WO2023169594A1 - 血液来源的样品在制备囊泡中的应用 - Google Patents

血液来源的样品在制备囊泡中的应用 Download PDF

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WO2023169594A1
WO2023169594A1 PCT/CN2023/085257 CN2023085257W WO2023169594A1 WO 2023169594 A1 WO2023169594 A1 WO 2023169594A1 CN 2023085257 W CN2023085257 W CN 2023085257W WO 2023169594 A1 WO2023169594 A1 WO 2023169594A1
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vesicles
cells
hours
heat treatment
hydrogen peroxide
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PCT/CN2023/085257
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English (en)
French (fr)
Inventor
寇晓星
施松涛
曹泽源
孟博文
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中山大学
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Priority claimed from CN202210220389.5A external-priority patent/CN116769708A/zh
Priority claimed from CN202210624896.5A external-priority patent/CN116763817A/zh
Priority claimed from CN202210625524.4A external-priority patent/CN116769709A/zh
Application filed by 中山大学 filed Critical 中山大学
Publication of WO2023169594A1 publication Critical patent/WO2023169594A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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

Definitions

  • the invention belongs to the field of biomedicine and relates to the application of blood-derived samples in preparing vesicles.
  • Extracellular vesicles are nanoscale carriers secreted by cells containing proteins, nucleic acids and various cytokines. Extracellular vesicles can act on target cells in an endocrine or paracrine manner and play an important role in the process of intercellular material transfer and information exchange. Research has found that information exchange mediated by extracellular vesicles plays an important regulatory role in physiological or pathological processes of the body, involving immune regulation, tumor growth, angiogenesis, damage repair, etc. Extracellular vesicles are an important medium for intercellular communication, allowing intracellular bioactive molecules to be transferred from one cell to another to function. Therefore, it is becoming increasingly clear that these vesicles are involved in many physiological processes, which will provide opportunities for applications in the treatment of disease.
  • Exosomes are extracellular vesicles with a diameter of about 30-150 nm, which contain components such as RNA, lipids, and proteins. Exosomes are widely involved in various physiological/pathological regulation of the body and can be used for diagnosis, treatment and prognosis assessment of various diseases. So far, mesenchymal stem cells (MSCs) are considered to be the cells with the strongest ability to produce exosomes. Numerous studies have found that exosomes derived from MSCs can simulate the biological functions of MSCs and play an important regulatory role in promoting cell growth and differentiation and repairing tissue defects.
  • MSCs mesenchymal stem cells
  • ARDS Acute respiratory distress syndrome
  • Intrapulmonary causes of ARDS include severe pneumonia, drowning, aspiration, pulmonary contusion, inhalation of toxic substances, etc.
  • Extrapulmonary causes include severe infection, severe trauma, shock, severe pancreatitis, poisoning, major surgery, and after cardiopulmonary resuscitation. They are common.
  • the pathological basis is acute lung injury.
  • ARDS In addition to well-known risk factors for ARDS, exposure to high ozone levels and low vitamin D plasma concentrations have also been found to be predisposing environments. ARDS treatment is expensive. However, apart from protective lung ventilation, treatment of the primary disease and supportive care, there is still a lack of targeted treatment. Based on the latest research, although overall survival in ARDS is improving, considering the in-hospital mortality in several observational studies, which can be explained by risk factors, availability of diagnosis, ability to identify ARDS, and some selection bias affecting clinical diagnosis, these tests Data indicate that ARDS remains underdiagnosed and undertreated worldwide, and current definitions of ARDS are inadequate in most clinical situations. Drug-based prevention strategies remain a major challenge, and some recent studies have focused on improving the prognosis of acute respiratory distress syndrome in this disease, but high mortality and disabling complications remain to be improved.
  • Colitis also known as non-specific ulcerative colitis, has many types and causes. The onset is often slow and the severity of the disease varies. The main clinical manifestations are diarrhea, abdominal pain, mucus and bloody purulent stools, tenesmus, and even constipation. , unable to pass stool within a few days, sometimes diarrhea and sometimes constipation, often accompanied by weight loss and fatigue, etc., often recurring. Abdominal pain is usually dull or crampy, often located in the left lower abdomen or lower abdomen. Other symptoms include loss of appetite, abdominal distension, nausea, vomiting, and hepatomegaly. There may be tenderness in the left lower abdomen, and sometimes the spasmodic colon can be palpated. Common systemic symptoms include weight loss, fatigue, fever, anemia, etc. In a small number of patients, during the chronic course of the disease, their condition suddenly worsens or becomes explosive for the first time, causing severe diarrhea and causing great suffering to people or animals.
  • Extracellular vesicles are nanoscale carriers secreted by cells containing proteins, nucleic acids and various cytokines. Extracellular vesicles can act on target cells in an endocrine or paracrine manner and play an important role in the process of intercellular material transfer and information exchange. Research has found that information exchange mediated by extracellular vesicles plays an important regulatory role in physiological or pathological processes of the body, involving immune regulation, tumor growth, angiogenesis, damage repair, etc.
  • Red blood cells especially normal mature red blood cells, have no nuclei or organelles. They are biconcave disk-shaped with a diameter of 7 to 8 ⁇ m, and the thinnest part in the center is only about 1 ⁇ m. This special structure is conducive to increasing its surface area for material exchange. Red blood cells are plastically deformable. When circulating in blood vessels throughout the body, they can pass through capillaries and sinusoids with smaller diameters after deformation.
  • the red blood cell membrane (RBCm) has osmotic fragility, and red blood cells can undergo hemolysis in hypotonic solutions, which is beneficial to the For the exchange of substances, the red blood cell membrane itself is suitable for transport within blood vessels.
  • Red Blood Cell Lysis Buffer or ACK Lysis Buffer
  • ACK Lysis Buffer is the simplest and easiest way to remove red blood cells. It uses lysis buffer to lyse red blood cells. It does not damage nucleated cells and can fully remove red blood cells.
  • Lysis solution lysis is a relatively mild method of removing red blood cells. It is mainly used for the separation and purification of tissue cells dispersed by enzyme digestion, the separation and purification of lymphocytes, and the removal of red blood cells in experiments such as tissue cell protein and nucleic acid extraction. The tissue cells obtained by lysis of red blood cell lysis solution do not contain red blood cells and can be further used for primary culture, cell fusion, flow cytometry analysis, separation and extraction of nucleic acids and proteins, etc.
  • the present invention provides a use of a blood-derived sample for preparing vesicles, which are inducible vesicles.
  • the blood includes plasma, whole blood.
  • the blood is peripheral blood.
  • the blood contains blood cells.
  • the sample comprises peripheral blood mononuclear cells.
  • PBMCs Peripheral blood mononuclear cells
  • lymphocytes and monocytes are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes. They are a useful source of cells because they are easy to collect and facilitate clinical application.
  • the volume, shape and specific gravity of mononuclear cells are different from other cells in peripheral blood.
  • the specific gravity of red blood cells and multinucleated leukocytes is around 1.092
  • the specific gravity of mononuclear cells is 1.075-1.090
  • the specific gravity of platelets is 1.030-1.035. Therefore, a solution between 1.075-1.092 that is close to isotonic is used for density gradient centrifugation, so that cells of a certain density are distributed according to the corresponding density gradient, and various blood cells and mononuclear cells can be separated.
  • the sample includes red blood cells.
  • the peripheral blood mononuclear cells are isolated or unisolated cells derived from blood.
  • the inducible vesicles are vesicles produced by inducing apoptosis by an external force during normal survival of cells in a sample derived from blood.
  • the external force includes addition of staurosporine, ultraviolet irradiation, starvation, or heat stress, or a combination of one or more thereof.
  • the external force is heat treatment.
  • the heat treatment is performed in the range of 38°C to 60°C. In some embodiments, the heat treatment is performed in the range of 40°C to 55°C.
  • the heat treatment is performed in the range of 40°C to 52°C. In some embodiments, the heat treatment is performed in the range of 42°C to 52°C. In some embodiments, the heat treatment is performed in the range of 42°C to 50°C. In some embodiments, the heat treatment time is 3 to 20 hours. In some embodiments, the heat treatment time is 3 to 15 hours. In some embodiments, the heat treatment time is 3 to 12 hours. In some embodiments, the heat treatment time is 3 to 10 hours. In some embodiments, the vesicles are positive for Annexin V, Integrin alpha 5 and Syntaxin 4 expression. In some embodiments, the vesicles are 0.03-6 ⁇ m in diameter.
  • the vesicles are 0.03-4.5 ⁇ m in diameter. In some embodiments, the vesicles are 0.03-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.04-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.05-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.1-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.15-1 ⁇ m in diameter. In some embodiments, the present invention provides a method of preparing vesicles, the method comprising subjecting a blood-derived sample to external force treatment to obtain the vesicles.
  • the present invention provides a method for preparing vesicles, the method comprising subjecting a sample derived from blood to external force treatment to obtain the vesicles, and the vesicles are inducible vesicles.
  • the invention provides a vesicle obtained by the above method.
  • the present invention provides the use of inducible vesicles derived from a blood sample or a pharmaceutical composition containing said vesicles in the preparation of a product for treating or preventing or ameliorating a disease or complications of said disease.
  • the invention provides a method of treating, preventing, or ameliorating a disease or complications of the disease, comprising administering the vesicles or the pharmaceutical composition to a patient or subject.
  • the products include pharmaceuticals, foods, health products, cosmetics, additives, or intermediate products.
  • the disease includes pulmonary disease or intestinal disease or diabetes.
  • the product is used to promote hair follicle repair and/or hair regeneration.
  • the lung disease is acute respiratory distress syndrome.
  • the intestinal disease is enteritis.
  • the enteritis is acute enteritis.
  • the medicament is used to improve symptoms of weight loss or colon shortening in mice caused by acute enteritis.
  • the diabetes is type 1 diabetes.
  • the medicament is used to promote type 1 diabetic wound healing.
  • the blood is peripheral blood.
  • the blood contains blood cells.
  • the sample comprises peripheral blood mononuclear cells.
  • the sample includes red blood cells.
  • the peripheral blood mononuclear cells are isolated or unisolated cells derived from blood.
  • the inducible vesicles are vesicles produced by inducing apoptosis by an external force during normal survival of cells in a sample derived from blood.
  • the inducible vesicles provided by the present invention using peripheral blood as a sample can be directly reused in the body of the original individual after being obtained in vitro. It has the advantages of convenient sampling and being derived from autologous sources, and therefore has excellent operability. sex and safety.
  • the external force includes one or more combinations of adding staurosporine, adding ethanol, adding hydrogen peroxide, ultraviolet irradiation, starvation method, lysis solution, thermal stress method, or mechanical force method.
  • the external force is heat treatment.
  • the heat treatment is performed in the range of 38°C to 60°C.
  • the heat treatment is performed in the range of 40°C to 55°C.
  • the heat treatment is performed in the range of 40°C to 52°C.
  • the heat treatment is performed in the range of 42°C to 52°C.
  • the heat treatment is performed in the range of 42°C to 50°C.
  • the heat treatment time is 3 to 20 hours.
  • the heat treatment time is 3 to 15 hours.
  • the heat treatment time is 3 to 12 hours.
  • the heat treatment time is 3 to 10 hours.
  • the concentration of hydrogen peroxide is 20-2000 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 50-1500 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 50-1200 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 50-1000 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 100-1200 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 100-1000 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 100-800 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 100-500 ⁇ M. In some embodiments, the concentration of hydrogen peroxide is 100-400 ⁇ M.
  • the concentration of hydrogen peroxide is 100-300 ⁇ M.
  • the hydrogen peroxide treatment time is 4 to 30 hours. In some embodiments, the hydrogen peroxide treatment time is 5 to 25 hours. In some embodiments, the hydrogen peroxide treatment time is 8 to 25 hours. In some embodiments, the hydrogen peroxide treatment time is 10 to 25 hours. In some embodiments, the hydrogen peroxide treatment time is 12 to 25 hours. In some embodiments, the hydrogen peroxide treatment time is 12 to 20 hours. In some embodiments, the vesicles are inducible vesicles.
  • the vesicles are positive for Annexin V, Integrin alpha 5 and Syntaxin 4 expression.
  • the vesicles are inducible vesicles. In some embodiments, the vesicles are 0.03-6 ⁇ m in diameter. In some embodiments, the vesicles are 0.03-4.5 ⁇ m in diameter. In some embodiments, the vesicles are 0.03-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.04-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.05-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.1-1 ⁇ m in diameter. In some embodiments, the vesicles are 0.15-1 ⁇ m in diameter.
  • the concentration of ethanol is 50-500 nM. In some embodiments, the concentration of ethanol is 100-500 nM. In some embodiments, the concentration of ethanol is 100-300 nM. In some embodiments, the ethanol treatment time is 4 to 30 hours. In some embodiments, the ethanol treatment time is 8 to 25 hours. In some embodiments, the ethanol treatment time is 12 to 20 hours.
  • the ultraviolet irradiation is treated with an ultraviolet cross-linker at 200-500 mJ/cm 2 for 1-8 hours. In some embodiments, the ultraviolet irradiation is treated with an ultraviolet cross-linker at 200-400 mJ/cm 2 for 2-6 hours. In some embodiments, the ultraviolet irradiation is treated with an ultraviolet cross-linker at 200-350 mJ/cm 2 for 2-5 hours.
  • PBMC can be obtained by heating PBMC at a temperature ranging from 30°C to 100°C for a predetermined time.
  • PBMC can be obtained by heating PBMC at a temperature ranging from 30°C to 100°C for a time period ranging from 1 minute to 1000 minutes.
  • PBMC can be obtained by heating PBMC at a temperature ranging from 30°C to 100°C for a time period ranging from 10 minutes to 100 minutes.
  • PBMC can be obtained by heating PBMC at a temperature ranging from 40°C to 70°C for a predetermined time.
  • PBMC can be obtained by heating stem cells at a temperature ranging from 40°C to 70°C for a period ranging from 1 minute to 1,000 minutes.
