WO2021228067A1 - 一种外周体液多标生物标志物检测中枢神经***疾病的方法和*** - Google Patents

一种外周体液多标生物标志物检测中枢神经***疾病的方法和*** Download PDF

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WO2021228067A1
WO2021228067A1 PCT/CN2021/092983 CN2021092983W WO2021228067A1 WO 2021228067 A1 WO2021228067 A1 WO 2021228067A1 CN 2021092983 W CN2021092983 W CN 2021092983W WO 2021228067 A1 WO2021228067 A1 WO 2021228067A1
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nervous system
central nervous
disease
protein
derived
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WO2021228067A8 (zh
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韩卿卿
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北京新源长青生物科技有限公司
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Priority to CN202180034267.0A priority Critical patent/CN116601305A/zh
Priority to JP2022568816A priority patent/JP2023540831A/ja
Priority to EP21805000.3A priority patent/EP4151747A1/en
Priority to US17/924,511 priority patent/US20230176075A1/en
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Definitions

  • the present invention relates to the field of biomedical technology, in particular to the technical field of in vitro diagnostic testing, and to methods and systems for detection using dual markers and/or multiple markers, preferably simultaneous detection using dual markers and/or multiple markers Method and system.
  • biomarker detection plays an increasingly important role in assisting disease diagnosis, differential diagnosis, or disease progression monitoring.
  • central nervous system disease biomarkers is mainly developed based on the detection of cerebrospinal fluid and the direct detection of peripheral body fluid.
  • the performance of cerebrospinal fluid detection is better, because cerebrospinal fluid is in direct contact with the brain and spinal cord and can better reflect the changes in the central nervous system.
  • due to the traumatic nature of cerebrospinal fluid acquisition and poor patient acceptance it greatly limits the in-depth development, clinical transformation and wide application of cerebrospinal fluid biomarkers.
  • peripheral body fluids are not ideal, and there are mainly two problems: On the one hand, due to the existence of the blood-brain barrier, not all components derived from the central nervous system will be detected in the peripheral body fluids, even if they exist in the peripheral body fluids. The abundance of central nervous system-derived biomarkers, especially proteins, is also very low; on the other hand, because many central nervous system disease-related markers are not specifically expressed in the central nervous system, other peripheral organs and/or systems can also produce them. Signals from the central nervous system are extremely susceptible to the influence of peripheral components.
  • central nervous system diseases have brain-specific features.
  • the main pathological changes of Alzheimer's disease are the atrophy of the cerebral cortex such as the parietal lobe, temporal lobe, hippocampus, etc.
  • Parkinson's disease is mainly due to dopamine caused by pathological changes or damage of the substantia nigra-striatum dopamine circuit of the basal nucleus Caused by insufficient secretion.
  • the brain biopsy method can specifically detect and analyze the markers in different brain regions.
  • the existing in vitro detection techniques are unable to distinguish the source.
  • the diagnostic effect of different diseases is different, and the accuracy of the disease diagnosis is also different. There is room for improvement.
  • this application first proposes the concept and detection method for the diagnosis of central nervous system diseases by identifying biomarkers derived from the central nervous system in peripheral body fluids.
  • this application involves the following:
  • a method for diagnosing diseases of the central nervous system comprising:
  • central nervous system-derived markers Based on the detection results of central nervous system-derived markers and central nervous system disease-related markers, whether the subject suffers from a central nervous system disease is diagnosed.
  • the detection of the central nervous system-derived markers in the subject's biological fluid and the detection of the central nervous system disease-related markers in the subject's biological fluid are performed simultaneously.
  • Detecting the central nervous system-derived markers in the subject’s biological fluid refers to detecting the central nervous system-derived markers in the subject’s enriched biological samples.
  • Detecting the central nervous system disease-related markers in the biological fluid of the subject refers to detecting the central nervous system disease-related markers in the enriched biological sample of the subject.
  • the biological sample is an extracellular vesicle
  • the extracellular vesicles are extracellular vesicles derived from neurons, extracellular vesicles derived from astrocytes, extracellular vesicles derived from oligodendrocytes, or extracellular vesicles derived from microglia Bubble.
  • central nervous system disease is selected from one or more of the following: cerebrovascular disease, spinal cord disease, central nervous system infection, nervous system Genetic diseases, dysplasia of the nervous system, autonomic nervous system diseases, demyelinating diseases of the nervous system, epilepsy, and degenerative diseases of the nervous system.
  • the cerebrovascular disease includes cerebral infarction, cerebral hemorrhage, insufficient blood supply to the brain, and transient ischemic attack;
  • the myelopathy includes acute myelitis, polio, and syringomyelitis;
  • the central nervous system infections include encephalitis and meningitis;
  • the genetic diseases of the nervous system include neurocutaneous syndrome, spinocerebellar ataxia;
  • the developmental abnormalities of the nervous system include congenital hydrocephalus and cerebral palsy;
  • the autonomic nervous system disease is autonomic insufficiency
  • the neurodegenerative diseases include degenerative dementia, movement disorders, and prion diseases (Prion disease).
  • the degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and Lewy body dementia (DLB);
  • the movement disorders include motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), small chorea (Sydenham chorea), hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD).
  • MND motor neuron disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • SD small chorea
  • WD hepatolenticular degeneration
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • CBD cortical basal ganglia degeneration
  • the central nervous system-derived marker is a biomarker derived from the following neurons or cells: mature neurons, oligodendrocytes, star Glia cells, microglia, immature neurons, Schwann cells, radial glial cells, intermediate precursor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic Neurons, cholinergic neurons.
  • central nervous system-derived markers and/or central nervous system disease-related markers are each selected from one of protein, nucleic acid, and lipid Or two or three.
  • the central nervous system-derived marker is selected from one or more of the following: G Protein-Coupled Receptor 162 (GPR162) ), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1, HCN1, Gamma-Aminobutyric Acid Type A Receptor Subunit Delta, GABRD), neuroadhesion molecule 3 (Neuroligin 3, NLGN3), SHISA9, contactin-1 (contactin-1, CNTN1), NeuN, MAP2, neurofilament-L (neurofilament light, NF-L), neurofilament- M (neurofilament medium, NF-M), neurofilament-H (neurofilament heavy, NF-H), synaptophysin, syntaxin, PSD95, L1CAM, doublecortin, ⁇ 3 -Beta III tubulin, NeuroD1, TBR1, Stathmin 1, MPZ, NCAM, GAP43, S100,
  • GPR162 G Protein-Co
  • the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system;
  • the central nervous system-derived marker is one or two or more selected from L1CAM, SHISA9, CNPase, and GLT1 (EAAT2).
  • the central nervous system disease-related marker is a protein
  • the protein is selected from one or more of the following: A ⁇ 1-40 (A ⁇ 40) protein, A ⁇ 1- 42 (A ⁇ 42) protein, oligomeric A ⁇ (oligo-A ⁇ ) protein, tau protein, phosphorylated tau (p-tau) protein, ⁇ -synuclein protein ( ⁇ -synuclein protein), phosphorylation at position 129 ⁇ -synuclein (pS129) protein, oligomeric ⁇ -synuclein (oligomeric ⁇ -synuclein, oligo- ⁇ -syn) protein, prion protein.
  • the central nervous system disease-related marker is a nucleic acid
  • the nucleic acid is selected from one or more of the following: as Alzheimer's disease (AD) DNA or RNA as biomarkers, Parkinson’s disease (PD) and Parkinson’s syndrome (e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD), Lewy body DNA or RNA as a biomarker of dementia (DLB).
  • AD Alzheimer's disease
  • PD Parkinson’s disease
  • Parkinson’s syndrome e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD), Lewy body DNA or RNA as a biomarker of dementia (DLB).
  • the DNA or RNA used as a biomarker of AD includes: and genes UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3, RNF7, RPLPO, SPARC, WTAP, HNRNPU, LINC00265, INMT, SLC35E2, CT60, SYNCRIP, RGS2, SMC6, ARSA, SPDYE7P, SMIM17, TRAF3IP2-AS1, KCNC2, SLC24A1, HLCS, GOSR1, MN1, MGAT5, NBPF14, FBXO31, WDR52, TBC1D2B, ZNF648 DNA associated with NBPF16, PAGR1, AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1, BPTF
  • the DNA or RNA used as a biomarker of PD includes: and genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP, HNRNPAB, NXN, DNA or RNA associated with SLC9A4, EIF2AK1, PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5.
  • the step of detecting the central nervous system-derived marker in the subject’s biological fluid comprises: subjecting the subject’s biological fluid, preferably to The subject’s extracellular vesicles react with labeled antibodies that specifically react with the central nervous system-derived markers, and the intensity of the labeled signal after the reaction is detected to determine the presence and content of the central nervous system-derived markers.
  • the step of detecting the central nervous system disease-related marker in the biological fluid of the subject comprises: making the subject biological Body fluids, preferably the subject’s extracellular vesicles are reacted with labeled antibodies that specifically react with the central nervous system disease-related markers, and the intensity of the labeled signal after the reaction is detected to determine the central nervous system disease-related markers Existence and content.
  • label is selected from one or more of the following: fluorescent label, isotope label, enzyme label, chemiluminescent agent label, quantum dot label or colloidal gold label .
  • the step of detecting the central nervous system-derived marker in the biological fluid of the subject comprises: lysing the biological fluid of the subject, preferably The subject’s extracellular vesicles use DNA or RNA probes or sequencing technology to detect the presence and content of nucleic acids derived from the central nervous system.
  • the step of detecting the central nervous system disease-related marker in the biological fluid of the subject comprises: lysing the subject’s Biological fluids, preferably extracellular vesicles of the subject, and using DNA or RNA probes or sequencing technology to detect the presence and content of nucleic acids related to central nervous system diseases.
  • a system for diagnosing diseases of the central nervous system comprising:
  • the first detection module is used to detect the central nervous system-derived markers in the biological fluid of the subject,
  • the second detection module is used to detect the central nervous system disease-related markers in the biological fluid of the subject.
  • the diagnosis module diagnoses whether the subject suffers from the central nervous system disease based on the detection results of the central nervous system source markers and the central nervous system disease-related markers obtained by the first detection module and the second detection module, respectively.
  • the first detection module and the second detection module simultaneously detect central nervous system-derived markers in the subject's biological fluid and detect central nervous system disease-related markers in the subject's biological fluid;
  • the first detection module and the second detection module are the same module, which simultaneously detects the central nervous system-derived markers in the subject's biological fluid and detects the central nervous system disease-related markers in the subject's biological fluid .
  • the enrichment module is used to obtain the biological fluid of the subject and preprocess it to enrich the biological sample in the biological fluid,
  • the first detection module and the second detection module detect the central nervous system-derived markers and the central nervous system disease-related markers in the enriched biological sample of the subject.
  • a quantitative statistics module which counts the number of biological samples that detect both central nervous system-derived markers and central nervous system disease-related markers, and/or
  • a particle size measurement module which measures the particle size of a biological sample in which both central nervous system-derived markers and central nervous system disease-related markers are detected, and
  • the diagnosis module diagnoses whether the subject suffers from a central nervous system disease based on the number of the biological sample and/or the particle size of the biological sample.
  • the biological sample is an extracellular vesicle
  • the extracellular vesicles are extracellular vesicles derived from neurons, extracellular vesicles derived from astrocytes, extracellular vesicles derived from oligodendrocytes, or extracellular vesicles derived from microglia Bubble.
  • central nervous system disease is selected from one or more of the following: cerebrovascular disease, spinal cord disease, central nervous system infection, nervous system Genetic diseases, dysplasia of the nervous system, autonomic nervous system diseases, demyelinating diseases of the nervous system, epilepsy, and degenerative diseases of the nervous system.
  • the cerebrovascular disease includes cerebral infarction, cerebral hemorrhage, insufficient blood supply to the brain, and transient ischemic attack;
  • the myelopathy includes acute myelitis, polio, and syringomyelitis;
  • the central nervous system infections include encephalitis and meningitis;
  • the genetic diseases of the nervous system include neurocutaneous syndrome, spinocerebellar ataxia;
  • the developmental abnormalities of the nervous system include congenital hydrocephalus and cerebral palsy;
  • the autonomic nervous system disease is autonomic insufficiency
  • the neurodegenerative diseases include degenerative dementia, movement disorders, and prion diseases (Prion disease).
  • the degenerative degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and Lewy body dementia (DLB);
  • the movement disorders include motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), small chorea (Sydenham chorea), hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD).
  • MND motor neuron disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • SD small chorea
  • WD hepatolenticular degeneration
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • CBD cortical basal ganglia degeneration
  • the central nervous system-derived marker is a biomarker derived from the following neurons or cells: mature neurons, oligodendrocytes, astrocytes Glia cells, microglia, immature neurons, Schwann cells, radial glial cells, intermediate precursor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic Neurons, cholinergic neurons.
  • central nervous system-derived markers and/or central nervous system disease-related markers are each selected from one of protein, nucleic acid, and lipid Or two or three.
  • the central nervous system-derived marker is selected from one or two or more of the following: G Protein-Coupled Receptor 162 (GPR162), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1, HCN1, Gamma-Aminobutyric Acid Type A Receptor Subunit Delta, GABRD), neural adhesion molecule 3 (Neuroligin 3, NLGN3), SHISA9, contactin-1 (contactin-1, CNTN1), NeuN, MAP2, neurofilament-L (neurofilament light, NF-L), neurofilament-M ( neurofilament medium (NF-M), neurofilament-H (neurofilament heavy, NF-H), synaptophysin, syntaxin, PSD95, L1CAM, doublecortin, ⁇ 3-micro Beta III tubulin, NeuroD1, TBR1, Stathmin 1, MPZ, NCAM, GAP43, S100, CNPa
  • GPR162 G Protein-Coupled
  • the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system;
  • the central nervous system-derived marker is one or two or more selected from L1CAM, SHISA9, CNPase, and GLT1 (EAAT2).
  • the central nervous system disease-related marker is a protein
  • the protein is selected from one or more of the following: A ⁇ 1-40 (A ⁇ 40) protein, A ⁇ 1- 42 (A ⁇ 42) protein, oligomeric A ⁇ (oligo-A ⁇ ) protein, tau protein, phosphorylated tau (p-tau) protein, ⁇ -synuclein protein ( ⁇ -synuclein protein), phosphorylation at position 129 ⁇ -synuclein (pS129) protein, oligomeric ⁇ -synuclein (oligomeric ⁇ -synuclein, oligo- ⁇ -syn) protein, prion protein.
  • the central nervous system disease-related marker is a nucleic acid
  • the nucleic acid is selected from one or two or more of the following: Alzheimer's disease (AD) DNA or RNA as biomarkers, Parkinson’s disease (PD) and Parkinson’s syndrome (eg, multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD), Lewy body DNA or RNA as a biomarker of dementia (DLB).
  • AD Alzheimer's disease
  • PD Parkinson’s disease
  • Parkinson’s syndrome eg, multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD), Lewy body DNA or RNA as a biomarker of dementia (DLB).
  • the DNA or RNA used as a biomarker of AD includes: and genes UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3, RNF7, RPLPO, SPARC, WTAP, HNRNPU, LINC00265, INMT, SLC35E2, CT60, SYNCRIP, RGS2, SMC6, ARSA, SPDYE7P, SMIM17, TRAF3IP2-AS1, KCNC2, SLC24A1, HLCS, GOSR1, MN1, MGAT5, NBPF14, FBXO31, WDR52, TBC1D2B, ZNF648 DNA associated with NBPF16, PAGR1, AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B, ARHGEF5, PKD1,
  • DNA or RNA as a biomarker of PD includes: and genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP, HNRNPAB, NXN, DNA or RNA associated with SLC9A4, EIF2AK1, PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5.
  • the enrichment module has one or more of the following sub-modules: centrifugation, ultracentrifugation, ultrafiltration tube filtration, polymerization sedimentation, specific antibody capture .
  • the central nervous system-derived marker is a protein
  • the first detection module is used to make the subject’s biological fluid, preferably the subject’s extracellular vesicles, and the labeled
  • the antibody that specifically reacts with the central nervous system-derived markers reacts, and the intensity of the labeled signal after the reaction is detected to determine the presence and content of the central nervous system-derived markers.
  • the central nervous system disease-related marker is a protein
  • the second detection module is used to make the subject’s biological fluid, preferably the subject’s extracellular vesicles, and the labeled
  • the antibody that specifically reacts with the central nervous system disease-related markers reacts, and the intensity of the labeled signal after the reaction is detected to determine the presence and content of the central nervous system disease-related markers.
  • label is selected from one or more of the following: fluorescent label, isotope label, enzyme label, chemiluminescent agent label, quantum dot label or colloidal gold label .
