CN111514166A - Application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in drugs for autoimmune diseases - Google Patents

Abstract

The invention relates to the application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the preparation of medicines for treating autoimmune diseases. The MSCs-derived sEVs overexpressing IL-10 of the present invention not only have the characteristics and advantages shared by sEVs, such as easy transportation and storage, repeated freezing and thawing, low immunogenicity, etc., but also combined with the strong immunosuppression of IL-10 It has great potential clinical application value, and has the potential to develop into new clinical biological preparations, and promote the innovation and development of ophthalmic drugs. In addition, it was found that sEVs have inflammatory chemotactic ability and can accumulate to diseased organs to exert immunosuppressive effects.

Description

Mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in autoimmune Application in Epidemic Disease Drugs

technical field

The invention belongs to the field of cell technology, in particular to the application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the treatment of autoimmune diseases.

Background technique

Autoimmune disease is a type of disease in which the body produces an immune response against self-antigens and causes damage to its own tissues. The occurrence of autoimmune diseases is mainly due to the destruction of the autoimmune tolerance mechanism, the body produces antibodies against self-antigens or autoimmune cells attack its own tissues, resulting in severe tissue damage and dysfunction. Common autoimmune diseases include autoimmune uveitis, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis.

At present, traditional drugs for the treatment of autoimmune diseases mainly include glucocorticoids and immunosuppressants. However, both have many toxic and side effects, which limit their clinical application. Various biological agents, such as a variety of anti-tumor necrosis factor and anti-interleukin-6 antibodies, have been used for the treatment of autoimmune diseases. Cytokines are not effective in all patients. The development of safer and more effective new clinical therapeutic drugs is still the focus of autoimmune disease research.

Mesenchymal stem cells (MSCs) provide new ideas for the treatment of autoimmune diseases. MSCs are adult stem cells derived from the mesoderm. In vitro-expanded MSCs have strong immunosuppressive effects and can simultaneously play a regulatory role in multiple links in the immune response. In addition, in the treatment of autoimmune diseases, MSCs also have neurotrophic properties, promote wound healing and other properties, so they have advantages unmatched by commonly used drugs. However, with the deepening of research, MSCs therapy also exposed some drawbacks. As a cell therapy, MSCs have potential tumorigenic potential and are not easy to store and transport. Animal experiments have shown that myocardial infarction-related symptoms appear after intravenous administration of MSCs to dogs. In addition, the study also found that after intravenous injection of MSCs, only a small number of cells could survive for a week, which also greatly limited the therapeutic effect of MSCs. Recent studies have shown that MSCs-derived small extracellular vesicles (small extracellular vesicles, EVs) can mediate the paracrine mechanism of MSCs, and are one of the main mediators for the effect of MSCs.

Exosomes are membranous microvesicles derived from late endosomes, with a diameter of 40-150 nm and a bilayer lipid membrane structure. Exosomes are rich in protein and RNA components that can be secreted or absorbed by almost all cells. Due to their bilayer lipid membrane structure, exosomes can cross the blood-brain and blood-eye barriers. The bilayer lipid membrane structure can also effectively protect the protein and RAN components of exosomes, so that they will not be degraded and remain active for a long time, and can exert biological effects locally or at a distance. Recent studies have confirmed that MSCs-derived exosomes have some similar functions to MSCs, including regulating immunity, neuroprotection, promoting damage repair, and affecting tumor growth. Compared with cells, exosomes have more stable biological properties, are easier to transport and store, can be frozen and thawed repeatedly, and have lower immunogenicity, so they have greater potential clinical application value. As a cell-independent therapy, the tumorigenicity of exosomes and the possibility of blocking blood vessels are also greatly reduced, and it has good biological safety. In addition, due to the protection of bilayer lipid membranes, exosomes can also be used as ideal drug carriers to carry macromolecular drugs across biological barriers to exert therapeutic effects. The applicant's previous research found that MSCs-derived exosomes can effectively treat autoimmune uveitis animal models, but the efficacy still needs to be further improved. Since the current exosome isolation methods cannot completely remove non-exosome components, such as microvesicles (MVs), and there is a lack of direct evidence that exosomes are the source of endosomes, according to the latest expert consensus, it is recommended to use vesicle-based The method of vesicle diameter names the isolated extracellular microvesicles. The applicant therefore uses the term "Small extracellular vesicles" (sEVs) to name the isolated extracellular vesicles with a diameter of less than 150 nm.

