Bioactivity-related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles as well as screening method and application of bioactivity-related characteristic marker

Abstract

The invention relates to a bioactivity-related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles and application of the bioactivity-related characteristic marker. The bioactivity-related characteristic marker of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles comprises one or a combination of more than two of a PSMB3 protein, an SAA4 protein, a VPS33B protein and an AKR7A2 protein. The invention provides a screening method of the characteristic markers, and particularly, the biological activity of the human umbilical cord mesenchymal stem cell source extracellular vesicles can be effectively judged by detecting the characteristic markers or the combination of the characteristic markers of the umbilical cord mesenchymal stem cell source extracellular vesicles. An evaluation means and an important reference are provided for product quality control, clinical transformation and the like based on the umbilical cord mesenchymal stem cell-derived small extracellular vesicles.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a bioactivity-related characteristic biomarker of small extracellular vesicles derived from umbilical cord mesenchymal stem cells, its screening method, and its application. Background Technology

[0002] Small extracellular vesicles (sEVs) are vesicles secreted by living cells, ranging in size from 30 to 200 nm, with a phospholipid bilayer membrane structure. These vesicles contain various bioactive substances derived from parental cells, such as nucleic acids, proteins, and metabolites, and play an important role in intercellular communication (R. Kalluri, et al. Science, 2020, 367, 6478). Stem cell-derived sEVs possess similar immunomodulatory and tissue regeneration capabilities to stem cells, representing one of the mechanisms of action in stem cell therapy. Stem cell-derived sEVs can reduce the risks of immune rejection and potential tumorigenesis associated with stem cell transplantation. They are non-cellular, non-tumorigenic, stable, easy to preserve, biocompatible, and readily cross the blood-brain barrier, making them a promising novel strategy for "cell-free" therapy (Bao, H., et al. Nat. Biomed. Eng, 2023).

[0003] Human umbilical cord mesenchymal stem cells (hUCMSCs) have advantages such as non-invasive collection, no ethical issues, high proliferation, and low immunogenicity. They can promote tissue repair and regulate immune responses, making them the first choice in cell therapy and regenerative medicine. However, after long-term implantation in the body, they may cause problems such as abnormal differentiation and tumor growth (Zhu, W., et al. Cell Cycle, 2011, 10(18), 3198–3207). Small extracellular vesicles derived from umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) inherit the therapeutic potential of their parent cells and have advantages such as high bioactivity, low immunogenicity, high safety, and easy storage. They are expected to become an alternative strategy for stem cell therapy and show unique advantages in clinical translational applications.

[0004] In recent years, scientific research and clinical translational applications of hUCMSCs-sEVs have developed rapidly. However, due to the lack of unified standards for the isolation, purification, characterization, and detection of sEVs, different vesicle preparation techniques result in differences in purity and yield, and extracellular vesicles from different stem cell lineages exhibit variations in passage stability. Furthermore, there is a lack of quality control evaluation methods. Currently, commonly used biomarkers for small extracellular vesicles, such as CD9, CD63, CD81, TSG101, and Alix, have been shown to exhibit high heterogeneity in composition and content among small extracellular vesicles from different cell sources. Therefore, it is essential to develop characteristic biomarkers for hUCMSCs-sEVs and establish a system for evaluating the bioactivity and passage stability of hUCMSCs-sEVs at different passages to promote the clinical application and translation of hUCMSCs-sEVs. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a bioactive biomarker for small extracellular vesicles derived from umbilical cord mesenchymal stem cells, along with its screening method and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a combination of biomarkers related to the bioactivity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells. These biomarkers include one or more of the following proteins: PSMB3, SAA4, VPS33B, and AKR7A2. Specifically, the expression levels of the protein biomarkers PSMB3, SAA4, and AKR7A2 increase with increasing bioactivity of the small extracellular vesicles derived from umbilical cord mesenchymal stem cells, while the expression level of the protein biomarker VPS33B decreases with increasing bioactivity of the small extracellular vesicles derived from umbilical cord mesenchymal stem cells.