  • PBMC can be obtained by heating PBMC at a temperature ranging from 40°C to 70°C for a time period ranging from 10 minutes to 100 minutes.
  • the method provided by the present invention uses peripheral blood as a sample and adopts a heat treatment method to obtain the inducible vesicles. Since the induction process does not require the addition of additional induction reagents, only heat treatment is required, and there is no need to worry about the problem of residual reagents. . It is convenient for sampling and comes from the autologous body. After the inducible vesicles are obtained in vitro, they can be directly used back into the body of the original individual, so it has excellent operability and safety.
  • the inventors of the present invention unexpectedly found that heat treatment is particularly suitable for human PBMC to induce inducible vesicles.
  • the vesicles obtained by heat treatment of human PBMC were significantly higher than those of PBMC of mouse origin. This further demonstrates that heat treatment of PBMC to obtain inducible vesicles has excellent industrial prospects and is conducive to large-scale production.
  • the method for preparing inducible vesicles includes the steps of: (1) culturing mesenchymal stem cells; (2) collecting culture supernatant of mesenchymal stem cells; (3) from step (2) Vesicles were isolated from the culture supernatant.
  • the method is selected from the group consisting of polymer precipitation, immunoseparation, magnetic immunocapture, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, and Methods in the group consisting of their combinations isolate the vesicles from the culture medium.
  • the method of isolating vesicles includes isolating with Annexin V, Integrin alpha 5 and Syntexin 4 as markers.
  • the step of culturing mesenchymal stem cells in step (1) includes: (4) isolating mesenchymal stem cells from tissue; (5) adding culture medium to culture mesenchymal stem cells; Mesenchymal stem cells are exposed to apoptosis-inducing agents in their culture medium.
  • step (3) the method of isolating the vesicles includes using ultracentrifugation to separate the vesicles.
  • the step of isolating the vesicles by the ultracentrifugation method includes: (a) centrifuging the collected culture supernatant for the first time and taking the supernatant; (b) centrifuging the collected culture supernatant in step (a) Centrifuge the collected supernatant for the second time and take the supernatant; (c) Centrifuge the supernatant received in step (b) for the third time and take the precipitate; (d) Centrifuge the supernatant received in step (c) for the third time and take the precipitate; The precipitate was centrifuged for the fourth time and the precipitate was collected.
  • the first centrifugation is at 500-1500 g for 5-30 minutes; or the first centrifugation is at 500-1000 g for 5-20 minutes; or the first centrifugation is at 500-900 g Centrifuge for 5-15 minutes; or the first centrifugation is 800g for 5-10 minutes.
  • the second centrifugation is 1000-3000g for 5-30 minutes; or the second centrifugation is 1500-2500g for 5-20 minutes; or the second centrifugation is 1500-2200g. Centrifuge for 5-15 minutes; or the second centrifugation is 2000g for 5-10 minutes.
  • the third centrifugation is at 10,000-30,000g for 15-60 minutes; or the third centrifugation is at 12,000-25,000g for 20-60 minutes; or the third centrifugation is at 12,000-20,000g. Centrifuge for 20-40 minutes; or the third centrifugation is 16000-16500g for 30-35 minutes.
  • the fourth centrifugation is at 10,000-30,000g for 15-60 minutes, or the fourth centrifugation is at 12,000-25,000g for 20-60 minutes; or the fourth centrifugation is at 12,000-20,000g. Centrifuge for 20-40 minutes; or the fourth centrifugation is 16000-16500g for 30-35 minutes.
  • the blood sample is derived from a mammal.
  • the mammal is selected from primates or mice.
  • the primate is a human.
  • the invention provides a vesicle obtained by the above method.
  • the present invention provides a pharmaceutical composition including the vesicle and pharmaceutically acceptable excipients.
  • the pharmaceutical composition is in a preparation form selected from freeze-dried powder injections, injections, tablets, capsules, or patches.
  • the present invention provides a use of a lysis solution in preparing vesicles.
  • the lysate produces the vesicles by inducing apoptosis.
  • the cells include stem cells and red blood cells.
  • the stem cells are mesenchymal stem cells.
  • the source of mesenchymal stem cells includes: bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or combinations thereof.
  • the lysis solution includes red blood cell lysis solution.
  • red blood cell lysis solution lysis is a relatively mild method of removing red blood cells. It is mainly used in experiments such as the separation and purification of tissue cells dispersed by enzyme digestion, the separation and purification of lymphocytes, and the extraction of tissue cell proteins and nucleic acids. Removal of red blood cells.
  • the tissue cells obtained by lysis of red blood cell lysis solution do not contain red blood cells and can be further used for primary culture, cell fusion, flow cytometry analysis, separation and extraction of nucleic acids and proteins, etc.
  • the principle of lysis is that ammonium chloride is the main effector substance of red blood cell lysate.
  • red blood cell lysis solution to lyse
  • the main purpose of currently using red blood cell lysis solution to lyse is to remove red blood cells.
  • the understanding of the working principle of red blood cell lysis solution in the research field is single, and it is unanimously believed that the low osmotic pressure of red blood cell lysis solution causes red blood cells to rupture and die.
  • the present invention found that the red blood cells in the lysis solution mainly undergo morphological shrinkage. Produce vesicles, that is, die in the form of releasing "vesicles” rather than bursting the cells; thus, red blood cell lysate can be used to induce RBCs to produce "vesicles.”
  • the present invention proposes a new method of obtaining vesicles derived from red blood cells, which has good application potential.
  • red blood cells are one of the most widely available cells, and autologous sources are abundant and easy to obtain.
  • the red blood cell membrane is also one of the most widely used biological membranes in the field of biomaterials. If the red blood cell vesicles produced by the "lysate" method in the present invention have excellent therapeutic application value, it will be helpful to develop "lysed" red blood cell vesicles for treatment. New strategies to advance red blood cell vesicles in targeted therapies, drugs Developments in areas such as delivery, and even more, have become a simple and efficient solution for clinical translation applications.
  • the induction time is 1-8 hours. In some embodiments, the induction time is 2-4 hours. In some embodiments, the invention provides a method for producing vesicles, using a lysis solution to induce cells to obtain the vesicles.
  • the cells include stem cells and red blood cells.
  • the stem cells are mesenchymal stem cells.
  • the source of mesenchymal stem cells includes: bone marrow, urine, oral cavity, fat, placenta, umbilical cord, periosteum, or combinations thereof.
  • the lysis solution includes red blood cell lysis solution. In some embodiments, the induction time is 1-8 hours. In some embodiments, the induction time is 2-4 hours.
  • the method includes: (1) treating red blood cells with a lysis solution; (2) isolating the vesicles.
  • the method for isolating the vesicles in step (2) includes using ultracentrifugation to separate the vesicles.
  • the step of isolating the vesicles by the ultracentrifugation method includes: (a) centrifuging the collected lysate supernatant for the first time and taking the supernatant; (b) centrifuging the collected lysate supernatant in step (a) Centrifuge the collected supernatant for the second time and take the supernatant; (c) Centrifuge the supernatant received in step (b) for the third time and take the precipitate; (d) Centrifuge the supernatant received in step (c) for the third time and take the precipitate; The obtained precipitate was centrifuged for the fourth time and the precipitate was collected.
  • the first centrifugation is 500-1500g for 5-30 minutes. In some embodiments, the first centrifugation is 500-1000 g for 5-20 minutes. In some embodiments, the first centrifugation is 500-900 g for 5-15 minutes. In some embodiments, the second centrifugation is 1000-3000 g for 5-30 minutes. In some embodiments, the second centrifugation is centrifugation at 1500-2500 g for 5-20 minutes. In some embodiments, the second centrifugation is 1500-2200 g for 5-15 minutes. In some embodiments, the third centrifugation is centrifugation at 10,000-30,000 g for 15-60 minutes. In some embodiments, the third centrifugation is 12,000-25,000 g for 20-60 minutes.
  • the third centrifugation is 12,000-20,000 g for 20-40 minutes.
  • the fourth centrifugation is centrifugation at 10,000-30,000 g for 15-60 minutes. In some embodiments, the fourth centrifugation is 12,000-25,000 g for 20-60 minutes. In some embodiments, the fourth centrifugation is 12,000-20,000 g for 20-40 minutes. In some embodiments, the invention provides vesicles obtained by the methods.
  • the invention provides a composition comprising the vesicle and pharmaceutically acceptable excipients.
  • the present invention provides the use of the vesicles or the composition in the preparation of medicaments for treating enteritis.
  • the invention provides a method of treating enteritis, comprising administering the vesicles or the composition to a patient or subject.
  • the enteritis is acute enteritis.
  • the drug is used to improve the symptoms of weight loss or colon shortening in mice caused by acute enteritis.
  • the present invention provides the use of the vesicles or red blood cell-derived vesicles or the composition or red blood cells in the preparation of drugs or health care products that promote hair follicle repair and/or hair regeneration.
  • the present invention provides a method for promoting hair follicle repair and/or hair regeneration, the method comprising administering to a patient or subject the vesicles or red blood cell-derived vesicles or the composition or red blood cells. .
  • the present invention provides the use of the vesicles or the composition in the preparation of drugs for treating acute respiratory distress syndrome. In some embodiments, the invention provides the use of the vesicles or the composition in the preparation of diabetes drugs.
  • the invention provides a method of treating acute respiratory distress syndrome, comprising administering the vesicles or the composition to a patient or subject.
  • the invention provides a method of treating diabetes, comprising administering the vesicles or the composition to a patient or subject.
  • the diabetes is type 1 diabetes.
  • the medicament is used to promote wound healing in type 1 diabetes.
  • Figure 1 shows the process of isolating human PBMC using Formax PBMC separation tubes.
  • Figure 2 shows the process of PBMC separation by Ficoll-hypaque density gradient centrifugation.
  • Figure 3 is a diagram of the apoptosis status of human PBMC.
  • Figure 4 shows the technical roadmap of the standardized process for IEVs extraction.
  • Figures 5A-5C illustrate the analysis of extracted IEVs using flow cytometry.
  • Figure 5A shows the particle diameter distribution diagram of IEVs.
  • Figure 5B shows the results of nanoparticle tracking analysis (NTA) of IEVs produced by human PBMCs under different induction conditions.
  • Figure 5C shows the results of nanoparticle tracking analysis (NTA) of IEVs produced by mouse PBMCs under different induction conditions.
  • Figure 6 shows the results of flow cytometry analysis of surface membrane proteins of isolated and extracted PBMCs and IEVs derived from PBMCs.
  • Figure 7 shows the results of using Western blot to verify the expression of protein content of PBMC-IEVs induced in different ways.
  • Figure 8 shows the HE staining results of lung tissue treated with acute respiratory distress syndrome using IEVs derived from PBMCs.
  • Figure 9 shows the wet/dry weight ratio results of lung tissue treated with PBMCs-derived IEVs for acute respiratory distress syndrome.
  • Figures 10A-10C show the results of treating enteritis with IEVs derived from PBMCs.
  • Figure 10A shows that IEVs treatment significantly alleviated the trend of weight loss in mice with DSS-induced colitis.
  • Figure 10B shows that IEVs treatment significantly alleviated DSS-induced symptoms resulting in significant reduction in colon length, severe colon epithelial and crypt destruction, and massive infiltration of inflammatory cells.
  • Figure 10C is a HE staining picture showing that IEVs can inhibit DSS-induced enteritis.
  • Figure 11 shows that PBMC-derived IEVs can induce apoptosis of CD3+ T cells in enteritis mice.
  • Figure 12 shows that PBMS-derived IEVs can inhibit the activation of Th17 cells in enteritis mice.
  • Figure 13 shows a state diagram of vesicles generated from red blood cells treated with red blood cell lysate.
  • Figure 14 shows a state diagram of vesicles generated from hBMSCs treated with red blood cell lysate.
  • Figures 15A-15B show treatment of cells with STS.
  • Figure 15A shows the situation of red blood cells treated by STS.
  • Figure 15B shows the situation of STS treating hBMSCs.
  • Figures 16A-16C show the preliminary detection of vesicle size by flow cytometry and analysis of the expression of the apoptosis surface marker Annexin V.
  • Figure 16A shows the results of STS treatment of hBMSCs.
  • Figure 16B shows the results of hBMSCs treated with red blood cell lysate.
  • Figure 16C shows the results of red blood cell lysis solution treatment.
  • Figures 17A-17B show the results of treating enteritis with vesicles produced by red blood cells.
  • Figure 17A shows the weight loss ratio (%) of mice;
  • Figure 17B shows photos and length measurements (cm) of mouse colon tissue.
  • Figure 18 shows the hair regeneration in the depilated areas of mice in each group.
  • Figure 19 shows the skin tissue structure of the hair removal area of mice in each group.
  • Figure 20 shows HE staining of mouse lung tissue in each group.
  • Figure 21 shows the skin wound healing status of mice in each group.
  • Figures 22A-22C are the results of nanoparticle tracking analysis (NTA) of IEVs produced by PBMC induced by different temperatures using flow cytometry technology.
  • Figure 22A is a graph showing the number of IEVs produced.
  • Figure 22B is a particle size diagram of produced IEVs.
  • Figure 22C is a potential diagram of the production of IEVs.
  • Figure 23 is a diagram showing the state of producing vesicles from red blood cells treated with red blood cell lysate.
  • Figure 24 shows NanoFCM analysis showing the Annexin V-positive particle fraction in vesicles and exosomes.
  • red blood cell lysate is from Wuhan Sevier Biotechnology Co., Ltd., product number G2015.
  • IEVs in the embodiment of the present invention is the abbreviation of inducible vesicles, which can be called induced vesicles or induced extracellular vesicles (Induced extracellular vesicles, IEVs).
  • Inducible extracellular vesicles refer to a type of subcellular product that is interfered with or induced to induce apoptosis during the normal survival of precursor cells (such as stem cells). Usually this type of subcellular product has a membrane structure, expresses apoptotic markers, and partially contains genetic material DNA.
