WO2023123216A1 - 囊泡在制备治疗肺部疾病的药物中的应用 - Google Patents

囊泡在制备治疗肺部疾病的药物中的应用 Download PDF

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WO2023123216A1
WO2023123216A1 PCT/CN2021/143113 CN2021143113W WO2023123216A1 WO 2023123216 A1 WO2023123216 A1 WO 2023123216A1 CN 2021143113 W CN2021143113 W CN 2021143113W WO 2023123216 A1 WO2023123216 A1 WO 2023123216A1
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vesicles
disease
inducible
ievs
lung
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French (fr)
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寇晓星
施松涛
谭灵屏
赵陆
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中山大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure belongs to the field of biomedicine, and relates to the application of vesicles in the preparation of medicines for treating lung diseases.
  • Acute lung injury is acute hypoxic respiratory insufficiency caused by direct lung injury or indirect systemic injury, characterized by alveolar edema and hypoxemia caused by inflammation and changes in lung permeability. , respiratory distress, organ failure.
  • the course of ALI develops rapidly and has a high mortality rate, accounting for about 30% of the mortality of patients in intensive care.
  • ALI is also one of the important clinical manifestations and causes of death in severe patients with new coronavirus pneumonia. So far, the treatment of ALI lacks clear methods and targeted drugs.
  • the present disclosure provides the use of an inducible vesicle in the preparation of a medicament for treating/preventing a lung disease or disorder.
  • the pulmonary disease comprises acute inflammatory pulmonary disease.
  • the pulmonary disease or condition includes asthma, acute bronchitis, emphysema, chronic obstructive pulmonary disease, smoker's disease, reactive airway disease, cystic fibrosis, bronchiectasis, katage Internal syndrome, atelectasis, pneumonia, essential thrombocythemia, Legionnaires' disease, psittacosis, fibrosis pneumoconiosis, hypersensitivity diseases of the lungs, spontaneous permeability diseases of the lungs, respiratory distress syndrome, Lung tumors and diseases caused by organic dust, irritating gases and chemicals, acute lung injury.
  • the pulmonary disease is acute respiratory distress syndrome, acute lung injury, or pulmonary fibrosis.
  • the disease or condition is an infection caused by a bacterial, viral or fungal infection.
  • the virus comprises 2019 coronavirus.
  • the bacteria include Streptococcus pneumoniae, Staphylococcus aureus.
  • the present disclosure provides a composition comprising inducible vesicles, the composition further comprising a prophylactic or therapeutic agent for a lung disease or disorder, the prophylactic or therapeutic agent being selected from the group consisting of anti- One or more of bacterial agents, antiviral agents, antifungal agents, antineoplastic agents, antihistamines, proteins, enzymes, hormones, non-steroidal anti-inflammatory substances, cytokines, steroids, nicotine, and insulin.
  • the pulmonary disease is acute respiratory distress syndrome, acute lung injury, or pulmonary fibrosis.
  • the therapeutic agent is an antiviral agent.
  • the virus comprises 2019 coronavirus.
  • the present disclosure provides a medicine set, comprising: (a) an inducible vesicle; (b) a preventive or therapeutic agent for a lung disease or disorder; in the medicine set, the inducible vesicle and the The prophylactic or therapeutic agent for the above-mentioned pulmonary disease or disorder is packaged separately.
  • the present disclosure provides a use of a pharmaceutical composition comprising inducible vesicles in the manufacture of a medicament for treating or preventing a pulmonary disease or disorder.
  • the present disclosure provides a method of treating and/or preventing a pulmonary disease or condition comprising: administering an inducible vesicle to a patient.
  • the pulmonary disease or disorder comprises acute inflammatory pulmonary disease.
  • the pulmonary disease or disorder is acute respiratory distress syndrome, acute lung injury, or pulmonary fibrosis.
  • the disease or condition is a pulmonary disease or condition caused by a bacterial, viral or fungal infection.
  • the virus comprises 2019 coronavirus.
  • the bacteria include Streptococcus pneumoniae, Staphylococcus aureus.
  • the inducible vesicles are vesicles produced by inducing apoptosis through external factors when the stem cells are in normal survival.
  • the inducible vesicles are produced by inducing stem cell apoptosis, and the inducing method includes adding Staurosporum, ultraviolet irradiation, starvation method, or heat stress method.
  • the stem cells are mesenchymal stem cells.
  • the inducible vesicles can also be derived from P62-P8 generation, but it is not limited thereto.
  • the source of mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, or peripheral blood.
  • the mesenchymal stem cells are bone marrow mesenchymal stem cells.
  • the mesenchymal stem cells are derived from mammals, but are not limited thereto.
  • the mammal is selected from primates or mice, but is not limited thereto.
  • the primate is a human.
  • the method for preparing the inducible vesicles comprises the steps of: (1) culturing mesenchymal stem cells; (2) collecting the medium supernatant of mesenchymal stem cells; (3) extracting from step (2) Vesicles were isolated from the culture supernatant.
  • the step of culturing mesenchymal stem cells in the step (1) includes: (4) isolating mesenchymal stem cells from tissues; (5) adding medium to culture mesenchymal stem cells; said mesenchymal stem cells Mesenchymal stem cells were exposed to apoptosis-inducing agents in culture medium.
  • the method for isolating the vesicles includes using ultracentrifugation to isolate the vesicles.
  • the step of isolating the vesicles by the ultracentrifugation method includes: (a) performing the first centrifugation on the collected culture supernatant, and taking the supernatant; (b) the step (a) The collected supernatant is centrifuged for the second time, and the supernatant is taken; (c) the supernatant received in the step (b) is centrifuged for the third time, and the precipitate is taken; (d) the supernatant received in the step (c) is The precipitate was centrifuged for the fourth time, and the precipitate was collected.
  • the drug is selected from injection, nebulized inhalation, spray, oral preparation or external preparation.
  • the drug is an injection.
  • the inducible vesicles can be optionally injected through the group selected from intravenous injection, intramuscular injection, subcutaneous injection, tracheal drip, sheath Administration is performed by the route in the group consisting of intra-injection or infusion and intra-organ infusion.
