Highly active neural stem cell exosome and preparation method and application thereof

Abstract

This invention discloses a highly active neural stem cell extravesicle, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method includes: using Laminin521-coated culture flasks, resuscitating hNSCs and culturing them in suspension culture medium for 7 days until the cells aggregate to form neurospheres, then seeding them into culture flasks and adhering to the culture medium containing composite regulatory factors until the cell density reaches 2~3×10⁻⁶. 7 The supernatant was collected per vial and purified by centrifugation, ultrafiltration, and washing. This invention specifically enriches five miRNAs—hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, and hsa-miR-125b-5p—in extracellular vesicles, significantly increasing their yield and activity, and can be used in the treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a highly active neural stem cell extravesicle, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment and memory decline. Its pathological mechanisms are complex, primarily related to β-amyloid (Aβ) deposition, tau protein hyperphosphorylation, neuroinflammation, and neuronal apoptosis. Currently, there are no effective drugs to cure AD. Therefore, developing novel targeted therapies is of significant clinical importance.

[0003] Extracellular vesicles derived from neural stem cells (NSCs) (NSC-EVs), as natural nanoscale vesicles, carry various functional microRNAs, proteins, and other bioactive substances. They can cross the blood-brain barrier, regulate nerve cell survival, inhibit Aβ deposition, and reduce neuroinflammation, showing promising application prospects in the treatment of Alzheimer's disease (AD). However, traditional methods for preparing NSC-EVs mostly employ suspension culture, which suffers from low EV yield, unstable activity, and insufficient expression levels of characteristic biomarkers (CD9 / CD81 / CD63), limiting their clinical translation and application.

[0004] In existing technologies, neural stem cell culture often relies on suspension culture in ultra-low adsorption flasks without the addition of targeted regulatory factors, resulting in low cell proliferation efficiency and limited functional activity of secreted extracellular vesicles. Furthermore, extracellular vesicles harvested using traditional methods show poor protective effects against Aβ-induced neurological damage when used in Alzheimer's disease (AD) treatment, failing to meet clinical needs. Therefore, developing a method to increase the yield, activity, and neuroprotective function of neural stem cell extracellular vesicles has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the problems of low yield, poor activity, and limited neuroprotective function in the preparation of extracellular vesicles of neural stem cells in existing technologies, the main objective of this invention is to provide a method for preparing highly active extracellular vesicles of neural stem cells. By adding compound regulatory factors, using a Laminin521-coated adherent culture process that differs from classic suspension culture, and optimizing culture conditions, the yield and activity of extracellular vesicles are improved.

[0006] Another objective of this invention is to provide a highly active neural stem cell extravesicle, which is prepared by the method described above and specifically enriches five functional microRNAs: hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, and hsa-miR-125b-5p.

[0007] Another object of the present invention is to provide the application of the aforementioned highly active neural stem cell extravesicles in the preparation of drugs for treating Alzheimer's disease, thereby achieving effective intervention in AD pathological damage.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing extravesicles of highly active neural stem cells, comprising the following steps:

[0010] (1) Laminin521 coated culture flasks were used as the adherent culture system;

[0011] (2) After the human neural stem cells (hNSCs) were revived, they were cultured in suspension culture medium for 7 days at 37°C, 5% CO2 and saturated humidity until the cells aggregated to form a neural sphere with a complete structure and clear boundaries.

[0012] (3) The neurospheres after suspension culture were inoculated into the adherent culture system and replaced with an adherent culture medium containing composite regulatory factors for adherent culture. The total culture period was 14 days, during which time fluid was replenished in stages on the 4th, 7th and 10th days.

[0013] (4) The cell density in the culture flask reaches 2~3×10 7 When the number of cells / bottle is reached, the supernatant is collected and purified by centrifugation, ultrafiltration and washing to obtain highly active neural stem cell extravesicles (N5-NSC-EV).

[0014] Preferably, in step (1), laminin521 is used on 182 cm. 2 The culture flasks were coated at a concentration of 5 μg / mL and incubated overnight in an incubator to ensure that the substrate evenly covered the bottom of the flask.

[0015] Preferably, in step (2), the suspension culture medium comprises: basal culture medium (DMEM / F12), stock solution; Neurobasal... TMCulture medium, mixed with DMEM / F12 at a volume ratio of 1:1; N2 cell culture additive, 0.5×; B27 serum-free additive, 1×; recombinant human basic fibroblast growth factor (BFGF), 10 ng / mL; recombinant human leukemia inhibitory factor (LIF), 10 ng / mL; recombinant human epidermal growth factor (EGF), 20 ng / mL; non-essential amino acids (NEAA), 1×; β-mercaptoethanol, 0.1 mM; Laminin 521, 5 μg / mL; PBS buffer (calcium and magnesium-free), 1× (stock solution).