  • inducible extracellular vesicles are a type of substance that is different from cells and conventional extracellular vesicles (such as exosomes, etc.).
  • the cells that are normally alive include, for example, non-apoptotic cells, non-senescent cells, non-aging cells with arrested proliferation, non-revived cells after cryopreservation, and non-malignant cells with abnormal proliferation. cells or non-damaged cells, etc.
  • the normally viable cells are obtained from cells at a time when the cell contact fusion is 80-100% during cell culture. In some embodiments, the normally viable cells are obtained from log phase cells. In some embodiments, the cells in normal survival are obtained from primary culture and subcultured cells derived from human or mouse tissue. In some embodiments, the normally viable cells are obtained from an established cell line or strain. In some embodiments, the precursor cells are derived from early cells.
  • PBMC peripheral blood mononuclear cells, which are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes.
  • red blood cell lysate is from Wuhan Sevier Biotechnology Co., Ltd., product number G2015.
  • RBC-EVs lysis refers to vesicles derived from red blood cells induced by lysis solution.
  • the "erythrocyte lysate" described in Examples 4-6 is from Wuhan Sevier Biotechnology Co., Ltd., product number G2015.
  • the "apoEV” also refers to “vesicles”.
  • RBC-EVs refers to vesicles derived from red blood cells induced by lysis solution.
  • hBMSCs human bone marrow mesenchymal stem cells
  • the trypsin digestion method of hBMSCs cells and the identification method of mesenchymal stem cells are the same as those in patent application 202110077486.9.
  • Centrifuge room temperature, 500g, 15min, ⁇ 4 ⁇ 0. After centrifugation, it is divided into 4 layers from the bottom of the tube to the liquid surface, which are red blood cells and granulocytes layer, separation liquid layer, mononuclear cell layer, and plasma layer.
  • Washing Add at least 3 times the volume of PBMC cells in PBS, room temperature, 400g, 5 min, twice.
  • Flow cytometry detection Take an appropriate amount of cells for CD11b and CD45 staining, and perform flow cytometry detection on the machine.
  • Cell counting Discard the supernatant, add 1mL of RPMI-1640 culture medium (containing 10% fetal calf serum), mix by pipetting, and make a PBMC cell suspension.
  • RPMI-1640 culture medium containing 10% fetal calf serum
  • Cell culture After counting the cells, adjust the cell concentration to 1X10 6 /mL culture medium and add it to a 10cm culture dish for culture.
  • 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 2 200 ⁇ M 16h, PBMC+H 2 O 2 800 ⁇ M 16h, PBMC+UV 4h, Apoptosis was induced in the culture dish of PBMC + 45°C for 6h).
  • the STS group, alcohol group and hydrogen peroxide group were cultured in a 37°C, 5% carbon dioxide incubator.
  • the UV group was treated with 300mJ/ cm2 of UV cross-linker for 4h, and the heating group was incubated at 37°C. 45°C Water bath treatment for 6 hours.
  • the culture medium of each group is RPMI-1640 medium (containing 10% fetal calf serum), for example, STS, ethanol or H 2 O 2 is added to it, or UV or heat treatment is performed.
  • IEVs after inducing apoptosis, check the cell status to confirm that most cells have undergone apoptosis ( Figure 3). IEVs are isolated according to the standardized process for IEVs extraction. The technical route is detailed in Figure 4:
  • the human PBMCs isolated and extracted in the above Example 1 were analyzed using flow cytometry technology. The results showed that the isolated and extracted PBMCs were CD45 positive, and it can be concluded that the isolated PBMCs are PBMCs (as shown in Figure 6). Further analysis of the surface membrane proteins of IEVs derived from PBMCs showed that IEVs can express the universal surface protein CD9 of extracellular vesicles ( Figure 6).
  • ARDS acute respiratory distress syndrome
  • ARDS Acute respiratory distress syndrome
  • PBMC peripheral blood mononuclear cell
  • the research drug is human PBMC-ApopEV suspension, and its preparation steps are as follows: Use Ficoll density gradient centrifugation to separate PBMC from fresh human whole blood (approximately 1 ⁇ 10 7 PBMCs are isolated from 8 ml of human whole blood), wash with PBS, and add starch. Cytosporin (STS) induces apoptosis.
  • ARDS mouse model 8-12 weeks old C57BL/6 mice were randomly divided into 4 groups, 3-5 mice in each group, namely the normal control group (Control) and the model group (LPS group) , human PBMC-ApopEV transtracheal infusion treatment group (PBMC-ApopEV-L group) and human PBMC-ApopEV tail vein injection treatment group (PBMC-ApopEV-S group).
  • Control normal control group
  • LPS group model group
  • PBMC-ApopEV-L group human PBMC-ApopEV transtracheal infusion treatment group
  • PBMC-ApopEV-S group human PBMC-ApopEV tail vein injection treatment group
  • PBMC-ApopEV approximately 1 ⁇ 10 6 ApopEV
  • mice were sacrificed 24 hours after modeling (the animals were sacrificed at the 24th hour starting from the time of LPS administration).
  • HE staining of lung tissue The HE stained sections of the normal group can be seen as Control in Figure 8; in the LPS group, it can be seen that the alveolar tissue structure is severely damaged, congestion and edema are obvious, a large number of inflammatory cells are infiltrated, and the alveolar intervals are significantly thickened and structurally disordered; PBMC -In the ApopEV treatment group, congestion and inflammatory infiltration in the lung tissue of mice were significantly reduced, lung tissue exudation was reduced, and alveolar septal thickening and structural disorder were also significantly improved.
  • the tracheal infusion group (PBMC-ApopEV-L group) and the systemic intravenous infusion group (PBMC-ApopEV-S group) had similar efficacy (Figure 8).
  • PBMC-ApopEV suspension Use Ficoll density gradient centrifugation to separate PBMC from fresh human whole blood (approximately 1 ⁇ 10 7 PBMCs are isolated from 8 ml of human whole blood), wash with PBS, and add 500 nM staurosporine (RPMI-1640 medium containing 500nM STS) to induce apoptosis, collect the liquid after 16 hours, centrifuge at 800g and 4°C for 10 minutes, collect the supernatant, centrifuge at 2000g and 4°C for 10 minutes, collect the supernatant, centrifuge at 16000g and 4°C for 30 minutes, discard Resuspend the supernatant in 1 mL of PBS, centrifuge at 16000 g for 30 minutes at 4°C, and discard the supernatant again. Add PBS to prepare PBMC-ApopEV suspension and inject 1 ⁇ 10 6 /200 ⁇ L intravenously. The suspension should be stored at 4°C before use.
  • mice 8-12 weeks old C57BL/6 mice were given 3% (w:v) dextran sulfate sodium sulfate (DSS, molecular weight: 36,000–50,000Da; MP Biochemicals, 160110) Drinking water for 10 days induces colitis.
  • DSS dextran sulfate sodium sulfate
  • the mice were injected with 200 ⁇ l of PBMC-ApopE (ApopEV approximately 1 ⁇ 10 6 ) via the tail vein, and the mice were sacrificed 10 days after modeling.
  • PBMC-ApopE ApopEV approximately 1 ⁇ 10 6
  • PBMC can inhibit DSS-induced enteritis.
  • the body weight of mice with colitis was significantly reduced, and IEVs treatment significantly alleviated the tendency of weight loss in mice with DSS-induced colitis (Fig. 10A).
  • DSS induction resulted in a significant reduction in colon length, severe colon epithelial and crypt destruction, and massive infiltration of inflammatory cells, while IEVs treatment significantly alleviated these symptoms ( Figures 10B, 10C).
  • PBMC IEVs can regulate the excessive activation of T cells in enteritis mice. IEVs treatment can significantly induce the apoptosis of overactivated CD3 + T cells in mice with enteritis. Among them, PBMS-derived IEVs are more effective than PBMS exosomes (exo or exosome).
  • PBMS-derived IEVs can inhibit the activation of Th17 cells in enteritis mice. IEVs treatment can significantly induce Th17 cells in the circulation of enteritis mice ( Figure 12).
  • Plasma-MV group, Plasma-exo low group, and Plasma exo high group respectively refer to the large vesicles in the plasma under the uninduced state, the exosomes (small vesicles) low-dose group, and the exosomes (small vesicles) high-dose group. Group.
  • Example 4 Obtaining vesicles derived from red blood cells
  • the tube can be divided into 4 layers: the uppermost layer is light yellow diluted plasma; the second layer is the cloudy white film layer, which is peripheral blood mononuclear cells (PBMC); the third layer is the lymphatic separation fluid layer; the fourth layer is the lymphatic separation fluid layer.
  • the layer is red blood cells, and the red blood cells sink to the bottom of the tube. Discard all the liquid above the red blood cell layer, add the red blood cells to 40mL of PBS buffer, centrifuge at 400g, 4°C for 10 minutes, wash away the remaining plasma, lymphatic separation fluid and low-speed centrifugation to remove some platelets. This centrifugation step can be repeated once.
  • the obtained red blood cells can be resuspended in an appropriate amount of PBS. 2.
  • Use red blood cell lysis solution to induce red blood cells (RBCs) to produce vesicles
  • Example 5 Isolation and identification of vesicles produced by RBCs induced by red blood cell lysate
  • Flow cytometry was used to initially detect the particle size of the vesicles and analyze the expression of the apoptosis surface marker phosphatidylserine (PS). The results are shown in Figures 16A-16C. Compared with the vesicles produced by STS-induced hBMSCs, the vesicles produced after the death of hBMSCs induced by lysate have a higher proportion of small-sized vesicles and highly express apoptosis surface markers. PS ( Figure 16A, Figure 16B). The vesicles produced after lysate-induced death of RBCs highly express PS (Figure 16C) and have strong PS-positive surface characteristics similar to those of stem cell-derived IEVs (14B). It is speculated that they have similar metabolic pathways and functions in vivo.
  • PS phosphatidylserine
  • M8 and M10 only represent the name of the "gate” in the flow chart.
  • the "gate” in the flow chart means “range”, that is, M8 and M10 each represent a range, and the number displayed by the M8 gate represents a small particle size range.
  • the ratio, M10 represents the ratio of a large particle size range.
  • STS Specific steps for STS to induce vesicles produced by hBMSCs: (1) STS induces for 9 hours, pipet the cells at the bottom of the culture dish, and collect all the shed cells and cell supernatant; (2) Centrifuge at 800g for 10 minutes at 4°C (to remove dead cells), and leave Supernatant; (3) 2000g, centrifuge at 4°C for 10min (remove cell debris), keep the supernatant; (4) Supernatant 16000g, centrifuge at 4°C for 30min, discard the supernatant, keep the vesicle sediment; (5) Add 1ml PBS and pipet Resuspend the pellet, centrifuge at 16000g for 30 minutes at 4°C, discard the supernatant, and keep the vesicle pellet (the purpose of this step is to wash away possible residual culture medium and STS); (6) The vesicle pellet can be resuspended in an appropriate amount of PBS and set aside.
  • red blood cell lysate is used to induce hBMSCs to produce vesicles: hBMSCs (8th passage) are treated with red blood cell lysate for 4 hours, centrifuged at 800g, 4°C for 10min, and the supernatant is retained; centrifuged at 2000g, 4°C for 10min, and the supernatant is retained; Centrifuge at 16000g for 30 minutes at 4°C, discard the supernatant and keep the vesicle pellet; add 1ml PBS and pipette to resuspend the pellet, centrifuge at 16000g for 30min at 4°C, discard the supernatant and keep the vesicle pellet; resuspend the vesicle pellet with an appropriate amount of PBS and wait until use.
  • Dextran Sulfate Sodium Salt (DSS) solution was used to induce ulcerative colitis in mice, and it was observed whether the red blood cell-derived vesicles (RBC-EVs) induced by the lysate had a therapeutic effect on enteritis in mice.
  • DSS Dextran Sulfate Sodium Salt
  • mice Eight-week-old, male, C57 mice were selected for experiments, with 2 mice in each group.
  • the control group (Control group) drank and fed normally.
  • the modeling group (DSS group) and the treatment group (hBMSC-ApoEVs group, RBC-EVs group) all drank 2.5% DSS solution freely and fed normally for 8 days, with daily records. Changes in weight and degree of bloody stools.
  • the treatment group was treated with tail vein injection of corresponding vesicles on the 3rd day, and the hBMSC-ApoEVs group was each injected with a 10cm dish of hBMSCs (approximately 3 ⁇ 10 ⁇ 6 hBMSCs)-derived vesicles (the method is the same as in the Chinese patent According to the vesicle obtaining method in application 202110077486.9)), each RBC-EVs group was injected with 10 ⁇ 8 vesicles produced by RBC lysis (obtained by the method of Example 5). The modeling group only injected the same volume of PBS. On the 8th day, the mouse colon tissue was taken, photographed, measured in length, fixed, embedded and sectioned.
  • the steps to obtain the hBMSC-ApoEVs group are:
  • STS induces hBMSCs (8th passage) to undergo apoptosis for 9 hours, pipette the cells at the bottom of the culture dish, and collect all the shed cells and cell supernatant;
  • the vesicle pellet can be resuspended in an appropriate amount of PBS and set aside.
  • Red blood cells or red blood cell vesicles (RBC-EVs) promote hair growth
  • mice Observe the hair growth of mice every day and take photos to record the area of new hair. The observation period is 4 weeks. After 4 weeks, the skin of the mouse hair removal area is taken, tissue sections are stained, and the number of hair follicles, hair follicle size, hair follicle cell proliferation, etc. are detected.
  • Tissue staining Take the skin tissue from the depilated area of the mouse, fix it and dehydrate it, then slice the skin tissue longitudinally, and perform immunofluorescence staining. Blue represents the nucleus (Nucleus), green represents the cytoskeleton (Actin), and the number of hair follicles is counted.
  • the method for obtaining vesicles in the RBC-EVs (lysis) group is the same as the method of inducing red blood cells (RBCs) to produce vesicles with red blood cell lysis solution in Example 4.