  • intra-injection or infusion for intravenous injection, by way of example, through the tail vein.
  • Intra-organ infusion includes infusion into anatomical spaces such as, by way of example, the gallbladder, gastrointestinal lumen, esophagus, pulmonary system (by inhalation) and/or bladder.
  • the medicament further includes a pharmaceutically acceptable carrier.
  • the pharmaceutical carrier includes one or more of diluents, excipients, fillers, binders, disintegrants, surfactants and lubricants.
  • the inducible vesicles described in this disclosure are essentially different from existing extracellular vesicles (such as exosomes, etc.).
  • the inducible vesicles IEVs described in this disclosure highly express Syntaxin 4, and the expression levels of Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 are significantly higher than that of exosomes (see Example 4).
  • the inducible vesicle IEVs also exhibit characteristics different from those of stem cells and other extracellular vesicles (such as exosomes) in terms of function or therapeutic effect.
  • IEVs can significantly shorten the in vitro clotting time of most plasma, and the effect of promoting coagulation is better than that of exosomes (see Test Example 4).
  • the mechanism of IEVs treating hemophilic mice has nothing to do with PS and TF, while in previous literature reports, the procoagulant effect of extracellular vesicles is highly dependent on PS and TF on their surface (see Test Example 4).
  • the inventors have found in some studies that the therapeutic effects between the inducible vesicles described in the present disclosure and the blast cells from which they are derived are not reciprocal.
  • mesenchymal stem cells can treat non-alcoholic steatohepatitis and damaged liver fibrosis, however, inducible vesicles cannot achieve therapeutic effects on these diseases (see test examples 1 and 2).
  • MSCs can treat Sjogren's syndrome, while the inducible vesicles described in the present disclosure have no therapeutic effect on Sjögren's syndrome (see Test Example 3). That is to say, if a certain stem cell can treat a certain disease, it cannot necessarily be inferred that the induced vesicles (IEVs) produced by it must also be able to treat this disease.
  • IEVs induced vesicles
  • exosomes derived from lung endothelial cells can alleviate lung injury by repairing the connection between lung epithelial cells (Yue, Zhou, Pengfei, et al. Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury .[J].Critical Care,2019.); Exosomes derived from mesenchymal stem cells can reduce the release of pro-inflammatory factors and the increase of anti-inflammatory factors by regulating the polarization of macrophages (Li, JW, Wei, L , Han, Z, Chen, Z.
  • 1A-1E are the results of flow cytometric detection of surface markers of isolated BMMSCs.
  • Fig. 2 is the HE staining section of mouse lung tissue in each group.
  • A is the HE slice of the normal control group (Control group);
  • B is the HE slice of the model group (LPS group);
  • C is the HE slice of the tail vein injection treatment group (LPS+IEVs-S group);
  • D is the IEVs transtracheal infusion treatment group (LPS+IEVs-L group) HE slice;
  • E is 5 ⁇ HE slice of hBMMSC tail vein treatment group (LPS+hBMMSC group);
  • F is 10 ⁇ HE slice of hBMMSC tail vein treatment group (LPS+hBMMSC group).
  • Figure 5 shows the results of flow cytometry detection of neutrophils in mouse lung tissue.
  • Figure 6 shows the results of immunohistochemistry and immunofluorescence detection of neutrophils in mouse lung tissue.
  • Figure 7 is the particle size diagram of IEVs NTA detection.
  • Fig. 8 is a transmission electron microscope examination diagram of the morphology of IEVs.
  • Figure 9A- Figure 9D is the content analysis of IEVs:
  • Figure 9A is the quantitative analysis results of MSCs, MSCs-Exosomes, MSCs-IEVs proteomics by DIA quantitative technology;
  • Figure 9C is the result of GO enrichment analysis of differential proteins for IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules;
  • Figure 9D is western blot verification of MSCs, MSCs-Exosomes, MSCs-IEVs expressing Annexin V, results of Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4.
  • Figure 10 is the data of MSCs-derived IEVs in the treatment of fatty liver disease fed with methionine-choline feed.
  • Figure 11 is the data of MSCs-derived IEVs in the treatment of damaged liver fibrosis induced by ammonium thioacetate.
  • Figure 12 is IEVs treatment of Sjögren's syndrome: A.IEVs treatment of Sjögren's syndrome (Sjögren's syndrome) salivary flow rate; B.IEVs treatment of Sjogren's syndrome submandibular gland HE staining results; C. treatment of Sjogren's syndrome on B Effects on cells.
  • Figure 13 is the procoagulant effect of IEVs in hemophilia A mice.
  • Figure 14A- Figure 14D is the change situation of various blood coagulation factor levels after injecting IEVs to hemophilia A mice: Fig. 14A is the change situation of blood coagulation factor VIII; Fig. 14B is the change situation of vWF factor; Fig. 14C is tissue factor ( TF) changes; Figure 14D shows the changes in prothrombin.
  • Figures 15A-15B show the effects of blocking IEVs with PS and TF on the therapeutic effect of IEVs in vivo in the hemophilia A mouse model.
  • Figure 16 is a comparison of the therapeutic effects of IEVs and Exosomes derived from the same MSCs on hemophilia A mice.
  • IEVs in the embodiments of the present disclosure is the abbreviation of induced vesicles, which may be called induced vesicles, and may also be called induced extracellular vesicles (Induced extracellular vesicles, IEVs).
  • Inducible extracellular vesicles refer to a type of subcellular product that is intervened or induced to induce apoptosis when precursor cells (such as stem cells) survive normally. Usually this type of subcellular product has a membrane structure, expresses apoptotic markers, and partially contains genetic material DNA.
  • inducible extracellular vesicles are a class of substances that are distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.).
  • the normal surviving cells are, for example, non-apoptotic cells, non-senescent cells, non-senescent cells with stagnant proliferation, non-resuscitated cells after cryopreservation, non-malignant cells with abnormal proliferation cells or non-damaged cells, etc.
  • the normally viable cells are obtained from the cells when the cells are 80-100% confluent during cell culture. In some embodiments, the normally viable cells are obtained from logarithmic phase cells. In some embodiments, the normal living cells are obtained from primary culture and subculture cells derived from human or mouse tissue. In some embodiments, the normally viable cells are obtained from established cell lines or strains. In some embodiments, the precursor cells are obtained from earlier cells.
