Mesenchymal stem cell exosome loaded with miR-206-3p inhibitor and application of mesenchymal stem cell exosome

Abstract

The invention discloses a mesenchymal stem cell exosome loaded with a miR-206-3p inhibitor and application of the mesenchymal stem cell exosome, and particularly belongs to the field of biological medicine. The invention discovers that the mesenchymal stem cell exosome loaded with the miR-206-3p inhibitor can effectively improve the symptoms of the Alzheimer's disease for the first time. Animal experiments show that the mesenchymal stem cell exosome drug loaded with the miR-206-3p inhibitor can effectively improve the learning and cognition functions of an Alzheimer disease model mouse and pathological symptoms (including Abeta deposition, neuron loss and glial cell activation) of the Alzheimer disease; through comparison, the improvement effect of the exosome medicine on the Alzheimer's disease is better than that of the mesenchymal stem cell exosome and the miR-206-3p inhibitor which are independently used, and the molecular mechanism of the exosome medicine for improving the Alzheimer's disease is related to inflammation inhibition and nutrient factor expression promotion.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the fifth leading cause of death worldwide. As the aging population continues to accelerate, the number of AD patients has risen sharply. However, there are currently few effective drugs for treating AD, and the development of new therapeutic drugs is urgent. Over the past 100 years, people have actively conducted exploratory research on the treatment of AD. Among them, failed experimental experience suggests that drugs for the treatment of AD need to have the following characteristics: (1) enter the brain through the blood-brain barrier in an active state, (2) effectively intervene in and improve the pathological characteristics of AD, and (3) have low toxic side effects.

[0003] The sequence of miR-206-3p is highly conserved in different species. Among them, it is completely consistent in tissues from rats, mice and humans. miR-206-3p is highly expressed in the frontotemporal cortex and plasma of AD patients, and it participates in the regulation of neural function by negatively regulating the target molecule, brain-derived neurotrophic factor. In our previous studies, we found that after stereotaxic injection of miR-206-3p inhibitors into the hippocampus of AD mice, the β-amyloid protein in the brain of AD mice was significantly reduced, and cognitive function was significantly improved. This suggests that miR-206-3p inhibitors have potential value in the treatment of AD. However, brain localization injection is traumatic and not conducive to the long-term application of drugs. Therefore, choosing a suitable delivery system is of great significance.

[0004] Exosomes (EXO) are membrane vesicles with a diameter of 50-150nm. They have strong penetrating power and can protect the encapsulated cargo molecules from degradation. In addition, exosomes have low toxicity and low immunogenicity. These characteristics indicate that exosomes are an excellent natural delivery system.

[0005] Mesenchymal stem cells (MSCs) are a member of the stem cell family, with strong proliferation and differentiation potential, and low immune rejection characteristics. Studies have shown that mesenchymal stem cells have a positive effect in the treatment of brain diseases, mainly reflected in the effect of inhibiting inflammatory responses. Due to the unknown risks of stem cell homing, angiogenesis, and immune rejection in the treatment of diseases, their application is hindered. Exosomes with stem cell functional characteristics can better circumvent this drawback.

[0006] Although many achievements have been made in the treatment of AD based on miRNA inhibitors, and mesenchymal stem cell exosomes have also made progress in inhibiting brain inflammation, how to realize the application of miRNA inhibitors and mesenchymal stem cell exosomes in the treatment of brain diseases is still a challenge. At present, the main delivery systems include viral and non-viral systems. There are safety issues in the loading of endogenous RNA in viral systems. Non-viral systems are mainly found in lipids, nanoparticles, etc. Although they have low immunogenicity and good biocompatibility, they are difficult to load miRNA inhibitors and have low endosomal escape rates. Therefore, it is of great significance to develop new delivery systems to form a "cocktail" combination of drugs, efficiently load miRNA inhibitors, and then deliver miRNA inhibitors to target cells to achieve the treatment of AD. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention aims to provide a mesenchymal stem cell exosome loaded with a miR-206-3p inhibitor as an exosome drug to achieve the therapeutic effect of improving cognitive dysfunction in AD patients.

