Application of novel cell-specific mitochondrial vesicles in brain diseases

Abstract

The invention provides an application of novel cell-specific mitochondrial-vesicles in brain diseases, which comprises markers of the novel cell-specific mitochondrial-vesicles for non-disease diagnosis and treatment of neurodegenerative diseases, and the markers comprise one or more of NMDAR2A, CNPase, alpha-syn and Cytochrome C. The invention further provides an application of the novel cell-specific mitochondrial-vesicles in brain diseases. Neuron and oligodendroglia specific markers are combined with mitochondrial related markers, and detection is performed through a nanoflow method, so that a better means is provided for diagnosis and treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of Parkinson's disease diagnosis, and in particular relates to the application of novel cell-specific mitochondrial vesicles in brain diseases. Background Art

[0002] The main clinical symptoms of Parkinson's disease are resting tremor, bradykinesia, muscle rigidity and postural balance disorders. The main pathological features of Parkinson's disease are degeneration and necrosis of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies in the cytoplasm of neurons. Misfolded and polymerized α-synuclein (α-syn) is the main component of Lewy bodies.

[0003] Multiple system atrophy (MSA) is a rare neurodegenerative disease, the main pathological manifestation of which is the formation of Lewy bodies in oligodendrocytes. Its clinical manifestations are similar to those of Parkinson's disease, including autonomic dysfunction, Parkinson's syndrome, cerebellar dysfunction, etc. Multiple system atrophy can include two subtypes, MSA-P and MSA-C. MSA has an early onset age and progresses rapidly. Due to its similar clinical symptoms to Parkinson's disease, the differential diagnosis between Parkinson's disease and multiple system atrophy is currently quite challenging.

[0004] At present, the development of diagnostic technology has made continuous breakthroughs, and imaging and genetic testing have become common solutions in clinical practice. However, current methods still have great difficulties in the early diagnosis of Parkinson's disease and multiple system atrophy, especially the differential diagnosis of the two. Extracellular vesicles (EVs), as a new auxiliary diagnostic solution, have great potential in the diagnosis and differential diagnosis of neurodegenerative diseases. Summary of the invention

[0005] In order to overcome the deficiencies in the prior art, the present invention provides a novel application of cell-specific mitochondrial vesicles in brain diseases, which is a novel extracellular vesicle detection technology that combines extracellular vesicles from the nervous system and mitochondrial markers for auxiliary diagnosis and differential diagnosis of Parkinson's disease and multiple system atrophy.

[0006] The method determined that the markers NMDAR2A, CNPase, α-syn, and Cytochrome C can be used to mark mitochondria-related vesicles derived from neurons and oligodendrocytes in the peripheral blood of the nervous system for auxiliary diagnosis of neurodegenerative diseases, especially for the diagnosis and differential diagnosis of Parkinson's disease and multiple system atrophy. The present application extracts extracellular vesicles from plasma, combines extracellular vesicle-specific markers of the nervous system with mitochondrial markers, and uses nanoflow cytometry to provide an efficient and sensitive diagnostic method for neurodegenerative diseases.

[0007] As one aspect of the present invention, the present invention provides novel cell-specific mitochondrial vesicle markers for non-disease diagnosis and treatment of neurodegenerative diseases, wherein the markers include one or more of NMDAR2A, CNPase, α-syn, and Cytochrome C.

[0008] Preferably, the neurodegenerative disease includes one or more of Parkinson's disease and multiple system atrophy.

[0009] Preferably, the marker is derived from one or more of mitochondria-associated large vesicles and mitochondria-associated exosomes of neurons and oligodendrocytes in peripheral blood.

[0010] Preferably, NMDAR2A and Cytochtome C in mitochondria-associated vesicles of neuronal origin can be used to diagnose Parkinson's disease and / or multiple system atrophy.

[0011] Preferably, CNPase and Cytochtome C in mitochondria-associated vesicles derived from oligodendrocytes can be used to diagnose Parkinson's disease and / or multiple system atrophy.

[0012] Preferably, Cytochtome C in mitochondria-associated large vesicles can be used to screen for Parkinson's disease and / or multiple system atrophy.

[0013] Preferably, CNPase and Cytochtome C in mitochondria-associated large vesicles derived from oligodendrocytes can be used to screen for Parkinson's disease and / or multiple system atrophy.

[0014] Preferably, CNPase and α-syn in mitochondria-associated large vesicles derived from oligodendrocytes can be used to screen for Parkinson's disease.

