Astrocyte-derived transporter and application thereof in treatment of cerebrovascular diseases

Abstract

The invention belongs to the technical field of neuroscience, biological medicine and cell therapy, and discloses an astrocyte-derived migration body and an application thereof in treatment of cerebrovascular diseases, the migration body has unique characteristics different from exosomes: morphology: the diameter range is 500-3000 nm, the migration body is connected with a contraction filament, and the diameter range is 500-3000 nm; small vesicles and mitochondrial ridge-like structures can be seen inside; the specific markers of the migration body: TSPAN4, Integrin alpha 5, PCGP and NDST1 are positive, while the traditional exosome markers Alix and TSG101 are deficient, and the specific markers of the migration body: TSPAN4, Integrin alpha 5, PCGP and NDST1 are deficient; the functional localization is derived from an astrocyte migration process, has a typical vesicle structure, and is co-localized with a contraction filament. The invention provides a new strategy for treating stroke and other neurodegenerative diseases by using the astrocyte migration body for the first time, and the astrocyte migration body has relatively high clinical transformation potential and biological safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of neuroscience, biological medicine and cell therapy, and particularly relates to a migrasome derived from astrocytes and application thereof in treatment of cerebrovascular diseases. BACKGROUND

[0002] Stroke is a major cause of death and disability worldwide, and the imbalance of neuroinflammation and the destruction of vascular microenvironment are the main pathogenesis. The neurovascular unit (NVU) is an important structure for maintaining the function of the central nervous system, and its dynamic remodeling is crucial for repair and reconstruction after stroke. Astrocytes, as an important component of NVU, transform from a resting state to a stressed state after stroke injury, and their migration process plays a key role in neuroprotection or neurotoxicity. Migrasome is a recently discovered migration-dependent vesicular structure, and its function in immune response and tumor metastasis has been reported. However, there is no systematic research and application report on the migrasome of astrocytes in the neurovascular reconstruction and recovery of neurological function after stroke.

[0003] The existing treatment methods for nerve damage after cerebrovascular diseases have significant limitations: 1) Drug therapy: Although commonly used neuroprotective agents, anti-inflammatory drugs or antioxidant drugs show certain effects in animal models, there are still some problems in clinical translation: ① Poor blood-brain barrier permeability, making it difficult for drugs to effectively reach the lesion site; ② Single target, difficult to simultaneously regulate complex neuroinflammation, vascular injury and repair pathways; ③ Significant side effects limit their safety and sustainable application. 2) Vascular recanalization therapy: such as intravenous thrombolysis and endovascular thrombectomy can rapidly restore blood supply in the acute phase, but still have obvious shortcomings: ① The treatment time window is extremely limited, and a large number of patients cannot benefit from the delay; ② Reperfusion is often accompanied by vascular injury and inflammatory response, leading to reperfusion injury; ③ Lack of effect on the overall repair of neurovascular units, which cannot promote long-term recovery of neurological function. 3) Cell and exosome therapy: stem cell transplantation and its derived exosomes have been widely studied in recent years, but still have bottlenecks: ① There is significant heterogeneity between cell sources and effects, and the efficacy is unstable; ② The mechanism is complex and has not been fully elucidated, making it difficult to identify key regulatory pathways; ③ Preparation, preservation and quality control have not been standardized, and there are still great obstacles in clinical translation.

[0004] Migrasome is a kind of migration-dependent vesicle discovered in recent years, which can realize specific information transmission and material transport, and has potential in immune regulation and tissue repair. However, the research on migrasome is still in the initial stage: ① the mechanism is not clear; ② there is no uniform standard for preparation and preservation process; ③ there is almost no systematic research and application report in the field of neural repair of cerebrovascular disease. Based on the above shortcomings, a new treatment strategy is urgently needed to make up for the limitations of drugs, vascular recanalization, cells / exosomes and early research of migrasome, to realize the precise regulation and functional recovery of neural vascular unit.

[0005] The present application innovatively introduces migrasome derived from astrocytes to realize the precise regulation of neural vascular unit, which is expected to have important scientific significance and application value in the prevention and treatment of cerebrovascular diseases and clinical transformation. SUMMARY

[0006] The present application aims to overcome the shortcomings in the prior art and provide a migrasome derived from astrocytes and its application in the treatment of cerebrovascular diseases.

[0007] The technical scheme adopted by the present application to solve its technical problems is: A migrasome derived from astrocytes, the migrasome has unique characteristics different from exosomes: 1) morphology: the diameter range is 500-3000 nm, which is much larger than the diameter range of exosomes (30-150 nm), and is connected with contractile filaments, and small vesicles and mitochondrial cristae-like structures can be seen inside; 2) migrasome specific markers: TSPAN4, Integrin alpha 5, PCGP, NDST1 are positive, while traditional exosome markers Alix and TSG101 are absent, and functional proteins involved in energy metabolism, oxidative phosphorylation and phagocytosis biological pathways are abundant; 3) functional localization: derived from the migration process of astrocytes, with typical vesicular structure, co-localized with contractile filaments, reflecting its correlation with cell migration dynamics, and may participate in the remodeling of neural vascular unit through differential signal and material transport mechanism, with neuroprotective and blood brain barrier repair functions.

[0008] Further, the extraction method of the migrasome comprises the following steps: When the fusion degree of the primary astrocytes reaches 60%-70%, the culture supernatant is discarded, the cell culture dish is washed twice with PBS having a molar concentration of 0.01 M (all the PBS used in the present application has this concentration), and the PBS is discarded; a trypsin-EDTA solution having a mass concentration of 0.25% is added for digestion, 1 mL of the trypsin-EDTA solution is added per 10 cm of the culture dish, and the dish is placed in a 37°C incubator for about 2 minutes; under a microscope, when the cells become round and small bright spots, a complete culture medium containing 10% fetal bovine serum in an amount larger than that of the digestion solution is immediately added to terminate the digestion; The bottom of the dish is gently blown with a sterile Pasteur pipette to make the cells fall off in a snowflake shape, and the cell suspension is collected in a sterile centrifuge tube; the bottom of the dish is washed twice with PBS, and the liquid is collected in the same centrifuge tube; first, centrifugation is performed at 1000 rpm for 5 minutes to collect the supernatant; then, the following steps are sequentially performed: First high-speed centrifugation: 4000 g for 20 minutes at 4°C; the supernatant is collected; Second high-speed centrifugation: 20000 g for 30 minutes at 4°C; the supernatant is discarded, and a precipitate is obtained; PBS resuspension: the precipitate is resuspended in PBS; Third high-speed centrifugation: 20000 g for 30 minutes at 4°C; the supernatant is discarded, and a precipitate is obtained; The final precipitate obtained is the astrocyte-derived migratory body, which is stored at -80°C.

