CN117965431A - Vesicle and application thereof - Google Patents

Vesicle and application thereof Download PDF

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Publication number
CN117965431A
CN117965431A CN202410165336.7A CN202410165336A CN117965431A CN 117965431 A CN117965431 A CN 117965431A CN 202410165336 A CN202410165336 A CN 202410165336A CN 117965431 A CN117965431 A CN 117965431A
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vesicle
vesicles
centrifugation
stem cells
syntaxin
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张晓�
寇晓星
施松涛
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Medical Micro Cell Biotechnology Guangzhou Co ltd
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Medical Micro Cell Biotechnology Guangzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Abstract

The invention belongs to the field of biological medicine, and relates to a vesicle and application thereof. The source of the vesicles comprises stem cells or somatic cells, the markers comprise Syntaxin 4, and the vesicles are inducible vesicles. Compared with Exosomes in MSCs, the vesicles of the invention can specifically and highly express Syntaxin 4, and can be used for distinguishing vesicles from MSCs and characteristic markers of Exosomes. The method for preparing the vesicle has the advantages of rich yield, simple preparation process, short time consumption, low requirements on reagents and equipment and good treatment effect, and 300-2000 vesicles can be produced by single MSCs. The vesicle can play a remarkable coagulation promoting role in vitro, can remarkably improve the bleeding tendency of hemophilia mice after in vivo injection, and can be used for treating the bleeding tendency of hemophilia. And the vesicles can be discharged through skin and hair, and have safety in vivo, so that the vesicles have good application prospect.

Description

Vesicle and application thereof
The application relates to a Chinese patent application with the application number 202110077486.9, priority date 2020-01-20, application date 2021-01-20 and the name of 'a vesicle and application thereof'.
Technical Field
The invention belongs to the field of biological medicine, and relates to a vesicle and application thereof.
Background
Extracellular vesicles (extracellular vesicles, EVs) are nanoscale vectors containing proteins, nucleic acids, and various cytokines secreted by cells. Extracellular vesicles can act on target cells by endocrine or paracrine means, playing an important role in intercellular mass transfer and information communication. The research shows that the information communication mediated by the extracellular vesicles plays an important role in the physiological or pathological process of the organism, and relates to immunoregulation, tumor growth, angiogenesis, injury repair and the like. Research in this field is currently focused mainly on exosome (exosomes) orientation. The exosomes are extracellular vesicles with diameters of about 30-150nm, and contain RNA, lipid, protein and other components. Exosomes are widely involved in various physiological/pathological regulation of the body, and can be used for diagnosis, treatment and prognosis evaluation of various diseases. To date, mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) are considered to be the most potent cells to produce exosomes. Numerous researches find that the exosomes derived from MSCs can simulate the biological functions of MSCs, and play an important role in promoting cell growth and differentiation, repairing tissue defects and the like. Therefore, cell vesicle therapies based on exosomes derived from MSCs have been significantly developed in recent years. However, there are still many problems in the current exosome-based cell vesicle therapy, mainly represented by complicated extraction and purification processes of exosomes, long time consumption, high requirements on equipment and reagents, low physiological exosome yield, etc., and these defects limit the clinical transformation and application of exosome therapy.
Hemophilia (hemophilia) is a group of hemorrhagic diseases with hereditary coagulation dysfunction, which is commonly characterized by an active thromboplastin disorder, prolonged clotting time, a tendency to bleed after minor trauma throughout the life, and "spontaneous" bleeding without significant trauma in critically ill patients. The first few rare diseases catalog, in which hemophilia is recorded, was jointly formulated by 5 departments such as the national health committee, 5 months, 11 days 2018. Hemophilia is largely divided into three categories, namely hemophilia a, hemophilia B and hemophilia C. Hemophilia a, a deficiency in the procoagulant component of factor viii (viii: C), is a sexually linked recessive genetic disorder, transmitted by females, and occurs in males. Hemophilia B, factor Ix (FIX) deficiency, is also a sexually linked recessive inheritance, with a smaller number of episodes than hemophilia a. Hemophilia C, factor xi (fci) deficiency, is an autosomal incomplete recessive inheritance, a rare hemophilia. The incidence rate is 80% -85% of hemophilia A at most, 15% -20% of hemophilia B and less common of hemophilia C. For a long time, aiming at the treatment of hemophilia, the injection of exogenous coagulation factors is clinically used as a main intervention measure, but the method has various problems of high treatment cost, short treatment effective period, easy generation of autoantibodies and the like, and cannot be a practical and effective treatment means.
Disclosure of Invention
In some embodiments, the invention provides a vesicle derived from a mesenchymal stem cell.
In some embodiments, the invention provides a vesicle composition.
In some embodiments, the invention provides a pharmaceutical composition comprising vesicles for hemophilia.
In some embodiments, the invention provides a kit for screening or identifying or extracting vesicles.
In some embodiments, the invention provides a marker for a vesicle.
In some embodiments, the invention provides a method for identifying or selecting vesicles using a marker.
In some embodiments, the invention provides a method of preparing a vesicle.
In some embodiments, the invention provides a vesicle derived from a somatic cell or stem cell, the vesicle being an inducible vesicle, the vesicle having a marker comprising Syntaxin 4.
In some embodiments, 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).
In some embodiments, the somatic cells comprise an osteoblast cell line.
In some embodiments, the cells may be primary cultured cells, or may be existing or established cell lines.
In some embodiments, the cell line refers to an immortalized cell culture that can proliferate indefinitely in the appropriate fresh medium and space.
In some embodiments, the cell may be an established cell line.
In some embodiments, the inducible vesicle is a vesicle that results from induction of apoptosis by external forces during normal survival of the stem cell or somatic cell.
In some embodiments, the inducible vesicle is produced by inducing stem cells or stem cell apoptosis by addition of staurosporine, ultraviolet irradiation, starvation, or thermal stress, or a combination of one or more thereof.
In some embodiments, the vesicles have markers that further include one or more of Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA.
In some embodiments, the vesicle has a combination of markers Syntaxin 4, annexin V, flotillin-1, cadherin 11, and INTEGRIN ALPHA.
In some embodiments, the vesicles highly express markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and Syntaxin 4.
In some embodiments, the vesicles have higher expression levels of markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and Syntaxin 4 than MSC or exosomes.
In some embodiments, the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and Syntaxin 4 are expressed in 1-2 fold, 2-3 fold, 1-3 fold, 3-4 fold, and 3-6 fold, respectively, relative to the expression of markers in exosomes derived from mesenchymal stem cells.
In some embodiments, the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and Syntaxin 4 are expressed in amounts of 1.5-2 fold, 2.5-3 fold, 1.5-2.5 fold, 3.5-4 fold, and 3.5-5 fold, respectively, relative to the expression of the markers in exosomes derived from mesenchymal stem cells.
In some embodiments, the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and Syntaxin 4 are expressed in amounts of 1.5-1.9 fold, 2.5-2.9 fold, 1.8-2.5 fold, 3.5-3.9 fold, and 4-5 fold, respectively, relative to the expression of the markers in exosomes derived from mesenchymal stem cells.
In some embodiments, the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and syncaxin 4 are expressed in 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold, and 4.45-fold relative to the expression of markers in exosomes derived from mesenchymal stem cells, respectively.
In some embodiments, the exosomes do not express Syntaxin 4, and the vesicles of the invention express Syntaxin 4.