[0008] To achieve the above object, the present invention discloses the following technical solutions:

[0009] In a first aspect, the present invention provides a modified mesenchymal stem cell exosome, wherein the modified mesenchymal stem cell exosome is a modified mesenchymal stem cell exosome obtained by loading the mesenchymal stem cell exosome with a miR-206-3p inhibitor;

[0010] The miR-206-3p inhibitor is a complementary single strand of miR-206-3p;

[0011] The nucleotide sequence of miR-206-3p is shown in SEQ ID NO: 1;

[0012] The nucleotide sequence of the miR-206-3p inhibitor is shown in SEQ ID NO:2.

[0013] Preferably, the mesenchymal stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

[0014] In a second aspect, the present invention provides a method for preparing the modified mesenchymal stem cell exosomes according to the first aspect, the preparation method comprising the steps of:

[0015] (1) obtaining animal tissue cells through culture and screening to obtain mesenchymal stem cells, and then isolating and extracting the mesenchymal stem cells to obtain mesenchymal stem cell exosomes;

[0016] (2) The miR-206-3p inhibitor is loaded into the mesenchymal stem cell exosomes by electroporation to obtain the modified mesenchymal stem cell exosomes.

[0017] Preferably, the culture screening in step (1) is subculture screening, and the mesenchymal stem cells obtained are mesenchymal stem cells with a purity of ≥90%.

[0018] Preferably, in step (2), the mesenchymal stem cell exosomes and miR-206-3p inhibitor are 6.53×10 8 Mesenchymal stem cell exosomes were mixed with ≤20 pmol of miR-206-3p inhibitor and then electroporated;

[0019] The pulse voltage of the electroporation is ≤1250V.

[0020] Further preferably, the animal tissue cells are cells in the bone cavity, and the mesenchymal stem cells are bone marrow mesenchymal stem cells.

[0021] Mesenchymal stem cells are a type of multipotent stem cell. They are adult stem cells derived from the mesoderm, have the ability to self-renew and differentiate, and are widely distributed in bone marrow, umbilical cord blood, fat, and peripheral blood. Under the action of different induction factors, they can eventually differentiate into bone, fat, muscle cells, etc. The mesenchymal stem cells described in the present invention are bone marrow mesenchymal stem cells, which are obtained by further purification through differential screening.

[0022] In a third aspect, the present invention provides an exosome drug, wherein the exosome drug comprises the modified mesenchymal stem cell exosomes described in the first aspect.

[0023] Preferably, the exosome drug further comprises a pharmaceutically acceptable carrier.

[0024] More preferably, the exosome drug is administered via the nasal cavity.

[0025] The inventors of the present application found that the prepared exosome drug can enter the brain through nasal administration and has the therapeutic effect of improving AD cognitive dysfunction and pathological symptoms. The inventors also found that the prepared mesenchymal stem cell exosomes loaded with miR-206-3p inhibitors did not produce toxic side effects on AD model mice.

[0026] In a fourth aspect, the present invention provides use of the modified mesenchymal stem cell exosomes described in the first aspect in the preparation of a drug for treating Alzheimer's disease.

[0027] In a fifth aspect, the present invention provides use of the modified mesenchymal stem cell exosomes described in the first aspect in the preparation of a drug for treating a neurodegenerative disease caused by low brain-derived neurotrophic factor.

[0028] Beneficial effects of the present invention:

[0029] 1. It was found that intranasal administration of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor can effectively improve the pathological symptoms of Alzheimer's disease mice, enhance the learning and cognitive functions of mice, and achieve good therapeutic effects.

[0030] 2. It was found that the exosome drug is superior to the efficacy of exosomes or miR-206-3p inhibitors alone, and the provided exosome drug has the effects of inhibiting inflammatory response in the brain, upregulating brain-derived neurotrophic factor, promoting neuronal survival and neurite remodeling, thereby improving the phenotype of Alzheimer's disease.

