CN115404205A

CN115404205A - Novel exosome and preparation method and application thereof

Info

Publication number: CN115404205A
Application number: CN202210193450.1A
Authority: CN
Inventors: 杨灵巧; 许震宇
Original assignee: Zhejiang Puhui Medical Technology Co ltd; Zhejiang Shengchuang Precision Medical Technology Co ltd
Current assignee: Zhejiang Puhui Medical Technology Co ltd; Zhejiang Shengchuang Precision Medical Technology Co ltd
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2022-11-29

Abstract

The invention provides a novel exosome and a preparation method and application thereof. Specifically, the invention provides an exosome TNF alpha-EXO, a preparation method and application thereof, and relates to the technical field of biology. The exosome TNF alpha-EXO is obtained by culturing MenSCs by using a culture medium containing TNF alpha, can promote polarization of M2 macrophage, reduces expression of proinflammatory factors, and has a remarkable curative effect on IBD. Meanwhile, the exosome TNF alpha-EXO can maintain stable and unmetabolized state under the resting state of macrophages, and plays a positive role in prevention.

Description

Novel exosome and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a novel exosome TNF alpha-EXO and a preparation method and application thereof.

Background

Inflammatory Bowel Disease (IBD) is a chronic inflammatory bowel disease characterized by an extended duration of chronic disease, including Ulcerative Colitis (UC) and Crohn's Disease (CD). The exact mechanism of IBD is not known, but has been shown to be associated with environmental, genetic and intestinal flora. The uncertainty of the pathology and mechanism of the disease has led to limited research into the treatment of IBD. Traditional treatment methods, including immunosuppressants and biological drugs, are prone to generate therapeutic drug resistance, and have poor curative effects and obvious side effects. Anti-tumor necrosis factor-alpha (TNF- α) antibodies are the most advanced treatment methods, but this method is not responsive in up to 40% of patients. Therefore, a more suitable alternative therapy is urgently needed.

Exosomes are vesicles of 30 to 150 nanometers, rich in proteins and complex ribonucleic acids. Exosomes have the advantages of small size, direct action, easy storage, and they do not face the safety issues associated with cell therapy. In the study of IBD, mesenchymal stem cell exosomes from bone marrow, adipose and umbilical cord have been shown to have therapeutic effects on IBD, but their therapeutic mechanisms are very different. Menstrual blood-derived stem cells (MenSCs) are a novel mesenchymal stem cell from female menstrual blood. Advantages of MenSCS include easy availability, high cell proliferation rate, and no ethical issues. However, there is currently no relevant study of MenSCs for treating IBD.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

It is an object of the present invention to provide a TNF α -EXO.

Another object of the present invention is to provide a process for producing the TNF α -EXO.

The invention also aims to provide application of the TNF alpha-EXO.

The fourth object of the present invention is to provide a pharmaceutical composition for preventing and/or treating IBD.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

an exosome TNF α -EXO obtained by culturing MenSCs in a TNF α -containing medium.

Further, the content of TNF alpha is 10-30ng/ml;

preferably, the MenSCs are P5-P8.

Further, the culturing comprises: adherent MenSCs were cultured to 80% -90% confluence using TNF α -containing medium.

Further, the miR-24-3p content of the exosome TNF alpha-EXO is increased;

preferably, after MenSCs are cultured in the TNF alpha-containing medium, the method further comprises the step of culturing for 36-60h in a serum-free medium, and collecting supernatant and separating to obtain exosome TNF alpha-EXO.

The preparation method of the exosome TNF alpha-EXO comprises the steps of culturing MenSCs by using a culture medium containing TNF alpha, then culturing for 36-60h by using a serum-free culture medium, collecting supernatant and separating to obtain the exosome TNF alpha-EXO.

Further, the content of TNF alpha is 10-30ng/ml.

Further, the culturing comprises: adherent MenSCs were cultured to 80% -90% confluence using a TNF α -containing medium.

Use of the above exosome TNF α -EXO in any of:

(a) Preparing a medicament for preventing and/or treating IBD;

(b) Promoting the conversion of M1 macrophages to M2 macrophages;

(c) Inhibiting IRF-1 gene expression.

An agent for preventing and/or treating IBD, comprising the above-mentioned exosome TNF α -EXO.

Further, the administration includes intraperitoneal injection, intravenous injection or subcutaneous injection.

Compared with the prior art, the invention has the technical effects that:

the inventor finds that exosome TNF alpha-EXO generated by MenSCs after being stimulated by TNF alpha can promote polarization of M2 macrophages, reduce expression of proinflammatory factors and have obvious curative effect on IBD. Meanwhile, the exosome TNF alpha-EXO can maintain stable and unmetabolized state under the resting state of macrophages, and plays a positive role in prevention.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following descriptions are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the results of detection of surface markers for CD29, CD34, CD45, CD73, CD90, CD105, CD117, HLA-DR for flow cytometry detection of MenSCs as provided in example 2, with green lines indicating isotype control of each marker and purple lines indicating surface markers;

FIG. 2 is an optical image of the trilineage differentiation and morphology of MenSCs provided in example 2, wherein a is a MenSCs microscope picture, b is adipogenic differentiation, c is osteogenic differentiation, and d is chondrogenic differentiation;

FIG. 3 is a transmission electron micrograph of exosomes provided in example 2;