  • STS in the present disclosure is staurosporine.
  • the inducible vesicles can also be derived from hBMMSCs of passage P2-P8.
  • treatment or/prevention means that it can be treatment or prevention.
  • the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term “and/or” means that any one of the listed items can be used alone, or any combination of two or more listed items can be used . For example, if a composition, combination, configuration, etc.
  • composition may comprise A alone; B alone; C alone; D alone ; Combination of A and B; Combination of A and C; Combination of A and D; Combination of B and C; Combination of B and D; Combination of C and D; Combination of A, B and C A combination; a combination comprising A, B, and D; a combination comprising A, C, and D; a combination comprising B, C, and D; or a combination of A, B, C, and D.
  • the method for isolating the inducible vesicles comprises isolating the vesicles by ultracentrifugation.
  • the ultracentrifugation includes four centrifugations.
  • 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 Centrifuge for 5-15 minutes.
  • the second centrifugation is 1000-3000g centrifugation for 5-30 minutes; or the second centrifugation is 1500-2500g centrifugation for 5-20 minutes; or the second centrifugation is 1500-2200g Centrifuge for 5-15 minutes.
  • the second centrifugation is at 1700-2200 g for 5-18 minutes.
  • the second centrifugation is at 1990-2100 g for 8-15 minutes.
  • the supernatant obtained by the second centrifugation is subjected to a third centrifugation, and the third centrifugation is 15-60 minutes at 10000-30000 g; in some embodiments, the third centrifugation Centrifuge at 15500-19000g for 20-40 minutes. In some embodiments, the third centrifugation is 15500-17000 g for 20-40 minutes. In some embodiments, the fourth centrifugation is centrifugation at 10000-18000 g for 15-60 minutes. In some embodiments, the fourth centrifugation is centrifugation at 10000-17500 g for 15-60 minutes. In some embodiments, the fourth centrifugation is centrifugation at 10000-17000 g for 15-60 minutes.
  • the fourth centrifugation is 11000-17000 g for 15-60 minutes. In some embodiments, the fourth centrifugation is centrifugation at 12000-17000 g for 15-60 minutes. In some embodiments, the fourth centrifugation is 10000-18000g centrifugation for 20-60 minutes. In some embodiments, the fourth centrifugation is 10000-18000g centrifugation for 20-50 minutes.
  • the diameter of the inducible vesicle can be 0.03-6 ⁇ M; it can be 0.03-4.5 ⁇ M; it can also be 0.03-1 ⁇ M; it can be 0.04-1 ⁇ M, it can also be 0.05-1 ⁇ M, it can also be 0.1- 1 ⁇ M, can also be 0.15-1 ⁇ M, can also be 0.15-0.45 ⁇ M, can also be 0.15-0.3 ⁇ M, can also be 0.2-0.3 ⁇ M.
  • the inducible vesicle has the marker Syntaxin 4. In some embodiments, the inducible vesicle highly expresses the marker Syntaxin 4. In some embodiments, the expression of the marker Syntaxin 4 of the inducible vesicles is higher than that of MSCs or exosomes. In some embodiments, the expression level of the marker Syntaxin 4 is 3-6 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the expression level of the marker Syntaxin 4 is 3.5-5 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells.
  • the expression level of the marker Syntaxin 4 is 4.45 times the expression level of Syntaxin 4 in exosomes derived from mesenchymal stem cells.
  • the markers further include one or more of Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5.
  • the marker is a combination of Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5.
  • the inducible vesicles highly express markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5.
  • the expression levels of the inducible vesicle pair markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 are higher than those of MSCs or exosomes. In some embodiments, the expression of markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the inducible vesicles is relative to the expression of markers in exosomes derived from mesenchymal stem cells 1-2 times, 2-3 times, 1-3 times and 3-4 times respectively.
  • the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5 in the inducible vesicles are 1.5-2 times, 2.5-3 times, 1.5-2.5 times and 3.5- 4 times. In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 in the vesicles are 1.76 times, 2.81 times, 2.41 times, and 3.68 times, respectively.
  • mice were killed with excessive CO 2 , and under aseptic conditions, the tibia and femur were removed, and the muscles and connective tissues attached to them were peeled off, and the metaphysis was further separated to expose the bone marrow cavity.
  • Extract PBS containing 10% fetal bovine serum with a sterile syringe to repeatedly rinse the bone marrow cavity, filter through a 70 ⁇ m pore cell strainer, centrifuge at 500g for 5min, remove the supernatant and collect the cell pellet at the bottom, resuspend in PBS, and centrifuge again at 500g After 5 min, the final cell pellet was collected.
  • the cells were sorted by flow cytometry, and BMMSCs were sorted out by using CD34- and CD90+ as sorting criteria. Finally, the cells were resuspended in Dex(-) medium and inoculated on a 10cm diameter cell culture dish, and cultured at 37°C and 5% CO 2 . After 24 hours, the non-adherent cells in the supernatant were sucked off, washed with PBS, and then added with Dex(-) medium to continue culturing. One week later, an equal amount of Dex(+) culture medium was added, and dense colonies of primary BMMSCs could be seen after another week.
  • the purity of isolated BMMSCs was assessed by flow cytometry analysis of surface markers.
  • For the identification of surface markers after trypsinization to collect BMMSCs of passage P2, wash them once with PBS, resuspend the cells at a density of 5 ⁇ 10 5 /mL in PBS containing 3% FBS, add 1 ⁇ L of PE fluorescence-conjugated CD29, CD44, CD90, CD45 and CD34 antibodies were not added to the blank group. Incubate at 4°C in the dark for 30 minutes, wash with PBS twice, and test on the machine. The results of flow cytometry are shown in FIGS. 1A-1E . It can be seen that the isolated cells are BMMSCs (bone marrow mesenchymal stem cells).
  • Example 2 The acquisition of inducible vesicles (IEVs)
  • serum-free medium ⁇ -MEM medium
  • STS staurosporine 500nM
  • mice C57BL/6 mice, regardless of gender, 8-12 weeks old, weighing about 18g-22g, SPF grade.
