CN113827615A - Application of vesicles in preparation of medicine for treating autoimmune diseases - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to application of vesicles in preparation of a medicine for treating autoimmune diseases. The vesicle is an induced vesicle which has a remarkable immunosuppressive effect and is used for treating CD4⁺The balance of T cells has a very good regulatory role. Can be used for regulating and controlling the steady state of an immune system and has good effect on preventing and treating diseases such as systemic lupus erythematosus and the like.

Description

Application of vesicles in preparation of medicine for treating autoimmune diseases

Technical Field

The invention belongs to the field of biomedicine, and relates to application of vesicles in preparation of a medicine for treating autoimmune diseases.

Background

Autoimmune diseases are caused by immune disorders of the body, such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). CD4⁺T helper cell imbalance plays an important role in immune disorders of RA and SLE, including CD4⁺The T helper cells are activated in a large quantity, the memory T cells are increased, the proportion of subgroups is disordered, and the increased Th17 cells play an important role in joint inflammation and damageAnd (5) preparing. RA and SLE are both influenced by multiple factors of heredity and environment, and about 0.5% of people in China suffer from RA and involve various organ systems such as blood, bone joints, lungs, skin and the like; SLE is suffered in approximately 0.07% of the population, affecting almost all organ systems in the body, with most of the patients having the most marked kidney damage, manifested as massive proteinuria and renal failure. The disease course of autoimmune diseases is long-lasting, continuous drug control for years in short term and for a lifetime in long term is often needed, and the possibility of repeated disease exists, which brings long-term burden to the health of Chinese population. Therefore, the research of new methods and strategies for preventing and treating autoimmune diseases aiming at the steady-state regulation mechanism of the immune system is a great medical and scientific problem which needs to be solved urgently.

Disclosure of Invention

In some embodiments, the invention provides the use of vesicles, which are inducible vesicles, in the manufacture of a modulator of T cell activity.

In some embodiments, the IEVs are abbreviated as inducing vesicles, which may be referred to as inducing vesicles, and may also be referred to as Induced Extracellular Vesicles (IEVs).

In some embodiments, the T cell is CD4⁺T helper cells.

In some embodiments, the T cell comprises Treg, Th1, Th17, Th 2.

In some embodiments, the modulator of activity inhibits Th1, Th17, Th 2.

In some embodiments, the activity modulator inhibits differentiation of Th 17.

In some embodiments, the modulator of activity inhibits inhibition of CD25⁺CD4⁺T cells.

In some embodiments, the vesicle is formed by contacting CD4 directly with phosphatidylserine on the surface of the vesicle⁺T cells achieve regulatory T cell activity.

In some embodiments, the invention provides the use of vesicles, which are inducing vesicles, in the manufacture of a medicament for the treatment or prevention of an autoimmune disease.

In some embodiments, the autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis.

In some embodiments, the autoimmune disease does not include sjogren's syndrome.

In some embodiments, the source of vesicles includes stem cells.

In some embodiments, the stem cell is a mesenchymal stem cell.

In some embodiments, the cells may be primary cultured cells, as well as existing or established cell lines.

In some embodiments, the cell line refers to an immortalized cell culture that can be propagated indefinitely in appropriate fresh media and space.

In some embodiments, the cell can be an established cell line.

In some embodiments, the inducing vesicles are vesicles produced by inducing apoptosis by an external factor when stem cells are in normal survival.

In some embodiments, the inducing vesicles are derived from stem cells by inducing apoptosis by methods including, but not limited to, staurosporine, uv irradiation, starvation, or heat stress.

In some embodiments, the stem cell is a mesenchymal stem cell.

In some embodiments, the source of mesenchymal stem cells includes, but is not limited to, bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, or peripheral blood.

In some embodiments, the inducing vesicles are vesicles produced by inducing apoptosis by an external factor when stem cells are in normal survival.

In some embodiments, the inducing vesicles are induced by inducing stem cell apoptosis by methods including the addition of staurosporine, uv irradiation, starvation, or heat stress.

The inventors have found that vesicles having a smaller particle size range have a superior effect, at least in treating lupus erythematosus. In some embodiments of the invention, vesicles having a diameter in the range of 0.45 μ M or less can reduce pathological spleen weight in MRL/lpr mice to 60% of the model group when treating systemic lupus erythematosus compared to the model group. It was also found that such small particle size inducing vesicles could also reduce the serum ds DNA levels in MRL/lpr mice to 40% of the model group.

In another series of previous experiments, the inventors used vesicles with a large particle size range (with a mean particle size of 4 μm to 10 μm as determined by the current detection technique) to treat Systemic Lupus Erythematosus (SLE), and the results showed that the spleen size of the treatment group (MRL/lpr + vesicles) was 75% of the spleen size of the model group (MRL/lpr). The ds DNA level in the serum of the treated group (MRL/lpr + vesicles) was 80% of that of the model group (MRL/lpr).

In some embodiments, the vesicle has a diameter of 0.05-0.45 μ M. In some embodiments, the vesicle has a diameter of 0.05-0.4 μ M. In some embodiments, the vesicle has a diameter of 0.05-0.3 μ M. In some embodiments, the diameter of the vesicle may also be 0.15-0.45. mu.M, may also be 0.15-0.3. mu.M, and may also be 0.2-0.3. mu.M.

