Application of non-inflammatory microvesicles secreted by macrophages induced by β-glucan in the treatment of inflammation

Abstract

The present invention provides the use of non-inflammatory microvesicles secreted by macrophages induced by β-glucan in the treatment of inflammation, relating to microvesicles secreted by macrophages induced by β-glucan, products that can be prepared from the microvesicles, and their uses. The microvesicles provided by the present invention can inhibit M1 polarization of macrophages and the secretion of inflammatory factors in cells, thereby reducing inflammation.

Description

Technical Field

[0001] The present invention relates to application of non-inflammatory microvesicles secreted by macrophages induced by beta-glucan in treating inflammation, and relates to the technical field of biomedicine. Background Art

[0002] Extracellular vesicles (EVs) are lipid-coated biological nanoparticles secreted by virtually all cells. EVs are membrane-bound vesicles surrounded by a lipid bilayer. EVs can be classified as exosomes, microvesicles, and apoptotic bodies based on their cellular protein markers, size, and derivatives. Microvesicles are approximately 100–1000 nm in diameter and secreted by living cells. They contain a rich array of proteins, lipids, and nucleic acids. Recently, it has been discovered that microvesicles, as naturally stable nanoscale vesicles, can penetrate biological barriers, protect their contents from degradation, and be efficiently taken up by recipient cells. They can serve as carriers for genes and drugs and participate in disease treatment.

[0003] Macrophages are innate immune cells and a crucial component of the immune system. They play a crucial role in maintaining homeostasis in the peritoneal cavity and defending against pathogen invasion. Under normal physiological conditions, peritoneal macrophages are in a quiescent state. They possess potent phagocytic functions, clearing apoptotic cells, bacteria, fungi, and other foreign matter from the peritoneal cavity. As research deepens, it has been discovered that peritoneal macrophages exhibit a high degree of heterogeneity and plasticity. Under different stimulation conditions, they can polarize into distinct phenotypes, such as the classically activated M1 type and the alternatively activated M2 type, exerting distinct functions.

[0004] Glucan, also known as β-glucan, is a polysaccharide composed of glucose molecules linked by glycosidic bonds. It is widely present in nature and has been found in microorganisms, plants, and animals. In the microbial field, certain bacteria and fungi can synthesize glucans as an important component of their cell walls, providing structural support and protection for the cells. For example, the β-glucan in yeast cell walls has a unique structure and biological activity. Glucan is also a common component in plants, such as in the cell walls of cereals, where it helps maintain the morphology and stability of plant cells. From a biological perspective, β-glucan plays an important role in regulating the immune system. However, existing technologies have not yet reported whether β-glucan can induce macrophage polarization and produce microvesicles, nor is it known whether it plays a role in regulating the immune system. Summary of the Invention

[0005] In order to make up for the deficiencies of the prior art, the present invention aims to provide a non-inflammatory microvesicle secreted by macrophages induced by β-glucan, and its application in treating inflammation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a microvesicle, wherein the microvesicle is produced by macrophages stimulated by β-glucan.

[0008] Furthermore, the size of the microvesicles ranges from 50 to 400 nm.

[0009] Furthermore, the surface markers of the microvesicles are HSP70, TSG101, CD63, CD9, and CD81 which are positive.

[0010] Furthermore, the macrophages include mouse macrophages and human macrophages.

[0011] Furthermore, the human macrophages are primary macrophages.

[0012] Furthermore, the human primary macrophages include macrophages derived from peripheral blood.

[0013] Furthermore, the mouse-derived macrophages include immortalized macrophages and primary macrophages.

[0014] Furthermore, the mouse-derived immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line.

[0015] Furthermore, the mouse primary macrophages include macrophages derived from pluripotent stem cells, macrophages derived from peripheral blood, macrophages derived from bone marrow, macrophages derived from peritoneal cavity, and macrophages derived from tumors.

[0016] Furthermore, the macrophages are J774A.1 cell line.

