＝0.0041)。这提示外泌体中α-突触核蛋白含量具有检测疾病进展的价值。
需要说明的是，以上实施例仅用以说明本发明的技术方案而非限制。尽管本文已经参考较佳实施方案对本发明进行了详细的说明，但是本领域的普通技术人员应当理解的是，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的保护范围中。

Claims (15)

  1. 一种基于外泌体突触核蛋白的帕金森病早期诊断系统，其特征在于，通过检测样品中血液来源外泌体中α-突触核蛋白含量，实现帕金森病的早期诊断，所使用的检测试剂包括总外泌体提取试剂、中枢来源外泌体分离试剂和α-突触核蛋白含量检测试剂。
  2. 根据权利要求1所述的帕金森病早期诊断系统，其特征在于，所检测的样品包括受试者的血清或血浆。
  3. 根据权利要求1或2所述的帕金森病早期诊断系统，其特征在于，所述检测包括提取外泌体，所提取的外泌体包括外周血来源的总外泌体和中枢来源外泌体。
  4. 根据权利要求1或2所述的帕金森病早期诊断系统，其特征在于，所述检测包括检测α-突触核蛋白、磷酸化α-突触核蛋白和α-突触核蛋白寡聚体。
  5. 根据权利要求1或2所述的帕金森病早期诊断系统，其特征在于，所述检测所使用的检测方法包括化学发光免疫分析技术、酶联免疫吸附测定技术以及其他免疫相关的检测方法。
  6. 根据权利要求1或2所述的帕金森病早期诊断系统，其特征在于，所述检测所使用的检测试剂包含α-突触核蛋白抗体，所述α-突触核蛋白包括单体、寡聚体和磷酸化抗体。
  7. 根据权利要求1或2所述的帕金森病早期诊断系统，其特征在于，其用于帕金森病早期诊断和/或帕金森病前驱期诊断，或者其用于鉴别快动眼睡眠障碍患者和/或帕金森病高风险人群。
  8. 检测外泌体中α-突触核蛋白含量的试剂在制备用于诊断早期帕金森病的系统中或者在制备用于鉴别快动眼睡眠障碍患者和/或帕金森病高风险人群的系统中的用 途。
  9. 根据权利要求8所述的用途，其中所述试剂盒的使用包括包括将受试者样品中的α-突触核蛋白含量与健康人群作比较，若α-突触核蛋白含量升高，则表明受试者处于帕金森病前驱期阶段。
  10. 根据权利要求8或9所述的用途，其中检测外泌体中α-突触核蛋白含量的试剂包括总外泌体提取试剂、中枢来源外泌体分离试剂和α-突触核蛋白含量检测试剂。
  11. 一种生物标记物，其中所述生物标记物选自包括α-突触核蛋白，磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组，并且所述生物标记物用于基于外泌体早期诊断帕金森病。
  12. 生物标记物在制备帕金森病早期诊断试剂盒、帕金森病前驱期诊断试剂盒、快动眼睡眠障碍诊断试剂盒或者帕金森病疾病进展预测试剂盒中的用途，其中所述生物标记物选自包括α-突触核蛋白、磷酸化α-突触核蛋白及α-突触核蛋白寡聚体的组。
  13. 根据权利要求12所述的用途，其中所述试剂盒包括总外泌体提取试剂、中枢来源外泌体分离试剂和α-突触核蛋白含量检测试剂。
  14. 一种早期诊断帕金森病的方法，该方法包括以下步骤：
    (a)收集受试者血液样品，所述受试者被怀疑患有快动眼睡眠障碍，任选地，所收集的血液样品为血浆、血清；
    (b)从所收集的血液样品中提取外泌体；
    (c)从所收集的血液样品中分离中枢来源外泌体；
    (d)检测(b)和(c)步骤中外泌体中α-突触核蛋白含量，计算α-突触核蛋白的蛋白表达值；
    (e)将所得到的蛋白表达值与健康受试者的α-突触核蛋白的蛋白表达值比较，如果α-突触核蛋白表达升高，则表明受试者患有快动眼睡眠障碍，并存在帕金森病高风险。
  15. 一种监测受试者疾病进展的方法，该方法包括以下步骤：
    (a)在不同时间点收集受试者血液样品，所述受试者患有快动眼睡眠障碍，任选地，所收集的血液样品为血浆、血清；
    (b)从所收集的血液样品中提取外泌体；
    (c)从所收集的血液样品中分离中枢来源外泌体；
    (d)检测(b)和(c)步骤中外泌体中α-突触核蛋白含量，计算α-突触核蛋白蛋白表达值；
    (e)如果在稍后时间点中收集的样品中α-突触核蛋白含量较稍早时间点收集的样品中升高，则表明疾病由快动眼睡眠障碍向帕金森病进展。
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