CN114073715A

CN114073715A - Application of extracellular vesicles in preparation of preparation for regulating ovarian function

Info

Publication number: CN114073715A
Application number: CN202010800914.1A
Authority: CN
Inventors: 傅钰; 寇晓星; 施松涛
Original assignee: Medical Micro Cell Biotechnology Guangzhou Co ltd
Current assignee: Medical Micro Cell Biotechnology Guangzhou Co ltd
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2022-02-22

Abstract

The application discloses the use of extracellular vesicles in the preparation of a formulation for modulating ovarian function. The present application discloses that MRL/lpr mice exhibit increased ovaries, increased secondary and mature follicles, and premature ovaries, e.g., in estrus, for a long period of time, as compared to wild-type mice. These characterizations of MRL/lpr mice returned to normal levels after treatment with induced extracellular vesicles, indicating that induced extracellular vesicles may ameliorate the symptoms of premature ovary. Meanwhile, the application also discloses an improvement effect of the induced extracellular vesicles on premature ovarian failure.

Description

Application of extracellular vesicles in preparation of preparation for regulating ovarian function

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of extracellular vesicles in preparation of a preparation for regulating ovarian function.

Background

Female sexual precocity is caused by premature activation of ovarian follicular development, which enters into a pattern of puberty development even though women are not at their actual age (before age 8). Premature ovaries will lead to premature follicular development and excessive follicular discharge. From the hormone level, premature ovary will result in increased estrogen, and too high estrogen will result in precocious puberty, obesity and excessive ovulation, and complications also include uterine fibroids and endometriosis.

Premature Ovarian Failure (POF) refers to premature ovarian failure before the age of 40, and specifically includes some symptoms of ovarian function reduction, such as menstrual cycle disorder, abnormal menstrual flow and genital tract inflammation, and is also accompanied by a series of climacteric symptoms, such as skin relaxation, sleep loss, emotional anxiety and the like, and seriously affects the normal life of women.

Chemotherapy is widely used in the treatment of various malignancies and autoimmune diseases, but patients receiving chemotherapy must face severe side effects, and for female patients, irreversible damage to ovarian tissue from chemotherapy remains a significant concern. Premature ovarian failure is characterized primarily as: the volume of the bilateral ovaries is obviously reduced, granular cells are apoptotic, follicles are locked, and the estrogen level is reduced. The hot flashes, osteoporosis, sexual dysfunction and infertility are all the consequences of premature ovarian failure caused by chemotherapy, and at present, hormone supplementation treatment is mostly adopted for POF caused by chemotherapy, but the method cannot actually recover the morphological function of ovaries.

In the aspect of premature ovary, GnRH-a treatment is widely applied clinically as a gold standard, but has the side effects of possible headache, rash, gastrointestinal discomfort and the like, and a medicine or a preparation more suitable for maintaining the homeostasis of a human body is still lacked. In the aspect of premature ovarian failure, estrogen supplementation therapy is the most common method, and in principle, reduced estrogen is generated by supplementing ovarian failure, but the method only improves symptoms and cannot radically recover the ovarian function, and meanwhile, the method has the side effect of aggravating symptoms after drug withdrawal due to the dependence on estrogen. At present, no medicament or biological agent which can simultaneously generate curative effect on premature ovary and premature ovarian failure exists in the aspect of clinical treatment.

Extracellular Vesicles (EVs) are nanoscale carriers containing proteins, nucleic acids, and various cytokines secreted by cells. Extracellular vesicles can act on target cells through endocrine or paracrine modes, and play an important role in the processes of intercellular substance transfer and information exchange. Researches show that the information exchange mediated by the extracellular vesicles plays an important role in regulation and control in the physiological or pathological process of an organism, and relates to immunoregulation, tumor growth, angiogenesis, injury repair and the like. Research in this area is currently focused mainly on the exosomes (exosomes) direction. Exosomes are extracellular vesicles with a diameter of about 30-150nm, and contain RNA, lipids, proteins, and other components. Exosomes are widely involved in various physiological/pathological regulation of the body and can be used as diagnostics, therapeutics and prognostic assessments of a variety of diseases. To date, Mesenchymal Stem Cells (MSCs) are considered to be the most potent cells for producing exosomes. A plurality of researches find that the exosome from the MSCs can simulate the biological function of the MSCs and plays an important regulation and control role in promoting the growth and differentiation of cells, repairing tissue defects and the like. Therefore, in recent years, cell vesicle therapy based on exosomes derived from MSCs has been remarkably developed. However, the current cell vesicle therapy based on exosome still has many problems, which are mainly shown in that the extraction and purification process of exosome is complex, time-consuming, high in requirement on equipment and reagents, low in physiological exosome yield and the like, and the clinical transformation and application of exosome therapy are limited by the defects.