In some embodiments, the exosomes do not express Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and syncaxin 4 simultaneously, and the vesicles of the invention express Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA, and syncaxin 4 simultaneously.
In some embodiments, the vesicles and the exosomes are derived from MSCs of homogeneous origin.
In some embodiments, analysis of IEVs's surface membrane protein using flow cytometry showed that MSCs-derived IEVs was capable of expressing surface proteins similar to MSCs, i.e., CD29, CD44, CD73, CD166 positive, CD34, CD45 negative; at the same time IEVs is able to express the general surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles.
In some embodiments, the inducible vesicle is produced by inducing apoptosis of mesenchymal stem cells by addition of staurosporine, ultraviolet irradiation, starvation, thermal stress, or a combination thereof.
In some embodiments, the vesicles are produced by induction of mesenchymal stem cells with staurosporine.
In some embodiments, the mesenchymal stem cells may have an algebra of 2 th to 5 th generations, but are not limited thereto.
In some embodiments, the concentration of staurosporine is 1nM to 10000nM. In some embodiments, the concentration of staurosporine is 100nM to 10000nM. In some embodiments, the concentration of staurosporine is 500nM to 10000nM. In some embodiments, the concentration of staurosporine is 500-1000nM. In some embodiments, the concentration of staurosporine is 500-900nM. In some embodiments, the concentration of staurosporine is 500-800nM.
In some embodiments, the vesicles have a diameter of 0.03 to 6. Mu.M. In some embodiments, the vesicles have a diameter of 0.03 to 4.5. Mu.M. In some embodiments, the vesicles have a diameter of 0.03 to 1. Mu.M. In some embodiments, the vesicles have a diameter of 0.04 to 1. Mu.M. In some embodiments, the vesicles have a diameter of 0.05 to 1. Mu.M. In some embodiments, the vesicles have a diameter of 0.1 to 1. Mu.M. In some embodiments, the vesicles have a diameter of 0.15 to 1. Mu.M.
In some embodiments, the invention also provides a vesicle combination comprising the above vesicles.
In some embodiments, the vesicle combinations further comprise other prior art vesicles, including, but not limited to, e.g., exosomes, migrates, microbubbles, and Ectosome.
In some embodiments, wherein the number of vesicles in the vesicle composition is 65-100%.
In some embodiments, the number of vesicles in the vesicle composition is 75-98%.
In some embodiments, the number of vesicles in the vesicle composition is 80-96%.
In some embodiments, the invention also provides a composition comprising the above vesicles or a combination of the above vesicles.
In some embodiments, the composition comprises a pharmaceutical, food, nutraceutical, cosmetic, additive, or intermediate.
In some embodiments, the composition is a pharmaceutical product.
In some embodiments, the composition further comprises a pharmaceutically or immunologically acceptable carrier.
In some embodiments, the formulation of the composition is selected from the group consisting of a lyophilized powder injection, an injection, a tablet, a capsule, a kit, or a patch.
In some embodiments, the vesicle is used as a pharmaceutical carrier.
In some embodiments, the invention further provides a screening or identification or extraction reagent or kit for said vesicles comprising one or more of the following marker detection reagents: detection reagents for markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4.
In some embodiments, the detection reagent for the marker detects the expression level of the marker gene.
In some embodiments, the detection reagent for the marker detects the amount of expression of the marker mRNA.
In some embodiments, the detection reagent for the marker detects the amount of expression of the marker protein.
In some embodiments, the detection reagent for the marker is one or more of a fluorescent quantitative PCR dye, a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody functional fragment, and a conjugated antibody.
In some embodiments, the kit is selected from one or more of a qPCR kit, an immunoblot detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, and an ELISA kit.
In some embodiments, the kit is selected from the group consisting of flow cytometric assay kits.
In some embodiments, the invention also provides the use of said vesicle or said vesicle composition or said pharmaceutical composition for the preparation of a product for the treatment or prevention or amelioration of a disease or a complication of said disease; the diseases include liver disease and hemophilia.
In some embodiments, the disease is hemophilia, the vesicle can play a remarkable coagulation promoting role in vitro, can remarkably improve bleeding tendency of hemophilia mice after in vivo injection, can be used for treatment for improving bleeding tendency of hemophilia, and has good application prospect.
In some embodiments, the disease is hemophilia a.
In some embodiments, the product comprises a pharmaceutical product, a food product, a nutraceutical product, a cosmetic product, an additive or an intermediate product.
The vesicles may optionally be administered by a route selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intra-organ infusion during use of the vesicles in the treatment of disease. For example, for intravenous injection, tail vein injection may be used as an example. Intra-organ infusion includes infusion into anatomical spaces such as, for example, the gallbladder, gastrointestinal lumen, esophagus, pulmonary system (by inhalation), and/or bladder.
As an example, for abdominal cavity injection in gastrointestinal cavity infusion, intraperitoneal injection can also achieve the same therapeutic effect as compared to tail vein injection. The safety and operability of intraperitoneal injection are superior to that of tail vein injection.
In some embodiments, the invention also provides a method of 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 and Syntaxin 4.
And when the detection result shows the positive result of the marker, judging the vesicle.
In some embodiments, the expression result of the marker may be compared to a control, and a positive result may be determined when the expression level is significantly higher than the control. The control may be an existing other vesicle or exosome (which may include one or more of exosomes, migratory bodies, microbubbles, and Ectosome); other vesicles or exosomes derived from mesenchymal stem cells are possible.
Among the markers mentioned, the marker Syntaxin is particularly preferred. In some embodiments, a vesicle (e.g., an inducible vesicle) is determined when the measured amount of Syntaxin 4 expression of the vesicle is greater than or equal to 2-6 times (more preferably 4-5 times) the amount of an exosome (e.g., an exosome of allogeneic origin).
In some embodiments, the invention provides the use of a detection reagent for the marker in the preparation of a reagent or kit for detecting or identifying the vesicle, wherein the marker comprises one or more of Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and syncaxin 4, and the reagent or kit further comprises a control reagent comprising one or more of exosomes, migrates, microbubbles and Ectosome, and is determined to be positive when the expression level of the marker in the sample to be tested is higher than the control reagent.
In some embodiments, the control agent is an exosome.
In some embodiments, the vesicle is determined when the amount of expression of Syntaxin 4 in the test sample is greater than or equal to 2-6 fold of exosomes.
In some embodiments, the vesicle (e.g., an inducible vesicle) is determined when the amount of Syntaxin 4 expression in the test sample is greater than or equal to 4-5 fold that of the exosome.
In some embodiments, the invention provides a method of preparing the vesicle, comprising the step of inducing stem cells or somatic cells to produce the vesicle by adding an apoptosis-inducing agent.
In some embodiments, the method comprises the steps of: (1) culturing mesenchymal stem cells; (2) collecting a culture medium supernatant of the mesenchymal stem cells; (3) Separating vesicles from the culture supernatant in step (2).
In some embodiments, the step of culturing the mesenchymal stem cells in step (1) comprises: (4) isolating mesenchymal stem cells from the tissue; (5) adding a culture medium to culture the mesenchymal stem cells; the culture medium of the mesenchymal stem cells is contacted with an apoptosis inducer.
In some embodiments, the apoptosis-inducing agent comprises staurosporine, ultraviolet radiation, starvation, or thermal stress, or a combination of one or more thereof.