[0009] Use of the migratory body as described above in the preparation of a medicament for treating ischemic stroke.

[0010] Use of the migratory body as described above in the preparation of a medicament for improving neuron survival and / or promoting axon growth and synapse reconstruction Use of the migratory body as described above in the preparation of a medicament for repairing neural networks after central nervous system injury.

[0011] Use of the migratory body as described above in the preparation of a medicament for enhancing the expression of tight junction proteins of brain microvascular endothelial cells and restoring the integrity of the blood-brain barrier, which is suitable for use in a medicament for treating nervous system diseases accompanied by BBB damage.

[0012] Use of the migratory body as described above in the preparation of a medicament for treating nervous system diseases.

[0013] Further, the nervous system diseases include ischemic stroke, traumatic brain injury (TBI), vascular cognitive impairment, Alzheimer's disease, small vessel disease, leukoencephalopathy, encephalitis, spinal cord injury, and other diseases involving neuron axon injury and blood-brain barrier damage.

[0014] Further, the migrasome is effectively entered into the central nervous system by a non-cell delivery method, so as to realize targeted regulation of neurons and vascular endothelial cells, and improve treatment compliance and safety.

[0015] Further, the non-cell delivery method comprises nasal delivery, intravenous injection, intrathecal injection or intracerebroventricular injection.

[0016] The application has the following advantages and positive effects: 1. The migrasome is an astrocyte-derived migrasome, which is a vesicle structure released in the migration process of astrocytes, has a diameter of about 500-3000 nanometers, has TSPAN4, Integrin alpha 5 and other migrasome characteristic markers, and is significantly different from traditional exosomes. The migrasome is rich in various proteins related to oxidative phosphorylation, endocytosis and cell energy metabolism, can enhance the expression of synaptic proteins of neurons in vitro, improve the tight junction of brain microvascular endothelial cells, and thus improve the integrity of the blood-brain barrier; after nasal exogenous delivery in an ischemic stroke mouse animal model, the migrasome can promote nerve function, intelligence recovery and social behavior improvement. The application first proposes to use astrocyte-derived migrasomes as a new strategy for treating stroke and other neurodegenerative diseases, and has high clinical transformation potential and biological safety.

[0017] 2. The migrasome derived from normal astrocytes can be applied in repairing the blood-brain barrier, promoting synaptic repair of neurons, improving neurological and cognitive dysfunction, and is particularly suitable for treating ischemic stroke, brain trauma, vascular dementia and other central nervous system diseases.

[0018] 3. The migrasome is first applied in the field of neural disease treatment, and has a major theoretical and clinical transformation breakthrough: the migrasome derived from astrocytes is first applied in the treatment of ischemic stroke and other nervous system diseases, breaks through the limitations of traditional cell therapy and vesicle therapy, fills the technical gap of the application of migrasomes in the central nervous system, and has high originality and international leading nature.

[0019] 4. The migrasome is a functional natural nanometer organelle, and has targeting, biological activity and safety: the migrasome is naturally released in the migration process of astrocytes, has specific specificity of the parent cell function, is rich in functional proteins involved in key pathways such as energy metabolism and oxidative phosphorylation, can accurately regulate the function of the neuron-vascular unit after ischemia, has good tissue targeting, biocompatibility and low immunogenicity, and is suitable for non-cell-derived precise treatment.

[0020] 5, Intervention time window is wide, suitable for intervention in acute and recovery period of stroke: In vivo experiments show that M Ctrl The migration body can obtain the effect of recovery of neural function at multiple time points after stroke, showing excellent time window flexibility and safety, supporting its wide application in emergency intervention and long-term rehabilitation.

[0021] 6, Strong cross-cell function, supporting multi-dimensional repair of neural vascular unit: Fluorescence tracking experiments show that the migration body can be actively taken up by neurons and brain microvascular endothelial cells, realizing the targeted delivery of maternal functional proteins. The migration body not only realizes the delivery of signal substances between cells, but also adjusts the cell microenvironment, promotes synaptic reconstruction and blood brain barrier repair, and has significant cross-cell and cross-system functional integration ability.

[0022] 7, The mechanism is highly consistent with the pathology of stroke, and the treatment is precise: M Ctrl The migration body is rich in oxidative phosphorylation, phagocytosis and energy metabolism related proteins, which exactly corresponds to the key pathological links such as energy crisis of neurons and blood brain barrier damage after stroke, realizes high matching pathological mechanism intervention, and is expected to greatly improve the intervention efficiency and treatment response.

[0023] 8, Can be widely used for vascular cognitive impairment and other neurodegenerative diseases: Based on its repair ability of synaptic structure of neurons, blood brain barrier function and social cognitive behavior, the migration body has good disease generalization application potential, and is suitable for the treatment of Alzheimer's disease, vascular cognitive impairment, small vessel disease, brain trauma and other brain dysfunction diseases.

[0024] 9, The present application first proposes and verifies: The migration body derived from astrocytes can significantly enhance the expression of tight junction protein, improve the integrity of blood brain barrier (BBB), and promote the repair of neuronal synapses and neuronal survival in the astrocyte-brain microvascular endothelial cell co-culture model in vitro. Further, M Ctrl In the in vivo intervention experiment in the mouse model of ischemic stroke, it shows significant neuroprotective effect, can improve motor and sensory function, reduce neurological deficit score, and at the same time improve social novelty preference, suggesting that it also has potential benefits for cognitive function.

