CN116763817A - Use of blood-derived samples for preparing vesicles - Google Patents
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- CN116763817A CN116763817A CN202210624896.5A CN202210624896A CN116763817A CN 116763817 A CN116763817 A CN 116763817A CN 202210624896 A CN202210624896 A CN 202210624896A CN 116763817 A CN116763817 A CN 116763817A
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Classifications
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- 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
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
Landscapes
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Abstract
The invention belongs to the field of biological medicine, and relates to application of a blood-derived sample in preparation of vesicles. The invention provides an application of a blood-derived sample in preparing vesicles, wherein the vesicles are inducible vesicles. The peripheral blood provided by the invention is taken as a sample, and the peripheral blood can be directly reused in the body of an original individual after the induction in vitro to obtain the induced vesicle, so that the peripheral blood has the advantages of convenience in sampling and self-body, and has excellent operability and safety.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to application of a blood-derived sample in preparation of vesicles.
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. Extracellular vesicles are important mediators of intercellular communication, allowing intracellular bioactive molecules to migrate from one cell to another to function. It is therefore becoming more and more clear that these vesicles are involved in many physiological processes, which would provide an opportunity for application in the treatment of diseases.
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.
Acute Respiratory Distress Syndrome (ARDS), which has been a major clinical problem in respiratory medicine since the first description, has been of great interest, with a high incidence and mortality rate. ARDS is a clinical syndrome that is mainly manifested by lung injury due to various causes inside and/or outside the lung, and is characterized by progressive dyspnea and refractory hypoxia. The ARDS is caused by severe pneumonia, drowning aspiration, pulmonary contusion, toxic substance inhalation and the like in the lung, and the ARDS is caused by serious infection, serious trauma, shock, severe pancreatitis, poisoning, major surgery, after cardiopulmonary resuscitation and the like in the lung, and the common pathological basis is acute lung injury. In addition to the well known risk factors for ARDS, exposure to high ozone levels and low vitamin D plasma concentrations have also been found to be a susceptible environment. ARDS treatment is expensive, but currently lacks targeted therapies in addition to protecting lung ventilation, treating primary disease and supporting therapeutic approaches. Based on the latest studies, although the overall survival rate of ARDS is increasing, this can be interpreted as risk factors, availability of diagnosis, ability to identify ARDS and some selection bias trial affecting clinical diagnosis, considering the inpatient mortality of several observational studies, which data indicate that ARDS is still in an under-diagnosed and under-treated state worldwide, and current definition of ARDS is inadequate in most clinical situations. Drug-based preventive strategies remain a significant challenge, and some recent studies have focused on improving the prognosis of this disease acute respiratory distress syndrome, but high mortality and high disabling complications remain to be improved.
The colonitis is also called non-specific ulcerative colitis, which is various in variety, causes and has various causes, slow onset of disease, mild and severe disease conditions, and is mainly characterized by diarrhea, abdominal pain, mucous stool, bloody stool, tenesmus, even constipation, incapacitation of passing stool in a plurality of days, diarrhea and constipation, emaciation and hypodynamia and the like, which are frequently repeated. Abdominal pain is usually a dull pain or colic, usually located in the left lower abdomen or lower abdomen. Other manifestations include inappetence, abdominal distension, nausea, vomiting, large liver, and sometimes cramps in the lower left abdomen. Common general symptoms include emaciation, hypodynamia, fever, anemia and the like. In the course of chronic disease, a small number of patients develop severe diarrhea due to sudden deterioration or initial onset, which brings great pain to humans or animals.
Disclosure of Invention
In some embodiments, the invention provides the use of a blood-derived sample in the preparation of a vesicle, said vesicle being an inducible vesicle.
In some embodiments, the blood comprises plasma, whole blood.
In some embodiments, the blood is peripheral blood.
In some embodiments, the blood contains blood cells.
In some embodiments, the sample comprises peripheral blood mononuclear cells.
Peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMCs) are mononuclear cells in peripheral blood, including lymphocytes and monocytes, and are a useful source of cells because of their ease of collection, providing convenience for clinical use. The volume, morphology and specific gravity of mononuclear cells are different from those of other peripheral blood cells, the specific gravity of red blood cells and polynuclear white blood cells is about 1.092, the specific gravity of mononuclear cells is 1.075-1.090, and platelets are 1.030-1.035. Thus, by using a solution which is approximately isotonic between 1.075 and 1.092 for density gradient centrifugation, cells with a certain density are distributed according to the corresponding density gradient, and various blood cells can be separated from mononuclear cells.
In some embodiments, the sample comprises red blood cells.
In some embodiments, the peripheral blood mononuclear cells are isolated or non-isolated cells derived from blood.
In some embodiments, the inducible vesicle is a vesicle that is produced by external force induced apoptosis during normal survival of cells in a sample derived from blood.
In some embodiments, the external force comprises the addition of staurosporine, ultraviolet radiation, starvation, or thermal stress, or a combination of one or more thereof.
In some embodiments, the external force is a heat treatment.
In some embodiments, the heat treatment is performed in the range of 38 ℃ to 60 ℃.
In some embodiments, the heat treatment is performed at a temperature in the range of 40 ℃ to 55 ℃.
In some embodiments, the heat treatment is performed at a temperature in the range of 40 ℃ to 52 ℃.
In some embodiments, the heat treatment is performed in the range of 42 ℃ to 52 ℃.
In some embodiments, the heat treatment is performed in the range of 42 ℃ to 50 ℃.
In some embodiments, the heat treatment is for a period of 3 to 20 hours.
In some embodiments, the heat treatment is for a period of 3 to 15 hours.
In some embodiments, the heat treatment is for a period of 3 to 12 hours.
In some embodiments, the heat treatment is for a period of 3 to 10 hours.
In some embodiments, the vesicles are positive for annexin V, intigrin alpha 5, and Syntaxin 4 expression.
In some embodiments, the vesicles have a diameter of 0.03 to 6 μm.
In some embodiments, the vesicles have a diameter of 0.03 to 4.5 μm.
In some embodiments, the vesicles have a diameter of 0.03 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.04 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.05 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.1 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.15 to 1 μm.
