WO2021147923A1 - 一种囊泡及其应用 - Google Patents

一种囊泡及其应用 Download PDF

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WO2021147923A1
WO2021147923A1 PCT/CN2021/072938 CN2021072938W WO2021147923A1 WO 2021147923 A1 WO2021147923 A1 WO 2021147923A1 CN 2021072938 W CN2021072938 W CN 2021072938W WO 2021147923 A1 WO2021147923 A1 WO 2021147923A1
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vesicle
vesicles
stem cells
exosomes
syntaxin
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PCT/CN2021/072938
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English (en)
French (fr)
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张晓�
寇晓星
施松涛
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医微细胞生物技术(广州)有限公司
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Priority to US17/793,860 priority Critical patent/US20230051925A1/en
Priority to JP2022569292A priority patent/JP2023513395A/ja
Publication of WO2021147923A1 publication Critical patent/WO2021147923A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4718—Lipocortins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM

Definitions

  • the present disclosure belongs to the field of biomedicine, and relates to a vesicle and its application.
  • Extracellular vesicles are nano-scale carriers secreted by cells that contain proteins, nucleic acids and various cytokines. Extracellular vesicles can act on target cells through endocrine or paracrine mode, and play an important role in the process of material transfer and information exchange between cells. Studies have found that information exchange mediated by extracellular vesicles plays an important regulatory role in the body's physiological or pathological processes, involving immune regulation, tumor growth, angiogenesis, damage repair, and so on. The current research in this field is mainly focused on exosomes. Exosomes are extracellular vesicles with a diameter of about 30-150nm, which contain components such as RNA, lipids, and proteins.
  • Exosomes are widely involved in various physiological/pathological regulation of the body, and can be used for diagnosis, treatment and prognostic evaluation of various diseases. So far, mesenchymal stem cells (MSCs) are considered to be the cells with the strongest ability to produce exosomes. Numerous studies have found that exosomes derived from MSCs can mimic the biological functions of MSCs, and play an important regulatory role in promoting cell growth and differentiation, repairing tissue defects, and so on. Therefore, in recent years, cell vesicle therapy based on MSCs-derived exosomes has achieved remarkable development.
  • MSCs mesenchymal stem cells
  • Hemophilia is a group of bleeding disorders with inherited coagulation dysfunction. Its common feature is the generation of active thromboplastin, prolonged coagulation time, and lifelong tendency to bleeding after minor trauma. It can also be used in severe patients without obvious trauma. "Spontaneous" bleeding occurred. On May 11, 2018, the National Health Commission and other five departments jointly formulated the "First List of Rare Diseases", and hemophilia was included in it. Hemophilia is mainly divided into three categories, namely hemophilia A, hemophilia B and hemophilia C.
  • Hemophilia A, or factor VIII procoagulant component (VIII: C) deficiency is a sex-linked recessive genetic disease that is transmitted by females and is onset by males.
  • Hemophilia B, or factor IX (FIX) deficiency is also a sex-linked recessive inheritance, and its incidence is less than that of hemophilia A.
  • Hemophilia C, or factor XI (FXI) deficiency is inherited in an autosomal incomplete recessive manner and is a rare hemophilia. The incidence of hemophilia A accounts for up to 80%-85%, hemophilia B accounts for 15%-20%, and hemophilia C is rare.
  • the present disclosure provides a vesicle derived from mesenchymal stem cells.
  • the present disclosure provides a vesicle composition.
  • the present disclosure provides a pharmaceutical composition containing vesicles for hemophilia.
  • the present disclosure provides a vesicle screening or identification or extraction kit.
  • the present disclosure provides a marker for vesicles.
  • the present disclosure provides a method for identifying or selecting vesicles using markers.
  • the present disclosure provides a method for preparing vesicles.
  • the present disclosure provides a vesicle derived from a somatic cell or a stem cell, the vesicle is an inducible vesicle, and the vesicle has a marker including Syntaxin 4.
  • the present disclosure provides a method for treating or preventing or ameliorating a disease or a complication of the disease in a subject, comprising administering to the subject an effective amount of the vesicle or the The combination of vesicles or the composition; the disease is a hemorrhagic disease.
  • the bleeding disorder includes bleeding caused by a deficiency of coagulation factors, a decrease in the number of platelets, and/or a functional defect.
  • the bleeding disorder includes hemophilia, lupus bleeding, or Cheet-Dong syndrome.
  • the hemophilia includes hemophilia A, hemophilia B, or hemophilia C.
  • the disease is hemophilia A.
  • the stem cells include totipotent stem cells and pluripotent stem cells. In some embodiments, the stem cells include mesenchymal stem cells and induced pluripotent stem cells (IPS).
  • IPS induced pluripotent stem cells
  • the somatic cells include osteoblast cell lines.
  • the cell may be a primary cultured cell, or an existing or established cell line.
  • the cell line refers to an immortalized cell culture that can proliferate indefinitely in a suitable fresh medium and space.
  • the cell may be an established cell strain.
  • the inducible vesicle is a vesicle produced by external force inducing apoptosis during the normal survival of the stem cell or somatic cell.
  • the inducible vesicles are produced by inducing stem cells or stem cell apoptosis by adding astrospora, ultraviolet irradiation, starvation, or thermal stress, or a combination of one or more of them.
  • the markers possessed by the vesicles further include one or more of Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5.
  • the vesicle has a combination of the markers Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5.
  • the vesicles highly express the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4.
  • the expression level of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 of the vesicle is higher than that of MSC or exosomes.
  • the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in the vesicle are relative to the expression of the markers in exosomes derived from mesenchymal stem cells The amount is about 1-2 times, 2-3 times, 1-3 times, 3-4 times and 3-6 times respectively.
  • the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in the vesicle are relative to the expression of the markers in exosomes derived from mesenchymal stem cells The amount is about 1.5-2 times, 2.5-3 times, 1.5-2.5 times, 3.5-4 times and 3.5-5 times respectively.
  • the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in the vesicle are relative to the expression of the markers in exosomes derived from mesenchymal stem cells The amount is about 1.5-1.9 times, 2.5-2.9 times, 1.8-2.5 times, 3.5-3.9 times and 4-5 times respectively.
  • the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in the vesicle are relative to the expression of the markers in exosomes derived from mesenchymal stem cells The amount is about 1.76 times, 2.81 times, 2.41 times, 3.68 times and 4.45 times respectively.
  • the exosomes do not express Syntaxin 4, and the vesicles of the present disclosure express Syntaxin 4.
  • the exosomes do not express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 at the same time, and the vesicles of the present disclosure simultaneously express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4.
  • the vesicles and the exosomes are derived from MSCs of the same origin.
  • flow cytometry is used to analyze the surface membrane proteins of IEVs, and the results show that IEVs derived from MSCs can express surface proteins similar to MSCs, namely CD29, CD44, CD73, CD166 positive, CD34, CD45 negative ; At the same time, IEVs can express the universal surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles.
  • the inducible vesicles are produced by inducing mesenchymal stem cell apoptosis by adding staurosporine, ultraviolet irradiation, starvation method, thermal stress method, or a combination thereof.
  • the vesicles are produced by inducing mesenchymal stem cells with staurosporine.
  • the generation number of the mesenchymal stem cells may be about 2 to 5 generations, but it is not limited thereto.
  • the concentration of the staurosporine is about 1 nM to 10000 nM. In some embodiments, the concentration of the staurosporine is about 100 nM to 10000 nM. In some embodiments, the concentration of the staurosporine is about 500 nM-10000 nM. In some embodiments, the concentration of the staurosporine is about 500-1000 nM. In some embodiments, the concentration of the staurosporine is about 500-900 nM. In some embodiments, the concentration of the staurosporine is about 500-800 nM.
  • the diameter of the vesicle is about 0.03-6 ⁇ M. In some embodiments, the diameter of the vesicle is about 0.03-4.5 ⁇ M. In some embodiments, the diameter of the vesicle is about 0.03-1 ⁇ M. In some embodiments, the diameter of the vesicle is about 0.04-1 ⁇ M. In some embodiments, the diameter of the vesicle is about 0.05-1 ⁇ M. In some embodiments, the diameter of the vesicle is about 0.1-1 ⁇ M. In some embodiments, the diameter of the vesicle is about 0.15-1 ⁇ M.
  • the present disclosure also provides a combination of vesicles, comprising the above-mentioned vesicles.
  • the vesicle combination also includes other vesicles in the prior art, including, but not limited to, exosomes, migratory bodies, microvesicles, and Ectosomes.
  • the number of the vesicles in the vesicle composition accounts for about 65-100%.
  • the number of the vesicles in the vesicle composition accounts for about 75-98%.
  • the number of the vesicles in the vesicle composition accounts for about 80-96%.
  • the present disclosure also provides a composition, the pharmaceutical composition comprising the above-mentioned vesicle or the above-mentioned combination of vesicles.
  • the composition includes medicines, foods, health products, cosmetics, additives, or intermediate products.
  • the composition is a pharmaceutical product.
  • the composition further comprises a pharmaceutically or immunologically acceptable carrier.
  • the preparation form of the composition is selected from lyophilized powder injection, injection, tablet, capsule, kit or patch.
  • the vesicle is used as a drug carrier.
  • the present disclosure also provides a reagent or kit for screening or identifying or extracting the vesicle, comprising one or more of the following marker detection reagents: marker Annexin V, Flotillin-1 , Cadherin 11, Integrin alpha 5 and Syntaxin 4 detection reagents.
  • the detection reagent for the marker detects the expression level of the marker gene.
  • the detection reagent for the marker detects the expression level of the marker mRNA.
  • the detection reagent for the marker detects the expression amount of the marker protein.
  • the detection reagent for the marker is one or more of fluorescent quantitative PCR dyes, fluorescent quantitative PCR primers, fluorescent quantitative PCR probes, antibodies, antibody functional fragments, and conjugated antibodies.
  • the kit is selected from one or more of qPCR kits, western blot detection kits, flow cytometry kits, immunohistochemical detection kits, and ELISA kits.
  • the kit is selected from flow cytometry kits.
  • the present disclosure also provides the use of the vesicle or the vesicle composition or the pharmaceutical composition in the preparation of products for treating or preventing or improving diseases or complications of the diseases;
  • the diseases include liver disease and hemophilia.
  • the disease is hemophilia
  • the vesicles can exert a significant procoagulant effect in vitro, and can significantly improve the bleeding tendency of hemophilia mice after in vivo injection, and can be used to improve hemophilia
  • the treatment of bleeding tendency has good application prospects.
  • the disease is hemophilia A.
  • the product includes medicines, foods, health products, cosmetics, additives, or intermediate products.
  • the vesicles can be optionally selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intraorgan infusion.
  • the route of administration for example, as an example, for intravenous injection, it can be injected through the tail vein.
  • Intra-organ infusion includes infusion into an anatomical space, such as, for example, the gallbladder, gastrointestinal cavity, esophagus, pulmonary system (by inhalation), and/or bladder.
  • intraperitoneal injection in gastrointestinal cavity infusion
  • intraperitoneal injection can also obtain the same therapeutic effect.
  • the safety and operability of intraperitoneal injection are better than those of tail vein injection.
  • the present disclosure also provides a method for selecting or identifying the vesicle, the method comprising detecting one or more of the following markers: markers Annexin V, Flotillin-1, Cadherin 11. Integrin alpha 5 and Syntaxin 4.
  • test result shows a positive result for the marker, it is judged to be the vesicle.
  • the expression result of the marker can be compared with the control, and when the expression amount is significantly higher than the control, it is judged as a positive result.
  • the control can be other existing vesicles or exosomes (which can include one or more of exosomes, migrating bodies, microvesicles and Ectosome); it can be other vesicles derived from mesenchymal stem cells Or exosomes.
  • Syntaxin is particularly preferred.
  • the present disclosure provides the application of the detection reagent of the marker in the preparation of a reagent or kit for detecting or identifying the vesicle, characterized in that the marker includes Annexin V, Flotillin-1 One or more of Cadherin 11, Integrin alpha 5 and Syntaxin 4, the reagent or kit further includes a control reagent, and the control reagent includes one of exosomes, migrating bodies, microvesicles, and Ectosomes, or There are several types. When the expression level of the marker in the sample to be tested is higher than that of the control reagent, it is judged as positive.