[0017] The second aspect of the present invention provides products prepared from the microvesicles of the first aspect of the present invention, including: 1) products that inhibit macrophage polarization; 2) products that inhibit the secretion of cellular inflammatory factors; and 3) products that reduce inflammation.

[0018] Furthermore, the products include pharmaceutical compositions and kits.

[0019] Furthermore, the inhibiting macrophage polarization includes inhibiting macrophage M1 polarization.

[0020] Furthermore, the inhibiting of macrophage polarization is inhibiting macrophage M1 polarization.

[0021] Furthermore, the inhibition of macrophage M1 polarization includes: 1) reducing the transcription of M1 macrophage-related genes; and / or 2) reducing the secretion of cellular inflammatory factors.

[0022] Furthermore, the cellular inflammatory factors include TNF-α, IL-6, iNOS, Arg-1, and CD206.

[0023] Furthermore, the macrophages include mouse macrophages and human macrophages.

[0024] Furthermore, the human macrophages are primary macrophages.

[0025] Furthermore, the human primary macrophages include macrophages derived from peripheral blood.

[0026] Furthermore, the mouse-derived macrophages include immortalized macrophages and primary macrophages.

[0027] Furthermore, the mouse-derived immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line.

[0028] Furthermore, the mouse primary macrophages include macrophages derived from pluripotent stem cells, macrophages derived from peripheral blood, macrophages derived from bone marrow, macrophages derived from peritoneal cavity, and macrophages derived from tumors.

[0029] Furthermore, the macrophages are primary mouse macrophages.

[0030] Furthermore, the macrophages are peritoneal-derived macrophages.

[0031] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0032] Furthermore, the manifestation of reducing inflammation includes: 1) reducing macrophage polarization; and / or 2) reducing the secretion of cellular inflammatory factors.

[0033] Furthermore, the cellular inflammatory factors include TNF-α, IL-6, iNOS, Arg-1, and CD206.

[0034] The third aspect of the present invention provides the use of the microvesicles described in the first aspect of the present invention in preparing the product described in the second aspect of the present invention, and / or in inhibiting macrophage polarization, inhibiting the secretion of cellular inflammatory factors, and alleviating inflammation.

[0035] A fourth aspect of the present invention provides a method for culturing the microvesicles according to the first aspect of the present invention, the method comprising adding β-glucan to a culture system of macrophages.

[0036] Furthermore, the macrophage culture system comprises DMEM complete culture medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin.

[0037] Furthermore, the β-glucan or its pharmaceutically acceptable salt has an action time of 24 hours.

[0038] The fifth aspect of the present invention provides a method for inhibiting macrophage polarization, inhibiting the secretion of cellular inflammatory factors or alleviating inflammation, which comprises adding the microvesicles described in the first aspect of the present invention to a macrophage culture system.

[0039] Furthermore, the macrophages include mouse macrophages and human macrophages.

[0040] Furthermore, the human macrophages are primary macrophages.

[0041] Furthermore, the human primary macrophages include macrophages derived from peripheral blood.

[0042] Furthermore, the mouse-derived macrophages include immortalized macrophages and primary macrophages.

[0043] Furthermore, the mouse-derived immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line.

[0044] Furthermore, the mouse primary macrophages include macrophages derived from pluripotent stem cells, macrophages derived from peripheral blood, macrophages derived from bone marrow, macrophages derived from peritoneal cavity, and macrophages derived from tumors.

[0045] Furthermore, the macrophages are primary mouse macrophages.

[0046] Furthermore, the macrophages are peritoneal macrophages.

[0047] Furthermore, the macrophage culture system comprises RPMI1640 complete culture medium.

[0048] Advantages and beneficial effects of the present invention: The microvesicles provided by the present invention can inhibit the M1 polarization of macrophages, inhibit the secretion of cellular inflammatory factors, and have the effect of alleviating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The figure shows the immunoblotting characterization of microvesicle proteins.

[0050] Figure 2 This is a characterization diagram of the size range of microvesicle nanoparticle tracking analysis. Figure 2 A in the figure is the Ctrl-EV size range diagram. Figure 2 Figure B shows the size range of Glucan-EVs.