In some embodiments, the invention provides a method of preparing vesicles, the method comprising subjecting a sample derived from blood to an external force to obtain the vesicles, the vesicles being inducible vesicles.
In some embodiments, the invention provides a vesicle, obtained by the above-described method.
In some embodiments, the invention provides the use of an induced vesicle of a blood-derived sample, or a pharmaceutical composition comprising said vesicle, for the preparation of a product for the treatment or prevention or amelioration of a disease or a complication of said disease.
In some embodiments, the product comprises a pharmaceutical product, a food product, a nutraceutical product, a cosmetic product, an additive or an intermediate product.
In some embodiments, the disease comprises a pulmonary disease or an intestinal disease or diabetes.
In some embodiments, the product is used to promote hair follicle repair and/or hair regeneration.
In some embodiments, the pulmonary disease is acute respiratory distress syndrome.
In some embodiments, the intestinal disorder is enteritis.
In some embodiments, the enteritis is acute enteritis.
In some embodiments, the medicament is for ameliorating symptoms of weight loss or shortened colon in mice caused by acute enteritis.
In some embodiments, the diabetes is type 1 diabetes.
In some embodiments, the medicament is for promoting wound healing in type 1 diabetes. In some embodiments, the blood is peripheral blood.
In some embodiments, the blood contains blood cells.
In some embodiments, the sample comprises peripheral blood mononuclear cells.
In some embodiments, the sample comprises red blood cells.
In some embodiments, the peripheral blood mononuclear cells are isolated or non-isolated cells derived from blood.
In some embodiments, the inducible vesicle is a vesicle that is produced by external force induced apoptosis during normal survival of cells in a sample derived from blood.
In some embodiments, the peripheral blood provided by the invention can be directly reused in the body of the original individual after the induced vesicle is obtained in vitro, has the advantages of convenient sampling and self-body, and thus has excellent operability and safety.
In some embodiments, the external force comprises one or more of addition of staurosporine, addition of ethanol, addition of hydrogen peroxide, ultraviolet irradiation, starvation, lysate, thermal stress, or mechanical force.
In some embodiments, the external force is a heat treatment process.
In some embodiments, the heat treatment is performed in the range of 38 ℃ to 60 ℃.
In some embodiments, the heat treatment is performed at a temperature in the range of 40 ℃ to 55 ℃.
In some embodiments, the heat treatment is performed at a temperature in the range of 40 ℃ to 52 ℃.
In some embodiments, the heat treatment is performed in the range of 42 ℃ to 52 ℃.
In some embodiments, the heat treatment is performed in the range of 42 ℃ to 50 ℃.
In some embodiments, the heat treatment is for a period of 3 to 20 hours.
In some embodiments, the heat treatment is for a period of 3 to 15 hours.
In some embodiments, the heat treatment is for a period of 3 to 12 hours.
In some embodiments, the heat treatment is for a period of 3 to 10 hours.
In some embodiments, the vesicles are positive for annexin V, intigrin alpha 5, and Syntaxin 4 expression.
In some embodiments, the vesicle is an inducible vesicle.
In some embodiments, the vesicles have a diameter of 0.03 to 6 μm.
In some embodiments, the vesicles have a diameter of 0.03 to 4.5 μm.
In some embodiments, the vesicles have a diameter of 0.03 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.04 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.05 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.1 to 1 μm.
In some embodiments, the vesicles have a diameter of 0.15 to 1 μm.
In some embodiments, the PBMCs may be obtained by heating the PBMCs at a temperature in the range of 30 ℃ to 100 ℃ for a predetermined time. Alternatively, PBMCs may be obtained by heating PBMCs at a temperature in the range of 30 ℃ to 100 ℃ for a period of time in the range of 1 minute to 1000 minutes. Alternatively, the PBMCs may be obtained by heating the PBMCs at a temperature in the range of 30 ℃ to 100 ℃ for a period of time in the range of 10 minutes to 100 minutes. Alternatively, the PBMCs may be obtained by heating the PBMCs at a temperature in the range of 40 ℃ to 70 ℃ for a predetermined time. Alternatively, PBMCs may be obtained by heating stem cells at a temperature in the range of 40 ℃ to 70 ℃ for a period of time in the range of 1 minute to 1,000 minutes. Alternatively, the PBMCs may be obtained by heating the PBMCs at a temperature in the range of 40 ℃ to 70 ℃ for a period of time in the range of 10 minutes to 100 minutes.
In some embodiments, the peripheral blood provided by the invention is taken as a sample, and the induction vesicle is obtained by adopting a heat treatment method, so that an additional induction reagent is not required in the induction process, only the heat treatment is required, and the problem of residual reagent is not required to be worried. The method has the advantages of convenient sampling, self-body, and capability of directly reusing the induced vesicle after obtaining the induced vesicle in vitro, thus having excellent operability and safety.
In some embodiments, the inventors of the present invention have unexpectedly found that it is particularly suitable for PBMCs of human origin to be induced to obtain inducible vesicles by such a method of heat treatment. Compared to PBMCs of murine origin, vesicles obtained with heat treatment of PBMCs of human origin are significantly higher than those obtained with heat treatment of PBMCs of murine origin. Thus, the induction vesicle obtained by heat treatment of PBMC is further embodied to have excellent industrialization prospect, and is beneficial to large-scale production.
In some embodiments, the method of preparing the inducible vesicle 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 vesicles are isolated from the medium by a method selected from the group consisting of polymer precipitation, immunoisolation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, and combinations thereof.
In some embodiments, the method of isolating vesicles comprises isolating with Annexin V, integrin alpha 5, and Syntexin 4 as markers.
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, in step (3), the method of isolating vesicles comprises isolating the vesicles by an ultracentrifugation method.
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 obtained in step (c) for the fourth time, and collecting the precipitate.