FIG. 4 is the Nanoparticle Tracking Analysis (NTA) results of exosomes provided in example 2;

FIG. 5 provides a Westernblot method for evaluating surface markers (CD 63, TSG101, CD81, and GAPDH) for Exo and TNF α -Exo as provided in example 2, with MenSCs as a control and GAPDH as a negative control;

fig. 6 shows DAI scores of mice provided in example 2 after intraperitoneal injection of EXO and TNF α -EXO (200 μ g/mouse) (n = 6) in mice with acute IBD;

fig. 7 shows the body weight change of mice provided in example 2 after intraperitoneal injection of EXO and TNF α -EXO (200 μ g/mouse) (n = 6) in mice with acute IBD;

FIG. 8 is the results of MPO activity measurements after intraperitoneal injection of EXO and TNF α -EXO (200 μ g/mouse) (n = 6) in mice with acute IBD as provided in example 2;

FIG. 9 is a graph of colon length data and a gross colon image obtained at day 8 as provided in example 2, at the sacrifice of mice;

fig. 10 is a photograph of dehydrated colon after immersion in 4% paraformaldehyde for 48 hours, stained with Hematoxylin and Eosin (HE) as provided in example 2, at original magnification, × 40 (top), × 100 (bottom), × p <0.05 and × p <0.01, × p <0.001 by one-way anova;

FIG. 11 is the H & E staining pathology score results in the double-blind case provided in example 2;

figure 12 shows the results of the detection of cytokine levels in the colon of mice by ELISA (n = 6) as provided in example 2;

FIG. 13 is the levels of FITC-dextran in serum (n = 6) 4 hours after oral administration of FITC-dextran (0.6 mg/g body weight) provided in example 2;

figure 14 is the immunohistochemical staining (IHC) provided in example 2, followed by optical images of the claudin ZO1, ZO2 and zonulin obtained, at original magnification x 400, p <0.05 and p <0.01, p <0.001 by one-way anova;

figure 15 is the results of recording the clinical symptoms of chronic IBD mice daily, DAI scoring based on weight loss, stool consistency, blood in stool, and mental status, as provided in example 2;

FIG. 16 is the measurement of the colon length of each mouse in example 2;

FIG. 17 shows the results of measuring the levels of TNF α, IFN- γ, IL-10, IL-1 β and IL-6 in colon tissue by ELISA method in example 2;

FIG. 18 is a photograph of H & E stained mouse colon and pathology score results in example 2;

FIG. 19 shows the ratio of M2 measured by flow cytometry after treatment with 100ng/mLLPS in example 2 and addition of 100ng/mIL-4 or 100ug/ml TNF α -EXO;

FIG. 20 shows the results of PCR detection of mRNA expression of TNF α, IL-1 β, IL-17, IFN-. Gamma., iNOS, and Arg1 in RAW264.7 after different treatments in example 2;

FIG. 21 shows the detection of PKH 26-labeled exosomes by RAW264.7, stained with FITC-piperadine and DAPI, and confocal microscopy observations in example 2;

FIG. 22 shows the measurement of Arg1 level of M2 macrophage and iNOS level of M1 macrophage in mouse colon by ELISA method in example 2;

FIG. 23 is a graph showing the proportion of PKH 26-labeled TNF α -EXO contained in PKH26+ cells in colon LPMC by flow cytometry in example 2;

FIG. 24 shows the immunofluorescence assay for DAPI, F4/80 and Arg1 in the colon of mice, observed with confocal microscopy, with PKH 26-labeled exosomes injected intraperitoneally into the mice in example 2;

FIG. 25 is a heat map of the differences in miRNA expression between EXO and TNF α -EXO in miRNA sequencing in example 2;

figure 26 is a scatter plot and a volcano plot of miRNA differential expression between samples in example 2;

FIG. 27 shows the results of KEGG pathway enrichment in example 2 for analyzing target genes differentially expressing miRNAs;

FIG. 28 is the detection result of the sequencing result of miRNA confirmed by PCR detection in example 2;

FIG. 29 is a graph showing the flow cytometry results of detecting apoptosis of MenSCs at 0, 1, 3, and 5 days after TNF α treatment in example 2;

FIG. 30 shows the results of prediction and verification of miR-24-3p targeting gene IRF1 and binding site in example 2;

FIG. 31 shows the detection of IRF1 protein expression by immunoblotting after intraperitoneal injection of TNF α -EXO in DSS-induced acute IBD mice in example 2;

FIG. 32 is a Wb image of RAW264.7 obtained after three treatments of LPS + PBS with control, LPS and LPS + TNF α -EXO in example 2;

FIG. 33 is a graph of flow cytometry images showing the ratio of M1 (F4/80 + iNOS +) and M2 (F4/80 + CD206 +) macrophages for RAW264.7 under different treatments in example 2;

FIG. 34 is a Western blot image of Arg1, iNOS, and IRF1 in lps-stimulated RAW264.7 cells 48h after transfection with a miR-24-3p mimic in example 2 and a Western blot image of miR-24-3p inhibitor transfected during MenSCs culture, supernatant collected, and miR-24-3p inhibitor TNF α -EXO isolated, and miR-24-3p inhibitor TNF α -EXO co-cultured with RAW 264.7;

FIG. 35 shows WB validation of the effect of transfection of SI-IRF1 and IRF1OE in RAW264.7 on IRF1 expression in example 2.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The present invention provides an exosome TNF alpha-EXO obtained by culturing MenSCs with a TNF alpha-containing medium.