  • the central nervous system-derived marker is a nucleic acid
  • the first detection module is used to lyse the subject’s biological fluid, preferably the subject’s extracellular vesicles and use DNA or RNA probe or sequencing technology to detect the presence and content of nucleic acids derived from the central nervous system.
  • the central nervous system disease-related marker is a nucleic acid
  • the second detection module is used to lyse the subject’s biological fluid, preferably the subject’s extracellular vesicles and use DNA or RNA probes or sequencing technology to detect the presence and content of nucleic acids related to central nervous system diseases.
  • composition for detecting central nervous system diseases comprising:
  • Antibodies used to target markers related to central nervous system diseases are used to target markers related to central nervous system diseases.
  • central nervous system disease is selected from one or more of the following: cerebrovascular disease, spinal cord disease, central nervous system infection, nervous system genetic disease, neurological disease Systemic dysplasia diseases, autonomic nervous system diseases, demyelinating diseases of the nervous system, epilepsy, and degenerative diseases of the nervous system.
  • the cerebrovascular disease includes cerebral infarction, cerebral hemorrhage, insufficient blood supply to the brain, and transient ischemic attack;
  • the myelopathy includes acute myelitis, polio, and syringomyelitis;
  • the central nervous system infections include encephalitis and meningitis;
  • the genetic diseases of the nervous system include neurocutaneous syndrome, spinocerebellar ataxia;
  • the developmental abnormalities of the nervous system include congenital hydrocephalus and cerebral palsy;
  • the autonomic nervous system disease is autonomic insufficiency
  • the neurodegenerative diseases include degenerative dementia, movement disorders, and prion diseases (Prion disease).
  • the degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and Lewy body dementia (DLB);
  • the movement disorders include motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), small chorea (Sydenham chorea), hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD).
  • MND motor neuron disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • SD small chorea
  • WD hepatolenticular degeneration
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • CBD cortical basal ganglia degeneration
  • the central nervous system-derived marker is a biomarker derived from the following neurons or cells: mature neurons, oligodendrocytes, Astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate precursor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonin Energetic neurons, cholinergic neurons.
  • the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system;
  • the central nervous system-derived marker is one or two or more selected from L1CAM, SHISA9, CNPase, and GLT1 (EAAT2).
  • a ⁇ 1-40 (A ⁇ 40) protein A ⁇ 1 -42 (A ⁇ 42) protein
  • oligomeric A ⁇ (oligo-A ⁇ ) protein tau protein, phosphorylated tau (p-tau) protein
  • ⁇ -synuclein protein ⁇ -synuclein protein
  • composition according to any one of items 45 to 51, wherein the antibody for targeting a marker derived from the central nervous system and the antibody for targeting a marker related to a central nervous system disease It is a labeled antibody, preferably the label is selected from one or more of the following: fluorescent label, isotope label, enzyme label, chemiluminescent agent label, quantum dot label or colloidal gold label.
  • a kit for detecting a central nervous system disease in a subject comprising:
  • Reagents for detecting central nervous system disease-related markers in the biological fluids of subjects are provided.
  • kit according to item 53 wherein the reagent for detecting central nervous system-derived markers in the biological fluid of the subject and the reagent for detecting central nervous system disease-related diseases in the biological fluid of the subject
  • the reagent for the marker is the composition described in any one of items 45 to 52.
  • the reagent for detecting the central nervous system-derived marker in the biological fluid of the subject is a primer or probe that targets the central nervous system-derived marker
  • the reagent for detecting the markers related to the central nervous system disease in the biological fluid of the subject is a primer or probe that targets the markers related to the central nervous system disease.
  • kit according to item 53 further comprising:
  • Reagents and devices for obtaining biological samples of subjects preferably reagents and devices for obtaining extracellular vesicles of subjects.
  • the central nervous system disease is selected from one or more of the following: cerebrovascular disease, spinal cord disease, central nervous system infection, nervous system genetic disease, neurological disease Systemic dysplasia diseases, autonomic nervous system diseases, demyelinating diseases of the nervous system, epilepsy, and degenerative diseases of the nervous system.
  • the cerebrovascular disease includes cerebral infarction, cerebral hemorrhage, insufficient blood supply to the brain, and transient ischemic attack;
  • the myelopathy includes acute myelitis, polio, and syringomyelitis;
  • the central nervous system infections include encephalitis and meningitis;
  • the genetic diseases of the nervous system include neurocutaneous syndrome, spinocerebellar ataxia;
  • the developmental abnormalities of the nervous system include congenital hydrocephalus and cerebral palsy;
  • the autonomic nervous system disease is autonomic insufficiency
  • the neurodegenerative diseases include degenerative dementia, movement disorders, and prion diseases (Prion disease).
  • the degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and Lewy body dementia (DLB);
  • the movement disorders include motor neuron disease (MND), Parkinson's disease (PD), Huntington's disease (HD), small chorea (Sydenham chorea), hepatolenticular degeneration (WD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia degeneration (CBD).
  • MND motor neuron disease
  • PD Parkinson's disease
  • HD Huntington's disease
  • SD small chorea
  • WD hepatolenticular degeneration
  • MSA multiple system atrophy
  • PSP progressive supranuclear palsy
  • CBD cortical basal ganglia degeneration
  • Using the method, system, kit and composition of the present application can effectively enrich and specifically label biomarkers that can reflect changes in central nervous system diseases from peripheral body fluids, compared to direct detection of disease-related markers in peripheral body fluids The diagnosis effect is better.
  • the method, system, kit and composition of the present application can effectively use peripheral body fluid for detection, thereby avoiding traumatic acquisition of the subject’s cerebrospinal fluid, greatly reducing the subject’s sampling risk and greatly reducing the difficulty of detection It greatly increases the clinical applicability and promotion of the test.
  • the core of this application is a method and system for detection using dual markers and/or multiple markers, which can realize simultaneous detection of dual markers and/or multiple markers, that is, using markers to confirm biological samples, especially extracellular
  • the part of the vesicle derived from the central nervous system uses disease-related markers to produce common detection results for disease diagnosis, differential diagnosis, tracking and drug evaluation services.
  • Figure 1A shows that in Example 1, the ratio of single-labeled SHISA9 protein-positive extracellular vesicles to the total number of extracellular vesicles in plasma was in the range of 60-100nm particle size, 100-150nm particle size, and full particle size range.
  • FIG. 1B shows that in Example 1, the ratio of the number of individually labeled p-tau217 protein-positive extracellular vesicles to the total number of extracellular vesicles in the plasma was in the range of 60-100nm particle size and the full particle size range.
  • Both AD group and CT group existed Very significant difference (****, p ⁇ 0.0001), in the 100-150nm particle size range, there is a significant difference between the AD group and the CT group (**, p ⁇ 0.01);
  • Figure 1C shows that in Example 1, the ratio of SHISA9 protein and p-tau217 protein double-positive extracellular vesicles to the total number of extracellular vesicles in the plasma was in the 100-150nm particle size range and the full particle size range.
  • the AD group and CT There are extremely significant differences between the groups (***, p ⁇ 0.001);
  • Figure 1D shows the receiver operating characteristic curve (ROC) analysis in Example 1:
  • the area under curve (AUC) of SHISA9 protein single label in the particle size range of 60-100nm is 0.749
  • SHISA9 The AUC of the double-labeled protein and p-tau217 protein in the 100-150nm particle size range is 0.890, using logistic regression analysis Enter method to include SHISA9 60-100nm and SHISA9+p-tau217 100-150nm data, AUC is 0.891;
  • Figure 1E shows the ROC analysis in Example 1:
  • the AUC of single-labeled p-tau217 protein in the particle size range of 60-100nm is 0.877
  • the AUC of SHISA9 protein and p-tau217 protein double-labeled in the particle size range of 100-150nm is 0.890.
  • Logistic regression was used to analyze the Enter method to include the data of p-tau217 60-100nm and SHISA9+p-tau217 100-150nm, and the AUC was 0.910.
  • Figure 2A shows the number of extracellular vesicles that are individually labeled for GLT1 protein and the number of extracellular vesicles that are individually labeled for ⁇ -syn protein (syn211 and MJFR14 are anti- ⁇ -syn protein antibodies) in Example 2;
  • Figures 2B and C show the number of extracellular vesicles that are individually labeled with GLT1 protein or ⁇ -syn protein in the plasma, and the number of extracellular vesicles that are double-positive for GLT1 protein and ⁇ -syn protein in the plasma.
  • Figures 3A and B show that in Example 2, the number of extracellular vesicles double-positive for GLT1 protein and ⁇ -syn protein in plasma was significantly higher in the PD group than in the MSA and CT groups (***, p ⁇ 0.001), and There was no significant difference between the MSA group and the CT group.
  • Figure 4A shows that in Example 3, the number of extracellular vesicles positive for CNPase protein in plasma has no significant difference between the MSA group and the PD group, but there is a significant difference between the MSA group and the HC group (*, p ⁇ 0.05) ;
  • Figure 4B shows that in Example 3, the number of extracellular vesicles double-positive for CNPase protein and ⁇ -syn protein (anti-MJFR14 antibody labeling) in plasma has no significant difference between MSA group, PD group and HC group;
  • Figure 4C shows that in Example 3, the number of extracellular vesicles double positive for CNPase protein and ⁇ -syn protein in plasma was normalized by the total number of extracellular vesicles in plasma. Difference (*, p ⁇ 0.05).
  • the present application relates to a method for diagnosing central nervous system diseases, which includes: detecting central nervous system-derived markers in the biological fluid of the subject, and detecting the central nervous system in the biological fluid of the subject Nervous system disease-related markers, and based on the detection results of central nervous system-derived markers and central nervous system disease-related markers to diagnose whether the subject suffers from a central nervous system disease.
  • the present application relates to a method for diagnosing central nervous system diseases, which includes: detecting central nervous system-derived markers in the biological fluid of the subject, and detecting the central nervous system in the biological fluid of the subject Nervous system disease-related markers, and based on the detection results of central nervous system-derived markers and central nervous system disease-related markers to diagnose whether a subject suffers from a central nervous system disease, wherein the detection of the subject’s biological fluid
  • the central nervous system-derived markers and the detection of the central nervous system disease-related markers in the subject's biological fluid are performed simultaneously.
  • the present application relates to a system for diagnosing central nervous system diseases, which includes: a first detection module, which is used to detect central nervous system-derived markers in a subject's biological fluid , The second detection module, which is used to detect the central nervous system disease-related markers in the biological fluid of the subject, and the diagnosis module, which is based on the central nervous system source markers obtained by the first detection module and the second detection module respectively.
  • the detection results of markers related to central nervous system diseases are used to diagnose whether the subject suffers from central nervous system diseases.
  • the present application relates to a system for diagnosing central nervous system diseases, which includes: a first detection module, which is used to detect central nervous system-derived markers in a subject's biological fluid , The second detection module, which is used to detect the central nervous system disease-related markers in the biological fluid of the subject, and the diagnosis module, which is based on the central nervous system source markers obtained by the first detection module and the second detection module respectively.
  • the detection results of markers related to central nervous system diseases are used to diagnose whether the subject suffers from central nervous system diseases.
  • the first detection module and the second detection module simultaneously detect central nervous system-derived markers in the subject's biological fluid and detect central nervous system disease-related markers in the subject's biological fluid; or
  • the first detection module and the second detection module are the same module, which simultaneously detects the central nervous system-derived markers in the subject's biological fluid and detects the central nervous system disease-related markers in the subject's biological fluid.
  • the inventor has developed a new method and system uniquely, that is, using dual markers and/or multiple markers with different functions for detection, especially At the same time, detection is performed by such dual markers and/or multiple markers, so that only peripheral body fluids can be used to effectively detect central nervous system diseases.
  • detection method and system do not need to extract the patient's cerebrospinal fluid, which greatly reduces the patient's sampling risk.
  • the accuracy of the detection can be greatly improved, and if the detection is performed at the same time, the overall detection efficiency can also be improved .
  • the method of the present application further includes: obtaining the biological fluid of the subject and preprocessing it to enrich the biological sample in the biological fluid, and detecting the central nervous system-derived marker in the biological fluid of the subject refers to detecting The central nervous system-derived markers in the subject’s enriched biological sample, and the detection of central nervous system disease-related markers in the subject’s biological fluid refers to the detection of central nervous system diseases in the subject’s enriched biological sample Related markers.
  • the method of the present application also includes counting the number of biological samples in which both the central nervous system-derived marker and the central nervous system disease-related marker are detected, and/or the measurement of the central nervous system-derived marker and the central nervous system Based on the particle size of the biological sample of both the system disease-related markers, whether the subject suffers from the central nervous system disease is diagnosed based on the number of the biological sample and/or the particle size of the biological sample.
  • biological samples in which both central nervous system-derived markers and central nervous system disease-related markers are detected are usually referred to as double-positive biological samples, and diagnosis is based on the number and/or particle size of double-positive biological samples. Whether the subject suffers from central nervous system disease.
  • system of the present application further includes: an enrichment module, which is used to obtain the biological fluid of the subject and preprocess it to enrich the biological sample in the biological fluid, wherein the first detection module and the second detection module Detect the central nervous system-derived markers and central nervous system disease-related markers in the enriched biological samples of the subject.
  • the system of the present application further includes: a quantity statistics module, which counts the number of biological samples for which both the central nervous system source marker and the central nervous system disease-related marker are detected, and/or the particle size measurement module, which Measure the particle size of the biological sample in which both the central nervous system-derived markers and the central nervous system disease-related markers are detected.
  • the diagnosis module diagnoses whether the subject suffers from a central nervous system disease based on the number of the biological sample and/or the particle size of the biological sample.
  • biological samples in which both central nervous system-derived markers and central nervous system disease-related markers are detected are usually referred to as double-positive biological samples, and diagnosis is based on the number and/or particle size of double-positive biological samples. Whether the subject suffers from central nervous system disease.
  • the present application also relates to a composition for detecting central nervous system diseases, which includes: an antibody used to target central nervous system-derived markers, and an antibody used to target central nervous system disease-related markers.
  • This application also relates to a kit for detecting central nervous system diseases in subjects, which includes reagents for detecting central nervous system-derived markers in biological fluids of subjects, and reagents for detecting biological fluids of subjects. Reagents for markers related to central nervous system diseases in body fluids.
  • a reagent for detecting a central nervous system-derived marker in a subject's biological fluid and a reagent for detecting a central nervous system disease-related marker in a subject's biological fluid are in this application The composition described above.
  • the reagent for detecting the central nervous system-derived marker in the biological fluid of the subject is a primer or probe that targets the central nervous system-derived marker, and the reagent is used for detecting the central nervous system-derived marker.
  • the reagents for markers related to central nervous system diseases in biological fluids are primers or probes that target the markers related to central nervous system diseases.
  • the biological fluid is blood, serum, plasma, saliva, urine, lymph, semen or milk.
  • Preferred biological fluids are blood, serum, plasma, saliva or urine.
  • the method for obtaining the biological fluid of the subject may be any method known to those skilled in the art. Those skilled in the art can obtain the selected biological fluid from the subject.
  • the biological sample of the present application is an extracellular vesicle.
  • extracellular vesicles EVs, also called extracellular vesicles
  • Extracellular vesicles refer to vesicle-like bodies with a double-layer membrane structure that are shed from the cell membrane or secreted by cells, with a diameter of 40nm It varies from 1000nm.
  • Extracellular vesicles are mainly composed of microvesicles (MVs) and exosomes (Exosomes, Exs).
  • MVs microvesicles
  • Exs exosomes
  • Microvesicles are small vesicles that fall off the cell membrane after cell activation, injury or apoptosis, with a diameter of about 100nm ⁇ 1000nm.
  • Exosomes are released outside the cell in the form of multivesicular bodies (Multivesicular Bodies) in the cell and the cell membrane after fusion, with a diameter of about 30-150nm, which can be released by many different types of cells, and Perform different cellular functions, including cell-to-cell communication, antigen presentation, and transfer of proteins and nucleic acids.
  • Extracellular vesicles are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), carrying a variety of proteins, lipids, DNA, mRAN, miRNA related to cell sources Etc., involved in the processes of cell-to-cell communication, cell migration, angiogenesis and immune regulation.
  • the extracellular vesicles are preferably neuron-derived extracellular vesicles, astrocyte-derived extracellular vesicles, oligodendrocyte-derived extracellular vesicles, or small Extracellular vesicles derived from glial cells.
  • a method for obtaining biological fluid of a subject and pretreating it to enrich the biological sample in the biological fluid, especially extracellular vesicles It can be centrifugation, ultracentrifugation, ultrafiltration tube filtration, polymerization sedimentation, or specific antibody capture.
  • centrifugation, ultracentrifugation, ultrafiltration tube filtration, polymerization sedimentation, or specific antibody capture all have meanings that can be understood by those skilled in the art, and those skilled in the art can select a suitable method according to the extracellular vesicles to be obtained .