[0016] Preferably, in step (3), the adherent culture medium includes the suspension culture medium and the composite regulatory factor, wherein the composite regulatory factor is composed of 1 mmol / L α-ketoglutarate, 10 mg / L insulin, 2 mg / L ethanolamine, 2.5 μg / L folic acid, 15 μM Y-27632, 0.8 μM A23187, 0.8 mM dbcAMP, and 30 ng / mL BDNF.

[0017] Preferably, in step (3), the amount of liquid replenished each time is 1 / 3 of the total volume of the adherent culture medium, and new culture medium is added to maintain the stability of nutrient components and factor concentrations in the culture system.

[0018] Preferably, in step (4), the centrifugation process includes: the supernatant is first centrifuged at 4°C and 300×g for 10 min, filtered to remove intact cells, and the supernatant is collected; then centrifuged at 4°C and 2000×g for 20 min, filtered to remove cell debris and large particulate impurities, and the supernatant is collected again.

[0019] The ultrafiltration separation process includes: slowly adding the supernatant collected after centrifugation into the rinsed ultrafiltration centrifuge tube, with the amount added to each tube not exceeding the maximum capacity of the ultrafiltration tube; after tightening the cap, place the tube in a refrigerated centrifuge and centrifuge at 4°C and 3000×g for 15-20 min; after centrifugation, discard the filtrate and retain the concentrate in the upper chamber of the ultrafiltration tube;

[0020] The washing and purification process includes: adding 5 mL of pre-cooled sterile PBS solution to the concentrate in the upper chamber of the ultrafiltration tube, gently inverting to mix, then centrifuging at 4°C and 3000×g for 15 min, and discarding the filtrate; repeating this washing step twice to remove residual culture medium components and small molecule impurities, thereby improving the purity of extracellular vesicles.

[0021] In a second aspect, the present invention provides a highly active neural stem cell extravesicle, prepared by the method for preparing the highly active neural stem cell extravesicle, which has a round or elliptical vesicle structure with an intact lipid bilayer membrane, and specifically enriched with five functional microRNAs: hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, and hsa-miR-125b-5p.

[0022] A third aspect of the invention provides the use of the highly active neural stem cell extravesicles in the preparation of a medicament for treating Alzheimer's disease.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] I. Unlike traditional suspension culture, this invention achieves efficient preparation of N5-NSC-EVs by adding compound regulatory factors and using a Laminin521-coated adherent culture process. Compared with traditional suspension culture of classic neural stem cell extracellular vesicles (classic NSC-EV), it significantly improves the yield and activity of extracellular vesicles.

[0025] Second, transcriptome differential analysis confirmed that the N5-NSC-EV prepared in this invention specifically enriched five functional microRNAs: hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, and hsa-miR-125b-5p. These microRNAs showed superior therapeutic effects on AD models, providing an efficient and feasible technology for the application of neural stem cell extravesicles in AD treatment, with broad market prospects and clinical value. Attached Figure Description

[0026] Figure 1 The expression levels of the top 20 genes in the KEGG-ALZHEIMER'SDISEASE enrichment pathway, as shown in the examples, represent the differentially expressed genes.

[0027] Figure 2 The results of cell viability detection (CCK-8 assay) for different groups in the examples are shown below; the P value for the AD model group (Aβ Model) vs. the N5 vesicle group (N5-NSC-EV) was 0.0017, P<0.01; the P value for the N5 vesicle group (N5-NSC-EV) vs. the negative control group (classical NSC-EV) was 0.0047, P<0.01.

[0028] Figure 3The cell viability test results for different groups in the examples are shown below; A: Blank control group (Control); B: Negative control group (classic NSC-EV, batch number: NSCE20250707F1); C: N5 vesicle group (N5-NSC-EV, batch number: N5NSCE20250623F1); D: AD model group (Aβ Model).

[0029] Figure 4 The results of transmission electron microscopy (TEM) morphological identification of the classic NSC-EV and N5-NSC-EV in the examples are shown.

[0030] Figure 5 The particle size and particle concentration detection results for the classic NSC-EV and N5-NSC-EV in the examples are shown.

[0031] Figure 6 The results of membrane protein marker detection and identification are shown in the examples.

[0032] Figure 7 The images show the immunofluorescence staining results of the classic NSC-EV and N5-NSC-EV dosing groups in the examples.

[0033] Figure 8 To illustrate the number and area of ​​Aβ plaques in the hippocampus and cortex in the examples, the one-way Anova test was used to measure the differences between the groups. The data represent mean ± sem, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

[0034] Figure 9 This is a schematic diagram illustrating the principle of the tailing method for quantitative real-time PCR in the examples.