In some embodiments, the apoptosis-inducing agent is staurosporine.
In some embodiments, the concentration of staurosporine is 500-1000nM. In some embodiments, the concentration of staurosporine is 500-900nM. In some embodiments, the concentration of staurosporine is 500-800nM.
In some embodiments, the time for treating the cells with the apoptosis-inducing agent in step (5) is 16-24 hours.
In some embodiments, in step (3), the method of isolating vesicles comprises isolating the vesicles by an ultracentrifugation method.
In some embodiments, a single MSCs in the present disclosure is capable of producing 300-2000 vesicles.
In some embodiments, the step of separating the vesicles by the ultracentrifugation method comprises: (a) Centrifuging the collected culture supernatant for the first time, and taking the supernatant; (b) Subjecting the supernatant collected in step (a) to a second centrifugation to obtain a supernatant; (c) Centrifuging the supernatant received in step (b) for a third time to obtain a precipitate; (d) Centrifuging the precipitate received in step (c) for the fourth time, and taking the precipitate; (e) Centrifuging the precipitate received in the step (d) for the fifth time, and taking the precipitate;
In some embodiments, the first centrifugation is 500-1500g centrifugation for 5-30 minutes. In some embodiments, the first centrifugation is 500-1000g centrifugation for 5-20 minutes. In some embodiments, the first centrifugation is 500-900g centrifugation for 5-15 minutes. In some embodiments, the second centrifugation is from 1000 to 3000g centrifugation for 5 to 30 minutes. In some embodiments, the second centrifugation is from 1500 to 2500g centrifugation for 5 to 20 minutes. In some embodiments, the second centrifugation is from 1500 to 2200g centrifugation for 5 to 15 minutes. In some embodiments, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes. In some embodiments, the third centrifugation is 12000-25000g centrifugation for 20-60 minutes. In some embodiments, the third centrifugation is 12000-20000g centrifugation for 20-40 minutes. In some embodiments, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes. In some embodiments, the fourth centrifugation is from 12000 g to 25000g for 20 to 60 minutes. In some embodiments, the fourth centrifugation is from 12000 to 20000g centrifugation for 20 to 40 minutes.
In some embodiments, vesicles with a particular label can be enriched by an enrichment method for the particular label. After sufficient vesicles are obtained, the medium is collected and the specific vesicles are purified and isolated from the medium. This may be accomplished by any suitable method known in the art. These 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), and the like.
Immunoaffinity purification is a method based on selective capture of specific vesicles by surface markers. High-efficiency capturing of vesicles is achieved by high-affinity coupling between streptavidin covalently coated magnetic beads and biotinylated capture antibodies. The captured vesicles are eluted and are structurally complete and biologically active. Based on the findings of the present invention, the vesicles are capable of specifically and highly expressing Annexin V, flotillin-1,Cadherin 11,Integrin alpha 5 and Syntaxin 4 molecules, and then the present invention can use the method to isolate, purify or enrich vesicles.
In some embodiments, the vesicles may also be enriched using immunomagnetic beads, which are derived from monoclonal antibodies coupled to magnetic beads; the monoclonal antibody comprises one or more of an Anti-Syntaxin 4 antibody, an Anti-Annexin V antibody, an Anti-Flotillin-1 antibody, an Anti-Cadherin 11 antibody and an Anti-INTEGRIN ALPHA antibody.
When the term "enriched" is used in the present application, it encompasses the isolation of one or more vesicles from any other vesicles present in the sample or it means that the vesicles are present in a composition comprising vesicles in a higher total percentage content than when found in the tissue of an organism.
In one embodiment, the enriched vesicles are not isolated from the sample, but rather any diagnosis of the vesicles is made while they are still present in the sample. The sample may then be presented on a slide and may be diagnosed using a microscope, and in this embodiment vesicles detected without isolation.
In some embodiments, the invention also provides an inducible vesicle derived from an IPS cell. It is a class of subcellular products that intervene or induce apoptosis of IPS cells when they are normally viable.
In another embodiment, the enriched vesicles are isolated from the sample.
Among them, immunomagnetic bead separation technology (Immunomagneticbead-basedseparation, IMS) is a new immunological technology developed in recent years. The immune magnetic beads (Immunomagneticbead, IMB) can be combined with active protein antibody and attracted by magnet, after treatment, the antibody can be combined on the magnetic beads to make them become antibody carrier, after the antibody on the magnetic beads is combined with specific antigen substance, antigen-antibody-magnetic bead immune complex is formed, and said complex can be mechanically moved under the action of magnetic force so as to make the complex be separated from other substances so as to attain the goal of separating specific antigen. Immune Magnetic Beads (IMB) are a platform, can be applied to all fields working by utilizing the antigen-antibody combination principle, and have achieved remarkable results in aspects of medical and biological bone marrow transplantation, stem cell separation, organelles, cancer cells, hormones, pathogenic bacteria, toxins and the like. In recent years, IMB is widely applied to the separation and detection of mycotoxins in food, water, biological samples, environmental samples and other specimens by virtue of high sensitivity and specificity, and has good development and application prospects.
The immune magnetic bead method of the invention uses magnetic beads combined by specific antibodies to combine with target vesicles with specific surface antigens, and then uses magnetic fields to adsorb the target vesicles.
In certain embodiments of the invention, the vesicle specific enrichment method is that one or more of an Anti-Syntaxin 4 antibody, an Anti-Syntaxin V antibody, an Anti-Flotillin-1 antibody, an Anti-Cadherin 11 antibody, an Anti-INTEGRIN ALPHA antibody coated with an immunomagnetic bead is added to a vesicle-containing cell culture supernatant, and then the vesicle specifically bound to one or more of the Anti-Syntaxin 4 antibody, the Anti-Syntaxin V antibody, the Anti-Flotillin-1 antibody, the Anti-Cadherin 11 antibody, and the Anti-INTEGRIN ALPHA antibody is separated, thereby achieving the purpose of enriching the specific vesicle. In some preferred embodiments, the vesicles isolated from the immunomagnetic beads coated simultaneously with Anti-Syntaxin 4 antibody, anti-Annexin V antibody, anti-Flotillin-1 antibody, anti-Cadherin 11 antibody and Anti-INTEGRIN ALPHA antibody are the most pure and most effective in the treatment of diseases such as hemophilia A.
In some preferred embodiments, the cell culture supernatant after effectively removing impurities such as cells and cell fragments is combined and optimized based on a centrifugation method, and then one or more of an Anti-syntenin 4 antibody, an Anti-Annexin V antibody, an Anti-Flotillin-1 antibody, an Anti-Cadherin 11 antibody and an Anti-INTEGRIN ALPHA antibody coated immunomagnetic beads are added to the cell culture supernatant, so that vesicles capable of specifically binding to the antibodies can be separated, and the purpose of enriching specific vesicles can be achieved.
In some embodiments, the mesenchymal stem cells are derived from human or mouse, but are not limited thereto.
In some embodiments, the mesenchymal stem cells include bone marrow-derived mesenchymal cells, urine-derived mesenchymal stem cells, oral cavity-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, periosteum-derived stem cells, or a combination thereof, but are not limited thereto.
In some embodiments, the mesenchymal stem cells are selected from bone marrow-derived mesenchymal cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and oral cavity-derived mesenchymal stem cells.