Disclosure of Invention

In one aspect, the application provides the use of induced extracellular vesicles in the preparation of a formulation for modulating ovarian function.

In some embodiments, the modulating ovarian function comprises one or more of the following uses:

a. improving premature ovarian development;

b. improving premature ovarian failure.

In some embodiments, the premature ovarian development is selected from premature ovarian development resulting from immunity.

In some embodiments, the premature ovarian failure is selected from premature ovarian failure resulting from immunological, pharmacological, or age-related increases.

In some embodiments, the formulation is a therapeutic or prophylactic precocious formulation.

In some embodiments, the formulation is a formulation for treating or preventing menoxenia.

In some embodiments, the formulation is a formulation for treating or preventing excessive ovulation.

In some embodiments, the formulation is a formulation for treating or preventing premature ovarian failure.

The IEVs in the embodiments of the present invention are simply referred to as Induced extracellular vesicles, which may be referred to as Induced 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 fused 80-100% of the time during the 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.

In some embodiments, the agent is selected from an agent that reduces ovarian size, reduces ovarian weight, or reduces the ovarian to body weight ratio.

In some embodiments, the formulation is a formulation that modulates the physiological cycle of the ovary. In some embodiments, when a mouse is used as the subject, the physiological cycle is an estrus cycle; when human is used as the subject, the physiological cycle refers to the menstrual cycle.

In some embodiments, the formulation is a formulation that modulates serum estradiol levels.

In some embodiments, the formulation treats or prevents premature ovarian failure by modulating the Wnt signaling pathway.

In some embodiments, the formulation treats or prevents premature ovarian failure by modulating the expression of β -Catenin.

In some embodiments, the formulation is selected from a pharmaceutical formulation or a nutraceutical formulation.

In some embodiments of the present application, it was found that MRL/lpr mice (MRL/lpr mice are Fas receptor mutant mice) exhibit increased ovaries, increased secondary and mature follicles, and premature ovarian maturation, characterized by long term estrus, as compared to wild-type mice. These characterizations of MRL/lpr mice returned to normal levels after injection of IEVs, indicating that IEVs can ameliorate symptoms of premature ovarian failure.

In some embodiments of the present application, injection of IEVs reduces follicular atresia in POF models and restores ovarian morphology in mice in premature ovarian failure models established with Cyclophosphamide (CP). Meanwhile, researches on the expression level of beta-Catenin in a POF group mouse discover that the CP group mouse has the condition that the expression level of the beta-Catenin in a Wnt signal channel is increased, and the injection of IEVs can reduce the expression level of the beta-Catenin to a normal level, which shows that the IEVs can improve premature ovarian failure by regulating the expression of the beta-Catenin.

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

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

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

In some embodiments, the mesenchymal stem cells are selected from one or more of blood mesenchymal stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, periosteal mesenchymal stem cells, skin mesenchymal stem cells.

In some embodiments, the mesenchymal stem cells are selected from one or more of blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, oral mesenchymal stem cells, skin mesenchymal stem cells.

In some embodiments, the stem cells are selected from one or both of blood stem cells or bone marrow mesenchymal stem cells.

In some embodiments, the induced extracellular vesicles are produced by inducing apoptosis of mesenchymal stem cells by addition of staurosporine.

In some embodiments, the concentration of staurosporine is greater than or equal to 1 nM; preferably, it is 1-15000 nM; preferably 200-; preferably, 250-1000 nM; preferably 500-1000 nM. In addition, the concentration of staurosporine can also be 280-9000 nM; may also be 230-; and can also be 500-1000 nM; and can also be 500-900 nM; and may be 500 and 800 nM.

In some embodiments, the diameter of the inducing extracellular vesicles is 0.45 μm or less. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.45 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.1-0.45 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.1-0.35 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.35 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.3 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.2 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.4 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.38 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.35 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.32 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.3 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.25 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.22 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.22 μm. In some embodiments, the diameter of the inducing extracellular vesicles may also be 0.15-0.45 μm, and may also be 0.2-0.3 μm.