  • control reagent is an exosome.
  • the expression level of Syntaxin 4 in the test sample is greater than or equal to 2-6 times that of exosomes, it is determined to be the vesicle.
  • the expression level of Syntaxin 4 in the sample to be tested is greater than or equal to 4-5 times that of exosomes, it is determined to be the vesicle (for example, an inducible vesicle).
  • the present disclosure provides a method for preparing the vesicle, including the following steps, the method is to induce stem cells or somatic cells to produce the vesicle by adding an apoptosis-inducing agent.
  • the method includes the following steps: (1) culturing mesenchymal stem cells; (2) collecting the medium supernatant of the mesenchymal stem cells; (3) removing the medium supernatant from step (2) The vesicles were isolated from it.
  • the step of culturing mesenchymal stem cells in step (1) includes: (4) separating mesenchymal stem cells from tissues; (5) adding culture medium to culturing mesenchymal stem cells; the mesenchymal stem cells Contact the apoptosis inducer in the culture medium of the stem cells.
  • the apoptosis-inducing agent includes staurosporine, ultraviolet irradiation, starvation, or thermal stress, or a combination of one or more of them.
  • the apoptosis inducer is staurosporine.
  • the concentration of the staurosporine is about 500-1000 nM. In some embodiments, the concentration of the staurosporine is about 500-900 nM. In some embodiments, the concentration of the staurosporine is about 500-800 nM.
  • the time for the apoptosis-inducing agent to treat the cells in step (5) is about 16-24 hours.
  • the method of separating the vesicles includes using an ultracentrifugation method to separate the vesicles.
  • a single MSCs in the present disclosure can produce 300-2000 vesicles.
  • the step of separating the vesicles by the ultracentrifugation method includes: (a) subjecting the collected culture supernatant to a first centrifugation, and taking the supernatant; (b) performing step (a) The collected supernatant is centrifuged for the second time and the supernatant is taken; (c) the supernatant received in step (b) is centrifuged for the third time, and the precipitate is taken; (d) the supernatant received in step (c) The obtained precipitate is centrifuged for the fourth time, and the precipitate is taken; (e) the precipitate received in step (d) is centrifuged for the fifth time, and the precipitate is taken;
  • the first centrifugation is about 500-1500g for 5-30 minutes. In some embodiments, the first centrifugation is about 500-1000 g for 5-20 minutes. In some embodiments, the first centrifugation is about 500-900 g for 5-15 minutes. In some embodiments, the second centrifugation is about 1000-3000g for 5-30 minutes. In some embodiments, the second centrifugation is about 1500-2500 g for 5-20 minutes. In some embodiments, the second centrifugation is about 1500-2200g for 5-15 minutes. In some embodiments, the third centrifugation is about 10,000-30,000 g for 15-60 minutes. In some embodiments, the third centrifugation is about 12000-25000g for 20-60 minutes.
  • the third centrifugation is about 12000-20000g for 20-40 minutes.
  • the fourth centrifugation is about 10,000-30,000 g for 15-60 minutes. In some embodiments, the fourth centrifugation is about 12000-25000g for 20-60 minutes. In some embodiments, the fourth centrifugation is about 12000-20000g for 20-40 minutes.
  • vesicles with specific markers can be obtained by enrichment methods for specific markers. After enough vesicles are obtained, the culture medium is collected and specific vesicles are purified and isolated from the culture medium. This can be achieved by any suitable method known in the art. These methods include, for example, the original method of separating exosomes by differential ultracentrifugation, as well as newer methods such as polymer precipitation (ExoQuickTM from SBI, Palo Alto, CA), immunoaffinity capture (Greening et al. 2015, Methods in Molecular Biology), immunomagnetic capture (Exo-FLOWTM, SBI), etc.
  • Immunoaffinity purification is a method of selectively capturing specific vesicles based on surface markers. High-affinity coupling between streptavidin-covalently-coated magnetic beads and biotinylated capture antibodies enables efficient capture of vesicles. After the captured vesicles are eluted, the structure is complete and biologically active. Based on the findings of the present disclosure, the vesicles can specifically and highly express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules, then the present disclosure can use this method to isolate or purify or enrich the vesicles .
  • the vesicles can also be enriched by using immunomagnetic beads.
  • the immunomagnetic beads are obtained by coupling a monoclonal antibody to a magnetic bead; the monoclonal antibody includes Anti-Syntaxin. 4 One or more of antibody, Anti-Annexin V antibody, Anti-Flotillin-1 antibody, Anti-Cadherin 11 antibody, Anti-Integrin alpha 5 antibody.
  • the present disclosure also provides an inducible vesicle, which is derived from an IPS cell. It is a type of subcellular product produced by intervention or induction when IPS cells are in normal survival to make IPS cells apoptotic.
  • enrichment covers the separation of one or more vesicles from any other vesicles present in the sample or it refers to the vesicles in a composition comprising vesicles It is present in a higher total percentage content than when it is found in the tissues of the organism.
  • the enriched vesicles are not isolated from the sample, but any diagnosis is made on the vesicles while the vesicles are still present in the sample.
  • the sample can then be presented on a glass slide and can be diagnosed using a microscope, and in this embodiment the vesicles are detected without being separated.
  • the enriched vesicles are isolated from the sample.
  • Immunomagnetic bead-based separation is a new immunological technology developed in recent years.
  • Immunomagnetic beads can not only bind to active protein antibodies, but also can be attracted by magnets. After treatment, antibodies can be bound to magnetic beads to make them a carrier for antibodies. The antibodies and specific antigen substances on the magnetic beads After binding, an antigen-antibody-magnetic bead immune complex is formed. This complex moves mechanically under the action of magnetic force to separate the complex from other substances and achieve the purpose of separating specific antigens.
  • Immune Magnetic Beads is a platform that can be applied to any field that uses the principle of antigen-antibody binding, and has been used in medical and biological bone marrow transplantation, isolation of stem cells, organelles, cancer cells, hormones, pathogens, and toxins. Achieved remarkable results.
  • IMB has been widely used in the separation and detection of mycotoxins in food, water, biological samples, and environmental samples with its high sensitivity and specificity, showing a good prospect for development and application.
  • the immunomagnetic bead method described in the present disclosure uses magnetic beads with specific antibody binding to bind to target vesicles with specific surface antigens, and then uses a magnetic field to adsorb and extract the target vesicles.
  • the specific enrichment method for vesicles is to combine Anti-Syntaxin 4 antibody, Anti-Annexin V antibody, Anti-Flotillin-1 antibody, Anti-Cadherin 11 antibody, Anti-Integrin
  • the immunomagnetic beads coated with one or more of the alpha 5 antibodies are added to the cell culture supernatant containing the vesicles, then, it will interact with the Anti-Syntaxin 4 antibody, Anti-Annexin V antibody, and Anti-Flotillin-1 antibody.
  • the vesicles that can specifically bind to one or more of Anti-Cadherin 11 antibody and Anti-Integrin alpha 5 are isolated, which can achieve the purpose of enriching specific vesicles.
  • Anti-Syntaxin 4 antibody when using Anti-Syntaxin 4 antibody, Anti-Annexin V antibody, Anti-Flotillin-1 antibody, Anti-Cadherin 11 antibody and Anti-Integrin alpha 5 antibody simultaneously coated immunomagnetic beads to separate Vesicles have the highest purity and have the best effect in treating diseases such as hemophilia A.
  • the cell culture supernatant after the combination optimization based on the centrifugation method effectively removes the cells and cell debris and other impurities, and then the Anti-Syntaxin 4 antibody, Anti-Annexin V antibody, Anti-Flotillin -1 antibody, Anti-Cadherin 11 antibody, Anti-Integrin alpha 5 antibody, one or more antibody-coated immunomagnetic beads are added to the cell culture supernatant, then the antibody can be specific
  • the bound vesicles can be separated out, and the purpose of enriching specific vesicles can be achieved.
  • the mesenchymal stem cells are derived from humans or mice, but are not limited thereto.
  • the mesenchymal stem cells include bone marrow-derived mesenchymal cells, urine-derived mesenchymal stem cells, oral-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and placental-derived mesenchymal stem cells.
  • the mesenchymal stem cells are selected from bone marrow-derived mesenchymal cells, fat-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and oral-derived mesenchymal stem cells.
  • Figures 1A-1E show the flow cytometric detection results of the surface markers of isolated BMMSCs.
  • Fig. 2 is a flowchart of the operation of the second embodiment.
  • 3 is the number of MSCs IEVs statistical results analyzed by flow cytometry (10 6 MSCs) produced.
  • Figure 4A-4F shows the diameter detection of IEVs particles:
  • Figure 4A shows the particle diameter distribution of IEVs by flow detection;
  • Figure 4B shows the scattered light intensity of IEVs analyzed by side scattered light (SSC), showing the particle diameter distribution of IEVs;
  • Figure 4C To analyze the scattered light intensity of IEVs with standardized small particle microspheres produced by Bangs Laboratories, showing the particle diameter distribution of IEVs;
  • Figure 4D shows the IEVs observed by transmission electron microscopy (TEM), showing the particle diameter distribution of IEVs;
  • Figure 4E shows the particle diameter distribution of IEVs.
  • Tracking analysis NTA
  • Figure 4F shows the particle diameter detection of IEVs at the single vesicle level using nanoflow detection technology, showing the particle diameter distribution of IEVs.
  • Figures 5A-5K show the analysis results of surface membrane proteins of IEVs by flow cytometry.
  • Figure 6A- Figure 6D are the content analysis of IEVs:
  • Figure 6A is the proteomic quantitative analysis results of MSCs, MSCs-Exosomes, MSCs-IEVs by DIA quantitative technology;
  • Figure 6B is the heat map drawn by screening IEVs-specific and highly expressed proteins
  • Figure 6C is the GO enrichment analysis of differential proteins and the results of IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules;
  • Figure 6D is the Western Blot verification MSCs, MSCs-Exosomes, MSCs-IEVs express Annexin The results of V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4.
  • FIG. 7 shows the procoagulant effect of IEVs in hemophilia A mice.
  • Figures 8A-8D show the changes in the levels of various coagulation factors after injection of IEVs into hemophilia A mice: Figure 8A shows the changes in coagulation factor VIII; Figure 8B shows the changes in vWF factor; Figure 8C shows the changes in tissue factor ( TF) changes; Figure 8D shows the changes of prothrombin.
  • Figures 9A-9B show the effects of PS and TF blocking on IEVs in vivo in the hemophilia A mouse model.
  • Figure 9C shows that IEVs injection treatment of lpr mice can significantly improve the bleeding tendency of lpr mice.
  • Figure 9D shows that the bleeding tendency of CHS mice was significantly improved after IEVs injection treatment to CHS mice.
  • Figure 10 shows the comparison of the therapeutic effects of IEVs and Exosomes derived from the same MSC on hemophilia A mice.
  • WT is wild-type mouse
  • HA group is hemophilia A mouse model
  • HA+IEVs is hemophilia A mouse model given IEVs treatment
  • HA+PS-IEVs is hemophilia A mouse model given PS negative IEVs
  • HA+TF-IEVs are hemophilia A mouse models given TF-negative IEVs
  • HA+Exosomes are hemophilia A mouse models given Exosomes treatment.
  • Figure 11 shows the morphology of MC3T3-E1 and hBMMSC-derived IEVs under light microscope.
  • Figure 12 shows the flow cytometric detection results of the diameter distribution of IEVs particles derived from MC3T3-E1 and hBMMSC.
  • Figures 13A-13C show that IEVs can be excreted through the skin and hair:
  • Figure 13A is a schematic diagram of the dynamic metabolism of IEVs on the skin surface.
  • Figure 13B shows that over time, IEVs gradually move from the subcutaneous tissue to the dermis and epidermis.
  • Figure 13C shows that PKH26-IEVs were found in the hair follicles in the plucked hair from the mouse body on day 7.
  • Figure 14 shows the death process of hiPSCs and hUCMSCs taken by the high-content cell imaging analysis system.
  • Figure 15 shows the apoptosis-inducing rate of hiPSCs and hUCMSCs using flow cytometry, which proves that most of the cells have undergone apoptosis.
  • Figure 16 shows that the positive rate of Annexin5 expression using flow cytometry analysis is above 80% for both hiPSCs and hUCMSCs.