[0031] 3. In one aspect of the present invention, the medicament is suitable for treating neurodegenerative diseases characterized by low brain-derived neurotrophic factor. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 To show the parameter optimization of preparing mesenchymal stem cell exosomes loaded with miR-206-3p inhibitors and the particle size and electron microscopy images before and after optimization, wherein Figure A shows the relative level of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitors under different parameters, Figure B shows the particle size distribution of mesenchymal stem cell exosomes before and after parameter optimization, and Figure C shows the morphology of mesenchymal stem cell exosomes before and after parameter optimization;

[0034] Figure 2 This is a graph showing the detection of miR-206-3p levels in the brain after nasal administration of miR-206-3p inhibitor and mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor;

[0035] Figure 3 The results of mesenchymal stem cell exosomes / miR-206-3p inhibitor / mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on the improvement of Alzheimer's disease mice behavior, wherein Figure A is the movement trajectory of mice in the escape latency and space exploration experiments, Figure B is the escape latency of mice in 6 consecutive days of training, Figure C is a statistical analysis of the number of times mice crossed the platform in the space exploration experiment, and Figure D is a statistical analysis of the time mice stayed in the target quadrant in the space exploration experiment;

[0036] Figure 4A diagram showing the effects of mesenchymal stem cell exosomes / miR-206-3p inhibitor / mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on amyloid deposition in the brain of Alzheimer's disease mice;

[0037] Figure 5 A diagram showing the inhibition of brain inflammation by mesenchymal stem cell exosomes / miR-206-3p inhibitor / mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor;

[0038] Figure 6 Figure 2 shows the effects of mesenchymal stem cell exosomes / miR-206-3p inhibitor / mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on the growth of hippocampal neurons in Alzheimer's disease mice

[0039] Figure 7 A diagram showing the regulation of mesenchymal stem cell exosomes / miR-206-3p inhibitor / mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on the expression of brain-derived neurotrophic factor in the hippocampus of Alzheimer's disease mice. DETAILED DESCRIPTION

[0040] The concept and technical effects of the present invention will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features and effects of the present invention.

[0041] Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work all fall within the scope of protection of the present invention.

[0042] In the present invention:

[0043] The nucleotide sequence of miR-206-3p is shown in SEQ ID NO: 1;

[0044] The nucleotide sequence of the miR-206-3p inhibitor is shown in SEQ ID NO: 2, which was purchased from Guangzhou Ruibo Guangzhou Ruibo Biotechnology Co., Ltd.;

[0045] The reagents used in the examples are all commercially available.

[0046] Example 1

[0047] Preparation of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor

[0048] The preparation steps are as follows:

[0049] (1) Take the femur of a 21-day-old SD rat and remove the muscle tissue. After the intact femur is disinfected with 75% alcohol and rinsed with PBS buffer, use sterile scissors to cut the two ends of the bone, inject DMEM culture medium to blow out the cells in the bone cavity. Culture for 3 days under the conditions of 37°C and 5% CO2. When the cell growth density reaches 90%, discard the culture medium, rinse once with PBS buffer, add 1mL of 0.25% pancreatic enzyme, digest for 2 minutes until the cells become round, add serum-containing culture medium to terminate the digestion and transfer the suspended cells to another clean culture dish. After three passages, bone marrow mesenchymal stem cells with a purity of more than 90% were screened.

[0050] (2) The isolated bone marrow mesenchymal stem cells were divided into 2×10 5 100 cells / dish were inoculated into a culture dish with a diameter of 10 cm. When the cell density reached 80%, the culture medium was aspirated, PBS buffer was added for rinsing 3 times, DMEM culture medium and 5% exosome-free serum were added, and cultured at 37°C for 72 hours.

[0051] (3) After 72 hours of culture, collect the culture supernatant. Centrifuge at 300×g for 10 minutes, take the supernatant to a centrifuge tube; centrifuge for 10 minutes (1000×g), take the supernatant to a centrifuge tube, and centrifuge for 30 minutes (10000×g); retain the supernatant and continue centrifuging for 120 minutes (100000×g), discard the supernatant, add PBS buffer to wash the precipitate, and finally centrifuge for 60 minutes (160000×g). The resulting precipitate is the exosome, and add PBS buffer to resuspend the precipitate.