[0051] Figure 3 Transmission electron microscopy (TEM) characterization of microvesicles. Figure 3 A in the figure is the result of Ctrl-EV electron microscopy. Figure 2 B in the figure is the electron microscopy result of Glucan-EV.

[0052] Figure 4 This is a graph showing the expression levels of inflammatory factors in the supernatant of Glucan-stimulated microvesicles and macrophages co-cultured by real-time fluorescence quantitative PCR. Figure 4 A in the figure is the expression level of TNF-α. Figure 4 B in the figure is the expression level of iNOS, Figure 4 C in the figure is the expression level of CD206, Figure 4 D in the figure is the expression level of Arg-1.

[0053] Figure 5 This is an ELISA test of the expression levels of inflammatory factors in the supernatant of co-cultured microvesicles derived from Glucan stimulation and macrophages. Figure 5 A in the figure is the expression level of TNF-α. Figure 5 B in the figure is the expression level of IL-6. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and do not constitute a limitation of the present invention in any way.

[0055] For the purposes of this invention, the term "microvesicle" refers to extracellular vesicles (EVs) with a diameter of 100 to 1000 nm, also known as microparticles. These are small vesicles that shed from the cell membrane following cell activation, damage, or apoptosis. They interact with cells through various mechanisms and exert diverse biological functions. The microvesicles described in the first aspect of the present invention encompass the entire bilayer membrane and all proteins, lipids, nucleic acids, and other substances contained within it.

[0056] In the present invention, the term "β-glucan" (Glucan) refers to a homopolysaccharide composed of glucose as a monosaccharide, with glucose units connected by glycosidic bonds, its CAS number is 9012-72-0, and its molecular formula is C 18 H 30 O 14 X2.

[0057] In the present invention, the term "immortalization" refers to the property of cells that acquire the ability to continue to grow and proliferate. Immortalized cells have unlimited proliferative growth, can be passaged for a long time, and are often accompanied by karyotype changes. Immortalized cell lines have great research significance in the fields of studying the growth and development mechanisms of organisms, regulating life activities, and diagnosing and treating diseases. In the context of the present invention, immortalization refers to immortalized macrophages of mouse origin, including commercial immortalized macrophages and self-induced immortalized macrophages. In a specific embodiment of the present invention, J774A.1 cells are a type of immortalized mouse mononuclear macrophages, which are taken from adult female BALB / CN mice with reticulum cell sarcoma, and are isolated from ascites produced during cell tumor induction therapy.

[0058] In the present invention, the term "primary" refers to cells obtained by digesting and dispersing animal tissue with trypsin or other proteases to obtain single cells, which are then grown in a culture dish. The cells retain the basic properties of the original cells and, if they are normal cells, still retain a diploid number. Cultured cells within the first to tenth generations are generally referred to as primary cell cultures. Commonly used primary cell cultures include tissue block culture and dispersed cell culture. In some embodiments, primary peritoneal macrophages can be obtained by flushing the peritoneal cavity with physiological saline or cell culture medium and then extracting the peritoneal lavage fluid.

[0059] In the present invention, the term "polarization" refers to the process by which macrophages change their phenotype under the influence of cytokines to enhance their ability to respond to changes in the microenvironment. This process is called macrophage polarization. Based on phenotype and function, macrophage polarization includes classically activated M1 macrophages and alternatively activated M2 macrophages. In specific embodiments of the present invention, the primary peritoneal macrophages obtained are mostly M1 macrophages.

[0060] For the purposes of this invention, the term "peritoneal macrophages" refers to macrophages extracted from the mouse peritoneal cavity. Macrophages naturally exist in the mouse peritoneal cavity, and most are free in the ascites, making them easy to obtain. Macrophages can be obtained by flushing the peritoneal cavity with saline or cell culture medium and then extracting the peritoneal lavage fluid. Peritoneal macrophages are typically cultured by intraperitoneal injection of 3% thioglycolate broth, a non-infectious inflammatory stimulus that promotes macrophage aggregation. Thioglycolate (thioglycolate) releases macrophages from the peripheral blood into the peritoneal cavity, thereby extracting more macrophages for subsequent culture.