[0025] 10, The present application also finds that M CtrlThe migration body has unique protein load characteristics, is rich in various proteins related to energy metabolism, oxidative phosphorylation and phagocytosis pathways, and shows important potential in improving cell energy metabolism disorders and stabilizing intracellular homeostasis. Therefore, the present application provides a novel organelle extraction and mediated intervention strategy, i.e. repairing damaged neurovascular units by exogenous delivery of astrocyte migration bodies. This method is not only suitable for ischemic stroke, but also can be widely used in other cerebrovascular diseases, brain trauma, vascular dementia and neurodegenerative diseases. The migration body of the present application can be used as a natural nanoscale organelle, has low immunogenicity, strong targeting and stable function, and is suitable for developing precise neuroprotective drugs or biological treatment products. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure A: co-staining of primary astrocytes using TSPAN4-GFP and WGA-594, laser confocal imaging of migration bodies and contractile filaments; Figure B: extraction of M Ctrl and M OGD / R for transmission electron microscopy observation of vesicle structure; Figure C: Western blot for marker protein identification of M Ctrl and M OGD / R ; wherein WGA: wheat germ agglutinin; TSPAN4-GFP: green fluorescent protein labeled TSPAN4; DAPI: 4', 6-diamidino-2-phenylindole (labeling nucleus); Merge: merged image; Figure 2 Figure A: functional analysis of proteins in M Ctrl and M OGD / R ; Figure B: heat map of differentially expressed proteins in M Ctrl and M OGD / R ; Figure C: KEGG pathway diagram of differentially expressed proteins; Figure 3 Figure A: uptake of astrocyte-derived migration bodies by neurons, promoting neuron survival and synaptic structure recovery after OGD / R; Figure B: in the directional migration model, WGA-AF594-labeled migration bodies and TSPAN4-GFP-labeled HT22 cells were co-localized to show that migration bodies preferentially enriched in the pseudopod contact area of neurons, suggesting that neurons have chemotactic phagocytosis of migration bodies; Figures C-D: migration bodies improve neuron survival after OGD / R: in the neuron OGD / R model, CCK8 and LDH detection showed that normal and OGD / R astrocyte-derived migration bodies (MCtrl M OD / R Both can enhance neuronal activity and reduce cell damage, and M Ctrl The effect is more significant. E:M Ctrl Instead of M OGD / R Migratory bodies can improve neuronal synaptic length after OGD / R. (*) P <0.05,** P <0.01, *** P <0.001, **** P <0.0001); Figure 4 M in this invention Ctrl Migratory bodies promote brain microangiogenesis and upregulate tight junction proteins (Diagram); where A: migratory bodies derived from normal astrocytes (M) Ctrl ), rather than the migrants originating from hypoxia / reoxygenation treatment (M OGD / R ), significantly enhances the lumen-forming ability of brain microvascular endothelial cells (*) P <0.05, ns = no significant difference); BC: Western blot and immunofluorescence staining showed that M Ctrl It can upregulate the expression of tight junction proteins Occludin and ZO-1 (*) P <0.05,** P <0.01); Figure 5 M in this invention Ctrl Diagram illustrating the enhancement of the blood-brain barrier function by migratory bodies; where A: an in vitro blood-brain barrier model established using astrocytes and endothelial cells combined with transwell chambers. B: M Ctrl It can significantly improve the transmembrane resistance (TEER) value after OGD / R, while M OGD / R This reduces the TEER value. C: OGD / R increases blood-brain barrier permeability, M Ctrl Reduced blood-brain barrier permeability after OGD / R, M OGD / R This enhances its permeability (*) P <0.05,** P <0.01, **** P <0.0001, ns = no significant difference); Figure 6 M in this invention Ctrl Instead of M OGD / R Migration body therapy significantly improved neurological function in stroke mice; A shows the technical route design for migration body intervention in a mouse model of cerebral infarction; B shows the contrast imaging of laser speckle before and after photochemical embolization in mice; C shows the mNSS score indicating M Ctrl Improved neurological deficit symptoms in pMCAO mice; D represents MCtrl Significantly prolonged the pMCAO mice' s rotating rod time on day 5; E is M Ctrl Significantly shortened the pMCAO mice' s time of tearing off the sticky note on day 7 after modeling; E is M Ctrl Intervention can significantly improve the social novelty of pMCAO mice. DETAILED DESCRIPTION

[0027] The present application is further illustrated below in conjunction with examples, which are descriptive rather than limiting, and the protection scope of the present application cannot be limited by the following examples.

[0028] The various experimental operations involved in the specific embodiments are all conventional techniques in the art, and the parts not specially noted herein can be implemented by referring to various commonly used tool books, scientific and technical literature or related instructions, manuals, etc. before the filing date of the present application.

[0029] A star-shaped glial cell-derived migratory body, the migratory body has unique characteristics distinguishing from exosomes: 1) morphology: the diameter ranges from 500-3000 nm, which is much larger than the diameter range of 30-150 nm of exosomes, and is connected with contractile filaments, and small vesicles and mitochondrial cristae-like structures can be seen inside; 2) migratory body-specific markers: TSPAN4, Integrin α5, PCGP, NDST1 are positive, while traditional exosome markers Alix and TSG101 are absent, and functional proteins involved in energy metabolism, oxidative phosphorylation, and phagocytosis biological pathways are abundant; 3) functional localization: derived from the migration process of star-shaped glial cells, with typical vesicular structure, co-localized with contractile filaments, reflecting its correlation with cell migration mechanics, and may participate in the remodeling of neurovascular units through differential signal and substance transport mechanisms, with functions of neuroprotection and blood-brain barrier repair.

[0030] Preferably, the extraction method of the migratory body comprises the following steps: When the confluence of primary star-shaped glial cells reaches 60%-70%, discard the culture supernatant, wash the cell culture dish with PBS twice and discard the PBS; add 0.25% trypsin-EDTA solution for digestion, add 1 mL of trypsin-EDTA solution per 10 cm culture dish, and place in a 37°C incubator for about 2 minutes; under a microscope, when the cells become round small bright spots, immediately add more than the volume of the digestion solution of complete culture medium containing 10% fetal bovine serum to terminate the digestion; Gently tap the bottom of the dish with a sterile Pasteur pipette to dislodge the cells as a snowstorm, collect the cell suspension in a sterile centrifuge tube; wash the bottom of the dish with PBS twice, combine the liquid in the same centrifuge tube; first centrifuge at 1000 rpm for 5 minutes, collect the supernatant; then follow the steps in turn: First high-speed centrifugation: supernatant 4000 g, 20 minutes, 4°C; collect the supernatant; Second high-speed centrifugation: supernatant 20000 g, 30 minutes, 4°C; discard the supernatant, and obtain the precipitate; Resuspend the precipitate in PBS; Third high-speed centrifugation: 20000 g, 30 minutes, 4°C; discard the supernatant, and obtain the precipitate; The final precipitate is the astrocyte-derived migratory body, which is stored at -80°C.

[0031] Use of the migratory body as described above in the preparation of a medicament for treating ischemic stroke.

[0032] Use of the migratory body as described above in the preparation of a medicament for improving neuron survival and / or promoting axon growth and synaptic reconstruction Use of the migratory body as described above in the preparation of a medicament for repairing neural networks after central nervous system injury.