[0052] (4) Exosomes loaded with miR-206-3p inhibitor. Take the 6.53×10 8 Exosomes were mixed with 10 and 20 pmol of miR-206-3p inhibitor, and the mixed liquid was added to a pre-cooled electroporation cup (100 μL test tube with a diameter of 0.2 cm). The sample in the cup was subjected to a single pulse electroporation using an electroporation system. The pulse voltage was set to 0V, 750V, 1000V, and 1250V, and the voltage was applied for 5 ms. The electroporation reagent was PBS. Then, the cells were incubated at 37°C for 30 minutes and 4°C overnight. Rnase enzyme was added for 30 minutes, and then the Rnase inhibitor was added to terminate the enzymatic hydrolysis. The mixture was evenly mixed, centrifuged at 4°C for 2 hours (100,000 × g), and the exosomes were resuspended in PBS buffer to obtain mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor (MSC-Exo-miR-206-3p inhibitor).

[0053] (5) RNA was extracted from MSC-Exo-miR-206-3p inhibitors and the RNA concentration was detected using a spectrophotometer. In addition, the particle size and particle distribution of exosomes were analyzed by nanoflow cytometry, and the morphology of exosomes was observed using a transmission electron microscope.

[0054] Result analysis:

[0055] The RNA in the MSC-Exo-miR-206-3p inhibitor complex was extracted. The results showed that 6.53×10 8 The mixture of 10 exosomes and 20 pmol miR-206-3p inhibitor had the highest loading amount. The exosomes and exosome-miR-206-3p inhibitor complexes were observed using transmission electron microscopy, and both showed a saucer-like structure with a diameter between 50-100 nm. The nanoparticle tracing results also showed that the number and particle size distribution of exosomes and exosome-miR-206-3p inhibitor complexes were concentrated at 70 nm, indicating that the loading of miR-206-3p inhibitor had no significant effect on the morphology and size of mesenchymal stem cell exosomes (MSC-Exo) ( Figure 1 ).

[0056] Example 2

[0057] Exosomes enhance the targeting of miR-206-3p inhibitor into the brain

[0058] The operating method of Example 2 is as follows:

[0059] 1) Take out the slices, fix them with 4% paraformaldehyde at room temperature for 15 minutes, and rinse with PBS buffer for 5 minutes;

[0060] 2) Treat with 0.2 mol / L hydrochloric acid for 12 minutes;

[0061] 3) After drying the hydrochloric acid, treat the tissue with a solution containing 0.5% Triton for 12 minutes;

[0062] 4) Aspirate the solution containing 0.5% Triton X-ray and rinse with PBS buffer (prepared with DEPC water) for 5 minutes;

[0063] 5) Add 20 μg / mL proteinase K to the slide, let it stand at room temperature for 12 minutes, and then rinse with PBS buffer (prepared with DEPC water) for 5 minutes;

[0064] 6) Incubate the sections with 3% H2O2 solution at room temperature for 15 minutes, then rinse the sections with 0.1% DEPC water for 5 minutes;

[0065] 7) After shaking off the residual DEPC water, add 4% paraformaldehyde on the tissue and fix for 10 minutes;

[0066] 8) Remove the paraformaldehyde and soak the slices in PBS buffer for 5 minutes;

[0067] 9) Prehybridization. Cover the tissue with 40 μL of miRNA Hybridization Buffer and cover with a cover glass treated with DEPC water, and incubate at 55°C in an incubator for 1.5 hours;

[0068] 10) Probe preparation. 10 minutes before the end of pre-hybridization, prepare and mix miR-206-3p probe labeled with digoxigenin:miRNA Hybridization Buffer at a volume ratio of 1:100, denature at 85°C for 3 minutes, and equilibrate at 37°C for 5 minutes;

[0069] 11) Hybridization. Add probe dilution dropwise, gently place in a wet box, close the lid and move into a 41°C incubator for 48 hours;

[0070] 12) After hybridization, wash with washing buffer (10×washing buffer:DEPC water=1:9, volume ratio) for 3 times, 5 minutes each time, wherein the 10×washing buffer is a 10-fold concentrated elution buffer;