In some embodiments, the inducing vesicle has the marker Syntaxin 4. In some embodiments, the inducible vesicle overexpression marker Syntaxin 4. In some embodiments, the inducible vesicles express the marker Syntaxin 4 more than MSCs or exosomes. In some embodiments, the marker Syntaxin 4 is expressed in an amount 3-6 times the amount of expression of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker Syntaxin 4 is expressed in an amount 3.5-5 times the amount of expression of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker Syntaxin 4 is expressed in an amount 4.45 times the amount of expression of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker further comprises one or more of Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the marker is a combination of Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the inducible vesicle overexpression markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5. In some embodiments, the inducible vesicles express the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 in an amount greater than the MSCs or exosomes. In some embodiments, the expression level of markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 in the induced vesicles is 1-2 fold, 2-3 fold, 1-3 fold, and 3-4 fold, respectively, relative to the expression level of the marker in exosomes derived from mesenchymal stem cells. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 are expressed in the inducing vesicles in an amount of 1.5-2 fold, 2.5-3 fold, 1.5-2.5 fold, and 3.5-4 fold, respectively. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 are expressed in 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold, respectively, in the vesicle.

The induced vesicles of the invention are substantially different from exosomes, for example, compared with exosomes, the induced vesicles IEVs of the invention highly express Syntaxin 4, and the expression levels of Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5 are obviously higher than those of exosomes (see example 3). In addition to having marker differences, the induced vesicles IEVs also exhibit characteristics that are distinct from stem cells and other extracellular vesicles (e.g., exosomes) in function or therapeutic effect. For example, IEVs significantly shorten the clotting time of most plasma in vitro, and the procoagulant effect is better than that of exosomes (see test example 2). For example, the mechanism of IEVs in treating hemophilia mice is independent of PS and TF, whereas in previous literature reports, the procoagulant action of extracellular vesicles is highly dependent on both PS and TF on their surface (see test example 2).

In some embodiments, the method of making the inducing vesicles includes the steps of: (1) culturing mesenchymal stem cells; (2) collecting a culture medium supernatant of the mesenchymal stem cells; (3) isolating vesicles from the culture supernatant of step (2).

In some embodiments, the step of culturing mesenchymal stem cells in step (1) comprises: (4) isolating mesenchymal stem cells from the tissue; (5) adding a culture medium to culture the mesenchymal stem cells; contacting an apoptosis-inducing agent in a culture medium of the mesenchymal stem cells.

In some embodiments, in step (3), the method for separating vesicles comprises separating vesicles by ultracentrifugation.

In some embodiments, the step of ultracentrifuging the method of isolating the vesicles comprises: (a) performing first centrifugation on the collected culture supernatant, and taking the supernatant; (b) centrifuging the supernatant collected in step (a) a second time, and taking the supernatant; (c) centrifuging the supernatant received in step (b) for a third time, and taking the precipitate; (d) and (c) centrifuging the precipitate received in the step (c) for the fourth time, and taking the precipitate to obtain the compound.

In some embodiments, the first centrifugation is a 1500g centrifugation for 5-30 minutes; or the first centrifugation is 500-1000g centrifugation for 5-20 minutes; or the first centrifugation is 500-900g centrifugation for 5-15 minutes.

In some embodiments, the second centrifugation is 1000-3000g centrifugation for 5-30 minutes; or the second centrifugation is performed for 5-20 minutes at 2500g of 1500-; or the second centrifugation is 1500-2200g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is 1550-2200g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is 1600-2200g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is 1650-2200g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is 1700-2200g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is 1550-2200g centrifugation for 5-30 minutes. In some embodiments, the second centrifugation is 1600-2200g centrifugation for 5-25 minutes. In some embodiments, the second centrifugation is 1650-2200g centrifugation for 5-20 minutes. In some embodiments, the second centrifugation is 1700-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1750-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1800-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1850-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1900-2200g for 5-18 minutes. In some embodiments, the second centrifugation is 1950-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1980-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1990-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1990-2100g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1990-2200g centrifugation for 5-18 minutes. In some embodiments, the second centrifugation is 1990-2100g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is 1990-2100g centrifugation for 8-15 minutes.

In some embodiments, the supernatant from the second centrifugation is subjected to a third centrifugation at 10000-; or the third centrifugation is performed at 12000-25000g for 20-60 minutes; or the third centrifugation is 12000-20000g centrifugation for 20-40 minutes. In some embodiments, the third centrifugation is 13000-20000g centrifugation for 20-40 minutes. In some embodiments, the third centrifugation is 14000-. In some embodiments, the third centrifugation is 15000-. In some embodiments, the third centrifugation is 15500-. In some embodiments, the third centrifugation is 15500-19000g centrifugation for 20-40 minutes. In some embodiments, the third centrifugation is 15500-18000g centrifugation for 20-40 minutes. In some embodiments, the third centrifugation is 15500-17000g centrifugation for 20-40 minutes.

In some embodiments, the third centrifugation pellet is resuspended in PBS followed by a fourth centrifugation, which is 10000-110000 g centrifugation for 5-60 minutes. In some embodiments, the fourth centrifugation is 10000-100000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-. In some embodiments, the fourth centrifugation is 10000-. In some embodiments, the fourth centrifugation is 10000-70000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-. In some embodiments, the fourth centrifugation is 10000-50000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-40000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-.

In some embodiments, the fourth centrifugation is 12000-25000g centrifugation for 20-60 minutes; or the fourth centrifugation is 12000-20000g centrifugation for 20-40 minutes.