  • the model group and the treatment group were given 30 ⁇ L LPS (5 mg/kg) by tracheal infusion first, and 2 hours later, the mice in the three treatment groups were infused with 30 ⁇ L hBMMSC-IEVs (about 3 ⁇ 10 8 /mouse) respectively through the trachea.
  • 30 ⁇ L hBMMSC-IEVs about 3 ⁇ 10 8 /mouse
  • 200 ⁇ L of hBMMSC-IEVs (about 3 ⁇ 10 8 cells/mouse) and 200 ⁇ L of hBMMSC cells (about 1 ⁇ 10 6 cells/mouse) were injected intravenously, and the mice were sacrificed 24 hours after modeling.
  • one BMMSCs produces approximately 300 IEVs.
  • the preparation method of the PBS suspension of hBMMSC cells is as follows: hBMMSCs of P5-P8 with a confluence rate of 90-100% are used, washed with PBS, digested with trypsin, centrifuged at 1500rpm for 5 minutes, and the cell pellet is collected, washed with PBS, centrifuged again, collected The cell pellet was resuspended in PBS, and the hBMMSC cell suspension was stored at 4°C before use.
  • HE stained slices showed that the alveolar tissue structure of the lung tissue slices in the Control group was complete, without obvious congestion and inflammatory cell infiltration (Fig. 2A); in the LPS group, the alveolar tissue structure was severely damaged, the congestion and edema were obvious, a large number of inflammatory cell infiltration, and the alveolar septum was obvious Thickened and structurally disordered (Fig. 2B); in the LPS+IEVs-L and LPS+IEVs-S groups, the congestion and inflammatory infiltration of the lung tissue of the mice were significantly reduced, the exudation of the lung tissue was reduced, and the alveolar septum was also thickened and structurally disordered.
  • the BCA method was used to measure the total protein concentration in the supernatant obtained by BALF centrifugation, and the OD value at 570 nm was measured by a microplate reader and then converted into protein concentration.
  • ARDS is accompanied by the exudation of macromolecules such as proteins.
  • the total protein concentration in BALF is an important indicator reflecting lung injury in ARDS.
  • the results are shown in Figure 4: LPS stimulation significantly increased the total protein concentration of BALF, while the two IEVs treated Both methods significantly reduced the total protein concentration of BALF.
  • mice After the mice were sacrificed, the lung tissue was removed, and the mouse lung tissue cells were collected by enzymatic digestion (the cells were dispersed into single cells), resuspended cells (1 ⁇ 10 5 ) in 100 ⁇ L PBS, and used CD11b, ly6G antibodies on ice After staining for 15 minutes, add 400 ⁇ L PBS, centrifuge at 1500rmp to discard the supernatant, resuspend in 100ul PBS, and perform flow cytometry detection.
  • the experimental results showed that after LPS stimulation, the number of neutrophils (CD11b+ly6G+ double positive cells) in the lungs of mice was significantly increased, and the number of neutrophils was significantly reduced after tracheal instillation of IEVs.
  • the mouse lung tissue was taken to prepare paraffin sections and frozen sections, and immunohistochemistry and immunofluorescence staining were performed with ly6G.
  • the experimental results show that after LPS stimulation, the number of neutrophils (ly6G positive cells) in the lungs of mice significantly increased, and the number of neutrophils decreased significantly after tracheal instillation of IEVs.
  • the IEVs pellet of Example 2 was resuspended in PBS, and after dilution, the concentration and particle size of IEVs were detected with a zetaview instrument.
  • Example 2 The IEVs precipitate of Example 2 was fixed with glutaraldehyde phosphate buffer solution, routinely dehydrated, soaked, embedded, and stained to make ultrathin sections, and the morphology and structure of IEVs were observed and recorded under an electron microscope.
  • MSCs-Exosomes refers to exosomes derived from MSCs.
  • MSCs-IEVs refers to the IEVs derived from MSCs, the specific method of obtaining: except that the cell generation is P2, other steps are the same as in Example 2.
  • the MSCs in the content analysis are the same cell line as the MSCs from which Exosomes and IEVs are extracted.
  • BMMSCs cultured to the second generation in Example 1 were continued to be cultured with the medium (Dex(+) culture fluid) in Example 1 until the cells were confluent 80%-90%, Rinse twice with PBS, add serum-free medium ( ⁇ -MEM medium), incubate at 37°C for 48h, and collect cell supernatant for separation and extraction of Exosomes.
  • medium Dex(+) culture fluid
  • ⁇ -MEM medium serum-free medium
  • the extraction steps include: centrifuge at 800g for 10 minutes—collect the supernatant—centrifuge at 2000g for 10 minutes—collect the supernatant—centrifuge at 16,000g for 30 minutes—collect the supernatant—centrifuge at 120,000g 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.
  • MSCs can treat non-alcoholic steatohepatitis (NASH) (Ezquer et al., J Hepatol, 2011; Winkler et al., Methods Mol Biol, 2014).
  • NASH non-alcoholic steatohepatitis
  • Detection steps or methods Take 8-week-old male C57 mice and feed them with normal diet (Normal chow diet, NCD) or methionine-choline deficiency diet (Methionine and choline deficiency diet, MCD). IEVs were infused through the tail vein and fed for 8 weeks. Liver tissues were sectioned with paraffin wax and stained with HE.
  • MSCs and exosomes can treat carbon tetrachloride or thioacetamide (Thioacetamide, TAA) induced injury liver fibrosis (Mehrabani et al., Arch Razi Inst, 2019; Sabry et al., Int J Stem Cells, 2019; Rong et al., Stem Cell Res Ther, 2019).
  • Thioacetamide, TAA thioacetamide
  • Detection steps or methods Take 8-week-old C57 mice and inject 100 mg/kg TAA intraperitoneally, 3 times a week, for 8 weeks to create a model of hepatic fibrosis. During TAA modeling period, IEVs were infused through the tail vein every 2 weeks, and liver tissues were collected after 8 weeks, paraffin sections and HE staining.
