阅读说明：本技术 一种具有抗炎作用的细胞外囊泡的应用 (Application of extracellular vesicle with anti-inflammatory effect ) 是由 刘敬平 王一卓 刘书云 于 2021-09-03 设计创作，主要内容包括：本发明涉及一种M2型巨噬细胞来源的胞外囊泡的应用,具体为M2型巨噬细胞来源的胞外囊泡在制备用于减轻炎症反应的产品中的应用。本发明通过实验证明来源的EVs具有与类似的免疫调节功能,发挥抗炎及促进组织修复的作用,在急性全身性炎症反应中具有潜在的治疗作用。相比于细胞疗法,EVs具有多项优势,如：EVs携带有亲代细胞功能分子,可以通过将这些分子传递给受体细胞从而发挥特定功能；EVs具有较好的生物相容性,低免疫原性,生物安全性较高；EVs的物理化学性质较为稳定,便于储存、运输；EVs不具有细胞活性,易于制备,进入体内后不易受体内环境影响再分化。(The invention relates to application of an M2 type macrophage-derived extracellular vesicle, in particular to application of an M2 type macrophage-derived extracellular vesicle in preparation of a product for relieving inflammatory reaction. The invention is proved by experiments EVs of origin having)

Use of macrophage-derived extracellular vesicles of the M2 type for the preparation of a product for reducing inflammatory responses.

2. Use according to claim 1, wherein the product is a reagent, kit, medicament or device.

3. The use according to claim 1, wherein the M2-derived extracellular vesicles have an average particle size of 140nm to 160 nm.

4. The use according to claim 1, wherein the process for the preparation of extracellular vesicles derived from macrophages of the type M2 comprises the following steps: culturing M2 type macrophages with a culture medium for 24-72 hours, collecting supernatant, centrifuging for 15min at 300g, centrifuging for 15min at 2000g, taking supernatant, and centrifuging for 90min at 110000g, and taking precipitate.

5. The use according to claim 4, wherein the culture medium is prepared by the following method: RPMI1640 and DMEM medium containing 20% FBS were prepared, and after centrifugation at 120000g for 16 hours, the supernatant was collected, and the precipitate was discarded, followed by filter sterilization through a 0.22 μm filter.

6. The use of claim 4, wherein the M2-type macrophages are M2-type peritoneal macrophages.

7. The use according to claim 6, wherein the preparation method of the M2 type peritoneal macrophage comprises the following steps: after the mice are anesthetized, the peritoneal dialysis solution is injected into the abdominal cavity and extracted after the abdomen is massaged; carrying out centrifugal cleaning on the obtained peritoneal dialysis solution, then purifying by using CD11bMicroBeads, and screening out abdominal cavity mononuclear cells; inducing abdominal cavity monocyte differentiation into macrophage by RPMI1640 containing M-CSF, inactivated serum and double antibody, removing suspension cells after overnight incubation, and adding IL-4 and IL-13 to continue culturing to induce and form M2 type abdominal cavity macrophage.

8. The use according to claim 7, wherein the concentration of M-CSF is 20ng/ml, the concentration of inactivated serum is 10 wt% and the concentration of diabody is 1 wt%.

9. The use according to claim 7, wherein the preparation method of the M2 type peritoneal macrophage cell further comprises the following identification steps: and detecting the expression conditions of cytokines Arg1, CCL17, Mrc1, Retnla, Chil3 and TGF-beta in the M2 abdominal cavity macrophages by qRT-PCR.

Use of macrophage-derived extracellular vesicles of the M2 type for the preparation of a product for reducing the number of macrophages of the M1 type.

Technical Field

The invention relates to the technical field of cell biology, in particular to application of an extracellular vesicle with an anti-inflammatory effect.

Background

Inflammation (Inflammation) refers to an adaptive immune response of the body in response to harmful stimuli or the environment (e.g., pathogenic infection or tissue damage). It is generally believed that a controlled inflammatory response is beneficial to protect the body from pathogenic infection or tissue damage, whereas when the inflammatory response is uncontrolled, immune cells are continuously activated and recruited, initiating a hyper-reactive inflammatory state in the body, leading to the development of cytokine storms (septicemia). At present, the definition of the cytokine storm is not unified, wherein a relatively wide view point considers that the cytokine storm is a general term for the severe increase of the level of the cytokine (such as TNF-alpha, IL-6, IL-1 beta, CCL2, CCL3 and the like) and the high activation of immune cells (such as neutrophils, macrophages, T cells and the like) in a short time, the occurrence of the cytokine storm can be caused by iatrogenic treatment, pathogen invasion and autoimmune disorder, if the cytokine storm is not cured in time, the tissue damage can be caused finally, and the organ dysfunction and the body death can be caused by severe cases. According to 2019 edition of Chinese clinical practice guidelines for acute septic shock, although large-dose hormone shock therapy is still used clinically, the serious side effects therewith largely prevent the wide application thereof. Thus, the high clinical mortality rate due to cytokine storm is not significantly reduced and the hyperreactive inflammatory state caused by severe infection or injury remains one of the problems that plague the world public health industry.