In some embodiments, the first centrifugation is 500-1500g centrifugation for 5-30 minutes; or the first centrifugation is 500-1000g centrifugation for 5-20 minutes; or the first centrifugation is 500-900g for 5-15 minutes; or centrifuging for 5-10min at 800 g. In some embodiments, the second centrifugation is from 1000 to 3000g centrifugation for 5 to 30 minutes; or the second centrifugation is 1500-2500g for 5-20 min; or the second centrifugation is 1500-2200g for 5-15 minutes; or the second centrifugation is for 5-10 minutes at 2000 g. In some embodiments, the third centrifugation is 10000-30000g centrifugation for 15-60 minutes; or the third centrifugation is 12000-25000g centrifugation for 20-60 minutes; or the third centrifugation is 12000-20000g for 20-40 min; or the third centrifugation is 16000-16500g for 30-35 min. In some embodiments, the fourth centrifugation is 10000-30000g centrifugation for 15-60 minutes, or the fourth centrifugation is 12000-25000g centrifugation for 20-60 minutes; or the fourth centrifugation is 12000-20000g for 20-40 min; or the fourth centrifugation is 16000-16500g for 30-35 min.
In some embodiments, the blood sample is derived from a mammal.
In some embodiments, the mammal is selected from a primate or a mouse.
In some embodiments, the primate is a human.
In some embodiments, the present invention provides a vesicle, obtained by the above-described method.
In some embodiments, the invention provides a pharmaceutical composition comprising said vesicle and a pharmaceutically acceptable adjuvant.
In some embodiments, the pharmaceutical composition is in a formulation selected from the group consisting of a lyophilized powder for injection, an injection, a tablet, a capsule, or a patch.
Drawings
FIG. 1 is a process for isolating human PBMC using a Fucus PBMC isolation tube.
FIG. 2 is a process of Ficoll-hypaque density gradient centrifugation to separate PBMC.
FIG. 3 is a graph of apoptosis status of human PBMC.
Fig. 4 is an IEVs extraction normalization flow technique roadmap.
Fig. 5 is an analysis of extracted IEVs using flow cytometry. Fig. 5A is a graph of particle diameter distribution of IEVs. FIG. 5B shows the results of Nanoparticle Tracking Analysis (NTA) of human PBMCs producing IEVs under different induction conditions. FIG. 5C shows the results of Nanoparticle Tracking Analysis (NTA) of murine PBMCs yielding IEVs under different induction conditions.
FIG. 6 shows the results of flow cytometry analysis of isolated PBMCs and surface membrane proteins of PBMCs-derived IEVs.
FIG. 7 is a graph showing the results of protein content expression of PBMC-IEVs induced in different ways using western blotting.
FIG. 8 shows HE staining of lung tissue from PBMCs-derived IEVs for treating acute respiratory distress syndrome.
FIG. 9 is a graph showing the wet/dry weight ratio results of the lung of PBMCs-derived IEVs treating acute respiratory distress syndrome in lung tissue.
FIG. 10 shows the results of PBMCs-derived IEVs for treating enteritis. Fig. 10A shows that the IEVs treatment significantly reduced the tendency of DSS-induced colitis mice to lose weight. FIG. 10B shows that IEVs treatment significantly alleviates the symptoms of DSS induction leading to significant reduction in colon length, severe colonic epithelial and crypt destruction, and massive infiltration of inflammatory cells. Fig. 10C is a HE staining pattern showing that the IEVs can inhibit DSS-induced enteritis.
Fig. 11 shows that PBMC-derived IEVs were able to induce apoptosis of enteritis mouse cd3+ T cells.
Fig. 12 shows that PBMS-derived IEVs inhibit activation of Th17 cells in enteritis mice.
FIG. 13 shows a state diagram of red blood cell lysate processing red blood cells to generate vesicles.
FIGS. 14A-14B are particle sizes of primary flow assay vesicles and analysis of expression of the apoptotic surface marker Annexin V. FIG. 14A is a graph showing the results of red blood cell lysate treatment of red blood cells. FIG. 14B shows the results of treatment of hBMSCs with red blood cell lysate.
FIG. 15 shows the results of red blood cell-derived vesicles for the treatment of enteritis. FIG. 15A is a graph showing the weight loss (%) of mice; FIG. 15B is a photograph of colon tissue and length measurement (cm) of the mice.
FIG. 16 shows hair regrowth in the depilatory areas of mice of each group.
FIG. 17 shows the skin tissue structure of the dehairing area of each group of mice.
FIG. 18 is HE staining of lung tissue from mice of each group.
Fig. 19 shows the skin wound healing of each group of mice.
FIGS. 20A-20C are results of IEVs Nanoparticle Tracking Analysis (NTA) using flow cytometry to induce PBMC production at different temperatures. Fig. 20A is a graph of the number of IEVs produced. FIG. 20B is a graph of particle size for the production of IEVs. Fig. 20C is a potential diagram for IEVs generation.
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.
Herein, the "erythrocyte lysate" is derived from the wuhansai wilfordii biotechnology company, cat# G2015.
The IEVs in the embodiments of the present invention are short for inducible vesicles, which may be referred to as inducible vesicles, and 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 taken 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.
As used herein, "PBMC" refers to peripheral blood mononuclear cells, which are cells having a single nucleus in peripheral blood, including lymphocytes and monocytes.
Herein, the "erythrocyte lysate" is derived from the wuhansai wilfordii biotechnology company, cat# G2015.
Herein, "ab" is the same as "IEVs" or "ApopEVs". RBC-EVs (lysis) refer to red blood cell-derived vesicles induced by lysates.
EXAMPLE 1 obtaining of PBMC-derived IEVs
1. Human PBMC isolation experiment
Anticoagulation tube for blood collection, dilution of blood with PBS (PBS: peripheral blood=1:1 diluted blood), adding into Fumaisi PBMC separation tube (preloaded, containing separation liquid), centrifuging for 10min at 800g, collecting the centrifuged white membrane layer (as shown in FIG. 1) to a new 50mL centrifuge tube, washing twice with appropriate amount of PBS 300g,10min, re-suspending cell mass with RPMI (without FBS) for cell count, and adjusting cell concentration to 1X10 after cell count 6 Culture medium/mL was added to a 10cm dish for cultivation.