  • Detection steps or methods take 8-week-old Sjögren syndrome (SS) model mice, inject MSCs and IEVs through the tail vein system, collect samples 4 weeks after injection, detect saliva flow rate, collect salivary gland samples and perform paraffin section HE Staining and B cell marker B220 staining.
  • SS Sjögren syndrome
  • Example 3 The in vitro coagulation-promoting effect of the IEVs obtained in Example 2 and the Exosomes extracted in Example 4 was detected by an in vitro coagulation test. The results are shown in Table 3. IEVs can significantly shorten the coagulation time of most plasma in vitro, and the coagulation-promoting effect is better than that of Exosomes.
  • IEVs cannot exert in vitro procoagulant effect, indicating that the in vitro procoagulant effect of IEVs is more concentrated in the upstream of the common coagulation pathway.
  • hemophilia A mice deficiency of coagulation factor VIII
  • 9 ⁇ 10 8 IEVs were injected through the tail vein to observe the procoagulant effect of IEVs in vivo.
  • the results are shown in Figure 13, after IEVs treatment, the bleeding tendency of hemophilic mice can be significantly improved, and the therapeutic effect can be maintained stably for 14 days.
  • the experimental results show that IEVs can play a significant role in promoting coagulation in vitro. Moreover, the injection in vivo can significantly improve the bleeding tendency, and can be used to improve the bleeding tendency caused by hemophilia A. At the same time, the levels of various coagulation factors in mouse plasma were detected, and it was found that coagulation factor VIII, vWF factor, tissue factor (tissue factor, TF) and prothrombin (prothrombin) did not change significantly (Fig. 14A, Fig. 14B, Fig. 14C , Figure 14D).

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Abstract

提供一种囊泡在制备治疗肺部疾病的药物中的应用。所述囊泡为诱导性囊泡，能够用于治疗/预防多种肺部疾病或病症。急性肺损伤或急性呼吸窘迫综合症的治疗目前缺乏明确的方法及针对性的药物，所述的诱导性囊泡对其具有较好的治疗效果。其中急性肺损伤或急性呼吸窘迫综合症也是新型冠状病毒肺炎重症患者的重要临床表现及致死原因之一，因而所述的诱导性囊泡对于治疗2019冠状病毒肺炎具有极大的应用前景。

Description

囊泡在制备治疗肺部疾病的药物中的应用 技术领域
本公开属于生物医药领域，涉及一种囊泡在制备治疗肺部疾病的药物中的应用。
背景技术
急性肺损伤(Acute lung injury，ALI)是由直接肺部损伤或间接全身性损伤所导致的急性低氧性呼吸功能不全，其特征在于炎症及肺通透性改变导致肺泡水肿，低氧血症，呼吸窘迫，器官衰竭。ALI病程发展迅速，致死率高，约占重症监护患者死亡率的30％；同时，ALI也是新型冠状病毒肺炎重症患者的重要临床表现及致死原因之一。迄今为止，ALI的治疗缺乏明确的方法及针对性的药物。
发明内容
一些实施方案中，本公开提供了一种诱导性囊泡在制备用于治疗/预防肺部疾病或病症药物中的应用。
一些实施方案中，所述肺部疾病包括急性炎症引发的肺部疾病。
一些实施方案中，所述肺部疾病或病症包括哮喘、急性支气管炎、肺气肿、慢性阻塞性肺病、吸烟者的疾病、反应性气道疾病、囊性纤维化、支气管扩张、卡塔格内综合征、肺膨胀不全、肺炎、原发性血小板增多症、军团病、鹦鹉热、纤维化尘病、肺部的超敏反应性疾病、肺的自发性渗透性疾病、呼吸窘迫综合征、肺部肿瘤以及由有机灰尘、刺激性气体和化学物质引起的疾病、急性肺损伤。
一些实施方案中，所述肺部疾病为急性呼吸窘迫综合征、急性肺损伤或肺纤维化。
一些实施方案中，所述疾病或病症是由细菌、病毒或真菌感染引起的感染。
一些实施方案中，所述病毒包括2019冠状病毒。
一些实施方案中，所述细菌包括肺炎链球菌，金黄色葡萄球菌。
一些实施方案中，本公开提供了一种组合物，所述组合物包含诱导性囊泡，所述组合物还包含肺部疾病或病症的预防或治疗剂，所述预防或治疗剂选自抗细菌剂、抗病毒剂、抗真菌剂、抗肿瘤剂、抗组胺剂、蛋白质、酶、激素、非甾体抗炎性物质、细胞因子、类固醇、尼古丁和胰岛素中的一种或多种。
一些实施方案中，所述肺部疾病为急性呼吸窘迫综合征、急性肺损伤或肺纤维化。
一些实施方案中，所述治疗剂为抗病毒剂。一些实施方案中，所述病毒包括2019冠状病毒。