  • Figure 17 shows the particle size of two IEVs detected by Nanoparticle Tracking Analysis (NTA).
  • Figure 18 shows the number of IEVs produced by the two IEVs detected by Nanoparticle Tracking Analysis (NTA).
  • NTA Nanoparticle Tracking Analysis
  • Figure 19 shows the potentials of two IEVs detected by Nanoparticle Tracking Analysis (NTA).
  • IEVs in the embodiments of the present disclosure are the abbreviations for induced vesicles, which may be called induced vesicles, and may also be called induced extracellular vesicles (IEVs).
  • Inducible extracellular vesicles refer to a type of subcellular product produced when precursor cells (such as stem cells) survive normally and are interfered or induced to cause apoptosis. Usually this type of subcellular product has a membrane structure, expresses apoptotic markers, and partly contains genetic material DNA.
  • inducible extracellular vesicles are a class of substances distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.).
  • the cells that survive normally are, for example, non-apoptotic cells, non-senescent cells, non-aging cells that have stagnated proliferation, cells that are not recovered after cryopreservation, or abnormally proliferating without malignant transformation. Cells or non-damaged cells, etc.
  • the normally viable cells are taken from cells that are in contact with 80-100% of the fusion during the cell culture process. In some embodiments, the cells that survive normally are taken from cells in the log phase. In some embodiments, the cells that survive normally are obtained from primary culture and subculture cells derived from human or murine tissue. In some embodiments, the cells that survive normally are taken from established cell lines or cell lines. In some embodiments, the precursor cells are taken from early cells.
  • the IEVs in this disclosure are the same as IEVs.
  • the STS in the present disclosure is staurosporine.
  • Exosomes refers to exosomes.
  • compositions e.g., medium
  • method include the listed elements, but do not exclude other elements.
  • Consisting essentially of means excluding other elements that have any significance for the combination for the stated purpose. Therefore, 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 features of the claimed disclosure.
  • Consisting of refers to the elimination of trace elements and substantial method steps of other components. The embodiments defined by each of these transition terms are within the scope of this disclosure.
  • an “effective amount” is an amount sufficient to achieve beneficial or desired results, such as enhanced immune response, treatment, prevention, or improvement of medical conditions (disease, infection, etc.).
  • the effective amount can be administered in one or more administrations, applications, or doses.
  • the appropriate dosage will vary depending on body weight, age, health, disease or condition to be treated, and route of administration.
  • high expression and the like are intended to include increasing the expression of nucleic acid or protein to a level higher than that contained in prior art vesicles (for example, exosomes).
  • the term "pharmaceutically acceptable carrier” refers to any standard pharmaceutical carrier, such as a lyophilized powder injection, injection, tablet, capsule, kit or patch. Usually this carrier contains excipients, such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable fats or oils, gums, Glycol or other known excipients. These carriers may also include flavoring and color additives or other ingredients. Examples of pharmaceutically acceptable carriers include, but are not limited to, the following: water, saline, buffer, inert non-toxic solids (e.g., mannitol, talc).
  • composition containing such a carrier is formulated by well-known conventional methods.
  • the composition may be in the form of solid, semi-solid or liquid dosage forms, such as powders, granules, crystals, liquids, suspensions, liposomes, pastes, creams, ointments, etc., And it can be in the form of a unit dose suitable for administering a relatively precise dose.
  • the components in the “composition” may exist in a mixed form, or they may be packaged separately. Separately packaged components may also contain their respective adjuvants.
  • the adjuvant refers to a means that can assist the curative effect of drugs in pharmacy.
  • the separately packaged components can be administered simultaneously or in any order, in which the patient is treated with one drug first, and then another drug is administered .
  • the said patient refers to a mammalian subject, especially a human being.
  • the "composition” may also exist in a form in which one component is wrapped by another component.
  • the inducible vesicle in the composition, is used as a drug carrier, and drugs for treating or preventing diseases are encapsulated in the inducible vesicle.
  • the corresponding reagent sources are as follows: penicillin/streptomycin solution (BIOSOURCE; P303-100); glutamine (BIOSOURCE; P300-100); dexamethasone sodium phosphate (Sigma; D-8893); ⁇ - MEM (Gibco; 12571-063); 2-ME (GIBCO; 21985-023).
  • mice were killed with excessive CO 2 according to the guidance of the animal ethics committee. Under aseptic conditions, the tibia and femur were removed, and the muscle and connective tissue attached to it were peeled off. The metaphysis was further separated and the bone marrow cavity was exposed. 10 mL was used. A sterile syringe draws PBS with a volume fraction of 10% fetal calf serum to repeatedly wash the bone marrow cavity. After filtering with a 70 ⁇ m pore size cell strainer, centrifuge at 500g for 5min, remove the supernatant and collect the cell pellet at the bottom, resuspend in PBS, and centrifuge again at 500g 5min, collect the final cell pellet.
  • the cells were sorted by flow cytometry, and BMMSCs were sorted using CD34- and CD90+ as sorting criteria. Finally, the cells were resuspended in Dex(-) medium and seeded in a 10 cm diameter cell culture dish, incubated at 37°C and 5% CO 2 . After 24 hours, the supernatant non-adherent cells were aspirated, washed with PBS, and Dex(-) medium was added to continue the culture. One week later, the same amount of Dex(+) culture medium was added, and one week later, dense primary BMMSCs colonies were seen. The BMMSCs were digested with trypsin at 37°C and subcultured and expanded. Then, the Dex(+) medium was changed every 3 days, and then subcultured after full growth. Use P2 generation BMMSCs for follow-up experiments.
  • composition of Dex(-) culture solution is shown in Table 1
  • composition of Dex(+) culture solution is shown in Table 2:
  • Flow cytometry was used to analyze the surface markers to evaluate the purity of the isolated BMMSCs.
  • surface marker identification after trypsin digestion to collect P2 generation BMMSCs, wash once with PBS, resuspend the cells in 3% FBS-containing PBS at a density of 5 ⁇ 10 5 /mL, and add 1 ⁇ L of PE fluorescence-conjugated CD29, CD44, CD90, CD45 and CD34 antibodies were not added to the blank group. Incubate at 4°C in the dark for 30 minutes, wash with PBS twice, and then test on the machine. The results of flow cytometry are shown in Figures 1A-1E. It can be seen that the isolated cells are BMMSCs (Bone Marrow Mesenchymal Stem Cells).
  • the MSCs (MSCs derived from bone marrow, BMMSCs) cultured in Example 1 to the second passage were cultured with the medium (Dex(+) medium) in Example 1 until the cells were 80%-90% confluent. Rinse twice with PBS, add 500nM STS-containing serum-free medium ( ⁇ -MEM medium) to induce apoptosis, incubate at 37°C for 24h, collect cell supernatant for separation and extraction of IEVs.
  • medium Dex(+) medium
  • ⁇ -MEM medium 500nM STS-containing serum-free medium
  • IEVs are separated and extracted from the collected culture supernatant.
  • the operation process is shown in Figure 2.
  • the specific steps include: centrifuge at 800g for 10 minutes, collect the supernatant; then centrifuge at 2000g for 10 minutes, and then collect the supernatant; After centrifugation at 16000g for 30 minutes, the supernatant was removed, and the IEVs were resuspended in sterile PBS; then after centrifugation at 16000g for 30 minutes, the supernatant was removed, and the IEVs were resuspended in 300-500 ⁇ L of sterile PBS.
  • the MSCs (MSCs derived from bone marrow, BMMSCs) cultured in Example 1 to the second generation were cultured with the medium in Example 1 until the cells were 80%-90% confluent, rinsed twice with PBS, and added serum-free Incubate the culture medium at 37°C for 48 hours, and collect the cell supernatant for separation and extraction of Exosomes.
  • the extraction steps include: 800g centrifugation for 10 minutes-collection of supernatant liquid-2000g centrifugation for 10 minutes-collection of supernatant liquid-16000g centrifugation for 30 minutes-collection of supernatant liquid-120,000g centrifugation for 90 minutes-remove the supernatant, and resuspend in sterile PBS Precipitation—Centrifuge again at 120,000g for 90 minutes, remove the supernatant, collect the Exosomes at the bottom, and resuspend in sterile PBS.
  • Example 2 Flow cytometry of IEVs obtained in Example 2 was quantitatively analyzed, IH measurement time point for the first, second 4h, the first 8h, and 16h of the 24h, 10 6
  • the results show the induction of MSCs through IH, first 4h, After 8h, 16h and 24h, 0.76 ⁇ 10 8 , 1.29 ⁇ 10 8 , 1.95 ⁇ 10 8 , 2.48 ⁇ 10 8 , 3.14 ⁇ 10 8 IEVs can be produced respectively. It can be seen from this that induced After 24h, a single MSC can produce 300 IEVs (Figure 3).
  • NTA Nanoparticle Tracking Analysis
  • IEVs The surface membrane proteins of the IEVs extracted in Example 2 were analyzed by flow cytometry. The results showed that IEVs derived from MSCs can express surface proteins similar to MSCs, namely CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. At the same time, IEVs can express the universal surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (Figure 5A-5K).
  • IEVs were screened for specific and highly expressed proteins, and a heat map was drawn (Figure 6B), and further combined with the GO enrichment analysis results of differential proteins, it is clear that IEVs can specifically and highly express Annexin V, Flotillin-1, and Cadherin 11 Integrin alpha 5 and Syntaxin 4 molecules.
  • MSCs-Exosomes Refers to exosomes derived from BMMSCs.
  • MSCs-IEVs Refers to IEVs derived from BMMSCs.
  • the MSCs in the content analysis and the MSCs from which Exosomes and IEVs were extracted are the same BMMSCs cell line.
  • the in vitro coagulation experiment was used to detect the in vitro coagulation effect of the IEVs obtained in Example 2 and the Exosomes extracted in Comparative Example 1.
  • the results are shown in Table 3, IEVs can significantly shorten the in vitro clotting time of most plasma, and the procoagulant effect is better than Exosomes.
  • IEVs cannot play an in vitro coagulation effect, indicating that the in vitro coagulation effect of IEVs is more concentrated in the upstream of the common blood coagulation pathway.
  • hemophilia A mice deficiency of coagulation factor VIII
  • 9 ⁇ 10 8 IEVs were injected through the tail vein to observe the procoagulant effect of IEVs in vivo.
  • the results are shown in Figure 7. After treatment with IEVs, the bleeding tendency of hemophilia mice can be significantly improved, and the therapeutic effect can be stably maintained for 14 days.
  • IEVs can play a significant role in promoting coagulation in vitro. And after in vivo injection, it can significantly improve the bleeding tendency, and can be used to improve the bleeding tendency caused by hemophilia A.
  • SLE systemic lupus erythematosus
  • IEVs (extracted in Example 2) were injected into lpr mice, and tail trimming experiments were performed 7 days later. The results showed that IEVs treatment can significantly improve the bleeding tendency of lpr mice, and the therapeutic effect can be sustained and stably maintained for 7 days (Figure 9C).
  • the lpr mouse is a representative animal model of SLE.
  • CHS syndrome is an autosomal recessive genetic disease, which is more common in the offspring of consanguineous marriages.
  • the pathogenic gene is the lysosomal transport regulator gene (LYST). Mutations in the LYST gene often lead to abnormal LYST protein production, which in turn causes platelet dysfunction. The patient will show obvious bleeding tendency clinically, and there is no effective prevention and treatment measures at present.
  • the IEVs expression markers Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5 with the diameters of the IEVs obtained in Example 2 in the range of 0.03 ⁇ m to 0.2 ⁇ m and 0.2 ⁇ m to 1 ⁇ m have strong coagulation effects in vitro;
  • Exosomes prepared in Example 1 have a diameter of 0.03 ⁇ m ⁇ 0.15 ⁇ m, and express markers Complement C1q, Complement C3, Thrombospondin-1 and Thrombospondin-2,
  • iPS cells Induced pluripotent stem cells (iPS cells, iPSC) cell culture
  • 5 ⁇ 10 4 induced cells were seeded into 10 cm petri dishes with feeder cells (mEFs). The next day, the medium was changed to Es medium containing bFGF (4ng/ml), and changed every other day. After 5 days, the cells began to clone. If there were no Es-like clones after 40 days, it was considered a failure.