MenSCs as a mesenchymal stem cell has the advantages of wide source, rapid proliferation and no ethical problem. The cell status of MenSCs changes with age, and the long-term passability of MenSCs provided by older donors (> 40 years) decreases. However, the immunophenotypic characteristics of the cells do not change significantly with age. MenSCs useful in the present invention are from 20-30 year old women in the first menstrual period.

TNF α, as a proinflammatory cytokine, plays an important role in the pathogenesis of colitis. Pretreatment of MenSCs with TNF α was to force exosomes secreted by MenSCs to tend to inhibit TNF α. In experiments, the inventors also found that TNF α -EXO reduced the expression of TNF α in vitro and in vivo. TNF α, generally secreted by M1 macrophages, plays an important role in the pathogenesis of inflammatory bowel disease. The excessive inflammation caused by M1 macrophage is a very important reason for causing IBD, and the exosome TNF alpha-EXO in the invention can promote the polarization of M2 macrophage, reduce the expression of proinflammatory factor and has obvious curative effect on IBD. Meanwhile, the exosome TNF alpha-EXO can maintain stable and unmetabolized state in the resting state of macrophage cells, and plays a positive role in prevention.

In a preferred embodiment, the miR-24-3p content of the exosome TNF α -EXO is increased. miR-24-3p changes polarization of macrophages by binding to 3' UTR region of downstream IRF1mRNA, leading to down-regulation of IRF1 expression and then changes polarization of M2 macrophages, thereby relieving inflammation and achieving the purpose of treating IBD.

The preparation method of the exosome TNF alpha-EXO provided by the invention comprises the following steps:

culturing MenSCs by using a culture medium containing TNF alpha, culturing for 36-60h by using a serum-free culture medium, collecting supernatant, and separating to obtain the exosome TNF alpha-EXO.

In preferred embodiments, the level of TNF α in the TNF α -containing medium can be, but is not limited to, 10ng/ml, 12ng/ml, 14ng/ml, 16ng/ml, 18ng/ml, 20ng/ml, 22ng/ml, 24ng/ml, 26ng/ml, 28ng/ml or 30ng/ml. MenSCs are preferably P5, P6, P7 or P8 cells.

Optionally, the TNF α -containing medium further comprises IL-6. Illustratively, the IL-6 content may be between 5 and 50ng/ml, e.g.IL-6 at 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 ng/ml.

In a preferred embodiment, the medium containing TNF α cultures MenSCs to 80% -90% confluence.

The advantages of exosomes are that they are small, more direct in effect, better in therapeutic effect, and able to freely pass through the blood circulation without being trapped in capillaries. When MenSCs-EXO was used to treat mice, no rejection-related symptoms were found, except for the symptoms of colon injury caused by DSS. The invention also provides application of the exosome TNF alpha-EXO, wherein the exosome TNF alpha-EXO can be used for preparing a medicament for preventing and/or treating IBD, can promote M1 macrophage to be converted into M2 macrophage, and can inhibit IRF-1 gene expression.

The invention also provides a medicament for preventing and/or treating IBD, which comprises the exosome TNF alpha-EXO provided by the invention.

In a preferred embodiment, the IBD drug may be administered by intraperitoneal injection, intravenous injection, or subcutaneous injection. Research shows that the administration mode does not affect the curative effect of the exosome TNF alpha-EXO, and the exosome TNF alpha-EXO can be quickly eliminated by a blood system and absorbed by macrophages to achieve the aim of treating diseases. Meanwhile, the macrophage can maintain stable and unmetabolized state under the resting state, and plays a positive role in prevention.

The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or directly commercially available, unless otherwise specified.

All animal care and experimental protocols in this invention were approved by the animal care and use committee of the university of zhejiang.

Male C57BL/6J mice (6-8 weeks old) were purchased from Slac laboratory animals Co., ltd (Shanghai, china). All animals are positioned 24 _± Food and water were available for free in a light/dark cycle room at 2℃, 60% humidity, 12/12 hours, with 5 animals per cage.

Example 1 Experimental method

1. Isolation and culture of MenSCs

MenSCs were obtained from menstrual blood of female volunteers. Menstrual blood samples of healthy young women (n = 3) aged 20 to 30 years were collected using Divacup (kitchen, ON). Fresh menstrual blood samples were incubated overnight in stock solutions containing amphotericin B, gentamicin sulfate, kanamycin sulfate, cephalexin, vancomycin hydrochloride, and heparin at 4 ℃. Centrifuging at 4 deg.C and 2000rpm for 10min, and detecting microorganism with supernatant.

Mononuclear cells from menstrual blood were separated by ficoll-Pague (DAKEWE, china) density gradient centrifugation. The sandwich cells were harvested and cultured in α -MEM medium (Gibco, USA) containing 15% Australian Fetal Bovine Serum (FBS). After culturing for 48 hours at 37 ℃ in a cell culture chamber, the cells were completely adhered and the medium was replaced with fresh one. Cells were completely digested with 0.25% trypsin-edta (Fisher Scientific, USA) for 5min, neutralized in complete medium, and centrifuged to complete subculture. Generations 5-8 of MenSCs (p 5-p 8) are commonly used for co-culturing with cells, harvesting exosomes and injecting into animals. To reduce cellular damage, we used 90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO) for cryopreservation of cells.