一些实施方案中，本公开提供了一种药品套装，包含：(a)诱导性囊泡；(b)肺部疾病或病症预防或治疗剂；在所述的药品套装中诱导性囊泡和所述肺部疾病或病症预防或治疗剂被分开包装。
一些实施方案中，本公开提供了一种药物组合物在制备用于治疗或预防肺部疾病或病症的药物中的应用，所述药物组合物包含诱导性囊泡。
一些实施方案中，本公开提供了一种治疗和/或预防肺部疾病或病症的方法，包括：给予患者诱导性囊泡。
一些实施方案中，所述肺部疾病或病症包括急性炎症引发的肺部疾病。
一些实施方案中，所述肺部疾病或病症为急性呼吸窘迫综合征、急性肺损伤或肺纤维化。
一些实施方案中，所述疾病或病症是由细菌、病毒或真菌感染引起的肺部疾病或病症。
一些实施方案中，所述病毒包括2019冠状病毒。
一些实施方案中，所述细菌包括肺炎链球菌，金黄色葡萄球菌。
一些实施方案中，所述诱导性囊泡是在干细胞处于正常存活时通过外加因素诱导凋亡而产生的囊泡。
一些实施方案中，所述诱导性囊泡是诱导干细胞凋亡产生的，所述诱导方法包括添加星形孢菌、紫外线照射、饥饿法、或热应力法。
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，所述诱导性囊泡也可以来源于P62-P8代，但又不限于此。
一些实施方案中，所述间充质干细胞来源包括骨髓、牙髓、尿液、口腔、脂肪、胎盘、脐带、骨膜、肌腱或外周血。
一些实施方案中，所述间充质干细胞为骨髓间充质干细胞。
一些实施方案中，所述间充质干细胞来源于哺乳动物，但又不限于此。
一些实施方案中，所述哺乳动物选自灵长类动物或鼠，但又不限于此。
一些实施方案中，所述灵长类动物为人。
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，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，即得。
一些实施方案中，所述的药物选自注射剂、雾化吸入剂、喷雾剂、口服制剂或外用制剂。
一些实施方案中，所述药物为注射剂。
在一些实施方式中，将所述诱导性囊泡用于疾病治疗的过程中，可以将所述诱导性囊泡任选地通过选自由静脉内注射、肌肉注射、皮下注射、气管滴注、鞘内注射或输注以及器官内输注所组成的组中的途径进行给药。例如，作为例子，对于静脉注射，可以通过尾静脉注射。器官内输注包括输注至解剖学空间中，例如，作为例子，胆囊、胃肠腔、食道、肺系统(通过吸入)和/或膀胱。
一些实施方案中，所述药物还包括药学上可接受的药用载体。
一些实施方案中，所述药用载体包括稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂和润滑剂中的一种或几种。
本公开中所述诱导性囊泡与现有的细胞外囊泡(例如外泌体等)有本质的不同，如与外泌体相比，本公开所述的诱导性囊泡IEVs高表达Syntaxin 4，以及其Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5的表达量明显高于外泌体(见实施例4)。除了具有的标志物有差别之外，所述诱导性囊泡IEVs在功能上或治疗效果上也呈现出有别于干细胞和其他的细胞外囊泡(如外泌体)的特性。例如IEVs能显著缩短大部分血浆的体外凝固时间，促凝效果好于外泌体(见试验例4)。例如IEVs治疗血友病小鼠的机制与PS和TF无关，而以往文献报道中，细胞外囊泡发挥促凝作用都高度依赖于其表面的PS和TF(见试验例4)。
另外，发明人在一些研究中发现，本公开所述的诱导性囊泡与其来源的母细胞之间的治疗效果也不具有互推性。例如间充质干细胞可以治疗非酒精性脂肪性肝炎和损伤性肝纤维化，然而，诱导性囊泡对这些疾病并不能实现治疗效果(见试验例1、2)。例如MSCs能够治疗干燥综合征，而本公开所述的诱导性囊泡对干燥综合征无治疗效果(见试验例3)。也就是说，某种干细胞能够治疗某种疾病，并不能够必然地推断出其产生的诱导性囊泡(IEVs)也一定能够这种疾病。
有研究表明肺内皮组细胞来源的外泌体可以通过修复肺上皮细胞间的连接来缓解肺损伤(Yue,Zhou,Pengfei,et al.Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury.[J].Critical Care,2019.)；间充质干细胞来源的外泌体可以通过调控巨噬细胞极化来减少促炎因子的释放及抗炎因子的增加(Li,JW,Wei,L,Han,Z,Chen,Z.Mesenchymal stromal cells-derived exosomes alleviate ischemia/reperfusion injury in mouse lung by transporting anti-apoptotic mir-21-5p.Eur J Pharmacol.2019；852:68–76.)。而发明人在一些实验研究中发现，LPS刺激后，小鼠肺部中性粒细胞数量显著增多，气管滴注本公开获得的诱导性囊泡治疗后中性粒细胞数量显著降低。本公开的诱导性细胞外囊泡可以直接作用于肺损伤过程中大量浸润的中性粒细胞，减少其趋化并促进中性粒细胞的凋亡。
附图说明
图1A-1E为分离的BMMSCs的表面标志物的流式检测结果。
图2为各组小鼠肺组织HE染色切片图。A为正常对照组(Control组)HE切片；B为模型组(LPS组)HE切片；C为尾静脉注射治疗组(LPS+IEVs-S组)HE切片；D为IEVs经气管滴注治疗组(LPS+IEVs-L组)HE切片；E为hBMMSC尾静脉治疗组(LPS+hBMMSC组)5×HE切片；F为hBMMSC尾静脉治疗组(LPS+hBMMSC组)10×HE切片。
图3为小鼠肺湿/干重比值(n＝4－5个/组，P<0.05有统计学差异，**：P<0.01；***:P<0.001)。
图4为小鼠BALF总蛋白含量测定(n＝4－5个/组，P<0.05有统计学差异，**：P<0.01；***:P<0.001；****:P<0.0001)。
图5为小鼠肺组织中性粒细胞流式检测结果。
图6为小鼠肺组织中性粒细胞免疫组化及免疫荧光检测结果。
图7为IEVs NTA检测的粒径图。
图8为IEVs形态透射电镜检测图。
图9A-图9D为IEVs的内容物分析：图9A为DIA定量技术对MSCs、MSCs-Exosomes、MSCs-IEVs蛋白组学定量分析结果；图9B为筛选IEVs特异性高表达的蛋白绘制的热图；图9C为差异蛋白的GO富集分析IEVs表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分 子的结果；图9D为western blot验证MSCs、MSCs-Exosomes、MSCs-IEVs表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4的结果。