[0008] This invention also provides a method for screening bioactive characteristic markers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells, characterized by comprising the following steps:

[0009] (1) Isolation and purification of small extracellular vesicles in culture medium of human umbilical cord mesenchymal stem cells of different generations;

[0010] (2) Perform proteomic analysis on the small extracellular vesicles obtained in step (1);

[0011] (3) Perform bioinformatics analysis on the proteomic data obtained in step (2) to screen for activity-related candidate protein biomarkers;

[0012] (4) The small extracellular vesicles obtained in step (1) were applied to the oxygen-glucose deprivation-reperfusion neural cell model to evaluate the cell viability of neural cells before and after vesicle treatment.

[0013] (5) Combine the bioinformatics analysis results of step (3) with the cell viability results of nerve cells after treatment with small extracellular vesicles from different generations in step (4) to screen for potential protein biomarkers related to the bioactivity of small extracellular vesicles from umbilical cord mesenchymal stem cells.

[0014] Furthermore, the method for separating small extracellular vesicles in step (1) includes one or more of the following methods that can achieve the separation of small extracellular vesicles: ultracentrifugation, polymer precipitation, and ultrafiltration. Among them, ultracentrifugation is to separate vesicles by centrifuging the sample with a centrifugal force of >100,000g for more than 70 minutes; polymer precipitation is to separate vesicles by using polymers such as polyethylene glycol based on hydrophobic interactions; and ultrafiltration is to separate vesicles using an ultrafiltration membrane with a pore size of 30-1000 nanometers.

[0015] The proteomic detection of small extracellular vesicles in step (2) refers to the process of extracting, denaturing, reducing, alkylating, and enzymatically digesting the small extracellular vesicle samples obtained in step (1) to obtain peptide samples, performing liquid chromatography-mass spectrometry analysis on the peptide samples, detecting the protein composition and protein expression levels in small extracellular vesicle samples of different generations, and obtaining proteomic data.

[0016] The liquid chromatography conditions were as follows: a C18 capillary column was used as the separation column; mobile phase A was water containing 0.1% by volume FA; mobile phase B was 80% acetonitrile and 20% water containing 0.1% by volume FA; the flow rate was 600 nL / min; a linear gradient was used: 3%-5% of phase B was increased to 90%-95% of phase B; and the elution time was 120 minutes.

[0017] The mass spectrometry employed a data-independent acquisition mode, with full MS scan resolution of 60,000 m / s from 400 to 1200 m / s, MS / MS scan resolution of 30,000 m / s, RFlens (%) of 50 m / s, and a separation window of 20 m / s.

[0018] Furthermore, in step (3), bioinformatics analysis is performed on the proteomic data.

[0019] First, DIANN software was used to search the mass spectrometry data to obtain the protein composition and intensity values ​​in the samples. Perseus was used to perform multiple t-tests on the mass spectrometry data. Differentially expressed proteins were screened based on p-values ​​less than 0.05. Cluster heatmaps and principal component analysis were then performed on the differentially expressed proteins according to their mass spectrometry intensities (https: / / www.bioinformatics.com.cn / ). Based on the grouping results of the cluster heatmaps and principal component analysis, vesicle samples of different generations were divided into three groups. Perseus was used to perform t-tests on the data between the two groups. Differentially expressed proteins were screened based on p-values ​​less than 0.05 and fold differences greater than 1.5. The intersection of the differentially expressed proteins from each group yielded activity-related candidate protein biomarkers.

[0020] Furthermore, in step (4), small extracellular vesicles are applied to an oxygen-glucose deprivation reperfusion neural cell model, and the cell survival rate is detected by the CCK-8 assay to evaluate cell viability before and after vesicle treatment.

[0021] The bioactivity-related characteristic markers described in this invention were applied to determine the bioactivity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells. Higher expression levels of PSMB3, SAA4, and AKR7A2 proteins in the vesicles indicated higher vesicle activity, while higher expression levels of VPS33B protein in the samples indicated lower vesicle activity.