Interleukin 10 (IL-10) is an important negative immunoregulatory factor, which participates in the negative regulation of various immune mechanisms. In addition to inhibiting the release of various inflammatory factors, it can also inhibit the expression of antigen-presenting cells and macrophages in the Activation and migration of a variety of immune cells within. However, some studies have shown that the effect of single injection of recombinant IL-10 in the treatment of autoimmune diseases is not ideal, and the half-life of recombinant IL-10 injected alone is short and expensive, which greatly limits its clinical application. If MSCs-derived sEVs can be used as the carrier of IL-10, the long-term stability of IL-10 can be maintained, which is beneficial for IL-10 to cross the biological barrier, and can simultaneously exert the immunomodulatory effects of exosomes and IL-10.

Through searching, no published patent documents related to this patent application have been found.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies in the prior art and provide an application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the treatment of autoimmune diseases.

The technical scheme adopted by the present invention to solve its technical problems is:

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of medicines for treating autoimmune diseases.

Moreover, the autoimmune diseases mainly include autoimmune uveitis and multiple sclerosis.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of drugs for inhibiting T cell proliferation.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of drugs for inhibiting the differentiation ability of Th1 cells.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of drugs for inhibiting the differentiation ability of Th17 cells.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of immunosuppressive drugs.

The advantages and positive effects obtained by the present invention are:

1. The MSCs-derived sEVs overexpressing IL-10 of the present invention have the advantages of MSCs-derived exosomes, including functions such as immune regulation and neuroprotection, easy transportation and storage, low immunogenicity, etc. and better biological safety. sex. In addition, it was found that sEVs have inflammatory chemotactic ability and can accumulate to diseased organs to exert immunosuppressive effect.

2. The present invention uses MSCs-derived sEVs as a carrier to selectively enrich IL-10, which can maintain the long-term stability of IL-10, facilitate its passage through biological barriers, and thereby enhance its immunosuppressive effect.

3. In the present invention, IL-10 and MSCs-derived sEVs play a synergistic immunosuppressive effect, so that MSCs-derived sEVs overexpressing IL-10 can effectively inhibit Th1 and Th17 immune responses at the same time, which is more effective than MSCs-derived sEVs. Stronger immunosuppressive effect, showed stronger therapeutic effect on autoimmune uveitis and meningitis.

4. The human umbilical cord MSCs-derived sEVs overexpressing IL-10 of the present invention can be used to prepare a new Cell-Free biological preparation for the treatment of autoimmune diseases, which has great potential clinical application value and can promote the development of clinical autoimmune disease drugs. Innovation development.

Description of drawings

Fig. 1 is the fluorescence image of lentivirus infection MSCs in the present invention;

Fig. 2 is the transmission electron microscope identification diagram of two kinds of sEVs in the present invention;

Fig. 3 is the Nanosight identification figure of two kinds of sEVs in the present invention;

Fig. 4 is the Western-blot identification diagram of two kinds of sEVs in the present invention;

Figure 5 is the ELISA assay chart of IL-10 in sEVs-10 in the present invention, ***: P<0.01;

Figure 6 is a graph showing the clinical scores of mice in the present invention, *: P<0.05; ***: P<0.01;

Figure 7 is an example diagram of retinal HE staining on the 18th day after modeling in the present invention;

Fig. 8 is a statistical histogram of pathological scores in the present invention, *: P<0.05; ***: P<0.01;

Figure 9 is a graph showing the infiltration of Th1 cells (IFN-γ positive) and Th17 (IL-17A positive) cells in the eyeballs of mice in each group of the present invention;

Figure 10 is a statistical histogram of ocular inflammatory cell infiltration in the present invention, *: P<0.05; ***: P<0.01;

Figure 11 is an example diagram of HE staining in each group of EAE in the present invention;

Figure 12 is a graph showing the inhibitory effect of the sEVs-N group and the sEVs-10 group on the in vitro differentiation of Th1 (IFN-γ positive) cells in the present invention;

Figure 13 is a statistical histogram of the differentiation of Th1 cells in the PBS group, sEVs-N group, and sEVs-10 group in the present invention, *: P<0.05; ***: P<0.01,