[0033] Use of the migratory body as described above in the preparation of a medicament for enhancing the expression of tight junction proteins in brain microvascular endothelial cells and restoring the integrity of the blood-brain barrier, which is suitable for treating neurological diseases with BBB damage.

[0034] Use of the migratory body as described above in the preparation of a medicament for treating neurological diseases.

[0035] Preferably, the neurological diseases include ischemic stroke, traumatic brain injury (TBI), vascular cognitive impairment, Alzheimer's disease, small vessel disease, leukoaraiosis, encephalitis, spinal cord injury, and other diseases involving neuronal axon injury and blood-brain barrier damage.

[0036] Preferably, the migratory body effectively enters the central nervous system through a non-cellular delivery method, thereby achieving targeted regulation of neurons and vascular endothelial cells and improving treatment compliance and safety.

[0037] Preferably, the non-cellular delivery method includes nasal delivery, intravenous injection, intrathecal injection, or intracerebroventricular injection.

[0038] Specifically, the preparation and detection are as follows: 1. Extraction and identification of migrasome: After transfection of TSPAN4-GFP plasmid and co-staining with Wheat Germ Agglutinin (WGA) fluorescent probe, the existence of primary astrocyte migrasome and contractile filaments was observed under laser confocal microscope (ZEISS LSM880). The migrasome produced by primary astrocytes was extracted by differential centrifugation, that is: When the confluence of primary astrocytes reached about 60%-70%, the culture supernatant was discarded, and the cell culture dish was washed twice with 0.01M PBS (the PBS used in the present application is of this concentration). An appropriate amount of 0.25% trypsin-EDTA solution was added for digestion (for example, 1 mL was added for a 10 cm culture dish, and other culture dishes were adjusted proportionally), and the dish was placed in a 37°C incubator for about 2 minutes. Under a microscope, when the cells became round and small bright spots, an appropriate amount of complete medium containing 10% fetal bovine serum was immediately added to terminate the digestion.

[0039] The cells were gently blown off the dish bottom with a sterile Pasteur pipette to form a "snowflake" shape, and the cell suspension was collected in a sterile centrifuge tube. The dish bottom was washed twice with PBS, and the collected liquid was combined in the same centrifuge tube. First, centrifuge at 1000 rpm for 5 minutes to collect the supernatant. Then, the following steps were performed in order: First high-speed centrifugation: 4000 g for 20 minutes at 4°C; collect the supernatant.

[0040] Second high-speed centrifugation: 20000 g for 30 minutes at 4°C; discard the supernatant.

[0041] PBS resuspension: Resuspend the precipitate in PBS.

[0042] Third high-speed centrifugation: 20000 g for 30 minutes at 4°C; discard the supernatant.

[0043] The final precipitate is the migrasome. Store it at -80°C.

[0044] If used for Western blot or PCR experiments, the precipitate can be used directly.

[0045] If used as subsequent treatment experiment material, resuspend the precipitate with sterile PBS and store it at -80°C.

[0046] As shown in Figure 1 , the migrasome was extracted from primary astrocytes and identified by Western blot. A: Laser confocal microscopy showed that normal and OGD / R treated (the specific treatment is the same as the content in "Proteomics analysis") astrocytes can produce migratory bodies expressing TSPAN4-GFP (four transmembrane protein 4-green fluorescent protein fusion protein), and WGA (wheat germ agglutinin) labeling showed that they coexist with contractile filaments.

[0047] B: After the migratory bodies were separated and purified by differential centrifugation, transmission electron microscopy showed typical vesicular structures (diameter about 500 nm-3 µm) and morphological characteristics of their connection with broken contractile filaments under low magnification; high magnification images showed that the large vesicles contained multiple small vesicles and mitochondrial cristae structures.

[0048] C: Western blot detection showed that the migratory body marker proteins PCGP, NDST1 and Integrin α5 (PCGP: proteoglycan peptide-associated protein; NDST1: N-deacetylase / N-sulfotransferase 1; Integrin α5: Integrin α5) were positively expressed; no obvious expression signal was observed for exosome marker proteins Alix and TSG101 (Alix (ALG-2 interacting protein X) and TSG101 (tumor susceptibility gene 101 protein)), suggesting that this vesicle type is different from traditional exosomes.

[0049] In summary, the migratory body described in the present application has unique characteristics that distinguish it from exosomes: 1) morphology: diameter range about 500-3000 nm, much larger than exosomes (30-150 nm), and connected with contractile filaments, with small vesicles and mitochondrial cristae-like structures visible inside; 2) markers: TSPAN4, Integrin α5, PCGP, NDST1 positive, and exosome markers Alix, TSG101 absent; 3) functional localization: derived from astrocyte migration, co-localized with contractile filaments, reflecting its relationship with cell migration dynamics, and may participate in the remodeling of neurovascular units through differential signaling and material transport mechanisms. Therefore, the migratory body of the present application is significantly different from exosomes in terms of diameter, substructure, molecular markers and functional localization, constituting a completely new vesicle type and providing a unique material basis for subsequent therapeutic applications.

[0050] 2. Proteomics analysis: In order to study the effect of ischemia and hypoxia on the migratory bodies produced by primary astrocytes in vitro, oxygen-glucose deprivation / reperfusion (OGD / R) modeling was performed on primary astrocytes.

[0051] (1) Experimental grouping: Ctrl group: astrocytes cultured under normal conditions.

[0052] OGD / R group: astrocytes that underwent hypoxia + glucose-free treatment (OGD) for 6 hours and then reoxygenated for 24 hours.

[0053] (2) Cell preparation When the primary astrocytes reach 60%–70% confluence, discard the culture supernatant. Wash the cell culture dish twice with PBS preheated to 37°C, then discard the PBS.

[0054] (3) Culture medium replacement 1) Ctrl group: Add complete culture medium (containing 10% FBS), add 6 mL to a 10 cm culture dish.

[0055] 2) OGD / R group: Add sugar-free and serum-free culture medium, 5.4 mL to a 10 cm culture dish. (The volume is adjusted proportionally to the culture area).

[0056] (4) Hypoxia treatment (OGD): The cell culture dishes of the OGD / R group were placed in a hypoxia incubator with the following conditions: 1% O2, 5% CO2, 37 ℃, treatment time: 6 hours. The cell culture dishes of the Ctrl group were placed in a conventional incubator (37 ℃, 5% CO2, treatment time: 6 hours).

[0057] Reoxygenation: After 6 hours of hypoxia, remove the OGD / R group cells and add the following components to the culture medium: 10% FBS (final mass concentration), 1% glucose (100×) (final mass concentration), 1% sodium pyruvate (100×) (final mass concentration). After gently shaking, put the cells back into the conventional incubator (37 ℃, 5% CO2).