[0022] The application of bioactivity-related characteristic biomarkers to assess the bioactivity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells includes the following steps:

[0023] (1) Isolation of small extracellular vesicles from umbilical cord mesenchymal stem cell culture medium. Isolation methods include ultracentrifugation, polymer precipitation, and ultrafiltration. Ultracentrifugation involves centrifuging the sample at a force >100,000g for more than 70 minutes to achieve vesicle separation. Polymer precipitation uses polymers such as polyethylene glycol to achieve vesicle separation based on hydrophobic interactions. Ultrafiltration uses an ultrafiltration membrane with a pore size of 30-1000 nanometers to separate vesicles.

[0024] (2) The expression levels of PSMB3, SAA4, AKR7A2, and VPS33B proteins in small extracellular vesicle samples were detected. Higher expression levels of PSMB3, SAA4, and AKR7A2 proteins indicate higher biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells; higher expression levels of VPS33B protein indicate lower biological activity. Methods for detecting protein expression levels included Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, luciferase labeling, and flow cytometry.

[0025] This invention isolates, purifies, and performs deep proteomic coverage analysis on small extracellular vesicles derived from different generations of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells), screening out a set of characteristic biomarkers—PSMB3, SAA4, AKR7A2, and VPS33B—to evaluate the activity of these vesicles. This overcomes the current quality control strategies that lack bioactivity and passage stability for umbilical cord mesenchymal stem cell-derived small extracellular vesicles. By detecting the expression levels of these four bioactivity-related protein biomarkers in vesicle samples, the bioactivity of umbilical cord mesenchymal stem cell-derived small extracellular vesicles can be assessed, providing an evaluation method and important reference for product quality control and clinical translation based on umbilical cord mesenchymal stem cell-derived small extracellular vesicles. Attached Figure Description

[0026] Figure 1 Screening process for bioactive biomarkers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells.

[0027] Figure 2 The particle size, particle number, purity, morphology, and commonly used biomarkers of small extracellular vesicles derived from different generations of umbilical cord mesenchymal stem cells were characterized. Among them, (A) is particle size, (B) is particle number, (C) is purity, (D) is morphology, and (E) is protein biomarker.

[0028] Figure 3 Proteomic detection and bioinformatics analysis of small extracellular vesicles derived from different generations of umbilical cord mesenchymal stem cells; including (A) total protein count, (B) cluster heatmap, (C) principal component analysis, (D) differentially expressed proteins (high-activity group P5, P7, P9, P11 vs low-activity group P19), (E) differentially expressed proteins (high-activity group P5, P7, P9, P11 vs medium-activity group P1, P3, P13, P15, P17), (F) differentially expressed proteins (medium-activity group vs low-activity group), (G) expression level of PSMB3 protein in different generations of small extracellular vesicles, (H) expression level of SAA4 protein in different generations of small extracellular vesicles, (I) expression level of VPS33B protein in different generations of small extracellular vesicles, and (J) expression level of AKR7A2 protein in different generations of small extracellular vesicles.

[0029] Figure 4 Cell viability of small extracellular vesicles derived from different generations of umbilical cord mesenchymal stem cells before and after application in an oxygen-glucose deprivation-reperfusion neural cell model.

[0030] Figure 5 Expression levels of bioactive biomarkers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells in different generations of umbilical cord mesenchymal stem cells. Detailed Implementation

[0031] Example 1: Isolation and purification of small extracellular vesicles from different generations of umbilical cord mesenchymal stem cells. Vesicles in umbilical cord mesenchymal stem cell culture medium were separated using an electric field-assisted tangential flow filtration device (Guoshan Hou, et al. Analytical Chemistry, 2024, 96(33), 13345-13351). The electric field-assisted tangential flow filtration device consisted of a sample container, a peristaltic pump (Longer Pump BT300-2J), two filter chambers, two control valves, two pressure gauges (Asmik MIK-Y190), a DC power supply (Maisheng MS-1001D), and a waste container connected to form a circulation path. Both filter chambers and the control valve are custom-made from medical-grade polyetheretherketone (PEEK) material using CNC machining. The filter chambers are assembled from two circular flanges. The upper flange has an outer diameter of 84 mm and a thickness of 20 mm, with a spiral groove (1.5 mm high, 3.5 mm wide) at the bottom for sample flow. The lower flange groove contains a perforated plate with an outer diameter of 43.5 mm, a thickness of 2.55 mm, a pore size of 1.0 mm, and a pore spacing of 3.0 mm. This perforated plate supports the membrane and allows waste liquid to flow out. An inorganic alumina (AAO) membrane (47 mm diameter, 0.02 μm pore size, Whatman) is located between the spiral groove and the perforated plate. The entire filter chamber is sealed with medical-grade silicone rubber gaskets and secured with stainless steel bolts. Copper electrode plates are installed on the outside of the upper and lower flanges, connected to a power source to provide an electric field within the filter chamber. A peristaltic pump head is connected to medical-grade silicone tubing (Biosicon) to pump the sample into the filter chamber and is connected to the entire system via PTFE tubing.