  • the purchased MC3T3-E1Subclone14 was quickly thawed and centrifuged at 500g for 5min. The supernatant was removed and the cell pellet at the bottom was collected.
  • the cells were resuspended in Dex(-) medium and seeded in a 10cm diameter cell culture dish at 37°C, 5% CO 2 culture. After it grows up, trypsin is used to incubate at 37°C for digestion and passage amplification. After that, the Dex(-) culture medium is changed every 3 days, and the cells can be used for multiple passages. Among them, the composition of Dex(-) culture medium is shown in Table 5:
  • the methods for obtaining IEVs from the three types of cells are the same as in Example 2.
  • the flow cytometry results show that the osteoblast cell line MC3T3-E1 and human bone marrow mesenchymal stem cells derived from hBMMSC have similar IEVs particle diameter distribution.
  • the Western Blot method was used for detection, and the experimental results are shown in Figure 13.
  • the surface markers of IEVs derived from iPSCs and human bone marrow mesenchymal stem cells (hBMMSC) are compared, and the IEVs derived from the two types of cells are both high. Express IEVs marker Anenexin V. Compared with hBMMSC, IEVs derived from iPS cells express higher levels of Syntaxin4.
  • Example 6 IEVs can be discharged through the skin and hair
  • IEVs prepared in Example 2 Take 4 ⁇ 10 6 IEVs prepared in Example 2 labeled with DIR, resuspend them in 200 ⁇ l PBS, and inject them into nude mice BALB/c-nu/nu systemically through the tail vein. Observe 1, 3, and 7 days later and use them in vivo
  • the imaging instrument detects the distribution of IEVs on the skin surface, and the results are shown in Figures 13A-13C.
  • Figure 13A shows that IEVs can reach the skin surface, the number is the largest on the 3rd day, and basically disappears on the 7th day, showing the dynamic metabolic process of IEVs on the skin surface (Figure 13A).
  • Immunofluorescence results showed that PKH26-IEV gradually moved from the subcutaneous tissue to the dermis and epidermis over time after systemic injection of C57 mice. A large number of IEVs were observed in the stratum corneum of the skin on the 7th day, suggesting that the systemically injected IEVs can be excreted as the stratum corneum of the skin falls off (Figure 13B).
  • PKH26-IEV was found in the hair follicles in the hair plucked from the surface of the mouse on the 7th day, indicating that the systemically injected IEVs can also be metabolized along with the hair loss (Figure 13C).
  • This example shows that IEVs can be excreted through the skin and hair, indicating that it is safe to inject or increase the content of IEVs in the body.
  • hiPSCs The cultivation of hiPSCs is the same as in Example 5.
  • hUCMSCs are also conventional cultivation methods in the field.
  • the hiPSCs can be from the 26th to the 29th generation, but are not limited to this.
  • the 26th generation is specifically used in this embodiment; the hUCMSCs can be from the 7th to the 9th generation, but it is not limited to this.
  • the example is the 7th generation when it is used.
  • step (2) Separate IEVs from the supernatant of apoptotic cells in step (1) and use flow cytometry to detect AnnexinV expression rate.
  • the steps include: 800g centrifugation for 10mins-2000g centrifugation for 5mins (except for this step, the rest of the extraction steps are the same as in Example 2)-16000g centrifugation for 30mins-16000g centrifugation for 30mins to obtain IEVs.
  • AnnexinV dyeing for 15mins flow on the machine.
  • NTA Nanoparticle Tracking Analysis
  • the particle size of IEVs derived from hiPSCs is about 100nm, and the particle size of IEVs derived from hUCMSCs is about 180nm;

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Abstract

提供一种囊泡及其应用。该囊泡为诱导性囊泡，来源包括干细胞或体细胞，具有的标志物包括Syntaxin 4。该囊泡与间充质干细胞中的外泌体相比，能特异性高表达Syntaxin 4，可用于区分MSCs来源的囊泡和外泌体的特征性标志物。该囊泡能够在体外发挥促凝作用，体内注射后能够改善血友病小鼠的出血倾向，可用于改善血友病出血倾向的治疗。且所述囊泡可以通过皮肤和毛发排出。

Description

一种囊泡及其应用 技术领域
本公开属于生物医药领域，涉及一种囊泡及其应用。
背景技术
细胞外囊泡(extracellular vesicles,EVs)是细胞分泌的含有蛋白质、核酸及各种细胞因子的纳米级载体。细胞外囊泡可以通过内分泌或旁分泌的方式作用于靶细胞，在细胞间物质传递和信息交流过程中发挥了重要作用。研究发现，细胞外囊泡所介导的信息交流在机体生理或病理过程中发挥了重要的调控作用，涉及到免疫调节、肿瘤生长、血管生成、损伤修复等。目前该领域的研究主要集中在外泌体(exosomes)方向。外泌体是直径在30-150nm左右的细胞外囊泡，其内含有RNA、脂质和蛋白质等成分。外泌体广泛参与了机体的各种生理/病理性调控，能够用作多种疾病的诊断、治疗和预后评估。迄今为止，间充质干细胞(mesenchymal stem cells，MSCs)被认为是产生外泌体能力最强的细胞。众多的研究发现MSCs来源的外泌体能模拟MSCs的生物学功能，在促进细胞生长和分化，修复组织缺损等方面发挥了重要的调控作用。因此，近年来以MSCs来源的外泌体为基础的细胞囊泡疗法取得了显著的发展。然而，目前以外泌体为基础的细胞囊泡治疗仍然存在诸多问题，主要表现在外泌体的提取和纯化过程复杂，耗时长，对设备和试剂的要求较高，生理性外泌体产量较低等等，这些缺陷都限制了外泌体治疗的临床转化和应用。
血友病(hemophilia)为一组遗传性凝血功能障碍的出血性疾病，其共同的特征是活性凝血活酶生成障碍，凝血时间延长，终身具有轻微创伤后出血倾向，重症患者没有明显外伤也可发生“自发性”出血。2018年5月11日，国家卫生健康委员会等5部门联合制定了《第一批罕见病目录》，血友病被收录其中。血友病主要分为三类，即血友病A，血友病B和血友病C。血友病A，即因子Ⅷ促凝成分(Ⅷ：C)缺乏症，是一种性联隐性遗传疾病，女性传递，男性发病。血友病B，即因子Ⅸ(FIX)缺乏症，亦为性联隐性遗传，其发病数量较血友病A少。血友病C，即因子Ⅺ(FⅪ)缺乏症，为常染色体不完全隐性遗传，是一种罕见的血友病。发病率以血友病A最多占80％-85％，血友病B占15％-20％，血友病C很少见。长期以来，针对血友病的治疗，临床上以注射外源性凝血因子做为主要干预措施，但该方法存在治疗成本高、治疗有效期短、易产生自身抗体等各种问题，无法成为切实有效的治疗手段。
发明内容
在一些实施方案中，本公开提供了一种来源于间充质干细胞的囊泡。
在一些实施方案中，本公开提供了一种囊泡组合物。
在一些实施方案中，本公开提供了一种针对血友病的、包含囊泡的药物组合物。
在一些实施方案中，本公开提供了一种囊泡的筛选或者鉴定或者提取试剂盒。
在一些实施方案中，本公开提供了一种囊泡的标志物。
在一些实施方案中，本公开提供了一种利用标志物对囊泡进行鉴定或者选择的方法。
在一些实施方案中，本公开提供了一种囊泡的制备方法。
在一些实施方案中，本公开提供了一种来源于体细胞或干细胞的囊泡，所述囊泡是诱导性囊泡，所述囊泡具有的标志物包括Syntaxin 4。
在一些实施方案中，本公开提供了一种治疗或预防或改善受试者的疾病或所述疾病并发症的方法，包括给予所述受试者有效量的所述的囊泡或所述的囊泡组合或所述的组合物；所述疾病为出血性疾病。在一些实施方案中，所述出血性疾病包括凝血因子匮乏、血小板数量减少和/或功能缺陷导致的出血。在一些实施方案中，所述出血性疾病包括血友病、狼疮性出血或契-东综合征。在一些实施方案中，所述血友病包括血友病A、血友病B或血友病C。在一些实施方案中，所述疾病为血友病A。
在一些实施方案中，所述干细胞包括全能干细胞和多能干细胞。在一些实施方案中，所述干细胞包括间充质干细胞和诱导性多能干细胞(IPS)。
在一些实施方案中，所述的体细胞包括成骨细胞系。
在一些实施方案中，所述细胞可以是原代培养的细胞，也可以是已有的或已确立的细胞系。
在一些实施方案中，所述细胞系指的是是指永生化的细胞培养物，在适当的新鲜培养基和空间下可以无限增殖。
在一些实施方案中，所述细胞可以是确立细胞株。
在一些实施方案中，所述诱导性囊泡是在所述干细胞或体细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡。