[0035] Figure 10 The results are shown in the examples, representing the qPCR detection results. Detailed Implementation

[0036] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] Example 1

[0039] I. Preparation of highly active neural stem cell extravesicles (N5-NSC-EV)

[0040] Using Laminin 521 to cover 182 cm 2 The culture flasks were used as an adherent culture system. The preparation process was as follows: Human neural stem cells were first revived and cultured in suspension for 7 days to form neurospheres. Then, they were seeded into coated culture flasks and cultured in a medium containing a complex regulatory factor (composed of 1 mmol / L α-ketoglutarate, 10 mg / L insulin, 2 mg / L ethanolamine, 2.5 μg / L folic acid, 15 μM Y-27632, 0.8 μM calcium ion carrier (A23187), 0.8 mM dbcAMP, and 30 ng / mL brain-derived neurotrophic factor (BDNF)) for 14 days, with fluid replenishment at days 4, 7, and 10. The culture was continued until the cell density reached 2-3 × 10⁻⁶ cells / mL. 7 When the number of samples per bottle is reached, the supernatant is collected and purified by centrifugation, filtration, ultrafiltration and other steps to obtain N5-NSC-EV.

[0041] II. Screening for MicroRNAs with Specific Differences

[0042] Screening for specific differentially expressed microRNAs between N5-NSC-EV and classic NSC-EV to identify core microRNAs associated with the pathological regulation of Alzheimer's disease, the specific steps are as follows:

[0043] 1. Sample Preparation and Detection: Two types of extracellular vesicle samples were selected for detection. Three independent samples were set up for each of the N5-NSC-EV group (experimental group) and the classic NSC-EV group (control group) (all derived from multiple batches of extracellular vesicle products to ensure sample representativeness), for a total of six samples. All samples were sent to a professional testing institution for microRNA transcriptome sequencing to obtain raw microRNA expression profile data for the two groups of extracellular vesicles.

[0044] 2. Differential MicroRNA Screening: The raw transcriptome sequencing data were standardized and differentially expressed using the R language LIMMA package. A screening threshold (|log2FC|>1 and P<0.05) was set to identify microRNAs with significantly different expression between the experimental and control groups, and to clarify the upregulation / downregulation trends of differentially expressed microRNAs.

[0045] 3. Target Gene Prediction of Differentially Occurred MicroRNAs: The multiMiR database and accompanying analysis tools were used to predict the target genes of the screened differentially occult microRNAs. This tool integrates results from multiple authoritative target gene databases (miRBase, TargetScan, miRanda, etc.), and improves the accuracy and reliability of target gene prediction through intersection analysis, ultimately obtaining the target gene set corresponding to each differentially occult microRNA.

[0046] 4. KEGG functional enrichment analysis of target genes: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis is performed on the predicted differentially expressed MicroRNA target gene set. Through this analysis, the biological pathways mainly enriched by target genes are clarified, with focus on the pathway closely related to AD pathological mechanism (KEGG -mmu05010-Alzheimer's disease), and target genes enriched in AD-related pathways are screened.

[0047] 5. Screening of core differentially expressed MicroRNA: Based on the correspondence between AD-related target genes and differentially expressed MicroRNA, MicroRNAs corresponding to each target gene are counted, and finally the TOP 20 differentially expressed MicroRNAs with the highest frequency of regulating AD-related target genes are screened as core candidate MicroRNAs for subsequent functional verification and mechanism research, as shown in Table 1 and Figure 1 shown below.

[0048] Table 1

[0049]

[0050] The gene expression of the above 20 AD-related MicroRNAs was determined from raw data, and they were arranged in ascending order of P value. It was found that the TOP 5 MicroRNAs with the largest expression difference in NSC-EV (0.001<P<0.01) are: hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, hsa-miR-125b-5p, as shown in Table 2.

[0051] Table 2

[0052]

[0053] Based on the above data, the five MicroRNAs hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, and hsa-miR-125b-5p have the strongest functional correlation with AD and relatively high significance of expression difference, and can be characteristic components of N5-NSC-EV cultured by adherent culture method.

[0054] III. Functional Verification

[0055] To verify the effect of N5-NSC-EV extracellular vesicles, Aβ 1-42 oligomer-induced pathological injury model of neural stem cells is established.

[0056] (1) Aβ 1-42 Oligomer preparation

[0057] Dissolve 1 mg of Aβ in HFIP. 1-42 The peptide was incubated at 1 mM at room temperature for 1 h, and then freeze-dried under vacuum to remove HFIP, yielding Aβ. 1-42 The membrane was resuspended to 5 mM with 44 μL of DMSO, aliquoted, and stored at -20°C. Before use, it was diluted to the target concentration with hNSCs blank medium and incubated at 37°C for 24 h to form stable oligomers.

[0058] (2) N5-NSC-EV pretreatment

[0059] Take out the frozen N5-NSC-EV and the control classic NSC-EV, thaw them at 4℃, and then dilute them with hNSCs blank medium to a working concentration of 5×10^11 / mL, avoiding repeated freeze-thaw cycles.