Drawings
FIGS. 1A-1E are flow-through assays of isolated BMMSCs surface markers.
Fig. 2 is a flowchart of the operation of embodiment 2.
FIG. 3 shows the statistics of IEVs numbers generated by MSCs (10 6 MSCs) analyzed by flow cytometry.
FIGS. 4A-4F show IEVs particle diameter measurements: FIG. 4A is a graph of particle diameter distribution for flow test IEVs; FIG. 4B is a Side Scatter (SSC) analysis IEVs of the scattered light intensity, showing IEVs particle diameter distribution; FIG. 4C is a graph showing the distribution of particle diameters of IEVs for the scattered light intensity of a standardized small particle microsphere assay IEVs manufactured by Bangs Laboratories; FIG. 4D is a view IEVs of a Transmission Electron Microscope (TEM) showing a particle diameter distribution of IEVs; FIG. 4E is a Nanoparticle Tracking Analysis (NTA) showing IEVs particle diameter distributions; fig. 4F is a nano-flow detection technique for particle size detection at the single vesicle level for IEVs, showing the particle diameter distribution for IEVs.
FIGS. 5A-5K show the results of analysis of surface membrane proteins by flow cytometry IEVs.
Fig. 6A-6D are IEVs content analyses: FIG. 6A shows the results of quantitative proteomics analysis of MSCs, MSCs-Exosomes, MSCs-IEVs using DIA quantification technique; FIG. 6B is a heat map drawn to screen IEVs for a specific, highly expressed protein; FIG. 6C is a graph showing the results of GO enrichment analysis IEVs for differential proteins expressing Annexin V, flotillin-1,Cadherin 11,Integrin alpha 5 and Syntaxin 4 molecules; FIG. 6D is a Western Blot showing the results of MSCs, MSCs-Exosomes, MSCs-IEVs expressing Annexin V, flotillin-1,Cadherin 11,Integrin alpha 5 and Syntaxin 4.
FIG. 7 is a graph showing the procoagulant effect of IEVs in vivo in hemophilia A mice.
Fig. 8A-8D show changes in various levels of clotting factors following injection IEVs into hemophilia a mice: FIG. 8A is a variation of factor VIII; fig. 8B is a change in vWF factor; FIG. 8C is a graph showing changes in Tissue Factor (TF); fig. 8D shows the change of prothrombin.
Fig. 9A-9B are effects of IEVs on the in vivo therapeutic effects of IEVs following PS and TF blocking in a hemophilia a mouse model.
FIG. 10 is a comparison of the therapeutic effects of homologous MSC derived IEVs and Exosomes on hemophilia A mice.
Note that: wherein WT is a wild-type mouse; the HA group is a hemophilia a mouse model; ha+ IEVs given IEVs treatment for the hemophilia a mouse model; ha+ps-IEVs gave PS negative IEVs for the hemophilia a mouse model; HA+TF-IEVs gave TF negative IEVs for the hemophilia A mouse model; ha+exosomes Exosomes treatment was given to the mouse model of hemophilia a.
FIG. 11 is a diagram showing the morphology of MC3T3-E1, hBMMSC-derived IEVs mirror.
FIG. 12 shows the results of diameter distribution flow assay of MC3T3-E1, hbMC-derived IEVs particles.
Figures 13A-13C show IEVs that can be discharged through the skin and hair: fig. 13A is a schematic diagram of dynamic metabolism of IEVs at the skin surface. Fig. 13B shows IEVs gradually moving from subcutaneous tissue to the dermis and epidermis over time. Fig. 13C shows that PKH26-IEVs was found in hair follicles in hair pulled from the body surface of mice on day 7.
Fig. 14 shows a plot of the death process of hiPSCs and hcmscs taken by the high content cell imaging analysis system.
Fig. 15 shows apoptosis rates of hiPSCs and hucsscs induced apoptosis using flow assays, demonstrating that most cells were apoptotic.
FIG. 16 shows that both positive rate hiPSCs and hUCMSCs expressed using flow analysis of Annexin5 expression were over 80%.
Fig. 17 shows Nanoparticle TrackingAnalysis (NTA) detected particle sizes of two IEVs.
FIG. 18 shows Nanoparticle TrackingAnalysis (NTA) detected amounts of both IEVs yields IEVs.
Fig. 19 shows Nanoparticle TrackingAnalysis (NTA) detected potentials of two IEVs.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
IEVs in the examples of the present invention is simply referred to as inducible vesicles, which may also be referred to as inducible extracellular vesicles (Induced extracellular vesicles, IEVs). An inducible extracellular vesicle refers to a class of subcellular products that are produced by precursor cells (e.g., stem cells) that are interfered with or induced to undergo apoptosis when they survive normally. Typically, this class of subcellular products has a membrane structure, expresses apoptotic markers, and contains in part the genetic material DNA. The inventors have found that inducible extracellular vesicles are a class of substances that are distinguished from cells and conventional extracellular vesicles (e.g., exosomes, etc.). In some embodiments, the cells that survive normally are, for example, non-apoptotic cells, non-senescent cells that proliferate arrested, cells that revive after non-cryopreservation, cells that do not become malignant and proliferate abnormally, or cells that do not become damaged, and the like. In some embodiments, the cells that survive normally are taken from cells that fuse 80-100% in contact during cell culture. In some embodiments, the cells that survive normally are obtained from log phase cells. In some embodiments, the normally viable cells are obtained from primary cultures of human or murine tissue origin and their subcultured cells. In some embodiments, the normally viable cells are taken from an established cell line or strain. In some embodiments, the precursor cells are taken from early cells.
The IEV in the invention is IEVs.
STS in the present invention is staurosporine.
Exosomes in the present invention refer to Exosomes.
"Comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of … …" means excluding other elements that have any significance to the combination for the purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "consisting of … …" means the process steps excluding trace elements and essential components of the other components. Embodiments defined by each of these transitional terms are within the scope of this invention.
As used herein, the term "high expression" and the like are intended to include increasing the expression of a nucleic acid or protein to a level higher than that contained by vesicles (e.g., exosomes) of the prior art.
As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as a lyophilized powder for injection, tablet, capsule, kit or patch. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols 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, buffers, inert non-toxic solids (e.g., mannitol, talc). Compositions comprising such carriers are formulated by well-known conventional methods. Depending on the intended mode of administration and the intended use, the composition may be in the form of a solid, semi-solid or liquid dosage form, such as a powder, granule, crystal, liquid, suspension, liposome, paste, cream, ointment, or the like, and may be in a form suitable for administration of a relatively precise dosage unit dose.
In the present invention, the components in the "composition" may be present in a mixed form or may be packaged separately. The separately packaged components may also contain their respective adjuvants. The adjuvant refers to a means for assisting the curative effect of the medicine in pharmacy. In the case of separate packages for the components of the composition, the individual components of the separate packages may be administered simultaneously or in any order, wherein the patient is treated with one drug and then administered with the other. The patient refers to a mammalian subject, particularly a human.
In the present invention, the "composition" may also be in the form of one component being encapsulated by another component. In some embodiments, the inducible vesicles act as drug carriers in compositions, encapsulating drugs that treat or prevent the disease in the inducible vesicles.
In the invention, the corresponding reagent sources are as follows: penicillin/streptomycin solution (BIOSOURCE; P303-100); glutamine (BIOSOURCE; P300-100); dexamethasone sodium phosphate (Sigma; D-8893); alpha-MEM (Gibco; 12571-063); 2-ME (GIBCO; 21985-023).