Figure 14 is a graph showing the inhibitory effect of the sEVs-N group and the sEVs-10 group on the in vitro differentiation of Th17 (IL-17A positive) cells in the present invention;

Figure 15 is a statistical histogram of the differentiation of Th17 cells in the PBS group, sEVs-N group, and sEVs-10 group in the present invention, *: P<0.05; ***: P<0.01;

Figure 16 is a graph showing the inhibitory effect of sEVs-N group and sEVs-10 group on T cell proliferation in vitro in the present invention;

Figure 17 is a statistical histogram of proliferation of T cells in vitro in the present invention, *: P<0.05; ***: P<0.01;

Figure 18 is a diagram showing the distribution of sEVs-N and sEVs-10 in the retina of EAU and Naive mice according to the present invention.

Detailed ways

The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are descriptive, not restrictive, and cannot limit the protection scope of the present invention.

The raw materials used in the present invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional methods in the art unless otherwise specified.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of medicines for treating autoimmune diseases.

Preferably, the autoimmune disease is autoimmune uveitis or multiple sclerosis.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of drugs for inhibiting T cell proliferation.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of drugs for inhibiting the differentiation ability of Th1 cells.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of drugs for inhibiting the differentiation ability of Th17 cells.

The mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 are used in the preparation of immunosuppressive drugs.

Specifically, the relevant preparation and detection are as follows:

Part 1: Isolation and culture of human umbilical cord MSCs

Human umbilical cord tissue was obtained from healthy fetuses delivered by caesarean section in a cooperative obstetrics and gynecology hospital. Select healthy donor umbilical cord tissue with a diameter of more than 15cm, cut the umbilical cord tissue into a small section with a diameter of 1-1.5mm, do not wash, so as to retain the original environment of the tissue, and then directly spread each umbilical cord tissue block evenly by 50 pieces/bottle Inoculated into 9 T75 culture flasks.

Incubate the flasks containing the umbilical cord tissue in a 5% _CO2 incubator for 5 h without the addition of cell culture medium. After the incubation, 5ml of complete cell culture medium (DMEM/F12 (DF-12) medium plus 10% fetal bovine serum (FBS)) was added to each group of culture flasks, and 10ml of complete culture medium was added after overnight culture. Change the culture medium every 3-5 days. After about 10 days, carefully observe the level of cells crawling out of each bottle. When the cell confluence is 80%, carefully remove the umbilical cord tissue block, and perform protease digestion and count the cells. New T75 flasks were seeded at a density of 1×10 ⁴ -2×10 ⁴ cells/cm ² (approximately 1:3 ratio), while adding 10 ml of complete medium to each flask to further expand the cells. When UC-MSCs reached ≥80% confluency again, passage expansion was performed with the same seeding density and culture conditions.

Part II: Preparation of IL-10 Lentivirus and Infection of Target Cells

(1) Construction of IL-10 overexpression lentiviral packaging plasmid: the CDS region of IL-10 has a full length of 1172 bp and was cloned into the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP.

(2) Packaging of lentivirus: Prepare 293T cells with a density of 80% in advance in a 10cm dish, replace DMEM + 10% FBS medium without dual antibody on the day of transfection, and transfer the total mass of 25μg of vector plasmid and packaging plasmids PAX2 and PMD2G according to 4:3:1 was fully mixed with transfection reagent (Lipofactamine2000) and added to the 293T culture flask, and the fresh culture medium was replaced in 6-10 hours. The virus supernatant was collected at 48h and 72h after transfection, filtered at 0.22 μm, concentrated by ultracentrifugation at 72000g/min for 120min at 4°C, and the virus pellet was resuspended in an appropriate amount of DMEM medium.

(3) Infection of human umbilical cord mesenchymal stem cells: The virus and transfection-promoting reagent (Polybrene, 10ng/ml) were added to 50% confluent human umbilical cord mesenchymal stem cells (passage P2) at a multiplicity of infection (MOI) of 50. , 24h liquid change. After 48 h, MSCs cells could be observed to express GFP green fluorescence under a fluorescence microscope (Figure 1). As shown in Figure 1: MSCs were extracted and cultured in vitro by digestion method. Under bright field, the cells showed a relatively uniform fibroblast-like structure and were arranged in a swirling pattern. The MSCs after lentivirus infection could observe obvious green (GFP) fluorescence under dark field, indicating that the target gene (IL-10) was successfully integrated into MSCs.