[0032] Vesicles in umbilical cord mesenchymal stem cell (UC-C) culture medium were separated using an electric field-assisted tangential flow filtration (EPCF) device. The specific steps were as follows: 20 ml of UC-C culture medium from passages 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 were taken, centrifuged at 500g for 5 min to remove dead cells, and then centrifuged at 4000g for 20 min to remove cell debris. Larger vesicles were removed by filtration through a 0.22 μm filter membrane. The medium was then diluted with 20 ml of PBS buffer. The diluted cell culture medium was then passed through the EPCF device using a peristaltic pump. Small extracellular vesicles were separated and purified based on the size sieving of the 20 nm ultrafiltration membrane and the electric field migration at 100 V. When 5 ml of liquid remained in the device, 10 ml of pre-cooled PBS buffer was added for further filtration. This process was repeated three times, and the vesicle fluid was recovered. Using an ultrafiltration tube with a molecular cutoff pore size of 100 kD, the vesicle fluid obtained above was concentrated to 200 μL to obtain small extracellular vesicle samples derived from umbilical cord mesenchymal stem cells of generations 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.

[0033] Example 2: Characterization of particle size, particle number, purity, morphology, and commonly used biomarkers of small extracellular vesicles derived from different generations of umbilical cord mesenchymal stem cells.

[0034] Small extracellular vesicles were diluted to 10 μL with PBS. 7 -10 9 The particle concentration and size distribution of small extracellular vesicles were determined using a NanoSight NS300 (Malvern, UK) equipped with a 488nm laser and a high-sensitivity sCMOS camera, within a concentration range of particles / mL. Each sample was analyzed three times, and the results were averaged. Figure 2 As shown in Figure A, most vesicles have a particle size distribution between 30 and 200 nanometers, which conforms to the standard size distribution of small extracellular vesicles. Figure 2 As shown in B, the total number of enriched particles in small extracellular vesicles from generations 1 to 19 ranged from 2.2 to 8.6 × 10⁻⁶. 11 The number of vesicles secreted by cells of different generations varies. Protein concentration was determined using the BCA (bicinchoninic acid) method. 5 μL of small extracellular vesicle samples from different generations of umbilical cord mesenchymal stem cells were taken, diluted 10-fold with PBS buffer, and the protein concentration was measured using a BCA protein concentration assay kit (Beyotime, P0011). The purity of the vesicle sample was determined by the ratio of the protein concentration obtained from BCA and the vesicle particle concentration obtained from NTA. Figure 2 As shown in B, the purity distribution of small extracellular vesicles is 2-3 × 10⁻⁶. 8 The enrichment stability and reproducibility are good within the particle / μg range.

[0035] The morphology of small extracellular vesicles was characterized using transmission electron microscopy (TEM). 10 μL of small extracellular vesicle fluid was dropped onto a copper grid and incubated at room temperature for 1 minute, after which excess fluid was aspirated. The small extracellular vesicles were negatively stained with phosphotungstic acid (2%, v / v) solution for 1 minute. The morphology of the small extracellular vesicles was then observed using a transmission electron microscope (TEM, Hitachi H-7650, Japan) at an accelerating voltage of 80 kV. Figure 2 As shown in D, small extracellular vesicles of different generations all exhibited a typical circular morphology between 30 and 200 nanometers, successfully enriching small extracellular vesicles of different generations.