2. Murine PBMC isolation experiments
Ficoll-hypaque density gradient centrifugation of PBMC cells (Solarbio various animal peripheral blood lymphocyte separation medium kit):
1. blood collection: the left thumb and index finger grasp the ears and the back skin of a mouse (C57 BL/6WT 12 Zhou Xiongxing mouse), the tail is fixed by the small finger, the left forelimb of the mouse is lightly pressed on the heart part of the sternum by the middle finger, the ring finger is pressed on the abdomen, the thumb is twisted, the eye skin on the blood taking side is lightly pressed, the eyeball is made to be hyperemic and protrudes, the eyeball is clamped by the elbow forceps, the directions of the thumb and the index finger are twisted according to the need, the blood vertically flows into a centrifuge tube from the eyeorbit at different speeds, the heart part of the mouse is lightly pressed by the middle finger at the same time, so as to accelerate the blood pumping speed of the heart, and when the blood flows out, the mouse is killed by an dislocation method. The method takes 0.8-1.2 mL of whole blood of the mice, and the whole blood is injected into an EDTA-K2 anticoagulation tube and is gently shaken.
2. Dilution: adding an equal volume of diluent or PBS at room temperature, and gently beating and shaking uniformly;
3. sample adding: taking a 15mL centrifuge tube, sucking the separating liquid with the same volume as that of the 1+2 step into the centrifuge tube, slowly spreading diluted blood on the liquid level of the separating liquid by a pipette tip along the tube wall, (adding 3mL of separating liquid when the volume of the blood after the 1+2 dilution is less than 3mL, and adding the separating liquid with the same volume when the volume of the blood is greater than or equal to 3 mL).
4. And (3) centrifuging: at room temperature, 500g,15min, +.4 ∈0, 4 layers from the bottom of the tube to the liquid surface after centrifugation, red blood cells and granulocyte layers, a separated liquid layer, a mononuclear cell layer and a plasma layer in sequence.
5. And (3) recycling: the pipette is inserted directly into the buffy coat (or the upper plasma is aspirated first) and the buffy coat (as in fig. 2) is gently aspirated into a new 15mL centrifuge tube.
6. Washing: at least 3 times the cell volume of PBMC were added with PBS at room temperature, 400g,5min, twice.
7. And (3) flow detection: appropriate amount of cells were taken for CD11b and CD45 staining and flow-on-machine detection.
8. Cell count: the supernatant was discarded, 1mLRPMI-1640 medium (containing 10% fetal bovine serum) was added, and the mixture was blown and mixed well to prepare PBMC cell suspensions.
9. Cell culture: cell concentration was adjusted to 1X10 after cell counting 6 Culture medium/mL was added to a 10cm dish for cultivation.
3. Acquisition of PBMC-derived IEVs
The extracted PBMC cells were isolated and inoculated to different stimuli (PBMC+STS 500nM 16h, PBMC+ethanol 200mM 16h, PBMC+ethanol 800mM 16h, PBMC+H) 2 O 2 200μM 16h，PBMC+H 2 O 2 Apoptosis is induced in a culture dish of 800 mu M16h,PBMC+UV 4h,PBMC+45 ℃ for 6 h), and STS group, alcohol group and hydrogen peroxide group are cultured in a 5% carbon dioxide incubator at 37 ℃; ultraviolet ray set ultraviolet crosslinking instrument 300mJ/cm 2 Treating for 4 hours, and treating the heated group in a water bath kettle at 45 ℃ for 6 hours.
The culture medium of each group is RPMI-1640 culture medium (containing 10% foetal calf serum), such as STS or ethanol or H is added 2 O 2 Or UV or heat treatment.
The following method is adopted to separate and extract IEVs: after induction of apoptosis, the cell status was checked to confirm that most cells were apoptotic (fig. 3), and the isolation of the IEVs was performed according to the IEVs extraction normalization procedure, and the technical route is shown in fig. 4:
(1) Blowing and beating the separated cells by a liquid transfer device, and collecting the cells and supernatant to a 15mL centrifuge tube;
(2)800g，10min，4℃；
(3) The supernatant was transferred to a 1.5mLEP tube (note that the supernatant was not discarded);
(4)2000g，10min，4℃；
(5) Collecting supernatant, applying to a high-speed refrigerated centrifuge, and discarding precipitate;
(6) 16,000g,30min,4 ℃ (pre-cooling in advance);
(7) The supernatant was discarded, resuspended in PBS and transferred to a 1.5mLEP tube;
(8) 16,000g,30min,4 ℃ (pre-cooling in advance);
(9) The supernatant was discarded, the AB pellet was resuspended in sterile PBS and stored in a refrigerator at 4 ℃.
EXAMPLE 2 analysis of PBMC-derived IEVs
Analysis of the extracted IEVs by flow cytometry showed that the particle diameter distribution of the IEVs was mainly concentrated below 1um from 1h to 24h after induction, accounting for about 95% (fig. 5A, which is the result of murine pbmc+sts 500nm 16h group in example 1). Nanoparticle Tracking Analysis (NTA) results showed that PBMCs of human (fig. 5B, table 1) and murine (fig. 5C, table 2) origin produced IEVs under different induction conditions, and that the produced IEVs were not significantly different in particle size and potential from those produced under STS induction.
Analysis of the isolated human PBMCs from example 1 above using flow cytometry showed that the isolated PBMCs were CD45 positive, resulting in isolated PBMCs (FIG. 6). Further analysis of the surface membrane proteins of PBMCs-derived IEVs showed that the IEVs were able to express the general surface protein CD9 of extracellular vesicles (fig. 6).
TABLE 1 human PBMC-derived IEVs
TABLE 2 murine PBMC derived IEVs
Protein content expression of the different induced PBMC-IEVs was verified by western blotting, and the results showed that the IEVs produced by the different induction modes were each capable of specifically expressing Annexin V, integrin alpha 5 and Syntexin 4 (FIG. 7). The above 3 proteins are characteristic protein markers that distinguish between MSCs-derived IEVs and exosomes.
