CN110312515A

CN110312515A - The extracellular vesica of new anti-angiogenesis

Info

Publication number: CN110312515A
Application number: CN201780086372.2A
Authority: CN
Inventors: 安东内拉·维奥拉; 毛里齐奥·穆拉卡
Original assignee: City Of Hope Foundation - Non-Profit Social Organizations; UNI DEGLI STUDI DI PADOVA
Current assignee: City Of Hope Foundation - Non-Profit Social Organizations; UNI DEGLI STUDI DI PADOVA
Priority date: 2016-12-14
Filing date: 2017-12-14
Publication date: 2019-10-08
Anticipated expiration: 2037-12-14
Also published as: WO2018109525A1; EP3554513A1; CN110312515B; CA3052739A1; AU2017374947B2; AU2017374947A8; WO2018109700A1; AU2017374947A1

Abstract

The invention discloses by bracket/matrix mesenchymal cell (also referred to as mescenchymal stem cell, MSC) new anti-angiogenesis extracellular vesica (the extracellular vesica formed, EV), the EV is used to treat the disease characterized by vascularization increase, the pharmaceutical composition comprising the EV and the method for being used to prepare the EV.

Description

The extracellular vesica of new anti-angiogenesis

Technical field

The invention discloses the new anti-blood formed by dry/matrix mesenchymal cell (also referred to as mescenchymal stem cell, MSC) The outer vesica (extracellular vesica, EV) of pipe cellulation, the EV are used to treat the disease characterized by vascularization increase, include The pharmaceutical composition of the EV and the method for being used to prepare the EV.

Background technique

Angiogenesis (angiogenesis) is caused from pre-existing vascular system (vascular system) formation New blood vessel/vasculolymphatic process, many physiology courses (such as during development of fetus and in regeneration) and It plays a crucial role in many pathological conditions (such as in ischemic, inflammatory, autoimmune disease and in cancer).

The process of angiogenesis includes the different phase continuously fine-tuned, these stages are with endothelium and extracellular matrix Modification is characterized.When the process starts, observe that the permeability of vascular increases as the connection between endothelial cell is reduced. Followed by the destruction of capillary beds, this is necessary for being able to carry out tissue intrusion by new vascular.It is interior in this stage Chrotoplast is by forming new vessel lumen come tissue.Finally, the capillary newly formed passes through building basement membrane and Cell tracking Stablize.When vascular insufficiency (such as apoplexy) occurs, or on the contrary, by hyper-proliferative, for example, hemangioma, tumour and In retinopathy, which may change.

Particularly, angiogenesis and inflammation are two processes being closely related.In inflammation, if inflammatory stimulus is persistently deposited Then angiogenesis in the endothelial cell migration to tissue of veinlet liner by causing.The generation of new blood vessel is inflammatory cell Necessary to surviving within the organization, therefore inhibit the factor for promoting angiogenesis that can reduce inflammation and prevent its pathological consequences, Such as inflammatory tissue damage, autoimmunity, fibrosis or tumour growth.

Therefore, control angiogenesis is the very attractive treatment method for a variety of symptom.For wherein new blood The formation of pipe has all symptom of therapeutic effect, it is expected that positive control (positive control), and for its medium vessels Generate all symptom (such as the inflammatory disease, proliferative diseases (view to play a crucial role in the triggering and/or maintenance of disease Film disease, cancer, tumor disease), autoimmune disease, fibrosis, graft rejection etc.) in, it is expected that negative sense control (negative control)。

The molecule of many interference angiogenesis has been developed that and in the test of preclinical and clinical stage, but its validity is logical Often it is limited.

Mescenchymal stem cell (MSCs) is the more of the potentiality for having self-regeneration ability and being divided into various mesodermal lineages It can progenitor cells.MSC is present in the base portion of many tissues, and wherein they are located near vessels, this is shared with pericyte Feature.In fact, MSC and pericyte show similar form and functional character when analyzing in vitro, although both Cell type may have the function of different in vivo.Although pericyte adjusts capillary stable state and structure, the internal function of MSC It can act on and be not clear, and may be tissue specificity.For example, MSC contributes to form candidate stem cell in marrow (HSC) " ecological niche ", to provide suitable microenvironment for hemoposieis.In other tissues, MSC may participate in stable state control System and tissue repair.

MSC has effective stabilization to blood vessel endothelium, has after traumatic brain injury and presses down in hemorrhagic shock The ability of endothelial permeability processed.Therefore, the specific target of vascular endothelium seemingly MSC bioactivity.

Recently, Zanotti et al. confirms blood vessel formation against function (Zanotti et al., the Mouse of MSC mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1,Leukemia(2016)30,1143– 1154), which is mediated by soluble factor.

However, although multiple tests report for using MSC treatment human inflammatory's disease more and more interested It is heterogeneous as a result, MSC response differs in the patient for the treatment of for 15% to 55%.

The reason of these contradictory results, is not known in the art, and wherein may include and keep super in patients When the difference of the quantity of MSC survived, this is the key factor for being very difficult to control so far.

Another critical aspects of cell therapy based on MSC are its safeties, especially when considering long-term complications: MSC may cause tumour formation or the abnormal differentiation after heterotopic transplantation.

Finally, another risk factors is related with administration method.In fact, although realizing needed for immunological regulation in vivo MSC quantity be it is substantially unknown, but low in view of cell retention and survival rate, inject a large amount of cells may be obtain maximum face Necessary to bed benefit.In these cases, MSC is likely to form aggregation, can lead to patient's pulmonary embolism or infarct.

The encapsulation (E-MSC) of MSC has been proposed to solve the problems, such as application.

From above it can be clearly seen that interference angiogenesis be a kind for the treatment of method with broad field of application, and And there is lasting meaning to the identification of new anti-angiogenesis tool useful in treatment.

Summary of the invention

Author of the invention provides the efficient tool for inhibiting angiogenesis, and there is no known with use MSC phase The shortcomings that pass.

As already discussed above, this field is it has been reported that the MSC cultivated in the presence of certain pro-inflammatory cytokines Cells show goes out anti-angiogenesis activity (referring to Zanotti et al. 2016).However, the paper for disclosing the information also confirms this Caused by activity is the protein secreted as the pre-adjusted MSC cell of pro-inflammatory cytokine.

, it is surprising that author of the invention has found from the MSC cell cultivated in the presence of pro-inflammatory cytokine Middle isolated EV MSC shows anti-angiogenesis activity, therefore due to the protein of cell secretion, maintains thin by MSC The anti-angiogenesis activity that born of the same parents show.

It should be noted that so far, all EV MCS described in the art only show Angiogensis activity.

The present invention provide for the first time by mesenchyma it is dry/the extracellular vesica (EV MSC) of stroma cell release, the EV MSC shows angiogenesis inhibiting activity.