  • an ultracentrifugation method is used to enrich extracellular vesicles from biological fluids, especially enriched extracellular vesicles derived from neurons and astrocytes.
  • ordinary centrifugation or ultrafiltration tube filtration is used to enrich extracellular vesicles, especially to enrich extracellular vesicles derived from neurons and cells derived from astrocytes.
  • the central nervous system disease is selected from one or more of the following: cerebrovascular disease, spinal cord disease, central nervous system infection, nervous system genetics sexual diseases, dysplasia of the nervous system, autonomic nervous system diseases, demyelinating diseases of the nervous system, epilepsy, and degenerative diseases of the nervous system.
  • the cerebrovascular disease includes cerebral infarction, cerebral hemorrhage, cerebral insufficiency, and transient ischemic attack;
  • the myelopathy includes acute myelitis , Polio, syringomyelia;
  • the central nervous system infections include encephalitis and meningitis;
  • the nervous system genetic diseases include neurocutaneous syndrome, spinocerebellar ataxia;
  • the nervous system developmental disorders include Congenital hydrocephalus and cerebral palsy;
  • the autonomic nervous system disease is autonomic insufficiency, and the neurodegenerative disease includes degenerative dementia, dyskinesia, and prion disease (Prion's disease).
  • the degenerative dementia includes Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease dementia (PDD), and Lewy body dementia (DLB);
  • the movement disorder includes motor nerves Primary disease (MND), Parkinson's disease (PD), Huntington's disease (HD), chorea (Sydenham chorea), hepatolenticular degeneration, multiple system atrophy (MSA), progressive supranuclear palsy (PSP) ), cortical basal ganglia degeneration (CBD).
  • the central nervous system disease is Alzheimer's disease.
  • the central nervous system disease is Parkinson's disease.
  • the central nervous system-derived markers and/or central nervous system disease-related markers are each selected from one of protein, nucleic acid, and lipid. kind or two or three.
  • the central nervous system-derived markers and the central nervous system disease-related markers are both proteins.
  • the central nervous system-derived markers and the central nervous system disease-related markers are both nucleic acids.
  • the central nervous system-derived markers and the central nervous system disease-related markers are all lipids.
  • the central nervous system-derived marker is a protein
  • the central nervous system disease-related marker is a nucleic acid
  • the central nervous system-derived marker is a nucleic acid
  • the central nervous system disease-related marker is a protein
  • the central nervous system-derived marker is a protein
  • the central nervous system disease-related marker is a lipid
  • the central nervous system-derived marker is a nucleic acid
  • the central nervous system disease-related marker is a lipid
  • the central nervous system-derived marker is lipid
  • the central nervous system disease-related marker is protein
  • the central nervous system-derived marker is lipid
  • the central nervous system disease-related marker is nucleic acid
  • the central nervous system-derived marker is a biomarker derived from the following neurons or cells: mature neurons, oligodendrocytes, Astrocytes, microglia, immature neurons, Schwann cells, radial glial cells, intermediate precursor cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonin Energetic neurons, cholinergic neurons.
  • the marker of central nervous system origin is a biomarker of neuron origin.
  • the central nervous system-derived marker is a biomarker derived from astrocytes and/or oligodendrocytes.
  • the central nervous system-derived marker is a marker located on the surface of extracellular vesicles derived from the central nervous system.
  • the marker derived from the central nervous system is selected from one or more of the following: G protein coupled receptor 162 (G Protein -Coupled Receptor 162, GPR162), Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1, HCN1, ⁇ -aminobutyric acid type A receptor subunit ⁇ (Gamma- Aminobutyric Acid Type A Receptor Subunit Delta, GABRD), neuroadhesion molecule 3 (Neuroligin 3, NLGN3), SHISA9, contact protein-1 (contactin-1, CNTN1), NeuN, MAP2, neurofilament-L (neurofilament light, NF- L), neurofilament-M (neurofilament medium, NF-M), neurofilament-H (neurofilament heavy, NF-H), synaptophysin, syntaxin, PSD95, L1CAM, bicortex Doublecortin
  • the central nervous system-derived marker is one or more selected from L1CAM, SHISA9, CNPase, and GLT1 (EAAT2).
  • L1CAM and SHISA9 are neuron-derived biomarkers
  • GLT1 (EAAT2) is astrocyte-derived biomarker
  • CNPase is oligodendrocyte-derived biomarker.
  • the biomarkers derived from mature neurons include L1CAM, SHISA9, contact protein-1 (contactin-1, CNTN1), NeuN, MAP2, neuron Silk-L (neurofilament light, NF-L), neurofilament-M (neurofilament medium, NF-M), neurofilament-H (neurofilament heavy, NF-H), synaptophysin (synaptophysin), synaptic fusion protein ( syntaxin), PSD95, etc.
  • the biomarkers derived from oligodendrocytes include CNPase, olig 1, olig 2, olig 3, MBP , OSP, MOG, SOX10, NG2, etc.
  • CNPase refers to 2', 3'-cyclic nucleotide 3'-phosphodiesterase.
  • the biomarkers derived from astrocytes include GFAP, GLAST (EAAT1), GLT1 (EAAT2), glutamine Synthetase (glutamine synthesis), S100- ⁇ , ALDH1L1, etc.
  • GLT1 (EAAT2) refers to glutamate transporter 1.
  • GLT1 (EAAT2) is a glutamate transporter specific to astrocytes in the central nervous system and a marker of astrocytes.
  • the biomarkers derived from Microglia include CD11b, CD45, Iba1, F4/80, CD68, CD40 and the like.
  • the biomarkers derived from immature neurons include doublecortin, ⁇ 3-tubulin (beta III tubulin). ), NeuroD1, TBR1, microtubule depolymerization protein 1 (stathmin 1), etc.
  • the biomarkers derived from Schwann cells include MPZ, NCAM, GAP43, S100 and the like.
  • the biomarkers derived from radial glia include vimentin, nestin, PAX6, HES1, HES5, GFAP, GLAST, BLBP, TN-C, N-cadherin, SOX2, etc.
  • the biomarkers derived from intermediate progenitors include TBR2, MASH1 (Ascl1), and the like.
  • biomarkers derived from glutamatergic neurons include vGluT1, vGluT2, NMDAR, glutaminase, Glutamine synthetase and so on.
  • the biomarkers derived from GABAergic neurons include GABA transporter 1 (GABA transporter 1, GAT1), GABA B receptor 1 (GABA B receptor 1), GABA B receptor 2 (GABA B receptor 2), GAD65, GAD67, etc.
  • the biomarkers derived from dopaminergic neurons include tyrosine hydroxylase (TH) and dopamine transporter. (dopamine transporter, DAT), FOXA2, GIRK2, Nurr1, LMX1B, etc.
  • the biomarkers derived from serotonergic neurons include tryptophan hydroxylase (TPH), serum Serotonin transporter, Pet1, etc.
  • the biomarkers derived from cholinergic neurons include choline acetyl transferase (ChAT), acetylcholine transferase Body (vesicular acetylcholine transporter, VAChT), acetylcholinesterase (AchE), etc.
  • the central nervous system disease-related marker is a protein
  • the protein is selected from one or more of the following: A ⁇ 1-40 (A ⁇ 40) protein, A ⁇ 1-42 (A ⁇ 42) protein, oligomeric A ⁇ (oligo-A ⁇ ) protein, tau protein, phosphorylated tau (p-tau) protein, ⁇ -synuclein protein ), phosphorylated ⁇ -synuclein (pS129) protein, oligomeric ⁇ -synuclein (oligomeric ⁇ -synuclein, oligo- ⁇ -syn) protein, and prion protein at position 129.
  • biomarkers related to Alzheimer's disease include, but are not limited to: A ⁇ 1-40 (A ⁇ 40) protein, A ⁇ 1-42 (A ⁇ 42) protein, oligomeric A ⁇ (oligo-A ⁇ ) protein, tau protein, phosphorylated tau (p-tau) protein.
  • Parkinson's disease and Parkinson's syndrome, including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), Cortical basal ganglia degeneration (CBD), Parkinson's disease dementia (PDD) and Lewy body dementia (DLB) related biomarkers, including but not limited to: ⁇ -synuclein protein, phosphorylated ⁇ -synuclein (pS129) protein at position 129 , Oligomeric ⁇ -synuclein (oligomeric ⁇ -synuclein, oligo- ⁇ -syn) protein, tau protein, phosphorylated tau (p-tau) protein.
  • ⁇ -synuclein protein ⁇ -syn protein
  • ⁇ -syn protein is the main protein component of Lewy body. Numerous studies have shown that it is closely related to the pathogenesis of Parkinson's disease.
  • the biomarker associated with prion disease is a prion protein.
  • the marker related to the central nervous system disease is a nucleic acid
  • the nucleic acid is selected from one or more of the following: DNA or RNA as a biomarker of Alzheimer’s disease (AD), as Parkinson’s disease (PD) and Parkinson’s syndrome (e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia DNA or RNA for biomarkers of degeneration (CBD), Lewy body dementia (DLB)).
  • AD Alzheimer’s disease
  • PD Parkinson’s disease
  • Parkinson’s syndrome e.g., multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical basal ganglia DNA or RNA for biomarkers of degeneration (CBD), Lewy body dementia (DLB)
  • the DNA or RNA used as a biomarker of AD includes: and genes UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3 , RNF7, RPLP0, SPARC, WTAP, HNRNPU, LINC00265, INMT, SLC35E2, CT60, SYNCRIP, RGS2, SMC6, ARSA, SPDYE7P, SMIM17, TRAF3IP2-AS1, KCNC2, SLC24A1, HLCS, GOSR1, MN1, MGAT5, NBPF14, FBXO31 , WDR52, TBC1D2B, ZNF648, NBPF16, PAGR1, AQP2, PRKCI, SCN2B, DPYSL3, TMEM26, TSPAN11, ELL2, FAM186A, CD59, THSD4, GOLGA6B
  • the DNA or RNA used as a biomarker of PD includes: and genes BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5 , HDLBP, HNRNPAB, NXN, SLC9A4, EIF2AK1, PAPOLA, TRIM50, SIX4, RAB3IP, VANGL2, DHRSX, FOXP4, SYNM, ZNF543, ATF6, LOC100132832, and BLOC1S1-RDH5 associated DNA or RNA.
  • the reagent for detecting the marker of central nervous system origin in the biological fluid of a subject is a primer or probe that targets the marker of central nervous system origin is a primer or probe that targets the aforementioned DNA or RNA.
  • the central nervous system-derived marker is a protein
  • the step of detecting the central nervous system-derived marker in the biological fluid of the subject includes: subjecting The subject’s biological fluid, preferably the subject’s extracellular vesicles are reacted with labeled antibodies that specifically react with the central nervous system-derived markers, and the intensity of the labeled signal after the reaction is detected to determine the central nervous system-derived markers
  • the presence and content of the central nervous system disease-related markers are proteins
  • the step of detecting the central nervous system disease-related markers in the subject’s biological fluid includes: making the subject’s biological fluid, preferably the subject’s
  • the extracellular vesicles react with labeled antibodies that specifically react with the central nervous system disease-related markers, and the intensity of the labeled signal after the reaction is detected to determine the presence and content of the central nervous system disease-related markers.
  • the extracellular vesicles of the subject simultaneously react with a labeled antibody that specifically reacts with a marker derived from the central nervous system and a labeled marker associated with a central nervous system disease.
  • the specific reaction antibody reacts to efficiently track the double-labeled and/or multi-labeled extracellular vesicles.
  • a biological sample for example, extracellular
  • a biological sample for example, extracellular
  • Vesicles and/or measure the size of biological samples (such as extracellular vesicles) that detect both central nervous system-derived markers and central nervous system disease-related markers, so that you can track the double-label And/or multi-mark positive biological samples, such as extracellular vesicles, can also fully consider the number and/or particle size of such biological samples, and combine the results of these indicators to analyze the subject’s risk of disease.
  • the label is selected from one or more of the following: fluorescent label, isotope label, enzyme label, chemiluminescent agent label, quantum dot Marker or colloidal gold marker.
  • the fluorescent marker is selected from one or more of the following: Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705, Qdot800, Alexa350, Alexa 405, Alexa488, Alexa 532, Alexa546, Alexa555, Alexa 568, Alexa 594, Alexa647, Alexa700, Alexa750.
  • isotope labeling enzyme labeling, chemiluminescent agent labeling, quantum dot labeling or colloidal gold labeling can all adopt methods well known to those skilled in the art.
  • the central nervous system-derived marker is a nucleic acid
  • the step of detecting the central nervous system-derived marker in the biological fluid of the subject includes: lysing the receptor The subject’s biological fluid, preferably the subject’s extracellular vesicles, and using DNA or RNA probes or sequencing technology to detect the presence and content of nucleic acids derived from the central nervous system;
  • the central nervous system disease-related markers are Nucleic acid
  • the step of detecting central nervous system disease-related markers in the subject’s biological fluid includes: lysing the subject’s biological fluid, preferably the subject’s extracellular vesicles, and using DNA or RNA probes or sequencing technology to Detect the presence and content of nucleic acids related to central nervous system diseases.
  • the subject’s extracellular vesicles simultaneously use DNA or RNA probes to detect the presence and content of nucleic acids derived from the central nervous system and to detect the presence and content of nucleic acids related to central nervous system diseases. Existence and content, so as to efficiently track double-labeled and/or multi-labeled extracellular vesicles.
  • detecting the intensity of the labeled signal after the reaction to determine the presence and content of the central nervous system-derived marker can be used, for example, immunoassays such as ELISA, Luminex, MSD, Quanterix, Singulex, eCL8000, Cobas are the preferred methods for measuring protein biomarkers.
  • the method or system or composition or kit of the present application it is used to detect the number of biological samples in which both the central nervous system-derived markers and the central nervous system disease-related markers are detected, and/ Or detection instruments that measure the particle size of biological samples that detect both central nervous system-derived markers and central nervous system disease-related markers, including but not limited to particle size analyzers and nano flow cytometers.
  • detection instruments that measure the particle size of biological samples that detect both central nervous system-derived markers and central nervous system disease-related markers, including but not limited to particle size analyzers and nano flow cytometers.
  • particle size analyzers and nano flow cytometers including but not limited to particle size analyzers and nano flow cytometers.
  • nanoparticle tracking technology to detect the size of extracellular vesicles
  • nanoflow analysis technology to detect the number of double-positive extracellular vesicles.
  • nucleic acid for nucleic acid to be used as a biomarker, the biological fluid or extracellular vesicles need to be lysed before measuring the nucleic acid biomarker.
  • the nucleic acid can be detected by any method known to those skilled in the art, for example, DNA or RNA probes, or any known sequencing technology.
  • composition for detecting central nervous system diseases which includes: antibodies for targeting central nervous system-derived markers and for targeting central nervous system diseases Antibodies to related markers.
  • the antibodies used to target markers derived from the central nervous system and the antibodies used to target markers related to central nervous system diseases are labeled antibodies, preferably
  • the label is selected from one or more of the following: fluorescent label, isotope label, enzyme label, chemiluminescent agent label, quantum dot label or colloidal gold label.
  • composition for detecting central nervous system diseases comprising: an antibody for targeting SHISA9 protein, and an antibody for targeting phosphorylated tau217 (p-tau217) protein Of antibodies.
  • a composition for detecting central nervous system diseases includes: an antibody for targeting GLT1 (EAAT2) protein, and an antibody for targeting tau protein.
  • a composition for detecting central nervous system diseases comprising: an antibody for targeting GLT1 (EAAT2) protein, and an antibody for targeting ⁇ -synuclein ( ⁇ -synuclein protein) antibody.
  • a composition for detecting central nervous system diseases includes: an antibody for targeting GLT1 (EAAT2) protein, and an antibody for targeting oligomeric ⁇ -synuclein ( oligomeric ⁇ -synuclein, oligo- ⁇ -syn) protein antibody.
  • a composition for detecting central nervous system diseases includes: an antibody for targeting GLT1 (EAAT2) protein, and an antibody for targeting phosphorylation ⁇ at position 129 -Synuclein (pS129) protein antibody.
  • a composition for detecting central nervous system diseases includes: an antibody for targeting GLT1 (EAAT2) protein, and an antibody for targeting phosphorylated tau(p- tau) protein antibody.
  • a composition for detecting central nervous system diseases includes: an antibody for targeting CNPase (CNPase) and an antibody for targeting tau protein.
  • a composition for detecting central nervous system diseases which includes: an antibody for targeting CNPase (CNPase), and an antibody for targeting ⁇ -synuclein ( ⁇ -synuclein protein) antibody.