Part III: Collection of IL-10-overexpressing MSC-derived extracellular vesicles

Before the P3 passage, the cells were conventionally cultured and passaged. When the fusion of P3-P5 generation MSCs reached about 60%, the original medium was replaced with human mesenchymal stem cell serum-free medium (BI, Israel) containing 0.6% MSC serum-free supplement (BI, Israel). Continue to culture for 24 hours, collect the culture supernatant of MSCs (passages 3-5) overexpressing IL-10, centrifuge at 4°C and 300g for 3 minutes to collect the supernatant; centrifuge at 4°C and 2000g for 10 minutes to collect the supernatant; Collect the supernatant; centrifuge at 110,000 g for 70 min at 4°C, discard the supernatant; resuspend the pellet with PBS; centrifuge again at 110,000 g for 70 min, discard the supernatant, resuspend the precipitate in a small amount of phosphate buffer, sterilize by filtration with a 0.22 μm filter, to obtain overexpressed IL -10 of MSCs-derived sEVs.

More specifically, relevant preparation and detection are as follows:

1. Identification of MSCs-derived sEVs (sEVs-10) overexpressing IL-10

(1) Transmission electron microscope: Dilute the sEVs sample to an appropriate concentration, fix it with paraformaldehyde for 5 minutes, take 20 μl drops on the copper mesh grid, let it stand for 5 minutes, and then add 2% uranyl acetate solution dropwise for negative staining and fix it for 3 minutes , after removing excess liquid, observe and take pictures on the machine after drying.

Transmission electron microscopy results showed that both sEVs-10 and sEVs-N had a diameter of 40-150 nm and a round cup-shaped double-membrane vesicle structure (Fig. 2).

(2) Nanosight particle size analysis: Dilute the sEVs with double pure water to an appropriate concentration, set the number of cycles and time, load a 1ml syringe, and measure the size of the sEVs with a Nanosight particle size analyzer. Data were analyzed using Nanosight NTA3.3 software.

Nanosight results showed that the average diameters of sEVs-10 and sEVs-N were 105 nm and 101 nm, respectively; D50 was 98.1 nm, 93.8 nm; D90 was 150.3 nm, 147.2 nm (Figure 3).

(3) Determination of exosome surface markers by Western Blot method: Western Blot method was used to detect exosome surface markers CD9 and CD81. After 20 μg of total protein samples were fully lysed, denatured at 95°C for 5 min, and electroporated at 85 V for 2 h. The protein was transferred to PVDF membrane, blocked with 5% nonfat milk in TBST solution at room temperature for 1 h, the primary antibody CD9, CD81 (Abcam, USA) was diluted 1:1000 and incubated at 4°C overnight, washed with TBST, incubated with secondary antibody at room temperature for 1 h, and washed the membrane After adding ECL luminescent substrate, dark room exposure.

Western-Blot results showed that CD9 and CD81 protein markers were highly expressed in sEVs-10 and sEVs-N (Fig. 4).

The above TEM, Nanosight particle size analysis and Western Blot results indicated that the isolated sEVs were mainly exosomes.

(4) ELISA method to detect the expression of target protein: the concentration of sEVs was diluted to 1 μg/μl, and the concentration of target protein in sEVs was determined according to the instructions of IL-10 ELISA assay kit (R&D Company, USA). Measure the absorbance value of each substrate at 450nm wavelength with a microplate reader, make a standard curve and calculate the concentration of each sample.

The results showed that compared with sEVs-N, the protein content of IL-10 in sEVs-10 was significantly increased (Figure 5). ELISA results showed that the target protein IL-10 in sEVs-10 was significantly higher than that in sEVs-N (P<0.01 ). (***: P < 0.01)

2. The therapeutic effect of sEVs-10 on experimental autoimmune uveitis (EAU)

(1) Model establishment: 7-week-old, SPF-grade healthy female C57BL/6 mice were used for model establishment. After examination, it was confirmed that the refractive interstitium was clear and the fundus had no abnormality. Dissolve 250 μg/per IRBP _651-670 polypeptide fragment in a final volume of 100 μl PBS, and then mix well with an equal volume of complete Freund’s adjuvant with a tuberculin concentration of 3.5 mg/ml until the mixture is milky, and then use it in the spare mice. 4 points were evenly injected into the bilateral groin and back of the tail, and 30 minutes before and 24 hours after the modeling, PBS containing 0.5 μg of purified pertussis toxin (PTX) was intraperitoneally injected as an adjuvant immune adjuvant. agent. Housing methods were in accordance with the Society for Vision and Ophthalmology Research Standards for the Care and Use of Animals in Ophthalmology and Vision Research.