Innate immune cells are the first line of defense in the immune defense response of the body, and the cells mainly involved in cytokine storms are neutrophils (neutrophiles), Macrophages (Macrophages,) And Natural killer cells (NKs). Macrophages play an indispensable role in the processes from antigen presentation, immune regulation, tissue repair and the like, and can secrete a large amount of proinflammatory inflammatory factors after being activated, so that the macrophages become one of the main sources of the inflammatory factors. Macrophages are derived from monocytes, are widely present in all tissues, are highly heterogeneous, and different stimuli can induce their differentiation into different subtypes. The traditional view points are thatCan be induced into 2 subtypes: macrophages of M1 subtype activated by LPS and/or Th1 type cytokines (e.g., gamma-IFN, GM-CSF, etc.) (M1-like macrophages,) Can produce proinflammatory cytokines such as TNF-alpha, IL-6, IL-1 beta and the like, and play a role in proinflammatory; m2-type macrophages activated by Th 2-type cytokines (e.g., IL-4/IL-13, etc.) (M2-like macrophages,) And the Mannose receptor (MrcMMR), the Arginase (Arg 1), the anti-inflammatory factor IL-10 and the like are highly expressed, and the effects of resisting inflammation, regulating immunity, promoting tissue repair and the like are exerted. Studies have shown that in a sepsis-induced acute lung injury model,the cells and the supernatant of the culture medium can effectively promote the proliferation of the lung vascular endothelial cells and can be transplanted in vivoCan effectively relieve pulmonary tissue edema, reduce vascular permeability and improve the survival rate of mice. In addition, the scholars point outAnti-inflammatory effects in colitis models, in vivo injectionIt is possible to reduce the severity of colitis by inducing the transformation of macrophages in the body, producing and releasing the anti-inflammatory factor IL-10.Has wide prospect for immune adjustment and tissue repair, but also has certain problems. For example, as a living cell,the storage and transport conditions of (a) may have some effect on cell activity; in addition to this, the present invention is,it is highly heterogeneous, and when it is administered into the body, the complex environment inside the body may induce redifferentiation.

Disclosure of Invention

Based on this, there is a need for a new application of extracellular vesicles derived from macrophages of the M2 type.

The invention provides application of M2 type macrophage-derived extracellular vesicles in preparation of products for relieving inflammatory response.

In one embodiment, the product is a reagent, kit, medicament or device.

In one embodiment, the M2-type macrophage-derived extracellular vesicle has an average particle size of 140nm to 160 nm.

In one embodiment, the method for preparing the M2-type macrophage-derived extracellular vesicle comprises the following steps: culturing M2 type macrophages with a culture medium for 24-72 hours, collecting supernatant, centrifuging for 15min at 300g, centrifuging for 15min at 2000g, taking supernatant, and centrifuging for 90min at 110000g, and taking precipitate.

In one embodiment, the culture medium is prepared by the following method: RPMI1640 and DMEM medium containing 20% FBS were prepared, and after centrifugation at 120000g for 16 hours, the supernatant was collected, and the precipitate was discarded, followed by filter sterilization through a 0.22 μm filter.

In one embodiment, the M2-type macrophage is a M2-type peritoneal macrophage.

In one embodiment, the preparation method of the M2 type peritoneal macrophage comprises the following steps: after the mice are anesthetized, the peritoneal dialysis solution is injected into the abdominal cavity and extracted after the abdomen is massaged; centrifuging and cleaning the obtained peritoneal dialysis solution, purifying by using CD11b MicroBeads, and screening out peritoneal mononuclear cells; inducing abdominal cavity monocyte differentiation into macrophage by RPMI1640 containing M-CSF, inactivated serum and double antibody, removing suspension cells after overnight incubation, and adding IL-4 and IL-13 to continue culturing to induce and form M2 type abdominal cavity macrophage.

In one embodiment, the concentration of M-CSF is 20ng/ml, the concentration of inactivated serum is 10 wt%, and the concentration of diabody is 1 wt%.

In one embodiment, the method for preparing M2-type peritoneal macrophages further comprises the following identification steps: and detecting the expression conditions of cytokines Arg1, CCL17, Mrc1, Retnla, Chil3 and TGF-beta in the M2 abdominal cavity macrophages by qRT-PCR.

The invention also provides application of the extracellular vesicles derived from M2 type macrophages in preparing products for reducing the number of M1 type macrophages.

Extracellular Vesicles (EVs) are nanovesicles secreted by cells and have a double-layer phospholipid membrane structure, and can participate in information exchange between cells and tissues by transmitting various bioactive substances. EVs have several advantages over cell therapy as a potential cell-free alternative therapy, such as: EVs carry parental cell functional molecules, such as lipids, proteins, nucleic acids, etc., that can be used to perform specific functions by delivering these molecules to recipient cells; EVs have good biocompatibility, low immunogenicity and high biological safety; the physical and chemical properties of EVs are stable, and the EVs are convenient to store and transport; the EVs serving as the nano vesicle with the double-layer membrane structure does not have cell activity, is easy to prepare, and is not easy to redifferentiate under the influence of the internal environment of a receptor after entering the body. Therefore, there is great promise in using EVs instead of cell therapy. At present, although a great deal of research has been shownTherapeutic effects in models of inflammation, but relatively few studies on the EVs from which they were derived, we speculated and further experimentally demonstratedEVs of origin havingAndsimilar immunoregulation function, plays the roles of resisting inflammation and promoting tissue repair, and has potential treatment effect in acute systemic inflammatory reaction.