[0036] Western blot was used to characterize small extracellular vesicles using commonly used protein markers (CD9, CD63, Alix, TSG101) and the negative control Calnexin. Small extracellular vesicle samples were mixed with 6× loading buffer (TransGen, DL101) and boiled at 95°C for 5 min. Samples were separated by 12.5% ​​SDS-PAGE (Epizyme, PG113) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was blocked in TBST containing 5% skim milk powder at room temperature for 1 h, and then incubated overnight at 4°C with primary antibody. The following antibodies were used for Western blot analysis: anti-CD9 (Abcam, ab263019), anti-CD63 (Abcam, ab134045), anti-Alix (Abcam, ab186429), anti-TSG101 (Abcam, ab125011), and anti-Calnexin (Abcam, ab133615). All primary antibodies were diluted 1:1,000. After incubation at room temperature for 30 minutes with HRP-conjugated anti-rabbit IgG secondary antibody (1:20,000, Invitrogen), imaging was performed using ultrasensitive enhanced chemiluminescence, and detection was performed by ChemiDoc XRS+ (Bio-Rad). Figure 2 As shown in E, protein markers could be identified in all small extracellular vesicles isolated from cell culture media of different passages, but Calnexin protein was not identified, proving that small extracellular vesicles of different passages were successfully isolated.

[0037] Example 3: Clustering and differential protein screening of extracellular vesicle proteomes from different generations of umbilical cord mesenchymal stem cells

[0038] Preparation of iodoacetic acid-functionalized silica spheres: Take 10g of silica microspheres (average particle size 5µm), add 80mL of concentrated hydrochloric acid (36-38% by mass), in an oil bath at 85℃ for 6h, wash with water until pH=7, wash three times with 50mL of anhydrous ethanol, and vacuum dry at 70℃; add 50mL of toluene and 0.75mL of methyl 3-trimethoxysilane-2-bromo-2-methylpropionate, in an oil bath at 90℃ for 12h, wash twice with 50mL of anhydrous toluene, wash six times with 50mL of anhydrous ethanol, and vacuum dry at 70℃; add 28mL of methanol, stir, and sonicate for 5min, add 3mL of glycidyl methacrylate and 32µL of N,N,N',N”,N”'-pentamethyldiethylenetriamine, purge with nitrogen for 5min, add 10mg of CuCl and 1.7mg of... CuCl2 was continuously purged with nitrogen for 5 minutes, refluxed under sealed conditions at 60°C for 6 hours, washed three times with 50 mL of methanol, twice with 50 mL of water, five times with 50 mL of saturated EDTA, three times with 50 mL of water, and three times with 50 mL of ethanol. The mixture was then dried under vacuum at 70°C. 100 mg of polyethyleneimine dissolved in 50 mL of H2O was added, and the mixture was placed in a water bath at 50°C for 6 hours. The mixture was washed with water until pH = 7, washed three times with 50 mL of anhydrous ethanol, and dried under vacuum at 70°C to obtain the final product.

[0039] Take 150 μg of small extracellular vesicle samples derived from umbilical cord mesenchymal stem cells of passages 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. Add 6 M guanidine hydrochloride solution, protease inhibitor (protein:protease inhibitor 1:100 (v / v)), and 10 mM tris(2-carboxyethyl)phosphine to a final concentration. Incubate at 95 °C for 5 min. After cooling the samples to room temperature, add 7 mg of iodoacetic acid-functionalized silica beads. Incubate in the dark at 40 °C with shaking at 1500 rpm for 2 h to allow the protein to fully bind to the silica beads. Centrifuge at 4000 g for 3 min and discard the supernatant. Wash once with 50 μL acetonitrile and twice with 50 μL 50 mM ammonium bicarbonate solution. Centrifuge at 4000 g for 3 min and discard the supernatant. Resuspend the silica beads in 50 μL 50 mM ammonium bicarbonate solution and add 6 μg of guanidine hydrochloride solution. Trypsin enzyme was used to hydrolyze the enzyme at 37°C with shaking at 1500 rpm for 12 hours. After hydrolysis, the mixture was centrifuged at 16,000 g for 10 minutes. The supernatant was collected, and the silica beads were washed twice with 50 μl of acetonitrile. The supernatants were combined, which yielded the final peptide fragments. The peptide solution was lyophilized and stored at -80°C.

