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

Abstract

The invention provides application of non-inflammatory microvesicles secreted by beta-glucan-induced macrophages in treatment of inflammation, and relates to microvesicles secreted by beta-glucan-induced macrophages, products capable of being prepared from the microvesicles and application of the microvesicles. The microvesicle provided by the invention can inhibit M1 polarization of macrophages and secretion of cell inflammatory factors, and has the effect of relieving inflammation.

Description

Technical Field

[0001] The 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 that are secreted by almost all cells. EVs are membrane-like vesicles surrounded by a lipid bilayer. EVs can be divided into exosomes, microvesicles, and apoptotic bodies based on their cell protein markers, size, and derivatives. Microvesicles are EVs with a diameter of about 100 to 1000 nm secreted by living cells, which contain rich proteins, lipids, and nucleic acids. In recent years, it has been discovered that microvesicles, as naturally stable nanoscale vesicles, can penetrate biological barriers, protect their contents from degradation, and are efficiently taken up by receptor cells. They can be used as carriers of genes and drugs and participate in disease treatment.

[0003] Macrophages are innate immune cells and an important component of the body's immune system. They play an important role in maintaining the homeostasis of the peritoneal environment and resisting the invasion of pathogens. Under normal physiological conditions, peritoneal macrophages are in a resting state. They have a strong phagocytic function and can clear apoptotic cells, bacteria, fungi and other foreign bodies in the peritoneal cavity. With the deepening of research, it has been found that peritoneal macrophages have a high degree of heterogeneity and plasticity. Under different stimulation conditions, they can polarize into different phenotypes, such as the classically activated M1 type and the alternatively activated M2 type, and play different functions.

[0004] Glucan, also known as β-glucan, is a polysaccharide composed of glucose molecules connected by glycosidic bonds. It is widely present in nature and is found in microorganisms, plants and animals. In the field of microorganisms, some bacteria and fungi can synthesize glucan as an important component of their cell walls, providing structural support and protection for cells. For example, b-glucan in yeast cell walls has a unique structure and biological activity. In plants, glucan is also a common component, such as in the cell walls of grains, which helps maintain the morphology and stability of plant cells. From the perspective of biological function, β-glucan plays an important role in the regulation of the immune system. The prior art has not reported whether β-glucan can induce macrophage polarization and produce microvesicles, nor is it known whether it plays a role in the regulation of the immune system. Summary of the invention

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

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a microvesicle, wherein the microvesicle is produced by macrophages stimulated by β-glucan.

[0007] Furthermore, the size of the microvesicles ranges from 50 to 400 nm.

[0008] Furthermore, the surface markers of the microvesicles are positive: HSP70, TSG101, CD63, CD9, and CD81.

[0009] Furthermore, the macrophages include mouse macrophages and human macrophages.

[0010] Furthermore, the human macrophages are primary macrophages.

[0011] Furthermore, the human primary macrophages include macrophages derived from peripheral blood.

[0012] Furthermore, the mouse-derived macrophages include immortalized macrophages and primary macrophages.

[0013] Furthermore, the mouse-derived immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line.

[0014] 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.

[0015] Furthermore, the macrophages are J774A.1 cell line.

[0016] The second aspect of the present invention provides products prepared by the microvesicles of the first aspect of the present invention, and the products include: 1) products that inhibit macrophage polarization; 2) products that inhibit the secretion of cellular inflammatory factors; 3) products that reduce inflammation.

[0017] Furthermore, the products include pharmaceutical compositions and kits.

[0018] Furthermore, the inhibiting of macrophage polarization includes inhibiting macrophage M1 polarization.

[0019] Furthermore, the inhibition of macrophage polarization is inhibition of macrophage M1 polarization.

[0020] 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.

[0021] Furthermore, the cellular inflammatory factors include TNF-α, IL-6, iNOS, Arg-1, and CD206.

[0022] Furthermore, the macrophages include mouse macrophages and human macrophages.

[0023] Furthermore, the human macrophages are primary macrophages.

[0024] Furthermore, the human primary macrophages include macrophages derived from peripheral blood.

[0025] Furthermore, the mouse-derived macrophages include immortalized macrophages and primary macrophages.

[0026] Furthermore, the mouse-derived immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line.

[0027] 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.

[0028] Furthermore, the macrophages are primary mouse macrophages.

[0029] Furthermore, the macrophages are peritoneal macrophages.

[0030] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0031] Furthermore, the manifestation of reducing inflammation includes: 1) reducing macrophage polarization; and / or 2) reducing the secretion of cellular inflammatory factors.