[0071] 13) drying the washing solution, adding Blocking buffer dropwise, and incubating at 37° C. for 1 hour. The Blocking buffer is a blocking solution, specifically containing maleic acid, sodium chloride, Tween 20 and 2% BSA;

[0072] 14) Discard the Blocking buffer, add anti-Digoxin HRP conjugate (anti-Digoxin HRP Conjugate: Blocking buffer = 1:100, volume ratio), and incubate at 37°C for 1 hour;

[0073] 15) Wash with PBS buffer (DEPC water) 3 times, 5 minutes each time;

[0074] 16) Add a solution containing TSA-488:TSA amplification Buffer:0.15% H2O2=1:100:1 (volume ratio); incubate at 37°C in the dark for 15 minutes;

[0075] 17) Wash with PBS buffer (DEPC water) three times in dark conditions, 5 minutes each time;

[0076] 18) Dry the PBS buffer, add DAPI-Antifade solution and cover with a coverslip, leave at room temperature for 20 minutes, and store away from light;

[0077] 19) Take photos under a confocal microscope.

[0078] Result analysis:

[0079] The efficiency of miR-206-3p inhibitor entering the brain was investigated by fluorescent labeling and tracing, and the level of miR-206-3p in the brain was detected by in situ hybridization. The results showed that mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor could effectively inhibit the level of miR-206-3p in the brain through nasal administration, and the effect was better than that of exosomes or miR-206-3p inhibitor alone ( Figure 2 ).

[0080] Example 3

[0081] Effects of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on learning and cognitive function in Alzheimer's disease mice

[0082] Experimental methods:

[0083] AD model mice were prepared and randomly divided into 5 groups, which were given exosomes, mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor negative control, mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor, miR-206-3p inhibitor and PBS buffer, and a normal control group was set up. The drug solution was prepared and administered nasally, and the dosage of miR-206-3p inhibitor and miR-206-3p inhibitor in mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor was 5nmol per mouse. The drug was administered once every other day for 3 weeks.

[0084] After 3 weeks of drug administration, the learning and memory ability of mice was evaluated by water maze experiment. The water maze process includes positioning navigation and spatial exploration. The positioning navigation experiment first trained the mice to obtain the orientation information of the platform above the water surface, and the learning ability of the mice in repeated training was evaluated by the time to climb onto the platform. After the positioning navigation was completed, the platform was removed, and the mouse was gently placed in the water with its head facing the wall of the tank, and the number of times it crossed the platform within a fixed 90 seconds was recorded to evaluate the memory ability of the mouse.

[0085] Result analysis:

[0086] like Figure 3As shown in the figure, after intranasal administration of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor, the time taken by mice to find the platform was significantly shorter than that of mice in other treatment groups, close to normal; in the spatial exploration experiment, the number of times mice crossed the platform and the time they stayed in the target quadrant increased significantly, both better than those of mice in other treatment groups. This result shows that compared with exosomes and miR-206-3p inhibitor alone, mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor can effectively improve the learning and memory ability of Alzheimer's disease mice.

[0087] Example 4

[0088] Effects of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on amyloid deposition in the brain of Alzheimer's disease mice

[0089] Experimental methods:

[0090] Mice were anesthetized by intraperitoneal injection of tribromoethanol (20 μL / g), and the limbs were fixed after coma, and the chest cavity was carefully opened to expose the heart. The right auricle of the mouse was cut open, and a needle was inserted from the left ventricle, and perfusion was performed with pre-cooled PBS buffer, and the whole body blood was replaced through the systemic circulation system. Then the head was fixed, and the skin of the mouse was cut to expose the skull. When cutting the skull, it was first cut horizontally along the neck, and then a small cut was carefully made along the midline, and it was cut horizontally again along the eyes, and finally the skull was opened with tweezers to expose the whole brain, and the brain was taken out with curved tweezers, placed on ice, and the right hemibrain was separated and placed in 4% paraformaldehyde solution, fixed at room temperature for 24 hours, and replaced with 30% sucrose solution the next day, and after dehydration twice, OCT embedding agent was used for tissue embedding, and frozen sections with a thickness of 10 μm were made and stored at -80°C.