2. Characterization and flow cytometry analysis of MenSCs

To verify the multipotentiality of MenSCs, we induced MenSCs cells into osteocytes, adipocytes and chondrocytes over 15-30 days. After the cells differentiated to some extent, they were stained with alizarin red, oil red O and acyclovir blue, respectively. Flow cytometry (ACEA novoCyte, ACEA Biosciences, USA) was used to identify MenSCs surface markers. After digestion of the cells with 0.25% trypsin-EDTA, the cells were suspended in staining buffer (BD Biosciences, san Jose, CA, USA) and washed twice before antibody incubation. After the final centrifugation, the cells were resuspended in 50. Mu.l of staining buffer and the antibodies were incubated for 15-30min at 4 ℃ in a shaking table protected from light. Antibodies that bind Phycoerythrin (PE) include CD29, CD34, CD45, CD73, CD90, CD105 and CD117, and HLA-DR (Biosciences, san Jose, USA).

3. Exosome isolation

Exosomes were isolated from the cell culture supernatant of MenSCs by ultracentrifugation. After the MenSCs were passaged to P5-P8, the cells adhered to the wall overnight and cultured with 20ng/ml TNF α or without TNF α to 80% -90% confluency. Then, the cells were cultured in serum-free medium for 48 hours, and the cell supernatant was collected. The collected cell supernatant was first centrifuged in several steps (300, 2000 and 10000g, respectively, at medium and low speed for 10, 20 and 30 min) to remove dead cells and cell debris. After centrifugation, the supernatant was filtered through a 0.22 μm filter (Millipore, USA), and the filtrate was collected. The filtrate was centrifuged at 10 ten thousand g for 70 min in a super speed centrifuge (Beckman Counter, USA), the supernatant was discarded, the PBS resuspended, and 10 ten thousand g was centrifuged again for 70 min. The pellet obtained by centrifugation is an exosome. After resuspension with a small amount of PBS, it was stored in a refrigerator at-80 ℃. Exosome lytic protein (Beyotime, china) was quantified using BCA assay kit (Beyotime, china). Both MenSCs-derived Exosomes (EXO) and TNF- α -pretreated MenSCs-derived exosomes (TNF α -EXO) can be used to function in vitro and in vivo by direct addition to cell culture medium or injection into animals. And (3) separating exosome miRNA by adopting a miRNA separation kit (Qiagen, USA), and detecting the relative expression quantity of miR-24-3p by adopting q-PCR.

4. Identification of exosomes

The exosome is mainly identified by a transmission electron microscope, nanoparticle tracer analysis and Western Blotting (WB). When the exosomes are identified by a Transmission Electron Microscope (TEM), the exosomes are firstly fixed, and then a copper mesh is placed on a hydrophobic membrane. Mu.l of the immobilization suspension was added to a copper mesh. After removing the side excess liquid, the film was stained with 1% uranyl acetate, allowed to stand at room temperature for 5 minutes, and washed with distilled water. After drying, observation was carried out under a transmission electron microscope (Thermo FEI, czech Reublic). The particle size distribution and concentration of exosomes were identified with nano-vision NS500 (malvin, england). After lysis of MenSCs with or without TNF α treatment and exosomes with RIPA lysate, the protein amount was corrected using the BCA kit to ensure that each group of samples was 20 μ g. WB experiments were performed with GAPDH, CD63, CD81 and TSG101 markers.

5. Marking of exosomes

EXO and TNF α -EXO were stained with the red fluorescent dye PKH26 (SigmaAldrich, USA). Mu.l of PKH26 was added to 250. Mu.l of diluent C and 25. Mu.l of exosomes were added to the mixture. After incubation at room temperature for 5min, 500. Mu.l of exosomes were depleted of fetal bovine serum (fetal bovine serum) to stop staining, and then exosomes were extracted by ultrafiltration centrifugation. RAW264.7 was cultured on 24-well plates, and PKH 26-labeled exosomes were added to the cells when cell fusion reached 70%. After 24 hours, cells were fixed with 4% paraformaldehyde for 30 minutes. After 3 washes with PBS, they were treated with amphetamine and DAPI, respectively, and then sealed with an anti-fluorescence quenching seal, and observed with an Olympus IX-83-FV3000-OSR instrument (Olympus Corporation, japan).

6. Cell transfection

miRNA mimics or inhibitors of miR-24-3p are synthesized by Ribobio corporation (Guangzhou, china). IRF1 small interfering RNA (siRNA) and IRF1 overexpression plasmids were synthesized by shanghai workers (shanghai, china). In addition, liposomes are also used ^TM 3000 transfection reagent (ThermoFisher, USA) according to the manufacturer's instructions for transfection. And (3) detecting the transfection efficiency by adopting real-time fluorescent quantitative PCR or WB.