图10为MSCs来源IEVs治疗蛋氨酸-胆碱饲料喂养的脂肪性肝病数据。A.肝病理组织学切片；B.脂肪性肝病评分。
图11为MSCs来源IEVs治疗硫代乙酸铵诱导的损伤性肝纤维化数据。A.肝病理组织学切片；B.脂肪性肝病评分。
图12为IEVs治疗干燥综合症：A.IEVs治疗干燥综合症(舍格伦综合征)唾液流率的影响；B.IEVs治疗干燥综合症下颌下腺HE染色结果；C.治疗干燥综合症对B细胞的影响。
图13为IEVs在血友病A小鼠的体内促凝作用。
图14A-图14D为给血友病A小鼠注射IEVs之后各种凝血因子水平的变化情况：图14A为凝血因子VIII的变化情况；图14B为vWF因子的变化情况；图14C为组织因子(TF)的变化情况；图14D为凝血酶原的变化情况。
图15A-图15B为血友病A小鼠模型中，IEVs分别进行PS和TF的封闭之后对IEVs的体内治疗效果的影响情况。
图16为同种MSCs来源的IEVs和Exosomes对血友病A小鼠治疗效果对比。
具体实施方式
以下通过具体的实施例进一步说明本公开的技术方案，具体实施例不代表对本公开保护范围的限制。其他人根据本公开理念所做出的一些非本质的修改和调整仍属于本公开的保护范围。
本公开实施例中的IEVs为诱导性囊泡的简称，可称为诱导性囊泡，也可称为诱导性细胞外囊泡(Induced extracellular vesicles,IEVs)。诱导性细胞外囊泡是指的是一种在前体细胞(例如干细胞)正常存活时，被干预或诱导，使其凋亡产生的一类亚细胞产物。通常这一类亚细胞产物，具有膜结构，表达凋亡性标志物，部分包含有遗传物质DNA。发明人发现诱导性细胞外囊泡是区分于细胞和常规细胞外囊泡(如外泌体等)的一类物质。在一些实施方式中，所述的正常存活时的细胞，例如是非凋亡的细胞、非衰老的细胞、非老化而增殖停滞的细胞、非冻存后复苏的细胞、非发生恶变而异常增殖的细胞或非出现损伤的细胞等。在一些实施方式中，所述的正常存活时的细胞取自细胞培养过程中，细胞接触融合80-100％的时候的细胞。一些实施方式中，所述的正常存活时的细胞取自对数期细胞。一些实施方式中，所述的正常存活时的细胞取自人或鼠组织来源的原代培养及其传代培养细胞。一些实施方式中，所述的正常存活时的细胞取自已确立的细胞系或细胞株。在一些实施方式中，所述的前体细胞取自早期的细胞。
本公开中的STS为星形孢菌素。
在一些实施例中，所述诱导性囊泡也可以来源于P2-P8代的hBMMSC。
文中“/”该符号指的是可以选择其一，例如“治疗或/预防”指的是可以为治疗，可以为预防。
如本文所使用的术语“和/或”是指并且涵盖一个或多个相关联的所列项目的任何和所有可能的组合。当在两个或多个项目的列表中使用时，术语“和/或”表示所列出的项目中的任何一个可以单独使用，或者可以使用两个或多个所列出的项目的任何组合。例如，如果组合物，组合，构造等被描述为包括(或包含)组分A，B，C和/或D，则该组合物可以单独包含A；单独包含B；单独包含C；单独包含D；包含A和B的组合；包含A和C的组合；包含A和D的组合；包含B和C的组合；包含B和D的组合；包含C和D的组合；包含A，B和C的组合；包含A，B和D组合；包含A，C和D的组合；包含B，C和D组合；或A，B，C和D组合使用。
在一些实施方案中，在一些实施方案中，分离所述诱导性囊泡的方法包括选用超速离心的方法分离所述囊泡。所述超速离心包括四次离心。
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。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。在一些实施方案中，在一些实施方案中，所述第二次离心为1700-2200g离心5-18分钟。在一些实施方案中，所述第二次离心为1990-2100g离心8-15分钟。在一些实施方案中，将所述第二次离心得到的上清进行第三次离心，所述第三次离心为10000-30000g离心15-60分钟；在一些实施方案中，所述第三次离心为15500-19000g离心20-40分钟。在一些实施方案中，所述第三次离心为15500-17000g离心20-40分钟。在一些实施方案中，所述第四次离心为10000-18000g离心15-60分钟。在一些实施方案中，所述第四次离心为10000-17500g离心15-60分钟。在一些实施方案中，所述第四次离心为10000-17000g离心15-60分钟。在一些实施方案中，所述第四次离心为11000-17000g离心15-60分钟。在一些实施方案中，所述第四次离心为12000-17000g离心15-60分钟。在一些实施方案中，所述第四次离心为10000-18000g离心20-60分钟。在一些实施方案中，所述第四次离心为10000-18000g离心20-50分钟。
在一些实施例中，所述诱导性囊泡的直径可以为0.03-6μM；可以是0.03-4.5μM；还可以是0.03-1μM；0.04-1μM，还可以是0.05-1μM，还可以是0.1-1μM，还可以是0.15-1μM，还可以是0.15-0.45μM，还可以是0.15-0.3μM，还可以是0.2-0.3μM。
在一些实施方式中，所述诱导性囊泡具有标志物Syntaxin 4。在一些实施例中，所述诱导性囊泡高表达标志物Syntaxin 4。在一些实施例中，所述诱导性囊泡对标志物Syntaxin 4的表达高于MSCs或外泌体。在一些实施例中，所述标志物Syntaxin 4的表达量为来 源于间充质干细胞的外泌体中Syntaxin 4的表达量的3-6倍。在一些实施例中，所述标志物Syntaxin 4的表达量为来源于间充质干细胞的外泌体中Syntaxin 4的表达量的3.5-5倍。在一些实施例中，所述标志物Syntaxin 4的表达量为来源于间充质干细胞的外泌体中Syntaxin 4的表达量的4.45倍。在一些实施例中，所述标志物还包括Annexin V、Flotillin-1、Cadherin 11和Integrin alpha 5中的一种或多种。在一些实施例中，所述标志物为Syntaxin 4、Annexin V、Flotillin-1、Cadherin 11和Integrin alpha 5的组合。在一些实施例中，所述诱导性囊泡高表达标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5。在一些实施例中，所述诱导性囊泡对标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5的表达量高于MSCs或外泌体。在一些实施例中，所述诱导性囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5的表达量相对于来源于间充质干细胞的外泌体中标记物的表达量分别为1-2倍、2-3倍、1-3倍和3-4倍。一些实施例中，所述诱导性囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5的表达量分别为1.5-2倍、2.5-3倍、1.5-2.5倍和3.5-4倍。在一些实施例中，所述囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5的表达量分别为1.76倍、2.81倍、2.41倍、3.68倍。