In some embodiments, the fourth centrifugation is 10000-. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-18500g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-18000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-17500g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 11000-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 12000-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 12500-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 13000-17000 g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is a 17000g centrifugation of 13500-60 minutes. In some embodiments, the fourth centrifugation is 14000-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 14500-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 15000-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 15500-17000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 15500-16500g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is 15500-17000g centrifugation for 15-55 minutes. In some embodiments, the fourth centrifugation is 15500-16500g centrifugation for 15-50 minutes. In some embodiments, the fourth centrifugation is 15500-16500g centrifugation for 15-40 minutes. In some embodiments, the fourth centrifugation is 15500-16500g centrifugation for 20-40 minutes. In some embodiments, the fourth centrifugation is 15800-16500g centrifugation for 20-35 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 15-80 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 15-70 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 20-70 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 20-60 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 20-50 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 20-40 minutes. In some embodiments, the fourth centrifugation is 10000-19000g centrifugation for 20-35 minutes. In some embodiments, the fourth centrifugation is 10000-18000g centrifugation for 20-60 minutes. In some embodiments, the fourth centrifugation is 10000-18000g centrifugation for 20-50 minutes.

In some embodiments, the number of generations of the mesenchymal stem cells may be from 2 to 5 generations, but is not limited thereto.

In some embodiments, the mesenchymal stem cells are derived from a mammal, but are not limited thereto.

In some embodiments, the mammal is selected from the group consisting of a human or a mouse, but is not limited thereto.

In some embodiments, the inducing vesicles are used in the treatment of disease, optionally by a route selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intraorgan infusion. For example, for intravenous injection, the injection may be via the tail vein, as an example. Intra-organ infusion includes infusion into anatomical spaces such as, by way of example, the gallbladder, gastrointestinal lumen, alimentary tract, pulmonary system (by inhalation), and/or bladder.

Drawings

FIG. 1 FIGS. 1A-1E show the results of flow assays of surface markers from isolated BMMSCs.

FIG. 2 is a flowchart of the operation of example 2.

FIG. 3 shows MSCs (10) analyzed by flow cytometry⁶Individual MSCs) produced statistics of the number of IEVs.

FIGS. 4A-4D are diameter measurements of IEVs particles: FIG. 4A is a graph showing the particle diameter distribution of IEVs by analyzing the scattered light intensity of IEVs using standardized small particle microspheres manufactured by Bangs Laboratories, Inc.; FIG. 4B is Transmission Electron Microscopy (TEM) observed IEVs showing the particle diameter distribution of the IEVs; FIG. 4C is a Nanoparticle Tracking Analysis (NTA) showing the distribution of IEVs particle diameter; fig. 4D is a single vesicle level particle size measurement of IEVs using nano-flow detection techniques, showing the particle diameter distribution of IEVs.

FIGS. 5A-5K are results of flow cytometry analysis of surface membrane proteins of IEVs.

FIGS. 6A-6D are content analyses of IEVs: FIG. 6A shows the results of quantitative analysis of proteomics of MSCs, MSCs-Exosomes and MSCs-IEVs by DIA quantitative technique; FIG. 6B is a heat map drawn screening for IEVs specific highly expressed proteins; FIG. 6C is the results of GO enrichment analysis of differential proteins for IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules; FIG. 6D is a result of verifying that MSCs, MSCs-Exosomes, MSCs-IEVs express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 by western blot.

FIG. 7: IEVs on CD4 in mouse spleen cells⁺The activation marker CD25 of T lymphocytes is dose-dependent inhibited; IEVs on CD4 in mouse spleen cells⁺The activation product IL-2 of T lymphocyte is inhibited in a dose-dependent way; IEVs showed more significant effect on the inhibition of high-intensity T Cell Receptor (TCR) signals; IEVs on multiple CDs 4⁺T cell subsets including Th1, Th17 and Th2 are all significantly inhibited; IEVs can remarkably inhibit secretion products of Th1, Th17 and Th2, such as IFN-gamma, IL17A and IL 10; IEVs do not significantly inhibit Foxp³⁺Treg cells; ievs induce activation-related apoptosis of T lymphocytes in a dose-dependent manner. StimRefers to anti-CD3/CD28 antibody activation; unstim means no activation.

Figure 8 is that the immunosuppressive effects of IEVs are superior to exosomes and are not completely independent of the soluble components released by IEVs.

FIG. 9: IEVs are able to directly inhibit purified naive CD4⁺T cell activation; a5 blocking PS treatment attenuates the inhibition of T cell activation and IL-2 secretion by IEVs; the direct close contact between the IEVs labeled by the PKH26 and the T cells can not be realized after the A5 blocks the PS; IEVs were able to significantly suppress TCR signaling, and IEVs after a5 blocking failed to suppress TCR signaling. Stim refers to anti-CD3/CD28 antibody activation; unstim means no activation

FIG. 10: ievs are dose-dependent on inhibition of Th17 differentiation; IEVs inhibit IL-17A secretion within 6 hours; ievs inhibited Th17 differentiation more significantly than the equal proportion of BMMSCs. Wherein

Refers to naive T cells; th0 refers to undifferentiated T cells; iTh17+ IEVs refers to IEVs treated under conditions that induce Th 17.