7. Dual luciferase reporter gene assay

Wild-type (WT) or 3' -UTR Mutant (MUT) sequences of IRF1mRNA, and recombinant pmirGLO plasmids containing WT sequences or MUT sequences were synthesized by Oligobio biotech (Beijing, china). The direct interaction between miR-24-3p and IRF1 is verified through dual-luciferase reporter gene detection. Human embryonic kidney cells (HEK 293T, ATCC) were cultured in 12-well plates at 37 ℃ in an incubator, and approximately 1X 10 cells per well were placed ⁵ And (4) one cell. When the degree of fusion reached 60%, miRNA mimics and plasmids were transfected. By usingSerum medium DMEM (Thermo Fisher, USA) and lipo3000 were co-transfected with 1. Mu.g/ml plasmid and 50nM miRNA mimic. After 6 hours, serum-free DMEM in the 12-well plates was replaced with complete medium containing 10% fetal bovine serum. 48 hours after transfection, the cells were lysed with luciferase reporter kit (Promega, USA) lysate, placed in white plates according to the manufacturer's instructions, and tested with a multifunctional microplate reader (Thermo, USA).

8. Real-time fluorescent quantitative PCR and immunoblotting method

Total RNA was extracted from RAW264.7 using an RNA extraction kit (Qiagen, USA) according to the instructions. The extracted RNA was immediately reverse transcribed into cDNA using prime script RT Kit (Takara), and quantitative polymerase chain reaction (qPCR) was performed on the cDNA using SYBR Premix TaqTM Kit (Takara) and CFX96 Rapid real-time PCR System Instrument (Biorad, USA). Quantitative analysis adopted-2 ^ΔΔCt A method is provided. The mRNA quantification was performed using GAPDH as an internal control, and the miRNA quantification was performed using u6 as an internal control. Will be 6X 10 ⁵ Cells were plated in 6-well plates. After each group was treated accordingly, the cells were lysed with RIPA lysate and protease inhibitors were added during lysis. The cells were scraped and centrifuged at 15000g for 15min to obtain the supernatant, i.e. the protein. Protein concentrations were determined using the BCA kit to balance protein concentrations among groups. Then, 4 Xloaded buffer was added and the mixture was kept in a metal bath at 100 ℃ for 10min. Proteins were then fractionated by 8-12% SDS-PAGE (5% gel 80V,8-12% gel 120V) and transferred to PVDF membrane (220mA, 90min). PVDF membrane was blocked with TBST and 5% skim milk for 1 hour at room temperature. Incubate primary antibody overnight at 4 ℃. The following day, after washing the membrane three times with TBST, the membrane was incubated with secondary anti-diluent (goat anti-rabbit IgG (HCL) -HRP conjugate (1.

In the experiment, data were statistically analyzed using graphpadprism8.0.2 (GraphPad, san Diego, CA). The difference in mean values was analyzed using one-way analysis of variance (LSD t-test) and Student's t-test. Data were from at least three independent experiments, and p-values <0.05 were considered significant.

Example 2 results of the experiment

1. Identification of MenSCs and exosomes

MenSCs are mainly identified by optical images, surface markers and trilineage differentiation. The results showed that MenSCs were positively expressed in CD29, CD73, CD90 and CD105, while MenSCs were less or even not expressed in CD34, CD45, CD117 and HLA-DR (FIG. 1).

Under microscope, menSCs are spindle-shaped and fibrous (FIG. 2, a).

Pluripotency of MenSCs can be assessed by trilinear differentiation, including adipogenic, osteogenic, and chondrogenic. After 21 to 30 days of culture in a special induction medium, adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation were observed by staining (b-d of FIG. 2).

The morphology of EXO and TNF α -EXO, oval bilayer lipid vesicles approximately 120nm in diameter, was visualized by transmission electron microscopy (FIG. 3).

In the nanoparticle tracking assay, EXO and TNF α -EXO both had diameters of 30-150nm. (FIG. 4). The average diameter of EXO was 166.8nm and the average diameter of TNF α -EXO was 163.9nm.

In WB-validated exosomes, CD63, CD81, TSG101 showed bands in EXO and TNF α -EXO, while the corresponding MSCs showed weak or no bands as controls (fig. 5).

2.TNF alpha-EXO can relieve Dextran Sodium Sulfate (DSS) induced acute IBD (inflammatory bowel disease) in mice

Acute IBD models were established using oral 5% Dextran Sodium Sulfate (DSS) 7d, and clinical symptoms were monitored daily using Disease Activity Index (DAI). Exosomes were orally administered the next day, and mice were replaced with plain drinking water on day 7. The method for chronic modeling comprises the following steps: mice were fed 3-vol DSS water for 1 week and normal water for 1 week, respectively, and one cycle was repeated with a model making time of 28 days. DAI and mortality were observed.

The next day after oral 5% DSS, the mice appeared flaccid and stooped. Intraperitoneal injection of exosomes relieved subsequent clinical symptoms, with TNF α -EXO being more potent than EXO (fig. 6).

Daily weight monitoring showed that the weight average of each group was reduced and the weight loss of TNF α -EXO group was significantly less than that of the other groups except the control group (FIG. 7).

Myeloperoxidase (MPO) activity may reflect neutrophil infiltration, and excessive MPO activity may also damage the tissue itself. Following exosome treatment, we found a decrease in MPO activity (fig. 8).

The colon length was not as short as in the untreated group (fig. 9). In particular, the colon length of the TNF α -EXO group was almost unchanged. From direct observation of the colon, it can be seen that the DSS group had red overall colon, increased permeability, loose colon contents, and severe shortening, while the exosome-treated group, including the EXO group and the TNF α -EXO group, was significantly improved.