[0060] (3) Aβ 1-42 Concentration screening

[0061] 96-well plate seeding with hNSCs (1×10⁶ per well) 4 (number of cells), cultured for 24 h; set Aβ 1-42 hNSCs were treated with concentration gradients (0, 2, 3, 5, 7 μM) for 24 h each; 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37℃ for 2 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated, as shown in Table 3.

[0062] Table 3

[0063]

[0064] Note: When the concentration is 5 μM, the cell survival rate is close to 50%, so this concentration was used for subsequent validations.

[0065] IV. Verifying the efficacy of N5-NSC-EV in an AD-like pathological lesion model

[0066] (1) Cell plating and AD model construction

[0067] 96-well plate seeding with hNSCs (2 × 10⁶ per well) 4 Each group (AD model group, N5-NSC-EV group, and negative control group) was incubated with a culture medium containing eight added compound regulatory factors at 37°C and 5% CO2 for 24 h. The old culture medium was then discarded. Aβ-containing media were added to the AD model group, N5-NSC-EV group, and negative control group. 1-42 The culture medium was prepared to achieve a final concentration of 5 μM. The blank control group was supplemented with a medium containing no Aβ. 1-42 The culture medium was used to incubate AD-like neural stem cells for 24 hours to induce them.

[0068] (2) N5-NSC-EV vesicle intervention treatment

[0069] N5-NSC-EV vesicles or classic NSC-EV vesicles were added to the groups as negative controls. The blank control group and AD model group were added with an equal amount of hNSCs culture medium and cultured for another 24 h. Cell morphology changes were observed during the period.

[0070] (3) Indicator detection and results

[0071] 1. CCK-8 assay for cell viability: Cells were simultaneously seeded and cultured in 96-well plates. After intervention, cell viability was assessed according to the pre-experimental procedure, and the cell viability of each group was calculated, as shown in Table 4. Figure 2 As shown.

[0072] Table 4

[0073]

[0074] 2. AO / PI method for detecting cell viability

[0075] Two additional 6-well plates were prepared, and the cell seeding density was 5 × 10^5. Other reagents were adjusted proportionally. Cell viability was detected using the AO / PI method, as shown in Table 5. Figure 3 As shown.

[0076] Table 5

[0077]

[0078] In summary, although both N5-NSC-EV and classic NSC-EV can provide some protection for neural stem cells, extracellular vesicles secreted by adherent culture method N5-NSC-EV have a better protective and repairing effect on neural stem cells.

[0079] Example 2: Preparation of N5-NSC-EV

[0080] I. Materials and Methods

[0081] Cell source: Primary neural stem cells, midbrain-derived neural stem cells, passage number P8, provided by Shanghai Anji Xiekang Biotechnology Co., Ltd.

[0082] reagents

[0083] a. The reagents used in the classic neural stem cell culture method are shown in Table 6.

[0084] Table 6

[0085]

[0086] b. The eight additional factors that need to be added in the improved culture method and their concentrations are shown in Table 7.

[0087] Table 7

[0088]

[0089] II. Preparation Method

[0090] (I) Experimental group culture method

[0091] The experimental group used an adherent culture method for neural stem cells, and the specific procedures are as follows:

[0092] Neurosphere preparation: The revived hNSCs were inoculated into a culture container containing basal culture medium and cultured in suspension for 7 days. During this period, the cell growth status was observed daily to ensure a stable culture environment (37℃, 5% CO2, saturated humidity) until the cells aggregated to form a neurosphere with a complete structure and clear boundaries.

[0093] Preparation for adherent culture: Coat the 182 cm² culture flask with laminin521 at a concentration of 5 μg / ml, and incubate overnight in an incubator to ensure that the substrate evenly covers the bottom of the flask.

[0094] Adherent culture and factor addition: Neurospheres cultured for 7 days were seeded into 182 cm² cells coated with laminin521. 2 Replace the culture medium in the culture flask with a special culture medium containing the above 8 factors, and start adherent culture. The total culture period is 14 days.

[0095] Replenishment of medium: Replenishment of medium was carried out on the 4th, 7th and 10th days of adherent culture. Each replenishment was 1 / 3 of the total volume of the culture medium. Fresh culture medium was added to maintain the stability of nutrient components and factor concentrations in the culture system.

[0096] Conditioned culture medium collection: When the cell density in the culture flask reaches 2~3×10⁻⁶ 7 When the sample is taken from one bottle, the supernatant is collected as a conditioned medium. Each bottle yields approximately 40 mL. After sterilization by filtration through a 0.22 μm filter membrane, the sample is stored at -80°C for later use.

[0097] (II) Control group culture method

[0098] The control group used the classic neural stem cell suspension culture method, and the specific procedures are as follows:

[0099] Culture container selection: Use ultra-low adsorption culture flasks (Corning®, Catalog Number: 3814), which can prevent cells from adhering to the wall and maintain the suspension growth of neural stem cells.