Author of the invention is also proved using the standard assay for assessing and measuring angiogenic activity, of the invention New EV MSC has measurable anti-angiogenesis activity in vitro and in vivo.EV is utilized as the treatment work of this field Tool, they are to transmit a series of signal and therefore can be in the complexity of both various horizontal disturbance angiogenesis and inflammation Biologic grain, with drug used at present on the contrary, the latter is selectively applied to specific approach or metabolic stage.

Therefore, new EV MSC of the invention, which is provided, can be used for treating the disease that wherein angiogenesis plays pathogenic effects New, safe and efficient tool.

Therefore, the purpose of the present invention is to

By mesenchyma it is dry/the extracellular vesica (EV MSC) of stroma cell release, the EV MSC shows angiogenesis Inhibitory activity；The EV MSC for using in the treatment；Include the EV MSC and at least one pharmaceutically acceptable load The pharmaceutical composition of body, wherein the EV MSC；Described pharmaceutical composition for using in the treatment；Include the EV MSC's Medical device；The method for being used to prepare the EV MSC, the EV MSC that can get or obtain by the method include the EV The pharmaceutical composition of MSC, purposes in the treatment, and, the EV MSC of the invention in any embodiment of description It is used to prepare the purposes and therapeutic treatment of drug comprising to this hair of subject with this need application therapeutically effective amount The step of bright EV MSC.

Detailed description of the invention

Fig. 1 inhibits angiogenesis from the EV that the MSC of sensitization is separated in vitro

The figure illustrates to from the MSC EV that obtains of MSC cell stimulated according to the method for the present invention with inflammatory agent and never piercing The result for the comparative test that the effect for the MSC EV that sharp MSC is obtained carries out.

The experiment carried out on mouse endothelial cell line (SVEC4-10) is described in detail in embodiment part.

The figure reports quantifying for the section length of the tube generated by SVEC4-10 cell (segment length), The SVEC4-10 cell is with the EV (EV st MSC-CM) that obtains of MSC stimulated according to the method for the present invention from inflammatory agent or never EV (the EV unst MSC-CM) culture obtained with the MSC that inflammatory agent stimulates.Also with the stimulation of inflammatory agent according to the method for the present invention On the culture medium of the medium supernatant of MSC (st MSC-CM) or the MSC (unst MSC-CM) from unused inflammatory agent stimulation Clear liquid culture SVEC4-10 cell has carried out identical analysis.

The EV that Fig. 2 is separated from the MSC of sensitization influences body vessel and generates.

The figure illustrates the results obtained on the vascularization model on matrix rubber plug (matrigel plug).In reality It applies and the experiment is described in detail in a part.

To anesthesia 12 week old male C57BL/6N mouse dorsal sc injection mixed with 500 μ l matrigels 5 × 10⁵Unst-MSC or st-MSC, or the EV obtained from same cell.Matrigel adds 50ng/ml VEGF and 100ng/ml bFGF As positive control.Pure matrigel (Bare Matrigel) is injected as negative control.Plug is taken out after 10 days, and passes through blood The vascularization of each plug is quantitatively evaluated in Lactoferrin.

The figure shows that the plug for being supplemented with EV st MSC-CM or st MSC shows vascularization even lower than negative right The vascularization observed according in, hence it is demonstrated that the internal blood vessel formation against function of EV MSC of the invention.

Fig. 3: the EV separated from the MSC of sensitization influences the angiogenesis in developmental Mouse Retina.

The figure illustrates after the EV that obtains from MSC that is stimulation or not stimulating carries out whole body processing, using in development Mouse Retina in vascularization model obtain result.The experiment is described in detail in embodiment part.

The 50 μ l EV to 1 age in days C57BL/6N mouse cub intraperitoneal injection from unst MSC or st MSC.Collect view Nethike embrane is simultaneously dissected the entire bracket of retina and is suitably dyed before laying flat.Number is captured using confocal microscope is inverted Image (A-B).Total retina and vessel area are measured using ImageJ.(C) and total branch retinal are relatively radially expanded in analysis Point (D).For retina radial dilatation, the retina radius (R, with from optic nerve to view membrane edge of each retina valve is measured The black arrow of edge indicates) and vessel radius (v is indicated with from optic nerve to the grey arrow of blood vessel front).Retinal vessel Expansion is calculated as the ratio between vessel radius (v) and retina radius (R).Data are expressed as relative to pair with vehicle treated Average value ± S.E.M., * P < 0.05, the T inspection being normalized according to mouse (n=4 mouse).

It is directly affected the figure illustrates how the systemic administration of EV of the present invention forms to have to retinal vessel.

EV of the Fig. 4 from MSC-CM simulates full terms culture medium completely

It is assessed by carrying out tube formation measurement by unst-MSC or st-MSC (unst MSC-CM or st MSC- CM) and the comparison of the complete medium of their extracellular vesica (EV unst MSC-CM or EV st MSC-CM) adjusting divides Analysis.By SVEC4-10 cell inoculation at the top of matrigel layer in the presence of appropriate stimulation.After 6 hours, it is inverted with difference aobvious Micro mirror obtains image under 4 times of object lens magnifications.A) the representative picture tested；Scale bar is 100 μm corresponding.B ImageJ) is used Angiogenesis analyzer carries out quantitative (being normalized relative to culture medium) of opposite tube length.3 independent experiments, number According to being expressed as average value ± SEM (* p b 0.05, * * p b 0.01, single factor test ANOVA).C) pass through Western blot analysis EV On TIMP-1 expression, use CD63 and CD9 as EV- marker.3 independent experiments；Data are expressed as average value ± SEM (* P b 0.05, * * p b 0.01, T- are examined.D) raw in the blood vessel mediated by the EV from st MSC-CM in order to study TIMP-1 At the effect in inhibition, adds TIMP-1 blocking antibody and carry out tube formation measurement.With ImageJ angiogenesis analyzer into Quantitative (being normalized relative to culture medium) of the opposite tube length of row.3 independent experiments, data be expressed as average value ± SEM (* p b 0.05, * * p b 0.01, single factor test ANOVA).

Fig. 5-inhibits the migration of the endothelial cell of VEGF stimulation from the EV of st-MSC-CM

The evaluation and test of endothelial cell migration is assessed by scratch test.By marked on the SVEC4-10 single layer converged across The line of culture dish bottom manufactures wound.With extracellular vesica (the EV unst separated from unst-MSC-CM or st-MSC-CM MSC-CM or EV st MSC-CM) processing cell.After 6 hours, with difference inverted microscope with 4 times of object lens magnifications to cell Imaging.A) representative picture；Scale bar is 100 μm corresponding.B) the quantization migrated；By initial (time 0) length for measuring scratch With final (6 hours time) length, analyzed with ImageJ.Data are expressed as average value ± S.E.M. (n=3).*P< 0.05, single factor test ANOVA.C) in order to study effect of the TIMP-1 in the blood vessel formation against function of the EV from st-MSC-CM, Identical experiment is carried out with TIMP-1 blocking antibody.Here, reporting with a μm quantization for the migration indicated.Data are expressed as average Value ± S.E.M. (n=3).* P < 0.05, single factor test ANOVA.