[0015] Beneficial effects of the present invention:

[0016] The present invention provides a method for detecting mitochondria-related vesicles derived from neurons and oligodendrocytes in peripheral blood, which can be used for auxiliary diagnosis and differential diagnosis of Parkinson's disease and multiple system atrophy. By combining neuron- and oligodendrocyte-specific markers with mitochondria-related markers and performing detection by nanoflow cytometry, a better diagnosis and treatment method for neurodegenerative diseases is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The screening results of the normal group and the disease group in Example 1, wherein A, mitochondria-related exosomes derived from neurons are used for the diagnosis of PD and MSA, and B, mitochondria-related exosomes derived from oligodendrocytes are used for the diagnosis of PD and MSA;

[0018] Figure 2 The screening results of the normal group and the disease group in Example 2, wherein A, mitochondria-associated large vesicles can be used for the diagnosis of Parkinson's disease, B, oligodendrocyte-derived large vesicles can be used for the diagnosis of Parkinson's disease, and C, α-syn in mitochondria-associated large vesicles derived from oligodendrocytes can be used for the diagnosis of Parkinson's disease;

[0019] Figure 3 The screening results of the normal group and the disease group in Example 3, wherein A. mitochondria-associated large vesicles can be used for the differential diagnosis of Parkinson's disease and multiple system atrophy, B. large vesicles derived from oligodendrocytes can be used for the differential diagnosis of Parkinson's disease and multiple system atrophy, and C. α-syn in mitochondria-associated large vesicles derived from oligodendrocytes can be used for the differential diagnosis of Parkinson's disease and multiple system atrophy. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further explained below by examples, but the protection scope of the present invention is not limited in any form by the examples.

[0021] Example 1: Diagnosis of Parkinson's disease and multiple system atrophy by detecting mitochondrial-associated exosomes in peripheral blood

[0022] ① EVs extraction from plasma samples:

[0023] Whole blood samples were centrifuged at 500-800 g for 10-15 minutes at 4°C to remove red blood cells, and at 1000-3000 g for 15-20 minutes at 4°C to remove platelets to obtain plasma samples. 80-100 μL of the supernatant was diluted 8-12 times with 800-1000 μL of phosphate-buffered saline (PBS; filtered with a 0.22 μm filter membrane) and centrifuged at 10000-20000 g for 60-90 minutes; the supernatant, about 80-100 μL, was taken and added with 800-1000 μL of phosphate-buffered saline (PBS; filtered with a 0.22 μm filter membrane). PBS, after strict balance, centrifuge at 80000-120000g for 60-90 minutes at 4℃, retain 100-200μL of precipitate, add 800-1000μL PBS, after strict balance, centrifuge at 80000-120000g for 60-90 minutes at 4℃, retain about 100-200μL of precipitate, and then aliquot and store at -80℃;

[0024] ② Sample blocking: Add 5-10 μL 2%-4% BSA to 5-10 μL exosome sample for blocking, store at 26°C for 1 hour, and then use 5-20 μL PBS to terminate the blocking;

[0025] ③ Antibody fluorescent labeling: Use PBS to adjust 1-2 μg of the corresponding antibody (NMDAR2A, CNPase, Cytochtome C) to a total volume of 5-10 μL, add 5-10 μL Alexa Fluor TM (NNMDAR2A, CNPase using Alexa Fluor TM 488 labeled, Cytochrome C using Alexa Fluor TM 647 labeling), place in a dark place at 26°C for 20-40 minutes, add 2-5 μL of solution B, place in a dark place at 26°C for 10-15 minutes, and finally add PBS to make the system up to a total volume of 50 μL. Add 3-5 μL of labeling solution (for each antibody) to each sample and place at 4°C, away from light, overnight;

[0026] ④ Flow cytometry: Add 15-30 μL 4% PFA to each sample, store in a dark place at 26°C for 10-40 minutes, add 100-150 μL PBS to each sample, and perform Cytoflex flow cytometry analysis.

[0027] The EVs in the plasma of the normal control group and the disease group (the disease group refers to Parkinson's disease and multiple system atrophy) were extracted through the above preparation steps and conditions, and the differences between the normal control group and the disease group were compared after using antibodies to label the biomarkers CNPase, Cytochtome C, and NMDAR2A. The results are shown in Figure 1 In the figure, the normal group is NC, Parkinson's disease is PD, and multiple system atrophy is MSA. NMDAR2A positivity indicates that it is derived from neural cells, and CnPase positivity indicates that it is derived from oligodendrocytes.

