CN-121995056-A - Biological activity related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, screening method and application thereof

CN121995056ACN 121995056 ACN121995056 ACN 121995056ACN-121995056-A

Abstract

The invention relates to a biological activity related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles and application thereof. The biological activity related characteristic markers of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles comprise one or more than two of PSMB3 protein, SAA4 protein, VPS33B protein and AKR7A2 protein. The invention provides a screening method of the characteristic markers, in particular to the screening method, which can effectively judge the bioactivity of the human umbilical cord mesenchymal stem cell-derived small extracellular vesicles by detecting the characteristic markers of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles or the combination thereof, and provides an evaluation means and important references for quality control, clinical transformation and the like of products based on the umbilical cord mesenchymal stem cell-derived small extracellular vesicles.

Inventors

ZHANG LIHUA
LIU JING
Hou Guopan
LIU LUKUAN
YUAN HUIMING
WANG FENGYA
ZHAO BAOFENG
GUAN XIN

Assignees

中国科学院大连化学物理研究所
大连医科大学附属第一医院

Dates

Publication Date: 20260508
Application Date: 20241105

Claims (10)

1. A biological activity-related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, characterized in that: The characteristic marker comprises one or more than two of PSMB3 protein, SAA4 protein, VPS33B protein and AKR7A2 protein.
2. The method of claim 1, wherein the protein markers PSMB3, SAA4 and AKR7A2 are expressed at an increased level with the increase of the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles, and the protein marker VPS33B is expressed at a decreased level with the increase of the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles.
3. A method for screening for a marker characteristic of biological activity of umbilical cord mesenchymal stem cell-derived small extracellular vesicles according to claim 1 or 2, comprising the steps of: (1) Separating and purifying small extracellular vesicles in the culture solution of the umbilical cord mesenchymal stem cells of different generations; (2) Performing proteome detection on the small extracellular vesicles obtained in the step (1); (3) Performing bioinformatics analysis on the proteome data obtained in the step (2), and screening characteristic candidate protein markers related to activity; (4) Applying the small extracellular vesicles obtained in the step (1) to an oxygen glucose deprivation reperfusion nerve cell model, and evaluating proliferation activity of nerve cells before and after the small extracellular vesicles are treated; (5) Combining the bioinformatics analysis result of the step (3) with the cell viability result after the small extracellular vesicles from different generations in the step (4) are treated, and screening to obtain the potential protein markers related to the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles.
4. A method of screening for a characteristic marker according to claim 3, wherein: The small extracellular vesicles separation method in the step (1) comprises an ultracentrifugation method, a polymer precipitation method, an ultrafiltration method and the like, wherein the ultracentrifugation method is a method capable of realizing vesicle separation by centrifuging a sample for more than 70 minutes by using a centrifugal force of more than 100,000g, the polymer precipitation method is a method for realizing vesicle separation by using polymers such as polyethylene glycol based on hydrophobic interaction, and the ultrafiltration method is a method for separating vesicles by using an ultrafiltration membrane with a pore diameter of 30-1000 nanometers.
5. The method for screening a characteristic marker according to claim 3, wherein the step (2) of proteomic detection of small extracellular vesicles is performed by subjecting the small extracellular vesicle sample separated in the step (1) to protein extraction, denaturation, reduction, alkylation and enzymolysis to obtain a peptide fragment sample, and subjecting the peptide fragment sample to liquid chromatography-mass spectrometry to detect the protein composition and the protein expression level in different generations of small extracellular vesicle samples, thereby obtaining proteomic data.
6. The method for screening a characteristic marker according to claim 3, wherein the proteomic data in the step (3) is analyzed bioinformatically, The method comprises the steps of searching a library for obtained mass spectrum data by DIANN software to obtain protein composition and intensity values in samples, performing multi-group t-test on the mass spectrum data by Perseus, screening differential proteins based on p values smaller than 0.05, drawing a cluster heat map and principal component analysis on the differential proteins according to mass spectrum intensity at a micro-organism information on-line drawing website (https:// www.bioinformatics.com.cn /), dividing different generation secondary vesicle samples into 3 groups according to grouping results of the cluster heat map and principal component analysis, performing t-test on data between the two groups by Perseus, screening the differential proteins based on p values smaller than 0.05 and difference multiples larger than 1.5, and acquiring intersections of the differential proteins of each group to obtain activity related candidate protein markers.
7. The method of screening for a characteristic marker according to claim 3, wherein the small extracellular vesicles in the step (4) are applied to an oxygen-deprived reperfusion nerve cell model, and the viability of the cells is measured by CCK-8 assay to evaluate the viability of the cells before and after the vesicle treatment.
8. The application of the characteristic markers related to the biological activity of umbilical cord mesenchymal stem cell-derived small extracellular vesicles according to claim 1 or 2 is characterized in that one or more than two of the characteristic markers in claim 1 are applied to judging the biological activity of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, the higher the expression level of PSMB3, SAA4 and AKR7A2 proteins in the vesicles is, the higher the vesicle activity is, and the higher the expression level of VPS33B proteins in a sample is, the lower the vesicle activity is.
9. The use according to claim 8, comprising the steps of: (1) Separating small extracellular vesicles from umbilical cord mesenchymal stem cell culture solution; (2) Detecting the expression level of one or more than two of PSMB3, SAA4, AKR7A2 and VPS33B proteins in a small extracellular vesicle sample, wherein the higher the expression level of the PSMB3, SAA4 and AKR7A2 proteins is, the higher the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicle is, and the higher the expression level of the VPS33B protein is, the lower the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicle is.
10. The method according to claim 9, wherein the separation of small extracellular vesicles in step (1) comprises an ultracentrifugation method, a polymer precipitation method, an ultrafiltration method, etc. which can achieve the separation of small extracellular vesicles; The protein expression level detection method in the step (2) comprises Western blotting, enzyme-linked immunosorbent assay, mass spectrometry, luciferase labeling technology, flow cytometry, radioimmunoassay and the like.