Drawings

FIG. 1 is a schematic view of an embodimentDetecting the purity of the product; detecting the F4/80+ ratio of the separated cells by flow cytometry;

FIG. 2 is a diagram showing the morphological examination of cells of M0 and M2 subtypes in the examples; respectively collectAndobserving the cell morphology under an inverted microscope;

FIG. 3 shows an embodiment(ii) phenotypic testing of; IL-4/IL-13 combination InductionExtracting total RNA of cells after 48h, and detecting by qRT-PCRExpression levels of the cytokines Arg1, CCL17, Mrc1 mRNA (andin comparison, P < 0.05, P < 0.01);

FIG. 4 shows the identification of M2-Evs characterization in the example; figure a is TEM characterization EVs morphology; FIG. B shows the size of EVs particle size detected by NTA; FIG. C is a Westernblot detection of EVs surface markers;

FIG. 5 shows an embodimentThe cell takes up the fluorescence verification result of M2-Evs;

FIG. 6 is a graph showing that M2-EVs reduced LPS/γ -IFN induction in exampleExpression data for inflammatory factor mRNA levels; using M2-EVs and interfering with LPS/gamma-IFN inductionAfter 4h, cells were harvested and the expression levels of the inflammatory factors TNF- α and IL-6mRNA (P < 0.05, P < 0.01 compared to LPS/γ -IFN group) were measured;

FIG. 7 shows anti-inflammatory function data of ELISA test M2-EVs in examples; intervention of LPS/gamma-IFN induction with M2-EVsCollecting cell supernatant after 4h, and detecting the protein expression level of inflammatory factors TNF-alpha and IL-6;

FIG. 8 shows the in vivo distribution of Cy 7-labeled M2-EVs in the examples; the graph A is the distribution of M2-EVs heart, liver, lung, spleen and kidney, and the graph B is the distribution statistical chart of M2-EVs in vivo;

FIG. 9 is the data of the mouse serum TNF-alpha and IL-6 expression measured by ELISA in the examples; after LPS is administrated to the abdominal cavity, M2-EVs (80 mu g/100 mu l/mouse) is injected into tail vein, and after 4h, the serum of the mice is collected to detect the protein expression level of inflammatory factors TNF-alpha and IL-6;

FIG. 10 is the data of the expression of TNF-alpha and IL-6 in mouse tissues measured by qRT-PCR in the examples; after LPS is administrated to the abdominal cavity, M2-EVs (80 mu g/100 mu l/mouse) is injected into tail vein, lung, liver and kidney tissues of mice are harvested after 4h, and the expression levels of inflammatory factors TNF-alpha and IL-6mRNA are detected;

FIG. 11 is the data of flow cytometry detection of the expression of F4/80 and CD11c in mouse spleen in the examples; p < 0.05, P < 0.01 compared to LPS group.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

In this example, we selected peritoneal macrophages based on therapeutic dose requirements and ease of preparationExtracellular vesicles derived from (M2-EVs) were used as a study to investigate their role in acute inflammation models.

1. Materials and instruments

1.1 Experimental animals

SPF-grade healthy male C57BL/6J mice, 20-25g in weight, purchased from Duoduoshu laboratory animals Co., Ltd, are raised in cages at room temperature of 20-25 ℃ and 70% humidity, are fed with free diet, and are alternately raised in light and dark for 12 hours, wherein the raising conditions meet the 'Sichuan medical laboratory animal raising and management regulations' (national standard GBI 4925-2001).

1.2 Main Experimental reagents and materials

1) Fetal Bovine Serum (FBS), Gibco, USA

2) Trypsin, Gibco, USA

3) DMEM Medium, Gibco, USA

4) RRMI 1640 Medium, Gibco, USA

5) Penicillin/streptomycin, Biyuntian, China

6) M-CSF, IL-4, IL-13, nearshore organisms, China

7) LPS, Sigma, USA

8) Gamma IFN, Sigma, USA

9) Anti-HSP70 antibodyy, Abcam, UK

10) Anti-TSG101 antibody, Proteintech, USA

11) Anti-ALIX antibody, Proteintech, USA

12) Anti-GM130 antibody, Cell Signaling Technology, USA

13) BD PharmingenTMACC-CyTM 7 Anti-mouse CD45, BD, USA

14) BD Pharmingen PE RatInti-Mouse F4/80, BD, USA

15) APC anti-mouse CD11cAntibody, Biolegend, USA

16) BD horizon Fixable visual Stain 700, BD, USA

17) IL-6ELISA test kit, Dake, China

18) TNF-alpha ELISA assay kit, Dake, China

19) HRP-labeled anti-rabbit secondary antibody in mice, Abbkine, China

20) DAPI, Thermo Fisher Scientific, USA

21) RIPA lysate, Biyunyan, China

22) Protease inhibitors, Calbiochem, USA

23) Phosphoprotease inhibitors Calbiochem, USA

24)4 × Loading buffer, Bio-Rad, USA

25) BCA protein detection kit, kang is century, China

26) Prestatinedprotein ladder, Thermo, USA

27) ECL ultrasensitive luminogenic substrate, Millipore, USA

28) Cell culture flasks, well plates, Petri dishes, BD, USA

29) CD11b + MicroBeads, Miltenyi Biotec, Germany

30) 10. mu.l, 200. mu.l, 1ml pipette tip, Eppendorf, USA

31)1.5ml, 4ml, 15ml, 50ml centrifuge tubes, BD, USA

32) Paraformaldehyde, KH2PO4, Tween-20, NaCl, KCl, Na2HPO 4.7H 2O, Triton X-100, Koran, China