FIG. 11: schematic of the 4-week experiment. Ievs injection effectively corrected relative apoptosis deficiency and lymphoproliferation in LPR circulating blood and spleen; the ApopEVs apoptotic extracellular vesicles EVs in the ordinate of the graph b are nano-sized small bodies with membrane structures secreted when cells undergo apoptosis in the normal culture process or undergo spontaneous apoptosis under in vivo physiological conditions, have diameters varying from 40 nm to 1000nm, mainly consist of Microvesicles (MVs) and Exosomes (Exosomes), and contain signal molecules such as various RNAs and proteins. IEVs corrected Foxp of spleen and mesenteric lymph node of experimental animal³⁺The proportion of Treg cells is unbalanced. IEVs correct the imbalance of Th1 cell proportion of spleen and mesenteric lymph node of experimental animal. IEVs improved CD4 in experimental animals by correcting memory cell accumulation and juvenile cell reduction phenotype of spleen of Lpr mouse⁺T cell homeostasis is deregulated. Ievs corrected the activation-related apoptotic defect of T lymphocytes in experimental animals. Ievs improved autoantibody content in circulating blood of experimental animals. i. Long termSchematic experimental diagram. Ievs corrected the pathological enlargement of spleen, mesenteric lymph nodes in experimental animals. Ievs corrected T lymphocyte infiltration in the lungs and colon of experimental animals. Ievs delayed the production of proteinuria in experimental animals, implying protection of kidney function. Renal HE staining, Trichrome staining suggested that IEVs improved immune cell infiltration and fibrotic changes in experimental locomotor kidneys. Ievs improved survival of experimental animals. Wherein MRL/wt is wild group; MRL/lpr is a model group; IEVs were the MRL/lpr treatment group.

FIG. 12: a. schematic experimental diagram. IEVs significantly inhibited arthritis activity, once injection effect was superior to BMMSCs, and effect was comparable to first line therapy TNFa, significantly superior to PBS control group. c. Affected joint maps of experimental animals; he, collagen fastgreen staining, and uCTt suggested that the pathological changes of joint tissues in IEVs treated groups were superior to BMMSCs, and the effect was comparable to first-line TNFi, which was significantly superior to PBS control group. The spleen and draining lymph node of experimental animals treated by IEVs have significantly less Th17 cells than BMMSCs and PBS treated animals.

Figure 13 is IEVs treatment of sjogren's syndrome: ievs treatment of the effects of sjogren's syndrome (sjogren's syndrome) salivary flow rate; staining results of IEVs in treating submandibular gland HE of dry syndrome; C. treatment of the effects of sjogren's syndrome on B cells.

FIG. 14 is an in vivo procoagulant effect of IEVs in hemophilia A mice.

Fig. 15A-15D are graphs showing the change in levels of various clotting factors following injection of IEVs into hemophilia a mice: FIG. 15A shows a variation of factor VIII; FIG. 15B shows the change in vWF factor; FIG. 15C shows changes in Tissue Factor (TF); FIG. 15D shows the prothrombin profile.

Fig. 16A-16B are graphs showing the effect of blockade of PS and TF, respectively, on the in vivo therapeutic efficacy of IEVs in a mouse model of hemophilia a.

FIG. 17 is a comparison of the therapeutic effect of IEVs and Exosomes from the same MSCs on hemophilia A mice.

Note: in the figures, WT is a wild-type mouse; the HA group is a hemophilia a mouse model; HA + IEVs treatment with IEVs for hemophilia a mouse model; HA + PS-IEVs PS-negative IEVs administered to hemophilia A mouse model; HA + TF-IEVs to TF-negative IEVs for hemophilia A mouse model; HA + Exosomes subjects haemophilia a mouse models were given Exosomes treatment.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others following the concepts of the present invention are within the scope of the invention.

IEVs in the present embodiments are simply referred to as inductive vesicles, which may also be referred to as Inductive Extracellular Vesicles (IEVs). Induced extracellular vesicles are a type of subcellular product produced by intervention or induction of apoptosis in a precursor cell (e.g., a stem cell) during its normal survival. Usually this class of subcellular products, with membrane structures, express apoptotic markers, and contain, in part, genetic material DNA. The inventors have found that induced extracellular vesicles are a class of substances that is distinguished from cells and conventional extracellular vesicles (e.g., exosomes, etc.). In some embodiments, the normally viable cells are, for example, non-apoptotic cells, non-senescent cells with arrest in proliferation, cells that have been revived after non-cryopreservation, cells that have not undergone malignant transformation with abnormal proliferation, or cells that have not undergone injury, etc. In some embodiments, the cells that survive normally are obtained from cells that have been exposed to fusion between 80-100% during cell culture. In some embodiments, the normally viable cells are taken from log phase cells. In some embodiments, the normally viable cells are obtained from primary cultures of human or murine tissue origin and subcultured cells thereof. In some embodiments, the normally viable cells are taken from an established cell line or cell strain. In some embodiments, the precursor cells are taken from early stage cells.

STS in the present invention is staurosporine.

The vesicle diameter size defined in the claims is the diameter size corresponding to the detection of the particle size at the level of single vesicle by the method of analyzing the scattered light intensity of IEVs using the standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) manufactured by Bangs Laboratories, the method of Transmission Electron Microscopy (TEM) observation, the method of Nanoparticle Tracking Analysis (NTA). In particular, the TEM method can be used.

Particle size display may be smaller when detected using another of the most advanced nano-flow detection techniques. In one example, IEVs were shown to have a vesicle diameter of 0.2 μm or less when analyzed for scattered light intensity measurements using standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) from Bangs Laboratories. In another example, when the particle size is measured by Transmission Electron Microscopy (TEM), the diameter of the vesicles is 200nm or less and 200nm or less. In one embodiment, the IEVs particles average 169nm in diameter when subjected to particle size detection using Nanoparticle Tracking Analysis (NTA).

