Application of human umbilical cord mesenchymal stem cell exosome in preparation of nasal delivery preparation for treating spinal cord injury

Abstract

The invention belongs to the technical field of biological medicine, and relates to application of human umbilical cord mesenchymal stem cell exosome in preparation of a nasal delivery preparation for treating spinal cord injury. The exosome used in the invention is derived from human umbilical cord mesenchymal stem cells, and is extracted from a cell culture solution after being stimulated by a culture medium. The exosome has typical structural characteristics of the exosome and expresses corresponding protein. The extracted umbilical cord mesenchymal stem cell exosome is applied to a mouse spinal cord semi-transection model in a nasal dripping manner, and can effectively cross a blood brain barrier to diffuse into the brain and spinal cord of a model mouse, so that the immune response after injury is relieved, and the motor function recovery is promoted.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of human umbilical cord mesenchymal stem cell exosomes in the preparation of a nasal administration preparation for treating spinal cord injury. Background Art

[0002] Spinal cord injury is a serious disease of the central nervous system. It is reported that about 250,000 to 300,000 people are affected by spinal cord injury every year. Common causes of spinal cord injury include traffic accidents, falling objects, tumors, inflammation, etc. Spinal cord injury often causes permanent and irreversible organic damage to the spinal cord tissue, causing the patient to lose all or part of the sensory and motor functions below the injury plane, often accompanied by urination and defecation dysfunction, which seriously reduces the patient's quality of life and imposes a heavy burden on the patient's family and even society. Therefore, the problem of injury treatment needs to be solved urgently.

[0003] At present, the main clinical treatments for spinal cord injury are surgical treatment and drug treatment. Surgical treatment mainly relieves the local mechanical compression after injury through surgery to reduce the secondary injury caused by compression. Drug treatment is generally a large dose of hormone shock represented by methylprednisolone to reduce the oxidative stress response after injury, thereby reducing secondary neuronal damage. However, these methods have very limited therapeutic effects on spinal cord injury, and there are potential risks in the implementation process. Therefore, spinal cord injury regeneration has always been a key problem that needs to be solved urgently in the biomedical field.

[0004] At present, a large number of studies have confirmed that mesenchymal stem cells (MSCs) can play a neuroprotective role in central nervous system injury. Umbilical cord mesenchymal stem cells are derived from the umbilical cord of newborns, have the advantages of low immunogenicity, easy access, no ethical controversy, and are easier to enter clinical transformation. A large number of studies have shown that MSCs exert their therapeutic effects by secreting a variety of nutritional factors and paracrine effects on the microenvironment. Therefore, on the one hand, MSCs-derived exosomes have the functions of regulating immunity and improving the microenvironment, and on the other hand, they can avoid the potential tumorigenic risks of stem cells. Therefore, the application of exosomes in the treatment of central nervous system diseases including spinal cord injury has received more and more attention. And due to the existence of the blood-brain barrier, there are certain obstacles in the entry of drugs into the central nervous system through intravenous injection and other methods, while exosomes can effectively bypass the blood-brain barrier through nasal administration, which is an efficient and non-invasive method of administration. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of human umbilical cord mesenchymal stem cell exosomes in the preparation of a nasal administration preparation for treating spinal cord injury. It provides a new administration method for exosome treatment of spinal cord injury and a new treatment strategy for clinical treatment of spinal cord injury. The exosomes can quickly enter the central nervous system through nasal administration, reduce the immune response after spinal cord injury, and promote the recovery of motor function after spinal cord injury.

[0006] The technical solution provided by the present invention is as follows:

[0007] The present invention provides the use of human umbilical cord mesenchymal stem cell exosomes in preparing a nasal administration preparation for treating spinal cord injury.

[0008] Furthermore, the number of exosome particles per milliliter of the nasal administration preparation is 3.3×10 11 .

[0009] Furthermore, the treatment of spinal cord injury is to administer human umbilical cord mesenchymal stem cell exosomes through the nasal cavity to cross the blood-brain barrier, inhibit the activation of microglia at the spinal cord injury site, reduce cell apoptosis at the injured site after spinal cord injury, and promote the recovery of motor function after spinal cord injury.

[0010] Furthermore, the nasal administration preparation is any one or more of nasal drops, sprays, powders, gels, microspheres, emulsions, and liposomes.

[0011] Furthermore, the nasal administration preparation is nasal drops.

[0012] Furthermore, the nasal drops are administered once every 3 days, and one course of treatment is 30 days.