  • This application relates to a kit for detecting central nervous system diseases in subjects, which includes: reagents for detecting central nervous system-derived markers in biological fluids of subjects, and reagents for detecting biological fluids of subjects Reagents for markers related to central nervous system diseases.
  • the reagent for detecting the central nervous system-derived marker in the biological fluid of the subject and the reagent for detecting the central nervous system disease-related marker in the biological fluid of the subject may be the above-mentioned composition involved in the present application .
  • This application relates to a kit for detecting central nervous system diseases in subjects, which includes: reagents for detecting central nervous system-derived markers in biological fluids of subjects, and reagents for detecting biological fluids of subjects Reagents for markers related to central nervous system diseases.
  • the reagent for detecting the central nervous system-derived marker in the biological fluid of the subject is a primer or probe that targets the central nervous system-derived marker, and the reagent is used to detect the central nervous system in the biological fluid of the subject
  • the reagents for disease-related markers are primers or probes that target the markers related to the central nervous system disease.
  • the kit involved in the present application also includes reagents and devices for obtaining a biological sample of a subject, preferably reagents and devices for obtaining extracellular vesicles of a subject.
  • the reagents and devices for obtaining extracellular vesicles may include commonly used reagent kits and devices known to those skilled in the art.
  • the method, system, kit and composition of the present application even if the subject's peripheral body fluid is used, it is possible to diagnose whether the subject suffers from a central nervous system disease. For example, it can effectively realize the diagnosis and differential diagnosis of diseases such as Alzheimer's disease, Parkinson's disease, and multiple system atrophy, and effectively distinguish it from healthy controls or other diseases.
  • diseases such as Alzheimer's disease, Parkinson's disease, and multiple system atrophy
  • Using the method, system, kit and composition of the present application can effectively enrich and specifically label biomarkers that can reflect changes in central nervous system diseases from peripheral body fluids, compared to direct detection of disease-related markers in peripheral body fluids The diagnosis effect is better.
  • these central nervous system-derived extracellular vesicle biomarkers such as L1CAM protein, SHISA9 protein, GLT1 protein, and CNPase protein can be effectively used, and combined with biomarkers that can reflect changes in central nervous system diseases, and are optimized
  • Diagnosis can also be made by simultaneously detecting the number of extracellular vesicles double-positive for two markers and/or the size of such extracellular vesicles. For example, it can also assist in calculating the ratio of extracellular vesicles double-positive for two markers to extracellular vesicles or total extracellular vesicles that are positive for a certain marker, thereby further improving the diagnostic effect.
  • the method, system, kit and composition of the present application can effectively use peripheral body fluid for detection, thereby avoiding traumatic acquisition of the subject’s cerebrospinal fluid, greatly reducing the subject’s sampling risk and greatly reducing the difficulty of detection It greatly increases the clinical applicability and promotion of the test.
  • it is possible to detect both central nervous system-derived markers and disease-related markers at the same time, reducing sample detection time, and being more efficient and convenient.
  • SHISA9 protein and phosphorylated tau217 (p-tau217) protein can be effectively used as dual-label detection to distinguish Alzheimer's disease patients from healthy people.
  • GLT1 protein and ⁇ -syn protein can be effectively used as dual-label detection to distinguish Parkinson's disease patients, patients with multiple system atrophy, and healthy people.
  • CNPase protein and ⁇ -syn protein can be effectively used as dual-label detection to distinguish Parkinson's disease patients, patients with multiple system atrophy, and healthy people.
  • Alexa fluorescent labeling to label antibodies targeting extracellular vesicle biomarkers from the central nervous system and antibodies targeting Alzheimer's disease biomarkers.
  • the antibody targeting the biomarker of extracellular vesicles from the central nervous system is SHISA9 polyclonal antibody.
  • the antibody targeting Alzheimer's disease biomarker is p-tau217 polyclonal antibody.
  • the labeled reagents are SHISA9 polyclonal antibody labeling purchased from Zenon TM Alexa Fluor TM 405, and p-tau217 polyclonal antibody labeling purchased from Zenon TM Alexa Fluor TM 488.
  • This experiment involves nerve-specific marker antibody SHISA9; disease-related marker antibody p-tau217; using Zenon TM Alexa Fluor TM 405 to label SHISA9, and Zenon TM Alexa Fluor TM 488 to label p-tau217;
  • step 3.2 Add 3 ⁇ L (3 ⁇ g) of blocking agent (IgG blocking agent diluted to 1 ⁇ g/ ⁇ L) to the solution of step 3.2, mix and incubate at room temperature (25°C) in the dark for 10 minutes;
  • blocking agent IgG blocking agent diluted to 1 ⁇ g/ ⁇ L
  • the NanoFCM nano flow analyzer is used to detect the intensity and particle size distribution of the labeling signal to determine the existence and content of relevant biomarkers.
  • Plasma samples of 98 subjects with Alzheimer's disease (AD group) and 12 healthy controls (CT group) with gender and age matching were obtained, and the experiment was carried out using the above-mentioned experimental method 1.
  • the ratio of single-labeled SHISA9 protein-positive extracellular vesicles to the total number of extracellular vesicles in plasma is in the range of 60-100nm particle size, 100-150nm particle size, and full particle size.
  • There are significant differences between the AD group and the CT group **, p ⁇ 0.01) ( Figure 1A); the ratio of the number of p-tau217 protein-positive extracellular vesicles in plasma to the total number of extracellular vesicles is in the range of 60-100nm particle size and the full size range.
  • the diagnostic efficiency was evaluated by receiver operating characteristic curve (ROC) analysis.
  • the area under curve (AUC) of SHISA9 protein single label in the particle size range of 60-100nm was 0.749, SHISA9 protein and p -tau217 protein double-labeled AUC in the 100-150nm particle size range is 0.890, using logistic regression analysis Enter method to incorporate SHISA9 60-100nm and SHISA9+p-tau217 100-150nm data, AUC is 0.891 ( Figure 1D); p-tau217
  • the AUC of protein single label in the particle size range of 60-100nm is 0.877, and the AUC of SHISA9 protein and p-tau217 protein in the particle size range of 100-150nm is 0.890.
  • Logistic regression analysis was used to include p-tau217 60-100nm and SHISA9+p-tau217 100-150nm data, AUC is 0.910 ( Figure 1E).
  • Example 1 show that SHISA9 protein-positive extracellular vesicles, p-tau217 protein-positive extracellular vesicles, and SHISA9 protein and p-tau217 protein double-positive extracellular vesicles all have good Alzheimer's disease diagnosis. Potential, especially the use of p-tau217 protein-positive extracellular vesicles and SHISA9 protein + p-tau217 protein double-positive extracellular vesicles for joint diagnosis.
  • Alexa fluorescent labeling to label antibodies targeting extracellular vesicle biomarkers from the central nervous system and antibodies targeting Alzheimer's disease biomarkers.
  • the antibody targeting the extracellular vesicle biomarker from the central nervous system is the GLT1 monoclonal antibody
  • the antibody targeting the Parkinson's disease biomarker is the ⁇ -synuclein ( ⁇ -syn) monoclonal antibody (syn211 and MJFR14).
  • the labeled reagents are GLT1 monoclonal antibody labeling purchased from Zenon TM Alexa Fluor TM 405, and ⁇ -synuclein ( ⁇ -syn) monoclonal antibody labeling purchased from Zenon TM Alexa Fluor TM 488.
  • the Apogee nano flow analyzer is used to detect the intensity of the labeled signal to determine the existence and content of disease-related biomarkers derived from the central nervous system in the peripheral body fluid.
  • GLT1 protein-positive extracellular vesicles and ⁇ -syn protein (syn211 and MJFR14 are anti- ⁇ -syn protein antibodies) positive extracellular vesicles ( Figure 2A)
  • extracellular vesicles double positive for GLT1 protein and ⁇ -syn protein can also be detected at the same time, with good reliability and repeatability (Figure 2B and C).
  • the number of extracellular vesicles double-positive for GLT1 protein and ⁇ -syn protein can be used as a clinical diagnosis, which can better distinguish PD patients from healthy controls, PD patients and MSA patients, and has the potential for clinical Parkinson's disease diagnosis and MSA differential diagnosis.
  • L1CAM is used as a marker to first enrich the exosomes derived from the central nervous system (a type of extracellular vesicles with a diameter in the range of about 30-150nm), and then use the Luminex detection platform to detect the exosomes Related markers.
  • the system or method of the present application can achieve similar PD diagnosis effects.
  • the method of the present application can detect two or more markers in biological fluids at the same time, and through multi-standard detection It is more time-saving, efficient, and has a high degree of clinical application, and has a good prospect.
  • Alexa fluorescent labeling to label antibodies targeting extracellular vesicle biomarkers from the central nervous system and antibodies targeting Alzheimer's disease biomarkers.
  • the antibody targeting the biomarker of extracellular vesicles from the central nervous system is CNPase monoclonal antibody.
  • the target multi-system atrophy biomarker is ⁇ -synuclein ( ⁇ -syn) monoclonal antibody.
  • the labeled reagent is CNPase monoclonal antibody labeling purchased from Zenon TM Alexa Fluor TM 488, and ⁇ -synuclein ( ⁇ -syn) monoclonal antibody labeling purchased from Zenon TM Alexa Fluor TM 405.
  • the Apogee nano flow analyzer is used to detect the intensity of the labeled signal to determine the existence and content of disease-related biomarkers derived from the central nervous system in the peripheral body fluid.
  • PD Parkinson’s disease
  • MSA multiple system atrophy