[0032] Furthermore, the cellular inflammatory factors include TNF-α, IL-6, iNOS, Arg-1, and CD206.

[0033] 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.

[0034] The fourth aspect of the present invention provides a method for culturing the microvesicles described in the first aspect of the present invention, the method comprising adding β-glucan to the culture system of macrophages.

[0035] 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 liquid.

[0036] Furthermore, the β-glucan or its pharmaceutically acceptable salt has an action time of 24 hours.

[0037] The fifth aspect of the present invention provides a method for inhibiting macrophage polarization, inhibiting the secretion of cellular inflammatory factors or alleviating inflammation, the method comprising adding the microvesicles described in the first aspect of the present invention to a macrophage culture system.

[0038] Furthermore, the macrophages include mouse macrophages and human macrophages.

[0039] Furthermore, the human macrophages are primary macrophages.

[0040] Furthermore, the human primary macrophages include macrophages derived from peripheral blood.

[0041] Furthermore, the mouse-derived macrophages include immortalized macrophages and primary macrophages.

[0042] Furthermore, the mouse-derived immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line.

[0043] 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.

[0044] Furthermore, the macrophages are primary mouse macrophages.

[0045] Furthermore, the macrophages are peritoneal macrophages.

[0046] Furthermore, the macrophage culture system comprises RPMI1640 complete culture medium.

[0047] 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

[0048] Figure 1 The figure shows the immunoblotting characterization of microvesicle proteins.

[0049] 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 is a diagram showing the size range of Glucan-EVs.

[0050] Figure 3 This is the transmission electron microscopy (TEM) characterization picture 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.

[0051] Figure 4 This is an ELISA test of the expression level of inflammatory factors in the supernatant of co-cultured microvesicles and macrophages derived from Glucan stimulation. Figure 4 A in the figure is the expression level of TNF-α. Figure 4 B in the figure is the expression level of IL-6.

[0052] Figure 5 This is a graph showing the expression level of inflammatory factors in the supernatant of co-cultured microvesicles and macrophages stimulated by Glucan by real-time fluorescence quantitative PCR. Figure 5 A in the figure is the expression level of TNF-α. Figure 5 B in the figure is the expression level of iNOS. Figure 5 C in the figure is the expression level of CD206. Figure 5 D in the figure is the expression level diagram of Arg-1. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0054] In the present invention, the term "microvesicle" refers to an extracellular vesicle with a diameter of 100-1000 nm, also known as a microparticle, which is a small vesicle shed from the cell membrane after cell activation, damage or apoptosis, and can interact with cells through different mechanisms to exert different biological functions. The microvesicle described in the first aspect of the present invention is a whole including a double membrane and all proteins, lipids, nucleic acids and other substances that may be contained in the membrane.

[0055] In the present invention, the term "β-glucan" (Glucan) refers to a homopolysaccharide composed of glucose as a monosaccharide, wherein the glucose units are connected by glycosidic bonds, and its CAS number is 9012-72-0, and its molecular formula is C 18 H 30 O 14 X2.

[0056] In the present invention, the term "immortalization" refers to the property of cells that they acquire the ability to continue to grow and proliferate. Immortalized cells have unlimited proliferation and 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 mechanism 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 immortalized macrophages cultured by self-induction. In a specific embodiment of the present invention, J774A.1 cells are an immortalized mouse mononuclear macrophage, which is taken from adult female BALB / CN mice with reticulum cell sarcoma, and ascites is produced during cell tumor induction therapy and isolated therefrom.

[0057] In the present invention, the term "primary" refers to cells grown in a culture dish after animal tissue is digested and dispersed by trypsin or the like to obtain single cells, which retain the basic properties of the original cells. If they are normal cells, they still retain the diploid number. Cultured cells within the first to tenth generations are usually 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 peritoneal lavage fluid.

[0058] In the present invention, the term "polarization" refers to the process in which macrophages change their phenotype under the action of cytokines to enhance their ability to cope with changes in the microenvironment. This process is called macrophage polarization. According to phenotype and function, polarization of macrophages includes classically activated M1 macrophages and alternatively activated M2 macrophages. In a specific embodiment of the present invention, the primary peritoneal macrophages obtained are mostly M1 macrophages.