33) PCR primer Synthesis, Productivity, China

34) Chloroform, isopropanol, anhydrous ethanol, methanol, normal saline, xylene, hydrochloric acid, Kelong chemical reagent factory, China

35) TRIzol Reagent, Thermo Fisher, USA

36) 10 mul of RNase free, 200 mul of RNase free and 1ml of RNase gun head; 0.2ml, 1.5ml, 2ml centrifuge tubes, GCS technologies, USA

37)Q RT Supermix for qPCR kit, Vazyme, China

38) AceQTM qPCR SYBR GreenMasterMix, Vazyme, China

39) Sodium pentobarbital, Mecrk, Germany

40) Fluorescent quantitative PCR eight-tube, Thermo Fisher, USA

41) SW32Ti rotor, Beckman, USA

42) Ultra-high speed centrifuge tubes, Beckman, USA

1.3 Main Experimental instruments

1) CO2 cell culture incubator Sanvall Legand RT, USA

2) Clean bench Nuaire NU-425-

3) SANYO, Japan, at-150 ℃ ultra-low temperature refrigerator

4) Centrifuge Sorvall, Legend RT, USA

5) Inverted phase contrast microscope Olympus IX 50, Japan

6) Fluorescence inverted phase contrast microscope Olympus IX 71, Japan

7) Full-automatic upright fluorescence microscope Zeiss ImagerZ2, Germany

8) Electronic thermostatic water bath Thermo Fisher Scientific, USA

9) 2.5. mu.l, 10. mu.l, 100. mu.l, 200. mu.l, 1ml sample application gun Eppendorf, USA

10) Full-automatic multifunctional microplate reader BioTek, USA

11) Vertical electrophoresis apparatus Bio-Rad, USA

12) Naimei Linbel of the rotating apparatus, China

13) Namen of vortex apparatus Linbel, China

14) Magnetic stirrer Haimanghei Linbel, China

15) Shanghai electronic balance, China

16) Ultra pure Water System Millipore, USA

17) Autoclave SANYO, Japan

18) Flow cytometer Backman coulter, USA

19) Fluorospectrophotometers molecular devices, USA

20) Ventilation kitchen Thermo Fisher, USA

21) PCR amplification apparatus Bio-Rad, USA

22) CFX96 qRT-PCR Instrument Bio-Rad, USA

23) Small animal Living body imager PerkinElmer, USA

24) Ultra high speed centrifuge Beckman, USA

1.4 preparation of the test reagents

1.4.1 preparation of the culture Medium

Preparing a culture medium: thawing the serum at 4 deg.C, and placing in 56 deg.C constant temperature water bath for 30min to obtain inactivated serum. RAW264.7 cell lines were cultured using RPMI1640 containing 10% inactivated FBS;RPMI1640 containing 20ng/ml MCS-F, 10% inactivated FBS was used for the culture.

1.4.2 preparation of macrophage inducing factor

1) And (3) LPS preparation: 1mg LPS powder is weighed and dissolved in 1ml sterile PBS, vortex and mix evenly to obtain 1mg/ml LPS stock solution. 100 μ l of stock was dissolved in 2.4ml sterile PBS to obtain 1000 X40 μ g/ml LPS stock, which was frozen at-80 ℃ after dispensing.

2) Preparing gamma-IFN: weighing 1mg of gamma-IFN powder, dissolving the powder in 1ml of sterile PBS, and uniformly mixing the powder by vortex to obtain 1mg/ml of gamma-IFN stock solution. 100 μ l of stock solution was dissolved in 4.9ml sterile PBS to obtain 1000 Xof 20 μ g/ml γ -IFN stock solution, which was frozen at-80 ℃ after packaging.

3) M-CSF, IL-4, IL-13 formulation: taking 50 mu g of dry powder, instantly separating the dry powder to the bottom of a tube, adding the dry powder into 250 mu l of sterile PBS to obtain 200 mu g/ml M-CSF solution, taking 10 mu l of the M-CSF solution, dissolving the 10 mu l of the M-CSF solution in 90 mu l of sterile PBS to obtain 1000 Xstock solution, subpackaging and freezing at-80 ℃.

1.4.3 preparation of sodium pentobarbital

Weighing 0.5g of pentobarbital sodium powder, diluting to 50ml with normal saline, filtering and sterilizing with 0.22 μm filter membrane, and temporarily storing at 4 ℃. It is used as it is.

1.4.4 Western blot related reagent preparation

1) PMSF stock solution:

weighing 0.174g of PMSF powder, fixing the volume of isopropanol to 10ml, and fully dissolving to obtain a final concentration of 100 mM; after dissolution, the solution was dispensed into 1.5ml centrifuge tubes and stored at-20 ℃.