[0058] The Ctrl group was kept in complete culture medium and cultured synchronously. Both groups were cultured for another 24 hours.

[0059] (5) Mitochondrial extraction: 24 hours later, cells from the Ctrl group and OGD / R group were collected and processed according to the aforementioned mitochondrial extraction procedure (digestion with 0.25% Trypsin-EDTA solution, washing with PBS, stepwise centrifugation, and ultracentrifugation at 20000 g). The extracted mitochondrial cells were named as follows: 1) Migrasome-Ctrl (M Ctrl Normal astral colloid migrants; 2) Migrasome-OGD / R (M OGD / R Hypoxic star colloidal migrants.

[0060] (6) Subsequent experiments: The two groups of migratory bodies were precipitated and stored at -80 °C for proteomics sequencing analysis.

[0061] like Figure 2 As shown, M Ctrl With M OGD / R Proteomics analysis was performed. A: 28 proteins were found in M. OGD / R 24 proteins were significantly upregulated in M OGD / R Significantly downgraded in the middle. B: Shows a correlation with M. Ctrl In comparison, M OGD / R The protein load was mainly concentrated in key pathways such as energy metabolism, oxidative phosphorylation, and endocytosis, suggesting that astrocytes may participate in the remodeling of the microenvironment after ischemia by delivering energy metabolism-related components through migration bodies.

[0062] 2. In vitro neuronal function verification: A large number of normal, OGD / R primary astrocytes were cultured under the aforementioned experimental conditions, and their migratory bodies M were extracted. Ctrl and M OGD / R The migration body precipitate was resuspended in sterile PBS to prepare a migration body suspension for subsequent treatment. Simultaneously, the protein concentration of the migration bodies was determined using a protein concentration kit. Previous experiments had established the experimental conditions in the primary neuronal cell (HT22 cell) OGD / R model as 1% O2, 5% CO2, 37℃, hypoxia for 1 hour, and reoxygenation for 24 hours. Migration bodies were placed at the beginning of reoxygenation after the end of hypoxia. Ctrl and M OGD / R The culture medium for primary neurons was added at a concentration of 20 μg / mL. Cell samples were collected after the reoxygenation process, and cell proliferation assays and immunofluorescence staining (MAP2, microtubule-associated protein 2-neuronal marker) were used to assess changes in cell viability and synaptic length after OGD / R injury. At the same time, cell culture supernatant samples were collected, and lactate dehydrogenase (LDH) cytotoxicity assays were performed using a detection kit. When cells are damaged or die, they can release LDH into the culture supernatant, meaning that the higher the LDH value, the more severe the cell damage.

[0063] like Figure 3 As shown, A: Whether astrocyte migration bodies extracted in vitro are added to the HT22 cell (mouse hippocampal neuron cell line) culture system for culture, or astrocytes are directly co-cultured with HT22 cells, HT22 cells are observed to gravitate towards and engulf the migration bodies. BC: M CtrlAfter the migratory bodies are invasively engulfed by neurons, they can significantly improve the survival rate of neurons after OGD / R, reduce LDH release, and decrease synaptic damage caused by OGD / R.

[0064] 2. In vitro Validation of Vascular Endothelial Cell Function: Previous experiments determined the OGD / R model conditions for the bend.3 cell line of brain vascular endothelial cells as 1% O2, 5% CO2, 37℃, hypoxia for 4 hours, and reoxygenation for 24 hours. The migration body intervention method was as described above. The migration ability of vascular endothelial cells was assessed by angiogenesis experiments, and the expression of tight junction proteins (ZO-1, Occludin) of the blood-brain barrier was studied by Western blot. At the same time, an in vitro blood-brain barrier (BBB) ​​OGD / R model was established using transwell chambers, and migration body intervention was performed. The changes in transepithelial electrical resistance (TEER) at different time points were measured using EndOhm electrodes, and the permeability was obtained by measuring the concentration of FITC-labeled dextran across the membrane at different time points, thereby determining the effect of migration bodies on BBB permeability.

[0065] Angiogenesis assay: The matrix gel was removed from -20°C overnight and thawed at 4°C. The pipette tips and 96-well plates were pre-cooled. Bend.3 cells were starved for 24 hours using DMEM medium containing 0.2% FBS. The matrix gel was placed on ice, and 60 μL of matrix gel was added to each well of the 96-well plate using a pipette, taking care to avoid air bubbles. The plate was placed in a 37°C cell culture incubator and incubated for 30 minutes to allow the matrix gel to solidify. The Bend.3 cells were digested and centrifuged. The cell pellet was resuspended in complete culture medium, mixed well, and counted. 5 × 10⁵ cells were added to each well of the 96-well plate. 4 Cells were cultured in wells at 37°C. Images were taken at 2, 4, 6, and 8 hours after seeding, and the images were analyzed using ImageJ.

[0066] Construction of the BBB (Blood-Brain Barrier) in vitro model: A 0.4µm transwell chamber was inverted in a sterile empty lunchbox, and 1×102 astrocytes were collected. 4 Seed 100 μL of cells per well on the lower surface of the Transwell chamber. After 12 hours, invert the chamber and place it in a 24-well plate, taking care not to let the cell suspension dry out. Add 1 mL of complete culture medium to the well plate and 300 μL of complete culture medium to the chamber. After culturing for 24 hours, transfer 5 × 10⁶ cells to the transwell chamber. 3One microvascular endothelial cell (bend.3) was seeded on the upper surface of the transwell chamber and placed in a cell culture incubator. The culture medium was changed every other day. After the cells adhered to the transwell chamber, their resistance was monitored daily using an EndOhm transwell resistance meter. The model was considered successful when the resistance stabilized.

[0067] TEER (Transepithelial / Transendothelial Electrical Resistance): After successful modeling, follow Ctrl, OGD / R, OGD+M. Ctrl OGD+M OGD / R The experiment was conducted in groups.

[0068] Experimental subject: brain vascular endothelial cell line bend.3.

[0069] Group design: (1) Ctrl group (normal control group): normal culture conditions. 300 µL of complete culture medium was added to the upper chamber and 1 mL of complete culture medium was added to the lower chamber. The culture was carried out in a conventional incubator.

[0070] (2) OGD / R group (hypoxia group): First, the medium (270 µL in the upper chamber and 900 µL in the lower chamber) was placed in a hypoxia incubator for 4 h; then reoxygenation was performed, and 30 µL of mixed medium (containing 10% FBS, 1% glucose and 1% sodium pyruvate, all of which are final mass concentrations) was added to the upper chamber and 100 µL was added to the lower chamber. The mixture was then cultured in a conventional incubator for 24 h.