EXAMPLE 3 use of PBMC-derived IEVs
1. Treatment of Acute Respiratory Distress Syndrome (ARDS)
The incidence and mortality of Acute Respiratory Distress Syndrome (ARDS) are high, the pathogenesis is not completely elucidated so far, and no specific treatment method exists at present. The present study observes the therapeutic effect of tracheal instillation and tail vein infusion of human Peripheral Blood Mononuclear Cell (PBMC) -derived IEVs (or "ApopEV") in mice with acute respiratory distress syndrome models.
(1) Experimental method
1) Experimental animals: c57BL/6 mice, with unlimited sex, 8-12 weeks of age, SPF grade.
2) The study drug was a human PBMC-ApopEV suspension prepared as follows: PBMC of human fresh whole blood (8 ml human whole blood separated by about 1X 10) were isolated by Ficoll density gradient centrifugation 7 PBMC), PBS wash, addition of staurosporineApoptosis was induced by bacteriocin (STS), liquid was collected 16 hours, centrifuged at 800g at 4℃for 10 minutes, supernatant was collected, centrifuged at 2000g at 4℃for 10 minutes, supernatant was collected, centrifuged at 16000g at 4℃for 30 minutes, supernatant was discarded, 1mL PBS was resuspended, and supernatant was discarded again after centrifugation at 16000g at 4℃for 30 minutes. Adding PBS to prepare PBMC-ApopEV suspension, and instilling 1X 10 in trachea 6 30 μl, 1×10 intravenous infusion 6 200. Mu.L of the suspension was stored at 4℃before use.
3) ARDS mouse model construction and treatment: c57BL/6 mice of 8-12 weeks old were randomly divided into 4 groups of 3-5 animals each, which were normal Control group (Control), model group (LPS group), human PBMC-ApopEV airway instillation treatment group (PBMC-ApopEV-L group) and human PBMC-ApopEV tail intravenous injection treatment group (PBMC-ApopEV-S group). The water is forbidden after 6 hours before operation, and 10ml/kg of 4% chloral hydrate is used for anesthesia. Both model and treatment groups were first tracheally instilled with 30 μl LPS (5 mg/kg), and after 4 hours, treatment groups were tracheally instilled with 30 μl of PBMC-ApopEV (ApopEV about 1×10) 6 By tail vein injection of 200. Mu.L of PBMC-ApopEV (ApopEV about 1X 10) 6 And) were sacrificed 24 hours after molding (animals were sacrificed from the start of LPS administration to 24 hours).
(2) Experimental results
1) HE staining of lung tissue: control in fig. 8 can be seen for HE stained sections of the normal group; the LPS group can be seen to have serious destruction of alveolar tissue structure, obvious congestion and edema, massive inflammatory cell infiltration, obvious thickening of alveolar space and structural disturbance; in the PBMC-ApopEV treatment group, the congestion and inflammatory infiltration of the lung tissues of the mice are obviously reduced, the exudation of the lung tissues is reduced, and the alveolar space thickening and the structural disorder are also obviously improved. Wherein the tracheal instillation group (PBMC-ApopEV-L group) had similar efficacy to the systemic intravenous infusion group (PBMC-ApopEV-S group) (FIG. 8).
2) Wet/dry weight ratio of lung: the lung wet weight is weighed, and the lung dry weight is obtained by baking the lung wet weight to constant weight. The lung W/D value was calculated according to the formula. The results showed that LPS stimulation significantly increased the lung wet/dry weight ratio in mice, whereas PBMC-ApopEV treatment by tracheal instillation or tail vein infusion significantly decreased this ratio, but there was no difference between the treatment groups (fig. 9).
2. Treating enteritis
(1) Research method
1) Experimental animals: c57BL/6 mice, with unlimited sex, 8-12 weeks of age, SPF grade.
2) Preparation of human PBMC-ApopEV suspension: PBMC of human fresh whole blood (8 ml human whole blood separated by about 1X 10) were isolated by Ficoll density gradient centrifugation 7 PBMC), PBS, 500nM staurosporine (RPMI-1640 medium with 500nM STS) was added to induce apoptosis, liquid was collected 16 hours, 800g was centrifuged at 4℃for 10 minutes, supernatant was collected, 2000g was centrifuged at 4℃for 10 minutes, supernatant was collected, 16000g was centrifuged at 4℃for 30 minutes, supernatant was discarded, 1mLPBS was resuspended, 16000g was centrifuged at 4℃for 30 minutes, and supernatant was discarded again. PBMC-ApopEV suspension was prepared by adding PBS and infused intravenously 1X 10 6 200. Mu.L, the suspension was stored at 4℃before use.
3) Construction and treatment of a DSS-induced mouse enteritis model: the 8-12 week old C57BL/6 mice were given 3% (w: v) dextran sulfate sodium (DSS, molecular weight: 36,000-50,000Da;MP Biochemicals,160110) drinking water for 10 days to induce colitis. On day 3 of DSS treatment, mice were injected with 200 μl PBMC-ApopE via tail vein (ApopEV about 1×10) 6 And, a) mice were sacrificed 10 days after molding.
(2) Experimental results
1) As shown in fig. 10, PBMCs were able to inhibit DSS-induced enteritis. The body weight of the colitis mice was significantly reduced and the IEVs treatment significantly reduced the tendency of DSS-induced weight loss in the colitis mice (fig. 10A). DSS induction resulted in a significant decrease in colon length, severe colonic epithelial and crypt destruction, and massive infiltration of inflammatory cells, whereas the IEVs treatment significantly alleviated these symptoms (fig. 10b,10 c).
2) The results of fig. 11 show that PBMC IEVs are able to modulate enteritis mouse T cell overactivation. CD3 capable of remarkably inducing excessive activation of enteritis mice by IEVs treatment + Apoptosis of T cells. Wherein the PBMS-derived IEVs are superior to PBMS exosomes (exo or exosomes).
3) PBMS-derived IEVs inhibit activation of Th17 cells in enteritis mice. The treatment with IEVs significantly induced Th17 cells in the circulation of enteritis mice (fig. 12).
The Plasma-MV group, the Plasma-exo low group and the Plasma exo high group refer to large vesicles in Plasma in an uninduced state, an exosome (small vesicles) low dose group and an exosome (small vesicles) high dose group respectively.