(2) Treatment method and grouping: C57BL/6 mice were divided into mesenchymal stem cell-derived sEVs treatment group (sEVs-N group) and IL-10-overexpressing mesenchymal stem cell-derived sEVs group (sEVs-N group). sEVs-10 group) and PBS control group, 6 mice in each group. The treatment intervention time was taken at the early stage of the onset of experimental autoimmune uveitis disease in mice (the 11th day after modeling). Mice in each group were injected with 50 μg (200 μl) of corresponding sEVs or the same volume of PBS via tail vein as blank control.

(3) Clinical observation and scoring: Mice in each group were dilated with compound tropicamide eye drops every other day from the 9th day after immunization, and then the fundus of mice was observed by head-mounted ophthalmoscope. Scoring and grading were based on the Caspi clinical grading, and the scoring criteria were as follows: 0 points: no changes compared with normal retina. 0.5 points: a small number of small, peripheral punctate lesions, mild vasculitis, vitreous inflammation. 1 point: moderate vasculitis, <5 small punctate lesions, ≤1 linear lesions. 2 points: multiple (>5) chorioretinal lesions or inflammatory exudation, severe vasculitis, and a few linear lesions (<5). 3 points: grid-like linear lesions, large confluent lesions, subretinal neovascularization, retinal hemorrhage, papilledema. 4 points: extensive retinal detachment, retinal atrophy.

The results showed that from 14 to 22 days after modeling, the clinical scores of mice in the sEVs-N group and sEVs-10 group were lower than those in the PBS control group, and the difference was statistically significant (p<0.05). From 16 to 22 days after modeling, the clinical scores of mice in the sEVs-10 group were lower than those in the sEVs-N and PBS control groups, and the difference was statistically significant, p<0.01 (Figure 6) (*: P<0.05; *** : P<0.01).

In conclusion, the clinical scores of mice show that: tail vein injection of MSCs-derived sEVs can achieve a certain therapeutic effect, but the effect is weak; while sEVs overexpressing IL-10 in the tail vein can achieve an ideal therapeutic effect.

(4) HE staining and scoring standard: On the 18th day after modeling, the mice were killed by de-neck method and the eyeballs were removed, fixed in 10% formalin, routinely dehydrated, and embedded in paraffin. Using a tissue microtome, the sagittal diameter of the optic disc was continuously cut into 6 μm thick sections for routine hematoxylin-eosin (HE) staining. The scoring criteria are as follows: 0 points: no changes compared with normal retina. 0.5 points: slight inflammatory cell infiltration, no tissue damage. 1 point: Inflammatory cell infiltration, retinal folds, a small number of small granulomas in the retina and choroid, peripheral vascular inflammation. 2 points: moderate inflammatory cell infiltration, retinal folds, local photoreceptor cell damage, mild to moderate granulation tissue. 3 points: moderate to severe inflammatory infiltration, extensive retinal fold detachment, moderate photoreceptor cell damage, moderate granuloma damage, and subretinal neovascularization. 4 points: Severe inflammatory infiltration, diffuse retinal detachment with severe exudation, subretinal hemorrhage, extensive photoreceptor cell damage, severe granulomatous damage, and subretinal neovascularization.

18 days after modeling, the pathological scores of the retinas of the mice showed that the pathological scores of the mice in the sEVs-N group and the sEVs-10 group were lower than those in the PBS control group, and the difference was statistically significant (p<0.05) (Figure 7). The infiltration of inflammatory cells in retinal tissue and vitreous cavity of mice in group -10 was significantly reduced, and no serious structural disorder was observed in the retina. The pathological scores of mice in the sEVs-10 group were lower than those in the sEVs-N group and the PBS control group, and the difference was statistically significant (p<0.01) (Fig. 8) (*: P<0.05; ***: P<0.01).