EXAMPLE 1 isolation culture of MSCs
Mice were sacrificed with excess CO 2 according to the guidelines of the animal ethics committee, the tibia and femur were removed under aseptic conditions, the muscles and connective tissue attached to them were stripped off, the metaphyseal, exposed bone marrow cavity was further isolated, the bone marrow cavity was repeatedly rinsed with PBS with a 10% fetal bovine serum volume fraction extracted with a 10mL sterile syringe, after filtration through a 70 μm pore size cell filter, 500g was centrifuged for 5min, the supernatant was removed, the bottom cell pellet was collected, resuspended in PBS, again 500g centrifuged for 5min, and the final cell pellet was collected. Cells were then flow sorted, using CD 34-and CD90+ as sorting criteria, to sort BMMSCs. Finally, the cells were resuspended in Dex (-) medium and inoculated into 10cm diameter cell culture dishes, cultured at 37℃with 5% CO 2. After 24h, the supernatant was aspirated off of non-adherent cells, and after PBS washing, dex (-) broth was added for continued culture. Equal amounts of Dex (+) medium were added after 1 week, and dense primary BMMSCs colonies were visible after 1 week. Digestion BMMSCs was incubated with trypsin at 37 ℃ and passaged for expansion, after which the Dex (+) medium was changed every 3 days and passaged after confluence. Subsequent experiments were performed using P2 generation BMMSCs.
Wherein, the composition of the Dex (-) culture solution is shown in Table 1, and the composition of the Dex (+) culture solution is shown in Table 2:
TABLE 1Dex (-) culture composition table
TABLE 2Dex (+) culture fluid formulation table
The purity of the isolated BMMSCs was assessed by flow cytometry analysis of surface markers. For surface marker identification, after collection of P2 generation BMMSCs by trypsin digestion, PBS was washed 1 time, cells were resuspended at a density of 5 x 10 5/mL in PBS containing 3% fbs, 1 μl of PE fluorescence conjugated CD29, CD44, CD90, CD45 and CD34 antibodies were added, and the blank was not added. Incubation for 30min at 4 ℃ in dark, PBS cleaning for 2 times, and detecting on a machine. The results of the flow assay are shown in FIGS. 1A-1E, and it is understood that the isolated cells are BMMSCs (bone marrow mesenchymal stem cells).
Example 2 acquisition of inducible vesicles
MSCs (bone marrow derived MSCs, BMMSCs) cultured to passage 2 in example 1 were washed 2 times with PBS when further cultured to 80% -90% confluence with the medium (Dex (+) medium) in example 1, apoptosis was induced by adding serum-free medium (alpha-MEM medium) containing 500nM STS, incubated at 37℃for 24h, and cell supernatants were collected for isolation and extraction IEVs.
IEVs separating and extracting from the collected culture supernatant, wherein the operation flow is shown in fig. 2, and the specific steps comprise: after centrifugation at 800g for 10 minutes, the supernatant was collected; then 2000g was centrifuged for 10 minutes and the supernatant was collected; then 16000g was centrifuged for 30 minutes, the supernatant removed and resuspended IEVs in sterile PBS; after centrifugation at 16000g for 30 minutes, the supernatant was removed and 300-500. Mu.L of sterile PBS was resuspended IEVs.
Comparative example 1 isolation and extraction of homogeneous MSC-derived exosomes
MSCs (bone marrow derived MSCs, BMMSCs) cultured to passage 2 in example 1 were washed 2 times with PBS when continued to culture to 80% -90% confluence with the medium in example 1, added with serum-free medium, incubated at 37℃for 48h, and cell supernatants were collected for isolation and extraction of Exosomes.
The extraction steps comprise: centrifugation at 800g for 10min, collection of supernatant, centrifugation at 2000g for 10min, collection of supernatant, centrifugation at 16000g for 30min, collection of supernatant, centrifugation at 120000g for 90min, removal of supernatant, suspension of pellet with sterile PBS, centrifugation at 120000g for 90min again, removal of supernatant, collection of bottom Exosomes, suspension with sterile PBS.
Analysis of example 3IEVs
1. IEVs quantification and analysis of Membrane proteins
The IEVs obtained in example 2 was quantitatively analyzed by flow cytometry at 1h, 4h, 8h, 16h and 24h, and it was shown that 10 6 MSCs produced 0.76X10 8, 1.29X10 8, 1.95X10 8, 2.48X10 8, 3.14X10 8 IEVs, respectively, after induction to 1h, 4h, 8h, 16h and 24h, from which it was seen that 300 IEVs could be produced by a single MSC after induction to 24h (fig. 3).
Further, the particle diameter distribution of IEVs was found to be concentrated to 1 μm or less by the flow assay, accounting for 94.97% (FIG. 4A), and the side scattered light (SSC) analysis results also showed that the intensity of IEVs scattered light was concentrated to 1 μm or less (FIG. 4B). Further, the scattered light intensity of IEVs was analyzed by standardized small particle microspheres (0.2 μm,0.5 μm,1 μm) produced by Bangs Laboratories company, which showed that the particle diameters of IEVs were all below 0.2 μm (FIG. 4C). The Transmission Electron Microscope (TEM) showed similar results to the flow type detection, and most vesicles were 200nm and less in diameter (FIG. 4D). The Nanoparticle Tracking Analysis (NTA) results were consistent with transmission electron microscopy observations, with IEVs particles averaging 169nm in diameter (fig. 4E). Particle size detection at the single vesicle level was performed using the most advanced nanofluidic detection technique, which also showed that IEVs had an average particle diameter of 100.63nm (fig. 4F).
Analysis of the IEVs surface membrane proteins extracted in example 2 using flow cytometry showed that MSCs derived IEVs was capable of expressing surface proteins similar to MSCs, i.e., CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. At the same time IEVs is capable of expressing the general surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (see fig. 5A-5K).
2. IEVs content analysis
Proteomic quantitative analysis of BMMSCs, MSCs-Exosomes (extracted from comparative example 1), MSCs-IEVs (obtained from example 2) was performed using protein DIA quantification technique. The results showed that the protein content expression of MSCs-Exosomes and MSCs-IEVs had a high overlap with the parent cells, and that 170 proteins were specifically highly expressed in IEVs (FIG. 6A).
Through bioinformatics analysis, IEVs specific high-expression proteins are screened, a heat map is drawn (figure 6B), and further the GO enrichment analysis result of the differential protein is combined, so that IEVs can specifically and high-express Annexin V, flotillin-1,Cadherin 11,Integrin alpha 5 and Syntaxin 4 molecules, and compared with Exosomes derived from the same MSCs, the expression quantity of 5 characteristic molecules of IEVs is obviously up-regulated, specifically: the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA 5 and Syntaxin 4 in IEVs were expressed 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold and 4.45-fold, respectively, relative to the corresponding markers in the Exosomes (FIG. 6C). Finally, the Western Blot technique is used for verification again, and the result is consistent with the DIA quantitative analysis result (FIG. 6D).
MSCs-Exosomes: refers to exosomes derived from BMMSCs.
MSCs-IEVs: refers to IEVs derived from BMMSCs.
Wherein the MSCs in the content analysis and the MSCs from which Exosomes and IEVs are extracted are the same BMMSCs cell line.