在一些实施方案中，所述诱导性囊泡是通过添加星形孢菌、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合诱导干细胞或干细胞凋亡产生。
在一些实施方案中，所述囊泡具有的标志物还包括Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5中的一种或几种。
在一些实施方案中，所述囊泡具有标志物Syntaxin 4、Annexin V、Flotillin-1、Cadherin 11和Integrin alpha 5的组合。
在一些实施方案中，所述囊泡高表达标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4。
在一些实施方案中，所述囊泡对标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量高于MSC或外泌体。
在一些实施方案中，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别约1-2倍、2-3倍、1-3倍、3-4倍和3-6倍。
在一些实施方案中，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别约1.5-2倍、2.5-3倍、1.5-2.5倍、3.5-4倍和3.5-5倍。
在一些实施方案中，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别约1.5-1.9倍、2.5-2.9倍、1.8-2.5倍、3.5-3.9倍和4-5倍。
在一些实施方案中，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别约1.76倍、2.81倍、2.41倍、 3.68倍和4.45倍。
在一些实施方案中，所述外泌体不表达Syntaxin 4，本公开的囊泡表达Syntaxin 4。
在一些实施方案中，所述外泌体不同时表达Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4，本公开的囊泡同时表达Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4。
在一些实施方案中，所述囊泡和所述外泌体来源于同种来源的MSCs。
在一些实施方案中，利用流式细胞技术对IEVs的表面膜蛋白进行分析，结果显示，MSCs来源的IEVs能够表达和MSCs相似的表面蛋白，即CD29,CD44,CD73,CD166阳性，CD34,CD45阴性；同时，IEVs能够表达细胞外囊泡的普遍性表面蛋白CD9,CD63,CD81和C1q。
在一些实施方案中，所述诱导性囊泡是通过添加星形孢菌素、紫外线照射、饥饿法、热应力法或其组合诱导间充质干细胞凋亡产生。
在一些实施方案中，所述的囊泡是用星形孢菌素诱导间充质干细胞产生的。
在一些实施方案中，所述间充质干细胞的代数可以约第2～5代，但又不限于此。
在一些实施方案中，所述星形孢菌素的浓度约1nM-10000nM。在一些实施方案中，所述星形孢菌素的浓度约100nM-10000nM。在一些实施方案中，所述星形孢菌素的浓度约500nM-10000nM。在一些实施方案中，所述星形孢菌素的浓度约500-1000nM。在一些实施方案中，所述星形孢菌素的浓度约500-900nM。在一些实施方案中，所述星形孢菌素的浓度约500-800nM。
在一些实施方案中，所述囊泡的直径约0.03-6μM。在一些实施方案中，所述囊泡的直径约0.03-4.5μM。在一些实施方案中，所述囊泡的直径约0.03-1μM。在一些实施方案中，所述囊泡的直径约0.04-1μM。在一些实施方案中，所述囊泡的直径约0.05-1μM。在一些实施方案中，所述囊泡的直径约0.1-1μM。在一些实施方案中，所述囊泡的直径约0.15-1μM。
在一些实施方案中，本公开还提供了一种囊泡组合，包含上述囊泡。
在在一些实施方案中，所述囊泡组合还包含其他现有技术中的囊泡，包括但不限于如外泌体、迁移体、微泡和Ectosome。
在一些实施方案中，其中所述的囊泡在所述囊泡组合物中的数目占比约65-100％。
在一些实施方案中，所述的囊泡在所述囊泡组合物中的数目占比约75-98％。
在一些实施方案中，所述的囊泡在所述囊泡组合物中的数目占比约80-96％。
在一些实施方案中，本公开还提供了一种组合物，所述药物组合物包含上述囊泡或者上述囊泡组合。
在一些实施方案中，所述组合物包括药品、食品、保健品、化妆品、添加剂或中间品。
在一些实施方案中，所述组合物为药品。
在一些实施方案中，所述组合物还包含药学上或免疫学上可接受的载体。
在一些实施方案中，所述组合物的制剂形式选自冻干粉针、注射剂、片剂、胶囊、试剂盒或贴剂。
在一些实施方案中，所述囊泡是作为一种药物载体使用。
在一些实施方案中，本公开还提供了一种所述囊泡的筛选或者鉴定或者提取试剂或试剂盒，包含以下标志物检测试剂中的一种或多种：标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的检测试剂。
在一些实施方案中，所述标志物的检测试剂检测所述标志物基因的表达量。
在一些实施方案中，所述标志物的检测试剂检测所述标志物mRNA的表达量。
在一些实施方案中，所述标志物的检测试剂检测所述标志物蛋白的表达量。
在一些实施方案中，所述标志物的检测试剂为荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针、抗体、抗体功能性片段和偶联抗体中的一种或多种。
在一些实施方案中，所述试剂盒选自qPCR试剂盒、免疫印迹检测试剂盒、流式细胞分析试剂盒、免疫组化检测试剂盒和ELISA试剂盒中的一种或多种。
在一些实施方案中，所述试剂盒选自流式细胞分析试剂盒。
在一些实施方案中，本公开还提供了所述囊泡或所述的囊泡组合物或所述的药物组合物在制备治疗或预防或改善疾病或所述疾病并发症的产品中的应用；所述疾病包括肝病、血友病。
在一些实施方案中，所述疾病为血友病，所述的囊泡能够在体外发挥显著的促凝作用，体内注射后能够显著改善血友病小鼠的出血倾向，可用于改善血友病出血倾向的治疗，具有良好的应用前景。
在一些实施方案中，所述疾病为血友病A。
在一些实施方案中，所述产品包括药品、食品、保健品、化妆品、添加剂或中间品。
将所述囊泡用于疾病治疗的过程中，可以将所述囊泡任选地通过选自由静脉内注射、肌肉注射、皮下注射、鞘内注射或输注以及器官内输注所组成的组中的途径进行给药。例如，作为例子，对于静脉注射，可以通过尾静脉注射。器官内输注包括输注至解剖学空间中，例如，作为例子，胆囊、胃肠腔、食道、肺系统(通过吸入)和/或膀胱。
作为例子，对于胃肠腔输注中腹腔注射，与尾静脉注射相比，腹腔注射也可以获得同等的治疗效果。腹腔注射的安全性和可操作性要优于尾静脉注射。
一些实施方案中，本公开还提供了一种选择或鉴定所述囊泡的方法，所述方法包括对以下标志物中的一种或多种进行检测：标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4。
当检测结果显示所述标志物阳性结果时，则判别为所述囊泡。
在一些实施方案中，可以将所述标志物的表达结果与对照相比，当表达量显著高于对照时，判断为阳性结果。所述的对照可以是现有其他囊泡或者外泌体(可以包括外泌体、迁移体、微泡和Ectosome中的一种或几种)；可以是来源于间充质干细胞的其他囊泡或者外泌体。
在所述的标志物中，尤其优选标志物Syntaxin。在一些实施方案中，当所测囊泡的Syntaxin 4表达量大于或等于外泌体(例如同种细胞来源的外泌体)的2-6倍(更优选4-5倍)时，判断为所述的囊泡(例如诱导性囊泡)。
在一些实施方案中，本公开提供了所述标志物的检测试剂在制备检测或鉴定所述囊泡的试剂或试剂盒中的应用，其特征在于，所述标志物包括Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4中的一种或多种，所述试剂或试剂盒还包括对照试剂，所述对照试剂包含外泌体、迁移体、微泡和Ectosome中的一种或几种，当所述待测样品中的所述标志物的表达量高于对照试剂时，则判断为阳性。
在一些实施方案中，所述对照试剂为外泌体。
在一些实施方案中，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的2-6倍时，则判断为所述囊泡。
在一些实施方案中，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的4-5倍时，则判断为所述囊泡(例如诱导性囊泡)。
在在一些实施方案中，本公开提供了一种制备所述囊泡的方法，包括以下步骤，所述方法为通过添加凋亡 诱导剂诱导干细胞或体细胞产生所述囊泡。
在一些实施方案中，所述方法包括以下步骤：(1)培养间充质干细胞；(2)收集间充质干细胞的培养基上清；(3)从步骤(2)中的培养基上清中分离出囊泡。
在一些实施方案中，步骤(1)中的培养间充质干细胞的步骤包括：(4)从组织中分离间充质干细胞；(5)添加培养基培养间充质干细胞；所述间充质干细胞的培养基中接触凋亡诱导剂。
在一些实施方案中，所述凋亡诱导剂包括星形孢菌素、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合。
在一些实施方案中，所述凋亡诱导剂为星形孢菌素。
在一些实施方案中，所述星形孢菌素的浓度约500-1000nM。在一些实施方案中，所述星形孢菌素的浓度约500-900nM。在一些实施方案中，所述星形孢菌素的浓度约500-800nM。
在一些实施方案中，步骤(5)中凋亡诱导剂的处理细胞的时间约16-24小时。
在一些实施方案中，所述步骤(3)中，分离囊泡的方法包括选用超速离心的方法分离所述囊泡。
在一些实施方案中，本公开中单个MSCs能够产出300-2000个囊泡。
在一些实施方案中，所述超速离心的方法分离所述囊泡的步骤包括：(a)将收集到的培养上清进行第一次离心，取上清；(b)将步骤(a)中收集到的上清进行第二次离心，取上清；(c)将步骤(b)中收到到的上清进行第三次离心，取沉淀；(d)将步骤(c)中收到到的沉淀进行第四次离心，取沉淀；(e)将步骤(d)中收到到的沉淀进行第五次离心，取沉淀；
在一些实施方案中，所述第一次离心约500-1500g离心5-30分钟。在一些实施方案中，所述第一次离心约500-1000g离心5-20分钟。在一些实施方案中，所述第一次离心约500-900g离心5-15分钟。在一些实施方案中，所述第二次离心约1000-3000g离心5-30分钟。在一些实施方案中，所述第二次离心约1500-2500g离心5-20分钟。在一些实施方案中，所述第二次离心约1500-2200g离心5-15分钟。在一些实施方案中，所述第三次离心约10000-30000g离心15-60分钟。在一些实施方案中，所述第三次离心约12000-25000g离心20-60分钟。在一些实施方案中，所述第三次离心约12000-20000g离心20-40分钟。在一些实施方案中，所述第四次离心约10000-30000g离心15-60分钟。在一些实施方案中，所述第四次离心约12000-25000g离心20-60分钟。在一些实施方案中，所述第四次离心约12000-20000g离心20-40分钟。
在一些实施方案中，带有特定标记物的囊泡，可以通过针对特定标记物的富集方法富集获得。在得到足够的囊泡后，收集培养基并从培养基中纯化和分离特定的囊泡。这可以通过本领域已知的任何合适的方法来实现。这些方法包括，例如，通过差异超速离心分离外泌体的原始方法，以及更新的方法，例如聚合物沉淀(来自SBI，Palo Alto，CA的ExoQuickTM)、免疫亲和捕获(Greening等人2015，Methods in Molecular Biology)、免疫磁捕获(Exo-FLOWTM，SBI)等。
免疫亲和纯化是基于表面标志物选择性捕获特定囊泡的方法。通过链霉亲和素共价包覆的磁珠和生物素化的捕获抗体之间高亲和力的偶联，实现囊泡的高效捕获。所捕获的囊泡经洗脱后，结构完整且具有生物活性。基于本公开的发现，所述囊泡能特异性高表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分子，那么本公开可以使用该方法对囊泡进行分离或纯化或富集。
在一些实施方案中，还可以使用免疫磁珠的方法对所述囊泡进行富集，所述免疫磁珠是由单克隆抗体与磁珠偶联得到的；所述单克隆抗体包括Anti-Syntaxin 4抗体、Anti-Annexin V抗体、Anti-Flotillin-1抗体、Anti-Cadherin 11抗体、Anti-Integrin alpha 5抗体中的一种或多种。
在一些实施方案中，本公开还提供了一种诱导性囊泡，该诱导性囊泡来源于IPS细胞。其是在IPS细胞处于正常存活时，进行干预或诱导，使IPS细胞凋亡产生的一类亚细胞产物。
当在本申请中使用术语“富集”时，它涵盖了一个或多个囊泡从存在于样品中的任何其它囊泡中的分离或者其是指包含囊泡的组合物中所述囊泡以更高的总百分比含量存在，与发现其存在生物体的组织中时相比。
在一种实施方式中，被富集的囊泡不从样品中分离，而是在囊泡仍存在于样品中时对囊泡进行任何诊断。所述样品则可呈现在载玻片上，并可使用显微镜进行诊断，并且在此实施方式中囊泡被检测而不被分离。
在另外一种实施方式中，被富集的囊泡从样品中分离。