Fig. 6-adenosine mediates the second blood vessel formation against function of the EV from st-MSC-CM

A it) uses CD63 and CD9 as EV marker, is measured by western blot and be responsible for ATP water on the research surface EV The expression of extracellular nucleotides enzyme (ectonucleotidase) CD39 and CD73 of solution.3 independent experiments；Data are expressed as average value ± SEM (* p b 0.05, * * p b 0.01, single factor test ANOVA).By with ARL 67156 (be used for CD39) B) and AMP-CP (be used for CD73) C) inhibit the activity of these enzymes to carry out scratch test, with study ATP metabolin (respectively AMP and adenosine) by Influence in the inhibition of the endothelial cell migration mediated from the EV of st-MSC-CM.Data endothelial cell migration is expressed as putting down Mean value ± S.E.M. (n=3).* P < 0.05, single factor test ANOVA.

Fig. 7-adenosine influences the endothelial cell of migration with the mechanism dependent on ROS accumulation

A) by being come from during scratch migration test using CM-H2DCFDA (general oxidative stress indicator) to study The horizontal increased ability of EV induction ROS of st-MSC-CM.It here is the presentation graphics of report.Fluorescence is calculated by ImageJ Average value is simultaneously normalized relative to culture medium.Data are expressed as average value ± S.E.M. (n=3).* P < 0.05, single factor test ANOVA.B) in order to assess the effect of adenosine in this process, by blocking CD73 activity (the final participant of ATP hydrolysis) weight Multiple experiment.Data are expressed as average value ± S.E.M. (n=2).* P < 0.05, single factor test ANOVA.C) general anti-oxidant by using By the EV st-MSC-CM oxidative stress induced during agent n-acetyl-L-cysteine (NAC) processing assessment scratch test Inhibit, to confirm the inhibiting effect of ROS.Data are expressed as average value ± S.E.M. (n=2).* P < 0.05, single factor test ANOVA.

Fig. 8-derives from interior therapeutic potentiality of the EV for control angiogenesis of MSC

A) in order to assess effect of the EV separated from MSC-CM in the angiogenesis of developmental Mouse Retina, The 50 μ l EV to 1 age in days C57BL/6N mouse cub intraperitoneal injection from unst MSC-CM or st MSC-CM.After 4 days, place Dead mouse is to collect retina.It dissects the entire bracket of retina and dyes (green, in left side) with isolectin B4.Using Set confocal microscope capture digital picture (right side).B) using ImageJ software measure total retina and vessel area with Calculate relatively radially expansion and opposed branch point (being normalized both with respect to the cub of vehicle treated).Data are expressed as putting down Mean value ± S.E.M. (n=10 cub/group in 3 different experiments).* P < 0.05, single factor test ANOVA.C) to anesthesia 12 week old male C57BL/6N mouse dorsal sc injection and matrigel add that VEGF mixes from unst-MSC-CM or st- The EV of MSC-CM.Pure matrigel is injected as negative control.After 7 days, mouse is put to death, harvests matrix rubber plug to carry out hemoglobin It is quantitative.Plug content of hemoglobin is measured using Drabkin kit 525 (Sigma-Aldrich), and is surveyed relative to by BCA The total protein concentration of amount is normalized.Data are expressed as average value ± SEM (n=8 mouse/group).* P < 0.05, T are examined.

Detailed description of the invention

Nomenclature in the sense of the present invention:

Mescenchymal stem cell (MSC) is the multipotency for the potentiality for having self-regeneration ability and being divided into various mesodermal lineages Progenitor cells.MSC according to the present invention is present in the base portion of many tissues, and wherein they are located near vessels.In this theory In the meaning of bright book, depending on the recipient for treatment, MSC can be animal or people source.Unless otherwise stated, Such as this field MSC is defined in this specification.MSC is considered as adult stem cell or somatic stem cell, and in its all one's life In most of the time in keep non-proliferative quiescent condition, until being updated by tissue, the signal of damage and remodeling process triggering Stimulation.When deriving from fetal membrane, such as when chorion and amnion, MSC is considered as human embryo stem cell (hESC) and thin at soma Intermediate between born of the same parents.When meaning is related to people MSC, non-embryonic MSC is considered as possible preferred embodiment of the invention.

In the meaning of this specification, by mesenchyma it is dry/the extracellular vesica of stroma cell release or " EV MSC " be by Mesenchyma is dry/the extracellular vesica of stroma cell secretion.According to the prior art and this specification, EV MSC include by MSC secretion/ The EV of all kinds of release.MSC secretes the extracellular vesica (EV) of a variety of different sizes, form, content and function, they with Neuron target cell interaction simultaneously changes its phenotype and function.According further to the prior art and this specification, EV can according to its size, Source and separation method are divided into three categories: (i) microcapsule bubble or the vesica that falls off (size, between 1000nm, is sprouted 50 from plasma membrane, And it is rich in CD40)；(ii) (for size between 800 and 5000nm, the fragment from dying cell is rich in a group egg to apoptotic body White and DNA)；And (iii) excretion body, it is the small (~30-120nm) membrane vesicle from endocytosis source (rich in late endosomal Film marker, including Tsg101, CD63, CD9 and CD81).In the meaning of this specification, according to the prior art, released by MSC The above-mentioned all types of vesicas put/generated all cover by mesenchyma it is dry/the extracellular vesica or " EV of stroma cell release In the statement of MSC ".

In the meaning of this specification, term " angiogenesis (angiogenesis) " can be covered from pre-existing vascular Form new blood vessel and/or new lymphatic vessel (this field is also referred to as angiogenesis and lymphatic vessel generation).In containing for this specification In justice, two kinds of meanings (blood vessel angiogenesis and lymphatic vessel angiogenesis) are covered by more generally angiogenesis.Therefore, when As used herein, which can refer to the formation of new blood vessel, the formation of new vasculolymphatic formation or both.

In the meaning of this specification, term anti-angiogenesis or " showing angiogenesis inhibiting activity " are considered same Compound that adopted word and indicating inhibits external as defined above and/or body vessel to generate, cell, vesica, composition, point Son, mixture, part, substance, product.In the meaning of this specification, inhibition can be it is at least part of, i.e., with unused test Anti-angiogenic compounds, cell, vesica, composition, molecule, mixture, part or substance processing positive control compare Reduce angiogenesis, or complete, i.e., with the anti-angiogenic compounds of unused test, cell, vesica, composition, point Son, mixture, part, product or the positive control of substance processing compare no detectable angiogenesis.

In the meaning of this specification, therapeutically effective amount is enough in patient or the disease model measured interested Position play the inhibiting effect of angiogenic activity as defined above and at least reduced so as to cause the symptom of treated disease Amount.In other words, therapeutically effective amount used herein refer to cause in organization system, animal or people researcher, animal doctor, Biology sought by doctor or other clinicians or medicinal response (symptom including mitigating treated disease or illness) Reactive compound or medicament amount.