(5) Detection of T cell infiltration in mouse eyeball by flow cytometry: Th1 and Th17 cells are the main pathogenic cells in the pathological process of EAU mice. The mice were sacrificed on the 15th day after immunization, and the eyes of the mice were taken, the optic nerve, cornea, and lens were cut off, and then digested with collagen D for 30 min to prepare a lymphocyte suspension. After stimulation for 5 h, the cells were fixed, permeabilized, stained with corresponding antibodies, and the proportions of IFN-γ ⁺ CD4 ⁺ T cells (representing Th1 cells) and IL-17 ⁺ CD4 ⁺ T cells (representing Th17 cells) were detected by flow cytometry.

The results showed that the proportions of Th1 cells and Th17 cells in the eyeballs of the mice in the sEVs-10 group were lower than those in the sEVs-N group and the PBS group, and the difference was statistically significant (p<0.01); the proportions of the two in the sEV-N group were lower than those in the PBS group , the difference was statistically significant (p<0.05) (Figure 9, Figure 10). As can be seen from Figure 9, the flow cytometry results showed that the infiltration of Th1 and Th17 cells in the eyeballs of the mice in the sEVs-10 group was significantly reduced. The statistical results in Figure 10 showed that the proportions of Th1 cells and Th17 cells in the overexpression sEVs-10 group were lower than those in the sEVs-10 group. -N group and PBS group, the difference was statistically significant (p<0.01). This result demonstrated that after tail vein injection of IL-10-overexpressing mesenchymal stem cell sEVs, the infiltration of pathogenic cells in the mouse eyeball was significantly reduced, indicating its therapeutic effect. (*: P<0.05; ***: P<0.01)

3. The therapeutic effect of sEVs-10 on experimental autoimmune myelitis (EAE)

(1) Model establishment: 7-week-old C57BL/6 female mice were taken, and the emulsion, which was thoroughly mixed with MOG _35-55 and complete Freund's adjuvant (CFA), was uniformly injected into the bilateral backs of spare mice At 4 points, on the day of immunization and 48 hours later, mice were injected intraperitoneally with PBS containing 500 ng of PTX for auxiliary immunization. Mice were randomly divided into PBS control group, sEVs-10 treatment group and sEVs-N treatment group, with 6 mice in each group. After 24 hours of autoimmunity, the onset of the mice was observed daily and the neurological dysfunction score was performed. On the 7th day after modeling, mice in each group were injected with 50 μg sEVs-10, sEVs-N and the same volume of PBS through tail vein respectively as a control.

(2) Neurological dysfunction score: The incidence of the mice was observed every day from the day of immunization and the neurological dysfunction score was carried out. The scoring standard is based on the international 5-point scale: 1 point = animal tail weakness; 2 points = animal hind limb weakness; 3 points = hind limb paralysis; 4 points = forelimb paralysis; 5 points = moribund state or death.

The scoring results showed that the scores of the sEVs-N group, the sEVs-10 group and the PBS group were (2.17±0.41), (1.17±0.41), and (3.5±0.48) in the peak period (17th day after modeling). The results showed that both sEVs and sEVs overexpressing IL-10 could alleviate the progression of EAE model (P<0.05), while sEVs overexpressing IL-10 had a stronger therapeutic effect (P<0.01).

(3) At the peak period of EAE onset (the 17th day after modeling), the mice in each group were sacrificed by cervical dislocation, and the cerebral and spinal cord tissues were taken out, fixed in 10% formalin, routinely dehydrated, embedded in paraffin, and subjected to routine hematoxylin Serum-eosin (HE) staining. The infiltration of inflammatory cells in the spinal cord was observed under an inverted microscope, and the internationally recognized 5-point method was used for pathological scoring: 0 = no inflammatory cells were observed; 1 = inflammatory cell infiltration was limited to the periphery of blood vessels and the spinal cord; 2 = inflammation in the spinal cord Slight infiltration of cells; 3 points = moderate infiltration of cells in the spinal cord; 4 points = severe infiltration of inflammatory cells in the spinal cord.