EXAMPLE 4 use and mechanism study of IEVs derived from MSCs for treatment of hemophilia mice
The in vitro procoagulant effect of IEVs obtained in example 2 and Exosomes extracted in comparative example 1 was examined using an in vitro coagulation assay. As shown in Table 3, IEVs significantly shortens the in vitro clotting time of most plasma, and the procoagulant effect is better than that of Exosomes. However, for factor II, V, X deficient plasma IEVs failed to exert an in vitro procoagulant effect, suggesting that the in vitro procoagulant effect of IEVs is more concentrated upstream of the common pathway of coagulation.
TABLE 3 Table 3
The procoagulant effect of IEVs in vivo was observed by tail vein injection of 9×10 8 IEVs using hemophilia a mice (factor VIII deficiency) as a model. The results are shown in fig. 7, and IEVs treatment was able to significantly improve bleeding tendency in hemophilia mice and the treatment effect was maintained stably for 14 days.
Experimental results show that IEVs can exert remarkable coagulation promoting effect in vitro. And the bleeding tendency can be obviously improved after in vivo injection, and the injection can be used for improving the bleeding tendency caused by hemophilia A.
At the same time, the levels of various coagulation factors in the plasma of mice were examined, and it was found that neither coagulation factor VIII, vWF factor, tissue Factor (TF) nor prothrombin (prothrombin) were significantly changed (fig. 8A, 8B, 8C, 8D).
In the hemophilia a mouse model, the tail-clipping experiments were performed 7 days after injection of normal IEVs, PS negative IEVs and TF negative IEVs, respectively, and as shown in fig. 9A and 9B, the blocking of PS and TF did not affect the in vivo therapeutic effect of IEVs, initially demonstrating that IEVs treatment of hemophilia mice was independent of PS and TF. In the previous literature, extracellular vesicles exert procoagulant effects highly depend on PS and TF on the surface, while the in vivo experimental results of IEVs are inconsistent with previous studies, suggesting that IEVs may have a new mechanism of action to exert procoagulant effects in vivo environments.
Comparative example 2
Injection treatment (9×10 8) of IEVs (obtained in example 2) and Exosomes (extracted in comparative example 1) of the same MSC source, respectively, was performed on the hemophilia a mouse model, showing that IEVs was able to significantly correct the bleeding tendency of the mice without significant therapeutic effect of Exosomes (fig. 10).
The in vitro procoagulant effect of IEVs obtained in example 2 was compared with that of Exosomes prepared in comparative example 1:
Wherein IEVs obtained in example 2 has a diameter of IEVs expression markers Syntaxin 4, annexin V, flotillin-1, cadherin 11 and INTEGRIN ALPHA 5in the range of 0.03 μm to 0.2 μm and 0.2 μm to 1 μm, and has a strong in vitro coagulation promoting effect; exosomes prepared in comparative example 1 had diameters of 0.03 μm to 0.15. Mu.m, expressed markers Complement C q, complement C3, thrombospondin-1 and Thrombospondin-2, and the in vitro coagulation promoting effect was relatively weak.
TABLE 4 Table 4
Example 5
1. Induced pluripotent stem cell (induced pluripotent STEM CELLS, iPS cell, iPSC) cell culture
(1) Lentivirus preparation:
1mL of DMEM was transferred into an EP tube, 5. Mu.g of gene expression plasmid and 5. Mu g vsvg of plasmid were added to 25. Mu.l of liposomes, and the mixture was gently stirred at room temperature for 20 minutes. The mixture was added drop-wise to cultured GP2-293 cells (95% mix) and the mixture was evenly distributed by spinning. After 12 hours the medium was changed (dmem+10% fbs (heat-inactivated) +glutamine). After 24 hours of medium exchange, the virus-containing medium was collected and after 48 hours the medium was collected again.
(2) Inducing cell reprogramming:
Each well (12-well plate) was inoculated with 5X 10 5 of the GP2-293 cells cultured in step (1), 100ng of virus was added to a medium (DMEM+10% FBS (heat-inactivated) +Glutamine) having a confluence of 500-1000. Mu.l/well, 4. Mu.g/ml of Polybrene was added, incubated for 12 hours, and medium was replaced, and this step was repeated. Within 7 days, 5×10 4 induced cells were seeded into 10cm dishes with feeder cells (mEFs). The next day, the medium was changed to Es medium containing bFGF (4 ng/ml), and after 5 days, the cells began to clone, and if after 40 days there were no Es-like clones, failure was considered.
(3) Cell passage:
After 60% confluence, 0.5 ml accutase was added to each dish and allowed to stand at room temperature for 1 minute. The isolated cell aggregates were transferred to a 15mL centrifuge tube and an additional 2mLmTeSR a1 was used to collect any remaining aggregates. The rinse was added to a 15ml tube. A15 mL tube containing the cell aggregates was centrifuged at 200g for 5 minutes at room temperature. The supernatant was aspirated. Resuspend the cells and ensure that the cells remain aggregated. Human iPS cells were aggregated with mTeSR1 on new plates coated with matrix gel. The culture dish was placed in a 37℃incubator and moved rapidly from side to evenly distribute the movement of the clumps of cell aggregates. Incubated at 37℃with 5% carbon dioxide and 95% humidity. The liquid is changed every day.
2. MC3T3-E1Subclone14 osteoblast cell line culture
The commercially available MC3T3-E1Subclone14 was rapidly thawed, centrifuged at 500g for 5min, the supernatant removed and the bottom cell pellet collected, and the cells resuspended in Dex (-) medium and inoculated into 10cm diameter cell culture dishes for culture at 37℃with 5% CO 2. After the cells grow up, the cells are subjected to incubation digestion and passage amplification by trypsin at 37 ℃, and then the Dex (-) culture solution is changed every 3 days, so that the cells can be used for multiple passages. Wherein, the Dex (-) culture medium composition is shown in Table 5:
TABLE 5Dex (-) culture composition table
3. IEVs analysis
Including a comparison of osteoblast MC3T3-E1 and iPS cells and IEVs derived from human bone marrow mesenchymal stem cells (hBMMSC). The method for obtaining IEVs of the three cell sources is the same as in example 2.
(1) Morphological detection
As shown in fig. 11, the results of the experiment were similar to, and irregular in morphology of, the iPS cells (ipscs) and osteoblasts MC3T 3-E1-derived IEVs and human BMMSC-derived IEVs under 400 x microscope.
(2) Diameter detection of IEVs particles
The results of the flow assay are shown in FIG. 12, and the results of the flow assay show that the particle diameter distribution of the osteoblast line MC3T3-E1 and the human bone marrow mesenchymal stem cells hBMMSC derived IEVs is similar.
(3) IEVs detection of surface markers
The detection by Western Blot method shows the experimental results as shown in FIG. 13, in which the surface markers of IEVs derived from iPS cells (iPSC) and human mesenchymal stem cells (hBMMSC) were compared, and the IEVs marker AnenexinV was highly expressed in IEVs derived from both cells. IEVs, which is derived from iPS cells, expressed higher levels of syncaxin 4 relative to hbmmscs.
Example 6IEVs percutaneous and Hair drainage
IEVs prepared in example 2 of 4X 10 6 was labeled with DIR, resuspended in 200. Mu.L PBS, and injected systematically into BALB/C-nu/nu nude mice via the tail vein, and the distribution of IEVs on the skin surface was examined with a biopsy instrument after 1,3,7 days of observation, and the results are shown in FIGS. 13A-13C.