In some embodiments, the induced extracellular vesicles have the marker Syntaxin 4. In some embodiments, the inducible extracellular vesicle high expression marker Syntaxin 4. In some embodiments, the marker Syntaxin 4 of the induced extracellular vesicles is expressed higher than MSC 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 extracellular vesicle overexpression markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 of the induced extracellular vesicles are expressed in higher amounts than the MSCs or exosomes. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 are expressed in the induced extracellular vesicles in an amount of 1-2 fold, 2-3 fold, 1-3 fold, and 3-4 fold, respectively, relative to the amount of expression of the markers 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 induced extracellular vesicles in an amount of 1.5-2 fold, 2.5-3 fold, 1.5-2.5 fold, and 3.5-4 fold, respectively, relative to the expression of the markers 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 induced extracellular vesicles in an amount of 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold, respectively, relative to the expression of the marker in exosomes derived from mesenchymal stem cells. The induced extracellular vesicles of the invention are substantially different from exosomes, for example, the induced extracellular 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 significantly higher than those of exosomes (see example 3). In addition to differential marker expression, the induced extracellular vesicles IEVs also exhibit characteristics that are distinct from stem cells and other extracellular vesicles such as exosomes, either functionally or therapeutically. In some embodiments, the inducing extracellular vesicles further express CD29, CD44, CD73, CD 166; and does not express CD34, CD 45. In some embodiments, the inducing extracellular vesicles further express one or more of CD9, CD63, CD81, and C1 q.

In some embodiments, the medicament is an injection, an oral formulation, or an external formulation.

In some embodiments, the drug is an injection.

In some embodiments, the drug is an intravenous, intramuscular, subcutaneous, or intrathecal injection.

In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants, and lubricants.

In some embodiments, there is also provided a method of preparing the induced extracellular vesicles, comprising the steps of:

1) culturing mesenchymal stem cells in vitro, and washing with PBS for 2-5 times when the cells are 80% -90% confluent;

2) adding the mesenchymal stem cells prepared in the step 1) into a serum-free culture medium containing 500-1000nM staurosporine, incubating for 16-24h at 37 ℃, and collecting cell supernatant;

3) centrifuging the cell supernatant collected in step 2) at 500-;

4) centrifuging the cell supernatant collected in step 3) at 1500-;

5) centrifuging the cell supernatant collected in the step 4) at 10000-;

preferably, a washing step of the induced extracellular vesicles is also included;

preferably, the cleaning step specifically comprises: 6) resuspending the induced extracellular vesicles prepared in step 5) with PBS, and centrifuging at 10000-30000g at 4 ℃ for 15-60 minutes to obtain precipitates which are the induced extracellular vesicles.

In the present invention, the "mesenchymal stem cell" refers to a pluripotent stem cell having all the commonalities of stem cells, i.e., self-renewal and multipotential differentiation ability. The mesenchymal stem cells can be derived from bone marrow, fat, blood (e.g., extrinsic blood), synovium, bone, muscle, lung, liver, pancreas, oral cavity, craniomaxillofacial (e.g., deciduous teeth, dental pulp, periodontal ligament, gingiva, apical tooth papilla, etc.), and amniotic fluid, umbilical cord, i.e., the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, skeletal mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreas mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc.

In the present invention, the term "preventing" means that when used in a disease or condition, the agent reduces the frequency of, or delays the onset of, symptoms of the medical condition in a subject as compared to a subject to which the agent is not administered.

In the present invention, the term "treating" refers to alleviating, alleviating or ameliorating the symptoms of a disease or disorder, ameliorating the underlying metabolic-induced symptoms, inhibiting a disease or symptom, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, causing regression of a disease or disorder, alleviating a condition caused by a disease or disorder, or arresting the symptoms of a disease or disorder.

Drawings

FIG. 1 is a flow cytometric assay of example 1.

FIG. 2 is a technical scheme for preparing IEVs according to example 2.

FIG. 3 is a statistical analysis of the number of IEVs produced by MSCs (106 MSCs) analyzed by flow cytometry.

FIGS. 4A-4D are diameter measurements of IEVs particles: FIG. 4A is a graph of the intensity of light scattered from IEVs analyzed using standardized small particle microspheres from Bangs Laboratories, Inc., showing the particle diameter distribution of IEVs; 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 assay techniques, showing the particle diameter distribution of IEVs.

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

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 quantification 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 shows that the ovarian weight was significantly elevated in 12-20 weeks MRL/lpr mice compared to C57BL/6 wild-type mice, and the ovarian-to-body weight ratio was significantly higher than in C57BL/6 wild-type mice, all of which were significantly down-regulated following IEVs injection.

FIG. 8 shows that the ovarian morphology of LPR mice at 20 weeks was significantly greater than that of C57BL/6 wild type mice, and that the ovarian morphology became smaller after injection of IEVs, but still larger than that of C57BL/6 wild type mice.