其中，免疫磁珠分离技术(Immunomagneticbead-basedseparation，IMS)是近年来发展起来的一项新的免疫学技术。免疫磁珠(Immunomagneticbead，IMB)既可结合活性蛋白质抗体，又可被磁铁吸引，经过处理后，可将抗体结合在磁珠上，使之成为抗体的载体，磁珠上抗体与特异性抗原物质结合后，则形成抗原-抗体-磁珠免疫复合物，这种复合物在磁力作用下发生力学移动，使复合物与其它物质分离，而达到分离特异性抗原的目的。免疫磁珠(IMB)是一个平台，凡是利用抗原抗体结合原理进行工作的领域都可以应用，并且已在医学和生物学的骨髓移植、分离干细胞、细胞器、癌细胞、激素、病原菌以及毒素等方面取得了显著成绩。近年来IMB以其高度的敏感性和特异性被广泛应用于食品、水、生物样品、环境等标本中真菌毒素的分离和检测工作中，显示出良好的开发应用前景。
本公开所述的免疫磁珠方法是使用有特异性抗体结合的磁珠去和有特异性表面抗原的靶囊泡相结合，再用磁场来吸附，抽取靶囊泡。
在本公开的某些具体实施方式中，所述囊泡具体富集方法为，将Anti-Syntaxin 4抗体、Anti-Annexin V抗体、Anti-Flotillin-1抗体、Anti-Cadherin 11抗体、Anti-Integrin alpha 5抗体中的一种或多种抗体包被的免疫磁珠加到含有囊泡的细胞培养上清中，那么，与Anti-Syntaxin 4抗体、Anti-Annexin V抗体、Anti-Flotillin-1抗体、Anti-Cadherin 11抗体、Anti-Integrin alpha 5中的一种或多种抗体能够特异性结合的囊泡就被分离出来，可以达到富集特定囊泡的目的。一些优选的实施方式中，当使用Anti-Syntaxin 4抗体、Anti-Annexin V抗体、Anti-Flotillin-1抗体、Anti-Cadherin 11抗体和Anti-Integrin alpha 5抗体同时包被的免疫磁珠分离出的囊泡其纯度最高，治疗疾病例如血友病A的效果最好。
一些优选的实施方式中，是基于离心方法进行了组合优化有效去除了细胞以及细胞碎片等杂物之后的细胞培养上清，然后再将Anti-Syntaxin 4抗体、Anti-Annexin V抗体、Anti-Flotillin-1抗体、Anti-Cadherin 11抗体、Anti-Integrin alpha 5抗体中的一种或多种抗体包被的免疫磁珠加到所述的细胞培养上清中，那么，与所述抗体能够特异性结合的囊泡就能够被分离出来，可以达到富集特定囊泡的目的。
一些实施方案中，所述间充质干细胞来源于人或鼠，但不限于此。
一些实施方案中，所述间充质干细胞包括骨髓来源的间充质细胞、尿液来源的间充质干细胞、口腔来源的间充质干细胞、脂肪来源的间充质干细胞、胎盘来源的间充质干细胞、脐带来源的间充质干细胞、骨膜来源的干细胞或其组合，但不限于此。
一些实施方案中，所述间充质干细胞选自骨髓来源的间充质细胞、脂肪来源的间充质干细胞、脐带来源的间充质干细胞和口腔来源的间充质干细胞。
附图说明
图1A-1E为分离的BMMSCs的表面标志物的流式检测结果。
图2为实施例2的操作流程图。
图3为用流式细胞分析技术分析的MSCs(10 6个MSCs)产生的IEVs数量统计结果。
图4A-图4F为IEVs颗粒的直径检测：图4A为流式检测IEVs的颗粒直径分布图；图4B为侧向散射光(SSC)分析IEVs的散射光强，显示IEVs颗粒直径分布；图4C为以Bangs Laboratories公司生产的标准化小颗粒微球分析IEVs的散射光强，显示IEVs的颗粒直径分布；图4D为透射电镜(TEM)观察的IEVs，显示IEVs的颗粒直径分布；图4E为纳米粒子跟踪分析(NTA)，显示IEVs颗粒直径分布；图4F为纳米流式检测技术对IEVs进行单囊泡水平的粒径检测，显示IEVs的颗粒直径分布。
图5A-图5K为流式细胞技术IEVs的表面膜蛋白进行分析结果。
图6A-图6D为IEVs的内容物分析：图6A为DIA定量技术对MSCs、MSCs-Exosomes、MSCs-IEVs蛋白组学定量分析结果；图6B为筛选IEVs特异性高表达的蛋白绘制的热图；图6C为差异蛋白的GO富集分析IEVs表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分子的结果；图6D为Western Blot验证MSCs、MSCs-Exosomes、MSCs-IEVs表达Annexin V,Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4的结果。
图7为IEVs在血友病A小鼠的体内促凝作用。
图8A-图8D为给血友病A小鼠注射IEVs之后各种凝血因子水平的变化情况：图8A为凝血因子VIII的变化情况；图8B为vWF因子的变化情况；图8C为组织因子(TF)的变化情况；图8D为凝血酶原的变化情况。
图9A-图9B为血友病A小鼠模型中，IEVs进行PS和TF的封闭之后对IEVs的体内治疗效果的影响情况。图9C为对lpr小鼠进行IEVs注射治疗后能够显著改善lpr小鼠的出血倾向。图9D为对CHS小鼠进行IEVs注射治疗后显著改善CHS小鼠的出血倾向。
图10为同种MSC来源的IEVs和Exosomes对血友病A小鼠治疗效果对比。
注：其中WT为野生型小鼠；HA组为血友病A小鼠模型；HA+IEVs为血友病A小鼠模型给予IEVs治疗；HA+PS-IEVs为血友病A小鼠模型给予PS阴性IEVs；HA+TF-IEVs为血友病A小鼠模型给予TF阴性IEVs；HA+Exosomes为血友病A小鼠模型给予Exosomes治疗。
图11为MC3T3-E1、hBMMSC来源的IEVs光镜下的形态图。
图12为MC3T3-E1、hBMMSC来源的IEVs颗粒的直径分布流式检测结果。
图13A-13C显示IEVs可经皮肤和毛发排出：图13A为IEVs在皮肤表面的动态代谢示意图。图13B显示随着时间推移，IEVs逐渐从皮下组织向真皮层和表皮移动。图13C显示第7天时在小鼠体表拔下的毛发中发现毛囊中存在PKH26-IEVs。
图14显示了高内涵细胞成像分析系统拍摄的hiPSCs和hUCMSCs死亡过程图。
图15显示诱导凋亡的hiPSCs和hUCMSCs使用流式分析凋亡率，证明绝大部分细胞发生了凋亡。
图16显示使用流式分析Annexin5表达的阳性率hiPSCs和hUCMSCs两者表达均在80％以上。
图17显示Nanoparticle Tracking Analysis(NTA)检测出的两种IEVs的粒径。
图18显示Nanoparticle Tracking Analysis(NTA)检测出的两种IEVs产IEVs数量。
图19显示Nanoparticle Tracking Analysis(NTA)检测出的两种IEVs的电位。
具体实施方式
以下通过具体的实施例进一步说明本公开的技术方案，具体实施例不代表对本公开保护范围的限制。其他人根据本公开理念所做出的一些非本质的修改和调整仍属于本公开的保护范围。
本公开实施例中的IEVs为诱导性囊泡的简称，可称为诱导性囊泡，也可称为诱导性细胞外囊泡(Induced extracellular vesicles,IEVs)。诱导性细胞外囊泡是指的是一种在前体细胞(例如干细胞)正常存活时，被干预或诱导，使其凋亡产生的一类亚细胞产物。通常这一类亚细胞产物，具有膜结构，表达凋亡性标志物，部分包含有遗传物质DNA。发明人发现诱导性细胞外囊泡是区分于细胞和常规细胞外囊泡(如外泌体等)的一类物质。在一些实施方式中，所述的正常存活时的细胞，例如是非凋亡的细胞、非衰老的细胞、非老化而增殖停滞的细胞、非冻存后复苏的细胞、非发生恶变而异常增殖的细胞或非出现损伤的细胞等。在一些实施方式中，所述的正常存活时的细胞取自细胞培养过程中，细胞接触融合80-100％的时候的细胞。一些实施方式中，所述的正常存活时的细胞取自对数期细胞。一些实施方式中，所述的正常存活时的细胞取自人或鼠组织来源的原代培养及其传代培养细胞。一些实施方式中，所述的正常存活时的细胞取自已确立的细胞系或细胞株。在一些实施方式中，所述的前体细胞取自早期的细胞。
本公开中的IEV同IEVs。本公开中的STS为星形孢菌素。本公开中Exosomes指的是外泌体。
“包含”或“包括”旨在表示组合物(例如介质)和方法包括所列举的要素，但不排除其他要素。当用于定义组合物和方法时，“基本上由……组成”意味着排除对于所述目的的组合具有任何重要意义的其他要素。因此，基本上由本文定义的元素组成的组合物不排除不会实质上影响要求保护的本公开的基本和新颖特征的其他材料或步骤。“由……组成”是指排除其他组成部分的微量元素和实质性的方法步骤。由这些过渡术语中的每一个定义的实施方案都在本公开的范围内。
“有效量”是足以实现有利或所需结果的量，例如增强的免疫应答，医学状况(疾病、感染等)的治疗、预防或改善。有效量可以在一次或多次施用、应用或剂量中施用。合适的剂量将依赖于体重、年龄、健康、待治疗的疾病或状况和施用途径而变。
如本文所用，术语“高表达”等旨在包括将核酸或蛋白质的表达增加至高于现有技术的囊泡(例如外泌体)所含有的水平。
如本文所用，术语“药学上可接受的载体”是指任何标准药物载体，例如冻干粉针、注射剂、片剂、胶囊、试剂盒或贴剂。通常这种载体含有赋形剂，例如淀粉、乳、糖、某些类型的粘土、明胶、硬脂酸或其盐、硬脂酸镁或硬脂酸钙、滑石、植物脂肪或油、树胶、二醇或其它已知的赋形剂。这些载体还可以包括调味剂和颜色添加剂或其他成分。药学上可接受的载体的例子包括但不限于以下：水、盐水、缓冲液、惰性的无毒固体(例如甘露醇、滑石)。通过众所周知的常规方法配制包含此类载体的组合物。取决于预期的施用方式和预期用途，组合物可以是固体、半固体或液体剂型的形式，例如粉末、颗粒、晶体、液体、悬浮液、脂质体、糊剂、乳膏、油膏等，并且可以是适于施用相对精确剂量的单位剂量的形式。
在本公开中，“组合物”中的组分可以是混合的形式存在，也可以被分开包装。分开的包装的组分也可以含有其各自的佐剂。所述的佐剂是指在药学中，可辅助药物疗效的手段。对于组合物中的组分在分开包装的情况下，各个分开包装的组分可以是同时施用或者是以任意的前后顺序施用，其中患者先用一种药物治疗，然后再给以另一种药物。所述的患者是指哺乳动物受治疗者，尤其是人类。
在本公开中，所述的“组合物”还可以是一种组分被另外一种组分包裹的形式存在。在一些实施方式中，在组合物中，所述的诱导性囊泡作为药物载体，将治疗或预防疾病的药物包裹在所述诱导性囊泡中。
本公开中，相应的试剂来源如下：青霉素/链霉素溶液(BIOSOURCE；P303-100)；谷氨酰胺(BIOSOURCE；P300-100)；地塞米松磷酸钠(Sigma；D-8893)；α-MEM(Gibco；12571-063)；2-ME(GIBCO；21985-023)。
实施例1 MSCs的分离培养
依据动物伦理委员会的指导用过量CO 2处死小鼠，在无菌条件下，取下胫骨和股骨，剥净附着在其上的肌肉和结缔组织，进一步分离干骺端、暴露骨髓腔，用10mL无菌注射器抽取喊体积分数为10％胎牛血清的PBS反复冲洗骨髓腔，70μm孔径细胞滤网过滤后，500g离心5min，去除上清液后收集底部的细胞沉淀，PBS重悬，再次500g离心5min，收集最终的细胞沉淀。而后对细胞进行流式分选，以CD34-和CD90+为分选标准，分选出BMMSCs。最后以Dex(-)培养液重悬细胞并接种于10cm直径细胞培养皿，37℃、5％CO 2培养。24h后，吸除上清液未贴壁细胞，PBS清洗后，加入Dex(-)培养液继续培养。1周后加入等量Dex(+)培养液，再1周后可见密集的原代BMMSCs集落。采用胰蛋白酶37℃孵育消化BMMSCs并传代扩增，之后每3日换Dex(+)培养液，长满后传代。使用P2代BMMSCs进行后续实验。
其中，Dex(-)培养液成分如表1所示，Dex(+)培养液成分如表2所示：
表1 Dex(-)培养液成分表
Figure PCTCN2021072938-appb-000001
表2 Dex(+)培养液配方表
Figure PCTCN2021072938-appb-000002
采用流式细胞术分析表面标志物的方法来评估分离的BMMSCs的纯度。对于表面标志鉴定，胰蛋白酶消化收集P2代BMMSCs后，PBS清洗1次，以5×10 5/mL密度重悬细胞于含3％FBS的PBS中，加入1μL PE荧光偶联的CD29、CD44、CD90、CD45和CD34抗体，空白组不加。4℃避光孵育30min，PBS清洗2遍后，上机检测。流式检测结果如图1A-1E所示，可知，分离的细胞为BMMSCs(骨髓间充质干细胞)。
实施例2诱导性囊泡的获得
将实施例1中培养至第2代的MSCs(骨髓来源的MSCs,BMMSCs)，用实施例1中的培养基(Dex(+)培养液)继续培养至细胞汇合80％-90％时，用PBS冲洗2遍，加入含500nM STS的无血清培养基(α-MEM培养 基)诱导凋亡，37℃孵育24h，收集细胞上清液，用于分离和提取IEVs。
从收集的培养上清中进行IEVs的分离与提取，操作流程如图2所示，具体步骤包括：800g离心10分钟后，收集上清液；接着2000g离心10分钟，再收集上清液；然后16000g离心30分钟，移除上清液，无菌PBS重悬IEVs；接着16000g离心30分钟后，移除上清液，300-500μL无菌PBS重悬IEVs。
对比例1同种MSC来源的外泌体分离和提取