[0013] Furthermore, the nasal drops need to be treated with 100U of hyaluronidase for 30 minutes before use.

[0014] Furthermore, when administering the drug to the nasal cavity, the drug was administered alternately through the left and right nostrils and dripped into the nasal cavity 6 times, with an interval of at least 5 minutes between each drip. The total number of exosome particles after 6 nasal drops was 1×10 10 .

[0015] The present invention also provides a method for preparing a human umbilical cord mesenchymal stem cell exosome preparation for nasal administration, comprising the following steps: S1. Culture and passage human mesenchymal stem cells 3-5 times; S2, harvesting the culture medium, extracting the exosomes in the culture medium, and obtaining exosomes derived from human mesenchymal stem cells; S3. Mixing the obtained human mesenchymal stem cell exosomes with pharmaceutically acceptable excipients to obtain a nasal administration preparation.

[0016] Furthermore, the pharmaceutically acceptable excipients include any one or more of preservatives, stabilizers, and absorption promoters.

[0017] Furthermore, the culture medium for culturing umbilical cord mesenchymal stem cells is a complete culture medium.

[0018] Furthermore, when culturing umbilical cord mesenchymal stem cells, when the cells grew to 80%, the culture medium was discarded, and after washing with PBS, the cells were replaced with a serum-free basal culture medium until the cells were confluent, and the supernatant was collected and filtered for exosome extraction.

[0019] Furthermore, the umbilical cord mesenchymal stem cells were cultured at 37°C and 5% CO2.

[0020] Furthermore, the filtering method is a 0.22 μm filter membrane.

[0021] Furthermore, umbilical cord mesenchymal stem cell exosomes were collected using the ion exchange method.

[0022] Further, the mixture was added to an ion exchange column and centrifuged at 500 g for 1 min.

[0023] Furthermore, the filtered cell culture fluid was added to an equal volume of XBP buffer, mixed and added to the ion exchange column for treatment, and the impurities such as proteins adsorbed on the column were eluted with XWP, and the exosomes adsorbed on the column were eluted with XE buffer.

[0024] The research of the present invention shows that umbilical cord mesenchymal stem cell exosomes can quickly enter the central nervous system after being administered by nasal drops, have the effect of improving the immune response after spinal cord injury, and can be used as a new treatment option after spinal cord injury.

[0025] Umbilical cord mesenchymal stem cell exosomes are delivered through the nasal cavity to treat spinal cord injury. The umbilical cord mesenchymal stem cell exosomes are produced by stimulating umbilical cord mesenchymal stem cells with serum-free culture medium. The exosomes are extracted from the culture medium and administered by nasal drops.

[0026] It should be noted that, based on the contents disclosed in the present invention, those skilled in the art may obtain umbilical cord mesenchymal stem cell exosomes by methods including but not limited to ultracentrifugation, ultrafiltration, kits, etc., regardless of the method used to obtain umbilical cord mesenchymal stem cell exosomes, and use nasal delivery for the treatment of spinal cord injury.

[0027] Beneficial Effects

[0028] The exosome nasal administration preparation of the present invention can enter the central nervous system within 3 hours after being dripped into the nasal cavity. After the nasal drop treatment, the mice with hemisection of the tenth thoracic spinal cord were found to have a reduced distribution range of microglia in the injured area by immunofluorescence, which has the effect of reducing the immune response after spinal cord injury, and the cell apoptosis in the injured area is also significantly reduced.

[0029] In addition, the motor function of spinal cord injured mice was significantly improved 15 days after exosome nasal drops treatment. And the nasal delivery method of the present invention can be used as a substitute for the traditional exosome in situ or intravenous injection method.

[0030] Umbilical cord mesenchymal stem cells are easy to obtain and have no ethical controversy. Their exosomes have similar therapeutic effects to stem cell therapy and avoid the risk of stem cell inhibition. The nasal administration method is non-invasive, less harmful to patients, and easier for patients and their families to operate. It is more accepted by the patient population and is a new strategy for the treatment of spinal cord injury with broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the extraction and identification of exosomes from umbilical cord mesenchymal stem cells (a, human umbilical cord mesenchymal stem cells cultured in vitro, scale bar is 100μm; b, transmission electron microscopy photo of exosomes from umbilical cord mesenchymal stem cells, scale bar is 150nm; c, nanoparticle tracking analyzer to identify the particle size and concentration of exosomes from umbilical cord mesenchymal stem cells; d, identification of marker proteins of exosomes from umbilical cord mesenchymal stem cells, cell: total protein sample of umbilical cord mesenchymal stem cells, EV: protein sample of umbilical cord mesenchymal stem cells; e, in vitro internalization identification of umbilical cord mesenchymal stem cells, green is Tuj1-labeled PC12 highly differentiated cells, red is PKH26 fluorescent dye-labeled exosomes, blue is DAPI-labeled cell nuclei, scale bar is 25μm).