FIG. 9 shows the change of ovarian follicles at all levels in ovaries after 12-20 weeks of injection of IEVs in C57BL/6 wild-type mice, MRL/lpr mice. The red arrow indicates the case of follicular atresia.

FIG. 10 shows statistics of the number of follicles at all levels in ovaries after 12-20 weeks of injection of IEVs in C57BL/6 wild-type mice, MRL/lpr mice, and MRL/lpr mice.

FIG. 11 shows the variation of the estrous cycle of 20-week C57BL/6 wild-type mice, MRL/lpr mice injected with IEVs; in the figure: estrus prophase (promestrus, P), Estrus (Estrus, E), Estrus postestrus (Metestrus, M), Estrus interphase (disestrus, D).

FIG. 12 shows the change in serum estradiol following injection of IEVs to C57BL/6 wild type mice, MRL/lpr mice from 12 weeks to 20 weeks.

In fig. 13, a: the ovary of the wild mouse is sliced, and all stages of follicles and corpus luteum can be seen; b: after injection of CP at 100mg/kg for 7d, the granular cell layer disappeared in the mouse ovary primordial follicles and primary follicles, the follicles were atretic (indicated by red arrows), and the number of mature follicles was reduced; c: after injection of IEVs into CP group mice, a reduction in ovarian atretic follicles was seen (indicated by red arrows), but no mature follicles were seen. D: panel A is a partial magnification showing mature follicles of normal ovaries; e: the diagram B is enlarged partially and shows a large number of locked follicles (indicated by red arrows) formed by the original and primary follicles. F: the plot C is enlarged partially and shows a significant reduction in the number of locked follicles in the CP + IEVs group compared to the CP group (indicated by the red arrows).

FIG. 14 shows the change in estradiol in serum of different groups.

In FIG. 15, A/B/C: in wild type mouse ovary, the expression level of beta-Catenin is low in mesenchymal cells (indicated by red arrow) and luteal granular cells (indicated by white arrow); D/E/F: in the ovary of the CP group mouse, the expression level of beta-Catenin is increased at interstitial cells (indicated by red arrows) and luteal granule cells (indicated by white arrows); G/H/I: in the ovary of the mouse CP + AB group, compared with the ovary of the mouse CP group, the expression level of beta-Catenin at interstitial cells (indicated by red arrows) and luteal granule cells (indicated by white arrows) is reduced, and is consistent with the wild type.

Figure 16A is a schematic representation of the dynamic metabolism of IEV at the skin surface in example 11.

Figure 16B shows that over time, IEV gradually moved from the subcutaneous tissue to the dermal layer and epidermis.

FIG. 16C shows that PKH26-IEV was found in hair follicles found in hairs pulled from mouse bodies at day 7.

Figure 17A shows the distribution of IEV throughout the body of mice on

days

1, 3, and 7 as measured by in vivo imaging techniques.

FIG. 17B shows distribution of IEV in each mouse organ as measured by in vivo imaging technique.

Figure 17C is a comparison of IEV distribution in mouse organs at day 7 with control.

FIG. 17D is a schematic illustration of the dynamic metabolism of IEV in the colon on

days

1, 3, and 7; wherein M represents the muscularis mucosae layer and V represents the chorion.

Figure 17E shows a schematic representation of the metabolism of IEVs in the nail and incisors of mice on day 3.

Figure 18 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 Sjogren syndrome submandibular gland HE; C. treatment of the effects of sjogren's syndrome on B cells.

Figure 19 is an in vivo procoagulant effect of IEVs in hemophilia a mice.

Fig. 20A-20D are graphs showing the change in levels of various clotting factors following injection of IEVs into hemophilia a mice: FIG. 20A is a variation of factor VIII; FIG. 20B shows the change in vWF factor; FIG. 20C shows changes in Tissue Factor (TF); FIG. 20D shows the prothrombin profile.

FIGS. 21A-21B are graphs of the effect of IEVs on the in vivo therapeutic efficacy of IEVs after PS and TF blockade, respectively, in a mouse model of hemophilia A.

FIG. 22 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 application are further illustrated by the following specific examples, which do not represent a limitation to the scope of the present application. Insubstantial modifications and adaptations of the concepts taught herein by others are intended to be covered by the present disclosure.

STS in the present invention is staurosporine.

"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding others. When used in defining compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any significance to the combination of the stated objects. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed application. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present application.