[0061] In the present invention, the term "reduced" refers to a decrease in measurable lesions compared to before treatment, which persists for at least four weeks, with no new lesions appearing during that period. In some embodiments, the severity or duration of the patient's inflammatory manifestations is reduced, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the absence of the active ingredient of the present invention (e.g., the microvesicles of the first aspect of the present invention).

[0062] In the context of this invention, the term "inflammation" refers to the body's defensive response to irritation, manifested as redness, swelling, heat, pain, and functional impairment. Inflammation can be acute or chronic, and can be infectious or non-infectious. Inflammation as described herein includes inflammation caused by physical factors, chemical factors, biological factors, tissue necrosis, allergic reactions, and foreign bodies. Physical factors include high and low temperatures, mechanical trauma, ultraviolet light, and radiation. Chemical factors include both endogenous and exogenous chemicals. Endogenous chemicals include decomposition products of necrotic tissue and metabolic products that accumulate in the body under pathological conditions, such as urea; exogenous chemicals include strong acids, strong bases, strong oxidants, and mustard gas. Improper use of drugs and other biological agents can also cause inflammation. Biological factors include viruses, bacteria, rickettsiae, protozoa, fungi, spirochetes, and parasites. Tissue necrosis caused by any cause is a potential inflammatory factor. An allergic reaction occurs when the body's immune response is abnormal, leading to an inappropriate or excessive immune response, tissue damage, and inflammation. Examples include allergic rhinitis and glomerulonephritis. Foreign bodies, such as surgical sutures, silica crystals, or fragments of material, can remain in tissues and cause inflammation.

[0063] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, oral administration is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, pharmaceutically acceptable acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may be administered to the recipient by any route that reaches the target tissue.

[0064] The pharmaceutical composition of the present invention may also be used in combination with other drugs for treating, preventing, alleviating, and / or alleviating diseases related to myelin damage in the nervous system. These other compounds for treating, preventing, alleviating, and / or alleviating diseases related to myelin damage in the nervous system may be administered simultaneously with the main active ingredient (e.g., the microvesicles described in the first aspect of the present invention), or even administered simultaneously in the same composition. The other therapeutic compounds may also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient.

[0065] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids such as water, saline, glycerol and ethanol. Auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in such vehicles.

[0066] In the present invention, the term "culture system" refers to a system that simulates the in vivo environment (sterility, suitable temperature, pH, and specific nutritional conditions) in vitro, allowing cells to survive, grow, reproduce, and maintain their primary structures and functions. It typically includes culture medium, nutrients, cytokines, gas exchange, and supports (e.g., glass or plastic culture dishes and flasks). In the context of the present invention, a culture system refers to a liquid culture system that does not include gas exchange or supports.

[0067] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials and reagents used in the following examples, unless otherwise specified, are all commercially available. Simple modifications to the present invention made according to the essence of the present invention fall within the scope of protection claimed in the present invention.

[0068] Example 1 Extraction and identification of microvesicles

[0069] 1. Main instruments and reagents: Low-speed horizontal centrifuge was purchased from Thermo Scientific, USA; PCR instrument was purchased from Bio-RAD, USA; agarose electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; chemiluminescence analyzer was purchased from Shanghai Qinxiang Science Co., Ltd.; cell ultra-clean bench was purchased from Beijing Ruiwot Biotechnology Co., Ltd.; carbon dioxide cell culture incubator was purchased from Thermo Scientific, USA; microplate reader was purchased from Thermo Scientific, USA; high-speed refrigerated centrifuge was purchased from Thermo Scientific, USA; real-time fluorescence quantitative PCR instrument was purchased from Roche, USA. Thioglycollate fluid medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd., fetal bovine serum was purchased from Kangyuan Biotechnology Co., Ltd., RPMI1640 medium and DMEM medium were purchased from Sewell, 0.25% trypsin was purchased from Thermo Fisher Scientific, USA, 2 ´ Taq Pro UniversialSYBR qPCR Master Mix was purchased from Nanjing Novozymes, 5 ´ ABScript III RT Mix was purchased from ABclonal, TRIZOL was purchased from Thermo Fisher Scientific, USA, Glucan was purchased from MCE, and NaOH was a product of Sinopharm Group.