[0028] from Figure 1 It can be seen that CNPase, Cytochtome C, and NMDAR2A can be used as markers for accurate diagnosis of two diseases: Figure 1 A, NMDAR2A and Cytochtome C double positive vesicles were significantly different between the disease group and the control group, so the neuronal mitochondrial associated vesicles (particle size

[0029] <150-200nm) can be used to diagnose Parkinson's disease or multiple system atrophy; Figure 1 B, CNPase and Cytochtome C double positive vesicles have significant differences in disease and control groups, so CNPase and Cytochtome C in mitochondria-associated vesicles derived from oligodendrocytes can be used to diagnose Parkinson's disease or multiple system atrophy.

[0030] Example 2 Diagnosis of Parkinson's disease by detecting mitochondrial-associated large vesicles in peripheral blood

[0031] ① EVs extraction from plasma samples:

[0032] Whole blood samples were centrifuged at 500-800g for 10-15 minutes at 4°C to remove red blood cells, and at 1000-3000g for 15-20 minutes at 4°C to remove platelets to obtain plasma samples. 80-100 μL of the supernatant was diluted 8-12 times with 800-1000 μL of phosphate buffered saline (PBS; filtered with 0.22 μm filter membrane), and centrifuged at 20000-50000g for 60-90 minutes. 100-200 μL of the bottom was resuspended and pipetted 200-300 times, and 800-1000 μL PBS was added. After strict balancing, the samples were centrifuged again at 20000-50000g for 60-90 minutes at 4°C. 100-200 μL of the precipitate was retained, resuspended and pipetted 200-300 times, and then aliquoted and stored at -80°C.

[0033] ② Sample blocking: Add 5-10 μL 2%-4% BSA to 5-10 μL large vesicle sample for blocking, store at 26°C for 1 hour, and then use 5-10 μL PBS to terminate the blocking;

[0034] ③ Antibody fluorescent labeling: Use PBS to adjust 1-2 μg of antibody (CNPase, Syn agg, Cytochtome C) to a total volume of 5-10ul, add 5-10uL Alexa Fluor TM 488, 561 or 647 were used to label α-syn, CNPase and Cytochrome C, respectively. After being stored in the dark at 26°C for 20-40 minutes, 2-5 μL of solution B was added and stored in the dark at 26°C for 10-15 minutes. Finally, 30-50 μL of PBS was added to a total volume of 50 μL. 3-5 μL of labeling solution (for each antibody) was added to each sample and incubated at 4°C, protected from light, overnight.

[0035] Flow cytometry: add 15-30 μL 4% PFA to each sample and store it in the dark at 26°C for 10-40 minutes. Add 100-150 μL PBS to each sample and perform Cytoflex flow cytometry to detect large vesicles (particle size greater than 200 nm).

[0036] The EVs in the plasma of the normal control group and the disease group (the disease group refers to Parkinson's disease) were extracted through the above preparation steps and conditions, and the mitochondria-related and oligodendrocyte-related biomarkers CNPase, α-syn, and Cytochtome C were labeled with antibodies to compare the differences between the normal control group and the disease group. The results are shown in Figure 2 .

[0037] according to Figure 2 The results show that CNPase, α-syn, and Cytochtome C can be used as markers for accurate diagnosis of this disease and can be used as the basis for diagnosis of this disease: Figure 2 A, Cytochtome C positivity leads to significant differences, so Cytochtome C can be used to screen for Parkinson's disease; Figure 2 B, CNPase and Cytochtome C positivity results in significant differences, so CNPase and Cytochtome C can be used to screen for Parkinson's disease; Figure 2 C, Antibody CNPase and α-syn positivity bring significant differences, so CNPase and α-syn can be used to screen for Parkinson's disease.

[0038] Example 3 Differential diagnosis of Parkinson's disease and multiple system atrophy by detecting mitochondrial-associated large vesicles in peripheral blood

[0039] ① EVs extraction from plasma samples:

[0040] Whole blood samples were centrifuged at 500-800g for 10-15 minutes at 4°C to remove red blood cells, and at 1000-3000g for 15-20 minutes at 4°C to remove platelets to obtain plasma samples. 80-100 μL of the supernatant was diluted 8-12 times with 800-1000 μL of phosphate buffered saline (PBS; filtered with 0.22 μm filter membrane), and centrifuged at 20000-50000g for 60-90 minutes. The bottom 80-100 μL was resuspended and pipetted 200-300 times, and 800-1000 μL PBS was added. After strict balancing, the samples were centrifuged again at 20000-50000g for 60-90 minutes at 4°C. 100-150 μL of the precipitate was retained, resuspended and pipetted 200-300 times, and then aliquoted and stored at -80°C.