The term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and/or" means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition, combination, construction, etc., is described as comprising (or comprising) components a, B, C, and/or D, the composition can comprise a alone; b alone; separately comprises C; separately contain D; a combination comprising A and B; a combination comprising A and C; a combination comprising A and D; a combination comprising B and C; a combination comprising B and D; a combination comprising C and D; a combination comprising A, B and C; comprising a combination of A, B and D; a combination comprising A, C and D; comprises a combination of B, C and D; or A, B, C and D are used in combination.

EXAMPLE 1 isolation and culture of BMMSCs

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, the metaphysis is further separated, the marrow cavity is exposed, PBS with the volume fraction of 10% fetal calf serum is extracted by a 10mL aseptic syringe to repeatedly wash the marrow cavity, after filtration by a 70-micron pore cell filter screen, 500g is centrifuged for 5min, the cell sediment at the bottom is collected after the supernatant is removed, PBS is resuspended, the PBS is centrifuged again for 5min by 500g, and the final cell sediment is collected. The cells were then flow sorted and Bone Marrow Mesenchymal Stem Cells (BMMSCs) were sorted using CD 34-and CD90+ as sorting criteria. 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. BMMSCs were digested by trypsin incubation at 37 ℃ and expanded by passage, after which Dex (+) culture medium was changed every 3 days and passaged after confluency. 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 1 Dex (-) composition of culture solution

TABLE 2 Dex (+) culture solution formulation Table

The purity of the isolated BMMSCs was assessed by flow cytometry analysis of surface markers. To pairAfter surface marker identification and collection of P2 BMMSCs by trypsinization, the BMMSCs were 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 results of the detection are shown in FIG. 1.

Example 2 preparation of BMMSC-derived IEV

MSCs (bone marrow-derived MSCs) cultured up to passage 2 in example 1 were cultured in the medium (Dex (+) culture) 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 16-24h, cell supernatant was collected, centrifuged at 4 ℃ at 800g for 10 min, supernatant was collected, centrifuged at 4 ℃ at 2000g for 10 min, collected at 4 ℃ at 16000g for 30min, and the resulting precipitate was IEV. 500 μ l PBS heavy suspension precipitation, 4 degrees C under 16000g centrifugation for 30 minutes, get the washing IEV.

The preparation route is shown in figure 2.

Comparative example 1 exosome isolation and extraction from homogeneous MSC

MSCs (bone marrow-derived MSCs, BMMSCs) 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 by 80% to 90%, serum-free medium was added, incubated at 37 ℃ for 48 hours, and cell supernatant was collected for isolation 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 with sterile PBS at 120000g for another 90 min, removal of supernatant, collection of bottom exosomes, and resuspension with 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 performed by flow cytometry at 1h, 4h, 8 th time pointsh. 16 th and 24 th hour, results display 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 IEVs, it can be seen that after 24h induction, a single MSC can yield 300 IEVs (fig. 3).

Furthermore, flow assays found particle diameter distributions of IEVs centered at 1 μm or less, representing 94.97%.

Side scatter light (SSC) analysis also shows IEVs scattered light intensity concentrated in the range below 1 μm.

Further, 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) observation were similar to those of flow-based assays, with most vesicles having diameters between 200nm and below 200nm (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 100.63 nm (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 quantitative analysis result of DIA (FIG. 6D).

MSCs-Exosomes: refers to exosomes derived from MSCs.

MSCs-IEVs: refer to IEVs derived from MSCs.

Wherein the MSCs in the assay of the contents are of the same cell line as the MSCs from which the exosomes and IEVs are extracted.

General Experimental methods for examples 4-7

1. Experimental Material

The 6 normal wild type C57BL/6, 12 MRL/lpr mice were divided into three groups, i.e., C57BL/6 group (wild type control group), MRL/lpr group, MRL/lpr + IEVs group, 6 mice per group.

IEVs were prepared for example 2 and MSCs were BMMSCs prepared for example 1.

2. Experimental methods

2.1 treatment of mice

MRL/lpr + IEVs group 68 week MRL/lpr mice tail vein injection 4X 10⁸One/one IEVs, two from each of the three groups after 4 weeks; the remaining 4 MRL/lpr + IEVs group continued tail vein injection 4X 10⁸One/one IEVs, two from each of the three groups after 4 weeks; the remaining 2 MRL/lpr + IEVs group continued tail vein injection of 4X 10⁸IEVs/one, 4 weeks (period vaginal smear, detection estrus cycle) three groups of two each;

i.e. the experiment sets 3 time gradients: each time gradient consisted of C57BL/6 (wild-type control), MRL/lpr + IEVs, 2 each at 12, 16, and 20 weeks. 2 mice of the MRL/lpr + IEVs group in the 12-week gradient were each injected with 1 IEVs, 2 mice of the MRL/lpr + IEVs group in the 16-week gradient were each injected with 2 IEVs, and 2 mice of the MRL/lpr + IEVs group in the 20-week gradient were each injected with 3 IEVs.