[0091] 1) Take out the tissue sections and place them at room temperature for 15 minutes.

[0092] 2) Use an immunohistochemistry pen to draw a circle around the tissue, add a drop of permeabilization solution (0.2% Triton X-100 solution), and let it stand at room temperature for 15 minutes.

[0093] 3) Remove the permeabilization solution, wash with PBS buffer for 3 times, add 5% BSA solution to cover the tissue, and block at room temperature for 1.5 hours.

[0094] 4) Dry the blocking solution, add the diluted primary antibody solution (6E10, 1:200), and incubate at 4°C for 14 hours.

[0095] 5) The next day, remove the incubation box, equilibrate at room temperature for 15 minutes, and wash with PBS buffer three times, 5 minutes each time.

[0096] 6) Incubate with secondary antibody. Add anti-mouse fluorescent secondary antibody solution to ensure that the tissue is completely covered, and incubate at 37°C for 1 hour.

[0097] 7) Wash with PBS buffer 3 times, 5 minutes each time.

[0098] 8) Add DAPI dye solution to cover the tissue and treat at room temperature for 10 minutes, then wash the tissue three times with PBS buffer, each time for 5 minutes.

[0099] 9) Add sealing medium and cover with coverslip.

[0100] Result analysis:

[0101] The stained tissue was scanned using a laser confocal microscope to detect the fluorescence signal imaging. Figure 4 As shown in the figure, compared with the model mice, the area and number of β-amyloid plaques in the hippocampus tissue of mice treated with mesenchymal stem cell exosomes loaded with miR-206-3p inhibitors were reduced by about 3.2 times and 2.3 times, respectively. Although exosomes and miR-206-3p inhibitors can also reduce the area and number of β-amyloid plaques in the hippocampus tissue of mice, the effect is far less than that of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitors.

[0102] Example 5

[0103] Effects of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on neuronal growth in the brain of Alzheimer's disease mice

[0104] Experimental methods:

[0105] Immunofluorescence was used to detect the number of neurons in the hippocampal tissue of mice in different drug treatment groups.

[0106] The specific steps are as follows:

[0107] 1) Take out the tissue sections from the -80℃ freezer and place them at room temperature for 15 minutes.

[0108] 2) Use an immunohistochemistry pen to draw a circle around the tissue, add permeabilization solution (0.2% TritonX-100 solution), and let it stand at room temperature for 15 minutes.

[0109] 3) Remove the permeabilization solution, wash with PBS buffer for 3 times, add 5% BSA solution to cover the tissue, and block at room temperature for 1.5 hours.

[0110] 4) Dry the blocking solution, add the diluted primary antibody solution (Dcx antibody, 1:200 dilution), and incubate at 4°C for 12 hours.

[0111] 5) The next day, remove the incubation box, equilibrate at room temperature for 15 minutes, and wash with PBS buffer three times, 5 minutes each time.

[0112] 6) Incubate with secondary antibody. Add anti-mouse fluorescent secondary antibody solution (1:300 dilution) to ensure complete coverage of the tissue and incubate at 37°C for 1 hour.

[0113] 7) Wash with PBS buffer 3 times, 5 minutes each time.

[0114] 8) Add DAPI dye solution to cover the tissue and treat at room temperature for 10 minutes, then wash the tissue three times with PBS buffer, each time for 5 minutes.

[0115] 9) Add sealing medium, cover with coverslip, and read the slide under laser confocal microscope.

[0116] Result analysis:

[0117] like Figure 5 As shown in the figure, the expression level of newborn neuron marker protein Dcx in the hippocampus tissue of mice treated with mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor was about 8 times that of mice in the model group. Although exosomes and miR-206-3p inhibitor alone can also increase Dcx protein-positive cells, their effect is not as good as that of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor.

[0118] Example 6

[0119] Effects of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on inflammatory response in the brain of Alzheimer's disease mice

[0120] Experimental methods:

[0121] Semi-quantitative RT-PCR was used to detect gene expression in exosomes, mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor negative control, mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor, and hippocampal tissue of Alzheimer's disease model mice after treatment with miR-206-3p inhibitor.