[0041] ② Sample blocking: Add 5-10 μL 4% BSA to 5-10 μL large vesicle sample for blocking, store at 26°C for 1 hour, and then use 5-10 μL PBS to terminate the blocking;

[0042] ② Antibody fluorescent labeling: Use PBS to adjust 1-2 μg of antibody (CNPase, α-syn, CytochtomeC) to a total volume of 5-10 μL, add 5-10 μL Alexa Fluor TM 488, 561 or 647 were used to label α-syn, CNPase and Cytochrome C, respectively. After being stored in the dark at 26°C for 20-40 minutes, 2-5 μL of LB solution was added and the mixture was stored in the dark at 26°C for 10-15 minutes. Finally, 30-50 μL of PBS was added to a total volume of 50 μL. 3-5 μL of labeling solution (for each antibody) was added to each sample and incubated at 4°C, protected from light, overnight.

[0043] ④ Flow cytometry: Add 15-30 μL 4% PFA to each sample, store in a dark place at 26°C for 10-20 minutes, and add 100-150 μL PBS to each sample for Cytoflex flow cytometry analysis.

[0044] The EVs in the plasma of the normal control group and the disease group (the disease group refers to Parkinson's disease and multiple system atrophy) were extracted through the above preparation steps and conditions, and the mitochondria-related and oligodendrocyte-related biomarkers CNPase, α-syn, and Cytochtome C were labeled with antibodies to compare the differences between the normal control group and the disease group. The results are shown in Figure 3 .

[0045] according to Figure 3 The results show that CNPase, α-syn, and Cytochtome C are markers for differential diagnosis of Parkinson's disease and multiple system atrophy, and can be used as the basis for diagnosis and differential diagnosis of these two diseases: Figure 3 A, Cytochtome C positivity results in significant differences, so Cytochtome C in mitochondrial-associated large vesicles can be used to screen for Parkinson's disease or multiple system atrophy; Figure 3 B, CNPase and Cytochtome C positivity of antibodies bring significant differences, so CNPase and Cytochtome C in oligodendrocyte-derived large vesicles can be used to screen for Parkinson's disease or multiple system atrophy; Figure 3 In C, CNPase and α-syn positivity brought significant differences, so CNPase and α-syn in mitochondria-associated large vesicles derived from oligodendrocytes can be used to screen for Parkinson's disease or multiple system atrophy.

[0046] The present invention determines that the markers NMDAR2A, CNPase, α-syn, and Cytochrome C can be used to mark mitochondria-related vesicles derived from neurons and oligodendrocytes of the nervous system in peripheral blood, and are used for auxiliary diagnosis of neurodegenerative diseases, especially for the diagnosis and differential diagnosis of Parkinson's disease and multiple system atrophy. It extracts extracellular vesicles from plasma, combines extracellular vesicle-specific markers of the nervous system with mitochondrial markers, and adopts a nanoflow detection method, thereby providing an efficient and sensitive diagnosis method for neurodegenerative diseases.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A novel cell-specific mitochondrial vesicle marker for non-disease diagnosis and treatment of neurodegenerative diseases, characterized by: The markers include one or more of NMDAR2A, CNPase, α-syn, and Cytochrome C.

2. The marker according to claim 1, characterized in that: The neurodegenerative diseases include one or more of Parkinson's disease and multiple system atrophy.

3. The marker according to claim 1, characterized in that: The marker is derived from one or more of mitochondria-associated large vesicles and mitochondria-associated exosomes of neurons and oligodendrocytes in peripheral blood.

4. The marker according to claim 1, characterized in that: NMDAR2A and Cytochtome C in mitochondria-associated vesicles of neuronal origin can be used to diagnose Parkinson's disease and / or multiple system atrophy.

5. The marker according to claim 1, characterized in that: CNPase and Cytochtome C in oligodendrocyte-derived mitochondria-associated vesicles can be used to diagnose Parkinson's disease and / or multiple system atrophy.

6. The marker according to claim 1, characterized in that: Cytochtome C in mitochondria-associated large vesicles can be used to screen for Parkinson's disease and / or multiple system atrophy.

7. The marker according to claim 1, characterized in that: CNPase and Cytochtome C in oligodendrocyte-derived mitochondria-associated large vesicles can be used to diagnose Parkinson's disease and / or multiple system atrophy.

8. The marker according to claim 1, characterized in that: CNPase and α-syn in oligodendrocyte-derived mitochondria-associated large vesicles can be used to diagnose Parkinson's disease.

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