2) Preparing a protein lysate:

taking RIPA lysate, adding PMSF according to 1%, fully and uniformly mixing, temporarily storing at 4 ℃, and preparing for use.

3) Preparation of separation gel and concentrated gel:

the following is a one-piece glue formulation method. Mixing the separated gel solution and the buffer solution with equal volume (2.7 ml each), adding 60 μ l of improved coagulant, mixing, injecting into a glass plate for preparing gel, adding appropriate amount of anhydrous ethanol to cover the lower layer of gel, when there is a fold line between the gel and the alcohol, coagulating the gel, and removing the upper layer of alcohol; mixing the concentrated gel and the color concentrated gel buffer solution (0.75 ml each), adding 15 μ l of improved coagulant, injecting into a glass plate, inserting into comb teeth, and removing the comb teeth after the upper gel is solidified.

4)10 × electrophoresis buffer:

glycine: 188 g; tris base: 30.2 g; SDS (sodium dodecyl sulfate): and 10g, adding triple-distilled water for dissolving, fixing the volume to 1L, and storing at room temperature. When in use, 100mL of 10 × electrophoresis buffer solution is taken, and diluted to 1L to obtain 1 × electrophoresis buffer solution.

5)10 × transfer membrane buffer:

glycine: 29 g; tris (Tris): 58g of the total weight of the mixture; SDS (sodium dodecyl sulfate): 3.7 g; adding three times of distilled water for dissolving, fixing the volume to 1L, and storing at room temperature. When in use, 80mL of 10 Xtransmembrane buffer and 200mL of methanol are taken, and the volume is determined to be 1L and diluted into 1 Xtransmembrane buffer.

6) 5% skimmed milk (100mL)

Weighing 5g of skimmed milk powder, adding PBS to dissolve, fixing the volume to 100mL, temporarily storing at 4 ℃, and preparing for use.

7)0.02mol/LPBS buffer:

na2HPO4 & 12H 2O: 7g of a mixture; NaH2PO4 & 2H 2O: 0.7g, dissolved by stirring with 900mL of double distilled water, adjusted to pH 7.4, and stored at room temperature with a constant volume of 1L.

8) 0.5% PBST buffer:

taking 0.5ml of Twenen-20, adding 1LPBS, stirring uniformly, and storing at room temperature for use. 0.02mol/L PBS: 1L; stirring and mixing evenly, and storing at room temperature.

1.4.5 preparation of ELISA-related reagents

1) And (4) standard product: prepared using Diluenbuffer according to the instruction and diluted to the detectable concentration in turn.

2) Sample dilution: the samples were diluted to the appropriate ratio using a Diluenbuffer.

3) Antibody working solution: the medicament is prepared according to a ratio of 1:100 by using Diluenbuffer and is prepared for use.

4) Washingbuffer working solution: diluted 50 times with deionized water and ready to use.

5) Streptavidin-HRP working solution: diluenbuffer was diluted 1: 100.

1.4.6 FCM-related reagent formulations

1) PBS formulation of 1% BSA: 1g of BSA powder was weighed and made up to 100ml with PBS buffer.

2) Preparing an antibody: the antibodies were diluted to the appropriate ratio according to the instructions for each antibody.

2. Experimental methods

2.1 cell culture and Induction identification

2.1.1 mouse peritoneal macrophage isolation identification

1) Peritoneal macrophage isolation

8-week-old C57BL/6 male mice were intraperitoneally injected with 1% pentobarbital sodium (10mg/kg), fixed in the supine position after anesthesia, sterilized with iodophor in the lower abdomen, deiodinated with 75% alcohol, then percutaneously passed into the left lower quadrant of the abdominal cavity using a 22G venous indwelling needle, injected with 7ml of preheated (37 ℃) dialysate (physiological saline), the abdomen was gently massaged, and the peritoneal dialysate was withdrawn after several minutes.

Centrifuging the obtained dialysate at 1250rpm for 3min, washing with sterile PBS, purifying the separated cells with CD11b + MicroBeads, screening out high-purity abdominal cavity mononuclear cells, inducing the cells to differentiate into macrophages with RPMI1640 containing M-CSF (20ng/ml), 10% inactivated serum and 1% P/S, incubating overnight, removing culture medium, washing with PBS, removing suspended cells, and collecting adherent cells. Subsequently, IL-4/IL-13(20ng/ml) was added to the basal medium and cultured for another 48 hours, after which it was induced to。

2) Peritoneal macrophage identification

After the cells are attached to the wall, the morphology of the cells is observed under an inverted microscope.

Detecting the purity of adherent cells by FCM, digesting the adherent cells by 0.25 percent of pancreatin, collecting the cells after centrifuging the suspension at 1250rpm for 3min, and resuspending cell precipitates after washing the cells once by PBS (phosphate buffer solution), wherein the grouping is as follows: blank; f4/80. Adding the antibody working solution, incubating on ice in dark for about 30min, washing with PBS buffer containing 1% BSA for 2 times after incubation, resuspending the obtained cell precipitate with 300 μ l PBS buffer, mixing, and detecting on a machine.

The expression of M2 type macrophage factors Arg1, CCL17, Mrc1, Retnla, Chil3 and TGF-beta is detected by qRT-PCR.