[0100] Culture conditions control: Except for not adding the above 8 regulatory factors, the other culture conditions were completely consistent with those of the experimental group, including the composition of the basal culture medium, culture temperature (37℃), CO2 concentration (5%), culture cycle and replenishment time.

[0101] (III) Preparation of extracellular vesicles by ultrafiltration

[0102] Sample pretreatment: Collect the supernatant of the neural stem cell culture from the adherent culture (N5-NSC-EV group) and suspension culture (classic NSC-EV group). First, centrifuge at 4℃ and 300×g for 10 min to remove intact cells and collect the supernatant. Then, centrifuge at 4℃ and 2000×g for 20 min to remove cell debris and large particulate impurities and collect the supernatant again for later use.

[0103] Ultrafiltration equipment preparation: Select ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 kDa. Before use, rinse the ultrafiltration membrane three times with pre-cooled sterile PBS solution. After each rinse, add 5 mL of PBS and centrifuge at 4°C and 3000×g for 5 min to ensure that the membrane channels are unobstructed and to remove any impurities that may be present on the membrane surface.

[0104] Fractional ultrafiltration concentration: Slowly add the pretreated culture supernatant to the rinsed ultrafiltration centrifuge tubes, with the amount added to each tube not exceeding the maximum capacity of the ultrafiltration tube (usually 15 mL). After tightening the cap, place the tubes in a refrigerated centrifuge and centrifuge at 4°C and 3000×g for 15-20 min. After centrifugation, discard the filtrate and retain the concentrate in the upper chamber of the ultrafiltration tube, concentrating it to 1-2 mL.

[0105] Washing and purification: Add 5 mL of pre-cooled sterile PBS solution to the concentrate in the upper chamber of the ultrafiltration tube, gently invert to mix, then centrifuge at 4°C and 3000×g for 15 min, discard the filtrate, and repeat the washing step twice to remove residual culture medium components and small molecule impurities, thereby improving the purity of extracellular vesicles.

[0106] Collection and storage: After washing, transfer the remaining concentrate (i.e., the solution rich in extracellular vesicles) in the upper chamber of the ultrafiltration tube to a sterile EP tube. Place the collected extracellular vesicles in a -80°C freezer for later use, avoiding repeated freeze-thaw cycles.

[0107] III. Quality Identification of Extracellular Vesicles

[0108] (1) TEM morphological identification

[0109] Sample preparation: N5-NSC-EV and classic NSC-EV were diluted to appropriate concentrations with pre-cooled PBS solution. 5 μL of the diluted extracellular vesicle suspension was added to a copper grid coated with a 300-mesh carbon membrane and allowed to stand at room temperature for 5 minutes to allow for full adsorption of the extracellular vesicles. Excess liquid was then blotted with filter paper, and 0.5% uranyl acetate solution was added for negative staining. Staining was performed at room temperature in the dark for 10 minutes. After blotting the stain again with filter paper, the copper grid was allowed to air dry at room temperature.

[0110] Observations and Results: The dried copper mesh was placed in a TEM and observed and images were taken under an accelerating voltage of 80 kV.

[0111] The results are as follows Figure 4 The results showed that both N5-NSC-EV and classic NSC-EV exhibited typical extracellular vesicle morphological characteristics, with complete bilayer lipid membrane structures, and were round or oval vesicles. There were no significant differences between the two types of extracellular vesicles in terms of morphological integrity and membrane structure clarity, indicating that N5-NSC-EV prepared by different methods did not change the basic morphological characteristics of extracellular vesicles.

[0112] (2) Detection of particle size and particle concentration

[0113] Using the same cell source, different culture methods were employed to compare the differences in particle size and concentration of EVs produced by adherent culture and suspension culture methods. A nanoparticle tracking analyzer (NTA) was used to determine particle size distribution and concentration, and the distribution and number of particles with a diameter of 100-150 nm were observed.

[0114] Adherent culture methods yield higher extracellular vesicle yields compared to suspension culture methods, such as... Figure 5 As shown.

[0115] (3) Detection of characteristic markers

[0116] Membrane protein markers were used for identification: Western blotting (WB) was used to detect extracellular vesicle markers in NSC-EV cells to verify the basic characteristics of extracellular vesicles. Experimental data were analyzed using t-tests to differentiate between groups. Results are as follows: Figure 6 As shown, the CD63 expression level in the N5-NSC-EV group was significantly higher than that in the NSC-EV group (* P<0.05); the expression of CD9 and CD81 showed an increasing trend, with no statistical difference (P>0.05), indicating that the N5-NSC-EV group had more expression of characteristic proteins such as CD9 / CD81 / CD63.