H & E staining showed that DSS resulted in incomplete colon structure, crypt loss, structural destruction and inflammatory cell infiltration. However, in the DSS + TNF α -EXO group, the colon tissue was more intact than the DSS + PBS group and the number of lymphocytes was not high (fig. 10).

Furthermore, we performed a semi-quantitative analysis of the H & E staining results in the double-blind case, which results are represented by pathology scores (fig. 11). The pathology score may reflect the status of the colon, the higher the pathology score, the more severe the colon damage. After the treatment of the exosome, the pathological score of the colon of the mouse is obviously reduced.

3. TNF alpha-EXO can affect the expression of inflammatory cytokines and the integrity of intestinal epithelial cells

Dysfunction of inflammatory mediators and gut barrier dysfunction are two prominent features of IBD. Expression of the pro-inflammatory cytokines TNF α, IFN- γ, IL-6 and IL-17 was down-regulated and the anti-inflammatory cytokine IL-10 was up-regulated after EXO or TNF α -EXO treatment in an enzyme-linked immunosorbent assay (ELISA) (FIG. 12). In order to investigate the integrity of the intestinal epithelium, the expression of zonulin ZO1, ZO2 and zonulin of the colon and the content of FITC-dextran in the serum after 4 hours of FITC-dextran were investigated by immunochemical methods. In the FITC dextran assay, the higher the FITC value, the higher the permeability of the intestinal epithelium. High intestinal permeability indicates that the intestinal epithelial cells are incomplete after injury. The fluorescence value of FITC in the serum of the mice is detected by a microplate reader to reflect the content of FITC-dextran. We found that there was almost no FITC fluorescence in the sera of the control mice, while the serum fluorescence of the TNF α -EXO group was much lower than that of the other groups (FIG. 13). In addition, the contents of zonulin ZO1, ZO2 and zonulin in colon of mice are detected through immunohistochemical experiments. As can be seen from the figure, the expression levels of these three proteins were significantly higher in the control group and TNF α -EXO group than in the other two groups (FIG. 14).

4. TNF alpha-EXO can reduce Dextran Sodium Sulfate (DSS) induced chronic IBD in mice

To investigate the role of TNF α -EXO in chronic and recurrent inflammatory bowel disease, two cycles of oral 3-th dss were followed, and mice receiving two cycles of treatment (day 7 and day 16, i.e., 200 μ g exosomes per mouse) were found to have more clinically manifested themselves in different degrees of bloody stools, diarrhea, weight loss and colon shortening after different treatments (fig. 15). The colon length of this group is closest to the unmodeled control group (fig. 16).

In ELISA, after TNF alpha-EXO is injected in the abdominal cavity twice, the levels of TNF alpha, IFN-gamma, IL-6 and IL-1 beta in colon are obviously reduced. Unexpectedly, the level of IL-10 appeared to be higher for the single dose than for the two doses (FIG. 17).

H & E staining showed that colon epithelium was intact, crypt and goblet cell structures were normal, and only mild inflammatory cell infiltration in two doses of mice. In the colon treated with exosomes only on day 7, both cranial fossa damage and inflammatory cell infiltration were evident (fig. 18). From some data, the expression of inflammatory cytokines in mice injected with exosomes only on day 7 was the same as that of mice injected on

days

7 and 16. However, colon length, H & E staining and DAI in mice all reflected better efficacy at both doses (day 7 and day 16).

Importantly, it was found experimentally that the addition of 5-50ng/ml of IL-6 in addition to 20ng/ml of TNF α during exosome production was significantly beneficial for the treatment and amelioration of chronic IBD in mice. Illustratively, exosomes prepared using 20ng/ml TNF α +50ng/ml IL-6 could achieve 16 days of effect on the seventh day with 20ng/ml TNF α, fast colon length recovery, and rapid and significant improvement in pit damage and inflammatory cell infiltration. In addition, in addition to 20ng/ml TNF α, exosomes prepared with 5 and 50ng/ml IL-6, respectively, were significantly superior to exosomes obtained from 20ng/ml TNF α culture alone, especially in colon length recovery, in treating chronic IBD in mice.

5. MenSCs-EXO in vitro conversion of macrophages to the M2 phenotype

To investigate whether TNF α -EXO could promote polarization of M2 macrophages, TNF α -EXO (100 μ g/mL) was added to RAW264.7 cultures. RAW264.7 requires stimulation with 100ng/ml LPS to expose macrophages to the inflammatory microenvironment prior to exosome therapy. Flow cytometry revealed F4/80 after 48 hours of co-culture with TNF α -EXO or IL-4 ⁺ CD206 ⁺ Cells exceeded 30% and were identified as M2 macrophages (fig. 19).

To further understand the secretion of inflammatory cytokines in cells after different treatments, we verified the expression of inflammatory cytokines such as TNF α, IFN- γ, IL-1 β, IL-17, etc. by PCR. However, both TNF α -EXO and EXO reduced the expression of proinflammatory factors compared to the LPS group, consistent with our hypothesis that exosomes could reduce inflammation. We suspect that EXO can also reduce inflammation by a different mechanism, although not as much as TNF α -EXO. TNF α -EXO down-regulated the M1 marker iNOS and up-regulated the M2 marker CD206 in RAW264.7, indicating that TNF α -EXO can convert M1 macrophages to M2 macrophages (FIG. 20).