[0122] The steps are as follows:

[0123] 1) Add 200 μL Trizol solution to the Eppendorf tube containing hippocampal tissue, put in a sterilized grinding rod, and lyse the tissues one by one with the help of the grinder. Grind each tissue for 3 seconds each time during lysis, and repeat in sequence, ensuring that each tissue is ground about 5 times. Stop lysis when there is no obvious tissue block.

[0124] 2) Add 800 μL of Trizol solution to the ground tissue to make the final volume of Trizol solution 1 mL, shake vigorously for 3 minutes, then add 200 μL of chloroform, shake vigorously and let stand for 5 minutes, then centrifuge for 15 minutes (12000 rpm).

[0125] 3) In The extracted RNA was reverse transcribed into cDNA according to the instructions of Ⅱ1st Strand cDNA Synthesis Kit (11119ES60 yeasen).

[0126] 4) Perform PCR amplification.

[0127] 5) Prepare agarose gel. Weigh 1g of agarose into a conical flask filled with 100mL of 1×TAE electrophoresis buffer, seal it and place it in a microwave oven, cook on medium heat for 2 minutes to completely dissolve the agarose; then, add nucleic acid dye at a ratio of 1 / 20000 (volume ratio), shake to mix, carefully inject the gel, and let it stand at room temperature for 30 minutes.

[0128] 6) Loading nucleic acid for electrophoresis. Carefully pull the comb out of the solidified agarose gel and slowly move the gel into the electrophoresis tank. Add 1×TAE electrophoresis buffer, making sure that the buffer covers the comb holes. Then load the samples one by one, adding 6 μL of amplification product to each well, and perform electrophoresis at 120V.

[0129] 10) After about 25 minutes, turn off the power, take out the gel, and place it in a gel imager for photography.

[0130] Result analysis:

[0131] like Figure 6 As shown, the inflammatory factors IL-1β, IL-6 and TNF-α in the hippocampus of model mice were about 1.5 times, 1 times and 1.1 times higher than those of normal mice, respectively. Mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor effectively inhibited the expression of inflammatory factors and reduced it to near normal levels. In addition, exosomes and miR-206-3p inhibitors can also inhibit inflammatory responses in the brains of Alzheimer's mice, but the effect is not as good as that of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitors.

[0132] Furthermore, immunofluorescence was used to detect the activation of astrocytes and microglia in the mouse brain. The method described in this example is the same as that in Examples 4 and 5, except that different primary antibodies were used, specifically Iba1 (1:300), CD68 (1:300), GFAP (1:300), and C3 (1:200). Figure 6 As shown, exosomes, miR-206-3p inhibitor and mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor can all inhibit the activation of proinflammatory glial cells in the hippocampus of Alzheimer's disease mice, and the inhibitory effect of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor is the most obvious.

[0133] Example 7

[0134] Effects of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor on brain-derived nutritional factor in the brain of Alzheimer's disease mice

[0135] Experimental methods:

[0136] Western blot was used to detect the expression level of brain-derived neurotrophic factor (BDNF) in the hippocampal tissue of mice in different treatment groups. Proteins in tissues were extracted using protein lysis buffer, and the protein concentration of each group of samples was detected using a protein quantification kit. 5× loading buffer was added to the protein solution to make the final concentration 1× loading buffer, heated at 100°C for 8 minutes, and stored in a -80°C refrigerator.

[0137] Next, test the samples:

[0138] 1) Prepare 12% separation gel and 5% stacking gel in advance, and load 30 μg of protein sample per well (30 μg / 10 μL);

[0139] 2) Perform electrophoresis at 60V for 30 minutes on the concentrated gel and 110V for 70 minutes on the separated gel;

[0140] 3) When the bromophenol blue indicator band reaches 5 mm from the bottom of the gel, stop electrophoresis, remove the gel, and install it in the order of negative electrode-sponge-filter paper-gel-activated PVDF membrane-filter paper-sponge-positive electrode, turn on the power and adjust it to 260 mA, start the transfer, and the duration is 90 minutes;

[0141] 4) Add 5% skim milk powder solution to cover the PVDF membrane and block at room temperature for 1 h;

[0142] 5) Incubate with primary antibody. Pour off skim milk powder, rinse twice with TBST buffer, dry, add diluted primary antibody solution (BDNF, 1:1000), and incubate overnight on a shaker at 4°C.