  • Example 8 Therapeutic application of red blood cell vesicles (RBC-EVs) in mouse acute respiratory distress syndrome (ARDS) and diabetic wound healing models
  • mice Eight-week-old C57BL/6 mice were randomly divided into 3 groups, namely the normal control group (control), the ARDS model group, and the RBC-EVs (lysis) tracheal instillation treatment group. No food or water was allowed 6 hours before surgery, and 4% chloral hydrate 10ml/kg was used for anesthesia. Both the model group and the treatment group were first administered 30 ⁇ l LPS (5 mg/kg based on mouse body weight) via tracheal instillation, and the normal group was instilled with an equal volume of PBS.
  • the treatment group was instilled with 50 ⁇ L of RBC-lysis-EV (approximately 3 ⁇ 10 10 cells) through the trachea, and the normal group and ARDS modeling group were instilled with an equal volume of PBS. Mice were sacrificed 48 hours after modeling, and lung tissue sections were fixed and stained.
  • the method for obtaining vesicles in the RBC-EVs (lysis) group is the same as the method of inducing red blood cells (RBCs) to produce vesicles with red blood cell lysis solution in Example 4.
  • HE staining of lung tissue As shown in Figure 20, compared with the control group, the ARDS group showed serious damage to the alveolar tissue structure, obvious congestion and edema, a large number of inflammatory cell infiltrates, and the alveolar septa were significantly thickened and structurally disordered.
  • the degree of congestion and inflammatory infiltration in the mouse lung tissue was significantly reduced, the lung tissue exudation was reduced, and the thickening of the alveolar septa and structural disorder were also significantly improved.
  • mice Six-week-old C57BL/6 mice were randomly divided into 3 groups: normal control group (control), type 1 diabetes (T1DM) model group, and RBC-EVs (lysis) local injection treatment group. All mice were fed adaptively for 1 week. Fasting and water deprivation were conducted for 12 hours before the start of modeling, and fasting body weight and blood glucose were measured and recorded. Calculate the required dose of streptozotocin (STZ) (50 mg/kg ⁇ d ⁇ mouse) based on the fasting body weight of the mice, and inject it into the left lower abdomen of the mice once/d for 5 consecutive days. On the 7th day after administration, blood was taken from the tail vein for 3 consecutive days to measure random blood sugar.
  • STZ streptozotocin
  • mice in each group were shaved, hair removed with depilatory cream, and the back skin was cut with scissors to make an open skin wound of 1 cm ⁇ 1 cm.
  • the treatment group was given a single local injection of 100 ⁇ L of RBC-lysis-EV (approximately 3 ⁇ 10 10 ), the normal group and the T1DM model group were locally injected with equal volumes of PBS.
  • the wound healing status of mice in each group was observed daily and photographed and recorded.
  • the method for obtaining vesicles in the RBC-EVs (lysis) group is the same as the method of inducing red blood cells (RBCs) to produce vesicles with red blood cell lysis solution in Example 4.
  • the method is the same as in Example 4.
  • the red blood cell lysis solution induces red blood cells (RBCs) to produce vesicles, except that the source of the lysis solution is different. It is from Kangwei Century. Biotechnology Co., Ltd. Catalog #CW0613S.
  • hBMSCs cultured to the second passage were cultured until the cells were 80%-90% confluent, rinsed twice with PBS, added serum-free medium, incubated at 37°C for 48 hours, and the cell supernatant was collected for isolation and extraction of Exosomes.
  • the extraction steps include: centrifuge at 800g for 10 minutes - collect the supernatant - centrifuge at 2000g for 10 minutes - collect the supernatant - centrifuge at 16000g for 30 minutes - collect the supernatant - centrifuge at 120000g for 90 minutes - remove the supernatant and resuspend in sterile PBS Precipitation—Centrifuge again at 120,000g for 90 minutes, remove the supernatant, collect the Exosomes at the bottom, and resuspend in sterile PBS.
  • red blood cells red blood cells
  • the preparation method is the same as Example 4.
  • Flow cytometry was used to detect the expression of the apoptosis surface marker phosphatidylserine (PS) in exosomes and vesicles (apoEV) produced by red blood cells (RBCs) obtained in this example. The results are shown in Figure 24.
  • PS apoptosis surface marker phosphatidylserine
  • apoEV vesicles
  • the expression level of exosome surface marker PS is 23.9%, and the PS expression level of apoEV is 70.1%.

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Abstract

提供了来源于血液的样品在制备诱导性囊泡中的应用，以及裂解液在制备囊泡中的应用。还提供了获得的囊泡和包含该囊泡的组合物，以及它们的应用。

Description

血液来源的样品在制备囊泡中的应用 技术领域
本发明属于生物医药领域，涉及一种血液来源的样品在制备囊泡中的应用。
背景技术
细胞外囊泡(extracellular vesicles,EVs)是细胞分泌的含有蛋白质、核酸及各种细胞因子的纳米级载体。细胞外囊泡可以通过内分泌或旁分泌的方式作用于靶细胞，在细胞间物质传递和信息交流过程中发挥了重要作用。研究发现，细胞外囊泡所介导的信息交流在机体生理或病理过程中发挥了重要的调控作用，涉及到免疫调节、肿瘤生长、血管生成、损伤修复等。细胞外囊泡是细胞间通讯的重要媒介，使得细胞内地生物活性分子从一个细胞转移到另一个细胞从而发挥功能。因此，越来越清楚的是，这些囊泡参与许多生理过程，这将为疾病的治疗提供了应用的机会。
目前该领域的研究主要集中在外泌体(exosomes)方向。外泌体是直径在30-150nm左右的细胞外囊泡，其内含有RNA、脂质和蛋白质等成分。外泌体广泛参与了机体的各种生理/病理性调控，能够用作多种疾病的诊断、治疗和预后评估。迄今为止，间充质干细胞(mesenchymal stem cells，MSCs)被认为是产生外泌体能力最强的细胞。众多的研究发现MSCs来源的外泌体能模拟MSCs的生物学功能，在促进细胞生长和分化，修复组织缺损等方面发挥了重要的调控作用。因此，近年来以MSCs来源的外泌体为基础的细胞囊泡疗法取得了显著的发展。然而，目前以外泌体为基础的细胞囊泡治疗仍然存在诸多问题，主要表现在外泌体的提取和纯化过程复杂，耗时长，对设备和试剂的要求较高，生理性外泌体产量较低等等，这些缺陷都限制了外泌体治疗的临床转化和应用。
急性呼吸窘迫综合征(ARDS)自首次被描述以来，一直受到人们的关注，被认为是呼吸医学的主要临床问题，具有较高的发病率和死亡率。ARDS是由于肺内或/和肺外各种原因引起的、以肺损伤为主要表现的临床综合征，临床表现的显著特征是进行性呼吸困难和顽固性低氧血症。引起ARDS的肺内原因有重症肺炎、溺水误吸、肺挫伤、有毒物质吸入等，肺外原因有严重感染、严重外伤、休克、重症胰腺炎、中毒、大手术、心肺复苏后等，它们共同的病理基础是急性肺损伤。除了众所周知的ARDS的危险因素外，暴露于高臭氧水平和低维生素D血浆浓度也被发现是易感环境。ARDS治疗费用昂贵,然而目前除保护性肺通气、治疗原发病及支持治疗方法外，仍缺乏针对性治疗手段。基于最新研究，尽管ARDS总体生存率正在提高，但考虑到几项观察研究的住院死亡率，这可以解释为危险因素、诊断的可用性、识别ARDS的能力以及影响临床诊断的一些选择偏差试验，这些数据表明ARDS在世界范围内仍处于诊断不足和治疗不足的状态，目前对ARDS的定义在大多数临床情况下是不充分的。基于药物的预防战略仍然是一个重大挑战，一些最近的研究致力于改善这种疾病急性呼吸窘迫综合征的预后，但高死亡率和高致残并发症仍然有待改善。
结肠炎又称非特异性溃疡性结肠炎，其种类繁多，引发原因多样，并且起病多缓慢，病情轻重不一，主要临床表现腹泻、腹痛、粘液便及脓血便、里急后重、甚则大便秘结、数日内不能通大便，时而腹泻时而便秘，常伴有消瘦乏力等，多反复发作。腹痛一般多为隐痛或绞痛，常位于左下腹或小腹。其它表现有食欲不振、腹胀、恶心、呕吐及肝大等，左下腹可有压痛，有时能触及痉挛的结肠。常见的全身症状有消瘦、乏力、发热、贫血等。有少部分病人在慢性的病程中，病情突然恶化或初次发病就呈暴发性，表现严重腹泻，给人或动物带来极大的痛苦。