As used herein, term " composition " be intended to cover comprising specific quantity special component product, and directly or Any product generated indirectly by the combination of the special component of specific quantity.

The term as used herein " subject " or " patient " refer to the animal as treatment, observation or experiment object, preferably Mammal, it is optimal to choose.

As described above, subject description discloses it is new by mesenchyma it is dry/the extracellular vesica (EV of stroma cell release MSC), the EV MSC shows angiogenesis inhibiting activity (or as described above throughout the specification as possible synonymous The anti-angiogenesis activity of word).

According to this specification, EV MSC of the invention is shown, the EV MSC shows described in vitro and/or in vivo Angiogenesis inhibiting activity.In a preferred embodiment, the activity shows in vitro and in vivo.

Therefore, according to the present invention, it is desirable that the anti-angiogenesis EV MSC of protection shows detectable and/or measurable blood Pipe generates inhibitory activity.An embodiment according to the present invention, the activity can be formed in standard body outer tubular object and be measured Middle measurement.Tube formed measurement be measurement/assessment product angiogenesis (positively or negatively) it is active it is known in the art simultaneously And the standard testing sufficiently reported in the literature.

According to the present invention, any tube disclosed in this field forms measurement and is suitable for measurement/detection EV of the invention The anti-angiogenesis activity of MSC.

For example, but and non-limiting purpose, can be such as Ponce, 2009.Methods Mol Biol.2009；467: 183-8.doi:10.1007/978-1-59745-241-0_10.Tube formation:an in vitro matrigel Progress tube described in angiogenesis assay forms measurement.

According to another non-limiting example, according to the tube of this specification formed measurement can as in experimental section in detail Progress as stating.

The angiogenesis inhibiting activity of another embodiment according to the present invention, EV MSC of the invention can pass through body Interior Matrigel plug angiogenesis forms measurement to assess.

It is formed and is measured as tube, Matrigel plug angiogenesis forms the mark that measurement is also the angiogenic activity of assessment product Quasi- measurement.

According to the present invention, any Matrigel plug angiogenesis disclosed in this field forms measurement and is suitable for measurement/detect this hair The anti-angiogenesis activity of bright EV MSC.

For example, but and non-limiting purpose, Matrigel plug angiogenesis formed measurement can such as Brown et al., 2016.Methods Mol Biol.2016；1430:149-57.doi:10.1007/978-1-4939-3628-1_9.Tube- It is carried out described in Forming Assays.Brown RM1, Meah CJ2, Heath VL2, Styles IB3, Bicknell R4.

According to another non-limiting example, forming measurement according to the Matrigel plug angiogenesis of this specification can be such as experiment portion The progress being described in detail in point.

As above defined, blood vessel angiogenesis (blood is covered according to the term angiogenesis of this specification Vessels angiogenesis) and/or lymphatic vessel angiogenesis (lymphatic vessels angiogenesis), because This, an embodiment according to the present invention, EV MSC described in above and any following embodiments can be shown The inhibitory activity of blood vessel angiogenesis and/or lymphatic vessel angiogenesis.

As described above, the EV MSC of any embodiment disclosed herein is suitable for for using in the treatment.Institute as above It states, angiogenesis is the feature of a variety of diseases.Inflammatory disease is characterized in that angiogenic activity, and the feature of proliferative diseases exists In angiogenic activity, autoimmune disease is further characterized in that angiogenic activity and graft rejection.

Therefore, EV MSC of the invention leads to the disease of therapeutic effect especially suitable for treating the inhibition of wherein angiogenesis And/or illness.Therefore, EV MSC of the invention is suitable for the treatment that wherein instruction inhibits angiogenesis.

According to an embodiment, EV MSC of the invention is particularly suitable for treating to pass through angiogenesis pathology Property formed in the symptom or illness that new blood vessel and/or new lymphatic vessel are characterized and use.

According to this specification, the symptom or illness are selected from proliferative, inflammatory and autoimmune disease or illness, eye And periodontal disease, fat and aging-related disorders.

In one embodiment of the invention, the proliferative diseases are selected from cancer；The inflammatory/autoimmune disease Disease is selected from psoriasis, arthritis, endometriosis, Crohn disease, inflammatory bowel disease；The eye disease is selected from diabetic keratopathy Retinopathy, retinopathy of prematurity, radioactivity and sun retinopathy, macular degeneration；The periodontal disease is selected from gingivitis And periodontitis；The obesity related disorders are selected from the neovascularization of fat driving and the inflammation of fat driving；The aging phase It closes illness and is selected from wrinkle and skin aging.

Another embodiment of the invention is pharmaceutical composition, it includes by mesenchyma it is dry/stroma cell release it is thin Extracellular vesica (EV MSC) and at least one pharmaceutically acceptable carrier, wherein the EV MSC shows Agiogenesis inhibition Activity.Any one embodiment of above-mentioned EV MSC is suitable for the invention pharmaceutical composition.Therefore, the medicine of claim 11 Compositions, it is characterised in that the angiogenesis inhibiting activity is showed in vitro and in vivo by the EV MSC, and can be with Measurement and/or the measurement of Matrigel plug angiogenesis formation in vivo are formed by external tube to measure.

According to the present invention, pharmaceutical composition will play anti-angiogenic life to blood vessel angiogenesis and/or lymphatic vessel angiogenesis It is Viability.

Pharmaceutical composition of the invention will be suitable for the form preparation of whole body (enteral and parenteral) and local application.

The non-limiting example of the method for application is in intravenous, intramuscular, organ, transdermal, rectum, eye drip, glass Internal and commonly used in the art other way.

Therefore, according to the present invention, pharmaceutical composition can be solution, suspension, cream, foaming agent, emulsion, paste Agent, lotion, shakes lotion, ointment, transdermal patch, powder, sponginum, eye drops, adhesive tape agent, suppository, enema at gelling agent Form.

Solution and emulsion are applicable to systemic administration or local application or both.

Every kind of form can be prepared by technical staff according to the best practices of medicine preparation, therefore, according to ordinary skill Knowledge, technical staff will know how the suitable carrier and other useful ingredients that selection is used to prepare.

EV MSC of the invention is directly administered alone, and various pharmaceutical preparations are generally preferably made.Pharmaceutical preparation can be with By conventional method of pharmacy, by the way that active constituent is mixed with the pharmacologically acceptable carrier of one or two or more To prepare.

Carrier can take many forms, this depends on applying desired dosage form.These pharmaceutical compositions are ideal Ground is preferably adapted in applied systemically or topically or the unit dosage form of parenteral injection.For example, preparing oral dose shape When the composition of formula, any commonly employed drug media can be used.For parenteral composition, carrier is at least largely Sterile water is generally comprised, although may include other ingredients, such as to help to dissolve.For example, Injectable solution can be prepared, Middle carrier includes the mixture of saline solution, glucose solution or salt water and glucose solution.Injectable can also be prepared Suitable liquid-carrier and suspending agent can be used in suspension in this case.In the composition for being suitable for transdermal administration, carry Body optionally includes penetration enhancers and/or suitable wetting agent, the optionally suitable addition with any property of small percentage Agent combination, the additive will not generate significant illeffects to skin.Such additive can help to apply skin With and/or can help to prepare required composition.These compositions can be applied in many ways, such as transdermal patch, As a paint, as ointment.