[0143] 6) Washing with primary antibody solution. After discarding the primary antibody solution, add an appropriate amount of TBST buffer to the incubation box and wash on a horizontal shaker at medium or low speed for 5 times, 7 minutes each time;

[0144] 7) Secondary antibody incubation: Add diluted secondary antibody solution (goat anti mouse HRP: 5% skim milk powder = 1:5000) and incubate at room temperature for 1 hour;

[0145] 8) Washing of secondary antibody: discard the secondary antibody solution, add TBST buffer to cover the PVDF membrane, and wash 5 times at medium speed on a horizontal shaker, 7 minutes each time;

[0146] 9) Develop the target band. Evenly drop the developer on the membrane, then transfer the membrane to the developer, capture the band signal with the help of the imaging system, and save the image.

[0147] Result analysis:

[0148] The results are as follows Figure 7 As shown in the results, exosomes, miR-206-3p inhibitor and mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor can all upregulate the expression level of brain-derived neurotrophic factor in hippocampal tissue, among which the upregulation effect of mesenchymal stem cell exosomes loaded with miR-206-3p inhibitor was the most obvious.

[0149] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A modified mesenchymal stem cell exosome, characterized in that: The modified mesenchymal stem cell exosomes are modified mesenchymal stem cell exosomes obtained by loading the mesenchymal stem cell exosomes with a miR-206-3p inhibitor; The miR-206-3p inhibitor is a complementary single strand of miR-206-3p; The nucleotide sequence of miR-206-3p is shown in SEQ ID NO: 1; The nucleotide sequence of the miR-206-3p inhibitor is shown in SEQ ID NO:

2.

2. The modified mesenchymal stem cell exosomes according to claim 1, characterized in that The mesenchymal stem cell exosomes are bone marrow mesenchymal stem cell exosomes.

3. The method for preparing modified mesenchymal stem cell exosomes according to claim 1 or 2, characterized in that: The preparation method comprises the steps of: (1) obtaining animal tissue cells through culture and screening to obtain mesenchymal stem cells, and then isolating and extracting the mesenchymal stem cells to obtain mesenchymal stem cell exosomes; (2) The miR-206-3p inhibitor is loaded into the mesenchymal stem cell exosomes by electroporation to obtain the modified mesenchymal stem cell exosomes.

4. The method for preparing modified mesenchymal stem cell exosomes according to claim 3, characterized in that: The culture screening in step (1) is subculture screening, and the mesenchymal stem cells obtained are mesenchymal stem cells with a purity of ≥90%.

5. The method for preparing modified mesenchymal stem cell exosomes according to claim 3, characterized in that: In step (2), the mesenchymal stem cell exosomes and miR-206-3p inhibitor were expressed at 6.53×10 8 Mesenchymal stem cell exosomes were mixed with ≤20 pmol of miR-206-3p inhibitor and then electroporated; The pulse voltage of the electroporation is ≤1250V.

6. The method for preparing modified mesenchymal stem cell exosomes according to any one of claims 3 to 5, characterized in that: The animal tissue cells are cells in the bone cavity, and the mesenchymal stem cells are bone marrow mesenchymal stem cells.

7. An exosome drug, characterized in that: The exosome medicine comprises the modified mesenchymal stem cell exosomes according to claim 1 or 2.

8. The exosome drug according to claim 7, characterized in that The exosome drug also includes a pharmaceutically acceptable carrier.

9. The exosome drug according to claim 7 or 8, characterized in that: The exosome drug is administered via the nasal cavity.

10. Use of the modified mesenchymal stem cell exosomes according to claim 1 or 2 in the preparation of a medicament for treating Alzheimer's disease.

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