[0070] 2. Experimental Methods and Results

[0071] 1. J774A.1 cell culture: J774A.1 cells were purchased from Shanghai Meiyan Biotechnology Co., Ltd. They were cultured in complete DMEM supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin. The cells were cultured in a 37°C, 5% CO2 cell culture incubator, with a 1:2 subculture ratio daily.

[0072] 2. Extraction of Microvesicles Secreted by J774A.1 Cells: J774A.1 cells were cultured in six 10 cm cell culture dishes until confluency reached 80%-90%. Fresh DMEM complete medium was then replaced. Three dishes were stimulated with 10 μg / mL Glucan, while the remaining three dishes were treated with NaOH solution as a solvent control. Culture was continued for 24 h. The supernatant was collected and centrifuged at 300 g for 5 min at 4°C. The supernatant was transferred to a fresh centrifuge tube and centrifuged at 2000 g for 10 min at 4°C. The supernatant was centrifuged again and centrifuged at 15,000 g for 30 min at 4°C. The supernatant was discarded and the pellet was resuspended in 10 mL of PBS and centrifuged at 15,000 g for 30 min at 4°C. The supernatant was discarded and the pellet was resuspended in 1 mL of PBS and centrifuged at 15,000 g for 30 min at 4°C. The pellet was resuspended in an appropriate volume of PBS, aliquoted, and frozen at -80°C.

[0073] 3. Characterization and identification of microvesicles: Microvesicle markers (HSP70, TSG101, CD63, CD9, CD81) were detected by Western blot. Figure 1 As shown, HSP70: an obvious specific band was observed at the expected molecular weight of approximately 70 kDa; TSG101: an obvious specific band was observed at the expected molecular weight of approximately 45 kDa; CD63: an obvious specific band was observed at the expected molecular weight of approximately 53 kDa; CD81: an obvious specific band was observed at the expected molecular weight of approximately 26 kDa; CD9: an obvious specific band was observed at the expected molecular weight of approximately 24 kDa, among which the band of the Ctrl-EVs group was very weak.

[0074] Nanoparticle tracking analysis (NTA) measured the size range of Ctrl-EV and Glucan-EV, and the results were as follows Figure 2 As shown, Figure 2 A in the text is the size range of Ctrl-EV. Figure 2 B in the figure represents the size range of Glucan-EVs.

[0075] Transmission electron microscopy (TEM) was used to visualize the morphology of Ctrl-EV and Glucan-EV, and the microvesicles were characterized and analyzed. Figure 3 As shown, the microvesicles secreted by J774A.1 cells were spherical and enclosed within a double membrane.

[0076] Example 2 Anti-inflammatory effect of microvesicles

[0077] 1. Experimental Materials

[0078] C57BL / 6N mice were purchased from Weitonglihua and housed in the SPF animal facility of the Military Medical Research Institute of the Academy of Military Sciences. They were provided with free access to food and water and maintained on a 12-h light and dark cycle. Six- to eight-week-old mice of the same sex were used for the experiments.

[0079] 2. Experimental Methods and Results

[0080] 1. Isolation and Culture of Peritoneal Macrophages: Mice were intraperitoneally injected with 1 mL of 3% thioglycolate broth once a day for 3 consecutive days and then sacrificed by cervical dislocation. Under sterile conditions, the abdominal skin of the mice was cut open to expose the peritoneum. The needle of the syringe was inserted into the subcutaneous space and into the peritoneal cavity with the right hand. 5-10 mL of pre-chilled 1° PBS was injected into the peritoneal cavity and the abdomen was massaged for 5 minutes. The xiphoid process was lifted with sterile forceps and a small incision was made there. The peritoneal lavage fluid was repeatedly flushed with a 1 mL pipette and the lavage fluid was collected into a test tube. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the test tube was flicked, and the cells were resuspended in 1 mL of complete RPMI1640 medium. 10 mL of the cell suspension was taken for cell counting. The cell suspension was diluted to 2-4°C / 10 6 / mL, 500 mL per well was seeded in a 24-well plate, and the plate was placed in a cell incubator at 37°C and 5% CO2 for 4-6 h. The non-adherent cells were washed with PBS, and the remaining adherent cells were peritoneal macrophages.