Description

Biological activity related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, screening method and application thereof Technical Field The invention belongs to the technical field of biology, and relates to a biological activity related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, a screening method and application thereof. Background Small extracellular vesicles (small extracellularvesicles, sEVs) are vesicles secreted by living cells with a size between 30-200nm and having a phospholipid bilayer membrane structure. Extracellular vesicles contain a variety of biologically active substances from the parent cell, such as nucleic acids, proteins, metabolites, etc., and play an important role in intercellular communication (r.kalluri, et al science,2020,367,6478). Among them, the stem cell-derived extracellular vesicles have similar immune regulation and tissue regeneration ability as stem cells, and are one of the action mechanisms of stem cell therapy. The stem cell-derived extracellular vesicles can reduce the risks of immune rejection, potential tumorigenicity and the like during stem cell transplantation, have the advantages of no cell structure, no tumorigenicity, stable performance, easy preservation, good biocompatibility, easy passage through blood brain barrier and the like, and are a novel strategy of 'cell-free' treatment with great prospect (Bao, H., et al Nat. Biomed. Eng, 2023). Human umbilical mesenchymal stem cells (human umbilical cord MESENCHYMAL STEM CELLS, huchmscs) have the advantages of non-invasive collection, no ethical problem, high proliferation, low immunogenicity and the like, can promote tissue repair, regulate immune response, are the first choice in the fields of cell therapy and regenerative medicine, but after being implanted in the body for a long time, can have the problems of abnormal differentiation, tumor growth and the like (Zhu, w., et al cell Cycle,2011,10 (18), 3198-3207). Umbilical mesenchymal stem cell-derived small extracellular vesicles (hUCMSCs-sEVs) inherit the therapeutic potential of parent cells, have the advantages of high bioactivity, low immunogenicity, high safety, easiness in storage and the like, are expected to become alternative strategies for stem cell treatment, and show unique advantages in clinical transformation application. In recent years, scientific research and clinical transformation application of hUCMSCs-sEVs are rapidly developed, but the existing sEVs separation and purification, characterization detection and the like still lack unified standards, different vesicle preparation technologies have differences in purity and yield, and different generation stem cell-derived extracellular vesicles have passage stability differences and lack quality control evaluation means. The common markers CD9, CD63, CD81, TSG101, alix currently directed against small extracellular vesicles demonstrate a high degree of heterogeneity in composition and content in small extracellular vesicles of different cell origin. Therefore, the characteristic markers aiming at the hUCMSCs-sEVs must be developed, and the biological activity and passage stability evaluation system of the hUCMSCs-sEVs of different generations is established to promote the clinical application and transformation of the hUCMSCs-sEVs. Disclosure of Invention In order to solve the problems, the invention aims to provide a biological activity related characteristic marker of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, and a screening method and application thereof. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the invention provides a biological activity related characteristic marker combination of umbilical cord mesenchymal stem cell-derived small extracellular vesicles, which comprises one or more than two of PSMB3 protein, SAA4 protein, VPS33B protein and AKR7A2 protein. Wherein, the expression level of the protein markers PSMB3, SAA4 and AKR7A2 is increased along with the increase of the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles, and the expression level of the protein marker VPS33B is reduced along with the increase of the biological activity of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles. The invention also provides a screening method of the bioactive characteristic marker of the umbilical cord mesenchymal stem cell-derived small extracellular vesicles, which is characterized by comprising the following steps of: (1) Separating and purifying small extracellular vesicles in the culture solution of the umbilical cord mesenchymal stem cells of different generations; (2) Performing proteome detection on the small extracellular vesicles obtained in the step (1); (3) Performing bioinformatics analysis on the proteome