To further explore how exosomes affect macrophages, we labeled exosomes with PKH26 and observed the red light location of PKH26 under a confocal microscope by immunofluorescence experiments. The results show that PKH 26-labeled exosomes are abundant in the cytoplasm of macrophages, with and without TNF α stimulation (fig. 21). Thus, exosomes are taken up into the cytoplasm by macrophages, promoting macrophage type change.

6. MenSCs-EXO in vivo converts M1 macrophages to the M2 phenotype

The above experiments demonstrate that TNF α -EXO can convert M1 macrophages to M2 macrophages in vitro, and this example further studies whether the same effect can be observed in mice. In a mouse model of acute IBD, sacrificed colons 7 days after oral DSS were removed and tested by ELISA for iNOS and Arg 1. The results showed that after intraperitoneal injection of TNF α -EXO, arg1 expression was increased and iNOS expression was decreased in the colon of mice (FIG. 22).

We further investigated whether exosomes are taken up by cells in colon tissue and whether exosome function promotes M2 macrophage polarization by flow cytometry and immunofluorescence. Mice were intraperitoneally injected with PKH 26-labeled exosomes, and mouse colons were obtained within one week. Lamina Propria Mononuclear Cells (LPMC) were then isolated and analyzed by flow cytometry. Flow cytometry showed that after oral DSS treatment, the number of lamina propria PKH26+ monocytes was more than 3 times that of the NC group (fig. 23). This indicates that monocytes in the lamina propria of the colonic mucosa have some uptake capacity for exosomes, which is enhanced in the inflammatory environment. In immunofluorescence assays of colon tissue, arg1 and PKH26 positive sites were nearly identical in the TNF α -EXO injected group, while Arg1 positive cells were rare in the other groups (fig. 24). This suggests that macrophages that take up TNF α -EXO can be converted to M2 macrophages, thereby reducing excessive inflammation of the colon.

TNF alpha-EXO relieves colonic inflammation by macrophages after intraperitoneal injection of TNF alpha-EXO in mice. To verify the important role of macrophages in this process, we attempted to eliminate macrophages in mice with chlorophosphate liposomes (clotrimones) in a mouse model of acute IBD. After a single intraperitoneal injection of 250. Mu.l of clotting fat, bone marrow cells of the mice (after red blood cell lysis) were collected 7 days later and examined by flow cytometry. Flow cytometry results showed that the percentage of F4/80+ cells decreased by about 80%, demonstrating that clotted lipids successfully cleared most of the macrophages in mice. Our findings failed to affect colitis phenotype after intraperitoneal injection of TNF α -EXO in an acute IBD model after macrophage depletion from mice with clotted lipids.

7. TNF alpha induces changes in miRNA in exosomes from MenSCs

MenSCs-EXO (EXO) and TNF α -MenSCs-EXO (TNF α -EXO) were sequenced on the Illumina HiSeqTM 2500 platform (Guangdong Ribobio Biotechnology Co. LTD). Differences between miRNAs sample expression were screened by difference ratio (| log2 (FoldChange) | > 1) and significance level (P value < 0.05). After TNF alpha stimulation, exosomes such as Hsa-miR-708-5p, hsa-miR-1260b, hsa-miR-24-3p and the like in MenSCs are remarkably up-regulated, while Hsa-miR-365A-5p, hsa-miR-19a-3p and Hsa-miR-490-5p are remarkably down-regulated (figure 25). The inter-sample miRNA differential expression scattergram and miRNA differential expression volcano map reflect differential miRNA expression: differential mirnas, 38 of which were up-regulated and 32 down-regulated (figure 26). We then performed a biological pathway analysis on all differentially expressed mirnas. The analysis of the biological pathway was based on Kyoto encyclopedia of genes (KEGG) biological pathway database (http:// www.genome.jp /). From the perspective of a complex regulatory network, the analysis of the biological pathway enrichment of biological pathways in a gene set was performed. Enrichment analysis results showed that differential expression of mirnas was associated with specific viral and bacterial infections, endocytosis, PI3K-Akt signaling pathways, and signaling pathways that regulate stem cell pluripotency (figure 27). These results indicate that the effect of TNF α on MenSCs alters the function of exosomes produced by MenSCs, and that different miRNAs from TNF α -MenSCs are involved in different biological processes. Through the analysis of differential miRNAs and target proteins downstream of the differential miRNAs, we only focus on miR-24-3p for further research.

To verify the sequencing results, we extracted exosomes from MenSCs treated with TNF α for 0, 1, 2, 3, and 4 days, and obtained the relative expression of miR-24-3p using PCR. We found that the longer the TNF α treatment time, the higher the content of miR-24-3p in the obtained exosomes (FIG. 28). The longer the TNF α treatment, the worse the cell status and the higher the number of apoptotic cells. Thus, menSCs were treated with 20ng/ml TNF α for 0, 1, 3, and 5 days and the number of apoptosis was confirmed by flow cytometry (FIG. 29). Finally, menSCs were treated with TNF α for 48h to obtain high quality supernatants. In addition to the duration of TNF α action on MenSCs, TNF α concentration also affects MenSCs cell viability. Quantitative determination of different concentrations of TNF alpha treated MenSCs with Cell Counting Kit 8 (Cell Counting Kit 8, CCK8) revealed that the number of MenSCs was significantly reduced 48h after 50ng/mL TNF alpha treatment. Thus, 20ng/ml of TNF α is a safe concentration of MenSCs to produce anti-inflammatory exosomes.