[0081] 2. Co-culture of microvesicles with macrophages: Discard the culture medium, wash once with 1´ PBS, add fresh RPMI1640 complete medium, and add EVs at final concentrations of 5, 10, and 20 mg / mL (Ctrl-EVs, EVs extracted from J774A.1 cells after 24 h of NaOH stimulation; Glucan-EVs, EVs extracted from J774A.1 cells after 24 h of Glucan stimulation). Place the 24-well plate in a cell incubator at 37°C, 5% CO2 for 24 h.

[0082] 3. Real-time fluorescence quantitative PCR: To extract RNA from the microvesicle-macrophage co-culture system, TRIZOL was used to extract RNA. Reverse transcription was performed using the ABclonal reverse transcription kit, and quantitative PCR was performed using Novozyme reagents. The expression of iNOS, TNF-a, Arg-1, CD206, and GAPDH genes was quantified using a Roche LightCycler® 480 real-time fluorescence quantitative PCR instrument. −ΔΔCt The relative expression of genes was calculated using the method. Figure 4 As shown, real-time fluorescence quantitative PCR showed that compared with the addition of control vesicles, the expression levels of M1 polarization-related indicators, such as TNF-a and iNOS genes, were lower in the microvesicle system stimulated by Glucan ( Figure 4 A and B in the figure), M2 polarization indicators, CD206 and Arg-1 expression levels are higher ( Figure 4 C and D in the figure), but the levels of the two groups were lower than those of the blank control group.

[0083] 4. ELISA cytokine test: The supernatant of the co-culture of microvesicles and peritoneal macrophages was collected for cytokine testing. Cytokines (TNF-a, IL-6, IL-10) were detected using ELISA kits. For specific steps, refer to the Invitrogen ELISA kit instructions. The expression levels of TNF-a and IL-6 in the supernatant of the co-culture of peritoneal macrophage microvesicles and macrophages were detected by ELISA. The results are as follows: Figure 5 As shown, compared with the addition of control vesicles, the expression levels of TNF-a and IL-6 in the supernatant of Glucan-stimulated microvesicles were very low ( Figure 5 A and B in the sentence are almost not expressed.

[0084] 5. Statistical analysis: The experimental results were expressed as x ± s, and the data were statistically analyzed using GraphPad Prism 8 software. One-way analysis of variance was used for comparisons between multiple groups of samples, and two independent sample comparisons were performed using two independent sample comparisons. t test, P A value < 0.05 was considered statistically significant.

[0085] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. A method for preparing microvesicles, characterized in that: The method comprises: culturing J774A.1 cells in a culture system until the confluence reaches 80-90%, replacing with fresh DMEM complete medium, adding 10 μg / ml β-glucan, culturing for 24 hours, collecting the supernatant, centrifuging at 300g and 4°C for 5 minutes; taking the supernatant, centrifuging at 2000g and 4°C for 10 minutes; taking the supernatant and repeating the centrifugation once; taking the supernatant, centrifuging at 15,000g and 4°C for 30 minutes, and discarding the supernatant; resuspending the precipitate in 10 mL of PBS, and centrifuging at 15,000g and 4°C for 30 minutes; discarding the supernatant, resuspending the precipitate in 1 mL of PBS, and centrifuging at 15,000g and 4°C for 30 minutes to obtain microvesicles; The size of the microvesicles ranges from 50 to 400 nm; The surface markers of the microvesicles are HSP70, TSG101, CD63, CD9, and CD81.

2. The method according to claim 1, characterized in that The culture system contains DMEM complete medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin.

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