细胞外囊泡(extracellular vesicles,EVs)是细胞分泌的含有蛋白质、核酸及各种细胞因子的纳米级载体。细胞外囊泡可以通过内分泌或旁分泌的方式作用于靶细胞，在细胞间物质传递和信息交流过程中发挥了重要作用。研究发现，细胞外囊泡所介导的信息交流在机体生理或病理过程中发挥了重要的调控作用，涉及到免疫调节、肿瘤生长、血管生成、损伤修复等。
红细胞，特别是正常的成熟红细胞无核、无细胞器，呈双凹圆碟形，直径7～8μm，中央最薄处仅约1μm，这种特殊的结构有利于增大其表面积以便进行物质交换。红细胞具有可塑变形性，在全身血管中循环运行时，经过变形才能通过口径比它小的毛细血管和血窦孔隙。红细胞膜(RBCm)存在渗透脆性，在低渗溶液中红细胞可发生溶血，利于物 质的交换，红细胞膜本身适合在血管内运输。
红细胞裂解液(Red Blood Cell Lysis Buffer,或称ACK Lysis Buffer)是一种去除红细胞最简便易行的方法，即用裂解液裂解红细胞，它既不损伤有核细胞又能充分的去除红细胞。裂解液裂解是一种比较温和的红细胞去除方法，主要用于经酶消化分散的组织细胞的分离纯化，淋巴细胞的分离纯化以及组织细胞蛋白与核酸提取等实验中红细胞的去除。经红细胞裂解液裂解得到的组织细胞中不含红细胞，可进一步用于原代培养、细胞融合、流式细胞分析、核酸与蛋白的分离和提取等。
发明内容
在一些实施方案中，本发明提供了一种来源于血液的样品在制备囊泡中的应用，所述囊泡为诱导性囊泡。
在一些实施方案中，所述血液包括血浆、全血。
在一些实施方案中，所述血液为外周血。
在一些实施方案中，所述血液含有血细胞。
在一些实施方案中，所述样品包含外周血单个核细胞。
外周血单个核细胞(Peripheral blood mononuclear cell，PBMCs)是外周血中具有单个核的细胞，包括淋巴细胞和单核细胞，是一个有用的细胞来源，因其收集容易，为临床应用提供了方便。单个核细胞的体积、形态和比重与外周血其他细胞不同，红细胞和多核白细胞的比重在1.092左右，单个核细胞的比重为1.075-1.090，血小板为1.030-1.035。因此利用一种介于1.075-1.092之间而近于等渗的溶液作密度梯度离心，使一定密度的细胞按相应密度梯度分布，可将各种血细胞与单个核细胞分离。
在一些实施方案中，所述样品包含红细胞。在一些实施方案中，所述外周血单个核细胞为来源于血液的经分离或未经分离的细胞。在一些实施方案中，所述诱导性囊泡是在来源于的血液的样品中的细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡。在一些实施方案中，所述外力包括添加星形孢菌素、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合。在一些实施方案中，所述外力为热处理。在一些实施方案中，所述热处理是在38℃～60℃范围内进行的。在一些实施方案中，所述热处理是在40℃～55℃范围内进行的。在一些实施方案中，所述热处理是在40℃～52℃范围内进行的。在一些实施方案中，所述热处理是在42℃～52℃范围内进行的。在一些实施方案中，所述热处理是在42℃～50℃范围内进行的。在一些实施方案中，所述热处理的时间为3～20小时。在一些实施方案中，所述热处理的时间为3～15小时。在一些实施方案中，所述热处理的时间为3～12小时。在一些实施方案中，所述热处理的时间为3～10小时。在一些实施方案中，所述囊泡对Annexin V,Integrin alpha 5和Syntaxin 4表达呈阳性。在一些实施方案中，所述囊泡的直径为0.03-6μm。在一些实施方案中，所述囊泡的直径为0.03-4.5μm。在一些实施方案中，所述囊泡的直径为0.03-1μm。在一些实施方案中，所述囊泡的直径为0.04-1μm。在一些实施方案中，所述囊泡的直径为0.05-1μm。在一些实施方案中，所述囊泡的直径为0.1-1μm。在一些实施方案中，所述囊泡的直径为0.15-1μm。在一些实施方案中，本发明提供了一种制备囊泡的方法，所述方法包括对来源于血液的样品进行外力处理从而获得所述囊泡。
在一些实施方案中，本发明提供了一种制备囊泡的方法，所述方法包括对来源于血液的样品进行外力处理从而获得所述囊泡，所述囊泡为诱导性囊泡。在一些实施方案中，本发明提供了一种囊泡，由上述方法获得。
在一些实施方案中，本发明提供了来源于血液的样品的诱导性囊泡或含有所述囊泡的药物组合物在制备治疗或预防或改善疾病或所述疾病并发症的产品中的应用。一些实施方案中，本发明提供了一种治疗或预防或改善疾病或所述疾病并发症的方法，包括给予患者或者受试者所述的囊泡或所述的药物组合物。在一些实施方案中，所述产品包括药品、食品、保健品、化妆品、添加剂或中间品。在一些实施方案中，所述疾病包括肺部疾病或肠道疾病或糖尿病。在一些实施方案中，所述产品用于促进毛囊修复和/或毛发再生。在一些实施方案中，所述肺部疾病为急性呼吸窘迫综合症。在一些实施方案中，所述肠道疾病为肠炎。在一些实施方案中，所述肠炎为急性肠炎。在一些实施方案中，所述药物用于改善急性肠炎导致的小鼠体重下降或结肠缩短的症状。在一些实施方案中，所述糖尿病为1型糖尿病。在一些实施方案中，所述药物用于促进1型糖尿病伤口愈合。在一些实施方案中，所述血液为外周血。在一些实施方案 中，所述血液含有血细胞。在一些实施方案中，所述样品包含外周血单个核细胞。在一些实施方案中，所述样品包含红细胞。在一些实施方案中，所述外周血单个核细胞为来源于血液的经分离或未经分离的细胞。
在一些实施方案中，所述诱导性囊泡是在来源于的血液的样品中的细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡。一些实施方案中，本发明提供的这种以外周血为样品在体外获得诱导性囊泡之后可以直接再用回原个体的体内，具有采样方便，来自自体的优点，因而具有极好的可操作性和安全性。
在一些实施方案中，所述外力包括添加星形孢菌素、添加乙醇、添加双氧水、紫外线照射、饥饿法、裂解液、热应力法、或机械力法中一种或多种的组合。在一些实施方案中，所述外力为热处理法。在一些实施方案中，所述热处理是在38℃～60℃范围内进行的。在一些实施方案中，所述热处理是在40℃～55℃范围内进行的。在一些实施方案中，所述热处理是在40℃～52℃范围内进行的。在一些实施方案中，所述热处理是在42℃～52℃范围内进行的。在一些实施方案中，所述热处理是在42℃～50℃范围内进行的。在一些实施方案中，所述热处理的时间为3～20小时。在一些实施方案中，所述热处理的时间为3～15小时。在一些实施方案中，所述热处理的时间为3～12小时。在一些实施方案中，所述热处理的时间为3～10小时。
一些实施方案中，所述双氧水的浓度为20～2000μM。一些实施方案中，所述双氧水的浓度为50～1500μM。一些实施方案中，所述双氧水的浓度为50～1200μM。一些实施方案中，所述双氧水的浓度为50～1000μM。一些实施方案中，所述双氧水的浓度为100～1200μM。一些实施方案中，所述双氧水的浓度为100～1000μM。一些实施方案中，所述双氧水的浓度为100～800μM。一些实施方案中，所述双氧水的浓度为100～500μM。一些实施方案中，所述双氧水的浓度为100～400μM。一些实施方案中，所述双氧水的浓度为100～300μM。一些实施方案中，所述双氧水的处理时间为4～30h。一些实施方案中，所述双氧水的处理时间为5～25h。一些实施方案中，所述双氧水的处理时间为8～25h。一些实施方案中，所述双氧水的处理时间为10～25h。一些实施方案中，所述双氧水的处理时间为12～25h。一些实施方案中，所述双氧水的处理时间为12～20h。一些实施方案中，所述囊泡为诱导性囊泡。
在一些实施方案中，所述囊泡对Annexin V,Integrin alpha 5和Syntaxin 4表达呈阳性。
在一些实施方案中，所述囊泡为诱导性囊泡。在一些实施方案中，所述囊泡的直径为0.03-6μm。在一些实施方案中，所述囊泡的直径为0.03-4.5μm。在一些实施方案中，所述囊泡的直径为0.03-1μm。在一些实施方案中，所述囊泡的直径为0.04-1μm。在一些实施方案中，所述囊泡的直径为0.05-1μm。在一些实施方案中，所述囊泡的直径为0.1-1μm。在一些实施方案中，所述囊泡的直径为0.15-1μm。
一些实施方案中，所述乙醇的浓度为50～500nM。一些实施方案中，所述乙醇的浓度为100～500nM。一些实施方案中，所述乙醇的浓度为100～300nM。一些实施方案中，所述乙醇的处理时间为4～30h。一些实施方案中，所述乙醇的处理时间为8～25h。一些实施方案中，所述乙醇的处理时间为12～20h。一些实施方案中，所述紫外照射为紫外交联仪200～500mJ/cm2处理1～8h。一些实施方案中，所述紫外照射为紫外交联仪200～400mJ/cm2处理2～6h。一些实施方案中，所述紫外照射为紫外交联仪200～350mJ/cm2处理2～5h。
在一些实施方案中，PBMC可通过30℃～100℃范围内的温度下加热PBMC持续预定时间来获得。或者，PBMC可通过在30℃至100℃范围内的温度下加热PBMC 1分钟至1000分钟范围内的时间段来获得。或者，PBMC可通过在30℃至100℃范围内的温度下加热PBMC10分钟至100分钟范围内的时间段来获得。或者，PBMC可通过在40℃至70℃范围内的温度下加热PBMC持续预定时间来获得。或者，PBMC可通过在40℃至70℃范围内的温度下加热干细胞1分钟至1,000分钟范围内的时间段来获得。或者，PBMC可通过在40℃至70℃范围内的温度下加热PBMC10分钟至100分钟范围内的时间段来获得。
一些实施方案中，本发明提供的这种以外周血为样品，采用热处理的方法获得所述的诱导性囊泡由于诱导过程无需额外添加诱导试剂，只需要加热处理，无需担心试剂残留残留的问题。具有采样方便，来自自体，在体外获得诱导性囊泡之后可以直接再用回原个体的体内，因而具有极好的可操作性和安全性。
在一些实施方案中，本发明的发明人意外地研究发现，对于人源的PBMC尤其适合采用热处理这种方法诱导获得诱导性囊泡。与鼠来源的PBMC相比，人源的PBMC采用热处理获得的囊泡极显著地高于鼠来源的PBMC采用热处理获得的囊泡。这样，进一步地体现了热处理PBMC获得诱导性囊泡具有极好的产业化前景，有利于大规模的生产。
在一些实施方案中，所述诱导性囊泡的制备方法包括步骤：(1)培养间充质干细胞；(2)收集间充质干细胞的培养基上清；(3)从步骤(2)中的培养基上清中分离出囊泡。
在一些实施方案中，通过选自由聚合物沉淀、免疫分离、磁免疫捕获、超速离心、密度梯度离心、尺寸排阻色谱、超滤、超速离心、密度梯度离心、尺寸排阻色谱、超滤和它们的组合所组成的组中的方法从培养基中分离所述囊泡。
在一些实施方案中，所述分离囊泡的方法包括以Annexin V、Integrin alpha 5和Syntexin 4为标志物进行分离。
在一些实施方案中，所述步骤(1)中的培养间充质干细胞的步骤包括：(4)从组织中分离间充质干细胞；(5)添加培养基培养间充质干细胞；所述间充质干细胞的培养基中接触凋亡诱导剂。
在一些实施方案中，所述步骤(3)中，分离囊泡的方法包括选用超速离心的方法分离所述囊泡。
在一些实施方案中，所述超速离心的方法分离所述囊泡的步骤包括：(a)将收集到的培养上清进行第一次离心，取上清；(b)将步骤(a)中收集到的上清进行第二次离心，取上清；(c)将步骤(b)中收到的上清进行第三次离心，取沉淀；(d)将步骤(c)中收到的沉淀进行第四次离心，取沉淀。在一些实施方案中，所述第一次离心为500-1500g离心5-30分钟；或者所述第一次离心为500-1000g离心5-20分钟；或者所述第一次离心为500-900g离心5-15分钟；或者第一次离心为800g离心5-10min。在一些实施方案中，所述第二次离心为1000-3000g离心5-30分钟；或者所述第二次离心为1500-2500g离心5-20分钟；或者所述第二次离心为1500-2200g离心5-15分钟；或者所述第二次离心为2000g离心5-10分钟。在一些实施方案中，所述第三次离心为10000-30000g离心15-60分钟；或者所述第三次离心为12000-25000g离心20-60分钟；或者所述第三次离心为12000-20000g离心20-40分钟；或者所述第三次离心为16000-16500g离心30-35分钟。在一些实施方案中，所述第四次离心为10000-30000g离心15-60分钟，或者所述第四次离心为12000-25000g离心20-60分钟；或者所述第四次离心为12000-20000g离心20-40分钟；或者所述第四次离心为16000-16500g离心30-35分钟。
在一些实施方案中，所述的血液样品来源于哺乳动物。在一些实施方案中，所述哺乳动物选自灵长类动物或鼠。在一些实施方案中，所述灵长类动物为人。在一些实施方案中，本发明提供了一种囊泡，所述囊泡由上述方法获得的。在一些实施方案中，本发明提供了一种药物组合物，包括所述的囊泡以及药学上可接受的辅料。在一些实施方案中，所述药物组合物的制剂形式选自冻干粉针、注射剂、片剂、胶囊、或贴剂。
一些实施方案中，本发明提供了一种裂解液在制备囊泡中的应用。
一些实施方案中，所述裂解液通过诱导细胞凋亡产生所述囊泡。一些实施方案中，所述细胞包括干细胞和红细胞。一些实施方案中，所述干细胞为间充质干细胞。一些实施方案中，所述间充质干细胞来源包括：骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或其组合。一些实施方案中，所述裂解液包括红细胞裂解液。
从现有技术公开显示，红细胞裂解液裂解是一种比较温和的红细胞去除方法，主要用于经酶消化分散的组织细胞的分离纯化，淋巴细胞的分离纯化以及组织细胞蛋白与核酸提取等实验中红细胞的去除。经红细胞裂解液裂解得到的组织细胞中不含红细胞，可进一步用于原代培养、细胞融合、流式细胞分析、核酸与蛋白的分离和提取等。其裂解原理是氯化铵是红细胞裂解液的主要效应物质，氨根离子不能通过细胞膜，而其他离子可以通过，造成细胞内外的离子浓度差异，形成了渗透压差，外部的水分会扩散至细胞内，使红细胞膨胀，达到裂解的效果。可见，目前使用红细胞裂解液裂解的主要目的是去除红细胞。目前研究领域内对红细胞裂解液的工作原理的认识单一，且一致认为红细胞裂解液的低渗透压使红细胞胀破而死亡，但本发明研究发现，在裂解液中的红细胞主要发生形态皱缩，产生囊泡，即以释放“囊泡”的形式死亡，而非细胞胀破；从而可以利用红细胞裂解液诱导RBC产生“囊泡”。