[0071] (3) OGD / R + M Ctrl Group (hypoxia + normal astrocyte migration bodies): Treatment was the same as the OGD / R group, but during reoxygenation, astrocyte migration bodies derived from normal conditions (M) were added to the upper and lower chambers respectively. Ctrl (20 µg / mL), continue culturing for 24 h.

[0072] (4) OGD / R + M OGD / R Group (Hypoxia + Hypoxia Astrocyte Migrations): Treatment was the same as the OGD / R group, except that during reoxygenation, astrocyte-derived migrations (M) from hypoxic / reoxygenated conditions were added to the upper and lower chambers respectively. OGD / R (20 µg / mL), continue culturing for 24 h.

[0073] (5) Measure and record the resistance of each chamber 24 hours after reoxygenation. Prepare a blank control transwell, which is treated the same except that no cells are inoculated.

[0074] Resistance calculation: R blank +R true tissue =R Total， That is, R true tissue =R Total -R blank R blank Blank cell resistance value R true tissue The tissue's own electrical resistance value R Total Sample measured resistance value Because the resistance value is inversely proportional to the tissue area, while the resistance value per unit area is independent of the membrane area and can be used to compare data obtained from transwells of different sizes, we use the resistance value per unit area for analysis.

[0075] Calculation formula: Resistance per unit area = Resistance (Ω) × Effective film area (cm²) 2 = Resistance value (Ω) × Πd 2 / 4, Permeability assay: After OGD in the BBB model, the culture medium in the upper transwell chamber was discarded, and reoxygenation supplementation solution was added to the lower chamber. Simultaneously, FITC-glucan stock solution was diluted to a working concentration of 100 μg / mL using complete culture medium and added to the upper transwell chamber. At 0, 3, 6, and 24 hours of reoxygenation, 100 μL of culture medium was aspirated from the lower chamber, and an equal volume of 100 μL of complete culture medium was added back to the lower chamber. In the Ctrl group, the upper chamber culture medium was discarded and replaced with culture medium containing FITC-glucan; the lower chamber remained unchanged, and other procedures were the same as above. The 100 μL of culture medium collected from the lower chamber at different time points was temporarily stored in light-protected centrifuge tubes at 4°C.

[0076] At 24 hours after reoxygenation, a standard curve was set up with FITC-glucan at concentrations of 20, 10, 5, 1, 0.5, 0.25, and 0.1 μg / mL. 80 μL was added to each well of a 96-well plate with a black bottom. 80 μL of the lower chamber culture medium collected at different time points was added to each well of the 96-well plate, taking care to avoid air bubbles. The plates were then placed in a microplate reader for reading, and the glucan concentration was determined based on the standard curve. like Figure 4 As shown in Figure A: Angiogenesis experiment results show that M Ctrl Treatment significantly promoted vascular endothelial cell migration, with an average migration distance of 113.6 ± 3.08 μm, significantly higher than the control group's 100 ± 4.783 μm (p < 0.05); while M OGD / R The treatment group's value was 106.1 ± 7.801 μm, showing an increasing trend compared to the control group, but this trend was not statistically significant (p = ns). This indicates that M CtrlIt has the effect of promoting angiogenesis and repair, while M OGD / R This effect is absent.

[0077] BC: Western blot results further showed that after OGD / R, the expression level of Occludin decreased to 78% of Ctrl, M Ctrl Significantly upregulated the expression of tight junction proteins; Occludin expression levels were approximately 1.3-fold higher than the control OGD / R group, while M... OGD / R After treatment, Occludin expression decreased to 0.85 times that of the OGD / R group. Immunofluorescence staining results showed that after OGD / R, ZO-1 expression level was 11% of Ctrl, and M... Ctrl The intervention increased ZO-1 expression levels by approximately 2.5 times compared to the OGD / R group, while M... OGD / R After treatment, the expression level of ZO-1 decreased to 0.91 times that of the control OGD / R group. These results indicate that M... Ctrl It can enhance the integrity of the blood-brain barrier structure by increasing the expression of tight junction proteins in the blood-brain barrier.

[0078] like Figure 5 As shown: AC: In the blood-brain barrier function test, M Ctrl The OGD / R treatment significantly increased the transendothelial resistance (TEER) of the model, from 69.34±5.54 Ω·cm² in the control group to 83.21±4.193 Ω·cm² (p<0.01), while the permeability at 12 hours after reoxygenation decreased to approximately 0.83 times that of the OGD / R group (p<0.05); conversely, M OGD / R The intervention reduced the TEER value to 60.46±4.37 Ω·cm² and increased the permeability to about 1.2 times that of the OGD / R group, both significantly worse than the control group (p<0.05). These results indicate that M Ctrl It has a protective effect on the blood-brain barrier, while M OGD / R It will worsen blood-brain barrier damage.

[0079] 5. Animal experiments: Three-month-old C57BL / 6J mice were randomly divided into Sham, pMCAO+PBS, and pMCAO+M Ctrl pMCAO+M OGD / R Four groups were used to establish a photothrombotic middle cerebral artery occlusion (pMCAO) model using photochemical embolization.

[0080] Modeling method (applicable to all groups): Rose red powder was dissolved in 0.9% physiological saline to prepare a 10 mg / mL solution, which was then injected intraperitoneally at a dose of 70 μg / g body weight. Subsequently, the mice underwent skin preparation, incision, and localization procedures: Sham group: only incision and localization were performed followed by suturing, without light exposure. The other three groups: a cold light source (intensity 4.5) was used to irradiate the localization site for 20 minutes, followed by suturing.

[0081] Group processing: 1) Sham group: No further treatment was given except for skin opening and suturing.

[0082] 2) pMCAO+PBS group: On postoperative days 1–3, PBS 20 μL / animal / day was administered via the nasal cavity (10 μL in each nostril).

[0083] 3) pMCAO+M Ctrl Group: M was administered via the nasal cavity on postoperative days 1–3. Ctrl Migratory body suspension (same as in vitro neuronal function verification): 20 μL / animal / day (10 μL each nasal cavity, concentration 1.5 μg / μL). M used... Ctrl The samples were derived from sterile migratory bodies prepared and purified during in vitro functional validation (see Method 2, “Proteomics Analysis”).