[0032] Figure 2 Schematic diagram of the distribution of umbilical cord mesenchymal stem cell exosomes in the central nervous system after intranasal drops (a, 3 hours after intranasal drops, the exosomes diffused into the brain; b, fluorescence imaging 9 hours after intranasal drops, the two mice on the left are from the exosome group, and the one on the right is from the PBS group; c, after taking out the spinal cord and brain of the exosome group that had been intranasal drops for 9 hours, it can be seen that the exosomes diffused into the spinal cord).

[0033] Figure 3Schematic diagram of the effect of exosomes on the immune response at the injured site after nasal drops (exosomes significantly improved the immune response in the injured area, as manifested by the reduction in the distribution range of microglia; red is CD68-labeled microglia, green is GFAP-labeled astrocytes, and blue is DAPI-labeled cell nuclei (a, 7 days after mouse spinal cord hemisection; b 15 days after mouse spinal cord hemisection, scale bar is 200μm; c and d, statistics of microglia distribution range, *p<0.05, **p<0.01, Student's t-test analysis)).

[0034] Figure 4 Schematic diagram of the analysis of motor function of mice after intranasal treatment with exosomes (a, statistics of maximum rotation speed in the rotarod test of mice; b, statistics of wrong step rate in irregular horizontal ladder; c, schematic diagram of wrong step in irregular horizontal ladder test; d, gait imprint of hind limbs of mice in Catwalk; e, statistics of PrintPosition of right hind limb; f, statistics of step sequence regularity index; g, Couplings RH-LH CStat statistics, *p<0.05, ***p<0.001, Student's t-test analysis).

[0035] Figure 5 Schematic diagram of reducing cell apoptosis in the damaged area after exosome nasal drops treatment (a, red is caspase3, blue is dapi, marking the cell nucleus, merge arrows point to apoptotic cells; b, statistics of apoptotic cell numbers ***p<0.001, Student's t-test analysis). DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments.

[0037] Example 1 Cultivation of human umbilical cord mesenchymal stem cells and preparation and characterization of exosomes

[0038] 1. Culture of human umbilical cord mesenchymal stem cells

[0039] Primary human umbilical cord mesenchymal stem cells were cultured in complete medium, which was DMEM-f12 cell culture medium with 10% fetal bovine serum, 1% penicillin and streptomycin. They were cultured in an incubator at 37°C with 5% CO2, and the medium was changed every two days. Primary human umbilical cord mesenchymal stem cells were passaged to P3-P5 and could be used for subsequent exosome extraction.

[0040] 2. Extraction of exosomes from umbilical cord mesenchymal stem cells

[0041] When the umbilical cord mesenchymal stem cells grew to a cell density of 80%, the culture medium was discarded and replaced with serum-free DMEM-12 basal medium supplemented with 1% penicillin and streptomycin. After starvation culture for 48 h, the cell supernatant was collected.

[0042] (1) Exoeasy kit extraction method

[0043] After filtering the cell supernatant with a 0.22μm filter, add an equal volume of XBP buffer and gently flip the test tube 5 times to mix the solution. Add the mixed solution to the spin column of the exosome extraction kit (exoEasy Kit) and centrifuge at 500g for 1 minute. Discard the filtrate in the lower tube and add the sample again until the mixed solution passes through the column. Add 10mL XWP buffer, centrifuge at 5000g for 5 minutes, remove the residual liquid in the column, and discard the lower layer of liquid. Move the column to a new collection tube, and elute the exosomes adsorbed on the column with XE buffer. The exosomes obtained were concentrated by ultrafiltration using Amicon Ultra 10 kDa tube (Millipore), and the concentrated exosomes were stored in a -80℃ refrigerator for subsequent experiments.

[0044] (2) Ultracentrifugation extraction method

[0045] The filtered umbilical cord mesenchymal stem cell supernatant was added to a centrifuge tube, balanced, and centrifuged at 10,000 g at 4°C for 2 hours. After centrifugation, the supernatant was discarded, 2 mL of PBS was added, and the exosome precipitate obtained by centrifugation was blown evenly. PBS was added again and balanced, and centrifuged at 10,000 g at 4°C for 2 hours. After centrifugation, the supernatant was discarded, PBS was added in an amount suitable for subsequent use, and the exosome precipitate was blown. The obtained exosomes were stored in a -80°C refrigerator for subsequent experiments.