Pharmaceutical composition of the invention is suitable for using in the treatment.The composition, which can be used for treating, to be had been described above Any disease or illness.Particularly, composition can be used for treating.

According to this specification, the inflammation related pathologies can be selected from proliferative diseases, inflammatory disease, autoimmune disease Disease, graft rejection proliferative, inflammatory and autoimmune disease or illness, eye and periodontal disease, fat and aging related diseases Disease.

In a specific embodiment, the present invention relates to the doctors comprising EV MSC as described herein or pharmaceutical composition Device is treated, in a specific embodiment, the medical device is the form of transdermal patch, in another embodiment, The medical device is the form of the distributor for intranasal administration.

The invention further relates to the methods for being used to prepare MSC EV comprising following steps:

A. MSC is cultivated in the culture medium for being supplemented with one or more inflammatory cytokines,

B. it removes the culture medium and cultivates institute in the culture medium without one or more pro-inflammatory cytokines MSC is stated, and collects EV from culture supernatants.

According to this specification, one or more pro-inflammatory cytokines can be selected from IL-1 β, IL-6, TNF α and chemotactic The factor.Therefore, in the step a) of the method for the present invention, one or more pro-inflammatory cytokines listed above can be used for supplementing Growth medium used.

In a specific embodiment, the pro-inflammatory cytokine is at least two or at least three kinds.

In a preferred embodiment, the pro-inflammatory cytokine by IL-1 β, IL-6 and TNF α mixture generation Table.

According to the present invention, about 30-70ng/ml can be used for carrying out the suitable amount of the pro-inflammatory cytokine of step a The total amount of cell factor indicate.It is dense using two kinds of cell factors when using more than one cell factor Degree can be the concentration of every kind of cell factor 15ng/ml to 40ng/ml, or using three kinds of cell factors, dense Degree can be the concentration of every kind of cell factor 15ng/ml to 30ng/ml.

According to the present invention, the step a and b invented is adapted to commonly used in cultivating any known culture medium of MSC.

In both step a and b, culture medium is also supplemented with other compound/objects of the standard of the MSC commonly used in culture Matter, such as suitable fetal calf serum (FBS), antibiotic, suitable amino acid etc..

Those skilled in the art can easily assess the concentration of other composition/substances according to standard scheme.

The unrestricted of suitable culture medium and suitable other composition/substances is additionally provided in following experimental section Property example.

For example, FBS can be 5% to 15%, for example, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.

Always for example, suitable antibiotic can be commonly used in any antibiotic of culture MSC, such as strepto- Or mixtures thereof element, penicillin, altri.The usual amounts of antibiotic can be used in the process of the present invention.Non-limiting example is total Antibiotic amount is 80-150U/ml (for example, about 100U/ml).In non-limiting example, the mould of about 100U/ml can be used Element/streptomysin.

In addition other supplementation materials can be one or more amino acid.In one non-limiting example, Ke Yixiang About 2mM glutamine is added in growth medium.

The suitable culture medium of step a and b for the method for the present invention are by any culture medium generation commonly used in culture MSC Table.The non-limiting example of the culture medium is represented by low glucose DMEM (DMEM low glucose) or similar culture medium.

According to the present invention, the MSC can be cultivated 18 to 30 hours during step a.Therefore, the MSC can be cultivated about 18,19,20,21,22,23,24,25,25,27,28,29,30 hours.

In one embodiment of the invention, the MSC cultivates about 22-26 hours during step a, for example, about 22, 23,24,25 or 26 hours.

According to this specification, culture medium upon removal of step a and in stepb using the training of not factor-containing Base is supported, then MSC cell can be cultivated 12 to 24 hours during step b.

According to the present invention, the MSC can be cultivated 12 to 24 hours during step b, and therefore, the MSC can be cultivated 12,13,14,15,16,17,18,19,20,21,22,23,24 hours.

In one embodiment of the invention, the MSC cultivates about 16-20 hours during step b, for example, about 16, 17,18,19 or 20 hours.

It is known in the art that EV is separated from medium supernatant.Any suitable method can be used to implement this hair Bright method.In one non-limiting embodiment, EV of the invention can be collected by carrying out ultrafiltration to supernatant.

Another object of the present invention is represented by obtainable anti-angiogenesis EV MSC from the above.

Obtainable anti-angiogenesis EV MSC (is herein also referred to as obtained from the MSC-CM of stimulation by means of the present invention EV) be characterized in that expression TIMP1, CD73 and CD39.

Particularly, and from the EV for not having the identical MSC-CM of stimulation to obtain during cell culture disclosed in the present specification (EV that the MSC-CM herein also referred to as never stimulated is obtained) is compared, and obtainable EV is by means of the present invention at least 2 times Every kind of the ratio expression marker.

According to an embodiment, phase is expressed with the TIMP1 of the EV MSC from the culture medium as described herein not stimulated Than EV MSC of the invention (EV obtained by the culture medium i.e. from stimulation) is characterized in that at least 2 to 10 times high expression TIMP1.According to the present invention, as described herein, compared with the EV MSC from the culture medium not stimulated, by EV's of the invention TIMP1 is expressed as at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times of height.

In addition, as described herein, compared with the CD73 expression of the EV MSC obtained by the culture medium never stimulated, the present invention EV MSC be characterized in that expressing CD73 at least 2 to 3 times high.

EV MSC of the invention is further characterized in that the CD39 spy for expressing that molecular weight is about 175kDa in western trace Anisotropic band, wherein the protein therefrom extracted is dyed with 9 specific antibody of AntiCD3 McAb.

As disclosed in this specification, when the marker in EV of the invention being known as x times expressing, 1 times is except culture Base does not supplement except one or more inflammatory cytokines under identical condition of culture, is obtained by identical cell colony identical The amount of the identical marker of the EV expression of amount.In the present specification, the every kind of EV culture medium for never stimulating and stimulating obtained The 3 μ g gross proteins of the EV MSC of middle extraction are compared, and it is quantitative to carry out marker to carry out western trace.

Present invention also contemplates that the therapeutical uses of the EV MSC, pharmaceutical composition and its use comprising the EV MSC On the way, and the medical device and application thereof comprising the anti-angiogenesis EV MSC.

It can refer to benefit in experimental section and claim above with reference to EV MSC of the invention all the elements described EV MSC that is obtainable in aforementioned manners or obtaining.

The invention further relates to the therapeutic treatments of above-mentioned all diseases and illness, wherein by the EV of the invention of therapeutically effective amount MSC or pharmaceutical composition of the invention are applied to subject in need.