Example 4 obtaining of red blood cell derived vesicles
1. Erythrocyte origin and separation procedure
A10 mL blood sample from a volunteer was placed in an EDTA anticoagulation tube, and an equal volume of PBS buffer was added and mixed 1:1 to prepare a cell suspension. Taking 4 new 15mL centrifuge tubes, adding 5mL human peripheral blood lymph separating liquid (Solarbio, #P8610) into each tube in advance, slowly adding 5mL mixed liquid of blood and PBS into the upper layer of lymph along the tube wall, centrifuging for 30min at 4 ℃ by using a low-speed horizontal centrifuge 550g, and reducing the speed to 0 at 4. After centrifugation, the tube interior can be divided into 4 layers: the uppermost layer is pale yellow diluted plasma; the second layer is cloud-like white membrane layer, which is Peripheral Blood Mononuclear Cells (PBMC); the third layer is a lymph separation liquid layer; the fourth layer is red blood cells which sink to the bottom of the tube. Removing all liquid above erythrocyte layer, sucking erythrocyte, adding into 40ml buffer solution, 400g, centrifuging at 4deg.C for 10min, washing off residual blood plasma, lymph separating liquid and centrifuging at low speed to remove part of blood platelet. This centrifugation step may be repeated 1 time. The red blood cells obtained may be resuspended in an appropriate amount of PBS.
2. Induction of Red Blood Cell (RBC) production vesicles using red blood cell lysates
10mL of red blood cell lysate was used for lysis per 1mL of whole blood-derived red blood cells. Treating the erythrocytes with the erythrocyte lysate for 4 hours, blowing cells at the bottom of the culture dish, and collecting all the detached cells and cell supernatants; centrifuging at 4deg.C for 10min under 800g to obtain supernatant; 2000g, centrifuging at 4 ℃ for 10min, and leaving a supernatant; centrifuging 16000g of supernatant at 4deg.C for 30min, discarding supernatant, and retaining vesicle sediment; adding 1mL PBS, blowing to resuspend the sediment, centrifuging at 4 ℃ for 30min, discarding the supernatant, and reserving vesicle sediment; the vesicle pellet can be resuspended in an appropriate amount of PBS for use.
As seen using a fluorescence microscope, as shown in fig. 13 (30 min of lysis), treatment of RBCs with red blood cell lysate revealed a morphological change in the apparent shrinkage of the cell membrane and the generation of vesicles (red arrows). Lysates can induce RBCs to die in the form of released "vesicles" rather than cell burst.
Example 5 isolation and identification of vesicles from red blood cell lysate induced RBC production
Identification of the collected vesicles: taking a proper amount of vesicles, re-suspending the vesicles with 100 mu L of Binding buffer according to the requirements of BD brand Annexin V/PI apoptosis staining specification, adding 2 mu LAnnexin V dye (a dye specifically Binding to Phosphatidylserine (PS) on the surface of a membrane), incubating for 15min at room temperature, and detecting the proportion of Annexin V positive vesicles, namely the proportion of PS positive vesicles expressed on the surface of the membrane by a flow cytometer.
The particle size of the vesicles is primarily detected by flow and the expression of the apoptosis surface marker Phosphatidylserine (PS) is analyzed. As shown in fig. 14, vesicles produced after RBC death induced by lysates highly expressed PS (fig. 14A), have surface features that are strongly positive for PS like stem cell-derived IEVs (14B), presumably with similar metabolic pathways and functions in vivo.
Wherein, the use of erythrocyte lysate induces hBMSCs to generate vesicles: hBMSCs (passage 8) were treated with the erythrocyte lysate for 4 hours, centrifuged at 800g at 4℃for 10min, and the supernatant was left; 2000g, centrifuging at 4 ℃ for 10min, and leaving a supernatant; centrifuging 16000g of supernatant at 4deg.C for 30min, discarding supernatant, and retaining vesicle sediment; adding 1ml PBS, blowing to resuspend the sediment, centrifuging at 4 ℃ for 30min, discarding the supernatant, and reserving vesicle sediment; the vesicle pellet can be resuspended in an appropriate amount of PBS for use.
Specific steps of the lysate inducing the generation of vesicles from RBC: the specific procedure is as described in example 4.
Example 6 application of red blood cell lysate to induce RBC production of vesicles
1. The experimental method comprises the following steps:
the ulcerative colitis of mice was induced with dextran sodium sulfate salt (Dextran Sulfate Sodium Salt, DSS) solution, and whether red blood cell derived vesicles (RBC-EVs) induced by lysate had therapeutic effect on enteritis of mice was observed.
8 weeks, male, C57 mice were selected for the experiment, 2 per group. The Control group (Control group) was normally fed with water and with food, and the modeling group (DSS group) and the treatment group (hBMSC-ApoEVs group, RBC-EVs group) were both free to drink 2.5% DSS solution, and were normally fed for 8 days, with daily recordings of body weight changes and hematochezia levels. Wherein the treatment group was treated by tail vein injection of the corresponding vesicles on day 3, the hBMSC-ApoEVs groups were each injected with one 10cm dish of hBMSCs (about 3X 10. Times.6 hBMSCs) derived vesicles (method similar to the vesicle acquisition method in China patent application 202110077486.9)), and the RBC-EVs groups were each injected with 10. Times.8 RBC lysed vesicles (method of example 5). The building block was injected with only an equal volume of PBS. On day 8, colon tissue of the mice was taken, photographed, measured for length, tissue fixed, and sections embedded.
2. Experimental results:
the results are shown in fig. 15, and it can be seen from the graph that the tail vein injection of RBC-EVs can obviously improve the symptoms of mice body weight reduction and colon shortening caused by acute enteritis, which indicates that vesicles derived from RBC generation have certain disease treatment and application value.