  • HC Plasma samples from gender-matched healthy controls
  • the number of extracellular vesicles double-positive for CNPase protein and ⁇ -syn protein has the potential for diagnosis of multiple system atrophy (MSA) and its differential diagnosis with Parkinson's disease (PD).
  • MSA multiple system atrophy
  • PD Parkinson's disease

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Abstract

本发明提供了一种诊断中枢神经***疾病的方法、***、组合物和试剂盒，其中诊断***包括：第一检测模块，其用于检测受试者生物体液中的中枢神经***来源标志物，第二检测模块，其用于检测受试者生物体液中的中枢神经***疾病相关的标志物，以及诊断模块，其基于第一检测模块和第二检测模块分别获得的中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病。

Description

一种外周体液多标生物标志物检测中枢神经***疾病的方法和*** 技术领域
本发明涉及生物医学技术领域，尤其是涉及体外诊断检测技术领域，涉及采用双标志物和/或多标志物进行检测的方法和***，优选采用双标志物和/或多标志物同时进行检测的方法和***。
背景技术
随着科学技术的发展，生物标志物检测在辅助疾病的诊断、鉴别诊断或者病情进展监测中起到越来越重要的作用。
目前，中枢神经***疾病生物标志物的检测主要基于脑脊液检测和外周体液直接检测进行开发。利用脑脊液检测的表现较好，这是因为脑脊液与脑和脊髓直接接触，能够较好地反映中枢神经***的改变。但是由于脑脊液获取的创伤性，患者接受度差，所以极大地限制了脑脊液生物标志物的深入开发、临床转化与广泛应用。利用外周体液进行检测的表现较不理想，主要存在以下两方面问题：一方面由于血脑屏障存在，并不是所有中枢神经***来源的成分都会在外周体液中被检测到，即使存在于外周体液中的中枢神经***来源生物标志物，尤其是蛋白，其丰度也非常低；另一方面由于许多中枢神经疾病相关标志物并非中枢神经***特异性表达，外周其他器官和/或***也能够产生，中枢神经***来源的信号极易受到外周成分的影响。
同时，中枢神经***疾病具有脑区特异性的特征。例如，阿尔茨海默病的主要病理变化是发生在顶叶、颞叶、海马等大脑皮层的萎缩，帕金森病主要由于基底核的黑质-纹状体的多巴胺回路病变或损伤引起的多巴胺分泌不足而致。目前仅有脑部活检的方式可以特异性针对不同脑区进行标志物检测分析，现有的体外检测技术均未能进行来源区分，对于不同疾病的诊断效果存在差异，同时疾病诊断的准确度也存在一定的提高空间。
因此，如何有效地从外周体液中富集且特异性地标记能够反映中枢神 经***疾病变化的生物标志物是本领域的研究焦点。
发明内容
如上所述，在本领域中还未存在利用外周体液有效地对中枢神经***疾病进行检测的***、方法以及试剂盒，本申请意在提供一种能够在外周体液中有效识别到不同中枢神经***来源的标志物，将其与疾病相关标志物联合检测，多重标记同时检测，以达到避免获取脑脊液，而通过外周体液样本就可以反应中枢神经***变化的效果。
即，本申请首先提出利用在外周体液中识别中枢神经***来源的生物标志物进行中枢神经***疾病诊断的概念以及检测方法。
具体来说，本申请涉及以下内容：
1.一种诊断中枢神经***疾病的方法，其包括：
检测受试者生物体液中的中枢神经***来源标志物，
检测受试者生物体液中的中枢神经***疾病相关的标志物，以及
基于中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病。
2.根据项1所述的方法，其中，
检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物同时进行。
3.根据项1或2所述的方法，其还包括：
获取受试者的生物体液，对其进行预处理以富集生物体液中的生物样本，
检测受试者生物体液中的中枢神经***来源标志物是指检测受试者的富集生物样本中的中枢神经***来源标志物，
检测受试者生物体液中的中枢神经***疾病相关的标志物是指检测受试者的富集生物样本中的中枢神经***疾病相关的标志物。
4.根据项1～3中任一项所述的方法，其还包括：统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的数量，和/或
测量检测到中枢神经***来源标志物和中枢神经***疾病相关的标志 物两者的生物样本的粒径，以及
基于所述生物样本的数量和/或所述生物样本的粒径来诊断受试者是否罹患中枢神经***疾病。
5.根据项3或4所述的方法，其中，
所述生物样本为胞外囊泡；
优选所述胞外囊泡为神经元来源的胞外囊泡、星形胶质细胞来源的胞外囊泡、少突胶质细胞来源的胞外囊泡或小胶质细胞来源的胞外囊泡。
6.根据项1～5中任一项所述的方法，其中，所述生物体液选自血液、血清、血浆、唾液、尿液、淋巴液、***或乳汁中的一种或两种以上。
7.根据项1～6中任一项所述的方法，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
8.根据项7所述的方法，其中，
所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
所述中枢神经***感染包括脑炎、脑膜炎；
所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
所述自主神经***疾病为自主神经功能不全；
所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
9.根据项8所述的方法，其中，
所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
10.根据项1～9中任一项所述的方法，其中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、 星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能神经元、5-羟色胺能神经元、胆碱能神经元。
11.根据项1～10中任一项所述的方法，其中，所述中枢神经***来源标志物和/或中枢神经***疾病相关的标志物各自选自蛋白质、核酸、脂类中的一种或两种或三种。
12.根据项10或11所述的方法，其中，所述中枢神经***来源标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA B receptor 1)、GABA B受体2(GABA B receptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE)；
优选所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物；
进一步优选所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。
13.根据项11所述的方法，其中，所述中枢神经***疾病相关的标志物为蛋白质，所述蛋白质选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
14.根据项11所述的方法，其中，所述中枢神经***疾病相关的标志物为核酸，所述核酸选自以下中的一种或两种以上：作为阿尔茨海默病(AD)的生物标志物的DNA或RNA、作为帕金森病(PD)及帕金森综合征(例如，多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)、路易体痴呆(DLB))的生物标志物的DNA或RNA。
15.根据项14所述的方法，其中，所述作为AD的生物标志物的DNA或RNA包括：与基因UBAP2L、YBX1、SERF2、UBE2B、RPL10A、H3F3AP4、PPP2CA、NMD3、RNF7、RPLP0、SPARC、WTAP、HNRNPU、LINC00265、INMT、SLC35E2、CT60、SYNCRIP、RGS2、SMC6、ARSA、SPDYE7P、SMIM17、TRAF3IP2-AS1、KCNC2、SLC24A1、HLCS、GOSR1、MN1、MGAT5、NBPF14、FBXO31、WDR52、TBC1D2B、ZNF648、NBPF16、PAGR1、AQP2、PRKCI、SCN2B、DPYSL3、TMEM26、TSPAN11、ELL2、FAM186A、CD59、THSD4、GOLGA6B、ARHGEF5、PKD1、BPTF、FLG、POM121L10P、NXPH3、H3F3A、SH3TC2、GGCX、和GREB1关联的DNA或RNA。
16.根据项14所述的方法，其中，所述作为PD的生物标志物的DNA或RNA包括：与基因BLOC1S1、UBE2L3、RNF149、FAM89B、LCE6A、NT5DC2、PPP1CC、CCL5、HDLBP、HNRNPAB、NXN、SLC9A4、EIF2AK1、PAPOLA、TRIM50、SIX4、RAB3IP、VANGL2、DHRSX、FOXP4、SYNM、ZNF543、ATF6、LOC100132832、和BLOC1S1-RDH5关联的DNA或RNA。
17.根据项3所述的方法，其中，获取受试者的生物体液并对其进行预 处理以富集生物样本的方法选自以下中的一种或两种以上：离心、超速离心、超滤管过滤、聚合沉降、特异性抗体捕获。
18.根据项11所述的方法，其中，所述中枢神经***来源标志物为蛋白质，检测受试者生物体液中的中枢神经***来源标志物的步骤包括：使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***来源标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***来源标志物的存在及含量。
19.根据项11所述的方法，其中，所述中枢神经***疾病相关的标志物为蛋白质，检测受试者生物体液中的中枢神经***疾病相关的标志物的步骤包括：使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***疾病相关标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***疾病相关标志物的存在及含量。
20.根据项18或19所述的方法，其中，所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
21.根据项11所述的方法，其中，所述中枢神经***来源标志物为核酸，检测受试者生物体液中的中枢神经***来源标志物的步骤包括：裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***来源的核酸的存在及含量。
22.根据项11所述的方法，其中，所述中枢神经***疾病相关的标志物为核酸，检测受试者生物体液中的中枢神经***疾病相关的标志物的步骤包括：裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***疾病相关的核酸的存在及含量。
23.一种用于诊断中枢神经***疾病的***，其包括：
第一检测模块，其用于检测受试者生物体液中的中枢神经***来源标志物，
第二检测模块，其用于检测受试者生物体液中的中枢神经***疾病相关的标志物，以及
诊断模块，其基于第一检测模块和第二检测模块分别获得的中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者 是否罹患中枢神经***疾病。
24.根据项23所述的***，其中，
所述第一检测模块和所述第二检测模块同时检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物；或者
所述第一检测模块和所述第二检测模块为同一模块，其同时检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物。
25.根据项23或24所述的***，其还包括：
富集模块，其用于获取受试者的生物体液并对其进行预处理以富集生物体液中的生物样本，
其中第一检测模块和第二检测模块检测受试者的富集生物样本中的中枢神经***来源标志物和中枢神经***疾病相关的标志物。
26.根据项23～25中任一项所述的***，其还包括：
数量统计模块，其统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的数量，和/或
粒径测量模块，其测量检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的粒径，以及
诊断模块基于所述生物样本的数量和/或所述生物样本的粒径来诊断受试者是否罹患中枢神经***疾病。
27.根据项25或26所述的方法，其中，
所述生物样本为胞外囊泡；
优选所述胞外囊泡为神经元来源的胞外囊泡、星形胶质细胞来源的胞外囊泡、少突胶质细胞来源的胞外囊泡或小胶质细胞来源的胞外囊泡。
28.根据项23～27中任一项所述的***，其中，所述生物体液选自血液、血清、血浆、唾液、尿液、淋巴液、***或乳汁中的一种或两种以上。
29.根据项23～28中任一项所述的***，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
30.根据项29所述的***，其中，
所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
所述中枢神经***感染包括脑炎、脑膜炎；
所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
所述自主神经***疾病为自主神经功能不全；
所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
31.根据项30所述的***，其中，
所述变性变性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
32.根据项23～31中任一项所述的***，其中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能神经元、5-羟色胺能神经元、胆碱能神经元。
33.根据项23～32中任一项所述的***，其中，所述中枢神经***来源标志物和/或中枢神经***疾病相关的标志物各自选自蛋白质、核酸、脂类中的一种或两种或三种。
34.根据项32或33所述的***，所述中枢神经***来源标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝 -M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA B receptor 1)、GABA B受体2(GABA B receptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE))，
优选所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物；
进一步优选所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。
35.根据项34所述的***，其中，所述中枢神经***疾病相关的标志物为蛋白质，所述蛋白质选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
36.根据项34所述的***，其中，所述中枢神经***疾病相关的标志物为核酸，所述核酸选自以下中的一种或两种以上：作为阿尔茨海默病(AD)的生物标志物的DNA或RNA、作为帕金森病(PD)及帕金森综合征(例如， 多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)、路易体痴呆(DLB))的生物标志物的DNA或RNA。
37.根据项36所述的***，其中，所述作为AD的生物标志物的DNA或RNA包括：与基因UBAP2L、YBX1、SERF2、UBE2B、RPL10A、H3F3AP4、PPP2CA、NMD3、RNF7、RPLP0、SPARC、WTAP、HNRNPU、LINC00265、INMT、SLC35E2、CT60、SYNCRIP、RGS2、SMC6、ARSA、SPDYE7P、SMIM17、TRAF3IP2-AS1、KCNC2、SLC24A1、HLCS、GOSR1、MN1、MGAT5、NBPF14、FBXO31、WDR52、TBC1D2B、ZNF648、NBPF16、PAGR1、AQP2、PRKCI、SCN2B、DPYSL3、TMEM26、TSPAN11、ELL2、FAM186A、CD59、THSD4、GOLGA6B、ARHGEF5、PKD1、BPTF、FLG、POM121L10P、NXPH3、H3F3A、SH3TC2、GGCX、和GREB1关联的DNA或RNA。
38.根据项36所述的***，其中，所述作为PD的生物标志物的DNA或RNA包括：与基因BLOC1S1、UBE2L3、RNF149、FAM89B、LCE6A、NT5DC2、PPP1CC、CCL5、HDLBP、HNRNPAB、NXN、SLC9A4、EIF2AK1、PAPOLA、TRIM50、SIX4、RAB3IP、VANGL2、DHRSX、FOXP4、SYNM、ZNF543、ATF6、LOC100132832、和BLOC1S1-RDH5关联的DNA或RNA。
39.根据项25所述的***，其中，所述富集模块具有进行以下步骤中的一种或两种以上的子模块：离心、超速离心、超滤管过滤、聚合沉降、特异性抗体捕获。
40.根据项33所述的***，其中，所述中枢神经***来源标志物为蛋白质，第一检测模块用于使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***来源标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***来源标志物的存在及含量。
41.根据项33所述的***，其中，所述中枢神经***疾病相关的标志物为蛋白质，第二检测模块用于使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***疾病相关标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***疾病相关标志物的存在及含量。
42.根据项40或41所述的***，其中，所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
43.根据项33所述的***，其中，所述中枢神经***来源标志物为核酸，第一检测模块用于裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***来源的核酸的存在及含量。
44.根据项33所述的***，其中，所述中枢神经***疾病相关的标志物为核酸，第二检测模块用于裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***疾病相关的核酸的存在及含量。
45.一种用于检测中枢神经***疾病的组合物，其包括：
用于靶向中枢神经***来源标志物的抗体，以及
用于靶向中枢神经***疾病相关的标志物的抗体。
46.根据项45所述的组合物，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
47.根据项46所述的组合物，其中，
所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
所述中枢神经***感染包括脑炎、脑膜炎；
所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
所述自主神经***疾病为自主神经功能不全；
所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
48.根据项47所述的组合物，其中，
所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
49.根据项45～48中任一项所述的组合物，其中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能神经元、5-羟色胺能神经元、胆碱能神经元。
50.根据项45～49中任一项所述的组合物，其中，所述中枢神经***来源标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA B receptor 1)、GABA B受体2(GABA B receptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、 胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE)；
优选所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物；
进一步优选所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。
51.根据项45～50中任一项所述的组合物，其中，所述中枢神经***疾病相关的标志物选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
52.根据项45～51中任一项所述的组合物，其中，所述用于靶向中枢神经***来源标志物的抗体和所述用于靶向中枢神经***疾病相关的标志物的抗体是经标记的抗体，优选所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
53.一种用于检测受试者中枢神经***疾病的试剂盒，其包括：
用于检测受试者生物体液中的中枢神经***来源标志物的试剂，以及
用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂。
54.根据项53所述的试剂盒，其中，所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂和用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为项45～52中任一项所述的组合物。
55.根据项53所述的试剂盒，其中，
所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂为靶向中枢神经***来源标志物的引物或探针，
所述用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为靶向中枢神经***疾病相关的标志物的引物或探针。
56.根据项53所述的试剂盒，还包括：
用于获取受试者的生物样本的试剂和装置，优选用于获取受试者胞外囊泡的试剂和装置。
57.根据项53所述的试剂盒，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
58.根据项57所述的试剂盒，其中，
所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
所述中枢神经***感染包括脑炎、脑膜炎；
所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
所述自主神经***疾病为自主神经功能不全；
所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
59.根据项58所述的试剂盒，其中，
所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
发明的效果
利用本申请的方法、***、试剂盒以及组合物，即使仅仅利用受试者的外周体液也能够诊断受试者是否罹患中枢神经***疾病。
利用本申请的方法、***、试剂盒以及组合物能够有效地从外周体液中富集且特异性地标记能够反映中枢神经***疾病变化的生物标志物，相比直接检测外周体液中的疾病相关标志物，诊断效果更优。
利用本申请的方法、***、试剂盒以及组合物可以有效地利用外周体液来进行检测，从而可以避免创伤性地获取受试者的脑脊液，大大降低受试者的取样风险、大大减轻检测的困难程度、大大增加检测的临床应用性和推广性。