[0040] Peptides were analyzed using an Orbitrap Exploris 480 mass spectrometer. Samples were packed with ReproSil-Pur C18-AQ (1.9 μm) medium. Separation was performed on a C18 capillary column (150 μm·d × 25 cm). Mobile phase A consisted of water containing 0.1% FA (fat per unit volume), and mobile phase B consisted of 80% acetonitrile and 20% water containing 0.1% FA. The flow rate was 600 nL / min, and the peptide separation gradients were 0–8 min (5–8% B), 8–48 min (8–20% B), 48–90 min (20–32% B), 90–108 min (32–45% B), and 108–120 min (95% B). Mass spectrometry was performed in a data-independent acquisition mode, with a full MS scan resolution of 60,000 m / s and an MS / MS scan resolution of 30,000 m / s. The RFlens (%) was 50, and the separation window was 20 m / s. The mass spectrometry data were analyzed using DIA.NN 1.8 software. Based on the human Swiss-Prot database (updated April 22, 2024), the mass deviation of the precursor ion was set to 10 ppm, the mass deviation of the fragment ion to 20 mmu, the number of missed cleavages by trypsin was set to 2, and the variable modifications were methionine oxidation and protein N-terminal acetylation. Peptide profile matching (PSM) and the false detection rate (FDR) of peptides and proteins were set to 0.01. Figure 3 As shown in Figure A, approximately 1500 proteins were identified in small extracellular vesicles derived from P1-P17, and 1214 proteins were identified in small extracellular vesicles derived from P19. Multiple t-tests were performed on the data using Perseus, and differentially expressed proteins were screened based on p-values ​​less than 0.05. The proteins were then plotted online using the MicroBio Information Network (MBIN) based on mass spectrometry intensity. https: / / www.bioinformatics.com.cn / ) Clustering heatmaps and principal component analysis were performed on the screened differentially expressed proteins, such as... Figure 3 As shown in B and C, P1-P19 can be divided into three groups: P5, P7, P9, P11; P1, P3, P13, P15, P17; and P19. A perceptual t-test was performed on the three groups. Differentially expressed proteins were selected based on a p-value less than 0.05 and a fold change greater than 1.5. Figure 3 As shown in D, P5, P7, P9, and P11 had a total of 533 differentially expressed proteins compared to P19, of which 379 proteins were upregulated and 154 proteins were downregulated. Figure 3 As shown in E, P5, P7, P9, and P11 showed a total of 110 differentially expressed proteins compared to P1, P3, P13, P15, and P17, with 51 proteins upregulated and 59 proteins downregulated. Figure 3 As shown in F, P1, P3, P13, P15, and P17 have a total of 515 differentially expressed proteins compared to P19, of which 363 proteins are upregulated and 152 proteins are downregulated. Taking the intersection of the differentially expressed proteins in the three groups yields 21 candidate protein biomarkers.

[0041] Example 4: Cell viability of small extracellular vesicles derived from different generations of umbilical cord mesenchymal stem cells before and after application in an oxygen-glucose deprivation-reperfusion neural cell model.