Experimental section

Anti-angiogenesis EV MSC preparation

It, will in the low glucose DMEM for being supplemented with 20%FBS, 2mM glutamine, 100U/ml penicillin/streptomycin MSC bed board simultaneously grows it in vent cap flask until converging.Then culture medium is changed to and is supplemented with 10%FBS, 2mM paddy Glutamine, 100U/ml penicillin/streptomycin, with or without 25ng/ml mIL1b, 20ng/ml mIL6,25ng/ml The low glucose DMEM of mTNF α continues 24 hours.Then culture medium is changed to and is supplemented with 2mM glutamine, 100U/ml blueness Mycin/streptomysin low glucose DMEM continues subsequent 18 hours.Therefore, it obtains from MSC (the unst MSC- not stimulated CM) or stimulation MSC (st MSC-CM) conditioned medium.It usesUltra 15mL Filters(Merck Millipore EV) is separated from unst MSC-CM or st MSC-CM by ultrafiltration.

Tube forms measurement

The result of the measurement is as shown in Figure 1.

In flat 96 orifice plate, by 1.3 × 10⁴A SVEC4-10 cell (CRL-2181^TM) 100 μ l's Matrigel coating is seeded in unst MSC-CM or st MSC-CM and its EV (EV unst MSC-CM or EV st MSC-CM) Hole (80 μ l matrigels/hole) in.Low glucose DMEM with 10% heat-inactivated FBS is used as positive control.37 DEG C, 10%CO₂After lower 4 hours, (A) is imaged to cell tubule under 4 times of object lens magnifications with difference inverted microscope.It uses ImageJ Angiogenesis Analyse plugin carries out quantitative (B) of section length.Data be expressed as average value ± S.E.M. (n=3).* P < 0.05, T are examined.

Matrigel plug angiogenesis forms measurement

The result of the measurement is as shown in Figure 2.

The EV separated according to the present invention from the MSC of sensitization influences body vessel and generates.To the 12 week old male of anesthesia C57BL/6N mouse mixed in dorsal sc injection with 500 μ l matrigels 5 × 10⁵A unst-MSC or st-MSC, or from phase The EV obtained with cell.Matrigel adds 50ng/ml VEGF and 100ng/ml bFGF to be used as positive control.Pure matrigel is injected to make For negative control.For every group, matrigel is supplemented with heparin (50 units/ml).After 10 days, mouse is put to death, collects matrix rubber plug, It weighs and takes pictures (A).The quantitative hemoglobin in filling in that homogenizes is carried out using Drabkin kit 525 (Sigma-Aldrich) (B).Average value ± S.E.M. (n=4 mouse/group) is expressed as relative to the normalized data of total protein content.*P< 0.05, T examines.

Retinal vessel, which is formed, to be inhibited

The result of the test is as shown in Figure 3.

The angiogenesis in developmental Mouse Retina is influenced from the EV that the MSC of sensitization is separated according to the present invention.

To 1 age in days C57BL/6N mouse cub intraperitoneal injection, 50 EV of the μ l from unst MSC or st MSC.After 4 days, Mouse is put to death to collect retina.Two eyes are extracted and are fixed with 4%PFA.The entire bracket of dissection retina simultaneously uses biotin The isolectin B4 (Vector Laboratories) of change is dyed, and streptavidin-is used before laying flat Alexa 488 (Invitrogen) dyeing.Digital picture (A-B) is captured using confocal microscope is inverted.It uses ImageJ measures total retina and vessel area.In detail, (C) and total branch retinal point (D) are relatively radially expanded in analysis.It is right In retina radial dilatation, the retina radius (R, with from optic nerve to the black of view film edge of each retina valve is measured Arrow indicates) and vessel radius (v is indicated with from optic nerve to the grey arrow of blood vessel front).Retinal blood enlargement of pipe calculates For the ratio between vessel radius (v) and retina radius (R).Data are expressed as relative to the control mice (n with vehicle treated =4 mouse) average value ± S.E.M., * P < 0.05 for being normalized, T examines.

Extracellular vesica summarises the phenotype of their MSC-CM of separation

Have evaluated influence of the extracellular vesica for deriving from MSC-CM to angiogenesis.The use of EV have the advantages that it is multiple, Such as high stability and extensive a possibility that propagating potentiality, supporting treatment of the attractive industrialization based on EV.

The mouse MSC-CM for never stimulating or stimulating by ultrafiltration obtains EV.By Nanosight analyze verify its quality and Quantity (data are not shown).In order to study influence of the EV to angiogenesis, tube is carried out using SVEC4-10 cell and forms survey It is fixed, and the EV (respectively EV unst-MSC-CM or EV st-MSC-CM) of the MSC never stimulated the or MSC of stimulation separation with The effect of full culture medium (CM) compares.As previously mentioned, compared with the MSC-CM for compareing and not stimulating, the MSC-CM of stimulation Reduce tube length.It is interesting that the EV from st-MSC-CM plays identical blood vessel formation against function, it is strong to press down SVEC4-10 processed forms the ability (Figure 14 A, B) of capillary sample tubular structure.

Since TIMP1 has the known outstanding role of the anti-angiogenesis regulatory factor as st-MSC, have studied Whether TIMP1 participates in the blood vessel formation against function showed from the EV of st-MSC-CM.Firstly, being tested by western blot The presence of TIMP1 in EV of the invention is demonstrate,proved.TIMP1 is highly enriched in EV st-MSC-CM, and extracellular vesica marker The expression of CD63 and CD9 is uninfluenced (Fig. 4 C).Then, tube is repeated using TIMP1 blocking antibody form measurement (Fig. 4 D), It is as the result is shown TIMP1 dependence from the blood vessel formation against function that the EV that st-MSC-CM is separated is shown.

In short, these are the result shows that EV shows that inhibition identical as the angiogenesis for the conditioned medium for separating them is made With.

Scratch wound healing measurement discloses second of the anti-angiogenesis mechanism of EV st-MSC-CM

Angiogenesis and anti-angiogenesis activity can be assessed by using a variety of external tests, these measurements are usually used In the different committed steps 124 of research physiological disposition.Particularly, it is formed and is measured by tube, had evaluated and sent out in tubulose network Matrix destroying and Endothelial Morphology between the duration of an exhibition occurs.However, in endothelial cell is assembled into vascular tube in vivo and also needs The coordination of chrotoplast migrates.Whether played regulatory role in this process to assess EV, scratch wound healing has been used to try in vitro It tests, the ability 126 moved in free space based on cellular response in angiogenic factors.

Inoculation SVECA4-10 cell generates cell-free gap to form single layer, and by scratch in converging layer.With rush Angiogenesis factor VEGF is stimulated induction of subsequent cell migration.Addition derives from the EV of unst-MSC or st-MSC.37 DEG C 10%CO₂After lower incubation 6 hours, the distance (Fig. 5 A) that migrating cell is covered is measured.As expected, with do not stimulate Cell (solvent without VEGF) is compared, and in response to VEGF, the transfer ability of SVEC4-10 cell increases.With from unst- The EV processing of MSC-CM does not influence the migration (Fig. 5 B) of endothelial cell.On the contrary, completely eliminating sound from the EV that st-MSC-CM is separated It should be in the SVEC4-10 transfer ability (Fig. 5 B) of VEGF.