2.2Culture Medium Collection, concentration and evaluation of anti-inflammatory Effect

Collecting the supernatant: after induced differentiationAnd M2-RAW264.7 cell supernatant, and the cells were counted using a cell counting plate.

And (3) supernatant concentration: adding sterile PBS into the sterile ultrafiltration tube to rinse the ultrafiltration membrane, allowing PBS to pass through the membrane completely, precooling at 4 deg.C for 3min, centrifuging at 2500g for 5min, and discarding PBS; cells floating in the supernatant were removed by centrifugation at 1250rpm for 3min, the centrifuged supernatant of the medium (15 ml/tube) was added to an ultrafiltration tube, centrifuged at 2500g for 45min, and the concentrate in the filter was carefully aspirated by a pipette tip. The BCA kit measures protein concentration and quantitates with total protein/cell number.

Evaluation of action: taking proper amount of the growth stateInoculation in six-well plates, overnight, fresh medium was replaced and LPS (40ng/ml) was used in combination with gamma-IFN (20ng/ml) treatmentImmediately, an equal amount of concentrated medium produced by the cells was taken. The experimental groups were as follows: control group (CON), model group of cytokine storm (LPS/gamma-IFN), model group + M2-Model group + M2-RAW 264.7. After further culturing for 4h, the treated cells were collected and the expression levels of inflammatory factors TNF-. alpha.and IL-6 were detected by qRT-PCR.

2.3 EVs isolation and characterization

2.3.1 EVs isolation and purification

1) Exo-free FBS preparation

Preparing RPMI1640 culture medium containing 20% FBS, centrifuging at 4 ℃ for 16h at 120,000g, collecting supernatant, and removing tube bottom precipitate to obtain Exos-free FBS. Subsequently, the mixture was sterilized by filtration through a 0.22 μm filter and stored at-80 ℃ until use.

2) Collecting cell culture supernatant

Is induced to obtainThen, the original medium was removed, washed once with PBS, and cultured for another 48 hours in a serum medium with Exos removed, and the supernatant was collected.

3) Ultra-high speed ultracentrifugation for extracting EVs

The collected supernatant was centrifuged at 4 ℃ at 300g for 15min and then at 2,000g for 15min to remove dead cells and cell debris, the supernatant was centrifuged at 4 ℃ at 110,000g for 90min, and the resulting precipitate was washed with sterile PBS and resuspended.

4) Quantification of EVs

And measuring the protein concentration by using the BCA kit and the particle concentration by using NTA, and freezing and storing at-80 ℃ for later use after subpackaging.

2.3.2 EVs identification

1) TEM detection of EVs morphological structure

And (3) taking 10 mu l of separated and purified EV, adding an equal volume of PBS buffer solution for dilution, slowly dripping the EV onto a sample-carrying copper net, standing at room temperature for a plurality of minutes, clamping filter paper by using tweezers to suck excessive liquid, then dripping 2% (w/v) uranyl acetate solution, carrying out negative dyeing at room temperature for 5min, drying, and observing the morphological structure of the EVs under a transmission electron microscope.

2) Size of EVs particle diameter by NTA detection

10 μ l of separated and purified EVs was diluted 2000-4000 times with pure water according to the EVs concentration, and about 1ml of diluted sample was slowly sucked (without air bubbles) with a sterile 1ml syringe, and slowly injected into the sample addition hole, and the particle size was analyzed with ZETAVIEW.

3) Westernblot detection of EVs markers

Taking a proper amount of EVs sample, adding 4 Xloading buffer according to the ratio of 3:1(v/v), shaking and uniformly mixing, carrying out boiling water bath for 15min, and detecting the expressions of EV positive markers ALIX, HSP70, TSG101 and GM130 by Westernblot.

2.4M 2-EVs PairIntervention experiment of inflammation model

2.4.1 EVs cellular uptake assay

EVs were labeled with the membrane dye PKH26 according to the instructions and free dye was removed using an Exosome Spin Column according to the instructions. Taking appropriate amount of the plant with good growth stateAfter overnight inoculation in six-well plates, fresh medium was replaced and PKH 26-labeled EVs were added, and experimental groups were as follows: normal control group (PBS); dye control group (PKH 26); PKH 26-M2-EVs. After 4h of culture, the cells were washed three times with PBS and fixed for 10min at room temperature with 4% paraformaldehyde, and then observed under an inverted fluorescence microscope after washing the cells with PBSUptake of EVs.

2.4.2M 2-EVs anti-inflammatory Effect in vitro evaluation experiment

Taking proper amount of the growth stateInoculation in six-well plates, overnight, fresh medium was replaced and LPS (40ng/ml) was used in combination with gamma-IFN (20ng/ml) treatmentEV intervention was immediately added at equal protein levels. The experimental groups were as follows: control group (CON), acute inflammation model group (LPS/gamma-IFN), model group + M2-EVs (10; 20. mu.g/ml). After continuously culturing for 4h, collecting the treated cells, and detecting the mRNA expression levels of inflammatory factors TNF-alpha and IL-6 by qRT-PCR; collecting the cell culture medium supernatant after treatment, and detecting the concentrations of inflammatory factors TNF-alpha and IL-6 in the culture medium supernatant by ELISA.