因而，本发明提出了一种新的获取红细胞来源的囊泡的方式，具有较好的应用潜能。
在一些实施方式中，在目前已有实验数据中发现，这种裂解液产生的囊泡可以有效治疗DSS诱导的小鼠肠炎模型，推测可能具有类似干细胞ApoEVs的治疗效果。红细胞是可获得的来源最广泛的细胞之一，且自体来源数量足够，获取方式便捷。红细胞膜也是生物材料领域应用最广泛的生物膜之一，若本发明中“裂解液”方式产生的红细胞囊泡具有优秀的治疗应用价值，将有助于开发“裂解”红细胞产囊泡治疗的新策略以及推动红细胞囊泡在靶向治疗，药物 递送等领域的发展，更甚者，成为一种待选的简单高效的临床转化应用方案。
一些实施方案中，所述诱导时间为1-8小时。一些实施方案中，所述诱导时间为2-4小时。一些实施方案中，本发明提供了一种产囊泡的方法，使用裂解液诱导细胞获得所述囊泡。一些实施方案中，所述细胞包括干细胞和红细胞。一些实施方案中，所述干细胞为间充质干细胞。一些实施方案中，所述间充质干细胞来源包括：骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或其组合。一些实施方案中，所述裂解液包括红细胞裂解液。一些实施方案中，所述诱导时间为1-8小时。一些实施方案中，所述诱导时间为2-4小时。
一些实施方案中，所述方法包括：(1)使用裂解液处理红细胞；(2)分离出囊泡。一些实施方案中，所述步骤(2)分离囊泡的方法包括选用超速离心的方法分离所述囊泡。一些实施方案中，所述超速离心的方法分离所述囊泡的步骤包括：(a)将收集到的裂解液上清进行第一次离心，取上清；(b)将步骤(a)中收集到的上清进行第二次离心，取上清；(c)将步骤(b)中收到到的上清进行第三次离心，取沉淀；(d)将步骤(c)中收到到的沉淀进行第四次离心，取沉淀。
一些实施方案中，所述第一次离心为500-1500g离心5-30分钟。一些实施方案中，所述第一次离心为500-1000g离心5-20分钟。一些实施方案中，所述第一次离心为500-900g离心5-15分钟。一些实施方案中，所述第二次离心为1000-3000g离心5-30分钟。一些实施方案中，所述第二次离心为1500-2500g离心5-20分钟。一些实施方案中，所述第二次离心为1500-2200g离心5-15分钟。一些实施方案中，所述第三次离心为10000-30000g离心15-60分钟。一些实施方案中，所述第三次离心为12000-25000g离心20-60分钟。一些实施方案中，所述第三次离心为12000-20000g离心20-40分钟。一些实施方案中，所述第四次离心为10000-30000g离心15-60分钟。一些实施方案中，所述第四次离心为12000-25000g离心20-60分钟。一些实施方案中，所述第四次离心为12000-20000g离心20-40分钟。一些实施方案中，本发明提供了所述方法获得的囊泡。
一些实施方案中，本发明提供了一种组合物，包含所述的囊泡和药学上可接受的辅料。一些实施方案中，本发明提供了所述的囊泡或所述组合物在制备治疗肠炎的药物中的应用。
一些实施方案中，本发明提供了一种治疗肠炎的方法，包括给予患者或受试者所述的囊泡或所述的组合物。
一些实施方案中，所述肠炎为急性肠炎。一些实施方案中，所述药物用于改善急性肠炎导致的小鼠体重下降或结肠缩短的症状。一些实施方案中，本发明提供了所述的囊泡或红细胞来源的囊泡或所述组合物或红细胞在制备促进毛囊修复和/或毛发再生的药物或保健品中的应用。一些实施方案中，本发明提供了一种促进毛囊修复和/或毛发再生的方法，所述方法包括给予患者或者受试者所述的囊泡或红细胞来源的囊泡或所述组合物或红细胞。
一些实施方案中，本发明提供了所述的囊泡或所述组合物在制备治疗急性呼吸窘迫综合征药物中的应用。一些实施方案中，本发明提供了所述的囊泡或所述组合物在制备糖尿病药物中的应用。
一些实施方案中，本发明提供了一种治疗急性呼吸窘迫综合征的方法，包括给予患者或者受试者所述的囊泡或所述组合物。
一些实施方案中，本发明提供了一种治疗糖尿病的方法，包括给予患者或者受试者所述的囊泡或者所述的组合物。
一些实施方案中，所述糖尿病为1型糖尿病。一些实施方案中，所述药物用于促进1型糖尿病伤口愈合。
附图说明
图1为使用福麦斯PBMC分离管分离人源PBMC的过程。
图2为Ficoll-hypaque密度梯度离心分离PBMC的过程。
图3为人源PBMC凋亡状态图。
图4为IEVs提取标准化流程技术路线图。
图5A-5C为利用流式细胞技术对提取的IEVs进行分析。图5A为IEVs的颗粒直径分布图。图5B为人源PBMCs在不同诱导条件下产出IEVs的纳米粒子跟踪分析(NTA)结果。图5C为鼠源PBMCs在不同诱导条件下产出IEVs的纳米粒子跟踪分析(NTA)结果。
图6为流式细胞技术对分离提取的PBMCs以及PBMCs来源的IEVs的表面膜蛋白进行分析的结果。
图7为利用蛋白印记验证不同方式诱导得到的PBMC-IEVs的蛋白内容物表达的结果。
图8为PBMCs来源的IEVs治疗急性呼吸窘迫综合症的肺组织的HE染色结果。
图9为PBMCs来源的IEVs治疗急性呼吸窘迫综合症的肺组织的肺的湿/干重比结果。
图10A-10C为PBMCs来源的IEVs治疗肠炎的结果。图10A显示IEVs治疗显著减轻了DSS诱导的结肠炎小鼠体重减轻的趋势。图10B显示IEVs治疗显著缓解DSS诱导导致结肠长度显着减少，严重的结肠上皮和隐窝破坏以及炎性细胞的大量浸润的症状。图10C为HE染色图显示IEVs能抑制DSS诱导的肠炎。
图11为PBMC来源的IEVs能诱导肠炎小鼠CD3+T细胞的凋亡。
图12为PBMS来源的IEVs能抑制肠炎小鼠Th17细胞的激活。
图13显示红细胞裂解液处理红细胞产生囊泡的状态图。
图14显示使用红细胞裂解液处理hBMSCs产生囊泡的状态图。
图15A-15B显示使用STS处理细胞的情况。图15A为STS处理红细胞的情况。图15B为STS处理hBMSCs的情况。
图16A-16C显示流式初步检测囊泡的粒径以及分析凋亡表面标志物Annexin V的表达情况。图16A为STS处理hBMSCs的结果。图16B为红细胞裂解液处理hBMSCs的结果。图16C为红细胞裂解液处理红细胞的结果。
图17A-17B为红细胞产生的囊泡的治疗肠炎的结果。图17A为小鼠体重降低比例(％)；图17B为小鼠结肠组织照片和长度测量(cm)。
图18为各组小鼠脱毛区毛发新生情况。
图19为各组小鼠脱毛区皮肤组织结构。
图20为各组小鼠肺组织HE染色。
图21为各组小鼠皮肤创口愈合情况。
图22A-22C为利用流式细胞技术对不同温度诱导PBMC产的IEVs纳米粒子跟踪分析(NTA)结果。图22A为产IEVs的数量图。图22B为产IEVs的粒径图。图22C为产IEVs的电位图。
图23为红细胞裂解液处理红细胞产生囊泡的状态图。
图24为NanoFCM分析显示囊泡和外泌体中Annexin V阳性颗粒部分。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
本文中，所述的“红细胞裂解液”来源于武汉赛维尔生物科技有限公司，货号G2015。
本发明实施例中的IEVs为诱导性囊泡的简称，可称为诱导性囊泡，也可称为诱导性细胞外囊泡(Induced extracellular vesicles,IEVs)。诱导性细胞外囊泡是指的是一种在前体细胞(例如干细胞)正常存活时，被干预或诱导，使其凋亡产生的一类亚细胞产物。通常这一类亚细胞产物，具有膜结构，表达凋亡性标志物，部分包含有遗传物质DNA。发明人发现诱导性细胞外囊泡是区分于细胞和常规细胞外囊泡(如外泌体等)的一类物质。在在一些实施方案中，所述的正常存活时的细胞，例如是非凋亡的细胞、非衰老的细胞、非老化而增殖停滞的细胞、非冻存后复苏的细胞、非发生恶变而异常增殖的细胞或非出现损伤的细胞等。在一些实施方案中，所述的正常存活时的细胞取自细胞培养过程中，细胞接触融合80-100％的时候的细胞。在一些实施方案中，所述的正常存活时的细胞取自对数期细胞。在一些实施方案中，所述的正常存活时的细胞取自人或鼠组织来源的原代培养及其传代培养细胞。在一些实施方案中，所述的正常存活时的细胞取自已确立的细胞系或细胞株。在一些实施方案中，所述的前体细胞取自早期的细胞。
本文中，所述的“PBMC”是指外周血单个核细胞，是外周血中具有单个核的细胞，包括淋巴细胞和单核细胞。
本文中，所述的“红细胞裂解液”来源于武汉赛维尔生物科技有限公司，货号G2015。
本文中，“ab”同“IEVs”或“ApopEVs”。RBC-EVs(lysis)指的是裂解液诱导产生的红细胞来源的囊泡。
本文中，实施例4-6所述的“红细胞裂解液”来源于武汉赛维尔生物科技有限公司，货号G2015。
本文实施例中，所述的“apoEV”也指“囊泡”。
RBC-EVs(lysis)指的是裂解液诱导产生的红细胞来源的囊泡。
本文实施例中，hBMSCs(人骨髓间充质干细胞)购买自美国ScienCell公司，货号#7500，培养所述hBMSCs培养基如下表所示：
注：培养hBMSCs时，细胞以1：3传代，细胞密度约80～90％时传代，当诱导细胞凋亡时，使用α-MEM基础培养基。
hBMSCs细胞胰酶消化方式和间充质干细胞鉴定方式同专利申请202110077486.9。
实施例1 PBMC来源的IEVs的获得
1、人源PBMC分离实验
抗凝管取血，用PBS稀释血液(PBS：外周血＝1：1稀释血液)，之后加入到福麦斯PBMC分离管(预装，含分离液)，800g离心10min，取离心后白膜层(如图1)到新的50mL离心管，用适量PBS 300g，10min洗两次，RPMI(不含FBS)重悬细胞团进行细胞计数，细胞计数后调整细胞浓度为1X106/mL培养基，加入10cm培养皿中进行培养。
2、鼠源PBMC分离实验
Ficoll-hypaque密度梯度离心分离PBMC细胞(Solarbio各种动物外周血淋巴细胞分离液试剂盒)：
1.采血：左手拇、食指抓取小鼠(C57BL/6 WT 12周雄性小鼠)双耳及颈后皮肤，小指固定尾部→中指将小鼠左侧前肢轻压在胸骨心脏部位，无名指按在腹部，捻动拇指，轻压取血侧眼部皮肤，使眼球充血突出→用弯头镊夹取眼球→根据需要捻动拇指与食指的方向，使血液从眼眶内以不同速度垂直流入离心管→同时用左手中指轻按小鼠心脏部位，以加快心脏泵血速度→当血液流尽时，用脱臼法处死小鼠。此法取小鼠全血0.8～1.2mL，注入EDTA-K2抗凝管中，轻轻摇匀。
2.稀释：室温下加入等体积的稀释液或PBS，轻轻吹打摇匀；
3.加样：取15mL离心管，吸取与1+2步骤等体积的分离液于离心管中，移液枪头贴着管壁缓慢将稀释后的血液平铺在分离液液面上面，(当1+2稀释后血液体积小于3mL时，加入3mL分离液；大于等于3mL时，加入等体积分离液)。
4.离心：室温，500g，15min，↑4↓0，离心后从管底至液面分4层，依次为红细胞和粒细胞层、分离液层、单个核细胞层、血浆层。
5.回收：将移液管直接插入白膜层(或先吸去上层的血浆)，轻轻吸出白膜层(如图2)至新的15mL离心管中。
6.洗涤：加入至少PBMC细胞体积3倍的PBS，室温，400g，5min，两次。
7.流式检测：取适量细胞进行CD11b和CD45染色，流式上机检测。
8.细胞计数：弃上清，加入1mL RPMI-1640培养基(含10％胎牛血清)，吹打混匀，制成PBMC细胞悬液。
9.细胞培养：细胞计数后调整细胞浓度为1X106/mL培养基，加入10cm培养皿中进行培养。
3、PBMC来源的IEVs的获得
分离提取的PBMC细胞，接种到不同刺激(PBMC+STS 500nM 16h，PBMC+乙醇200mM 16h，PBMC+乙醇800mM 16h，PBMC+H2O2 200μM 16h，PBMC+H2O2 800μM 16h，PBMC+UV 4h，PBMC+45℃6h)的培养皿中诱导凋亡，STS组、酒精组和双氧水组于37℃，5％二氧化碳培养箱培养；紫外线组于紫外交联仪300mJ/cm2处理4h，加热组于45℃ 水浴锅处理6h。
各组的培养基为RPMI-1640培养基(含10％胎牛血清)，如在其基础上添加STS或乙醇或H2O2，或进行UV或加热处理。
采用以下方法分离提取IEVs：诱导凋亡以后，检查细胞状态，确认大部分细胞发生凋亡(图3)，按照IEVs提取标准化流程进行IEVs的分离，技术路线详见图4：
(1)用移液器吹打分离细胞，收集细胞及上清液至15mL离心管；
(2)800g，10min，4℃；
(3)收上清转移至1.5mL EP管(注意，勿弃上清)；
(4)2000g，10min，4℃；
(5)收上清至高速冷冻离心机适用玻璃管，弃沉淀；
(6)16,000g，30min,4℃(提前预冷)；
(7)弃上清，PBS重悬，转移至1.5mL EP管中；
(8)16,000g，30min,4℃(提前预冷)；
(9)弃上清，无菌PBS重悬AB沉淀，4℃冰箱保存。
实施例2 PBMC来源IEVs的分析
利用流式细胞技术对提取的IEVs进行分析，结果显示从诱导后的1h至24h，IEVs的颗粒直径分布主要都集中在1um以下，占95％左右(图5A，其为实施例1中鼠源PBMC+STS 500nM 16h组的结果)。纳米粒子跟踪分析(NTA)结果显示人源(图5B，表1)和鼠源(图5C，表2)PBMCs在不同诱导条件下均可以产出IEVs，且产生的IEVs在粒径和电位方面与STS诱导下产生的IEVs无显著差异。
利用流式细胞技术对上述实施例1分离提取的人源PBMCs进行分析，结果显示分离提取的PBMCs CD45阳性，可得出分离得到的就是PBMCs(如图6)。进一步对PBMCs来源的IEVs的表面膜蛋白进行分析，结果显示，IEVs能够表达细胞外囊泡的普遍性表面蛋白CD9(图6)。
表1人源PBMC来源的IEVs
表2鼠源PBMC来源的IEVs
利用蛋白印记进行验证不同方式诱导的PBMC-IEVs的蛋白内容物表达，结果表明，不同诱导方式产生的IEVs均能特异性表达Annexin V,Integrin alpha 5和Syntexin 4(图7)。以上3种蛋白为区分MSCs来源的IEVs和 exosomes的特征性蛋白标志物。