[0059] In the present invention, the term "peritoneal macrophages" refers to macrophages extracted from the peritoneal cavity of mice. Macrophages naturally exist in the peritoneal cavity of mice. Peritoneal macrophages mostly exist freely in the ascites and are easy to obtain: the peritoneal lavage fluid is extracted after flushing the peritoneal cavity with normal saline or cell culture medium to obtain the macrophages therein. The culture method of peritoneal macrophages is usually intraperitoneal injection of 3% thioglycolate broth, wherein the broth is a non-infectious inflammatory stimulus that can promote the aggregation of macrophages; the role of thioglycolate (thioglycolate) is to release macrophages from peripheral blood into the peritoneal cavity, thereby extracting more macrophages for subsequent culture.

[0060] In the present invention, the term "relieve" refers to a reduction in measurable lesions compared to before treatment, which lasts for at least four weeks, and no new lesions appear during the period. In some embodiments, the severity or duration of the patient's inflammatory manifestations is reduced by, for example, 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 (such as the microvesicles of the first aspect of the present invention).

[0061] In the context of the present invention, the term "inflammation" refers to a defensive reaction of the body to stimulation, which manifests as redness, swelling, heat, pain and dysfunction. Inflammation can be acute inflammation or chronic inflammation, and can be infectious inflammation caused by infection or non-infectious inflammation not caused by infection. The inflammation described in the present invention includes inflammation caused by physical factors, chemical factors, biological factors, tissue necrosis, allergic reaction and foreign matter. Physical factors include high temperature, low temperature, mechanical trauma, ultraviolet light and radiation. Chemical factors include endogenous and exogenous chemical substances, endogenous chemical substances such as decomposition products of necrotic tissue, metabolites accumulated in the body under pathological conditions such as urea, etc.; exogenous chemical substances include strong acids, strong bases, strong oxidants and mustard gas, etc. Improper use of drugs and other biological agents may also cause inflammation. Biological factors include viruses, bacteria, rickettsia, protozoa, fungi, spirochetes and parasites. Tissue necrosis caused by any reason is a potential inflammatory factor. Allergic reaction refers to the abnormal immune response of the body, which may cause inappropriate or excessive immune response, tissue damage and inflammatory response, such as allergic rhinitis and glomerulonephritis. Foreign body refers to surgical sutures, silica crystals or material fragments that remain in the body tissue and cause inflammation.

[0062] In the present invention, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implanted drug storage devices. In some embodiments, oral administration is preferred. The pharmaceutical composition of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, a pharmaceutical acid, base or buffer 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. As long as the target tissue can be reached, the pharmaceutical composition of the present invention may be administered to the receptor by any route.

[0063] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating, preventing, alleviating and / or relieving diseases related to myelin damage of the nervous system. Other compounds for treating, preventing, alleviating and / or relieving diseases related to myelin damage of the nervous system can 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. Other therapeutic compounds can also be administered separately in a separate composition or in a dosage form different from the main active ingredient.

[0064] In the present invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not itself induce the production of antibodies that are 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., can also be present in such vehicles.

[0065] In the present invention, the term "culture system" refers to a system that simulates the in vivo environment (sterility, suitable temperature, pH and certain nutritional conditions, etc.) in vitro to enable survival, growth, reproduction and maintenance of major structures and functions, usually including culture medium, nutrients, cytokines, gas exchange and supports (such as glass, plastic culture dishes, culture bottles, etc.). In the context of the present invention, the culture system refers to a liquid culture system that does not include gas exchange and supports.

[0066] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present 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 obtained from commercial sources. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention.

[0067] Example 1 Extraction and identification of microvesicles 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 instrument was purchased from Shanghai Qinxiang Science Co., Ltd.; cell ultra-clean bench was purchased from Beijing Ruiwote 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 culture medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd., fetal bovine serum was purchased from Kangyuan Biotechnology Co., Ltd., RPMI1640 culture medium and DMEM culture medium were purchased from Sewell, 0.25% trypsin was purchased from Thermo, 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, USA, Glucan was purchased from MCE, and NaOH was a product of Sinopharm Group.

[0068] 2. Experimental Methods and Results 1. J774A.1 cell culture: J774A.1 cells were purchased from Shanghai Meiyan Biotechnology Co., Ltd. The culture medium was DMEM complete medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin. The culture conditions were 37°C, 5% CO2 carbon dioxide cell culture incubator, and the cells were passaged at a rate of 1:2 every day.