2.5 animal experiments

2.5.1 establishment of acute inflammation model of mouse induced by LPS

C57BL/6 mice are injected with LPS (10mg/kg) with different concentrations in the abdominal cavity, 1% pentobarbital sodium (10mg/kg) is injected in the abdominal cavity, the blood is taken from the heart after the mice are anesthetized, the blood sample is kept stand for 15-20min at room temperature, the upper layer transparent liquid is taken out in another centrifuge tube after being centrifuged at 4 ℃ and 3000rpm for 15min, and the concentrations of inflammatory factors TNF-alpha and IL-6 in the serum are detected by ELISA.

2.5.2 intervention and evaluation of M2-EVs on LPS-induced acute inflammation model in mice

EVs in vivo distribution experiments in mice: EVs were labeled with the fluorescent dye Cy7 according to the instructions and free dye was removed using an Exosome Spin Column, as in 2.5.5.1. C57BL/6 mice were injected via tail vein with Cy 7-labeled EVs (60. mu.g/100. mu.l/mouse), and the experiments were divided into the following six groups: normal control group (PBS); dye control (Cy 7); cy7-M2-EVs (2, 6 h); . After treatment, the mouse is euthanized by injecting excessive anesthetic, and heart, lung, liver, spleen and kidney tissues of the mouse are taken to be imaged in a living body imaging system of the small animal to detect fluorescent signals.

M2-EVs anti-inflammatory effect in vivo evaluation experiment: treatment of tail vein with EVs (80. mu.g/100. mu.l/mouse) after intraperitoneal LPS (10mg/kg) administration, experimental groups were as follows: control group (CON), acute systemic inflammation model group (LPS), model group + M2-EVs (80. mu.g/100. mu.l/mouse). After 4h, 1% sodium pentobarbital (10mg/kg) was injected, the mice were anesthetized and blood was taken from the heart, followed by cervical dislocation and lung, liver and kidney tissue frozen at-80 ℃ and fresh spleen tissue was taken for FCM testing. Standing blood sample at room temperature for 15-20min, centrifuging at 4 deg.C and 3000rpm for 15min, and freezing at-80 deg.C.

2.6 evaluation of Effect

2.6.1 ELISA detection

1) Preparation of the experiment: equilibration at room temperature for 20min before use of Dilutionbuffer (1X), precoated antibody plate, Washing buffer (50X), TMB and Stop solution;

2) adding 100 mul/well of the standard substance into the standard substance hole, adding 100 mul/well of the sample into the sample hole, adding 50 mul of antibody working solution into each hole, covering a sealing plate membrane, and incubating for 90min at 37 ℃;

3) adding 200 μ l Washing buffer working solution into each well, reversing after 1min to remove the liquid, and repeatedly Washing for 3 times;

4) adding 100 μ l of Streptavidin-HRP working solution into each well, covering with a sealing plate membrane, and incubating at 37 deg.C for 30 min;

5) repeating the step 4) for 3 times;

6) mu.l TMB was added to each well, incubated at 37 ℃ for several minutes in the dark, 100. mu.l of stop buffer was added to each well, and OD450 readings were measured using a microplate reader.

2.6.2 qRT-PCR detection

Total RNA extraction from cells

1) Preparation of the experiment: consumables (RNase-free gun head and EP tube) are involved in the RNA extraction process;

2) removing the culture medium from the treated cells by suction, washing the cells twice by using PBS precooled at 4 ℃, and sucking off the residual PBS;

3) adding 1ml of Trizol separating reagent into each hole, and adding the Trizol separating reagent into an RNase-free centrifuge tube;

4) adding chloroform extract protein into 200 μ l chloroform/ml Trizol, reversing, mixing well for 2min, standing at room temperature for 10 min;

5) after centrifugation at 12,000rmp for 15min at 4 ℃, the sample was divided into three layers (upper layer: RNA, intermediate layer: DNA, lower layer: protein);

6) taking the upper aqueous phase (about 600 mu l) to a new RNase-free 1.5ml centrifuge tube, adding equal volume of isopropanol, reversing and mixing uniformly for 2min, standing at room temperature for 10min to precipitate RNA;

7) centrifuging at 4 deg.C and 12000g for 10min, and removing supernatant;

8) adding 1ml of 75% ethanol/ml of Trizol into precooled 75% ethanol, inverting the centrifuge tube for 2min, and suspending and precipitating;

9) centrifuging at 4 deg.C for 5min at 7500g, and discarding supernatant as much as possible;

10) repeat 8), 9) once to improve RNA quality

11) Drying the RNA precipitate at room temperature, adding a proper amount of RNase-free water to melt the RNA sample

12) And (3) measuring the OD260/280 ratio and the OD260 value by using a micro spectrophotometer, wherein the OD260/280 ratio is 1.8-2.0, and the RNA quality is good. RNA concentration was calculated from OD 260.

Tissue total RNA extraction

1) Preparation of the experiment: consumables (RNase-free gun head and EP tube) are involved in the RNA extraction process;

2) weighing about 50mg of tissue sample, adding grinding beads, adding 200 mul of Trizol separation reagent, oscillating and homogenizing, then supplementing 800 mul of Trizol separation reagent, and standing for 10min at room temperature;

3) mixing, centrifuging at 4 deg.C and 12,000rpm for 5min, discarding precipitate, and collecting supernatant to new RNase-free 1.5ml centrifuge tube;

the rest is the same as the total RNA extraction steps 4) -12).