The IEVs of a particular range of particle sizes may be obtained by controlling the centrifugation conditions, or the filtration means or enrichment means of the pore size.

EXAMPLE 1 isolation and culture of MSCs

Excess CO was used according to the guidance of the animal ethics Committee₂The mice are sacrificed, the tibia and the femur are taken off under the aseptic condition, the muscle and connective tissue attached to the tibia and the femur are stripped off, the metaphysis is further separated, the marrow cavity is exposed, PBS containing 10% fetal bovine serum by volume fraction is extracted by a 10mL aseptic syringe to repeatedly wash the marrow cavity, after filtration by a 70 mu m pore size cell filter screen, 500g is centrifuged for 5min, the cell sediment at the bottom is collected after the supernatant is removed, PBS is resuspended, 500g is centrifuged again for 5min, and the final cell sediment is collected. Then, the cells are subjected to flow sorting, and the BMMSCs are sorted by using CD34 & lt- & gt and CD90 & lt + & gt as sorting standards. Finally, cells were resuspended in Dex (-) medium and seeded on 10cm diameter cell culture dishes at 37 ℃ with 5% CO₂And (5) culturing. After 24h, the non-adherent cells in the supernatant were aspirated off, washed with PBS, and then cultured by adding Dex (-) culture medium. After 1 week, an equal amount of Dex (+) medium was added, and after 1 week, dense colonies of primary BMMSCs were observed. Digesting BMMSCs by incubating at 37 ℃ with trypsin and carrying out passage amplification, and then changing Dex (+) culture solution every 3 days for growingAnd (5) carrying out passage after the full. Subsequent experiments were performed using P2 generation BMMSCs.

The components of the Dex (-) culture medium are shown in Table 1, and the components of the Dex (+) culture medium are shown in Table 2:

TABLE 1Dex (-) composition of culture solution

TABLE 2Dex (+) culture solution formulation Table

The purity of the isolated BMMSCs was assessed by flow cytometry analysis of surface markers. For surface marker identification, after collection of P2 generation BMMSCs by trypsinization, the BMS was washed 1 time with PBS at 5X 10⁵Resuspend cells at density/mL in PBS containing 3% FBS, add 1 μ L PE fluorescently conjugated CD29, CD44, CD90, CD45 and CD34 antibodies, and blank group does not. Incubating at 4 ℃ in dark for 30min, washing for 2 times by PBS, and detecting on a machine. The flow assay results are shown in FIGS. 1A-1E, and it is found that the isolated cells are BMMSCs (bone marrow mesenchymal stem cells).

Example 2 acquisition of induced vesicles (IEVs)

MSCs (bone marrow-derived MSCs) cultured up to passage 2 in example 1 were cultured in the medium (Dex (+) medium) of example 1 until the cells reached 80% -90% confluence, washed 2 times with PBS, added with 500nM STS-containing serum-free medium (500 nM STS was added to the medium of example 1), incubated at 37 ℃ for 24h, and cell supernatants were collected for separation and extraction of IEVs.

The IEVs are isolated and extracted from the collected culture supernatant, the operation flow is shown in fig. 2, and the specific steps include: 800g centrifugation 10 minutes-collection of supernatant-2000 g centrifugation 10 minutes-collection of supernatant-16000 g centrifugation 30 minutes-removal of supernatant, sterile PBS heavy suspension IEVs-16000 g centrifugation 30 minutes-removal of supernatant, 300-.

Comparative example 1 exosome separation and extraction from homogeneous MSCs

MSCs (bone marrow-derived MSCs) cultured up to passage 2 in example 1 were cultured with the medium in example 1, washed 2 times with PBS when the cells were confluent to 80% -90%, serum-free medium was added, incubated at 37 ℃ for 48h, and cell supernatant was collected for separation and extraction of Exosomes.

The extraction step comprises: centrifugation at 800g for 10 min-collection of supernatant-centrifugation at 2000g for 10 min-collection of supernatant-centrifugation at 16000g for 30 min-collection of supernatant-centrifugation at 120000g for 90 min-removal of supernatant, resuspension of pellet in sterile PBS-centrifugation at 120000g for 90 min, removal of supernatant, collection of bottom Exosomes, and resuspension in sterile PBS.

Example 3 analysis of IEVs

1. Quantification of IEVs and analysis of Membrane proteins

Quantitative analysis of the IEVs obtained in example 2 was carried out by flow cytometry at the measurement time points 1h, 4h, 8h, 16h and 24h, and the results showed 10⁶The MSCs can respectively produce 0.76 multiplied by 10 after induction to 1h, 4h, 8h, 16h and 24h⁸1.29X 10⁸1.95 × 10⁸2.48 x 10⁸3.14 x 10⁸From the individual IEVs, it can be seen that a single MSCs can produce 300 IEVs after 24h induction (FIG. 3).

The scattering intensity of IEVs was analyzed by standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) manufactured by Bangs Laboratories, Inc., and the results showed that the particle diameters of IEVs were all below 0.2 μm (FIG. 4A).

The results of Transmission Electron Microscopy (TEM) showed that most of the vesicles had diameters between 200nm and 200nm or less (fig. 4B).

The Nanoparticle Tracking Analysis (NTA) results were consistent with transmission electron microscopy observations, with IEVs having an average particle diameter of 169nm (FIG. 4C).