利用本申请的方法、***、试剂盒以及组合物，能够同时检测中枢神经***来源标志物与疾病相关标志物，降低样本检测时间，更加高效、便捷。
本申请的核心是采用双标志物和/或多标志物进行检测的方法和***，可以实现双标志物和/或多标志物的同时检测，即利用标志物来确认生物样本，尤其是胞外囊泡中来源于中枢神经***的部分，在此基础上利用与疾病相关的标志物，产生的共同检测结果为疾病的诊断、鉴别诊断、追踪和药物评估服务。
附图说明
图1A显示实施例1中，血浆中单独标记SHISA9蛋白阳性胞外囊泡占总胞外囊泡数量的比值在60-100nm粒径、100-150nm粒径、以及全粒径范围，AD组和健康对照组(CT组)均存在显著性差异(**,p<0.01)；
图1B显示实施例1中，血浆中单独标记p-tau217蛋白阳性胞外囊泡占总胞外囊泡数量的比值在60-100nm粒径、以及全粒径范围，AD组和CT组均存在极显著性差异(****,p<0.0001)，在100-150nm粒径范围，AD组和CT组存在显著性差异(**,p<0.01)；
图1C显示实施例1中，血浆中SHISA9蛋白和p-tau217蛋白双阳性胞外囊泡占总胞外囊泡数量的比值在100-150nm粒径范围、以及全粒径范围，AD组和CT组均存在极显著性差异(***,p<0.001)；
图1D显示实施例1中，受试者工作特征曲线(receiver operating characteristic curve,ROC)分析：SHISA9蛋白单标在60-100nm粒径范围的曲线下面积(area under curve,AUC)为0.749，SHISA9蛋白和p-tau217蛋白双标在100-150nm粒径范围的AUC为0.890，使用逻辑回归分析Enter方法纳入SHISA9 60-100nm及SHISA9+p-tau217 100-150nm数据，AUC为0.891；
图1E显示实施例1中，ROC分析：p-tau217蛋白单标在60-100nm粒径范围的AUC为0.877，SHISA9蛋白和p-tau217蛋白双标在100-150nm粒径范围的AUC为0.890，使用逻辑回归分析Enter方法纳入p-tau217 60-100nm及SHISA9+p-tau217 100-150nm数据，AUC为0.910。
图2A显示实施例2中，血浆中单独标记GLT1蛋白阳性的胞外囊泡数量和单独标记α-syn蛋白(syn211与MJFR14均为抗α-syn蛋白抗体)阳性的胞 外囊泡数量；
图2B和C显示实施例2中，血浆中单独标记GLT1蛋白或α-syn蛋白的阳性胞外囊泡数量，GLT1蛋白与α-syn蛋白双阳性胞外囊泡数量。
图3A和B显示实施例2中，血浆中GLT1蛋白与α-syn蛋白双阳性的胞外囊泡数量，PD组极显著高于MSA组和CT组(***,p<0.001)，而MSA组和CT组之间没有显著差异。
图4A显示实施例3中，血浆中CNPase蛋白阳性的胞外囊泡，其数量在MSA组与PD组间无显著性差异，而MSA组与HC组间具有显著差异(*,p<0.05)；
图4B显示实施例3中，血浆中CNPase蛋白与α-syn蛋白(抗MJFR14抗体标记)双阳性的胞外囊泡数量，在MSA组、PD组与HC组间无显著差异；
图4C显示实施例3中，血浆中CNPase蛋白与α-syn蛋白双阳性的胞外囊泡数量通过血浆中胞外囊泡总数标准化后，MSA组与HC组、MSA组与PD组间具有显著差异(*,p<0.05)。
具体实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异来作为区分组件的方式，而是以组件在功能上的差异来作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。
在本申请的一个具体实施方式中，本申请涉及一种诊断中枢神经***疾病的方法，其包括：检测受试者生物体液中的中枢神经***来源标志物，检测受试者生物体液中的中枢神经***疾病相关的标志物，以及基于中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病。
在本申请的一个具体实施方式中，本申请涉及一种诊断中枢神经***疾病的方法，其包括：检测受试者生物体液中的中枢神经***来源标志物，检测受试者生物体液中的中枢神经***疾病相关的标志物，以及基于中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病，其中，检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物同时进行。
在本申请的一个具体实施方式中，本申请涉及一种用于诊断中枢神经***疾病的***，其包括：第一检测模块，其用于检测受试者生物体液中的中枢神经***来源标志物，第二检测模块，其用于检测受试者生物体液中的中枢神经***疾病相关的标志物，以及诊断模块，其基于第一检测模块和第二检测模块分别获得的中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病。
在本申请的一个具体实施方式中，本申请涉及一种用于诊断中枢神经***疾病的***，其包括：第一检测模块，其用于检测受试者生物体液中的中枢神经***来源标志物，第二检测模块，其用于检测受试者生物体液中的中枢神经***疾病相关的标志物，以及诊断模块，其基于第一检测模块和第二检测模块分别获得的中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病。所述第一检测模块和所述第二检测模块同时检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物；或者所述第一检测模块和所述第二检测模块为同一模块，其同时检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物。
如上所述，在本申请的方法和***中，发明人独创性地开发出一种新的 方法和***，即采用具有不同作用的双标志物和/或多标志物来进行检测，尤其是可以同时通过这样的双标志物和/或多标志物来进行检测，从而仅仅利用外周体液也能够有效地检测中枢神经***疾病。这样的检测方法和***不需要提取患者的脑脊液，大大减轻了患者的取样风险。同时由于采用具有标识中枢***来源的标志物和与待检测疾病相关的标志物的双重标记和/或多重标记，可以大大提高检测的准确程度，如果进一步同时进行检测，还可以提高整体检测的效率。而现有的其它方法往往需要更为复杂的提取、富集、或纯化步骤等才能够获取用于诊断的生物样本。本申请则简化了操作步骤，独创性地利用来源于中枢神经***的标志物来标注来源，并结合与疾病相关的标志物，从而实现了疾病检测准确度和效率的提升。
进一步地，本申请的方法还包括：获取受试者的生物体液并对其进行预处理以富集生物体液中的生物样本，检测受试者生物体液中的中枢神经***来源标志物是指检测受试者的富集生物样本中的中枢神经***来源标志物，检测受试者生物体液中的中枢神经***疾病相关的标志物是指检测受试者的富集生物样本中的中枢神经***疾病相关的标志物。
进一步地，本申请的方法还包括统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的数量，和/或测量检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的粒径，基于所述生物样本的数量和/或所述生物样本的粒径来诊断受试者是否罹患中枢神经***疾病。具体来说，通常将检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本称为双阳性生物样本，基于双阳性生物样本的数量和/或粒径来诊断受试者是否罹患中枢神经***疾病。
进一步地，本申请的***还包括：富集模块，其用于获取受试者的生物体液并对其进行预处理以富集生物体液中的生物样本，其中第一检测模块和第二检测模块检测受试者的富集生物样本中的中枢神经***来源标志物和中枢神经***疾病相关的标志物。
进一步地，本申请的***还包括：数量统计模块，其统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的数量、和/或粒径测量模块，其测量检测到中枢神经***来源标志物和中枢神经 ***疾病相关的标志物两者的生物样本的粒径。以及诊断模块基于所述生物样本的数量和/或所述生物样本的粒径来诊断受试者是否罹患中枢神经***疾病。具体来说，通常将检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本称为双阳性生物样本，基于双阳性生物样本的数量和/或粒径来诊断受试者是否罹患中枢神经***疾病。
本申请还涉及一种用于检测中枢神经***疾病的组合物，其包括：用于靶向中枢神经***来源标志物的抗体，以及用于靶向中枢神经***疾病相关的标志物的抗体。
本申请还涉及一种用于检测受试者中枢神经***疾病的试剂盒，其包括：用于检测受试者生物体液中的中枢神经***来源标志物的试剂，以及用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂。
在一个具体的实施方式中，用于检测受试者生物体液中的中枢神经***来源标志物的试剂和用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为本申请所描述的上述组合物。
在一个具体的实施方式中，所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂为靶向中枢神经***来源标志物的引物或探针，所述用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为靶向中枢神经***疾病相关的标志物的引物或探针。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，生物体液为血液、血清、血浆、唾液、尿液、淋巴液、***或乳汁。优选的生物体液是血液、血清、血浆、唾液或尿液。用于获取受试者生物体液的方法可以是任何本领域技术人员已知的方法。本领域技术人员可以针对所选用的生物体液的方法来从受试者中获取。
在一个具体的实施方式中本申请的生物样本为胞外囊泡。如在本文中所涉及的细胞外囊泡(Extracellular Vesicles，EVs，也称为胞外囊泡)是指从细胞膜上脱落或者有细胞分泌的双层膜结构的囊泡状小体，直径从40nm到1000nm不等。胞外囊泡主要由微囊泡(Microvesicles，MVs)和外泌体(Exosomes，Exs)组成，微囊泡是细胞激活、损伤或凋亡后从细胞膜脱落的小囊泡，直径约为100nm～1000nm。外泌体(Exosomes)由细胞内的多泡小体(Multivesicular Bodies)与细胞膜融合后以外泌体的形式释放到细胞外，直径 约为30～150nm，可以由众多不同类型的细胞所释放，并且执行不同的细胞功能，包括细胞间通讯、抗原呈递、和蛋白质以及核酸的转移。胞外囊泡广泛存在于细胞培养上清以及各种体液(血液、淋巴液、唾液、尿液、***、乳汁)中，携带有细胞来源相关的多种蛋白质、脂类、DNA、mRAN、miRNA等，参与细胞间通讯、细胞迁移、血管新生和免疫调节等过程。在一个具体的实施方式中，优选所述胞外囊泡为神经元来源的胞外囊泡、星形胶质细胞来源的胞外囊泡、少突胶质细胞来源的胞外囊泡或小胶质细胞来源的胞外囊泡。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，获取受试者的生物体液并对其进行预处理以富集生物体液中的生物样本，尤其是胞外囊泡的方法可以为离心、超速离心、超滤管过滤、聚合沉降或特异性抗体捕获。在本文中，离心、超速离心、超滤管过滤、聚合沉降或特异性抗体捕获均具有本领域技术人员可以理解的含义，本领域技术人员可以根据待获取的胞外囊泡来选择合适的方法。
在本申请的方法或***的一个实施方式中，采用超速离心的方法来从生物体液中富集胞外囊泡，尤其是富集神经元来源的胞外囊泡、星形胶质细胞来源的胞外囊泡、少突胶质细胞来源的胞外囊泡或小胶质细胞来源的胞外囊泡。
在本申请的方法或***的一个实施方式中，采用普通离心或超滤管过滤来富集胞外囊泡，尤其是富集神经元来源的胞外囊泡、星形胶质细胞来源的胞外囊泡、少突胶质细胞来源的胞外囊泡或小胶质细胞来源的胞外囊泡。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；所述中枢神经***感染包括脑炎、脑膜炎；所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；所述 自主神经***疾病为自主神经功能不全，所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。进一步，所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***疾病为阿尔茨海默病。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***疾病为帕金森病。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物和/或中枢神经***疾病相关的标志物各自选自蛋白质、核酸、脂类中的一种或两种或三种。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物和中枢神经***疾病相关的标志物均为蛋白质。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物和中枢神经***疾病相关的标志物均为核酸。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物和中枢神经***疾病相关的标志物均为脂类。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为蛋白质，中枢神经***疾病相关的标志物为核酸。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为核酸，中枢神经***疾病相关的标志物为蛋白质。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为蛋白质，中枢神经***疾病相关的标志物为脂类。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为核酸，中枢神经***疾病相关的标志物为脂类。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为脂类，中枢神经***疾病相关的标志物为蛋白质。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中 枢神经***来源标志物为脂类，中枢神经***疾病相关的标志物为核酸。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能神经元、5-羟色胺能神经元、胆碱能神经元。在本申请的方法或***或组合物或试剂盒的具体实施方式中，优选中枢神经***来源标志物是神经元来源的生物标志物。在本申请的方法或***或组合物或试剂盒的具体实施方式中，优选中枢神经***来源标志物是星形胶质细胞和/或少突胶质细胞来源的生物标志物。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源的标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、 GABA B受体1(GABA B receptor 1)、GABA B受体2(GABA B receptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE))。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。在这些标志物中，L1CAM和SHISA9是神经元来源的生物标志物，GLT1(EAAT2)是星形胶质细胞来源的生物标志物，CNP酶是少突胶质细胞来源的生物标志物。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于成熟神经元的生物标志物包括L1CAM、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于少突胶质细胞(Oligodendrocytes)的生物标志物包括CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2等。在本文中CNP酶是指2’,3’-环状核苷酸3’-磷酸二脂酶。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于星形胶质细胞(Astrocytes)的生物标志物包括GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1等。在本文中，GLT1(EAAT2)是指谷氨酸转运蛋白1。GLT1(EAAT2)是一种中枢神经***内星形胶质细胞特异的谷氨酸转运体，是一种星形胶质细胞的标志物。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于小胶质细胞(Microglia)的生物标志物包括CD11b、CD45、Iba1、F4/80、CD68、CD40等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于 未成熟神经元(Immature neurons)的生物标志物包括双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于施旺细胞(Schwann cells)的生物标志物包括MPZ、NCAM、GAP43、S100等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于放射状胶质细胞(Radial glia)的生物标志物包括波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于中间前体细胞(Intermediate progenitors)的生物标志物包括TBR2、MASH1(Ascl1)等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于谷氨酸能神经元(Glutamatergic neurons)的生物标志物包括vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于GABA能神经元(GABAergic neurons)的生物标志物包括GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA B receptor 1)、GABA B受体2(GABA B receptor 2)、GAD65、GAD67等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于多巴胺能神经元(Dopaminergic neurons)的生物标志物包括酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于5-羟色胺能神经元(Serotonergic neurons)的生物标志物包括色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，来源于胆 碱能神经元(Cholinergic neurons)的生物标志物包括胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE)等。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***疾病相关的标志物为蛋白质，所述蛋白质选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，与阿尔茨海默病(AD)相关的生物标志物，包括但不限于：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，与帕金森病(PD)以及帕金森综合征，包括多***萎缩(MSA)，进行性核上性麻痹(PSP)，皮质基底节变性(CBD)，帕金森病痴呆(PDD)以及路易体痴呆(DLB)相关的生物标志物，包括但不限于：α-synuclein蛋白、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白。其中，α-synuclein蛋白(α-syn蛋白)是路易小体(Lewy body)的主要蛋白质成分，众多研究表明，其与帕金森病的发病关系密切。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，与朊病毒病相关的生物标志物是朊病毒蛋白。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***疾病相关的标志物为核酸，所述核酸选自以下中的一种或两种以上：作为阿尔茨海默病(AD)的生物标志物的DNA或RNA、作为帕金森病(PD)及帕金森综合征(例如，多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)、路易体痴呆(DLB))的生物标志物的DNA或RNA。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述作为AD的生物标志物的DNA或RNA包括：与基因UBAP2L、YBX1、SERF2、 UBE2B、RPL10A、H3F3AP4、PPP2CA、NMD3、RNF7、RPLP0、SPARC、WTAP、HNRNPU、LINC00265、INMT、SLC35E2、CT60、SYNCRIP、RGS2、SMC6、ARSA、SPDYE7P、SMIM17、TRAF3IP2-AS1、KCNC2、SLC24A1、HLCS、GOSR1、MN1、MGAT5、NBPF14、FBXO31、WDR52、TBC1D2B、ZNF648、NBPF16、PAGR1、AQP2、PRKCI、SCN2B、DPYSL3、TMEM26、TSPAN11、ELL2、FAM186A、CD59、THSD4、GOLGA6B、ARHGEF5、PKD1、BPTF、FLG、POM121L10P、NXPH3、H3F3A、SH3TC2、GGCX、和GREB1关联的DNA或RNA。用于检测受试者生物体液中的中枢神经***来源标志物的试剂为靶向中枢神经***来源标志物的引物或探针为靶向上述DNA或RNA的引物或探针。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述作为PD的生物标志物的DNA或RNA包括：与基因BLOC1S1、UBE2L3、RNF149、FAM89B、LCE6A、NT5DC2、PPP1CC、CCL5、HDLBP、HNRNPAB、NXN、SLC9A4、EIF2AK1、PAPOLA、TRIM50、SIX4、RAB3IP、VANGL2、DHRSX、FOXP4、SYNM、ZNF543、ATF6、LOC100132832、和BLOC1S1-RDH5关联的DNA或RNA。用于检测受试者生物体液中的中枢神经***来源标志物的试剂为靶向中枢神经***来源标志物的引物或探针为靶向上述DNA或RNA的引物或探针。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为蛋白质，检测受试者生物体液中的中枢神经***来源标志物的步骤包括：使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***来源标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***来源标志物的存在及含量；所述中枢神经***疾病相关的标志物为蛋白质，检测受试者生物体液中的中枢神经***疾病相关的标志物的步骤包括：使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***疾病相关标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***疾病相关标志物的存在及含量。进一步，在具体的实施方式中，优选时受试者的胞外囊泡同时与经标记的、与中枢神经***来源标志物特异性反应的抗体和经标记的、与中枢 神经***疾病相关标志物特异性反应的抗体进行反应，从而高效地追踪到双标和/或多标阳性的胞外囊泡。