8. MiR-24-3p in TNF α -EXO by downregulation of IRF1 to convert M1 to M2

According to the bioinformatics website Targetscan analysis, miR-24-3p targeted downstream IRF1 has a great correlation with macrophage cell polarization. In addition, the website predicts that two binding sites may exist between the 3' -UTR of miR-24-3p and IRF 1. And (3) detecting and verifying the two binding sites by adopting dual-luciferase reporter genes, and discussing whether the miR-24-3p can reduce the expression of IRF 1. The wild type or mutant 3' -UTR of IRF1 site 1 was used for the detection of luciferase reporter gene in the presence or absence of miR-24-3p overexpression. The binding of the transfected miR-24-3p to the wild-type 3'-UTR results in a decrease in luciferase activity, while the mutant 3' -UTR sequence prevents the binding of miR-24-3p, so that luciferase activity remains unchanged. These results indicate that miR-24-3p can act directly on IRF1 at position 1 of the 3' -UTR. In contrast, there was no change in luciferase activity at site 2 in the experiment, which was predicted to be unable to bind to miR-24-3p or unstable after binding (FIG. 30).

In mice, it was found by WB experiments that intraperitoneal injection of TNF α -EXO also down-regulated IRF1 expression in mouse colon (fig. 31). This suggests that miRNA in TNF α -EXO may play an important regulatory role in vivo. WB also demonstrated that co-culture with TNF α -EXO induced down-regulation of the M1 marker and up-regulation of the M2 marker in RAW264.7 cells (FIG. 32).

The mechanism by which TNF α -EXO converts macrophages from M1 to M2 can be verified by in vitro flow cytometry and WB experiments. After RAW264.7 is treated differently by flow cytometry, LPS stimulation is found to increase the proportion of M1 macrophages remarkably, TNF alpha-EXO is added to reduce the proportion of M1 and increase M2.TNF α -EXO acts in RAW264.7 as does miR-24-3pmimic, both of which are able to increase M2 macrophages by about 30%. To demonstrate that miR-24-3p functions due to binding to IRF1mRNA, IRF1 was overexpressed, and it was found that overexpression of IRF1 (IRF 1 oe) can partially offset the effect of miR-24-3p mimetics on macrophage polarization (FIG. 33). The same is true in WB, when TNF α -EXO is added to RAW264.7 or transfected with miR-24-3p, the M2 marker is up-regulated and M1 is down-regulated. When miRNA is over-expressed (miR-24-3 p mimic transfection), the downstream target protein is also reduced, and when miRNA is inhibited (miR-24-3 p inhibitor transfection), the downstream target protein is simultaneously increased. Knocking out (si-IRF 1) or overexpressing a downstream target protein IRF1 can partially rescue the effects of miR-24-3p mimetics or inhibitors. After transfection of TNF alpha-MenSCs with NC inhibitors or miR-24-3p inhibitors, the obtained exosomes were co-cultured with RAW 264.7. The research shows that after miR-24-3p is inhibited, TNF alpha-EXO can not promote the polarization of macrophages any more. This demonstrates that miR-24-3p does influence M2 production by interfering with expression of downstream IRF1 (fig. 34). The effect of si-IRF1 and IRF1oe on IRF1 expression was verified by Western blot (FIG. 35).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications or substitutions do not depart from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. An exosome TNF α -EXO, obtained by culturing MenSCs with a TNF α -containing medium.

2. Exosome TNF α -EXO according to claim 1, characterized in that the content of TNF α is 10-30ng/ml;

preferably, the MenSCs are P5-P8,

optionally, the TNF α -containing medium further comprises IL-6.

3. Exosome TNF α -EXO according to claim 1 or 2, characterized in that said culturing comprises: adherent MenSCs were cultured to 80% -90% confluence using a TNF α -containing medium.

4. An exosome TNF α -EXO according to any of claims 1-3, characterized in that its miR-24-3p content is increased;

5. A method for producing an exosome TNF α -EXO according to any one of claims 1 to 4, wherein the exosome TNF α -EXO is obtained by culturing MenSCs in a TNF α -containing medium, then culturing the MenSCs in a serum-free medium for 36 to 60 hours, collecting the supernatant, and isolating the supernatant.

6. The method of claim 5, wherein the amount of TNF α is about 10ng/ml to about 30ng/ml.

7. The production method according to claim 5 or 6, wherein the culturing comprises: adherent MenSCs were cultured to 80% -90% confluence using TNF α -containing medium.

8. Use of the exosome TNF α -EXO of any one of claims 1-4 in any one of:

(a) Preparing a medicament for preventing and/or treating IBD;

(b) Promoting the conversion of M1 macrophages to M2 macrophages;

(c) Inhibiting IRF-1 gene expression.

9. A medicament for the prophylaxis and/or treatment of IBD, comprising the exosome TNF α -EXO of any of claims 1-4.

10. The medicament for preventing and/or treating IBD according to claim 9, wherein the administration mode comprises intraperitoneal injection, intravenous injection or subcutaneous injection.