[0046] 3. Exosome markers of umbilical cord mesenchymal stem cells

[0047] (1) Dir dye labeling of exosomes

[0048] The Dir dye storage solution was prepared into a working solution with a concentration of 100 μM. The dye working solution was added to the exosome suspension at a ratio of 1:10. After adding the dye, cover the lid and mix well. Then incubate at 37°C for 30 minutes. 5-10 times the volume of PBS was added to the incubated exosome-dye complex and mixed well. After centrifugation at 100,000 g for 1-1.5 hours, the excess dye solution was removed and the bottom precipitate was resuspended with PBS. The labeled exosomes were stored in a -80°C refrigerator for subsequent experiments.

[0049] (2) PKH26-labeled exosomes

[0050] A. Prepare a concentration of 1× 10 11Particles / mL of exosome solution (100μl). B. Add exosomes to 250μl Diluent C and mix gently. C. Take a new EP tube and add 2μl PKH26 to 250μl Diluent C. D. Add B to C and mix. E. Leave at room temperature for 4 minutes and mix with a pipette every 1 minute. F. Add an equal volume of 1% BSA to terminate staining. Centrifuge at 100000g for 1-1.5h to remove excess dye, resuspend the bottom precipitate with PBS, and store the labeled exosomes in a -80℃ refrigerator for subsequent experiments.

[0051] 4. Identification of umbilical cord mesenchymal exosomes

[0052] (1) Exosomes were resuspended in PBS, and the particle size and concentration (Particles / mL) of exosomes were determined by Nanoparticle tracking analyses (NTA).

[0053] (2) The exosome solution was deposited on a 300-mesh copper grid coated with polyvinyl acetate and carbon, left at room temperature for 3 min, and then stained with 1.5% uranyl acetate. The grid was imaged, observed, and photographed using a transmission electron microscope.

[0054] (3) Western Blot was used to detect the expression of exosome-related proteins of umbilical cord mesenchymal stem cells, including CD81 and TSG101.

[0055] 5. Identification of exosome internalization in vitro

[0056] First, PKH26 dye was used to label exosomes. The specific process was as follows: A. The concentration of PKH26 dye was 1×10 11 Particles / mL of exosome solution (100μl). B. Add the exosome solution to 250μl Diluent C and mix gently for later use. C. Take a new EP tube and add 2μl PKH26 to 250μl Diluent C and mix gently for later use. D. Add the exosome solution in step B to the dye solution in step C and mix gently. F. Leave at room temperature for 4 minutes and mix with a pipette every 1min. G. Add an equal volume of 1% BSA to terminate staining. H. Concentrate the solution with a 50kda protein concentrator and set aside. I. Dilute the concentrated exosomes with serum-free culture medium to a final concentration of 1.0×10 9Particles / mL of exosome solution. J. After PC12 highly differentiated cells were seeded in a 24-well plate for 4 hours, PKH26-labeled exosome solution was added and co-cultured for 24 hours. K. Discard the culture medium and add 4% paraformaldehyde to fix for 30 minutes. L. Discard the formaldehyde and add PBS to wash twice, 10 minutes each time; M. Add 100μl blocking solution to block at room temperature for 1 hour, N. Discard the blocking solution and add immunostaining primary antibody to incubate at 4℃ overnight. O. Take out the plate and warm it up for 15 minutes, discard the primary antibody, wash it 3 times with PBS, 10 minutes each time. P. Add immunostaining secondary antibody and incubate it at room temperature for 2 hours in the dark. Q. Discard the secondary antibody and wash it 3 times with PBS, 10 minutes each time. R. Seal the slide. S. Observe and take pictures with a confocal microscope.

[0057] Example 2 Nasal delivery of umbilical cord mesenchymal stem cell exosomes to treat spinal cord injury in mice

[0058] The spinal cord injury was hemisection of the 10th thoracic spinal cord of C57BL / 6 mice.