进一步，在本申请的方法或***或组合物或试剂盒的具体实施方式中，还包括统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本(例如胞外囊泡)的数量，和/或测量检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本(例如胞外囊泡)的粒径，这样可以在追踪到双标和/或多标阳性的生物样本，例如胞外囊泡之后，还可以充分地考虑这样的生物样本的数量和/或其粒径，结合这些指标的结果来分析受试者的患病风险。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，荧光标记物选自以下中的一种或两种以上：Qdot525、Qdot545、Qdot565、Qdot585、Qdot605、Qdot625、Qdot655、Qdot705、Qdot800、Alexa350、Alexa 405、Alexa488、Alexa 532、Alexa546、Alexa555、Alexa 568、Alexa 594、Alexa647、Alexa700、Alexa750。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记均可以采用本领域技术人员所熟知的方法。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，所述中枢神经***来源标志物为核酸，检测受试者生物体液中的中枢神经***来源标志物的步骤包括：裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***来源的核酸的存在及含量；所述中枢神经***疾病相关的标志物为核酸，检测受试者生物体液中的中枢神经***疾病相关的标志物的步骤包括：裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***疾病相关的核酸的存在及含量。进一步，在具体的实施方式中，优选时受试者的胞外囊泡同时利用DNA或RNA探针来检测与中枢神经***来源的核酸的存在及含量和检测与中枢神经***疾病相关的核酸的存在及含量，从而 高效地追踪到双标和/或多标阳性的胞外囊泡。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，检测反应后标记信号的强度以判断中枢神经***来源标志物的存在及含量可以采用，例如免疫测定，如ELISA、Luminex、MSD、Quanterix、Singulex、eCL8000、Cobas是用于测量蛋白质生物标志物的优选方法。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，用于检测统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的数量，和/或测量检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的粒径的检测仪器，包括但不限于粒度分析仪、纳米流式细胞仪。例如，利用纳米颗粒追踪技术检测胞外囊泡的粒径，以及利用纳米流式分析技术检测上述双阳性的胞外囊泡的数量。
在本申请的方法或***或组合物或试剂盒的具体实施方式中，对于核酸用作生物标志物，在测量核酸生物标志物以前，需要裂解生物体液或胞外囊泡。可以通过本领域技术人员已知的任何方法，例如，DNA或RNA探针，或任何已知的测序技术来检测核酸。
在本申请的一个具体的实施方式中，涉及一种用于检测中枢神经***疾病的组合物，其包括：用于靶向中枢神经***来源标志物的抗体，以及用于靶向中枢神经***疾病相关的标志物的抗体。
在本申请的一个具体的实施方式中，所述用于靶向中枢神经***来源标志物的抗体和所述用于靶向中枢神经***疾病相关的标志物的抗体是经标记的抗体，优选所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向SHISA9蛋白的抗体，以及用于靶向磷酸化tau217(p-tau217)蛋白的抗体。
在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向GLT1(EAAT2)蛋白的抗体，以及用于靶向tau蛋白的抗体。在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向GLT1(EAAT2)蛋白的抗体，以及用于靶向α-突触核蛋白(α-synuclein蛋白)的抗体。在本申请的一个具体的实施方 式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向GLT1(EAAT2)蛋白的抗体，以及用于靶向寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白的抗体。在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向GLT1(EAAT2)蛋白的抗体，以及用于靶向第129位磷酸化α-synuclein(pS129)蛋白的抗体。在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向GLT1(EAAT2)蛋白的抗体，以及用于靶向磷酸化tau(p-tau)蛋白的抗体。
在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向CNP酶(CNPase)的抗体，以及用于靶向tau蛋白的抗体。在本申请的一个具体的实施方式中，一种用于检测中枢神经***疾病的组合物，其包括：用于靶向CNP酶(CNPase)的抗体，以及用于靶向α-突触核蛋白(α-synuclein蛋白)的抗体。
本申请涉及一种用于检测受试者中枢神经***疾病的试剂盒，其包括：用于检测受试者生物体液中的中枢神经***来源标志物的试剂，以及用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂。所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂和用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂可以为上述本申请涉及的组合物。
本申请涉及一种用于检测受试者中枢神经***疾病的试剂盒，其包括：用于检测受试者生物体液中的中枢神经***来源标志物的试剂，以及用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂。所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂为靶向中枢神经***来源标志物的引物或探针，所述用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为靶向中枢神经***疾病相关的标志物的引物或探针。
在本申请涉及的试剂盒中，还包括用于获取受试者的生物样本的试剂和装置，优选用于获取受试者胞外囊泡的试剂和装置。其中，用于获取胞外囊泡的试剂和装置可以包括本领域技术人员所知的常用的试剂盒和装置。
利用本申请的方法、***、试剂盒以及组合物，即使仅仅利用受试者的 外周体液就能够诊断受试者是否罹患中枢神经***疾病。例如，可以有效地实现对于阿尔茨海默病、帕金森病、多***萎缩等疾病进行诊断与鉴别诊断，有效地与健康对照或其他疾病进行区分。
利用本申请的方法、***、试剂盒以及组合物能够有效地从外周体液中富集且特异性地标记能够反映中枢神经***疾病变化的生物标志物，相比直接检测外周体液中的疾病相关标志物，诊断效果更优。例如，可以有效地利用L1CAM蛋白、SHISA9蛋白、GLT1蛋白、CNPase蛋白等这些中枢神经***来源的胞外囊泡生物标志物，并与能够反映中枢神经***疾病变化的生物标志物结合，并且在优选的方式中，可以同时检测L1CAM蛋白、SHISA9蛋白、GLT1蛋白、CNPase蛋白等这些中枢神经***来源的胞外囊泡生物标志物和Aβ1-40蛋白、Aβ1-42蛋白、寡聚Aβ(oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-synuclein(α-syn)蛋白等能够反映中枢神经***疾病变化的生物标志物，从而依据于同时检测的结果来进行诊断。还可以通过同时检测到两种标志物双阳性的胞外囊泡的数量和/或这样的胞外囊泡的粒径进行诊断。例如还可以辅助计算两种标志物双阳性的胞外囊泡占某一种标志物阳性的胞外囊泡或总胞外囊泡中的比值，从而能够进一步提高诊断的效果。
利用本申请的方法、***、试剂盒以及组合物可以有效地利用外周体液来进行检测，从而可以避免创伤性地获取受试者的脑脊液，大大降低受试者的取样风险、大大减轻检测的困难程度、大大增加检测的临床应用性和推广性。利用本申请的方法、***、试剂盒以及组合物，能够同时检测中枢神经***来源标志物与疾病相关标志物，降低样本检测时间，更加高效、便捷。
当利用本申请的方法、***、组合物或试剂盒时，可以有效地通过SHISA9蛋白和磷酸化tau217(p-tau217)蛋白作为双标进行检测，以区分阿尔茨海默病患者和健康人群。
当利用本申请的方法、***、组合物或试剂盒时，可以有效地通过GLT1蛋白和α-syn蛋白作为双标进行检测，以区分帕金森病患者、多***萎缩患者和健康人群。
当利用本申请的方法、***、组合物或试剂盒时，可以有效地通过CNPase蛋白与α-syn蛋白作为双标进行检测，以区分帕金森病患者、多***萎缩患者和健康人群。
实施例
实验方法1：
(a)外周体液预处理——超速离心胞外囊泡富集
1.来自受试者的血浆在37℃恒温培养箱溶解10-15分钟，涡旋混匀；
2.在4℃条件下，10,000g转速离心30分钟；
3.吸取100μL离心后的血浆至超离管中，并加入900μL PBS缓冲液(0.22μm过滤)混匀；
4.在4℃条件下，100,000g转速超速离心1小时；
5.弃去上清，保留约100μL的沉淀；
6.加入200μL PBS缓冲液(0.22μm过滤)，使用200μL量程移液器吹吸200次重悬；
7.向重悬后的液体中加入700μL PBS缓冲液(0.22μm过滤)混匀；
8.在4℃条件下，100,000g转速超速离心1小时；
9.弃去上清，保留约50μL的沉淀；
10.加入100μL PBS缓冲液(0.22μm过滤)，使用200μL量程移液器吹吸200次重悬，待用。
(b)利用标记方法标记用于检测生物标志物的抗体
使用的试剂：
利用Alexa荧光标记来标记靶向中枢神经***来源胞外囊泡生物标志物的抗体和靶向阿尔茨海默病生物标志物的抗体。其中靶向中枢神经***来源胞外囊泡生物标志物的抗体为SHISA9多克隆抗体。其中靶向阿尔茨海默病生物标志物的抗体为p-tau217多克隆抗体。其中，标记后的试剂为SHISA9多克隆抗体标记购买自Zenon TM Alexa Fluor TM 405，p-tau217多克隆抗体标记购买自Zenon TM Alexa Fluor TM 488。
1.样本封闭
1.1取10μL利用上述方法经超速离心分离的胞外囊泡至1.5mL离心管或者PCR管；
1.2加入等体积2％BSA溶液(0.22μm过滤)混匀；
1.3室温孵育封闭1小时；
1.4加入10μL PBS缓冲液(0.22μm过滤)稀释封闭后的样本；
2.样本标记
本实验涉及神经特异性标志物抗体SHISA9；疾病相关标志物抗体p-tau217；使用Zenon TM Alexa Fluor TM 405标记SHISA9，Zenon TM Alexa Fluor TM 488标记p-tau217；
2.1准备1μgPBS缓冲液中的抗体(0.2μg/μL于5μLPBS中)；
2.2加入5μL染料在抗体(1μg)中，混匀后室温(25℃)避光孵育20分钟；
2.3向3.2步骤的溶液中加入3μL(3μg)阻断剂(IgG阻断剂稀释至1μg/μL)，混匀后室温(25℃)避光孵育10分钟；
2.4用PBS缓冲液(0.22μm过滤)稀释上述混合物至总体积50μL；
2.5加入5μL Zenon TM Alexa Fluor TM 488标记的p-tau217于步骤(a)制备完毕的样本中，混匀后室温(25℃)避光孵育5分钟；
2.6加入3μL Zenon TM Alexa Fluor TM 405标记的SHISA9于步骤2.5样本中，混匀后4℃避光孵育过夜；
3.样本固定
3.1在标记完毕的样本中加入20μL 4％PFA，混匀后室温(25℃)避光孵育20分钟；
3.2使用170μL PBS缓冲液(0.22μm过滤)稀释固定后的样本，待用。
(c)检测
通过NanoFCM纳米流式分析仪进行检测标记信号强度与粒径分布，从而确定相关生物标志物的存在及含量。
实施例1
分别获取性别、年龄匹配的阿尔茨海默病受试者(AD组)98例、健康对照(CT组)12例的血浆样本，利用上述实验方法1进行实验。
利用NanoFCM纳米流式分析仪分别检测SHISA9抗体单荧光标记、p-tau217抗体单荧光标记、SHISA9和p-tau217抗体双荧光标记的胞外囊泡数量，计算其占总胞外囊泡数量的比值，分析不同粒径范围下的各组差异。
血浆中单独标记SHISA9蛋白阳性胞外囊泡占总胞外囊泡数量的比值在60-100nm粒径、100-150nm粒径、以及全粒径范围，AD组和CT组均存在 显著性差异(**,p<0.01)(图1A)；血浆中单独标记p-tau217蛋白阳性胞外囊泡占总胞外囊泡数量的比值在60-100nm粒径、以及全粒径范围，AD组和CT组均存在极显著性差异(****,p<0.0001)，在100-150nm粒径范围，AD组和CT组存在显著性差异(**,p<0.01)(图1B)；血浆中SHISA9蛋白和p-tau217蛋白双阳性胞外囊泡占总胞外囊泡数量的比值在100-150nm粒径范围、以及全粒径范围，AD组和CT组均存在极显著性差异(***,p<0.001)(图1C)。
通过受试者工作特征曲线(receiver operating characteristic curve,ROC)分析进行诊断效率评估，SHISA9蛋白单标在60-100nm粒径范围的曲线下面积(area under curve,AUC)为0.749，SHISA9蛋白和p-tau217蛋白双标在100-150nm粒径范围的AUC为0.890，使用逻辑回归分析Enter方法纳入SHISA9 60-100nm及SHISA9+p-tau217 100-150nm数据，AUC为0.891(图1D)；p-tau217蛋白单标在60-100nm粒径范围的AUC为0.877，SHISA9蛋白和p-tau217蛋白双标在100-150nm粒径范围的AUC为0.890，使用逻辑回归分析Enter方法纳入p-tau217 60-100nm及SHISA9+p-tau217 100-150nm数据，AUC为0.910(图1E)。
实施例1的结果显示SHISA9蛋白阳性胞外囊泡、p-tau217蛋白阳性胞外囊泡、以及SHISA9蛋白和p-tau217蛋白双阳性胞外囊泡均具有较好的阿尔茨海默病诊断的潜力，尤其是采用p-tau217蛋白阳性胞外囊泡与SHISA9蛋白+p-tau217蛋白双阳性胞外囊泡进行联合诊断。
实验方法2：
(a)外周体液预处理——超速离心胞外囊泡富集
1.让受试者的血浆在37℃水浴1分钟快速溶解，在4℃条件下2,000g离心15min，转移至新1.5mL离心管后在4℃，12,000g离心30分钟；
2.将100μL血浆转移至1.5ml超速离心管中，并加入900μL 0.22μm过滤后的PBS稀释，4℃，100,000g离心30分钟；
3.小心弃去800μL上清后加入等体积0.22μm过滤后的PBS，混匀后再次4℃，100,000g离心30分钟，弃去上清至剩下200μL，用枪头反复吹打管壁让胞外囊泡重悬并混匀，分装后可保存在-80℃；
4.取出20μL胞外囊泡转移至100kDa超滤管中，加入200μL 0.1％Triton X-100/PBS，室温孵育30分钟；
5.将超滤管在4℃，12,000g离心3分钟，并加入200μL 0.22μm过滤后的PBS，再次离心；
6.将超滤管中剩余液体转移至新的1.5ml离心管中，待用；
(b)利用标记方法标记用于检测生物标志物的抗体
利用Alexa荧光标记来标记靶向中枢神经***来源胞外囊泡生物标志物的抗体和靶向阿尔茨海默病生物标志物的抗体。其中靶向中枢神经***来源胞外囊泡生物标志物的抗体为GLT1单克隆抗体，靶向帕金森病生物标志物的抗体为α-synuclein(α-syn)单克隆抗体(syn211和MJFR14)。其中，标记后的试剂为GLT1单克隆抗体标记购买自Zenon TM Alexa Fluor TM 405，α-synuclein(α-syn)单克隆抗体标记购买自Zenon TM Alexa Fluor TM 488。
1.按照产品说明书使用Zenon TM Alexa Fluor TM 405试剂盒标记GLT1抗体：5μL荧光二抗+1μg GLT1抗体，室温避光孵育5分钟后，再加入5μL IgG室温避光孵育5分钟；
2.每个样本加入0.2μg GLT1抗体，室温避光孵育30min；
3.按照产品说明书使用Zenon TM Alexa Fluor TM 488试剂盒标记α-synuclein单克隆抗体(syn211或MJFR14)：5μL荧光二抗+1μgα-syn抗体，室温避光孵育5分钟后，再加入5μL IgG室温避光孵育5分钟；
4.每个样本加入0.2μgα-syn抗体(syn211或MJFR14)，室温避光孵育30min；
5.用0.22μm过滤后的PBS稀释样本至400μL，待用。
(c)检测
通过Apogee纳米流式分析仪进行检测标记信号强度，从而确定外周体液中中枢神经***来源的疾病相关生物标志物的存在及含量。
实施例2
利用上述实验方法2，收集临床队列(103位帕金森病(PD)受试者，45位多***萎缩(MSA)受试者和106位年龄和性别匹配的健康对照(CT))的血浆样品并利用Apogee纳米流式细胞仪免疫荧光标记技术检测。
采用Apogee纳米流式分析仪免疫荧光标记技术，可以分别检测到GLT1蛋白阳性的胞外囊泡，以及α-syn蛋白(syn211与MJFR14均为抗α-syn蛋白抗体)阳性的胞外囊泡(图2A)，也可同时检测到GLT1蛋白与α-syn蛋白双阳性的胞外囊泡，具有良好的可靠性和可重复性(图2B和C)。
利用上述实验方法2，收集临床队列(103位帕金森病(PD)受试者，45位多***萎缩(MSA)受试者和106位年龄和性别匹配的健康对照(CT))的血浆样品并利用Apogee纳米流式细胞仪免疫荧光标记技术检测血浆中GLT1蛋白与α-syn蛋白双阳性的胞外囊泡数量，PD组极显著高于MSA组和CT组(***,p<0.001)，而MSA组和CT组之间没有显著差异(图3A和B)。
GLT1蛋白与α-syn蛋白双阳性的胞外囊泡数量作为临床诊断，能够较好地区分PD患者与健康对照、PD患者与MSA患者，具有临床帕金森病的诊断和MSA鉴别诊断的潜力。
在CN106062559B报道了以L1CAM为标志物，先富集中枢神经***来源的外泌体(胞外囊泡的一种，直径为约在30-150nm范围)，然后使用Luminex检测平台检测外泌体中的相关标志物。
使用267名帕金森患者(PD组)与215名年龄、性别相匹配的正常人对照(CT组)的队列进行血浆相关蛋白标志物α-syn的检测：直接检测血浆中的α-syn蛋白，PD组和CT组之间无统计学差异(参见CN106062559B的图1和图2)，而通过抗L1CAM捕获的血浆外泌体中的α-syn蛋白，以及其与血浆总α-syn蛋白的比值，PD组在统计学上有显著升高(**,p<0.01)(参见CN106062559B的图1和图2)，与脑脊液中α-syn蛋白的差异显著性相当(临床队列：100例帕金森患者，100例正常对照)(**,p<0.01)(参见CN106062559B的图1和图2)。
进一步评估抗L1CAM捕获的血浆外泌体中的α-syn蛋白协助临床帕金森病诊断的潜力，对其进行了Pearson相关性分析和受试者工作曲线(Characteristic Operation Curve，ROC)分析。发现抗L1CAM捕获的血浆外泌体中的α-syn蛋白水平与帕金森UPDRS运动评分和疾病严重程度具有相关性(r＝0.176,p＝0.004,Pearson相关)(参见CN106062559B的图3C)。外泌体中α-syn蛋白浓度(AUC＝0.654,灵敏度＝70.1％，特异度＝52.9％)(参见CN106062559B的图3A)及该浓度与血浆总α-syn蛋白的比值(AUC＝0.657， 灵敏度＝71.2％，特异度＝50.0％)均可以提供中等能力的诊断效率，与脑脊液中α-syn蛋白的诊断效率相当(AUC＝0.724，灵敏度＝76.8％,特异度＝53.5％)(参见CN106062559B的图3B)。
在申请号201810136747.8的中国发明专利申请中，收集临床队列(46位多***萎缩患者(MSA组)，49位帕金森病患者(PD组)和50位年龄和性别匹配的健康对照(CT组))的血浆样品，使用Nanosight纳米颗粒跟踪分析仪来测量荧光标记GLT1(EAAT2)的胞外囊泡浓度，评估在含有GLT1荧光标记的胞外囊泡浓度用于PD诊断的潜力。进行受试者工作曲线分析(Receiver Operating Characteristic Analysis，ROC analysis)。观察多***萎缩与帕金森病的鉴别诊断效率，发现对帕金森病的诊断效率适中(AUC＝0.6750，敏感性＝66.00％，特异性＝70.45％)，帕金森病与多***萎缩的鉴别诊断效率适中(AUC＝0.6948，敏感性＝65.91％，特异性＝78.57％)(参见201810136747.8申请的图3)。
如上所述，与已经报道的方法相比，本申请的***或方法可以达到相似的PD诊断效果，而本申请的方法由于同时检测生物体液中的两种或以上的标志物，通过多标检测更省时、高效、临床应用度高，具有良好的前景。
实验方法3：
(a)外周体液预处理——超滤离心
1.将约100μL受试者的血浆37℃水浴融化，在4℃条件下2,000g离心15min，转移至新1.5mL离心管后，在4℃条件下，12,000g离心30分钟；
2.将离心后的血浆上清转移至1.5ml新离心管中，混匀；
3.向100kDa超滤管加入200μL 0.22μm过滤后的0.1％Triton X-100/PBS，再加入50μL离心后的血浆，室温孵育30分钟；
4.孵育完毕后，在4℃条件下，12,000g离心3.5分钟；
5.在向超滤管加入200μL 0.22μm过滤后的0.1％Triton X-100/PBS，再次4℃条件下，12,000g离心3.5分钟；
6.将过滤余下的部分(超滤管内芯部分)用移液器吸出，20μL/管进行每个反应单元所需体积的分装，待用；
(b)利用标记方法标记用于检测生物标志物的抗体
使用的试剂：
利用Alexa荧光标记来标记靶向中枢神经***来源胞外囊泡生物标志物的抗体和靶向阿尔茨海默病生物标志物的抗体。其中靶向中枢神经***来源胞外囊泡生物标志物的抗体为CNPase单克隆抗体。其中靶向多***萎缩生物标志物为α-synuclein(α-syn)单克隆抗体。其中，标记后的试剂为CNPase单克隆抗体标记购买自Zenon TM Alexa Fluor TM 488，α-synuclein(α-syn)单克隆抗体标记购买自Zenon TM Alexa Fluor TM 405。
1.按照产品说明书使用Zenon TM Alexa Fluor TM 488试剂盒标记CNPase抗体：5μL荧光二抗+1μg CNPase抗体，室温避光孵育15分钟后，再加入5μL IgG室温避光孵育5分钟；
2.每个样本(20μL)加入0.2μg CNPase抗体，室温避光孵育30min；
3.按照产品说明书使用Zenon TM Alexa Fluor TM 405试剂盒标记α-synuclein单克隆抗体：5μL荧光二抗+1μgα-syn抗体，室温避光孵育15分钟后，再加入5μL IgG室温避光孵育5分钟；
4.每个样本(20μL)加入0.2μgα-syn抗体，室温避光孵育30min；
5.用0.22μm过滤后的PBS稀释样本至400μL，待用。
(c)检测
通过Apogee纳米流式分析仪进行检测标记信号强度，从而确定外周体液中中枢神经***来源的疾病相关生物标志物的存在及含量。
实施例3
采用Apogee纳米流式细胞仪免疫荧光标记技术，按照上述实验方法3对小型临床队列(15位帕金森病(PD)受试者，17位多***萎缩(MSA)受试者和17位年龄和性别匹配的健康对照(HC))的血浆样本进行检测，能够检测到血浆中CNPase蛋白阳性的胞外囊泡，其数量在MSA组与PD组间无显著性差异，而MSA组与HC组间具有显著差异(*,p<0.05)(图4A)；CNPase蛋白与α-syn蛋白(抗MJFR14抗体标记)双阳性的胞外囊泡数量，在MSA组、PD组与HC组间无显著差异(图4B)，将其通过血浆中胞外囊泡总数标准化后，MSA组与HC组、MSA组与PD组间具有显著差异(*,p<0.05)(图4C)。
CNPase蛋白与α-syn蛋白双阳性的胞外囊泡数量，具有多***萎缩 (MSA)诊断及其与帕金森病(PD)鉴别诊断的潜力。