Particle size measurements at the level of single vesicles were performed using state-of-the-art nano-flow detection techniques and also showed that the average particle diameter of the IEVs was 100.63nm (fig. 4D).

Analysis of the surface membrane proteins of the IEVs extracted in example 3 using flow cytometry showed that the iecs from MSCs were able to express similar surface proteins as MSCs, i.e. CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. Meanwhile, IEVs were able to express the ubiquitous surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (see fig. 5A-5K).

2. Content analysis of IEVs

Proteomic quantitative analysis of MSCs, MSCs-Exosomes (extracted in comparative example 1), MSCs-IEVs (obtained in example 2) was performed using protein DIA quantification technique. The results showed that the protein content expression of MSCs-Exosomes and MSCs-IEVs had higher overlap with the mother cells, and 170 proteins were specifically highly expressed in IEVs (fig. 6A). By bioinformatics analysis, the specific high-expression protein of IEVs is screened, a heat map is drawn (FIG. 6B), and further combined with the GO enrichment analysis result of differential protein, the specific high-expression molecules of Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 of IEVs are confirmed (FIG. 6C). Compared with Exosomes from the same MSCs, the expression levels of 5 characteristic molecules of IEVs are all significantly up-regulated, specifically: the expression levels of markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in IEVs were 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold and 4.45-fold, respectively, relative to the corresponding markers in Exosomes. Finally, the western blot technique is used to perform the verification again, and the result is consistent with the DIA quantitative analysis result (FIG. 6D).

MSCs-Exosomes: refers to exosomes derived from MSCs.

MSCs-IEVs: refer to IEVs derived from MSCs.

Wherein the MSCs in the content analysis are the same cell line as the MSCs from which the Exosomes and IEVs were extracted.

Example 4IEVs by modulating multiple CD4 in the spleen⁺T helper cell activation effects immune homeostasis regulation.

1. The detection step or method comprises: taking spleens of 8-week-old Wild Type (WT) mice, grinding and filtering on a 70-micron screen to obtain a spleen single cell suspension, using PBS with 20 times volume to neutralize after red blood cells are lysed for 3 minutes by using an ACK solution, and obtaining T-containing cells after centrifugation and heavy suspension twiceSplenic leukocytes including lymphocytes. IEVs were extracted by the method of Experimental example 2, and counted at 1X 10⁷Splenocytes were added to anti-CD3/CD28 coated dishes and co-cultured with IEVs at IEVs/splenocyte ratios of 0, 1/10(0.1), 1/5(0.2), 1:1(1), respectively, 3 days later cells and culture medium supernatants were harvested and examined for cell activation, subpopulation distribution and apoptotic phenotype, and cytokine levels using flow cytometry and ELISA, respectively.

2. As a result: as shown in FIG. 7, IEVs were administered in a dose-dependent manner against CD4 in mouse spleen cells⁺The activation of the T helper cell is inhibited, and the inhibition effect is more obvious on high-strength T cell receptor signals; IEVs vs CD4⁺The T helper cell inhibition is mainly performed on functional CD4 including Th1, Th17, Th2 and the like⁺Significant suppression of the T cell subpopulation, but not regulatory Foxp3+ Treg cells, suggested that IEVs were able to preferentially suppress the CD4+ T helper cell subpopulation that promotes inflammation without suppressing immune-regulatory Treg cells. Furthermore, IEVs also induce T lymphocytes to activate associated apoptosis in a dose-dependent manner.

Example 5 the immunomodulatory effects of IEVs are superior to exosomes in that they act in a manner independent of their soluble components

1. The detection step or method comprises: equal amounts of IEVs and exosomes were extracted as in Experimental example 2 and comparative example 1, respectively, and the IEVs supernatants were harvested from equal amounts of IEVs placed in cell culture medium for 72h to release their soluble components sufficiently and centrifuged. Splenocytes were co-cultured with IEVs, exosomes or IEVs supernatant, respectively, at a volume ratio of 1/5(0.2) as in Experimental example 4, and the cell activation phenotype was examined using flow cytometry after 3 days of cell harvest.

2. As a result: as shown in FIG. 8, IEVs vs CD25⁺CD4⁺T cells have an immunosuppressive effect superior to exosomes and are not completely independent of the soluble components released by IEVs.

Example 6IEVs were contacted directly with CD4 via Phosphatidylserine (PS)⁺T cells exert immunomodulatory effects

1. The detection step or method comprises: magnetic separation of splenocytes from non-activated CD25^-Juvenile CD4⁺And (3) co-culturing the IEVs or the IEVs with Annexin 5(A5) closed PS and the naive T cells according to the proportion of 1/5(0.2), collecting the cells after 1 hour, detecting a TCR signal path by using a western blot method, collecting the cells after 3 days and culture medium supernatant, and detecting the cell activation phenotype and the cytokine content by using flow cytometry and ELISA respectively. IEVs labeled with PKH26 or with A5-blocked PS were also used, and contact of the IEVs with T cells was observed under a fluorescent microscope.

2. As a result: as shown in FIG. 9, a shows that IEVs are able to directly inhibit purified naive CD4⁺T cell activation; b-c. shows that a5 blocks PS processing impairing inhibition of T cell activation and IL-2 secretion by IEVs; d-e shows that PKH 26-labeled IEVs were in direct intimate contact with T cells, whereas A5-blocked PS did not achieve intimate contact with T cells; f shows that IEVs can significantly inhibit TCR signal, and IEVs blocked by A5 cannot inhibit TCR signal.