[0117] In summary, this embodiment comprehensively confirmed, through TEM morphological identification, NTA particle size and concentration detection, and WB characteristic marker detection, that N5-NSC-EVs obtained by adherent culture possess the core characteristics of extracellular vesicles. Morphologically, N5-NSC-EVs are consistent with classic NSC-EVs, both possessing a complete lipid bilayer and round or elliptical vesicle structure. Functionally, they not only meet the extracellular vesicle standard in particle size distribution (target range of 100-150 nm), but also significantly improve the yield of extracellular vesicles compared to the classic suspension culture method, and enhance the expression of characteristic membrane proteins of extracellular vesicles such as CD9 / CD81 / CD63. The CD63 expression level in the N5-NSC-EV group was significantly higher than that in the NSC-EV group (*P<0.05). This demonstrates that the adherent culture method can optimize the yield and expression of marker proteins of extracellular vesicles without altering their basic morphological characteristics.

[0118] Example 3: The role of N5-NSC-EV in AD model mice (in vivo validation)

[0119] (1) Laboratory animals

[0120] To verify the effects of N5-NSC-EV extracellular vesicles and classic NSC-EV on Aβ protein deposition in APP / PS1 transgenic mice, APP / PS1 transgenic mice were selected as a commonly used model mouse for studying the pathological development mechanism of AD and drug development.

[0121] (2) Grouping situation

[0122] Table 8

[0123]

[0124] (3) Route of administration

[0125] Route of administration: Nasal administration

[0126] Dosage frequency and duration: Nasal administration once daily for one week.

[0127] 3.1 Nasal administration method

[0128] Anesthesia: Mice were placed in an induction chamber and anesthetized with isoflurane. The mouth and nose were covered with a mask to maintain the anesthesia. The mice were then fixed in a supine position.

[0129] Nasal cavity pretreatment: Before drug administration, the nasal cavity was treated with hyaluronidase (Sigma, catalog number H3884) at a concentration of 100 U / 10 μL, with 4 μL administered to each nasal cavity; after pretreatment, the nasal cavity was left to stand for 30 minutes before extracellular vesicle drug administration.

[0130] Administration: Attach a 0.5-0.7 cm long thin tube to the needle of a microsyringe for later use; adjust the extracellular vesicle concentration (N5-NSC-EV or classic NSC-EVs) to 5 × 10⁻⁶. 11 / mL, with a total dose of 4×10⁹ / mL administered bilaterally to each mouse's nasal cavity. 9 Each EV (total volume 8 μL) was administered. Mice were kept supine, and 4 μL of vesicle suspension was drawn up using a prepared microsyringe and slowly injected into the left nostril. This position was maintained for 5 minutes after injection. After a 15-minute interval, 4 μL of vesicle suspension was administered into the right nostril using the same method. After bilateral administration, the mice were kept in a supine anesthetized state for another 5 minutes before being returned to their cages.

[0131] Dosage frequency: Once daily for 7 consecutive days, as shown in Table 8.

[0132] (4) Sample collection and immunofluorescence staining

[0133] Mice administered isoflurane and deeply anesthetized until corneal reflex and limb voluntary movement cease. They are then rapidly placed in pre-cooled PBS solution (4°C) and immobilized in a supine position. The skin is incised along the midline of the mouse's skull to expose the skull. The skull is carefully cut open with ophthalmic scissors (avoiding damage to brain tissue), and the entire brain is completely dissected. The brain tissue is immediately rinsed twice in pre-cooled PBS solution (5 minutes each time to remove blood). The brain tissue is embedded in embedding gel and sectioned serially at 7 μm using a cryostat (LEICA-CM3050S). Sections are then attached to slides treated with anti-detachment agents and allowed to air dry at room temperature for 30 minutes before use.

[0134] The immunofluorescence staining procedure is as follows:

[0135] 1. Fixation and rinsing: The dried brain slices were fixed in 4% paraformaldehyde solution for 15 minutes, and then rinsed 3 times with 1×PBS buffer for 5 minutes each time.

[0136] 2. Antigen retrieval: Immerse the brain slices in citrate antigen retrieval solution (pH 6.0), place them in a microwave oven for antigen retrieval, heat to boiling on medium heat and maintain for 5 minutes, turn off the microwave oven and let cool naturally to room temperature, then rinse 3 times with 1×PBS, 5 minutes each time.

[0137] 3. Blocking: Add blocking solution containing 5% bovine serum albumin (BSA) and 0.3% Triton X-100 to the brain slices, and place them in a humidified chamber at room temperature for 1 hour to block nonspecific binding.

[0138] 4. Primary antibody incubation: Remove the blocking solution, add diluted anti-Aβ antibody 6E10 (Covance Catalog# SIG-39155, diluted with blocking solution at a ratio of 1:500), ensuring that the antibody evenly covers the brain slice, and incubate overnight at 4°C in a humidified chamber.

[0139] 5. Secondary antibody incubation: The next day, remove the brain slices from 4°C and allow them to warm to room temperature for 30 minutes. Rinse three times with 1×PBS for 5 minutes each time. After removing excess liquid, add diluted fluorescent secondary antibody Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (ThermoFisher catalog number A-31571, diluted with blocking buffer at a ratio of 1:1000). Incubate in a humidified chamber at room temperature in the dark for 1 hour.