[0069] 2. Extraction of microvesicles secreted by J774A.1 cells: J774A.1 cells were cultured in 6 10 cm cell culture dishes until the confluence reached 80%-90%, and fresh DMEM complete medium was replaced. Three dishes were stimulated with 10 μg / ml Glucan, and the other three dishes were added with NaOH solution as the solvent control group. The culture was continued for 24 h, and the supernatant was collected and centrifuged at 300 g for 5 min at 4°C; the supernatant was transferred to a new centrifuge tube and centrifuged at 2000 g for 10 min at 4°C; the supernatant was taken and centrifuged again; the supernatant was centrifuged at 15,000 g for 30 min at 4°C, and the supernatant was discarded; the precipitate was resuspended in 10 mL PBS and centrifuged at 15000 g for 30 min at 4°C; the supernatant was discarded, and the precipitate was resuspended in 1 mL PBS and centrifuged at 15000 g for 30 min at 4°C; the supernatant was resuspended in an appropriate volume of PBS, aliquoted, and frozen at -80°C.

[0070] 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 about 70 kDa; TSG101: an obvious specific band was observed at the expected molecular weight of about 45 kDa; CD63: an obvious specific band was observed at the expected molecular weight of about 53 kDa; CD81: an obvious specific band was observed at the expected molecular weight of about 26 kDa; CD9: an obvious specific band was observed at the expected molecular weight of about 24 kDa, among which the band of the Ctrl-EVs group was very weak.

[0071] Nanoparticle tracking analysis (NTA) measured the size range of Ctrl-EV and Glucan-EV. Figure 2 As shown, Figure 2 A in the figure is the size range of Ctrl-EV. Figure 2 B in the figure is the size range of Glucan-EV.

[0072] Transmission electron microscopy (TEM) visualized the morphology of Ctrl-EV and Glucan-EV, and characterized the microvesicles. Figure 3 As shown, the microvesicles secreted by J774A.1 cells were spherical and enclosed in a double membrane.

[0073] Example 2 Anti-inflammatory effect of microvesicles 1. Experimental Materials C57BL / 6N mice were purchased from Vital River Company and kept in the SPF animal room of the Military Medical Research Institute of the Academy of Military Sciences. They were given free access to food and water and the light and dark periods were 12 h each. Mice of the same sex aged 6 to 8 weeks were selected for the experiment.

[0074] 2. Experimental Methods and Results 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 euthanized by dislocation of the neck. 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 tissue and into the abdominal cavity with the right hand. 5-10 mL of pre-cooled 1 ´ PBS was injected into the abdominal cavity and the abdomen of the mice was massaged for 5 min. 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 washing fluid was recovered into a test tube. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the test tube was flicked, and the cells were resuspended and mixed with 1 mL of complete RPMI1640 medium. 10 mL of cell suspension was taken for cell counting. The cell suspension was diluted to 2-4´10 6 / mL, 500 mL per well was seeded in a 24-well plate, and the plate was placed in a 37°C, 5% CO2 cell incubator for 4-6 h. The non-adherent cells were washed away with PBS, and the remaining adherent cells were peritoneal macrophages.

[0075] 2. Co-culture of microvesicles and macrophages: discard the culture medium, wash once with 1 ´ PBS, add fresh RPMI1640 complete culture medium, add EVs with final concentrations of 5, 10, and 20 mg / mL respectively (Ctrl-EVs, EVs extracted from J774A.1 cells after NaOH stimulation for 24 h; Glucan-EVs, EVs extracted from J774A.1 cells after Glucan stimulation for 24 h), and place the 24-well plate in a cell incubator at 37°C, 5% CO2 for 24 h.

[0076] 3. Real-time fluorescence quantitative PCR: In order to extract RNA from the microvesicle and 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 the Novozyme reagent. The Roche LightCycler® 480 real-time fluorescence quantitative PCR instrument was used to quantify the gene expression of iNOS, TNF-a, Arg-1, CD206, and GAPDH. −ΔΔCt The relative expression of genes was calculated using the method. Figure 5 As shown, through real-time fluorescence quantitative PCR, it was found that compared with the addition of control vesicles, the expression levels of M1 polarization-related indicators, TNF-a, iNOS and other genes were lower in the microvesicle system stimulated by Glucan ( Figure 5 A and B in Figure 2), M2 polarization indicators, CD206 and Arg-1 expression levels are higher ( Figure 5 C and D in the figure), but the levels of the two groups were lower than those of the blank control group.

[0077] 4. ELISA cytokine test: Collect the supernatant of co-culture of microvesicles and peritoneal macrophages for cytokine testing. Use ELISA kit to detect cytokines (TNF-a, IL-6, IL-10). For specific steps, refer to the Invitrogen ELISA kit instructions. ELISA was used to detect the expression levels of TNF-a and IL-6 in the supernatant of co-culture of peritoneal macrophage microvesicles and macrophages. The results are as follows: Figure 4 As shown, compared with the addition of control vesicles, the expression levels of TNF-a and IL-6 in the supernatant of microvesicles derived from Glucan stimulation were very low ( Figure 4 A and B in the text are almost not expressed.