Material taking: weighing and recording the mice before the mice are sacrificed; anesthetizing the mouse with pentobarbital, collecting blood from eyeball, precipitating, centrifuging, collecting supernatant, and measuring sex hormone; weighing ovaries, fixing with paraformaldehyde, embedding in paraffin, performing paraffin section, and performing HE staining.

2.2 staining, sectioning, how to count the number of follicles at each level

After the ovary is fixedly embedded, carrying out paraffin continuous section, after HE staining, counting all levels of follicles of the ovary at intervals of 5 slices, and taking follicles of the oocyte which is wrapped by a monolayer of flat granular cells as original follicles (primordial follicle); the follicles in which the oocytes are surrounded by the multi-layered cuboidal granulocytes but which do not form the follicular cavity are Primary follicles (Primary follicles); the multilayered cuboidal granulosa cells that wrap the oocyte and initially form the cavity are the Secondary follicles (Secondary follicles); granulocytosis, and the enlarged cavity is called as the cavitated follicle (Antral follicule); the follicles form cumulus, i.e. the follicles that are to be expelled are called Preovulatory follicles (Preovulatory follicles). Both luminal and preovulatory follicles are Mature follicles (Mature follicles).

Example 4 BMMSCs-derived IEVs reduce ovarian size, weight, ovarian weight to weight ratio in MRL/Lpr mice

As shown in FIG. 7, the ovarian weight of 12-20 weeks MRL/lpr mice was significantly increased compared to C57BL/6 wild-type mice, the ratio of ovarian weight to body weight was significantly higher than that of C57BL/6 wild-type mice, and the ovarian weight, ovarian weight to body weight ratios were all significantly down-regulated after injection of IEVs. As the mice age (12 weeks-20 weeks), the ratio of ovary to body weight of MRL/lpr + IEVs group mice gradually returned to the level of wild type control mice, and the premature ovary was reversed, indicating that there was a trend toward better treatment of IEVs as the mice age increased.

As shown in FIG. 8, the ovarian morphology was significantly greater in the 20-week MRL/lpr mice than in the C57BL/6 wild-type mice, and after IEVs injection (i.e., MRL/lpr + IEVs group mice), the ovarian morphology became smaller, but still larger than the ovaries of the C57BL/6 wild-type mice.

Example 5 Effect of BMMSCs-derived IEVs on ovarian follicular MRL/Lpr mice

As shown in FIGS. 9 and 10, the 12-week MRL/lpr mice showed an increased number of follicles at each level and a tendency to precocity, as compared with the C57BL/6 wild-type mice; after IEVs injection (i.e., MRL/lpr + IEVs group mice), follicular numbers were restored at all levels (at the same level as in the C57BL/6 group), suggesting that IEVs may alleviate the tendency of premature ovaries.

Compared with C57BL/6 wild-type mice, the MRL/lpr mice at 16-20 weeks have reduced numbers of primary and primary follicles and appear with a large number of follicular atresia, but the numbers of secondary and mature follicles are still large, and show a premature senility trend; after IEVs injection (i.e., MRL/lpr + IEVs group mice), there was a return in primary and primary follicular numbers and a decrease in secondary and mature follicular numbers as compared to the MRL/lpr group of mice, indicating a trend toward a remission of premature ovarian failure.

The results of this example demonstrate that IEVs have therapeutic effects on premature ovarian failure and premature ovarian failure, and that IEVs injection can restore the state of the ovary to normal levels in individuals showing either of premature ovarian failure and premature ovarian failure.

Example 6 Effect of BMMSCs-derived IEVs on estrus cycle in MRL/Lpr mice

Vaginal smear estrous cycle tests were performed on MRL/lpr mice, which were in estrous for most of the time in both estrous cycles (FIG. 11), and which returned to normal estrous cycles after IEVs injection.

Example 7 Effect of BMMSCs-derived IEVs on serum estradiol of MRL/Lpr mice

The serum estradiol concentration of MRL/lpr mice at 12-20 weeks is detected, as shown in figure 12, the estradiol content of the MRL/lpr mice is obviously higher than that of C57BL/6 wild-type mice; the serum estradiol content of mice in the IEVs injection treatment group (namely MRL/lpr + IEVs group mice) is reduced along with time from 16 weeks to 20 weeks, which shows that the symptoms of ovarian hypersecretion of estrogen are relieved after multiple IEVs injections, and the IEVs can restore the symptoms of premature ovarian failure of the organism by regulating the ovarian estrogen secretion.