[0059] 1. Establishment of the spinal cord hemisection model in C57BL / 6 mice

[0060] C57BL / 6 mice were provided by the Experimental Animal Center of Nantong University. They were divided into two groups, the surgery group and the surgery + exosome group. The mice were weighed and anesthetized with mouse anesthetic (tribromoethanol), and then placed in a prone position on the operating table. After skin preparation and disinfection with iodine, a midline dorsal incision was made to separate the skin and muscles to fully expose the spine. After accurately locating the T10 lamina, the tissue at the junction of the T10 lamina and the T9 lamina was carefully cut with microscissors to fully expose the spinal cord while avoiding damage to the spinal cord. The iris knife was used to cross the midline of the spinal cord and perform a right hemisection of the spinal cord to establish a mouse spinal cord hemisection model. After sufficient hemostasis, the wound was sutured layer by layer. 24 hours after the hemisection surgery, the right hind limbs of the mice were checked for complete paralysis, and the mice that were not completely paralyzed were removed.

[0061] 2. Umbilical cord mesenchymal stem cell exosomes nasal drops

[0062] After the T10 spinal cord of mice was hemi-transected, exosomes were dripped into the nasal cavity every 3 days starting from the day of surgery in the surgery + exosome group, and the same volume of PBS was dripped into the surgery group. The specific method is as follows: exosome nasal drops were prepared, and the concentrated or frozen exosomes were added with an appropriate amount of PBS according to their concentration, and the exosome concentration in the nasal drops was adjusted to 3.3× 10 11Particles / mL. All mice were anesthetized by isoflurane breathing and placed on a heating pad to maintain body temperature. They were in the supine position throughout the nasal drip. Before the exosome / PBS drip, 5 μl (100 U) hyaluronidase was dripped into each nostril for 30 min to enhance the permeability of the nasal mucosa. After the hyaluronidase action, 5 μl of exosomes or PBS was dripped into each nostril at a time. Each nasal drip was separated by 5 minutes to ensure full absorption of the exosomes. A total of 30 μl of exosomes or PBS was dripped alternately into the left and right nostrils. The total number of exosome particles received by each mouse each time was 1×10 10 .

[0063] 3. In vivo fluorescence imaging of mice

[0064] After the spinal cord injury mice were nasally dripped with Dir dye-labeled exosomes, the mice were kept in anesthesia, their hair was shaved, and the remaining hair was removed with a depilatory cream. After the depilation was completed, the mice were placed on the imaging stage of the Perkinelmer In Vivo Imaging System, and the head of the mouse was aligned with the anesthesia mask to maintain its anesthesia state and body temperature. The excitation light wavelength was set to 748nm and the emission light wavelength was set to 780nm, and images were taken and analyzed.

[0065] 4. Frozen sections

[0066] After the end of the intranasal treatment, the mice were anesthetized with mouse anesthetic (tribromoethanol) and PBS and 4% paraformaldehyde were perfused through the aorta, and then the spinal cord was taken out together with the spine and soaked in PFA at 4°C overnight. After 16 hours, the spinal cord was peeled off from the spine, washed thoroughly with PBS, and then placed in 15% and 30% sucrose solutions for gradient dehydration. After dehydration, the spinal cord was trimmed to a suitable size and placed in a mold, OCT was added, the position of the spinal cord was adjusted, and it was left to stand for several minutes, and then transferred to a -80°C refrigerator for freezing and storage. The tissue was cut into 16μm slices using a freezing microtome for staining or transferred to a -80°C refrigerator for temporary storage.

[0067] 5. Immunofluorescence staining

[0068] Bake the tissue sections at 50℃ for 20 minutes. Select tissue sections with complete morphology, no wrinkles and no bubbles and put them into the washing tank, add PBST and shake at low speed for 15 minutes. Dry the washed tissues and circle all the tissues in a circle with a brush. Add 200μl of blocking solution to the circle to cover all the tissues and block at room temperature for 1h. Discard the blocking solution and add 200μl of primary antibody to the circle and incubate overnight at 4℃. Take the tissue out of the 4℃ refrigerator and let it stand at room temperature for 1h. Discard the primary antibody and wash it 3 times with PBS, 5 min each time. Add 200μl of secondary antibody to the circle and incubate it in a moisturizing dark box at room temperature for 3h away from light. Discard the secondary antibody and wash it 3 times with PBS, 5 min each time. Take out the tissue and dry it slightly. Add 50μl of sealing solution to each slide and cover it with a coverslip. If it needs to be stored for a long time, seal the edge with nail polish.