以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
Figure PCTCN2021092983-appb-000001

Claims (53)

  1. 根据权利要求1～6中任一项所述的方法，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
  2. 根据权利要求7所述的方法，其中，
    所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
    所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
    所述中枢神经***感染包括脑炎、脑膜炎；
    所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
    所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
    所述自主神经***疾病为自主神经功能不全；
    所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
  3. 根据权利要求8所述的方法，其中，
    所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
    所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
  4. 根据权利要求1～9中任一项所述的方法，其中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能神经元、5-羟色胺能神经元、胆碱能神经元。
  5. 根据权利要求1～10中任一项所述的方法，其中，所述中枢神经***来源标志物和/或中枢神经***疾病相关的标志物各自选自蛋白质、核酸、脂类中的一种或两种或三种。
  6. 根据权利要求10或11所述的方法，其中，所述中枢神经***来源标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道 1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA Breceptor 1)、GABA B受体2(GABA Breceptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE)；
    优选所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物；
    进一步优选所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。
  7. 根据权利要求11所述的方法，其中，所述中枢神经***疾病相关的标志物为蛋白质，所述蛋白质选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomeric α-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
  8. 根据权利要求11所述的方法，其中，所述中枢神经***疾病相关的标志物为核酸，所述核酸选自以下中的一种或两种以上：作为阿尔茨海默病(AD)的生物标志物的DNA或RNA、作为帕金森病(PD)及帕金森综合征(例如，多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)、路易体痴呆(DLB))的生物标志物的DNA或RNA。
  9. 根据权利要求14所述的方法，其中，所述作为AD的生物标志物的DNA或RNA包括：与基因UBAP2L、YBX1、SERF2、UBE2B、RPL10A、H3F3AP4、PPP2CA、NMD3、RNF7、RPLP0、SPARC、WTAP、HNRNPU、LINC00265、INMT、SLC35E2、CT60、SYNCRIP、RGS2、SMC6、ARSA、SPDYE7P、SMIM17、TRAF3IP2-AS1、KCNC2、SLC24A1、HLCS、GOSR1、MN1、MGAT5、NBPF14、FBXO31、WDR52、TBC1D2B、ZNF648、NBPF16、PAGR1、AQP2、PRKCI、SCN2B、DPYSL3、TMEM26、TSPAN11、ELL2、FAM186A、CD59、THSD4、GOLGA6B、ARHGEF5、PKD1、BPTF、FLG、POM121L10P、NXPH3、H3F3A、SH3TC2、GGCX、和GREB1关联的DNA或RNA。
  10. 根据权利要求14所述的方法，其中，所述作为PD的生物标志物的DNA或RNA包括：与基因BLOC1S1、UBE2L3、RNF149、FAM89B、LCE6A、NT5DC2、PPP1CC、CCL5、HDLBP、HNRNPAB、NXN、SLC9A4、EIF2AK1、PAPOLA、TRIM50、SIX4、RAB3IP、VANGL2、DHRSX、FOXP4、SYNM、ZNF543、ATF6、LOC100132832、和BLOC1S1-RDH5关联的DNA或RNA。
  11. 根据权利要求3所述的方法，其中，获取受试者的生物体液并对其进行预处理以富集生物样本的方法选自以下中的一种或两种以上：离心、超速离心、超滤管过滤、聚合沉降、特异性抗体捕获。
  12. 根据权利要求11所述的方法，其中，所述中枢神经***来源标志物为蛋白质，检测受试者生物体液中的中枢神经***来源标志物的步骤包括：使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***来源标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***来源标志物的存在及含量。
  13. 根据权利要求11所述的方法，其中，所述中枢神经***疾病相关的 标志物为蛋白质，检测受试者生物体液中的中枢神经***疾病相关的标志物的步骤包括：使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***疾病相关标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***疾病相关标志物的存在及含量。
  14. 根据权利要求18或19所述的方法，其中，所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
  15. 根据权利要求11所述的方法，其中，所述中枢神经***来源标志物为核酸，检测受试者生物体液中的中枢神经***来源标志物的步骤包括：裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***来源的核酸的存在及含量。
  16. 根据权利要求11所述的方法，其中，所述中枢神经***疾病相关的标志物为核酸，检测受试者生物体液中的中枢神经***疾病相关的标志物的步骤包括：裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***疾病相关的核酸的存在及含量。
  17. 一种用于诊断中枢神经***疾病的***，其包括：
    第一检测模块，其用于检测受试者生物体液中的中枢神经***来源标志物，
    第二检测模块，其用于检测受试者生物体液中的中枢神经***疾病相关的标志物，以及
    诊断模块，其基于第一检测模块和第二检测模块分别获得的中枢神经***来源标志物和中枢神经***疾病相关的标志物的检测结果来诊断受试者是否罹患中枢神经***疾病。
  18. 根据权利要求23所述的***，其中，
    所述第一检测模块和所述第二检测模块同时检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物；或者
    所述第一检测模块和所述第二检测模块为同一模块，其同时检测受试者生物体液中的中枢神经***来源标志物和检测受试者生物体液中的中枢神经***疾病相关的标志物。
  19. 根据权利要求23或24所述的***，其还包括：
    富集模块，其用于获取受试者的生物体液并对其进行预处理以富集生物体液中的生物样本，
    其中第一检测模块和第二检测模块检测受试者的富集生物样本中的中枢神经***来源标志物和中枢神经***疾病相关的标志物。
  20. 根据权利要求23～25中任一项所述的***，其还包括：
    数量统计模块，其统计检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的数量，和/或
    粒径测量模块，其测量检测到中枢神经***来源标志物和中枢神经***疾病相关的标志物两者的生物样本的粒径，以及
    诊断模块基于所述生物样本的数量和/或所述生物样本的粒径来诊断受试者是否罹患中枢神经***疾病。
  21. 根据权利要求25或26所述的方法，其中，
    所述生物样本为胞外囊泡；
    优选所述胞外囊泡为神经元来源的胞外囊泡、星形胶质细胞来源的胞外囊泡、少突胶质细胞来源的胞外囊泡或小胶质细胞来源的胞外囊泡。
  22. 根据权利要求23～27中任一项所述的***，其中，所述生物体液选自血液、血清、血浆、唾液、尿液、淋巴液、***或乳汁中的一种或两种以上。
  23. 根据权利要求23～28中任一项所述的***，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
  24. 根据权利要求29所述的***，其中，
    所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
    所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
    所述中枢神经***感染包括脑炎、脑膜炎；
    所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
    所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
    所述自主神经***疾病为自主神经功能不全；
    所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
  25. 根据权利要求30所述的***，其中，
    所述变性变性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
    所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
  26. 根据权利要求23～31中任一项所述的***，其中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能神经元、5-羟色胺能神经元、胆碱能神经元。
  27. 根据权利要求23～32中任一项所述的***，其中，所述中枢神经***来源标志物和/或中枢神经***疾病相关的标志物各自选自蛋白质、核酸、脂类中的一种或两种或三种。
  28. 根据权利要求32或33所述的***，所述中枢神经***来源标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、 巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA Breceptor 1)、GABA B受体2(GABA Breceptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE))，优选所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物；
    进一步优选所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。
  29. 根据权利要求34所述的***，其中，所述中枢神经***疾病相关的标志物为蛋白质，所述蛋白质选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
  30. 根据权利要求34所述的***，其中，所述中枢神经***疾病相关的标志物为核酸，所述核酸选自以下中的一种或两种以上：作为阿尔茨海默病(AD)的生物标志物的DNA或RNA、作为帕金森病(PD)及帕金森综合征(例如，多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)、路易体痴呆(DLB))的生物标志物的DNA或RNA。
  31. 根据权利要求36所述的***，其中，所述作为AD的生物标志物的DNA或RNA包括：与基因UBAP2L、YBX1、SERF2、UBE2B、RPL10A、H3F3AP4、PPP2CA、NMD3、RNF7、RPLP0、SPARC、WTAP、HNRNPU、LINC00265、INMT、SLC35E2、CT60、SYNCRIP、RGS2、SMC6、ARSA、SPDYE7P、SMIM17、TRAF3IP2-AS1、KCNC2、 SLC24A1、HLCS、GOSR1、MN1、MGAT5、NBPF14、FBXO31、WDR52、TBC1D2B、ZNF648、NBPF16、PAGR1、AQP2、PRKCI、SCN2B、DPYSL3、TMEM26、TSPAN11、ELL2、FAM186A、CD59、THSD4、GOLGA6B、ARHGEF5、PKD1、BPTF、FLG、POM121L10P、NXPH3、H3F3A、SH3TC2、GGCX、和GREB1关联的DNA或RNA。
  32. 根据权利要求36所述的***，其中，所述作为PD的生物标志物的DNA或RNA包括：与基因BLOC1S1、UBE2L3、RNF149、FAM89B、LCE6A、NT5DC2、PPP1CC、CCL5、HDLBP、HNRNPAB、NXN、SLC9A4、EIF2AK1、PAPOLA、TRIM50、SIX4、RAB3IP、VANGL2、DHRSX、FOXP4、SYNM、ZNF543、ATF6、LOC100132832、和BLOC1S1-RDH5关联的DNA或RNA。
  33. 根据权利要求25所述的***，其中，所述富集模块具有进行以下步骤中的一种或两种以上的子模块：离心、超速离心、超滤管过滤、聚合沉降、特异性抗体捕获。
  34. 根据权利要求33所述的***，其中，所述中枢神经***来源标志物为蛋白质，第一检测模块用于使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***来源标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***来源标志物的存在及含量。
  35. 根据权利要求33所述的***，其中，所述中枢神经***疾病相关的标志物为蛋白质，第二检测模块用于使受试者生物体液，优选受试者的胞外囊泡与经标记的、与中枢神经***疾病相关标志物特异性反应的抗体进行反应，以及检测反应后标记信号的强度以判断中枢神经***疾病相关标志物的存在及含量。
  36. 根据权利要求40或41所述的***，其中，所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
  37. 根据权利要求33所述的***，其中，所述中枢神经***来源标志物为核酸，第一检测模块用于裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***来源的核酸的存在及含量。
  38. 根据权利要求33所述的***，其中，所述中枢神经***疾病相关的标志物为核酸，第二检测模块用于裂解受试者的生物体液，优选受试者的胞外囊泡并利用DNA或RNA探针或测序技术来检测与中枢神经***疾病相关的核酸的存在及含量。
  39. 一种用于检测中枢神经***疾病的组合物，其包括：
    用于靶向中枢神经***来源标志物的抗体，以及
    用于靶向中枢神经***疾病相关的标志物的抗体。
  40. 根据权利要求45所述的组合物，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
  41. 根据权利要求46所述的组合物，其中，
    所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
    所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
    所述中枢神经***感染包括脑炎、脑膜炎；
    所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
    所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
    所述自主神经***疾病为自主神经功能不全；
    所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
  42. 根据权利要求47所述的组合物，其中，
    所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
    所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
  43. 根据权利要求45～48中任一项所述的组合物，其中，所述中枢神经***来源标志物是来源于如下神经元或细胞的生物标志物：成熟神经元、少突胶质细胞、星形胶质细胞、小胶质细胞、未成熟神经元、施旺细胞、放射状胶质细胞、中间前体细胞、谷氨酸能神经元、GABA能神经元、多巴胺能 神经元、5-羟色胺能神经元、胆碱能神经元。
  44. 根据权利要求45～49中任一项所述的组合物，其中，所述中枢神经***来源标志物选自以下中的一种或两种以上：G蛋白偶联受体162(G Protein-Coupled Receptor 162,GPR162)、超极化激活环核苷酸门控钾通道1(Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1,HCN1)、γ-氨基丁酸A型受体亚基δ(Gamma-Aminobutyric Acid Type A Receptor Subunit Delta,GABRD)、神经黏附分子3(Neuroligin 3,NLGN3)、SHISA9、接触蛋白-1(contactin-1,CNTN1)、NeuN、MAP2、神经丝-L(neurofilament light,NF-L)、神经丝-M(neurofilament medium,NF-M)、神经丝-H(neurofilament heavy,NF-H)、突触素(synaptophysin)、突触融合蛋白(syntaxin)、PSD95、L1CAM、双皮质素(doublecortin)、β3-微管蛋白(beta III tubulin)、NeuroD1、TBR1、微管解聚蛋白1(stathmin 1)、MPZ、NCAM、GAP43、S100、CNP酶(CNPase)、olig 1、olig 2、olig 3、MBP、OSP、MOG、SOX10、NG2、GFAP、GLAST(EAAT1)、GLT1(EAAT2)、谷氨酰氨合成酶(glutamine synthetase)、S100-β、ALDH1L1、CD11b、CD45、Iba1、F4/80、CD68、CD40、波形蛋白(vimentin)、巢蛋白(nestin)、PAX6、HES1、HES5、GFAP、GLAST、BLBP、TN-C、N钙粘附蛋白(N-cadherin)、SOX2、TBR2、MASH1(Ascl1)、vGluT1、vGluT2、NMDAR、谷氨酰胺酶(glutaminase)、谷氨酰胺合成酶(glutamine synthetase)、GABA转运蛋白1(GABA transporter 1,GAT1)、GABA B受体1(GABA Breceptor 1)、GABA B受体2(GABA Breceptor 2)、GAD65、GAD67、酪氨酸羟化酶(tyrosine hydroxylase,TH)、多巴胺转运蛋白(dopamine transporter,DAT)、FOXA2、GIRK2、Nurr1、LMX1B、色氨酸羟化酶(tryptophan hydroxylase,TPH)、血清素转运体(serotonin transporter)、Pet1、胆碱乙酰转移酶(choline acetyl transferase,ChAT)、乙酰胆碱转运体(vesicular acetylcholine transporter,VAChT)、乙酰胆碱酯酶(acetylcholinesterase,AchE)；
    优选所述中枢神经***来源标志物为位于中枢神经***来源的胞外囊泡表面的标志物；
    进一步优选所述中枢神经***来源标志物为选自L1CAM、SHISA9、CNP酶、GLT1(EAAT2)中的一种或两种以上。
  45. 根据权利要求45～50中任一项所述的组合物，其中，所述中枢神经 ***疾病相关的标志物选自以下中的一种或两种以上：Aβ1-40(Aβ40)蛋白、Aβ1-42(Aβ42)蛋白、寡聚Aβ(oligomeric Aβ,oligo-Aβ)蛋白、tau蛋白、磷酸化tau(p-tau)蛋白、α-突触核蛋白(α-synuclein蛋白)、第129位磷酸化α-synuclein(pS129)蛋白、寡聚α-synuclein(oligomericα-synuclein,oligo-α-syn)蛋白、朊病毒蛋白。
  46. 根据权利要求45～51中任一项所述的组合物，其中，所述用于靶向中枢神经***来源标志物的抗体和所述用于靶向中枢神经***疾病相关的标志物的抗体是经标记的抗体，优选所述标记选自以下中的一种或两种以上：荧光标记、同位素标记、酶标记、化学发光剂标记、量子点标记或胶体金标记。
  47. 一种用于检测受试者中枢神经***疾病的试剂盒，其包括：
    用于检测受试者生物体液中的中枢神经***来源标志物的试剂，以及
    用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂。
  48. 根据权利要求53所述的试剂盒，其中，所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂和用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为权利要求45～52中任一项所述的组合物。
  49. 根据权利要求53所述的试剂盒，其中，
    所述用于检测受试者生物体液中的中枢神经***来源标志物的试剂为靶向中枢神经***来源标志物的引物或探针，
    所述用于检测受试者生物体液中的中枢神经***疾病相关的标志物的试剂为靶向中枢神经***疾病相关的标志物的引物或探针。
  50. 根据权利要求53所述的试剂盒，还包括：
    用于获取受试者的生物样本的试剂和装置，优选用于获取受试者胞外囊泡的试剂和装置。
  51. 根据权利要求53所述的试剂盒，其中，所述中枢神经***疾病选自以下中的一种或两种以上：脑血管病、脊髓病变、中枢神经***感染、神经***遗传性疾病、神经***发育异常性疾病、自主神经***疾病、神经***脱髓鞘性病变、癫痫、和神经***变性疾病。
  52. 根据权利要求57所述的试剂盒，其中，
    所述脑血管病包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作；
    所述脊髓病变包括急性脊髓炎、脊髓灰质炎、脊髓空洞；
    所述中枢神经***感染包括脑炎、脑膜炎；
    所述神经***遗传性疾病包括神经皮肤综合征、脊髓小脑性共济失调；
    所述神经***发育异常性疾病包括先天性脑积水、脑性瘫痪；
    所述自主神经***疾病为自主神经功能不全；
    所述神经***变性疾病包括变性病性痴呆、运动障碍性疾病、朊病毒病(Prion病)。
  53. 根据权利要求58所述的试剂盒，其中，
    所述变性病性痴呆包括阿尔茨海默病(AD)、额颞叶痴呆(FTD)、帕金森病痴呆(PDD)、路易体痴呆(DLB)；
    所述运动障碍性疾病包括运动神经元病(MND)、帕金森病(PD)、亨廷顿舞蹈病(HD)、小舞蹈病(Sydenham舞蹈病)、肝豆状核变性(WD)、多***萎缩(MSA)、进行性核上性麻痹(PSP)、皮质基底节变性(CBD)。
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