[0078] 5. Statistical analysis: The experimental results were expressed as x±s, and the data were analyzed using GraphPad Prism 8 software. One-way analysis of variance was used for comparison between multiple groups of samples, and two independent sample analysis was used for comparison between two groups. t test, P The difference was considered statistically significant when the value was less than 0.05.

[0079] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A microvesicle, characterized in that The microvesicles are produced by macrophages stimulated by β-glucan; Preferably, the size of the microvesicles ranges from 50 to 400 nm; Preferably, the surface markers of the microvesicles are positive for HSP70, TSG101, CD63, CD9, and CD81.

2. The microvesicle according to claim 1, characterized in that The macrophages include mouse macrophages and human macrophages; Preferably, the human macrophages are primary macrophages; Preferably, the human primary macrophages include macrophages derived from peripheral blood; Preferably, the murine macrophages include immortalized macrophages and primary macrophages; Preferably, the mouse immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line; Preferably, the mouse primary macrophages include pluripotent stem cell-derived macrophages, peripheral blood-derived macrophages, bone marrow-derived macrophages, peritoneal cavity-derived macrophages, and tumor-derived macrophages; Preferably, the macrophages are J774A.1 cell line.

3. The product prepared by the microcapsule according to claim 1 or 2, characterized in that The products include: 1) Products that inhibit macrophage polarization; 2) Products that inhibit the secretion of cellular inflammatory factors; 3) Products that reduce inflammation; Preferably, the product includes a pharmaceutical composition and a kit.

4. The product according to claim 3, characterized in that The inhibiting of macrophage polarization is inhibiting macrophage M1 polarization; Preferably, the inhibiting macrophage polarization is inhibiting macrophage M1 polarization; Preferably, 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; Preferably, the cellular inflammatory factors include TNF-α, IL-6, iNOS, Arg-1, and CD206; Preferably, the macrophages include mouse macrophages and human macrophages; Preferably, the human macrophages are primary macrophages; Preferably, the human primary macrophages include macrophages derived from peripheral blood; Preferably, the murine macrophages include immortalized macrophages and primary macrophages; Preferably, the mouse immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line; Preferably, the mouse primary macrophages include pluripotent stem cell-derived macrophages, peripheral blood-derived macrophages, bone marrow-derived macrophages, peritoneal cavity-derived macrophages, and tumor-derived macrophages; Preferably, the macrophages are primary mouse macrophages; Preferably, the macrophages are peritoneal macrophages; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. The product according to claim 3, characterized in that The manifestations of reducing inflammation include: 1) reducing macrophage polarization; and / or 2) reducing the secretion of cellular inflammatory factors; Preferably, the cellular inflammatory factors include TNF-α, IL-6, iNOS, Arg-1, and CD206.

6. Use of the microvesicles according to claim 1 or 2 in the preparation of the product according to any one of claims 3 to 5, and / or in inhibiting macrophage polarization, inhibiting the secretion of cellular inflammatory factors, and alleviating inflammation.

7. A method for culturing the microvesicles according to claim 1 or 2, characterized in that: The method comprises adding β-glucan to a culture system of macrophages.

8. The method according to claim 7, characterized in that The macrophage culture system comprises a DMEM complete medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin; Preferably, the action time of the β-glucan is 24 hours.

9. A method for inhibiting macrophage polarization, inhibiting the secretion of cellular inflammatory factors or alleviating inflammation, characterized in that: The method comprises adding the microvesicles according to claim 1 or 2 to a culture system of macrophages.

10. The method according to claim 9, characterized in that The macrophages include mouse macrophages and human macrophages; Preferably, the human macrophages are primary macrophages; Preferably, the human primary macrophages include macrophages derived from peripheral blood; Preferably, the murine macrophages include immortalized macrophages and primary macrophages; Preferably, the mouse immortalized macrophages include THP-1 cell line, RAW264.7 cell line, U937 cell line, and J774A.1 cell line; Preferably, the mouse primary macrophages include pluripotent stem cell-derived macrophages, peripheral blood-derived macrophages, bone marrow-derived macrophages, peritoneal cavity-derived macrophages, and tumor-derived macrophages; Preferably, the macrophages are primary mouse macrophages; Preferably, the macrophages are peritoneal macrophages; Preferably, the macrophage culture system comprises RPMI1640 complete medium.

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