将实施例1中培养至第2代的MSCs(骨髓来源的MSCs,BMMSCs)，用实施例1中的培养基继续培养至细胞汇合80％-90％时，用PBS冲洗2遍，加入无血清培养基，37℃孵育48h，收集细胞上清液，用于分离和提取Exosomes。
提取步骤包括：800g离心10分钟—收集上清液—2000g离心10分钟—收集上清液—16000g离心30分钟—收集上清液—120000g离心90分钟—移除上清液，无菌PBS重悬沉淀—120000g再次离心90分钟，移除上清，收集底部的Exosomes，无菌PBS重悬。
实施例3 IEVs的分析
1、IEVs的定量和膜蛋白的分析
利用流式细胞技术对实施例2获得的IEVs进行定量分析，测量时间点为第1h、第4h、第8h、第16h和第24h，结果显示10 6个MSCs在诱导至第1h、第4h、第8h、第16h和第24h后分别可以产出0.76×10 8个、1.29×10 8个、1.95×10 8个、2.48×10 8个、3.14×10 8个IEVs，从中可以看出，诱导至24h后，单个MSC可以产出300个IEVs(图3)。
此外，流式检测发现IEVs的颗粒直径分布都集中在1μm以下，占94.97％(图4A)，侧向散射光(SSC)分析结果同样显示IEVs散射光强集中在1μm以下范围(图4B)。进一步的，通过Bangs Laboratories公司生产的标准化小颗粒微球(0.2μm，0.5μm，1μm)分析IEVs的散射光强，结果显示IEVs的颗粒直径都在0.2μm以下(图4C)。透射电镜(TEM)观察的结果与流式检测结果相似，大部分囊泡的直径都在200nm及200nm以下(图4D)。纳米粒子跟踪分析(NTA)结果与透射电镜观察结果相符，IEVs颗粒直径平均为169nm(图4E)。利用最先进的纳米流式检测技术进行单囊泡水平的粒径检测，结果也显示IEVs的平均颗粒直径在100.63nm(图4F)。
利用流式细胞技术对实施例2提取的IEVs的表面膜蛋白进行分析，结果显示，MSCs来源的IEVs能够表达和MSCs相似的表面蛋白，即CD29,CD44,CD73,CD166阳性，CD34,CD45阴性。同时，IEVs能够表达细胞外囊泡的普遍性表面蛋白CD9,CD63,CD81和C1q(如图5A-5K)。
2、IEVs的内容物分析
利用蛋白DIA定量技术完成BMMSCs，MSCs-Exosomes(对比例1提取的)，MSCs-IEVs(实施例2获得的)的蛋白组学定量分析。结果显示，MSCs-Exosomes和MSCs-IEVs的蛋白内容物表达与母细胞具有较高的重叠性，170种蛋白在IEVs中特异性高表达(图6A)。
通过生物信息学分析，筛选IEVs特异性高表达的蛋白，绘制热图(图6B)，进一步结合差异蛋白的GO富集分析结果，明确IEVs能特异性高表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分子，与同种MSCs来源的Exosomes相比，IEVs的5种特征性分子的表达量均显著上调，具体为：IEVs中的 标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4相对于Exosomes中相应标记物的表达量分别为1.76倍、2.81倍、2.41倍、3.68倍和4.45倍(图6C)。最后利用Western Blot技术再次进行验证，结果与DIA定量分析结果相符(图6D)。
MSCs-Exosomes：指的是来源于BMMSCs的外泌体。
MSCs-IEVs：指的是来源于BMMSCs的IEVs。
其中所述的内容物分析中的MSCs和与提取Exosomes和IEVs的MSCs为同一BMMSCs细胞株。
实施例4 MSCs来源的IEVs治疗出血性疾病小鼠的应用和机制研究
(1)IEVs治疗血友病小鼠的应用
利用体外凝血实验检测实施例2获得的IEVs和对比例1提取的Exosomes的体外促凝效果。结果如表3显示，IEVs能显著缩短大部分血浆的体外凝固时间，促凝效果好于Exosomes。但对于因子II,V,X缺乏的血浆，IEVs无法发挥体外促凝作用，说明IEVs的体外促凝作用更多集中于凝血共同途径的上游。
表3
Figure PCTCN2021072938-appb-000003
利用血友病A小鼠(凝血因子VIII缺乏)为模型，通过尾静脉注射9×10 8个IEVs，观察IEVs的体内促凝作用。结果如图7显示，IEVs治疗后能够显著改善血友病小鼠的出血倾向，治疗作用可以稳定地维持14天。
实验结果表明，IEVs能够在体外发挥显著的促凝作用。且体内注射后能够显著改善出血倾向，可用于改善血友病A导致的出血倾向。
同时检测小鼠血浆中各种凝血因子的水平，发现凝血因子VIII、vWF因子、组织因子(tissue factor，TF)和凝血酶原(prothrombin)均没有发生显著变化(图8A，图8B，图8C，图8D)。
在血友病A小鼠模型中，分别注射正常IEVs，PS阴性IEVs和TF阴性IEVs，7天后进行剪尾实验，结果如图9A和图9B显示，PS和TF的封闭并没有影响IEVs的体内治疗效果，初步说明IEVs治疗血友病小鼠的机制与PS和TF无关。以往文献报道中，细胞外囊泡发挥促凝作用都高度依赖于其表面的PS和TF，而IEVs的体内实验结果与以往研究不一致，这提示在体内环境下，IEVs可能有新的作用机制发挥促凝作用。
(2)IEVs治疗狼疮性出血小鼠的应用
在临床上，系统性红斑狼疮(SLE)的病人往往存在出血倾向，目前具体机制不明，现有的文献报道多认为是与SLE伴发的血小板减少等因素有关。在治疗措施上，多采用输血或输血小板的方法，效果不甚理想。
本发明对lpr小鼠进行IEVs(实施例2提取的)注射，7天后进行剪尾实验。结果显示，IEVs治疗后能够显著改善lpr小鼠的出血倾向，治疗作用可以持续稳定地维持7天(图9C)。其中lpr小鼠是SLE的代表性动物模型。
实验结果表明，IEVs可以用于改善红斑狼疮性出血倾向。
(3)IEVs治疗契-东综合征小鼠的应用
契-东综合征(CHS syndrome)是常染色体隐性遗传性疾病，多见于近亲结婚的后代。致病基因为溶酶体运输调节因子基因(LYST)。LYST基因的突变常导致异常LYST蛋白的生成，进而引起血小板功能障碍。患者在临床上会表现为明显的出血倾向，目前没有有效的预防和治疗措施。
本发明对CHS小鼠进行IEVs(实施例2获得的)注射，10天后进行剪尾实验。结果显示，IEVs治疗后能够显著改善CHS小鼠的出血倾向，治疗作用可以持续稳定地维持10天(图9D)。
实验结果表明，IEVs可用于改善契-东综合征小鼠的出血倾向。
对比例2
对血友病A小鼠模型，分别进行同种MSC来源的IEVs(实施例2获得的)和Exosomes(对比例1提取的)的注射治疗(9×10 8个)，结果显示，IEVs能够显著纠正小鼠的出血倾向，而Exosomes没有明显的治疗效果(图10)。
实施例2获得的IEVs与对比例1制备的Exosomes进行体外促凝效果比较：
其中实施例2获得的IEVs直径在0.03μm～0.2μm和0.2μm～1μm范围内的IEVs表达标志物Syntaxin 4、Annexin V、Flotillin-1、Cadherin 11和Integrin alpha 5，体外促凝效果强；对比例1制备的Exosomes直径为0.03μm～0.15μm，表达标记物Complement C1q、Complement C3、Thrombospondin-1和Thrombospondin-2，
体外促凝效果相对较弱。
表4
Figure PCTCN2021072938-appb-000004
实施例5
1、诱导性多能干细胞(induced pluripotent stem cells，iPS细胞，iPSC)细胞培养
(1)慢病毒制备：
将1mL DMEM移入EP管，加入5μg基因表达质粒和5μg vsvg质粒加入25微升的脂质体，轻轻搅拌，室温20分钟。将混合物逐滴加入培养的GP2-293细胞(95％混匀)中，旋转使混合物均匀分布。12小时后更换培养基(DMEM+10％FBS(热灭活)+谷氨酰胺)。更换培养基24小时后，收集含有病毒的培养基，48小时后再次收集培养基。
(2)诱导细胞重编程：
每孔(12孔板)接种步骤(1)中培养的GP2-293细胞5×10 5个，80％汇合在500-1000微升/孔的培养基(DMEM+10％FBS(热灭活)+谷氨酰胺)中加入病毒100ng，加入4微克/毫升聚布伦，孵育12h，换新培养 基，重复此步骤。在7天内，5×10 4个经诱导的细胞接种到带有饲养细胞的10cm培养皿中(mEFs)。第二天，将培养基改为Es培养基，含bFGF(4ng/ml)，每隔一天换一次，5天后，细胞开始出现克隆，如果40天后没有Es样克隆，则认为失败。
(3)细胞传代：
60％汇合后，每皿添加0.5毫升的accutase，在室温下静置1分钟。将分离的细胞聚集物转移到15mL离心管中，并用另外的2mLmTeSR1以收集任何剩余的聚集物。将冲洗液加入15毫升试管中。在室温下200g离心含有细胞聚集物的15mL试管5分钟。吸出上清液。使细胞再悬浮并确保细胞保持聚集状态。将人iPS细胞与mTeSR1聚集在涂有基质凝胶的新板上。将培养皿放入37℃培养箱中，快速左右移动培养皿使细胞聚集物均匀分布团块的运动。在37℃下用5％的二氧化碳和95％湿度培养。每天换液。
2、MC3T3-E1Subclone14成骨细胞系培养
将购买来的MC3T3-E1Subclone14，快速解冻后500g离心5min，去除上清液后收集底部的细胞沉淀，以Dex(-)培养液重悬细胞并接种于10cm直径细胞培养皿，37℃、5％CO 2培养。长满后采用胰蛋白酶37℃孵育消化并传代扩增，之后每3日换Dex(-)培养液，可多次传代使用细胞。其中，Dex(-)培养液成分如表5所示：
表5 Dex(-)培养液成分表
Figure PCTCN2021072938-appb-000005
3、IEVs的分析
其中包括成骨细胞MC3T3-E1和iPS细胞以及人骨髓间充质干细胞(hBMMSC)来源的IEVs的比较。所述的三种细胞来源的IEVs的获得方法同实施例2。
(1)形态检测
实验结果如图11所示，400倍光镜下，iPS细胞(iPSC)、成骨细胞MC3T3-E1来源的IEVs与人BMMSC来源的IEVs的形态相似，较不规则。
(2)IEVs颗粒的直径检测
用流式进行检测，实验结果如图12所示，流式检测结果显示，成骨细胞系MC3T3-E1、人骨髓间充质干细胞hBMMSC来源的IEVs颗粒直径分布相似。
(3)IEVs的表面标记物检测
用Western Blot的方法进行检测，实验结果如图13所示，iPS细胞(iPSC)、人骨髓间充质干细胞(hBMMSC)来源的IEVs的表面标记物进行比较，两种细胞来源的IEVs中均高表达IEVs标记物Anenexin V。相对于hBMMSC而言，iPS细胞来源的IEVs表达较高水平的Syntaxin4。
实施例6 IEVs可经皮肤和毛发排出
取4×10 6的实施例2制备的IEVs用DIR标记，200微升PBS重悬，通过尾静脉系统性注射于裸鼠 BALB/c-nu/nu体内，观察1，3，7天后用活体成像仪器检测IEVs在皮肤表面的分布，结果如图13A-13C所示。
图13A显示IEVs可到达皮肤表面，在第3天时数量最多，第7天基本消失，显示IEVs在皮肤表面的动态代谢过程(图13A)。免疫荧光结果显示PKH26-IEV系统性注射C57小鼠后，随着时间推移，逐渐从皮下组织向真皮层和表皮移动。第7天在皮肤表面角质层观测到IEVs大量存在，提示系统性注射的IEVs可以随着皮肤角质层的脱落而排泄出去(图13B)。同时，第7天时在小鼠体表拔下的毛发中发现毛囊中存在PKH26-IEV，说明系统性注射的IEVs还可随着毛发的脱落而代谢出去(图13C)。本实施例表明IEVs可以通过皮肤和毛发排出，说明注射或增加IEVs在体内的含量具有安全性。
实施例7
hiPSCs的培养同实施例5，hUCMSCs也为本领域常规的培养方法。
所述的hiPSCs可以为第26-29代，但又不限于此，本实施例具体使用的为第26代；所述的hUCMSCs可以为第7代-9代，但又不限于此，本实施例具体使用时为第7代。
1、实验方法
(1)使用星形环孢菌素(500nM)诱导hiPSCs和hUCMSCs约9h使细胞发生凋亡(其余步骤同实施例2)，染AnnexinV(15mins)和7AAD(3mins)，流式检测细胞凋亡率。
(2)从步骤(1)的凋亡细胞的上清中分离IEVs并用流式检测AnnexinV表达率。使用差数离心法提取IEVs，步骤包括：800g离心10mins—2000g离心5mins(除了这步，其余提取步骤与实施例2相同)—16000g离心30mins—16000g离心30mins获取IEVs。
AnnexinV染色15mins，流式上机。
2、实验结果
(1)我们使用高内涵拍摄了hiPSCs和hUCMSCs死亡过程，发现两者死亡过程存在差异，hiPSCs以核和质多个中心进行收缩，继而发出树枝状分支伴随出泡；而hUCMSCs是以核为中心的单中心收缩，同时伴随发出分支和出泡等活动。结果如图14所示。
(2)对诱导凋亡的hiPSCs和hUCMSCs使用流式分析凋亡率，证明绝大部分细胞发生了凋亡。结果如图15所示。
(3)使用流式分析Annexin5表达的阳性率hiPSCs和hUCMSCs两者表达均在80％以上。结果如图16所示。
(4)使用Nanoparticle Tracking Analysis(NTA)对两种IEVs进行表征：