This shows the EV separated from st-MSC-CM the not only digestion of negative sense control angiogenesis mesostroma and Endothelial Morphology Occur (Fig. 4), there are also the transfer abilities of the endothelial cell in response to VEGF.

In order to assess the influence of TIMP1 in this process, scratch wound healing is carried out in the presence of TIMP1 blocking antibody It measures (Fig. 5 C).It is interesting that with being observed in tube is formed and measured on the contrary, TIMP-1 blocking processing does not save quilt The transfer ability (Fig. 5 C) that EV from st-MSC-CM inhibits shows to come from st- there is the mechanism independent of TIMP1 The EV of MSC passes through its blood vessel formation against function of the mechanisms play.

CD39 and CD73 assigns EV st-MSC-CM anti-angiogenesis activity

In order to characterize the mechanism independent of TIMP1 for the blood vessel formation against function that EV is mediated, has studied and reported and can press down The possibility of the adenosine of various kinds of cell type migration processed participates in.Adenosine is a kind of purine nucleosides, can pass through the water of effect of extracellular ATP Solution generates.The reaction is played by two kinds of enzyme extracellular nucleotides enzyme CD39 and CD73 being limited on cytoplasma membrane, is catalyzed ATP respectively It is hydrolyzed to AMP and AMP and is hydrolyzed to adenosine 125.

In order to study the accumulation whether EV from st-MSC-CM induces this purine, pass through Western blot analysis sheet The expression (Fig. 6 A) of CD39 and CD73 on the surface EV of invention.Obtain the results show that the vesica separated with the MSC never stimulated It compares, the horizontal of both protein increases in EV st-MSC-CM.In addition, only being observed in the EV from st-MSC-CM To the CD39 specific band of higher molecular weight (about 175kDa).

In order to probe into whether adenosine may be the original for being isolated from the blood vessel formation against function that the EV of st-MSC-CM is showed Cause, in CD39 or CD73 inhibitor (respectively trisodium salt hydrate (ARL 67156) and adenosine 5 '-(α, β-methylene) two phosphorus Hydrochlorate (AMP-CP) (Fig. 6 B, C)) in the presence of carried out previously described (Fig. 5 A) scratch wound healing measurement.It is worth noting , in the presence of VEGF, the inhibition of every kind of enzyme has restored the SVEC4-10 cell institute with EV st-MSC-CM processing completely The distance of covering.

Therefore, these statistics indicate that, by be isolated from st-MSC-CM EV express CD39 and CD73 generate ATP be metabolized In the endothelial cell migration of VEGF, mechanism is completely independent of TIMP1 activity for object inhibition response.

EV st-MSC-CM induced activity oxygen (ROS) in the endothelial cell of migration

Recently, wide coverage adenosine participates in the adjusting that reactive oxygen species (ROS) is generated.Extracellular adenosine and four kinds The G-protein coupled cell surface receptor (A1R, A2AR, A2BR and A3R) of hypotype interacts.Although adenosine receptor and ROS are produced The raw mechanism connected also illustrates far away completely, but has proposed NADPH (nicotinamide-adenine dinucleotide phosphate) oxidizing ferment (NOX) it plays a crucial role.In fact, generate by electronics from NADPH be transferred to molecular oxygen ROS NOX activity with The activation of adenosine receptor is related.

Oxidative stress is considered as effective inducible factor of the cytoskeleton rearrangement of endothelial senility and functional disturbance, this It is the two kinds of mechanism that may cause the transport reaction of change.Therefore, author assumes anti-angiogenesis EV (EV st-MSC-CM) water Solution effect of extracellular ATP simultaneously causes the adenosine on endothelial cell surface to accumulate, and then the oxidative stress in inducing endothelial cell, causes The inhibition of cell migration.In order to verify this it is assumed that the ROS in the endothelium SVEC4-10 cell of detection migration is horizontal (Fig. 7).Especially Ground carries out scratch wound healing measurement by using CM-H2DCFDA (general oxidative stress indicator) and assessment ROS is horizontal. Use antimycin A (Antymycin A) (AA) as positive control, antimycin A be by with mitochondrial complex III phase interaction For inhibiting the compound of electron transport, causes mitochondria to collapse and accumulate (Fig. 7 A) with subsequent ROS.

In the SVEC4-10 cell of the migration with VEGF processing that is individual or being combined with the EV from unst-MSC-CM In, ROS accumulation does not dramatically increase (Fig. 7 A).On the contrary, the EV induced strong endothelial cell migration forward position from st-MSC-CM ROS is generated.These results support ours it is assumed that i.e. ROS participates in the endothelial cell migration that plays by EV st-MSC-CM Inhibition.In addition, by handling the migration for blocking adenosine accumulation not only to save endothelial cell with AMP-CP (CD73 inhibitor) (Fig. 6 C), and the ROS also reduced in the migration endothelial cell handled from the EV of st-MSC-CM is horizontal (Fig. 7 B).Cause This, these numbers are it was demonstrated that the part of adenosine caused by the presence of the extracellular nucleotides enzyme on the EV of st-MSC release generates induction ROS in endothelial cell is generated, to influence its motility.

In order to finally verify this mechanism, is reduced and aoxidized using general antioxidant n-acetyl-L-cysteine (NAC) It stress.It is worth noting that, having restored SVEC4-10 cell completely by the inhibition of the EV st-MSC-CM oxidative stress induced Transfer ability (Fig. 7 C).

These results highlight EV st-MSC-CM to the crucial inhibiting effect of the endothelial cell of migration, and mechanism depends on The ROS accumulation of induction is generated by the part of adenosine.

By inhibiting body vessel to generate with the EV processing from st-MSC-CM

In order to develop the new treatment generated for pathologic vessels, the internal of the EV from st-MSC-CM is demonstrated Effect.Therefore, retina mouse model has been used, this is the side of a kind of widely used studying physiological and pathologic vessels generation Method.In fact, with the mankind on the contrary, mouse cub has jejune retinal vasculature at birth；It was developed in several weeks Complete, by closely adjust with it is organized in a manner of carry out, reliably detect any defect.

Therefore, the EV of MSC source do not stimulated to cub intraperitoneal injection or stimulation, and put to death after 5 days to collect Retina.Dissection sample is simultaneously dyed with isolectin-B4 to measure the blood of developmental retina by Laser Scanning Confocal Microscope Guard system forms (Fig. 8 A).It is worth noting that, observing the retinal vessel tree of the cub of the EV processing with the source st-MSC The reduction of dendriticization, but the influence (Fig. 8 B) for the counterpart not stimulated is not observed.According in vitro results, from st-MSC-CM points From EV also inhibit angiogenesis in vivo.