The pathological score results showed that the pathological scores of the sEVs-N group, the sEVs-10 group and the PBS group at the peak period were (2.17±0.52), (1.08±0.38), and (3.42±0.38) respectively; the inflammatory cell infiltration in the sEVs-10 group Lightest (Figure 11). It can be seen from Figure 11 that the IL-10-overexpressing sEVs treatment group can significantly reduce the infiltration of inflammatory cells around the spinal cord. The results also showed that both sEVs and sEVs overexpressing IL-10 could alleviate the progression of the EAE model, while sEVs overexpressing IL-10 had a stronger therapeutic effect.

4. sEVs overexpressing IL-10 inhibit the proliferation of T cells and the differentiation of Th1 cells in vitro

(1) In vitro differentiation of Th1/Th17 cells: ①Isolation of mouse spleen Naive T cells by magnetic bead negative separation method: Take normal mouse spleen in the sterile barrier area, grind with a cell sieve, filter and lyse red blood cells, and count them for later use. The experimental steps were carried out according to the instructions of the magnetic bead negative sorting Total CD4 ⁺ T/Naive CD4 ⁺ T cell kit. Adjust the lymphocyte concentration to 1×10 ⁸ /ml, take 500 μl into a centrifuge tube, add 100 μl of FBS after heat inactivation (65°C, 30 min), add 100 μl of mixed antibodies, incubate at 4°C for 20 min on a shaker, add 10 ml of separation solution and centrifuge , 4ml of separation solution was resuspended, 1ml of pre-washed magnetic beads were added, 15ml of incubator was shaken at room temperature, 5ml of separation solution was added, and after mixing, they were placed in a flow tube in batches, inserted into a magnet and allowed to stand for 2min, and the supernatant was collected. The solution is transferred to a centrifuge tube (the cells contained in the supernatant are the target cells), centrifuged in an appropriate amount and resuspended, and the cells are counted for use. ②Th1 cell differentiation: After separation, 2x10 ⁵ cells/well were plated in a 96-well plate pre-incubated with CD3 antibody, and cultured in a cell incubator according to the following differentiation conditions: X-VIVO 15 serum-free medium + 2μg/ml CD28 antibody +10ng/ml IL-12+10mg/ml IL-4 antibody was collected after 5 days, and the cells were collected by flow cytometry, and the Th1 cell ratio was detected. Th17 cell differentiation: After separation, 2x10 ⁵ /well cells were plated in 96-well plates pre-incubated with CD3 antibody, and cultured in a cell incubator according to the following differentiation conditions: X-VIVO 15 serum-free medium + 2 μg/ml CD28 antibody +30ng/ml IL-6+2.5ng/ml TGF-β1+10mg/ml IL-4 antibody+10mg/ml IFN-γ antibody. After 5 days, the cells were collected by flow cytometry, and the ratio of Th17 cells was detected.

The results showed that in the in vitro differentiation of Th1 cells, sEVs overexpressing IL-10 could inhibit the in vitro differentiation of Th1 cells, and the proportion of Th1 cells in the sEVs-N group and the blank control group was significantly lower (p<0.01); Inhibits Th1 cell differentiation in vitro (Figure 12-13). The results in Figure 12 show that the sEVs-N group and the sEVs-10 group have inhibitory effects on the differentiation of Th1 (IFN-γ positive) cells in vitro; Figure 13 is the result of statistical analysis, the results show that the sEVs-10 group has a strong inhibitory effect Th1 cell differentiation in vitro. Regarding the differentiation of Th17 cells in vitro, both the sEVs-10 group and the sEVs-N group had the ability to inhibit the differentiation of Th17, and there was no significant difference between the two groups (P>0.05) (Figure 14-15). The results in Figure 14 show the inhibitory effects of the sEVs-N group and the sEVs-10 group on the differentiation of Th17 (IL-17A positive) cells in vitro. Figure 15 shows the results of statistical analysis. The results show that: the sEVs-10 group and the sEVs-N group Both have strong inhibitory effect on Th17 cell differentiation in vitro.