2.7 statistical analysis

All data were statistically analyzed using GraphPad 7.0 software, data are expressed as Mean ± SEM, and comparisons of Mean between groups were performed using one-way variance analysis (. P < 0.05,. P < 0.01).

3. Results

3.1M 2-EVs isolation and Induction identification

3.1.1Isolation and Induction identification

Inoculating the monocyte separated from abdominal cavity of mouse into T25 cell culture bottle, inducing it to differentiate into macrophage under M-CSF (20ng/ml), incubating overnight, removing culture medium, washing off suspended cells with PBS, and collecting the residual adherent cells. Flow results show that the positive rate of the macrophage cell positive marker F4/80 separated by us is 95.70%, and the successful separation is prompted to obtainAnd has a higher purity (fig. 1).

The cells were larger and irregular in shape, triangular or elongated as seen under an inverted microscope (FIG. 2). Subsequently, the process of the present invention,under the condition of IL-4/IL-13, the induction time is 48hMost cells were seen to be oval, spindle-shaped, etc. under an inverted microscope (FIG. 2).

qRT-PCR result extractionShows that the cell surface after 48h of IL-4/IL-13 combined induction highly expresses Arg1, CCL17, Mrc1, Retnla, CCL17 and TGF-beta (figure 3), and the results show that the cells successfully isolate and induce。

3.1.2 isolation and identification of M2-EVs

CollectingThe cell culture medium supernatant was subjected to ultracentrifugation to obtain EVs, and the separated products were identified from their morphology, size and surface molecular markers using TEM, NTA and WB, respectively. TEM results suggest that the isolated product is circular or elliptical with a lipid bilayer structure (fig. 4A). NTA results show that M2-EVs fit the EVs particle size distribution. The average particle size of M2-EVs was about 150nm (FIG. 4B). The Western blot results showed that both isolated products and cells expressed the positive markers HSP70, TSG101 and ALIX for EVs, and did not express the negative marker GM130 (fig. 4C). The above results indicate that we successfully isolated EVs from cell supernatants.

3.2 in vitro anti-inflammatory function study of M2-EVs

3.2.1Can take M2-EVs

To verifyUptake of EVs, treatment with M2-EVs labeled with the membrane fluorescent dye PKH26After 4h, the EVs labeled with PKH26 were able to enter as shown in FIG. 5 when observed under an inverted fluorescence microscopeAnd is mainly concentrated in the cytoplasm. The above results suggest that,can take M2-EVs.

3.2.2 inhibition of LPS-induced by M2-EVsInflammatory factor release

To evaluate the anti-inflammatory function of M2-EVs, LPS and gamma-IFN induced by treatment with different concentrations of EVsModels of inflammation. The qRT-PCR detection result shows (figure 6) that M2-EVs can inhibit the increase of TNF-alpha and IL-6mRNA expression level induced by LPS/gamma-IFN, and the result is in a concentration-dependent mode, and has statistical significance, which indicates that M2-EVs can effectively inhibit LPS-induced TNF-alpha and IL-6mRNA expression levelExpression of the inflammatory factors TNF-alpha and IL-6.

In addition, ELISA results showed (FIG. 7), TNF-. alpha.and IL-6 expression at the protein level was also inhibited. The results together show that M2-EVs can reduce the expression level of inflammatory factors in vitro.

3.3 in vivo anti-inflammatory function study of M2-EVs

3.2.1 in vivo distribution of EVs

To understand the distribution characteristics of M2-EVs after entering the mice, the fluorescent dye Cy7 labeled M2-EVs was injected into the mice through the tail vein, the mice were killed at the time points of 2h and 6h, and the major organs were taken to observe the distribution. The results are shown in the figure, and red fluorescence signals are detected in the heart, liver, spleen, lung and kidney, wherein the fluorescence signal in the liver is the strongest (figure 8), which indicates that M2-EVs can reach the main organs through blood circulation after entering the body.

3.2.2 evaluation of anti-inflammatory function in vivo of 2-EVs

Based on the results of the previous studies, we evaluated the anti-inflammatory function of M2-EVs using the LPS-induced C57BL/6 mouse acute inflammation model. Shown is the effect on the level of inflammation in the body 4h after intravenous administration of EVs. The ELISA results suggested that both serum TNF-. alpha.and IL-6 levels were significantly reduced in the M2-EVs treated group compared to the model group (FIG. 9).

In addition, as shown in FIG. 10, the qRT-PCR results suggested that the expression levels of mouse liver, lung and kidney tissue inflammatory factors TNF-alpha and IL-6 were also significantly reduced in the M2-EVs treated group. The results together show that M2-EVs can reduce the level of inflammation in the body.

Spleen tissue flow results As shown in FIG. 11, model group LPS treatment significantly increased pro-inflammatory typesThe ratio of the marker F4/80+ CD11c + in the M2-EVs intervention group reduced the expression of F4/80+ CD11c + with statistical differences (FIG. 11). Suggesting that M2-EVs may decrease pro-inflammatoryThe ratio exerts an anti-inflammatory effect.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.