Example 7 Hair growth promotion by Red Blood Cells (RBC) or Red blood cell vesicles (RBC-EVs)
1. The experimental steps are as follows:
balb/c mice are selected, the experiment is started in the telogen period (week 6), the back shaving is carried out by taking the parallel vertebrae as the long axis direction, the dehairing agent dehairing is carried out, and a dehairing area of 2cm multiplied by 4cm is manufactured for modeling; the next day of modeling intervention, 1X 10 weekly tail intravenous injection 9 EV (total 3X 10) generated by RBC lysis of mice 10 EV), PBS group was injected with equal RBC-EVs (lysis) group volume PBS, RBC group was injected once weekly by 1 x 10 tail vein 9 Mouse RBCs. Mice were observed daily for hair growth and photographed to record the area of new hair. The observation period is 4 weeks, the skin of the dehairing area of the mouse is taken after 4 weeks, the tissue section is dyed, and the number, the size, the proliferation condition and the like of hair follicles are detected.
Tissue staining: skin tissue of a dehairing area of a mouse is taken, fixed and dehydrated, longitudinal sections of the skin tissue are taken, immunofluorescent staining is carried out, blue color indicates cell Nucleus (nucleuses), green color indicates cytoskeleton (action), and the number of hair follicles is counted.
The method for obtaining vesicles from the RBC-EVs (lysis) group was the same as that for the red blood cell lysate induced Red Blood Cell (RBC) production in example 4.
2. Experimental results:
as shown in fig. 16, the neonatal hair area of RBC group and RBC-EVs (lysis) group mice was significantly higher than PBS group at day 21. As shown in fig. 17, the number of hair follicles in the dehairing area was significantly increased in the RBC group and RBC-EVs (lysis) group mice. Experimental results indicate that RBC or RBC-EVs (lysis) have the effects of obviously promoting hair regeneration in the depilatory area of mice and increasing the number of hair follicles.
Example 8 therapeutic use of red blood cell vesicles (RBC-EVs) in mice Acute Respiratory Distress Syndrome (ARDS) and diabetic wound healing models
1. ARDS model
1.1 experimental procedure:
8 week old C57BL/6 mice were randomly assigned to 3 groups, a normal control group (control), an ARDS model group, and a RBC-EVs (lysis) tracheal instillation treatment group, respectively. The water is forbidden after 6 hours before operation, and 10ml/kg of 4% chloral hydrate is used for anesthesia. The model and treatment groups were each first air instilled with 30 μl of LPS (5 mg/kg based on the weight of the mice) and the normal group instilled with an equal volume of PBS. After 4 hours, the treatment group instilled 50. Mu.L of RBC-lysis-EV (about 3X 10) 10 And a normal set and ARDS building block instill equal volumes of PBS. Mice were sacrificed 48 hours after molding and fixed sections of lung tissue were stained.
Among them, the method for obtaining vesicles of RBC-EVs (lysis) group was the same as the method for inducing Red Blood Cells (RBC) to generate vesicles by using the red blood cell lysate of example 4.
1.2 experimental results:
HE staining of lung tissue: as can be seen in fig. 18, the ARDS group showed severe destruction of alveolar tissue structure, obvious congestion edema, massive inflammatory cell infiltration, obvious thickening of alveolar spaces and structural disturbance, compared to the control group. RBC-EVs (lysis) trachea instillation group, the congestion and inflammatory infiltration degree of the lung tissue of the mice are obviously reduced, the exudation of the lung tissue is reduced, and the conditions of alveolar space thickening and structural disorder are also obviously improved.
2. Model for healing type 1 diabetes wound
2.1 experimental procedure:
6 week old C57BL/6 mice were randomly divided into 3 groups, namely a normal control group (control), a type 1 diabetes mellitus (T1 DM) model group and a RBC-EVs (lysis) local injection treatment group. All mice were fed adaptively for 1 week. Fasted, water-inhibited for 12 hours before the modeling started, fasting body weight and blood glucose were measured and recorded. The mice were given a dose of Streptozotocin (STZ) (50 mg/kg.d.times.1/d for 5 consecutive days) calculated on an empty stomach basis. And on the 7 th day after administration, continuous 3d tail vein blood sampling is carried out to measure random blood sugar, and the average value of the blood sugar is more than or equal to 16.7mmol/L, thus the modeling is successful. The backs of the mice were shaved, the depilatory cream was removed, the back skin was cut off with scissors, an open skin wound of 1cm×1cm was made, and 100 μl of RBC-lysis-EV (about 3×10) was locally injected once in the treatment group after wound modeling 10 Individual), normal and T1DM model groups were locally injected with equal volumes of PBS. Each group of mice was observed daily for wound healing and photographed for recording.
Among them, the method for obtaining vesicles of RBC-EVs (lysis) group was the same as the method for inducing Red Blood Cells (RBC) to generate vesicles by using the red blood cell lysate of example 4.
2.2 experimental results:
As can be seen in fig. 19, the wound healing rate was slower in the T1DM group compared to the control group. On day 7 after wound molding, the degree of wound healing of the RBC-EVs (lysis) local injection treatment group is obviously increased, which shows that the RBC-EVs (lysis) can obviously promote the skin wound healing of the diabetic mice.
EXAMPLE 9 analysis of PBMC-derived IEVs
1. Separation of human PBMC (Fumeis PBMC separation tube (preloaded, containing separation liquid)
Anticoagulation tube for blood collection, dilution of blood with PBS (PBS: peripheral blood=1:1 diluted blood), adding into Fumaisi PBMC separating tube, centrifuging for 10min at 800g, collecting the centrifuged white membrane layer, adding into new 50mL centrifuge tube, washing with appropriate amount of PBS 300g for 10min twice, re-suspending cell mass with RPMI (without FBS) for cell count, and adjusting cell concentration to 1×10 after cell count 6 Culture medium/mL was added to a 10cm dish for cultivation.
2. Induction of PBMC apoptosis:
PBMC+STS 500nM 12h，PBMC+42℃6h，PBMC+43℃6h，PBMC+45℃6h，PBMC+48℃6h。
3. IEVs extraction:
the procedure is as in example 1.
Zetaview detection: ddH for precipitation of IEVs 2 O was resuspended, diluted to the appropriate magnification, and run on-machine (PMX-420-12F-R5) with the results shown in FIGS. 20A-20C.