实施例3 PBMC来源IEVs的应用
1、治疗急性呼吸窘迫综合症(ARDS)
急性呼吸窘迫综合症(ARDS)发病率及死亡率均高，发病机制迄今尚未完全阐明，目前也无特效的治疗方法。本研究观察经气管滴注与尾静脉输注人外周血单个核细胞(PBMC)来源的IEVs(或称“ApopEV”)对急性呼吸窘迫综合症模型小鼠的治疗效果。
(1)实验方法
1)实验动物：C57BL/6小鼠，性别不限，8-12周龄，SPF级。
2)研究药物为人PBMC-ApopEV悬液，其制备步骤如下：采用Ficoll密度梯度离心法分离人新鲜全血的PBMC(8ml人全血约分离出1×107个PBMC)，PBS洗涤，加入星形孢菌素(STS)诱导凋亡，16小时候收集液体，800g 4℃离心10分钟，收集上清，2000g 4℃离心10分钟，收集上清，16000g 4℃离心30分钟，弃上清，1mL PBS重悬，16000g 4℃离心30分钟，再次弃上清。加PBS配置PBMC-ApopEV悬液，气管滴注1×106/30μL，静脉输注1×106/200μL，悬液在使用前4℃保存。
3)ARDS小鼠模型构建及治疗：取8-12周龄C57BL/6小鼠，随机分为4组，每组3-5只，分别是正常对照组(Control)，模型组(LPS组)，人PBMC-ApopEV经气管滴注治疗组(PBMC-ApopEV-L组)及人PBMC-ApopEV尾静脉注射治疗组(PBMC-ApopEV-S组)。术前6小时禁食禁水，麻醉使用4％水合氯醛10ml/kg。模型组及治疗组均先气管滴注给予30μl LPS(5mg/kg)，4小时后，治疗组分别经气管滴注30μL的PBMC-ApopEV(ApopEV约1×106个)，经尾静脉注射200μL的PBMC-ApopEV(ApopEV约1×106个)，造模24小时后处死小鼠(从LPS给予后开始计算，到第24小时处死动物)。
(2)实验结果
1)肺组织的HE染色：正常组的HE染色切片可见图8中的Control；LPS组可见肺泡组织结构破坏严重，充血水肿明显，大量炎性细胞浸润，肺泡间隔明显增厚且结构紊乱；PBMC-ApopEV治疗组，小鼠肺组织的充血及炎症浸润明显减轻，肺组织渗出减少，肺泡间隔增厚和结构紊乱也有明显改善。其中气管滴注组(PBMC-ApopEV-L组)与全身静脉输注组(PBMC-ApopEV-S组)疗效相似(图8)。
2)肺的湿/干重比：称量肺湿重，烤箱烤干到恒重称量得到肺干重。根据公式计算肺部W/D值。结果表明，LPS刺激显著增加了小鼠肺湿/干重比值，而经气管滴注或尾静脉输注的PBMC-ApopEV治疗则显著降低了这一比值，但各个治疗组之间没有差异(图9)。
2、治疗肠炎
(1)研究方法
1)实验动物：C57BL/6小鼠，性别不限，8-12周龄，SPF级。
2)人PBMC-ApopEV悬液的制备：采用Ficoll密度梯度离心法分离人新鲜全血的PBMC(8ml人全血约分离出1×107个PBMC)，PBS洗涤，加入500nM星形孢菌素(含有500nM STS的RPMI-1640培养基)诱导凋亡，16小时候收集液体，800g 4℃离心10分钟，收集上清，2000g 4℃离心10分钟，收集上清，16000g 4℃离心30分钟，弃上清，1mL PBS重悬，16000g 4℃离心30分钟，再次弃上清。加PBS配置PBMC-ApopEV悬液，静脉输注1×106/200μL，悬液在使用前4度保存。
3)DSS诱导的小鼠肠炎模型构建及治疗：取8-12周龄C57BL/6小鼠，给予3％(w：v)硫酸葡聚糖硫酸钠(DSS，分子量：36,000–50,000Da；MP Biochemicals，160110)饮水10天诱发结肠炎。在DSS处理第3天时，小鼠经尾静脉注射200μl的PBMC-ApopE(ApopEV约1×106个)，造模10天时处死小鼠。
(2)实验结果
1)如图10A-10C所示，PBMC能抑制DSS诱导的肠炎。结肠炎小鼠的体重显著降低，IEVs治疗显著减轻了DSS诱导的结肠炎小鼠体重减轻的趋势(图10A)。DSS诱导导致结肠长度显著减少，严重的结肠上皮和隐窝破坏以及炎性细胞的大量浸润，而IEVs治疗显著缓解这些症状(图10B，10C)。
2)图11的结果显示，PBMC IEVs能调节肠炎小鼠T细胞过度激活。IEVs治疗可显著诱导肠炎小鼠过度激活的CD3+T细胞的凋亡。其中PBMS来源IEVs效果优于PBMS外泌体(exo或exosome)。
3)PBMS来源的IEVs能抑制肠炎小鼠Th17细胞的激活。IEVs治疗可显著诱导肠炎小鼠循环中的Th17细胞(图12)。
Plasma-MV组，Plasma-exo low组，Plasma exo high组分别指未诱导状态下血浆中的大囊泡，外泌体(小囊泡)低剂量组，外泌体(小囊泡)高剂量组。
实施例4红细胞来源的囊泡的获得
1、红细胞来源及分离步骤
静脉抽取志愿者10mL血液样本置于EDTA抗凝管内，加入等体积的PBS缓冲液1:1混合制成细胞悬液。取4个新的15mL离心管，预先在每管中加入5mL人外周血淋巴分离液(Solarbio,#P8610)，将5mL血与PBS的混合液沿管壁缓慢加入到淋巴液上层，用低速水平离心机550g，4℃，离心30min，升速为4降速为0。离心后管内可见分为4层：最上层是淡黄色稀释的血浆；第二层为云雾状的白膜层，为外周血单个核细胞(PBMC)；第三层为淋巴分离液层；第四层为红细胞，红细胞沉于管底。弃掉红细胞层以上所有液体，吸取红细胞加入40mL PBS缓冲液中，400g，4℃，离心10min，洗去残余的血浆，淋巴分离液以及低速离心去除部分血小板。该离心步骤可重复1次。获得的红细胞可用适量PBS重悬。2、使用红细胞裂解液诱导红细胞(RBC)产生囊泡
每1mL全血来源的红细胞使用10mL红细胞裂解液裂解。使用红细胞裂解液处理红细胞4小时，吹打培养皿底细胞，收集所有脱落的细胞和细胞上清；将其进行800g，4℃离心10min，留上清；2000g，4℃离心10min，留上清；上清16000g，4℃离心30min，弃上清，保留囊泡沉淀；加入1mL PBS吹打重悬沉淀，16000g，4℃离心30min，弃上清，保留囊泡沉淀；囊泡沉淀可用适量PBS重悬，待用。
使用荧光显微镜进行观察，结果如图13(裂解第30min观察到的)所示，从图13可以看出，使用红细胞裂解液处理RBC可发现细胞膜出现明显皱缩的形态改变以及产生囊泡(红色箭头)。裂解液可诱导RBC以释放“囊泡”的形式死亡，而非细胞胀破。
3、使用红细胞裂解液诱导hBMSCs产生囊泡
使用红细胞裂解液处理hBMSCs(第8代)4小时，800g，4℃离心10min，留上清；2000g，4℃离心10min，留上清；上清16000g，4℃离心30min，弃上清，留囊泡沉淀；加入1ml PBS吹打重悬沉淀，16000g，4℃离心30min，弃上清，留囊泡沉淀；囊泡沉淀可用适量PBS重悬，待用。
使用荧光显微镜进行观察，结果如图14所示，从图14可以看出，使用红细胞裂解液处理hBMSCs发现细胞皱缩变圆，可见较多大泡(红色箭头)形成。
对比例1
(1)使用STS(星形孢菌素)诱导hBMSCs的具体步骤：待hBMSCs生长至90％时加入含500nM STS的α-MEM无血清培养基诱导3h。
(2)使用STS(星形孢菌素)诱导RBC的具体步骤：用含5000nM STS的α-MEM无血清培养基培养RBC 6h。
使用荧光显微镜进行观察，结果如图15A-15B所示，表明使用STS处理RBC后未见形态改变，表明STS不能诱导RBC死亡。STS可明显诱导hBMSCs凋亡，表现为细胞皱缩，产生大小不一的囊泡。
实施例5红细胞裂解液诱导RBC产生的囊泡的分离鉴定
收集到的囊泡的鉴定步骤：按BD品牌的Annexin V/PI凋亡染色说明书要求，取适量囊泡用100μL Binding buffer重悬，加入2μL Annexin V染料(一种特异性结合膜表面磷脂酰丝氨酸(phosphatidylserine，PS)的染料)，室温孵育15min，流式细胞仪检测Annexin V阳性囊泡比例，即膜表面表达PS阳性囊泡的比例。
流式初步检测囊泡的粒径以及分析凋亡表面标志物磷脂酰丝氨酸(PS)的表达情况。结果如图16A-16C所示，相比于STS诱导hBMSCs产生的囊泡，裂解液诱导hBMSCs死亡后产出的囊泡具有更高比例的小粒径囊泡，且高表达凋亡表面标志物PS(图16A、图16B)。裂解液诱导RBC死亡后产出的囊泡高表达PS(图16C)，具有类似干细胞来源的IEVs的PS强阳性的表面特征(14B)，推测在体内具有类似的代谢途径和功能。
备注：M8和M10仅代表流式图中“门”的名字，流式中“门”就是“范围”的意思，即M8和M10各表示一个范围，M8门显示的数字代表一个小粒径范围的比例，M10代表一个大粒径范围的比例。
STS诱导hBMSCs产生的囊泡的具体步骤：(1)STS诱导9h，吹打培养皿底细胞，收集所有脱落的细胞和细胞上清；(2)800g，4℃离心10min(去除死细胞)，留上清；(3)2000g，4℃离心10min(去除细胞碎片)，留上清；(4)上清16000g，4℃离心30min，弃上清，留囊泡沉淀；(5)加入1ml PBS吹打重悬沉淀，16000g，4℃离心30min，弃上清，留囊泡沉淀(此步骤目的洗掉可能残余的培养基和STS)；(6)囊泡沉淀可用适量PBS重悬，待用。
其中，使用红细胞裂解液诱导hBMSCs产生囊泡：使用红细胞裂解液处理hBMSCs(第8代)4小时，800g，4℃离心10min，留上清；2000g，4℃离心10min，留上清；上清16000g，4℃离心30min，弃上清，留囊泡沉淀；加入1ml PBS吹打重悬沉淀，16000g，4℃离心30min，弃上清，留囊泡沉淀；囊泡沉淀可用适量PBS重悬，待用。
裂解液诱导RBC产生的囊泡的具体步骤：具体步骤如实施例4中所述。
实施例6红细胞裂解液诱导RBC产生的囊泡的应用
1、实验方法：
使用葡聚糖硫酸钠盐(Dextran Sulfate Sodium Salt,DSS)溶液诱导小鼠溃疡性结肠炎，观察裂解液诱导产生的红细胞来源的囊泡(RBC-EVs)对小鼠肠炎是否有治疗作用。
选取8周，雄性，C57小鼠进行实验，每组2只。对照组(Control组)正常饮水和喂食，造模组(DSS组)和治疗组(hBMSC-ApoEVs组，RBC-EVs组)均自由饮用2.5％DSS溶液，正常喂食，持续8天，每日记录体重变化及便血程度。其中，治疗组于第3日予以尾静脉注射相应的囊泡进行治疗，hBMSC-ApoEVs组每只注射一个10cm dish的hBMSCs(大约3×10^6个hBMSCs)来源的囊泡(方法同中国专利申请202110077486.9中的囊泡获得方法))，RBC-EVs组每只注射10^8个RBC裂解产生的囊泡(实施例5的方法获得的)。造模组仅注射同等体积的PBS。第8日，取小鼠结肠组织，拍照，测量长度，组织固定，包埋切片。
2、实验结果：
结果如图17A-17B所示，从图中可以看出，尾静脉注射RBC-EVs可明显改善急性肠炎导致的小鼠体重下降和结肠缩短的症状，表明来源于RBC产生的囊泡具有一定的疾病治疗和应用价值。
其中，hBMSC-ApoEVs组获得步骤：
(1)STS诱导hBMSCs(第8代)凋亡9h，吹打培养皿底细胞，收集所有脱落的细胞和细胞上清；
(2)800g，4℃离心10min(去除死细胞)，留上清；
(3)2000g，4℃离心10min(去除细胞碎片)，留上清；
(4)上清16000g，4℃离心30min，弃上清，留囊泡沉淀；
(5)加入1mL PBS吹打重悬沉淀，16000g，4℃离心30min，弃上清，留囊泡沉淀(此步骤目的洗掉可能残余的培养基和STS)；
(6)囊泡沉淀可用适量PBS重悬，待用。
实施例7红细胞(RBC)或红细胞囊泡(RBC-EVs)促毛发生长
1、实验步骤：
选择Balb/c小鼠，雄性，毛发生长休止期(第6周)开始实验，以平行脊椎为长轴方向进行背部剃毛，脱毛剂脱毛，制作2cm×4cm的脱毛区进行造模；造模次日进行干预，每周尾静脉注射一次1×109个鼠RBC裂解产生的EV(共3×1010个EV)，PBS组注射等RBC-EVs(lysis)组体积的PBS，RBC组每周尾静脉注射一次1×109个鼠RBC。每日观察小鼠毛发生长情况并拍照记录新生毛发面积。观察周期为4周，4周后取小鼠脱毛区皮肤，组织切片染色，检测毛囊数量，毛囊大小，毛囊细胞增殖情况等。
组织染色：取小鼠脱毛区皮肤组织，经固定脱水后行皮肤组织纵切片，免疫荧光染色，蓝色表示细胞核(Nucleus)，绿色表示细胞骨架(Actin)，统计毛囊数量。
RBC-EVs(lysis)组的囊泡获得方法同实施例4中红细胞裂解液诱导红细胞(RBC)产生囊泡的方法。
2、实验结果：
如图18所示，第21天时RBC组和RBC-EVs(lysis)组小鼠的新生毛发面积明显高于PBS组。如图19所示，RBC组和RBC-EVs(lysis)组小鼠脱毛区毛囊数量明显增加。实验结果表明,RBC或RBC-EVs(lysis)具有明显促进小鼠脱毛区毛发新生和增加毛囊数量的作用。
实施例8红细胞囊泡(RBC-EVs)在小鼠急性呼吸窘迫综合征(ARDS)和糖尿病伤口愈合模型中的治疗应用
1、ARDS模型
1.1实验步骤：
取8周龄C57BL/6小鼠，随机分为3组，分别是正常对照组(control)，ARDS模型组，RBC-EVs(lysis)气管滴注治疗组。术前6小时禁食禁水，麻醉使用4％水合氯醛10ml/kg。模型组及治疗组均先气管滴注给予30μl LPS(按小鼠体重计算给药，5mg/kg)，正常组滴注等体积的PBS。4小时后，治疗组经气管滴注50μL的RBC-lysis-EV(约3×1010个)，正常组和ARDS造模组滴注等体积的PBS。造模48小时后处死小鼠，取肺组织固定切片染色。
其中，RBC-EVs(lysis)组的囊泡获得方法同实施例4中红细胞裂解液诱导红细胞(RBC)产生囊泡的方法。
1.2实验结果：
肺组织的HE染色：如图20可见，与control组相比，ARDS组可见肺泡组织结构破坏严重，充血水肿明显，大量炎性细胞浸润，肺泡间隔明显增厚且结构紊乱。RBC-EVs(lysis)气管滴注组，小鼠肺组织的充血及炎症浸润程度明显减轻，肺组织渗出减少，肺泡间隔增厚和结构紊乱情况也有明显改善。
2、1型糖尿病伤口愈合模型
2.1实验步骤：