[0042] Human neuronal HPP cell lines were revived and cultured in adherent DMEM complete medium (Gibco) containing 10% FBS and 1% penicillin-streptomycin solution (100×) in a 37°C, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were harvested and counted at 5×10⁶ cells / year. 3 Cells were inoculated into 96-well plates with five replicates, labeled as control, oxygen-glucose deprivation (OGD) group, and oxygen-glucose deprivation plus vesicle treatment group. The 96-well plates were incubated at 37°C and 5% CO2 for 48 hours until complete adhesion. In the OGD group, DMEM complete medium was removed and replaced with glucose-free DMEM complete medium, and the cells were starved. The 96-well plates were then incubated at 37°C and 95% N2 and 5% CO2 for 6 hours. In the OGD plus vesicle treatment group, after the same OGD treatment, the medium was replaced with high-glucose DMEM complete medium, and the cells were reoxygenated at 37°C and 5% CO2 for 24 hours. Small extracellular vesicles derived from different passages of umbilical cord mesenchymal stem cells were then added and co-cultured for 24 hours. After reoxygenation, cell proliferation activity was detected using a CCK-8 assay kit (Beyotime, C0037). Figure 4 As shown, after oxygen-glucose deprivation treatment, HPP cell activity decreased. After the addition of small extracellular vesicles, HPP cell proliferation activity gradually increased with increasing passage number of small extracellular vesicles, reaching a peak at P11. Subsequently, HPP cell proliferation activity decreased with increasing passage number of small extracellular vesicles, demonstrating that vesicles derived from P5, P7, P9, and P11 have high biological activity, while those derived from P1, P3, P13, P15, and P17... The activity of P19-derived vesicles was weaker than that of vesicles derived from P5, P7, P9, and P11. Compared with small extracellular vesicles from other generations, P19-derived vesicles showed the weakest biological activity, consistent with the results of proteomics cluster analysis. Therefore, based on activity levels, small extracellular vesicles derived from P5, P7, P9, and P11 were designated as the high-activity group, those derived from P1, P3, P13, P15, and P17 as the medium-activity group, and those derived from P19 as the low-activity group.

[0043] Mass spectrometry intensity analysis was performed on the 21 candidate protein biomarkers obtained in Example 3, such as... Figure 3As shown in GJ, the expression levels of PSMB3, SAA4, and AKR7A2 increased with the increase of biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells, while the expression level of VPS33B decreased with the increase of biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells. Bioactivity-related characteristic markers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells were obtained through screening.

[0044] Example 5: Application of bioactivity-related characteristic biomarkers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells

[0045] 15 μg of protein was taken from each of the high-activity, medium-activity, and low-activity vesicle samples and mixed with 6× loading buffer (TransGen, DL101), then boiled at 95°C for 5 min. The samples were separated by 12.5% ​​SDS-PAGE (Epizyme, PG113) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was blocked in TBST containing 5% skim milk powder at room temperature for 1 h, and then incubated overnight at 4°C with primary antibody. The following antibodies were used for Western blot analysis: anti-PSMB3 (Proteintech, 15983-1-AP), anti-SAA4 (Abcam, ab92540), anti-AKR7A2 (Proteintech, 66677-1-Ig), anti-VPS33B (Proteintech, 12195-1-AP), and anti-GAPDH (Proteintech, 60004-1-Ig). All primary antibodies were diluted 1:1,000. After incubation with HRP-conjugated secondary antibodies for 30 minutes at room temperature, imaging was performed using ultrasensitive enhanced chemiluminescence and detected by ChemiDoc XRS+ (Bio-Rad). Figure 5 As shown, the expression levels of PSMB3, SAA4, and AKR7A2 decreased sequentially in the high-activity group, medium-activity group, and low-activity group, while the expression level of VPS33B increased.

Claims

1. Bioactivity-related characteristic markers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells, characterized by: The characteristic biomarkers include one or more of the following: PSMB3 protein, SAA4 protein, VPS33B protein, and AKR7A2 protein.

2. The bioactivity-related characteristic markers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells as described in claim 1, characterized in that: The expression levels of protein markers PSMB3, SAA4, and AKR7A2 increased with the increase in the biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells, while the expression level of protein marker VPS33B decreased with the increase in the biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells.

3. A method for screening bioactive characteristic markers of small extracellular vesicles derived from umbilical cord mesenchymal stem cells as described in claim 1 or 2, characterized in that, The method includes the following steps: (1) Isolation and purification of small extracellular vesicles in culture medium of human umbilical cord mesenchymal stem cells of different generations; (2) Perform proteomic analysis on the small extracellular vesicles obtained in step (1); (3) Perform bioinformatics analysis on the proteome data obtained in step (2) to screen for activity-related characteristic candidate protein biomarkers; (4) The small extracellular vesicles obtained in step (1) were applied to the oxygen-glucose deprivation-reperfusion neural cell model to evaluate the proliferation activity of neural cells before and after treatment with small extracellular vesicles. (5) Combine the bioinformatics analysis results of step (3) with the cell viability results of small extracellular vesicles from different generations in step (4) to screen for potential protein biomarkers related to the bioactivity of small extracellular vesicles from umbilical cord mesenchymal stem cells.