The results of examples 4-7 demonstrate that MRL/Lpr mice exhibit premature ovarian maturation, and that the premature ovarian models of the present application, when injected with IEVs, reduce the ovarian size, weight, ovarian weight to weight ratio, reduce secondary and mature follicles, restore abnormal estrus cycles and serum estradiol levels, and ameliorate premature ovarian maturation.

General Experimental methods for examples 8-10

1. Experimental Material

The 18C 57BL/6 mice were divided into three groups, namely WT group (wild type control group), CP group (cyclophosphamide treated group), CP + IEVs group, 6 mice per group.

Cyclophosphamide (Cylcophosphamide, CP)

IEVs were prepared for example 2.

2. Experimental methods

2.1 treatment of mice

C57BL/6 mice were treated differently: wild Type (WT) no treatment; and (3) CP group: c57BL/6 mice were injected intraperitoneally at a CP dose of 100mg/kg once; CP + IEVs group: c57BL/6 mice were injected intraperitoneally once with a CP dose of 100mg/kg, seven days later at 4X 10⁸One/one IEV dose was administered once in tail vein and seven days later.

Material taking: anesthetizing the mouse with pentobarbital, collecting blood from eyeball, precipitating, centrifuging, collecting supernatant, and measuring estradiol; fixing ovary paraformaldehyde, embedding paraffin, performing paraffin section, and performing HE staining; fixing ovary paraformaldehyde, dehydrating sucrose, freezing, embedding, freezing, slicing, and performing immunofluorescence.

2.2 staining, sectioning, how to count the number of follicles at each level

Establishment of POF model

As shown in fig. 13, granular cell layers disappeared from the ovary primordial follicles and primary follicles in the CP group mice, the follicles were atretic (indicated by red arrows), and the number of mature follicles was decreased, indicating that the POF model was successfully established.

Example 8 Effect of BMMSCs-derived IEVs on ovarian follicular fluid in POF mice

After injection of IEVs into CP group mice, a reduction in ovarian atretic follicles was seen (indicated by red arrows), but no mature follicles were seen. (FIG. 13)

The experimental results indicate that IEVs can restore ovarian morphology in CP group mice.

Example 9 Effect of BMMSCs-derived IEVs on serum estradiol in POF mice

As shown in fig. 14, serum estradiol concentrations decreased in established POF mice injected with CP. By injecting IEVs into the CP group mice, the recovery of serum estradiol in the mice was observed compared to the CP group.

Example 10 BMMSCs-derived IEVs ameliorate premature ovarian failure by modulating beta-Catenin

As shown in FIG. 15, A/B/C: in wild type mouse ovary, the expression level of beta-Catenin is low in mesenchymal cells (indicated by red arrow) and luteal granular cells (indicated by white arrow); D/E/F: in the ovary of the CP group mouse, the expression level of beta-Catenin is increased at interstitial cells (indicated by red arrows) and luteal granule cells (indicated by white arrows); G/H/I: in the mouse ovaries of CP + IEVs group, compared with the CP group, the expression level of beta-Catenin in interstitial cells (indicated by red arrows) and luteal granule cells (indicated by white arrows) is reduced, and is consistent with the wild type.

The result shows that the CP group mice have the condition that the expression level of the beta-Catenin in the Wnt signal pathway is increased, and the injection of the IEVs can reduce the expression level of the beta-Catenin to a normal level, which shows that the IEVs can improve the premature ovarian failure by regulating the expression of the beta-Catenin.

Example 11 IEVs can be discharged transdermally and through hair

Take 4X 10⁶The IEV prepared in example 2 was re-suspended with DIR, 200. mu.l PBS, and systemically injected into the nude mice BALB/C-nu/nu via the caudal vein, and distribution of IEVs on the skin surface was observed after 1, 3, and 7 days using a live imaging device, and the results are shown in FIGS. 16A-16C.

Fig. 16A shows that IEVs are accessible to the skin surface, at the most abundant on day 3, and substantially disappeared on day 7, showing the dynamic metabolic process of IEVs at the skin surface (fig. 16A). Immunofluorescence results show that PKH26-IEV systemically injected C57 mice gradually moved from subcutaneous tissue to dermal and epidermal layers over time. The presence of a large number of IEVs was observed on the stratum corneum layer on the skin surface at day 7, suggesting that the systemically injected IEVs could be excreted as the stratum corneum of the skin sloughs (fig. 16B). Meanwhile, PKH26-IEVs were found in hair follicles in mice drawn down on the body surface at day 7, suggesting that IEVs injected systemically were also metabolized as the hair was shed (FIG. 16C).