IEVs achieve immunosuppressive effects by inhibiting TCR signaling through direct, intimate contact with T cells.

Example 7IEVs inhibit Th17 differentiation

1. The detection step or method comprises: magnetic separation of splenocytes from non-activated CD25^-Juvenile CD4⁺And (3) co-culturing IEVs or BMMSCs and naive T cells in an equal proportion and under the condition of Th17 differentiation according to the proportion of IEV/splenocytes of 0, 1/10(0.1), 1/5(0.2) and 1:1(1) to obtain T cells, collecting cells and culture medium supernatant after 3 days, and detecting the cell activation phenotype and the content of cytokines by using flow cytometry and ELISA respectively.

2. As a result: as shown in fig. 10, a is the dose dependence of IEVs on inhibition of Th17 differentiation; b is that IEVs inhibit the secretion of IL-17A within 6 hours; c is that IEVs inhibit Th17 differentiation more significantly than the equal proportion of BMMSCs.

Example 8IEVs to regulate the immune homeostasis of MRL/lpr lupus mice, effectively control SLE disease in experimental animals

1. The detection step or method comprises: adopting MRL/lpr spontaneous lupus model with Fas gene mutation, collecting 8-week-old MRL/lpr female mice, and repeating the steps once a week at a rate of 1 × 10⁶IEVs from individual MSCs (prepared in example 2) were resuspended in 200. mu.L PBS + 20. mu.LL heparin sodium solution (0.2% w/v). Mixing, standing on ice, and performing tail vein injection within 30 min. Control groups were injected with PBS. The organ system involvement and immunophenotype of mice were tested by drawing materials at 12 weeks of age or at the end of life, respectively, and the experimental schematic is shown in fig. 11, panel a.

2. As a result: as shown in FIG. 11, panels b-c show that IEVs injection effectively corrected relative apoptotic deficiency and lymphoproliferation in LPR circulating blood and spleen. Panel d shows IEVs corrected Foxp of spleen, mesenteric lymph nodes of experimental animals³⁺The proportion of Treg cells is unbalanced. Panel e shows that IEVs corrected the imbalance in the proportion of Th1 cells in the spleen, mesenteric lymph nodes of experimental animals. Panel f shows that IEVs improved CD4 in experimental animals by correcting memory cell accumulation, a na iotave cell-sparing phenotype in the spleen of Lpr mice⁺T cell homeostasis is deregulated. Panel g shows that IEVs corrected the activation-related apoptotic defect of experimental animal T lymphocytes. Panel h shows that IEVs improve autoantibody levels in the circulating blood of experimental animals. FIG. i is a schematic diagram of a long-term experiment. Panel j shows that IEVs corrected pathological enlargement of spleen, mesenteric lymph nodes in experimental animals. Panel k shows that IEVs corrected T lymphocyte infiltration in the lungs and colon of experimental animals. Panel l shows that IEVs delay the production of proteinuria in experimental animals, implying protection of renal function. Panel m shows renal HE staining, Trichrome staining, suggesting that IEVs improve immune cell infiltration and fibrotic changes in experimental locomotor kidneys. Panel n shows that IEVs improve survival in experimental animals. From FIG. 11, IEVs regulated the immune homeostasis of MRL/lpr lupus mice, effectively controlling SLE disease in experimental animals.

Example 9IEVs have an excellent effect of preventing and treating rheumatoid arthritis.

(1) The detection step or method comprises: a collagen-induced arthritis model is adopted, a DBA/1 mouse with the age of 8 weeks is taken, the emulsified II type collagen and Freund's adjuvant are injected subcutaneously into D0, and the immunity is enhanced in D7, so that an experimental animal generates autoantibodies to the II type collagen, and RA attack is induced. 1X 106 MSCs-derived IEVs (prepared in example 2) were resuspended in 200. mu.L PBS + 20. mu.L heparin sodium solution (0.2% w/v) at D14. Mixing, standing on ice, and performing tail vein injection within 30 min. ControlThe groups were injected with PBS, 1X 10 respectively⁶MSCs, or tumor necrosis factor inhibitor (TNFa, 5mg/kg) were intraperitoneally injected twice a week. Arthritis activity was recorded daily and mice were tested for joint pathology and immunophenotype by taking material at D42. Th17 cells play a key role in the pathogenesis of this RA model. The experimental schematic is shown in fig. 12, panel a.

(2) As a result: as shown in fig. 12, panel b is the clinical score of the joints of mice in each treatment group, and shows that IEVs significantly inhibit the mobility of arthritis, the effect of one injection is superior to that of BMMSCs, and the effect is comparable to that of TNFi, which is significantly superior to that of PBS control group.

Panel c is a comparison of the pre-treatment and post-treatment day 42 treatment affected joints in groups of experimental animals, and it can be seen that the therapeutic effect of IEVs is comparable to TNFi.

The results of the HE, collagen fastgreen staining and the uCTt experiment show that the pathological change of the joint tissues of the IEVs treatment group is superior to that of the BMMSCs, and the effect of the IEVs treatment group is comparable to that of TNFa of the first-line therapy and is significantly superior to that of the PBS control group.

Panel f is a flow cytometry assay showing that Th17 cells in spleen, draining lymph nodes of experimental animals of IEVs treated group were significantly less than BMMSCs and PBS treated group.

From fig. 12, it can be seen that a single injection of IEVs can significantly inhibit arthritis activity, with better effect than BMMSCs, comparable to first-line therapy TNFi, significantly better than PBS control group.