[0140] 6. Mounting and observation: Aspirate excess liquid around the brain slice, add anti-fluorescence quenching mounting medium, cover with a coverslip to avoid air bubbles, and observe and acquire images under a fluorescence microscope.

[0141] The results are as follows Figure 7 As shown, the N5-NSC-EV group significantly reduced Aβ deposition compared to the classic NSC-EV group. Figure 8 As shown, based on the statistical results of the area and number of ABETA plaque deposits in the cortex and hippocampus, the N5-NSC-EV has better protective and repair effects than the classic NSC-EV.

[0142] Example 4: Quality control of extracellular vesicles of neural stem cells (detection by qPCR method)

[0143] I. Validation of multiple batches of samples

[0144] (1) Experimental objective

[0145] We developed and validated a qPCR detection method suitable for extracellular vesicle microRNAs, and sequentially tested the functional microRNAs of screened neural stem cell extracellular vesicles.

[0146] (2) Experimental grouping

[0147] In this embodiment, the sample was designed into two groups: the experimental group (N5-NSC-EV) and the control group (classic NSC-EV). Both groups were derived from the same cell source, differing only in their culture methods. Three samples were randomly selected from each group across multiple batches of the product for testing. Detailed sample information is shown in Table 9.

[0148] Table 9

[0149]

[0150] II. Experimental Methods

[0151] (1) Extraction of total RNA from extracellular vesicles

[0152] Extracellular vesicle concentration: Extracellular vesicles were concentrated using the BeyoExo™ Enhanced Cell Supernatant Extracellular Vesicle Extraction Kit (precipitation method); 1 mL of the extracellular vesicle sample to be concentrated was added to 190 μL of BeyoExo™ Enhanced Cell Supernatant Extracellular Vesicle Extraction Kit, and the mixture was pipetted and mixed; the mixture was incubated at 4°C for 2-4 hours or overnight; the mixture was centrifuged at 10,000 g at 4°C for 30 minutes; the supernatant was carefully aspirated and the precipitate was collected, which is the extracellular vesicle.

[0153] (2) Total RNA was extracted from extracellular vesicles using the TRIzol method.

[0154] Add 1 mL of Trizol to the concentrated extracellular vesicle precipitate and dissolve by repeated pipetting; add 200 μL of chloroform and mix thoroughly for 15 seconds (the sample is an emulsion, let stand for 2-3 minutes); centrifuge at 17000g, 2-8℃ for 15 minutes (after centrifugation, the sample separates into three layers: the upper layer is a colorless, transparent aqueous phase containing RNA, the middle layer is a protein precipitate, and the lower layer contains DNA and lipids); carefully transfer the upper colorless aqueous phase to a new 1.5 mL EP tube (avoiding aspirating the middle protein layer); add 10 μg of Glycogen to aid precipitation (-20℃, 30 minutes); add 500 μL of isopropanol and let stand at room temperature for 10 minutes; centrifuge at 17000g, 2-8℃ for 10 minutes, white speckled precipitate will be visible at the bottom, discard the supernatant; add 1 mL of precipitate to the tube. Mix 75% ethanol by vortexing; centrifuge at 17000g and 2-8℃ for 5 min, and discard the supernatant (this step can be repeated once); open the EP tube cap and let stand for 5 min to evaporate the ethanol; add 30-50 uL of DEPC water to redissolve the precipitate (if necessary, incubate at 55℃ for 10 min to fully dissolve).

[0155] (3) RNA concentration detection

[0156] The concentration and purity of the extracted RNA samples were detected using a micro spectrophotometer, and the average value was obtained after repeated testing. Qualified samples were used as reverse transcription templates for subsequent steps.

[0157] (4) MicroRNA reverse transcription

[0158] The principle of microRNA tailing-based quantitative PCR: Mature microRNAs are very short, typically only about 21 nt, and cannot be directly reverse transcribed using conventional methods. Therefore, a special reverse transcription method must be used. Tailing is suitable for broad-based screening of differentially expressed microRNAs. The principle of tailing-based quantitative PCR is as follows: Figure 9 As shown.

[0159] 4.1 Reverse transcription reaction system:

[0160] Add the reaction mixture shown in Table 10 below to the centrifuge tubes in an ice bath:

[0161] Table 10

[0162]

[0163] 4.2 Reverse transcription procedure:

[0164] Inactivate the enzyme by heating at 37℃ for 60 min, then heating at 85℃ for 5 min, and store at 4℃.

[0165] Note: It is recommended to dilute the obtained cDNA reaction solution 50 times before using it as the first gradient template for quantitative fluorescence detection.

[0166] Primer synthesis: Sangon Biotech (Shanghai) Co., Ltd.