[0084] 4) pMCAO+M OGD / R Group: M was administered via the nasal cavity on postoperative days 1–3. OGD / R Migratory body suspension 20 μL / animal / day (10 μL in each nasal cavity, concentration 1.5 μg / μL). M used OGD / R The samples were derived from sterile migratory bodies prepared and purified during in vitro functional validation (see Method 2, “Proteomics Analysis”).

[0085] Mice in each group were assessed on postoperative days 1, 3, 5, 7, 10, and 14 using the modified Neurological Severity Score (mNSS), rotarod fatigue test, and sticky tag test. A three-box social test was performed on day 10. The experimental procedures are as follows: Modified Neurological Impairment Score (mNSS):

[0086] Rotary bar fatigue test: Mice were trained with a rotarod one week before modeling. The rotation speed of the rotarod was increased from 4 rpm to 40 rpm within 5 minutes. When the mice fell, the time was not recorded, but training continued to allow them to adapt to the rotation speed. The baseline level of the mice was recorded before modeling. In the experimental phase after modeling, the fall time of the mice was recorded.

[0087] Sticky tag test: One week before modeling, mice were trained to remove sticky tags. Mice were placed in an observation cage for 120 seconds to allow for acclimatization and reduce exploration time. Two tags were attached to the front paws, and the mice were observed until they removed both tags. During the experimental phase after modeling, the time it took for the mice to remove the two tags was recorded, with a maximum observation period of 120 seconds.

[0088] Three-compartment socialization experiment: Before the experiment, a three-compartment setup was constructed. Each of the left and right compartments contained an empty cage for a new mouse, while the middle compartment was empty. A passageway connected the compartments, allowing mice to move freely. Phase 1: Each mouse was placed in the middle compartment and allowed to explore freely for 5 minutes to adapt to the new environment. Phase 2: After a 1-hour interval, new mouse #1 was placed in the empty cage in the left compartment, while the right compartment remained empty. Then, each mouse was placed in the middle compartment. The time it took for each mouse to approach the new mouse and the empty cage within 5 minutes was observed and recorded. Approach was defined as being within 3 cm of the cage's outer diameter. Phase 3: New mouse #1 remained in the left compartment. New mouse #2 was placed in the right compartment. Then, each mouse was placed in the middle compartment. The time it took for each mouse to approach both new mice within 5 minutes was observed and recorded. Note that the experimental setup should be sprayed with alcohol and wiped clean after each mouse change to minimize the influence of the previous mouse's odor. The experimental environment should be quiet.

[0089] The results are as follows Figure 6 As shown, behavioral experimental results indicate that it can effectively improve motor function, sensory function, and social behavior indicators.

[0090] A: Technical route design for migratory intervention in pMCAO cerebral infarction model mice.

[0091] B: Laser speckle imaging results showed clear changes in local blood flow in the cerebral cortex before and after pMCAO, indicating successful model establishment.

[0092] C: Neurological function score (mNSS) results indicate that M Ctrl The intervention significantly improved the neurological deficits in mice on postoperative day 14.

[0093] D: Rotator assay showed that M on day 5 after pMCAO... Ctrl The treated mice spent significantly longer time on the rotundus, suggesting that their motor coordination was improved.

[0094] E: The results of the adhesive tag test showed that on the 7th day after surgery, M Ctrl The reaction time of the mice in the group to remove the adhesive tag was significantly shortened, indicating the recovery of sensory and motor function in the forelimbs.

[0095] F: In the social novelty test on postoperative day 14, pMCAO+M Ctrl The social novelty index of the group mice was significantly increased, suggesting that M Ctrl Migration interventions effectively promote the recovery of social cognitive function.

[0096] like Figure 6 As shown, the astrocyte-derived migratory organism (M) provided by this invention... Ctrl It showed significant neurological function improvement in a mouse model of ischemic stroke (pMCAO), with the following specific results: Fig. 6C. Improvement of neurological deficits: On postoperative day 14, the mNSS score of the control group mice remained at 3.6 ± 0.24 points, indicating severe neurological dysfunction. However, the M... Ctrl The mNSS score of the intervened mice decreased significantly to 2.5 ± 0.22 points, an improvement of nearly 31% (p<0.05). This result indicates that the M-type migration mechanism of the present invention... Ctrl The intervention significantly reduced neurological damage caused by cerebral ischemia, and was superior to the control group.

[0097] Fig. 6D. Recovery of motor coordination: In the rotarod experiment, on day 5 post-surgery, the dwell time of mice in the control group was only 179.15 ± 35.93 seconds, while that of pMCAO+M Ctrl The recovery time in the group was significantly prolonged to 264.19 ± 31.83 seconds (p<0.05), with a recovery level close to 88% of that in normal mice. The results showed that M... Ctrl The intervention significantly improved motor coordination and balance.

[0098] Fig. 6E. Improved sensorimotor function: In the adhesive tag experiment, on the 7th day after surgery, the reaction time for the control group mice to remove the adhesive tag was 41.17 ± 4.61 seconds, while M... Ctrl The intervention group experienced a significant reduction in time to 18.17 ± 3.34 seconds (p<0.05), representing an improvement of nearly 56%. This result demonstrates that the transfer body can effectively promote the recovery of sensory and motor function in the forelimbs.

[0099] Fig. 6F: Improved social cognitive function: In the social novelty experiment, on day 14 post-surgery, the social novelty index of the control group mice was only 44.13% ± 3.6%, showing significant cognitive impairment. However, after M... Ctrl The mice that underwent intervention showed a significant increase in their index to 60.97 ± 2.68 (p<0.001), with a recovery rate of 38%. These results indicate that the transference body intervention of this invention not only significantly improves basic motor and sensory functions but also promotes the recovery of higher-level social cognitive functions.

[0100] In summary, the transference body intervention of this invention, in an animal model of ischemic stroke, can simultaneously improve neurological deficits, motor coordination, sensorimotor function, and social cognitive function, with significantly greater improvements than the control group. Specifically, neurological function scores decreased by nearly half, motor coordination recovered to near-normal levels, sensorimotor reaction time was shortened by nearly half, and social cognitive function improved by nearly 70%. These results fully demonstrate the multidimensional, significant, and superior technical effects of transference bodies in stroke rehabilitation, exhibiting outstanding innovation and broad clinical application prospects.

[0101] This invention provides an unprecedented new strategy for the intervention of neurological diseases based on astrocyte-derived migrasomes.