In addition, both in vivo imaging data and immunofluorescence results showed that IEVs were distributed in large numbers in internal organs such as liver, spleen, bone marrow, lymph, etc., and were not distributed in heart, kidney, brain, but in colon, teeth and nails, further demonstrating that IEVs were excreted in vitro with metabolism (FIGS. 17A-17E).

This example shows that IEVs can be expelled through the skin and hair, indicating safety in injecting or increasing the level of IEVs in the body.

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. 18A-18C, the results showed that, compared to the effect of 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. 19, 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 can remarkably improve bleeding tendency after in vivo injection, and can be used for improving bleeding tendency caused by hemophilia 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. 20A, 20B, 20C, 20D).

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. 21A and 21B, 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 hemophilia A mouse model, injection treatments (9X 10) of IEVs (obtained in example 2) and Exosomes (extracted in comparative example 1) from the same MSCs were performed, respectively⁸One), the results show that IEVs are able to significantly correct bleeding tendencies in mice, while Exosomes have no significant therapeutic effect (fig. 22).

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the application and should not be taken as limiting the scope of the application. Rather, the scope of the application is defined by the appended claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. Use of an induced extracellular vesicle in the manufacture of a formulation for modulating ovarian function.

2. The use of claim 1, wherein modulating ovarian function comprises one or more of the following:

a. improving premature ovarian development;

b. improving premature ovarian failure;

preferably, the premature ovarian development is selected from premature ovarian development due to immunity;

preferably, the premature ovarian failure is selected from premature ovarian failure caused by immunity, drug or age increase.

3. The use of claim 1, wherein the formulation is a therapeutic or prophylactic precocious formulation;

preferably, the formulation is a formulation for treating or preventing menoxenia;

preferably, the formulation is one for the treatment or prevention of excessive ovulation;

preferably, the preparation is a preparation for treating or preventing premature ovarian failure;

4. The use of claim 1, wherein the agent is selected from the group consisting of an agent that reduces ovarian size, reduces ovarian weight, or reduces the ovarian to body weight ratio;

preferably, the formulation is one that modulates the physiological cycle of the ovary;

preferably, the formulation is a formulation that modulates serum estradiol content;

preferably, the formulation treats or prevents premature ovarian failure by modulating the Wnt signaling pathway;

preferably, the formulation treats or prevents premature ovarian failure by modulating the expression of β -Catenin;

preferably, the preparation is selected from a pharmaceutical preparation or a health product preparation.

5. The use of any one of claims 1 to 4, wherein the induced extracellular vesicles are vesicles produced by inducing apoptosis by an external agent when stem cells are in normal survival;

preferably, the induced extracellular vesicles are induced by stem cell apoptosis by adding staurosporium, ultraviolet irradiation, starvation, or thermal stress;

preferably, the stem cells are selected from mesenchymal stem cells;

preferably, the mesenchymal stem cells are selected from one or more of blood mesenchymal stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells, umbilical cord mesenchymal stem cells, periosteal mesenchymal stem cells and skin mesenchymal stem cells;

preferably, the mesenchymal stem cells are selected from one or more of blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, oral mesenchymal stem cells and skin mesenchymal stem cells;

preferably, the stem cell is selected from one or two of a blood stem cell or a bone marrow mesenchymal stem cell.

6. The use according to any one of claims 1 to 4, wherein preferably the stem cell extracellular vesicles are produced by induction of apoptosis of mesenchymal stem cells by addition of staurosporine.

7. The use according to any one of claims 1 to 4, wherein the concentration of staurosporine is greater than or equal to 1 nM;

preferably, it is 1-15000 nM;

preferably 200-; preferably, 250-1000 nM; preferably 500-1000 nM.

8. The use according to any one of claims 1 to 4, wherein the inducing extracellular vesicles have a diameter of 0.45 μm or less;

preferably, the diameter of the inducing extracellular vesicles is 0.05-0.45 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.1-0.45 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.1-0.35 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.15-0.35 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.15-0.3 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.15-0.2 μm.

9. The use of any one of claims 1 to 4, wherein the formulation is an injection, an oral formulation or an external formulation;

preferably, the formulation is an injection;

preferably, the formulation is an intravenous, intramuscular, subcutaneous or intrathecal injection;

preferably, the formulation further comprises a pharmaceutically acceptable carrier;

preferably, the pharmaceutical carrier comprises one or more of diluent, excipient, filler, binder, disintegrant, surfactant and lubricant.