Claims (10)
1. Use of a sample derived from blood for the preparation of vesicles, wherein the vesicles are inducible vesicles.
2. The use according to claim 1, wherein the blood comprises plasma, whole blood;
Preferably, the blood is peripheral blood;
or preferably, the blood contains blood cells;
more preferably, the sample comprises peripheral blood mononuclear cells;
or more preferably, the sample comprises red blood cells;
preferably, the peripheral blood mononuclear cells are isolated or non-isolated cells derived from blood.
3. The use according to claim 2, wherein the inducible vesicles are vesicles produced by induction of apoptosis by external forces during normal survival of cells in a sample derived from blood;
preferably, the external force comprises at least one of staurosporine addition, ethanol addition, hydrogen peroxide addition, ultraviolet irradiation, starvation, lysate, thermal stress, or mechanical force;
preferably, the external force is heat treatment;
preferably, the heat treatment is carried out in the range of 38 ℃ to 60 ℃;
preferably, the heat treatment is carried out in the range of 40 ℃ to 55 ℃;
preferably, the heat treatment is carried out in the range of 40 ℃ to 52 ℃;
preferably, the heat treatment is carried out in the range of 42 ℃ to 52 ℃;
preferably, the heat treatment is carried out in the range of 42 ℃ to 50 ℃;
Preferably, the time of the heat treatment is 3 to 20 hours;
preferably, the time of the heat treatment is 3 to 15 hours;
preferably, the time of the heat treatment is 3 to 12 hours;
preferably, the time of the heat treatment is 3 to 10 hours.
4. The use of claim 3, wherein said vesicles are positive for annexin v, interserin alpha 5 and syncaxin 4 expression;
preferably, the vesicles have a diameter of 0.03-6 μm;
preferably, the vesicles have a diameter of 0.03-4.5 μm;
preferably, the vesicles have a diameter of 0.03-1 μm;
preferably, the vesicles have a diameter of 0.04-1 μm;
preferably, the vesicles have a diameter of 0.05-1 μm;
preferably, the vesicles have a diameter of 0.1-1 μm;
preferably, the vesicles have a diameter of 0.15-1 μm.
5. A method of preparing vesicles, comprising subjecting a sample derived from blood to external force to obtain the vesicles;
preferably, the blood is peripheral blood;
or preferably, the blood contains blood cells;
more preferably, the sample comprises peripheral blood mononuclear cells;
or more preferably, the sample comprises red blood cells;
Preferably, the peripheral blood mononuclear cells are isolated or non-isolated cells derived from blood.
Preferably, the inducible vesicles are vesicles produced by induction of apoptosis by external forces during normal survival of cells in a sample derived from blood;
preferably, the external force comprises at least one of staurosporine addition, ethanol addition, hydrogen peroxide addition, ultraviolet irradiation, starvation method, lysate, thermal stress method or mechanical force method;
preferably, the external force is a heat treatment process;
preferably, the heat treatment is carried out in the range of 38 ℃ to 60 ℃;
preferably, the heat treatment is carried out in the range of 40 ℃ to 55 ℃;
preferably, the heat treatment is carried out in the range of 40 ℃ to 52 ℃;
preferably, the heat treatment is carried out in the range of 42 ℃ to 52 ℃;
preferably, the heat treatment is carried out in the range of 42 ℃ to 50 ℃;
preferably, the time of the heat treatment is 3 to 20 hours;
preferably, the time of the heat treatment is 3 to 15 hours;
preferably, the time of the heat treatment is 3 to 12 hours;
preferably, the time of the heat treatment is 3 to 10 hours;
Preferably, the vesicles are positive for Annexin V, intigrin alpha 5 and Syntaxin 4 expression;
preferably, the vesicle is an inducible vesicle;
preferably, the vesicles have a diameter of 0.03-6 μm;
preferably, the vesicles have a diameter of 0.03-4.5 μm;
preferably, the vesicles have a diameter of 0.03-1 μm;
preferably, the vesicles have a diameter of 0.04-1 μm;
preferably, the vesicles have a diameter of 0.05-1 μm;
preferably, the vesicles have a diameter of 0.1-1 μm;
preferably, the vesicles have a diameter of 0.15-1 μm.
6. The method of claim 5, further comprising extracting said vesicles;
preferably, the method comprises the steps of:
(1) Obtaining peripheral blood mononuclear cells and culturing;
(2) Performing heat treatment on the peripheral blood mononuclear cells in a culture medium to induce release of the vesicles;
(3) Collecting the culture medium supernatant of step (2), and isolating the vesicles;
preferably, the vesicles are isolated from the medium by a method selected from the group consisting of polymer precipitation, immunoisolation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, and combinations thereof;
Preferably, the method for separating vesicles comprises the steps of separating by taking Annexin V, integrin alpha 5 and Syntexin 4 as markers;
preferably, the vesicles are isolated by a method selected from ultracentrifugation.
7. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 6, wherein the sample is derived from a mammal;
preferably, the mammal is selected from primate or murine;
preferably, the primate is a human.
8. A vesicle, characterized in that it is obtained by the method of claim 5 or 6.
9. A pharmaceutical composition comprising the vesicle of claim 8 and a pharmaceutically acceptable adjuvant;
preferably, the pharmaceutical composition is in a form selected from the group consisting of lyophilized powder for injection, tablet, capsule, or patch.
10. Use of a vesicle according to claim 8 or a pharmaceutical composition according to claim 9 for the preparation of a product for the treatment or prevention or amelioration of a disease or complications of said disease;
preferably, the product comprises a medicament, a food product, a health product, a cosmetic product, an additive or an intermediate product;
preferably, the disease comprises a pulmonary disease or an intestinal disease or diabetes;
Preferably, the product is for promoting hair follicle repair and/or hair regeneration;
more preferably, the pulmonary disease is acute respiratory distress syndrome;
or more preferably, the intestinal disorder is enteritis;
preferably, the enteritis is acute enteritis;
preferably, the medicament is for ameliorating symptoms of weight loss or colon shortening in mice caused by acute enteritis;
preferably, the diabetes is type 1 diabetes;
preferably, the medicament is for promoting type 1 diabetic wound healing.
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