(2) Proliferation of T cells in vitro (CFSE method): The steps of magnetic beads negative sorting Total CD4 ⁺ T are the same as above. Put Total CD4 ⁺ T cells in 5 ml of a centrifuge tube, add 1 μl of CFSE stock solution (5 mmol/L), mix quickly, incubate at room temperature for 10 min in the dark, and add the same volume of RPMI 1640 medium containing 10% FBS to stop staining, After centrifugation, complete medium (configuration: 90% 1640 basal medium + 10% FBS + 2 μg/ml CD28 antibody + 100U/ml double antibody + 50 μM β-mercaptoethanol) was resuspended, and plated in a 96-well plate pretreated with CD3 antibody (cell density 5× ^{10 5} /well), placed in a cell incubator for 72 hours, and after staining with CD4 antibody by flow cytometry, the fluorescence expression of CFSE was detected by flow cytometry.

The experimental results showed that compared with the PBS group, the sEVs-10 group and the sEVs-N group could inhibit the proliferation of T cells in vitro, and the proportion of proliferating cells was lower than that of the PBS group, and the difference was statistically significant (p<0.01, p<0.05). Compared with the sEVs-N group, the -10 group had a lower proportion of proliferating cells, and the difference was statistically significant (p<0.01) (Figure 16-17). The results showed that sEVs-10 had a stronger function of inhibiting T cell proliferation in vitro (Figure 16-17). Figure 16. The CFSE results show that both the sEVs-10 group and the sEVs-N group have the effect of inhibiting the differentiation of T cells in vitro, and the sEVs-10 group has a stronger inhibitory ability; Figure 17 is the statistical analysis results, the results show: sEVs-10 The proportion of proliferating cells in the group was significantly lower than that in other groups, and the difference was statistically significant (P<0.01).

4. MSCs-derived sEVs have chemotactic effects on inflammation.

(1) PKH-26-labeled sEVs: According to the above ultracentrifugation method, sEVs were separated, and the last step was resuspended in diluent C, and stained with PKH26 red fluorescent labeling kit (Sigma-Aldrich). Dilute the PKH26 dye concentration to 8 μM with 500ul of Diluent C, add the sEVs resuspended in the same volume of Diluent C, and incubate for 5 min at room temperature. Mix gently during the incubation period. Use the excess dye in DMEM containing 10% exosome-free FBS. And, centrifuged at 11000g for 70min, washed twice, resuspended in appropriate amount of PBS, and filtered with a 0.22 μm filter.

(2) Tail vein injection of mice: 2 EAU model C57 mice were taken, and 100 μg of PKH-26-labeled sEVs-N/sEVs-10 were injected through the tail vein with a 29G syringe (BD, USA) at 11 d after modeling. Mice were sacrificed 24 hours after tail vein injection, and the eyeballs were isolated. Unmodeled mice were used as controls.

(3) Frozen section and immunofluorescence: After the mouse tissue was taken out, it was embedded with OCT, and immediately snap-frozen in liquid nitrogen, and then cut into 8 μm tissue sections with a cryostat and spread on glass slides. The slides covered with retinal tissue sections were fixed with 4% paraformaldehyde for 30 min, blocked with PBS containing 5% goat serum and 0.3% Triton for 1 h, washed 3 times, incubated with DAPI for 20 min, washed 3 times with PBST, and aired in a dark box. After drying, anti-fluorescence decay mounting medium was added dropwise, and the coverslips were mounted, dried in the dark, and observed under a confocal microscope (Zeiss, Germany).

The results showed that at 24h, red fluorescence representing sEVs could be observed in the eye sections of the two groups of EAU mice. In contrast, no red fluorescence representing sEVs was observed in normal mouse retinal sections. The results showed that sEVs had certain inflammatory chemotaxis ability, and could quickly reach the lesion site to exert immunosuppressive effect (as shown in Figure 18). 24h after tail vein injection of PKH-26-labeled sEVs, the retinas of un-modeled mice did not appear to be unaffected. Red fluorescence was found representing sEVs. In the retina of EAU model mice, red fluorescence could be observed in both the sEVs-10 treatment group and the sEVs-N treatment group, indicating that sEVs successfully reached the retina of the diseased tissue.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims (6)

1. The application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the preparation of medicines for the treatment of autoimmune diseases. 2. The use according to claim 1, wherein the autoimmune disease is autoimmune uveitis or multiple sclerosis. 3. The application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the preparation of drugs for inhibiting T cell proliferation. 4. The application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the preparation of drugs for inhibiting the differentiation ability of Th1 cells. 5. The application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the preparation of drugs for inhibiting the differentiation ability of Th17 cells. 6. The application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in the preparation of immunosuppressive drugs.

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