[0167] The primer information for synthesizing the microRNA obtained through KEGG data analysis is shown in Table 11.

[0168] Table 11

[0169]

[0170] Prepare the qPCR reaction system on ice as shown in Table 12:

[0171] Table 12

[0172]

[0173] The qPCR reaction procedure is shown in Table 13:

[0174] Table 13

[0175]

[0176] 4.3 qPCR test results

[0177] In qPCR data analysis, the ΔCt value is the difference between the Ct values ​​of the target gene and the reference gene (ΔCt = Ct target gene - Ct reference gene). The Ct value represents the number of cycles when the fluorescence signal exceeds the threshold. The smaller the ΔCt value, the higher the expression level of the target gene.

[0178] The results are as follows Figure 10As shown, the levels of the five key microRNAs in multiple batches of N5-NSC-EV samples were consistently higher than those in classic NSC-EV, and the differences were statistically significant using a Student's t-test. This indicates that these five microRNAs can be used as key quality control indicators for detecting neural stem cell vesicles produced by the culture method of this invention, and that qPCR is also a suitable detection method.

[0179] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing highly active neural stem cell extravesicles, characterized in that, Includes the following steps: (1) Laminin521 coated culture flasks were used as the adherent culture system; (2) After the human neural stem cells were revived, they were placed in suspension culture medium at 37°C, 5% CO2 and saturated humidity for 7 days until the cells aggregated to form a neural sphere with a complete structure and clear boundaries. (3) The suspended neurospheres were inoculated into the adherent culture system, replaced with an adherent culture medium containing composite regulatory factors and cultured adherently. The total culture period was 14 days, during which time fluid was replenished in stages on the 4th, 7th and 10th days. (4) When the number of cells in the culture flask reaches 2~3×10 7 When the number of cells / bottle is reached, the supernatant is collected and purified by centrifugation, ultrafiltration and washing to obtain highly active neural stem cell extravesicles. In step (2), the suspension culture medium comprises: basal culture medium DMEM / F12, stock solution; Neurobasal TM Culture medium, mixed with DMEM / F12 at a volume ratio of 1:1; N2 cell culture additive, 0.5×; B27 serum-free additive, 1×; recombinant human basic fibroblast growth factor, 10 ng / mL; recombinant human leukemia inhibitory factor, 10 ng / mL; recombinant human epidermal growth factor, 20 ng / mL; non-essential amino acids, 1×; β-mercaptoethanol, 0.1 mM; In step (3), the adherent culture medium includes the suspension culture medium and the composite regulatory factor, which is composed of 1 mmol / L α-ketoglutarate, 10 mg / L insulin, 2 mg / L ethanolamine, 2.5 μg / L folic acid, 15 μM Y-27632, 0.8 μM A23187, 0.8 mM dbcAMP and 30 ng / mL BDNF.

2. The method for preparing highly active neural stem cell extravesicles according to claim 1, characterized in that, In step (1), laminin521 was used on 182 cm 2 The culture flasks were coated at a concentration of 5 μg / mL and incubated overnight in an incubator until the substrate evenly covered the bottom of the flask.

3. The method for preparing highly active neural stem cell extravesicles according to claim 1, characterized in that, In step (3), the amount of liquid replenished each time is 1 / 3 of the total volume of the adherent culture medium.

4. The method for preparing highly active neural stem cell extravesicles according to claim 1, characterized in that, In step (4), the centrifugation process includes: the supernatant is first centrifuged at 4°C and 300×g for 10 min to remove intact cells, and the supernatant is collected; then it is centrifuged at 4°C and 2000×g for 20 min to remove cell debris and large particulate impurities, and the supernatant is collected again; The ultrafiltration process includes: slowly adding the supernatant collected after centrifugation into the rinsed ultrafiltration centrifuge tube, with the amount added to each tube not exceeding the maximum capacity of the ultrafiltration tube; after tightening the cap, place the tube in a refrigerated centrifuge and centrifuge at 4°C and 3000×g for 15-20 min; after centrifugation, discard the filtrate and retain the concentrate in the upper chamber of the ultrafiltration tube. The washing and purification process includes: adding 5 mL of pre-cooled sterile PBS solution to the concentrate in the upper chamber of the ultrafiltration tube, gently inverting to mix, then centrifuging at 4°C and 3000×g for 15 min to discard the filtrate, and repeating this washing step twice.

5. A highly active neural stem cell extravesicle, characterized in that, The highly active neural stem cell extravesicles are prepared by the method described in any one of claims 1 to 4. They have a round or oval vesicle structure with an intact lipid bilayer membrane and are specifically enriched with five functional microRNAs: hsa-let-7c-5p, hsa-miR-135b-5p, hsa-miR-340-5p, hsa-miR-92b-3p, and hsa-miR-125b-5p.

6. The use of the highly active neural stem cell extravesicles of claim 5 in the preparation of a medicament for treating Alzheimer's disease.

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