Test example 1

(1) The detection step or method comprises: taking 8-week-old Sjogren Syndrome (SS) model mice, injecting MSCs and IEVs through a tail vein system, taking materials 4 weeks after injection, detecting the flow rate of saliva, collecting salivary gland samples, and performing paraffin section HE staining and B cell marker B220 staining.

(2) As a result: as shown in fig. 13A-fig. 13C, the results showed that, compared to the effect of the mice bone marrow mesenchymal stem cells and their derived IEVs on the salivary flow rate of sjogren's syndrome, there was a slight recovery of salivary flow rate after mesenchymal stem cell treatment, and no improvement in salivary flow rate after IEVs treatment was seen (. # p <0.05 compared to WT group, # p <0.001 compared to MSCs group). IEVs injection did not alter inflammatory infiltration of salivary glands and B cell accumulation.

Test example 2

The IEVs obtained in example 2 and the Exosomes extracted in comparative example 1 were tested for their in vitro procoagulant effects using an in vitro clotting assay. The results are shown in Table 3, where IEVs significantly reduced the in vitro clotting time of most plasma, and the procoagulant effect was better than that of Exosomes.

However, for plasma deficient in factors II, V, X, IEVs failed to exert in vitro procoagulant effects, suggesting that the in vitro procoagulant effects of IEVs are more focused upstream of the common pathway of coagulation.

TABLE 3

Hemophilia A mice (factor VIII deficient) were used as a model and injected by tail vein with 9X 10⁸IEVs, observed for in vivo procoagulant effects of IEVs. The results are shown in fig. 14, which shows that after IEVs treatment, the bleeding tendency of hemophilia mice can be significantly improved, and the treatment effect can be sustained and stably maintained for 14 days.

Experimental results show that IEVs are able to exert a significant procoagulant effect in vitro. And the composition can remarkably improve the bleeding tendency after in vivo injection, and can be used for improving the bleeding tendency caused by hemosiderosis A. The levels of various coagulation factors in the plasma of mice were also measured, and no significant change was observed in any of coagulation factor VIII, vWF factor, Tissue Factor (TF), and prothrombin (fig. 15A, 15B, 15C, 15D).

In hemophilia a mouse model, normal IEVs, PS negative IEVs and TF negative IEVs were injected, respectively, and after 7 days, tail-clipping experiments were performed, and the results are shown in fig. 16A and fig. 16B, where the blockade of PS and TF did not affect the in vivo therapeutic effect of IEVs, which primarily indicates that the mechanism of IEVs in treating hemophilia mice is independent of PS and TF. In the past literature reports, the coagulation promoting effect of extracellular vesicles is highly dependent on PS and TF on the surface of the extracellular vesicles, and the in vivo experimental results of IEVs are inconsistent with the previous researches, which suggests that the IEVs may have a new action mechanism to exert the coagulation promoting effect under the in vivo environment.

For blood friendDisease A mouse model, injection treatments (9X 10) of IEVs (obtained in example 2) and Exosomes (extracted in comparative example 1) from the same MSCs, respectively⁸One), the results show that IEVs are able to significantly correct bleeding tendencies in mice, while Exosomes have no significant therapeutic effect (fig. 17).

Claims (10)

1. Use of vesicles in the manufacture of a modulator of T cell activity, wherein the vesicles are inductive vesicles; the diameter of the vesicle is 0.45 μ M or less.

2. The use of claim 1, wherein the vesicle has a diameter of 0.05-0.45 μ Μ.

3. The use of claim 1, wherein said T cells comprise CD4⁺A T helper cell;

preferably, the T cell comprises Treg, Th1, Th17, Th 2.

4. The use of claim 1, wherein the activity modulator inhibits the activity of Th1, Th17, Th 2;

preferably, the activity modulator inhibits differentiation of Th 17;

preferably, the activity modulator inhibits inhibition of CD25⁺CD4⁺T cells.

5. The use of claim 1, wherein the vesicle is prepared by contacting CD4 directly with phosphatidylserine on the surface of the vesicle⁺T cells achieve regulatory T cell activity.

6. Use of vesicles in the manufacture of a medicament for the treatment or prevention of an autoimmune disease, wherein the vesicles are inducing vesicles; the diameter of the vesicle is 0.45 μ M or less.

7. The use of claim 7, wherein the autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis.

8. The use of claim 7, wherein the autoimmune disease does not include Sjogren's syndrome.

9. The use of claims 1-8, wherein the inducing vesicles are vesicles produced by inducing apoptosis by an external agent when stem cells are in normal survival;

preferably, the inducing vesicle induces the generation of stem cell apoptosis, and the inducing method comprises adding staurosporine, ultraviolet irradiation, starvation, or thermal stress;

preferably, the stem cell is a mesenchymal stem cell;

preferably, the source of mesenchymal stem cells comprises bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon or peripheral blood.

10. The use of claims 1-9, wherein the vesicles have a diameter of 0.05-0.4 μ Μ;

preferably, the diameter of the vesicle is 0.05-0.38 μ M;

preferably, the diameter of the vesicle is 0.05-0.35 μ M;

preferably, the diameter of the vesicle is 0.05-0.32 μ M;

preferably, the diameter of the vesicle is 0.05-0.3 μ M;

preferably, the diameter of the vesicle is 0.05-0.25 μ M;

preferably, the diameter of the vesicle is 0.05-0.22 μ M;

preferably, the diameter of the vesicle is 0.55-0.22 μ M.

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