Fig. 13A shows IEVs that can reach the skin surface, the highest number at day 3, and the substantial disappearance at day 7, showing the dynamic metabolic process of IEVs at the skin surface (fig. 13A). Immunofluorescence results showed that PKH26-IEV was gradually moved from subcutaneous tissue to dermis and epidermis over time following systemic injection of C57 mice. The presence of IEVs in large amounts at the stratum corneum at the skin surface was observed on day 7, suggesting that the systematically injected IEVs could be excreted as the stratum corneum sloughed off (fig. 13B). Meanwhile, PKH26-IEV was found in hair follicles in mice at day 7, suggesting that the systemic injection IEVs may also be metabolized away as the hair falls off (fig. 13C). This example demonstrates IEVs can be expelled through the skin and hair, demonstrating the safety of injecting or increasing IEVs in vivo.
Example 7
Culture of hiPSCs the same procedure as in example 5 is used for the culture of hiucmscs as is conventional in the art.
The hiPSCs may be, but are not limited to, generations 26-29, and the embodiment specifically uses generation 26; the huchmscs may be 7 th to 9 th generation, but are not limited thereto, and the embodiment is specifically used in the 7 th generation.
1. Experimental method
(1) Cells were apoptosis induced for about 9h using staurosporine (500 nM) in hiPSCs and hUCMSCs (the remainder of the procedure was as in example 2), and the apoptosis rates were measured by flow-through using annexin V (15 mins) and 7AAD (3 mins).
(2) IEVs was isolated from the supernatant of apoptotic cells in step (1) and assayed for annexin v expression using a flow assay. Extracting IEVs using differential centrifugation, the steps comprising: centrifugation at 800g for 10 min-2000 g for 5min (except for this step, the extraction steps are the same as in example 2) -centrifugation at 16000g for 30 min-16000 g for 30min for IEVs.
Annexin v stained for 15mins and flow-on-machine.
2. Experimental results
(1) We used high connotation to shoot the death process of hiPSCs and hucsscs, found that there was a difference in the death process between them, and hiPSCs contracted with multiple centers of nuclei and masses, and then sent out dendritic branches with bleb; hUCMSCs are single-center contractions with nuclei as centers, and are accompanied by activities such as branching and blebbing. The results are shown in FIG. 14.
(2) Apoptosis rates of hiPSCs and huchmscs induced apoptosis were analyzed using a flow assay, demonstrating that most cells were apoptotic. The results are shown in FIG. 15.
(3) Both positive rate hiPSCs and hucss expressed by using flow analysis of Annexin5 expression were above 80%. The results are shown in FIG. 16.
(4) Two IEVs were characterized using Nanoparticle TrackingAnalysis (NTA):
1) As a result, as shown in FIG. 17, IEVs derived from hiPSCs had a particle size of about 100nm and IEVs derived from hUCMSCs had a particle size of about 180nm;
2) As a result, as shown in FIG. 18, IEVs derived from hiPSCs gave 21971 partics/hiPSCs, and 886 partics/hUCMSCs derived from hUCMSCs;
3) As a result, FIG. 19 shows that IEVs derived from hiPSC had a potential of about-12 mV and IEVs derived from hUCMSC had a potential of about-45 mV.

Claims (12)

1. A vesicle, wherein the vesicle is an inducible vesicle, and the cellular source of the vesicle comprises a stem cell or a somatic cell; the vesicle has a marker including Syntaxin 4.
2. The vesicle of claim 1, wherein said somatic cells comprise primary cultured cells or cell lines;
preferably, the somatic cells comprise an osteoblast cell line;
More preferably, the cell line is an immortalized cell culture that can proliferate indefinitely in the appropriate fresh medium and space;
preferably, the stem cells include totipotent stem cells and pluripotent stem cells;
preferably, the stem cells include mesenchymal stem cells and induced pluripotent stem cells;
Preferably, the inducible vesicle is a vesicle produced by inducing apoptosis by external force during normal survival of the stem cell or somatic cell; preferably, the induction vesicle is produced by inducing stem cells or stem cell apoptosis by addition of staurosporine, ultraviolet irradiation, starvation, or thermal stress, or a combination of one or more thereof;
Preferably, the vesicle has a marker which further comprises one or more of Annexin V, flotillin-1, cadherin 11 and INTEGRIN ALPHA;
Further preferred, the vesicles have the combination of markers Syntaxin 4, annexin V, flotillin-1, cadherin 11 and INTEGRIN ALPHA;
further preferred, the vesicle high expression markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4;
Or preferably, the vesicles have higher expression levels of markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4 than MSC or exosomes;
or preferably, the expression levels of markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4 in the vesicles are 1-2 fold, 2-3 fold, 1-3 fold, 3-4 fold and 2-6 fold, respectively, relative to the expression levels of markers in exosomes derived from mesenchymal stem cells;
More preferably, the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4 in the vesicles are expressed in amounts of 1.5-2 times, 2.5-3 times, 1.5-2.5 times, 3.5-4 times and 3.5-5 times, respectively, relative to the expression amounts of the markers in exosomes derived from mesenchymal stem cells;
Still more preferably, the markers Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4 in the vesicles are expressed in amounts of 1.5 to 1.9 times, 2.5 to 2.9 times, 1.8 to 2.5 times, 3.5 to 3.9 times and 4 to 5 times, respectively, relative to the expression amounts of the markers in exosomes derived from mesenchymal stem cells;
still more preferably, the vesicles and the exosomes are derived from MSCs of homogeneous origin;
Or preferably, the vesicle also expresses CD29, CD44, CD73, CD166; and does not express CD34, CD45;
or preferably, the vesicle also expresses one or more of CD9, CD63, CD81 and C1 q;
or preferably, the vesicles are produced by inducing mesenchymal stem cells with astromycin;
preferably, the concentration of staurosporine is 1nM to 10000nM;
Preferably, the concentration of staurosporine is 100nM-10000nM;
Preferably, the concentration of staurosporine is 500nM-10000nM;
preferably, the concentration of staurosporine is 500-1000nM;
further preferably, the concentration of staurosporine is 500-900nM;
further preferably, the concentration of staurosporine is 500-800nM.
3. The vesicle of claim 1, wherein said vesicle has a diameter of 0.03-6 μm;
Preferably, the vesicles have a diameter of 0.03-4.5 μm;
further preferably, the vesicles have a diameter of 0.03 to 1. Mu.M;
further preferably, the vesicles have a diameter of 0.04 to 1. Mu.M;
further preferably, the vesicles have a diameter of 0.05 to 1. Mu.M;
further preferably, the vesicles have a diameter of 0.1 to 1. Mu.M;
Further preferably, the vesicles have a diameter of 0.15 to 1. Mu.M.
4. A vesicle assembly comprising a vesicle according to any one of claims 1-3;
Preferably, the number of vesicles according to any one of claims 1 to 3 in the vesicle combination is 65-100%;
Further preferably, the number of vesicles according to any one of claims 1 to 3 in the vesicle combination is 75-98%; more preferably, the number of vesicles according to any one of claims 1 to 3 in the vesicle composition is 80-96%;
or preferably, the vesicle combination further comprises one or more of exosomes, migrates, microbubbles and Ectosome.