1)结果如图17所示，来源于hiPSCs的IEVs的粒径约100nm，来源于hUCMSCs的IEVs的粒径约180nm；
2)结果如图18所示，来源于hiPSCs的IEVs的产量为21971particles/hiPSCs，来源于hUCMSCs的产量为886particles/hUCMSCs；
3)结果如图19所示，来源于hiPSC的IEVs的电位约-12mV,来源于hUCMSC的IEVs电位约-45mV。

Claims (13)

  1. 一种囊泡，其特征在于，所述囊泡是诱导性囊泡，所述囊泡的细胞来源包括干细胞或体细胞；所述的囊泡具有的标志物包括Syntaxin 4。
  2. 如权利要求1所述的囊泡，其特征在于，所述体细胞包括原代培养的细胞或细胞系；
    优选地，所述的体细胞包括成骨细胞系；更为优选地，所述细胞系指的是是指永生化的细胞培养物，在适当的新鲜培养基和空间下可以无限增殖；
    优选地，所述干细胞包括全能干细胞和多能干细胞；
    优选地，所述干细胞包括间充质干细胞和诱导性多能干细胞；
    优选地，所述诱导性囊泡是在所述干细胞或体细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡；
    优选地，所述诱导性囊泡是通过添加星形孢菌、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合诱导干细胞或干细胞凋亡产生；
    优选地，所述囊泡具有的标志物还包括Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5中的一种或几种；
    进一步优选地，所述囊泡具有标志物Syntaxin 4、Annexin V、Flotillin-1、Cadherin 11和Integrin alpha 5的组合；
    进一步优选地，所述囊泡高表达标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4；
    或优选地，所述囊泡对标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量高于MSC或外泌体；
    或优选地，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别为1-2倍、2-3倍、1-3倍、3-4倍和2-6倍；
    更为优选地，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别为1.5-2倍、2.5-3倍、1.5-2.5倍、3.5-4倍和3.5-5倍；
    更进一步优选地，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别为1.5-1.9倍、2.5-2.9倍、1.8-2.5倍、3.5-3.9倍和4-5倍；
    更进一步优选地，所述囊泡和所述外泌体来源于同种来源的MSCs；
    或优选地，所述囊泡还表达CD29、CD44、CD73、CD166；且不表达CD34、CD45；
    或优选地，所述囊泡还表达CD9、CD63、CD81和C1q中的一个或多个；
    或优选地，所述的囊泡是用星形菌素诱导间充质干细胞产生的；
    优选地，所述星形孢菌素的浓度约1nM-10000nM；
    优选地，所述星形孢菌素的浓度约100nM-10000nM；
    优选地，所述星形孢菌素的浓度约500nM-10000nM；
    优选地，所述星形孢菌素的浓度约500-1000nM；
    进一步优选地，所述星形孢菌素的浓度约500-900nM；
    进一步优选地，所述星形孢菌素的浓度约500-800nM。
  3. 如权利要求1-2所述的囊泡，其特征在于，所述囊泡的直径约0.03-6μM；
    优选地，所述囊泡的直径约0.03-4.5μM；
    进一步优选地，所述囊泡的直径约0.03-1μM；
    进一步优选地，所述囊泡的直径约0.04-1μM；
    进一步优选地，所述囊泡的直径约0.05-1μM；
    进一步优选地，所述囊泡的直径约0.1-1μM；
    进一步优选地，所述囊泡的直径约0.15-1μM。
  4. 一种囊泡组合，其特征在于，包含权利要求1-3任一所述的囊泡；
    优选地，权利要求1-3任一所述的囊泡在所述囊泡组合中的数目占比约65-100％；
    进一步优选地，权利要求1-3任一所述的囊泡在所述囊泡组合中的数目占比约75-98％；更为优选地，权利要求1-3任一所述的囊泡在所述囊泡组合物中的数目占比约80-96％；
    或优选地，所述囊泡组合还包含外泌体、迁移体、微泡和Ectosome中的一种或几种。
  5. 一种组合物，其特征在于，包含权利要求1-3任一所述的囊泡；或者包含权利要求4所述囊泡组合；
    优选地，所述组合物包括药品、食品、保健品、化妆品、添加剂或中间品；
    优选地，所述组合物为药品；
    优选地，所述组合物还包含药学上或免疫学上可接受的载体；
    更为优选地，所述组合物的制剂形式选自冻干粉针、注射剂、片剂、胶囊、试剂盒或贴剂。
  6. 一种囊泡的筛选或者鉴定或者提取试剂或试剂盒，其特征在于，包含标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4中的一种或多种的检测试剂；
    优选地，所述检测试剂检测所述标志物基因的表达量；
    进一步优选地，所述检测试剂检测所述标志物mRNA的表达量；
    更为优选地，所述标志物的检测试剂检测所述标志物蛋白的表达量；
    或优选地，所述标志物的检测试剂为荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针、抗体、抗体功能性片段和偶联抗体中的一种或多种；
    或优选地，所述试剂盒选自qPCR试剂盒、免疫印迹检测试剂盒、流式细胞分析试剂盒、免疫组化检测试剂盒和ELISA试剂盒中的一种或多种；
    更为优选地，所述试剂盒选自流式细胞分析试剂盒；
    优选地，所述囊泡为诱导性囊泡。
  7. 一种选择或鉴定权利要求1-3任一所述囊泡的方法，其特征在于，所述方法包括以Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4中的一种或多种为标志物利用检测试剂对待测样品进行检测，
    优选地，所述方法中使用了对照试剂，所述对照试剂包含外泌体、迁移体、微泡和Ectosome中的一种或几种，当所述待测样品中的标志物的表达量高于对照试剂时，则判断为阳性；
    优选地，所述对照试剂包含外泌体；
    优选地，所述标志物为Syntaxin 4；
    优选地，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的2-6倍时，则判断为所述囊泡；更优选地，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的3-6倍时、更优选为4-5倍时，则判断为所述囊泡。
  8. 标志物的检测试剂在制备检测或鉴定权利要求1-3任一所述囊泡的试剂或试剂盒中的应用，其特征在于，所述标志物包括Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4中的一种或多种，所述试剂或试剂盒还包括对照试剂，所述对照试剂包含外泌体、迁移体、微泡和Ectosome中的一种或几种，当所述待测样品中的所述标志物的表达量高于对照试剂时，则判断为阳性；
    优选地，所述对照试剂为外泌体；
    优选地，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的2-6倍时，则判断为所述囊泡；
    更优选地，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的3-6倍时，则判断为所述囊泡；
    更优选地，当所述待测样品中的Syntaxin 4表达量大于或等于外泌体的4-5倍时，则判断为所述囊泡。
  9. 权利要求1-3任一所述囊泡或权利要求4所述的囊泡组合在制备治疗或预防或改善疾病或所述疾病并发症的产品中的应用；所述疾病为出血性疾病；
    优选地，所述出血性疾病包括凝血因子匮乏、血小板数量减少和/或功能缺陷导致的出血；
    更为优选地，所述出血性疾病包括血友病、狼疮性出血或契-东综合征；
    更为优选地，所述血友病包括血友病A、血友病B或血友病C；
    更为优选地，所述疾病为血友病A；
    或优选地，所述产品包括药品、食品、保健品、化妆品、添加剂或中间品。
  10. 一种制备权利要求1-3任一所述囊泡的方法，其特征在于，所述方法为通过添加凋亡诱导剂诱导干细胞或体细胞产生所述囊泡；
    优选地，所述方法包括以下步骤：
    (1)培养间充质干细胞；
    (2)收集间充质干细胞的培养基上清；
    (3)从步骤(2)中的培养基上清中分离出囊泡；
    优选地，步骤(1)中的培养间充质干细胞的步骤包括：
    (4)从组织中分离间充质干细胞；
    (5)添加培养基培养间充质干细胞；所述间充质干细胞的培养基中接触凋亡诱导剂；
    进一步优选地，所述凋亡诱导剂包括星形孢菌素、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合；
    更为优选地，所述凋亡诱导剂为星形孢菌素；
    优选地，所述星形孢菌素的浓度约1-10000nM；
    优选地，所述星形孢菌素的浓度约100-10000nM；
    优选地，所述星形孢菌素的浓度约500-10000nM；
    或更为优选地，所述星形孢菌素的浓度约500-1000nM；
    更进一步优选地，所述星形孢菌素的浓度约500-900nM；
    最为优选地，所述星形孢菌素的浓度约500-800nM；
    进一步优选地，步骤(5)中凋亡诱导剂的处理细胞的时间约16-24小时；
    或优选地，所述步骤(3)中，分离囊泡的方法包括选用超速离心的方法分离所述囊泡；
    进一步优选地，所述超速离心的方法分离所述囊泡的步骤包括：
    (a)将收集到的培养上清进行第一次离心，取上清；
    (b)将步骤(a)中收集到的上清进行第二次离心，取上清；
    (c)将步骤(b)中收到到的上清进行第三次离心，取沉淀；
    (d)将步骤(c)中收到到的沉淀进行第四次离心，取沉淀；
    进一步优选地，所述第一次离心约为500-1500g离心约5-30分钟；
    更为优选地，所述第一次离心约为500-1000g离心约5-20分钟；
    更进一步优选地，所述第一次离心约为500-900g离心约5-15分钟；
    进一步优选地，所述第二次离心约为1000-3000g离心约5-30分钟；
    更为优选地，所述第二次离心约为1500-2500g离心约5-20分钟；
    更进一步优选地，所述第二次离心约为1500-2200g离心约5-15分钟；
    进一步优选地，所述第三次离心约为10000-30000g离心约15-60分钟；
    更为优选地，所述第三次离心为约12000-25000g离心约20-60分钟；
    更进一步优选地，所述第三次离心约为12000-20000g离心约20-40分钟；
    进一步优选地，所述第四次离心约为10000-30000g离心约15-60分钟；
    更为优选地，所述第四次离心约为12000-25000g离心约20-60分钟；
    更进一步优选地，所述第四次离心约为12000-20000g离心约20-40分钟。
  11. 一种权利要求1-3所述囊泡的富集方法，其特征在于，使用免疫磁珠的方法对所述囊泡进行富集，所述免疫磁珠是由抗体与磁珠偶联得到的；所述抗体包括Anti-Syntaxin 4抗体；
    优选地，所述抗体还包括Anti-Annexin V抗体、Anti-Flotillin-1抗体、Anti-Cadherin 11抗体、Anti-Integrin alpha 5抗体中的一种或多种。
  12. 如权利要求1-3所述的囊泡或权利要求4所述的囊泡组合或权利要求5所述的组合物或权利要求6所述的提取试剂或提取试剂盒或权利要求8或9所述的应用或权利要求10或11所述的方法，其特征在于，所述间充质干细胞来源于哺乳动物；
    优选地，所述哺乳动物选自人或鼠；
    或优选地，所述间充质干细胞来源包括：骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或其组合；
    更为优选地，所述间充质干细胞来源选自骨髓、脂肪、脐带和口腔中的一种或多种。
  13. 一种治疗或预防或改善受试者的疾病或所述疾病并发症的方法，包括给予所述受试者有效量的权利要求1-3所述的囊泡或权利要求4所述的囊泡组合或权利要求5所述的组合物；所述疾病为出血性疾病；
    优选地，所述出血性疾病包括凝血因子匮乏、血小板数量减少和/或功能缺陷导致的出血；
    更为优选地，所述出血性疾病包括血友病、狼疮性出血或契-东综合征；
    更为优选地，所述血友病包括血友病A、血友病B或血友病C；
    更为优选地，所述疾病为血友病A。
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