[0102] In summary, it can be seen that: 1. Multi-level Functional Improvement: This invention comprehensively verifies the protective effect of transfer body intervention on ischemic brain injury through systematic assessment of neurological functional scores (mNSS), motor coordination (rotarod test), sensorimotor fine motor function (sticky tag test), and social cognitive behavior (social novelty test). Experimental results show that the transfer body can reduce mNSS scores by approximately 47%, restore motor coordination to 82% of the control group, shorten sensorimotor reaction time by nearly half, and increase the social novelty index by approximately 69%. These multi-dimensional and quantitative indicators fully demonstrate that this invention has significant therapeutic effects in both the acute and recovery phases, reflecting the comprehensive neuroprotective and repairing effects of the transfer body.

[0103] 2. Mechanism Innovation: Unlike traditional exosomes, migrasomes possess unique ultrastructure and molecular load characteristics, ranging in diameter from 500 to 3000 nm. They are rich in mitochondrial-related components and proteins involved in energy metabolism and signal transduction. Combined with the results of this study, it is suggested that migrasomes may remodel the microenvironment of ischemic brain tissue by delivering metabolic regulatory molecules and repair factors, thereby improving neuronal activity, promoting the recovery of vascular endothelial cell function, and ultimately achieving the overall repair of the neurovascular unit (NVU). This mechanism overcomes the limitations of traditional exosomes and constructs a novel therapeutic modality at the organelle level.

[0104] 3. Advantages of the route of administration: This invention uses nasal instillation to deliver the migrating organism, avoiding the physiological limitations of the blood-brain barrier (BBB). Compared with intravenous or intracerebral injection, this route has significant advantages such as ease of operation, minimal trauma, and strong targeting, making it closer to clinically applicable scenarios and providing a practical intervention for post-stroke treatment.

[0105] 4. Clinical Translation Potential: This invention is the first to validate the therapeutic effect of astrocyte-derived migration bodies in an animal model of cerebral ischemia. It not only significantly improves motor and sensory functions but also restores higher cognitive-level social behavioral abilities. Compared with existing drug or cell therapies, the migration body intervention of this invention improves neurological function while simultaneously restoring cognitive function, meeting the clinical needs of stroke rehabilitation and showing broad application prospects in post-stroke neurological repair and cognitive impairment intervention.

[0106] 5. Distinctiveness and Patent Value: This invention differs from existing exosome or stem cell therapies in its origin, structure, and functional characteristics. First, the migratory bodies originate from the migration process of astrocytes, possessing a unique formation mechanism and ultrastructure. Second, the molecular and signaling characteristics they carry endow them with unique repair potential. Finally, this invention, through multiple in vitro and in vivo experimental evidence, is the first to clearly demonstrate the unique therapeutic effect of migratory bodies in ischemic brain injury. These results not only provide new organelle therapy strategies but also lay a solid technical support and innovative foundation for the clinical development and industrialization of related products, possessing high patent patent value.

[0107] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. An astrocyte-derived migratory organism, characterized in that: The migratory bodies have unique characteristics that distinguish them from exosomes: 1) Morphology: The diameter ranges from 500 to 3000 nm, which is much larger than the diameter range of exosomes (30 to 150 nm). They are connected to contractile filaments and contain small vesicles and mitochondrial cristae-like structures; 2) Migratory body-specific markers: They are positive for TSPAN4, Integrin α5, PCGP, and NDST1, while they lack traditional exosome markers Alix and TSG101. They are rich in functional proteins involved in energy metabolism, oxidative phosphorylation, and phagocytic pathways; 3) Functional localization: They originate from the migration process of astrocytes, have typical vesicle structures, and are co-localized with contractile filaments, reflecting their association with cell migration dynamics. They may participate in the remodeling of neurovascular units through differentiated signal and substance transport mechanisms, and have neuroprotective and blood-brain barrier repair functions.

2. The migrant according to claim 1, characterized in that: The method for extracting the migrants includes the following steps: When the primary astrocytes reach 60%-70% confluence, discard the culture supernatant, wash the cell culture dish twice with 0.01M PBS and discard the PBS; add 0.25% Trypsin-EDTA solution for digestion, adding 1 mL of Trypsin-EDTA solution to each 10 cm culture dish, and incubate at 37℃ for about 2 minutes; observe under a microscope, and when the cells become small round bright spots, immediately add more than the volume of digestion solution in complete culture medium containing 10% fetal bovine serum to stop the digestion; Gently pipette the bottom of the dish with a sterile Pasteur pipette to detach the cells in a snowflake-like pattern, and collect the cell suspension in a sterile centrifuge tube. Wash the bottom of the dish twice with PBS, and combine the collected liquids in the same centrifuge tube. Centrifuge at 1000 rpm for 5 minutes and collect the supernatant. Then proceed with the following steps: First high-speed centrifugation: 4000 g of supernatant, 20 minutes, 4°C; Collect the supernatant; Second high-speed centrifugation: 20,000 g of supernatant, 30 minutes, 4°C; discard the supernatant to obtain the precipitate; PBS resuspending: Resuspend the precipitate in PBS; Third high-speed centrifugation: 20000 g, 30 minutes, 4℃; discard the supernatant and obtain the precipitate; The final precipitate is the astrocyte-derived migratory body, which is stored at -80°C.

3. The use of the migration body as described in claim 1 or 2 in the preparation of a medicament for treating ischemic stroke.

4. The use of the migration body as described in claim 1 or 2 in the preparation of a medicament for improving neuronal survival and / or promoting axonal growth and synaptic remodeling.

5. The use of the migration body as described in claim 1 or 2 in the preparation of a neural network repair drug after central nervous system injury.

6. The use of the migration bodies as described in claim 1 or 2 in the preparation of a medicament that enhances the expression of tight junction proteins in brain microvascular endothelial cells and restores the integrity of the blood-brain barrier, is applicable to the treatment of neurological diseases accompanied by BBB disruption.

7. The use of the migration body as described in claim 1 or 2 in the preparation of a medicament for treating nervous system diseases.

8. The application according to claim 8, characterized in that: The neurological diseases mentioned include ischemic stroke, traumatic brain injury (TBI), vascular cognitive impairment, Alzheimer's disease, small vessel disease, leukoencephalopathy, encephalitis, spinal cord injury, and other diseases involving neuronal axonal damage and blood-brain barrier disruption.

9. The application according to any one of claims 3 to 8, characterized in that: The migratory organisms effectively enter the central nervous system via non-cellular delivery, thereby achieving targeted regulation of neurons and vascular endothelial cells, improving treatment compliance and safety.

10. The application according to claim 9, characterized in that: The non-cellular delivery methods include nasal delivery, intravenous injection, intrathecal injection, or intraventricular injection.

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