5. A composition comprising a vesicle according to any one of claims 1-3; or comprising a vesicle combination of claim 4;
preferably, the composition comprises a pharmaceutical, food, health product, cosmetic, additive or intermediate;
preferably, the composition is a pharmaceutical product;
preferably, the composition further comprises a pharmaceutically or immunologically acceptable carrier;
more preferably, the formulation of the composition is selected from the group consisting of lyophilized powder for injection, tablet, capsule, kit or patch.
6. A screening or identification or extraction reagent or kit for vesicles, comprising one or more of markers Annexin V, flotillin-1, cadherein 11, INTEGRIN ALPHA and syncaxin 4;
preferably, the detection reagent detects the expression level of the marker gene;
further preferably, the detection reagent detects the expression amount of the marker mRNA;
More preferably, the detection reagent for the marker detects the expression level of the marker protein;
or preferably, the detection reagent of the marker is one or more of fluorescent quantitative PCR dye, fluorescent quantitative PCR primer, fluorescent quantitative PCR probe, antibody functional fragment and coupled antibody;
or preferably, the kit is selected from one or more of qPCR kit, immunoblot detection kit, flow cytometry kit, immunohistochemical detection kit and ELISA kit;
more preferably, the kit is selected from the group consisting of flow cytometric assay kits;
Preferably, the vesicle is an inducible vesicle.
7. A method for selecting or identifying a vesicle according to any one of claims 1-3, comprising detecting a sample to be detected with a detection reagent using one or more of Annexin V, flotillin-1, cadherin 11, INTEGRIN ALPHA and Syntaxin 4 as markers,
Preferably, a control reagent is used in the method, wherein the control reagent comprises one or more of exosomes, migrates, microbubbles and Ectosome, and is positive when the expression level of the marker in the sample to be detected is higher than that of the control reagent;
Preferably, the control reagent comprises an exosome;
preferably, the marker is Syntaxin 4;
Preferably, when the expression level of Syntaxin 4 in the sample to be detected is greater than or equal to 2-6 times that of the exosomes, determining that the vesicle; more preferably, the vesicle is determined when the expression level of Syntaxin 4 in the sample to be tested is 3-6 times or more, more preferably 4-5 times or more that of the exosome.
8. Use of a detection reagent for a marker for the preparation of a reagent or kit for detecting or identifying a vesicle according to any one of claims 1-3, wherein the marker comprises one or more of Annexin V, flotillin-1, cadherein 11, INTEGRIN ALPHA and syncaxin 4, the reagent or kit further comprising a control reagent comprising one or more of exosomes, migrates, microbubbles and Ectosome, and the marker in the sample to be tested is judged positive when the expression level of the marker is higher than the control reagent;
preferably, the control agent is an exosome;
Preferably, when the expression level of Syntaxin 4 in the sample to be detected is greater than or equal to 2-6 times that of the exosomes, determining that the vesicle; more preferably, when the expression level of Syntaxin 4 in the sample to be tested is 3-6 times or more that of the exosomes, the vesicle is determined;
More preferably, the vesicle is determined when the expression level of Syntaxin 4 in the sample to be tested is 4-5 times or more that of the exosome.
9. Use of a vesicle according to any one of claims 1-3 or a vesicle combination according to claim 4 for the preparation of a product for the treatment or prevention or amelioration of a disease or a complication of said disease; the disease is hemophilia;
More preferably, the disease is hemophilia a;
Or preferably, the product comprises a pharmaceutical, food, nutraceutical, cosmetic, additive or intermediate product.
10. A method of preparing a vesicle according to any one of claims 1-3, wherein the method is to induce stem cells or somatic cells to produce the vesicle by adding an apoptosis-inducing agent;
Preferably, the method comprises the steps of:
(1) Culturing mesenchymal stem cells;
(2) Collecting a culture medium supernatant of the mesenchymal stem cells;
(3) Separating vesicles from the culture supernatant in step (2);
Preferably, the step of culturing the mesenchymal stem cells in step (1) comprises:
(4) Isolating mesenchymal stem cells from a tissue;
(5) Culturing the mesenchymal stem cells by adding a culture medium; contacting an apoptosis inducer in a culture medium of the mesenchymal stem cells;
Further preferably, the apoptosis-inducing agent comprises staurosporine, ultraviolet radiation, starvation, or thermal stress or a combination of one or more thereof;
more preferably, the apoptosis-inducing agent is staurosporine;
preferably, the concentration of staurosporine is 1-10000nM;
Preferably, the concentration of staurosporine is 100-10000nM;
preferably, the concentration of staurosporine is 500-10000nM;
Or more preferably, the concentration of staurosporine is 500-1000nM;
Still more preferably, the concentration of staurosporine is 500-900nM;
Most preferably, the concentration of staurosporine is 500-800nM;
further preferably, the cells are treated with the apoptosis-inducing agent in step (5) for a period of 16-24 hours;
Or preferably, in the step (3), the method for separating vesicles comprises separating vesicles by an ultracentrifugation method;
further preferably, the step of separating the vesicles by the ultracentrifugation method comprises:
(a) Centrifuging the collected culture supernatant for the first time, and taking the supernatant;
(b) Subjecting the supernatant collected in step (a) to a second centrifugation to obtain a supernatant;
(c) Centrifuging the supernatant received in step (b) for a third time to obtain a precipitate;
(d) Centrifuging the precipitate received in step (c) for the fourth time, and taking the precipitate;
further preferably, the first centrifugation is 500-1500g centrifugation for 5-30 minutes;
more preferably, the first centrifugation is 500-1000g centrifugation for 5-20 minutes;
still more preferably, the first centrifugation is 500-900g centrifugation for 5-15 minutes;
further preferably, the second centrifugation is between 1000 and 3000g centrifugation for 5 to 30 minutes;
more preferably, the second centrifugation is from 1500 to 2500g for 5 to 20 minutes;
still more preferably, the second centrifugation is from 1500 to 2200g centrifugation for 5 to 15 minutes;
Further preferably, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes;
more preferably, the third centrifugation is 12000-25000g centrifugation for 20-60 minutes;
Still more preferably, the third centrifugation is 12000-20000g centrifugation for 20-40 minutes;
further preferably, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes;
more preferably, the fourth centrifugation is 12000-25000g centrifugation for 20-60 minutes;
still more preferably, the fourth centrifugation is for 20-40 minutes at 12000-20000 g.
11. A method of enriching a vesicle according to claims 1-3, characterized in that the vesicle is enriched by means of immunomagnetic beads, which are obtained by coupling antibodies with magnetic beads; the antibodies include Anti-Syntaxin 4 antibodies;
preferably, the antibody further comprises one or more of an Anti-Annexin V antibody, an Anti-Flotillin-1 antibody, an Anti-Cadherin 11 antibody, an Anti-INTEGRIN ALPHA antibody.
12. The vesicle of any one of claims 1-3 or the vesicle combination of claim 4 or the composition of claim 5 or the extraction reagent or extraction kit of claim 6 or the use of claim 7 or the method of claim 8 or the method of claim 9, wherein the mesenchymal stem cells are derived from a mammal;
preferably, the mammal is selected from human or murine;
Or preferably, the mesenchymal stem cell source comprises: bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or combinations thereof;
more preferably, the mesenchymal stem cell source is selected from one or more of bone marrow, fat, umbilical cord and oral cavity.
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