4. The method for screening characteristic markers as described in claim 3, characterized in that: The small extracellular vesicle separation methods in step (1) include ultracentrifugation, polymer precipitation, and ultrafiltration, which can achieve the separation of small extracellular vesicles. Among them, ultracentrifugation is to separate vesicles by centrifuging the sample with a centrifugal force of >100,000g for more than 70 minutes; polymer precipitation is to separate vesicles by using polymers such as polyethylene glycol based on hydrophobic interactions; and ultrafiltration is to separate vesicles using an ultrafiltration membrane with a pore size of 30-1000 nanometers.

5. The method for screening characteristic markers as described in claim 3, characterized in that: The proteomic detection of small extracellular vesicles in step (2) refers to the process of extracting, denaturing, reducing, alkylating, and enzymatically digesting the small extracellular vesicle samples obtained in step (1) to obtain peptide samples, performing liquid chromatography-mass spectrometry analysis on the peptide samples, detecting the protein composition and protein expression levels in small extracellular vesicle samples of different generations, and obtaining proteomic data.

6. The method for screening characteristic markers as described in claim 3, characterized in that: The step (3) involves performing bioinformatics analysis on the proteomic data. First, the DIANN software was used to search the library for the obtained mass spectrometry data to obtain the protein composition and intensity values ​​in the sample. Perseus was used to perform multiple t-tests on the mass spectrometry data. Differentially expressed proteins were screened based on p-values ​​less than 0.

05. Cluster heatmaps and principal component analysis were performed on the differentially expressed proteins using the Bioinformatics online plotting website (https: / / www.bioinformatics.com.cn / ) based on the mass spectrometry intensity. Based on the grouping results of the cluster heatmaps and principal component analysis, vesicle samples of different generations were divided into three groups. Perseus was used to perform t-tests on the data between the two groups. Differentially expressed proteins were screened based on p-values ​​less than 0.05 and fold differences greater than 1.

5. The intersection of the differentially expressed proteins in each group was used to obtain activity-related candidate protein biomarkers.

7. The method for screening characteristic markers as described in claim 3, characterized in that: In step (4), small extracellular vesicles are applied to an oxygen-glucose deprivation reperfusion neural cell model, and the cell survival rate is detected by the CCK-8 assay to evaluate cell viability before and after vesicle treatment.

8. The application of a bioactivity-related characteristic marker of small extracellular vesicles derived from umbilical cord mesenchymal stem cells as described in claim 1 or 2, characterized in that: One or more of the characteristic markers in claim 1 are used to determine the bioactivity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells. The higher the expression level of PSMB3, SAA4, and AKR7A2 proteins in the vesicles, the higher the vesicle activity. The higher the expression level of VPS33B protein in the sample, the lower the vesicle activity.

9. The application as described in claim 8, characterized in that, Includes the following steps: (1) Isolation of small extracellular vesicles from umbilical cord mesenchymal stem cell culture medium; (2) Detect the expression levels of one or more of the following proteins in small extracellular vesicle samples: PSMB3, SAA4, AKR7A2 and VPS33B. The higher the expression levels of PSMB3, SAA4 and AKR7A2 proteins, the higher the biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells. The higher the expression level of VPS33B protein, the lower the biological activity of small extracellular vesicles derived from umbilical cord mesenchymal stem cells.

10. The application as described in claim 9, characterized in that, The methods for separating small extracellular vesicles in step (1) include: ultracentrifugation, polymer precipitation, ultrafiltration and other methods that can separate small extracellular vesicles; The protein expression level detection methods in step (2) include Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, luciferase labeling, flow cytometry, and radioimmunoassay, which can all achieve protein expression level detection.

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