[0069] Umbilical cord mesenchymal stem cells ( Figure 1 a), after stimulation with the culture medium, the cell culture supernatant was collected. After the cell culture supernatant was filtered through a 0.22 μm filter membrane to remove impurities such as cell debris, the exosome extraction kit was used to extract the exosomes. After obtaining the umbilical cord mesenchymal stem cell exosomes, the exosome structure was first observed by transmission electron microscopy, and it was found that the obtained exosomes had the typical structural characteristics of a double-layered cup ( Figure 1 b) The particle size and concentration were further analyzed using a nanoparticle tracking instrument. The results showed that the concentration of the extracted exosomes was 1.2×10 11 Particles / mL, average particle size 133.3nm ( Figure 1 c); The proteins in the exosomes were extracted, and Western Blot analysis showed that the extracted exosomes expressed typical exosome proteins CD63 and TSG101. After completing the identification of the exosomes, we tested the ability of the exosomes to be internalized in vivo and enter the central nervous system. The extracted exosomes were labeled with Dir fluorescent dye and instilled into the nose for in vivo imaging. The results of in vivo imaging showed that nasal administration of umbilical cord mesenchymal stem cells can effectively cross the blood-brain barrier, and the fluorescence of the exosomes can be detected in the brain after 3 hours ( Figure 2 a), Exosome fluorescence can be detected in the spinal cord after 9 hours ( Figure 2 b). Further in vivo experiments used a mouse thoracic 10 spinal cord hemisection model. Immunofluorescence results showed that intranasal exosomes can reduce the immune response after spinal cord injury, specifically manifested by a significant reduction in the distribution of microglia near the injury after spinal cord hemisection. This phenomenon was observed 7 days after injury ( Figure 3 a, b) and 15 days ( Figure 3 c, d) were observed. Behavioral experiments showed that after nasal administration of exosomes, the motor function of the injured hind limb was significantly restored ( Figure 4 a, b, c), hind limb movement coordination was significantly improved ( Figure 4 d, e). Caspase-3 staining results showed that after nasal administration of exosomes, cell apoptosis at the injury site was significantly reduced compared with the control group on the third day of injury ( Figure 5 a, b).

[0070] The above experiments have proved that exosomes extracted from umbilical cord mesenchymal stem cells cultured in vitro can be administered through nasal delivery, which clearly shows that they can cross the blood-brain barrier through the nasal cavity, enter the brain and spinal cord, promote the repair of the damaged area after spinal cord injury, alleviate immune response, reduce cell apoptosis, protect the damaged tissue, and promote the recovery of motor function.

[0071] Although the above embodiments have described the present invention in detail, it is not limited to the above implementation modes. Practitioners in this field may make other modifications and improvements based on the inspiration of the present invention, which all fall within the protection scope of the present invention.

Claims

1. Application of human umbilical cord mesenchymal stem cell exosomes in the preparation of nasal administration preparations for the treatment of spinal cord injury.

2. The use according to claim 1, characterized in that: The number of exosome particles per milliliter of the nasal administration preparation is 3.3×10 11 .

3. The use according to claim 1, characterized in that: The treatment of spinal cord injury is to administer human umbilical cord mesenchymal stem cell exosomes through the nasal cavity to cross the blood-brain barrier, inhibit the activation of microglia at the spinal cord injury site, reduce cell apoptosis at the injury site after spinal cord injury, and promote the recovery of motor function after spinal cord injury.

4. The use according to claim 1, characterized in that: The nasal administration preparation is any one or more of nasal drops, sprays, powders, gels, microspheres, emulsions, and liposomes.

5. The use according to claim 4, characterized in that: The nasal administration preparation is nasal drops.

6. The use according to claim 5, characterized in that: The administration interval of the nasal drops is once every 3 days, and every 30 days is a course of treatment.

7. The use according to claim 5, characterized in that: The nasal drops need to be treated with 100U hyaluronidase nasal drops for 30 minutes before use.

8. The use according to claim 5, characterized in that: For nasal administration, the left and right nostrils were alternately administered and the nasal cavity was dripped 6 times, with an interval of at least 5 minutes between each drip. The total number of exosome particles after 6 nasal drops was 1×10 10 .

9. A method for preparing a human umbilical cord mesenchymal stem cell exosome preparation for nasal administration, characterized in that: The following steps are involved: S1. Culture and passage human mesenchymal stem cells 3-5 times; S2, harvesting the culture medium, extracting the exosomes in the culture medium, and obtaining exosomes derived from human mesenchymal stem cells; S3. Mixing the obtained human mesenchymal stem cell exosomes with pharmaceutically acceptable excipients to obtain a nasal administration preparation.

10. The preparation method according to claim 9, characterized in that: The pharmaceutically acceptable excipients include any one or more of preservatives, stabilizers, and absorption promoters.

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