This showed by the EV for being isolated from st-MSC-CM is further consolidated with the measurement of another vivo approaches matrix rubber plug Kind blood vessel formation against function.It includes that analysis is supplemented with then matrix rubber plug that EV is implanted into the back of C57BL/6-N mouse in advance Angiogenesis (Fig. 8 C).Quantifying for the content of hemoglobin of plug discloses only with the mouse of the EV processing from st-MSC-CM In vascular system reduce, it is therefore evident that the anti-angiogenesis characteristics of these products.

It is controlled in short, all these data provide the extracellular vesica from MSC for what control pathologic vessels generated Treat the strength evidence of potentiality.

Bibliography

Brown et al., 2016.Methods Mol Biol.2016；1430:149-57.doi:10.1007/978-1- 4939-3628-1_9.Tube-Forming Assays.Brown RM1,Meah CJ2,Heath VL2,Styles IB3, Bicknell R4

Ponce,2009.Methods Mol Biol.2009；467:183-8.doi:10.1007/978-1-59745- 241-0_10.Tube formation:an in vitro matrigel angiogenesis assay.

Zanotti et al., Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1, Leukemia(2016)30,1143–1154

Claims

1. by mesenchyma it is dry/the extracellular vesica (EV MSC) of stroma cell release, the EV MSC shows angiogenesis suppression System activity.

2. EV MSC according to claim 1, wherein the EV MSC shows the blood vessel life in vitro and in vivo At inhibitory activity.

3. EV MSC according to any one of claim 1 or 2, wherein tube forms measurement to the EV MSC in vitro In show the angiogenesis inhibiting activity.

4. EV MSC according to any one of claim 1 to 3, wherein EV MSC Matrigel plug angiogenesis shape in vivo At showing the angiogenesis inhibiting activity in measurement.

5. EV MSC according to any one of claim 1 to 4, wherein the angiogenesis be blood vessel angiogenesis and/ Or lymphatic vessel angiogenesis.

6. EV MSC according to any one of claim 1 to 5, the EV MSC for using in the treatment.

7. the EV MSC used according to claim 6, the EV MSC wherein need therapeutic inhibition blood vessel for treating The symptom of the illness of generation.

8. the EV MSC that any one of according to claim 6 or 7 uses, the EV MS are for treating with raw by blood vessel At and pathologic forms the symptom or illness that new blood vessel and/or new lymphatic vessel are characterized.

9. the EV MSC used according to claim 8, wherein the symptom or illness are selected from proliferative, inflammatory and itself exempt from Epidemic disease disease or illness, eye and periodontal disease, fat and aging-related disorders.

10. the EV MSC used according to claim 9, wherein the proliferative diseases are selected from cancer；The inflammatory/itself Immunity disease is selected from psoriasis, arthritis, endometriosis, Crohn disease, inflammatory bowel disease；The eye disease is selected from Diabetic retinopathy, retinopathy of prematurity, radioactivity and sun retinopathy, macular degeneration；The periodontal disease choosing From gingivitis and periodontitis；The obesity related disorders are selected from the neovascularization of fat driving and the inflammation of fat driving；Institute It states aging-related disorders and is selected from wrinkle and skin aging.

11. a kind of pharmaceutical composition, comprising by mesenchyma it is dry/the extracellular vesica (EV MSC) and at least one of stroma cell release Kind pharmaceutically acceptable carrier, wherein the EV MSC shows angiogenesis inhibiting activity.

12. 1 described in any item pharmaceutical compositions according to claim 1, wherein the angiogenesis be blood vessel angiogenesis and/ Or lymphatic vessel angiogenesis.

13. pharmaceutical composition described in any one of 1 or 12 according to claim 1, described pharmaceutical composition is adapted for systemic Enteral or parenteral administration or the form of local application.

14. pharmaceutical composition according to claim 13, described pharmaceutical composition is solution, suspension, cream, bubble Foam agent, paste, gelling agent, lotion, shakes lotion, ointment, transdermal patch, powder, sponginum, eye drops, adhesive tape at emulsion Agent, suppository, enema form.

15. pharmaceutical composition described in any one of 1 to 14 according to claim 1, described pharmaceutical composition is in the treatment It uses.

16. 5 described pharmaceutical composition used according to claim 1, described pharmaceutical composition wherein needs to treat for treating Property inhibit angiogenesis illness symptom.

17. any one of 5 or 16 described pharmaceutical composition used according to claim 1, described pharmaceutical composition is for treating Symptom or illness characterized by forming new blood vessel and/or new lymphatic vessel by angiogenesis pathologic.

18. 7 described pharmaceutical composition used according to claim 1, wherein the symptom or illness be selected from proliferative, inflammatory and Autoimmune disease or illness, eye and periodontal disease, fat and aging-related disorders.

19. 8 described pharmaceutical composition used according to claim 1, wherein the proliferative diseases are selected from cancer；The inflammation Property/autoimmune disease be selected from psoriasis, arthritis, endometriosis, Crohn disease, inflammatory bowel disease；The eye Disease is selected from diabetic retinopathy, retinopathy of prematurity, radioactivity and sun retinopathy, macular degeneration；The tooth All diseases are selected from gingivitis and periodontitis；The obesity related disorders are selected from the neovascularization and fat driving of fat driving Inflammation；The aging-related disorders are selected from wrinkle and skin aging.

20. a kind of medical device, including appointing in EV MSC described in any one of claims 1 to 5 or claim 11 to 14 Composition described in one.

21. medical device according to claim 20, the medical device is for the form of transdermal patch or for intranasally applying The form of distributor.

22. a kind of method for being used to prepare MSC EV, comprising the following steps:

B. the culture medium is removed and in the culture medium without one or more pro-inflammatory cytokines described in culture MSC, and EV is collected from culture supernatants.

23. according to the method for claim 22, wherein one or more pro-inflammatory cytokines are selected from IL-1 β, IL- 6, TNF α and chemotactic factor (CF).

24. the method according to any one of claim 22 or 23, wherein the pro-inflammatory cytokine is at least two Or at least three kinds.

25. the method according to any one of claim 22 to 24, wherein the pro-inflammatory cytokine is IL-1 β, IL- 6 and TNF α.

26. according to the method for claim 25, wherein each in the pro-inflammatory cytokine is in the culture medium In concentration be 15ng/ml to 30ng/ml.

27. the method according to any one of claim 25 to 29, wherein to cultivate 18 to 30 in step a small by the MSC When.

28. the method according to any one of claim 25 to 30, wherein to cultivate 12 to 24 in stepb small by the MSC When.

29. the method according to any one of claim 25 to 31, wherein being received by carrying out ultrafiltration to the supernatant Collect the EV.

30. the anti-angiogenesis EV MSC that the method as described in any one of claim 25 to 32 can obtain.

31. anti-angiogenesis EV MSC according to claim 33, the anti-angiogenesis EV MSC are in the treatment It uses.

32. a kind of pharmaceutical composition pharmaceutically may be used comprising the anti-angiogenesis EV MSC described in claim 33 at least one The carrier of receiving.

33. a kind of medical device includes the anti-angiogenesis EV MSC described in claim 33.