取6周龄C57BL/6小鼠，随机分为3组，分别是正常对照组(control)，1型糖尿病(T1DM)模型组，RBC-EVs(lysis)局部注射治疗组。所有小鼠适应性喂养1周。建模开始前禁食、禁水12h，测量空腹体重及血糖并记录。按空腹体重计算小鼠所需链脲佐菌素(streptozotocin，STZ)剂量(50mg/kg·d·只)，于小鼠左下腹注射，1次/d，连续5d。给药后第7天，连续3d尾静脉取血测随机血糖，血糖平均值≥16.7mmol/L即为建模成功。各组小鼠背部剃毛，脱毛膏脱毛，剪刀剪去背部皮肤，制作1cm×1cm的开放性皮肤创口，创口模型建立后治疗组单次局部注射100μL的RBC-lysis-EV(约3×1010个)，正常组和T1DM模型组局部注射等体积的PBS。每日观察各组小鼠创口愈合情况并拍照记录。
其中，RBC-EVs(lysis)组的囊泡获得方法同实施例4中红细胞裂解液诱导红细胞(RBC)产生囊泡的方法。
2.2实验结果：
如图21可见，与control组相比，T1DM组创口愈合速度减慢。创口造模后第7天，RBC-EVs(lysis)局部注射治疗组创口愈合程度明显增加，表明RBC-EVs(lysis)可明显促进糖尿病小鼠的皮肤创口愈合。
实施例9 PBMC来源IEVs的分析
1、人源PBMC分离(福麦斯PBMC分离管(预装，含分离液)
抗凝管取血，用PBS稀释血液(PBS：外周血＝1：1稀释血液)，之后加入到福麦斯PBMC分离管，800g离心10min，取离心后白膜层到新的50mL离心管，用适量PBS 300g，10min洗两次，RPMI(不含FBS)重悬细胞团进行细胞计数，细胞计数后调整细胞浓度为1×106/mL培养基，加入10cm培养皿中进行培养。
2、诱导PBMC凋亡：
PBMC+STS 500nM 12h，PBMC+42℃ 6h，PBMC+43℃ 6h，PBMC+45℃ 6h，PBMC+48℃ 6h。
3、IEVs提取：
方法同实施例1。
4.Zetaview检测：IEVs沉淀沉淀用ddH2O重悬，稀释至合适倍数，上机检测(PMX-420-12F-R5)，结果如图22A-22C所示。
实施例10囊泡的制备方法
方法如同实施例4中的红细胞裂解液诱导红细胞(RBC)产生囊泡，只是其中裂解液来源不同，其来源于康为世纪 生物科技有限公司，货号#CW0613S。
结果如图23所示，同实施例4的红细胞裂解液诱导红细胞(RBC)产生囊泡，也表现为细胞膜皱缩，细胞以产生囊泡的形式死亡。
对比例2
1、外泌体分离和提取
将培养至第2代的hBMSCs，培养至细胞汇合80％-90％时，用PBS冲洗2遍，加入无血清培养基，37℃孵育48h，收集细胞上清液，用于分离和提取Exosomes。
提取步骤包括：800g离心10分钟—收集上清液—2000g离心10分钟—收集上清液—16000g离心30分钟—收集上清液—120000g离心90分钟—移除上清液，无菌PBS重悬沉淀—120000g再次离心90分钟，移除上清，收集底部的Exosomes，无菌PBS重悬。
2、红细胞裂解液诱导红细胞(RBC)产生囊泡的分离和提取
制备方法同实施例4。
流式检测此实施例获得的外泌体(exosome)和红细胞(RBC)产生的囊泡(apoEV)凋亡表面标志物磷脂酰丝氨酸(PS)的表达情况，结果如图24所示。
从图24可以看出，exosome表面标志物PS表达量为23.9％，apoEV的PS表达量为70.1％。

Claims (20)

  1. 来源于血液的样品在制备囊泡中的应用，其特征在于，所述囊泡为诱导性囊泡。
  2. 如权利要求1所述的应用，其特征在于，所述血液包括血浆、全血；
    优选地，所述血液为外周血；
    或优选地，所述血液含有血细胞；
    更为优选地，所述样品包含外周血单个核细胞；
    或更为优选地，所述样品包含红细胞；
    优选地，所述外周血单个核细胞为来源于血液的经分离或未经分离的细胞。
  3. 如权利要求2所述的应用，其特征在于，所述诱导性囊泡是在来源于的血液的样品中的细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡；
    优选地，所述外力包括添加星形孢菌素、添加乙醇、添加双氧水、紫外线照射、饥饿法、裂解液、热应力法、或机械力法中的至少一种；
    优选地，所述外力为热处理；
    优选地，所述热处理是在38℃～60℃范围内进行的；
    优选地，所述热处理是在40℃～55℃范围内进行的；
    优选地，所述热处理是在40℃～52℃范围内进行的；
    优选地，所述热处理是在42℃～52℃范围内进行的；
    优选地，所述热处理是在42℃～50℃范围内进行的；
    优选地，所述热处理的时间为3～20小时；
    优选地，所述热处理的时间为3～15小时；
    优选地，所述热处理的时间为3～12小时；
    优选地，所述热处理的时间为3～10小时。
  4. 如权利要求3所述的应用，其特征在于，所述囊泡对Annexin V,Integrin alpha 5和Syntaxin 4表达呈阳性；
    优选地，所述囊泡的直径为0.03-6μm；
    优选地，所述囊泡的直径为0.03-4.5μm；
    优选地，所述囊泡的直径为0.03-1μm；
    优选地，所述囊泡的直径为0.04-1μm；
    优选地，所述囊泡的直径为0.05-1μm；
    优选地，所述囊泡的直径为0.1-1μm；
    优选地，所述囊泡的直径为0.15-1μm。
  5. 一种制备囊泡的方法，其特征在于，所述方法包括对来源于血液的样品进行外力处理从而获得所述囊泡；
    优选地，所述血液为外周血；
    或优选地，所述血液含有血细胞；
    更为优选地，所述样品包含外周血单个核细胞；
    或更为优选地，所述样品包含红细胞；
    优选地，所述外周血单个核细胞为来源于血液的经分离或未经分离的细胞。
    优选地，所述诱导性囊泡是在来源于的血液的样品中的细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡；
    优选地，所述外力包括添加星形孢菌素、添加乙醇、添加双氧水、紫外线照射、饥饿法、裂解液、热应力法或或机械力法中的至少一种；
    优选地，所述外力为热处理法；
    优选地，所述热处理是在38℃～60℃范围内进行的；
    优选地，所述热处理是在40℃～55℃范围内进行的；
    优选地，所述热处理是在40℃～52℃范围内进行的；
    优选地，所述热处理是在42℃～52℃范围内进行的；
    优选地，所述热处理是在42℃～50℃范围内进行的；
    优选地，所述热处理的时间为3～20小时；
    优选地，所述热处理的时间为3～15小时；
    优选地，所述热处理的时间为3～12小时；
    优选地，所述热处理的时间为3～10小时；
    优选地，所述双氧水的浓度为20～2000μM；
    优选地，所述双氧水的浓度为50～1500μM；
    优选地，所述双氧水的浓度为50～1200μM；
    优选地，所述双氧水的浓度为50～1000μM；
    优选地，所述双氧水的浓度为100～1200μM；
    优选地，所述双氧水的浓度为100～1000μM；
    优选地，所述双氧水的浓度为100～800μM；
    优选地，所述双氧水的浓度为100～500μM；
    优选地，所述双氧水的浓度为100～400μM；
    优选地，所述双氧水的浓度为100～300μM；
    优选地，所述双氧水的处理时间为4～30h；
    优选地，所述双氧水的处理时间为5～25h；
    优选地，所述双氧水的处理时间为8～25h；
    优选地，所述双氧水的处理时间为10～25h；
    优选地，所述双氧水的处理时间为12～25h；
    优选地，所述双氧水的处理时间为12～20h。
    优选地，所述囊泡对Annexin V,Integrin alpha 5和Syntaxin 4表达呈阳性；
    优选地，所述囊泡为诱导性囊泡；
    优选地，所述囊泡的直径为0.03-6μm；
    优选地，所述囊泡的直径为0.03-4.5μm；
    优选地，所述囊泡的直径为0.03-1μm；
    优选地，所述囊泡的直径为0.04-1μm；
    优选地，所述囊泡的直径为0.05-1μm；
    优选地，所述囊泡的直径为0.1-1μm；
    优选地，所述囊泡的直径为0.15-1μm；
    优选地，所述乙醇的浓度为50～500nM；
    优选地，所述乙醇的浓度为100～500nM；
    优选地，所述乙醇的浓度为100～300nM；
    优选地，所述乙醇的处理时间为4～30h；
    优选地，所述乙醇的处理时间为8～25h；
    优选地，所述乙醇的处理时间为12～20h；
    优选地，所述紫外照射为紫外交联仪200～500mJ/cm2处理1～8h；
    优选地，所述紫外照射为紫外交联仪200～400mJ/cm2处理2～6h；
    优选地，所述紫外照射为紫外交联仪200～350mJ/cm2处理2～5h。
  6. 如权利要求5所述的方法，其特征在于，所述方法还包括对所述的囊泡进行提取；
    优选地，所述的方法包括以下步骤：
    (1)获得外周血单核细胞，进行培养；
    (2)在培养基中，对所述的外周血单核细胞进行热处理，诱导所述囊泡的释放；
    (3)收集步骤(2)的培养基上清，分离出所述囊泡；
    优选地，通过选自由聚合物沉淀、免疫分离、磁免疫捕获、超速离心、密度梯度离心、尺寸排阻色谱、超滤、超速离心、密度梯度离心、尺寸排阻色谱、超滤和它们的组合所组成的组中的方法从培养基中分离所述囊泡；
    优选地，所述分离囊泡的方法包括以Annexin V、Integrin alpha 5和Syntexin 4为标志物进行分离；
    优选地，通过选用超速离心的方法分离所述囊泡。
  7. 如权利要求1-4任一所述的应用或权利要求5-6任一所述的方法，其特征在于，所述样品来源于哺乳动物；
    优选地，所述哺乳动物选自灵长类动物或鼠；
    优选地，所述灵长类动物为人。
  8. 一种囊泡，其特征在于，由权利要求5-7任一-所述的方法获得。
  9. 一种药物组合物，其特征在于，包括权利要求8所述的囊泡以及药学上可接受的辅料；
    优选地，所述药物组合物的制剂形式选自冻干粉针、注射剂、片剂、胶囊、或贴剂。
  10. 权利要求8所述的囊泡或权利要求9所述的药物组合物在制备治疗或预防或改善疾病或所述疾病并发症的产品中的应用；
    优选地，所述产品包括药物、食品、保健品、化妆品、添加剂或中间品；
    优选地，所述疾病包括肺部疾病或肠道疾病或糖尿病；
    优选地，所述产品用于促进毛囊修复和/或毛发再生；
    更为优选地，所述肺部疾病为急性呼吸窘迫综合症；
    或更为优选地，所述肠道疾病为肠炎；
    优选地，所述肠炎为急性肠炎；
    优选地，所述药物用于改善急性肠炎导致的小鼠体重下降或结肠缩短的症状；
    优选地，所述糖尿病为1型糖尿病；
    优选地，所述药物用于促进1型糖尿病伤口愈合。
  11. 一种裂解液在制备囊泡中的应用。
  12. 如权利要求11所述的应用，其特征在于，所述裂解液通过诱导细胞凋亡产生所述囊泡；
    优选地，所述诱导时间为1-8小时；
    优选地，所述诱导时间为2-4小时；
    优选地，所述细胞包括干细胞和红细胞；
    优选地，所述干细胞为间充质干细胞；
    优选地，所述间充质干细胞来源包括：骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或其组合。
  13. 如权利要求11或12所述的应用，其特征在于，所述裂解液包括红细胞裂解液。
  14. 一种产囊泡的方法，其特征在于，使用裂解液诱导细胞获得所述囊泡。
    优选地，所述细胞包括干细胞和红细胞；
    优选地，所述干细胞为间充质干细胞；
    优选地，所述间充质干细胞来源包括：骨髓、尿液、口腔、脂肪、胎盘、脐带、骨膜或其组合；
    优选地，所述裂解液包括红细胞裂解液。
    优选地，所述诱导时间为1-8小时；
    优选地，所述诱导时间为2-4小时；
    优选地，所述方法包括：(1)使用裂解液处理红细胞；(2)分离出囊泡；
    优选地，所述步骤(2)分离囊泡的方法包括选用超速离心的方法分离所述囊泡；
    优选地，所述超速离心的方法分离所述囊泡的步骤包括：(a)将收集到的裂解液上清进行第一次离心，取上清；(b)将步骤(a)中收集到的上清进行第二次离心，取上清；(c)将步骤(b)中收到到的上清进行第三次离心，取沉淀；(d)将步骤(c)中收到到的沉淀进行第四次离心，取沉淀；
    优选地，所述第一次离心为500-1500g离心5-30分钟；
    优选地，所述第二次离心为1000-3000g离心5-30分钟；
    优选地，所述第三次离心为10000-30000g离心15-60分钟；
    优选地，所述第四次离心为10000-30000g离心15-60分钟。
  15. 权利要求14所述的方法获得的囊泡。
  16. 一种组合物，其特征在于，包含权利要求15所述的囊泡和药学上可接受的辅料。
  17. 权利要求15所述的囊泡或权利要求16所述组合物在制备治疗肠炎的药物中的应用；
    优选地，所述肠炎为急性肠炎；
    优选地，所述药物用于改善急性肠炎导致的小鼠体重下降或结肠缩短的症状。
  18. 权利要求15所述的囊泡或红细胞来源的囊泡或权利要求16所述组合物或红细胞在制备促进毛囊修复和/或促进毛发再生的药物或保健品中的应用。
  19. 权利要求15所述的囊泡或权利要求16所述组合物在制备治疗急性呼吸窘迫综合征药物中的应用。
  20. 权利要求15所述的囊泡或权利要求16所述组合物在制备治疗糖尿病药物中的应用；
    优选地，所述糖尿病为1型糖尿病；
    优选地，所述药物用于促进1型糖尿病伤口愈合。
PCT/CN2023/085257 2022-03-08 2023-03-30 血液来源的样品在制备囊泡中的应用 WO2023169594A1 (zh)

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