实施例1 MSCs的分离培养
依据动物伦理委员会的指导用过量CO 2处死小鼠，在无菌条件下，取下胫骨和股骨，剥净附着在其上的肌肉和结缔组织，进一步分离干骺端、暴露骨髓腔，用10mL无菌注射器抽取含体积分数为10％胎牛血清的PBS反复冲洗骨髓腔，70μm孔径细胞滤网过滤后，500g离心5min，去除上清液后收集底部的细胞沉淀，PBS重悬，再次500g离心5min，收集最终的细胞沉淀。而后对细胞进行流式分选，以CD34-和CD90+为分选标准，分选出BMMSCs。最后以Dex(-)培养液重悬细胞并接种于10cm直径细胞培养皿，37℃、5％CO 2培养。24h后，吸除上清液未贴壁细胞，PBS清洗后，加入Dex(-)培养液继续培养。1周后加入等量Dex(+)培养液，再1周后可见密集的原代BMMSCs集落。采用胰蛋白酶37℃孵育消化BMMSCs并传代扩增，之后每3日换Dex(+)培养液，长满后传代。其中，Dex(-)培养液成分如表1所示，Dex(+)培养液成分如表2所示：
表1 Dex(-)培养液成分表
Figure PCTCN2021143113-appb-000001
表2 Dex(+)培养液配方表
Figure PCTCN2021143113-appb-000002
采用流式细胞术分析表面标志物的方法来评估分离的BMMSCs的纯度。对于表面标志鉴定，胰蛋白酶消化收集P2代BMMSCs后，PBS清洗1次，以5×10 5/mL密度重悬细胞于含3％ FBS的PBS中，加入1μL PE荧光偶联的CD29、CD44、CD90、CD45和CD34抗体，空白组不加。4℃避光孵育30min，PBS清洗2遍后，上机检测。流式检测结果如图1A-1E所示，可知，分离的细胞为BMMSCs(骨髓间充质干细胞)。
实施例2诱导性囊泡(IEVs)的获得
采用实施例1培养的汇合率90-100％的P5代的hBMMSC，PBS洗涤，加入含有星形孢菌素500nM(STS)的无血清培养基(α-MEM培养基)诱导凋亡，9小时后收集培养液，800g离心10分钟，收集上清，2000g离心10分钟，收集上清，16000g离心30分钟，弃上清，1mL的PBS重悬，16000g离心30分钟，再次弃上清，得IEVs。
实施例3 IEVs治疗急性肺损伤
1、ALI小鼠模型构建及治疗分组
(1)实验动物：C57BL/6小鼠，性别不限，8-12周龄，体重约18g-22g，SPF级。
(2)疾病模型构建及治疗分组：取8-12周龄C57BL/6小鼠，随机分为5组，每组3-5只，分别是正常对照组(Control组)，模型组(LPS组)，IEVs(实施例2制备的IEVs)经气管滴注治疗组(LPS+IEVs-L组)、尾静脉注射治疗组(LPS+IEVs-S组)、hBMMSC尾静脉治疗组(LPS+hBMMSC组)。术前6小时禁食禁水，麻醉使用4％水合氯醛10mL/kg。模型组及治疗组先气管滴注给予30μL LPS(5mg/kg)，2小时后，三个治疗组小鼠分别经气管滴注30μL的hBMMSC-IEVs(约3×10 8个/只)，尾静脉注射200μL的hBMMSC-IEVs(约3×10 8个/只)、尾静脉注射200μL的hBMMSC细胞(约1×10 6个/只)，造模24小时后处死小鼠。
注：本公开中，按照实施例2获得的IEVs，一个1个BMMSCs约产生300个IEVs。
其中，hBMMSC细胞PBS悬液的制备方法如下：采用汇合率90-100％的P5-P8的hBMMSC，PBS洗涤，胰蛋白酶消化，1500rpm，离心5分钟，收集细胞沉淀，PBS洗涤，再次离心，收集细胞沉淀，PBS重悬，hBMMSC细胞悬液在使用前4℃保存。
2、IEVs治疗急性肺损伤的有效性评价
(1)肺组织的HE染色
HE染色切片可见Control组肺组织切片肺泡组织结构完整，未见明显充血及炎性细胞浸润(图2A)；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(图2B)；LPS+IEVs-L及LPS+IEVs-S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，同时两个IEVs治疗组肺中央区及周围区组织病理变化均明显改善，疗效相似(图2C，2D),LPS+hBMMSC组小鼠肺组织的炎症浸润减轻，肺组织渗出减少，肺泡间隔增厚和结构紊乱也稍有改善，肺中央区组织病理变化改善，但边缘肺组织炎症改善不明显(图2E，F)。
(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。如图3所示，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IEVs治疗显著降低了这一比值，各个治疗组之间未见显著性差异。
(3)肺泡灌洗液(BALF)中总蛋白浓度的测定
采用BCA法，测定BALF离心所得上清中的总蛋白浓度，酶标仪测定其在570nm的OD值后换算成蛋白浓度。
ARDS伴随着蛋白质等大分子物质渗出，BALF中总蛋白浓度是反映ARDS中肺损伤的一项重要指标，结果如图4所示：LPS刺激显著增加了BALF总蛋白浓度，而两种IEVs治疗方式均显著降低了BALF总蛋白浓度。
3、IEVs治疗急性肺损伤的机制研究
(1)肺组织中性粒细胞流式检测
小鼠处死后，摘取肺组织，用酶消化法收集小鼠肺组织细胞(所述细胞被分散为单个细胞)，100μL PBS重悬细胞(1×10 5个)，使用CD11b、ly6G抗体冰上染色15分钟，加400μL PBS，1500rmp离心弃上清，100ul PBS重悬，流式上机检测。
如图5，实验结果显示，LPS刺激后，小鼠肺部中性粒细胞数量(CD11b+ly6G+双阳细胞)显著增多，气管滴注IEVs治疗后中性粒细胞数量显著降低。
(2)肺组织中性粒细胞免疫组织化学及免疫荧光染色
取小鼠肺组织制备石蜡切片和冰冻切片，用ly6G进行免疫组化及免疫荧光染色。如图6，实验结果显示，LPS刺激后，小鼠肺部中性粒细胞数量(ly6G阳性细胞)显著增多，气管滴注IEVs治疗后中性粒细胞数量显著降低。
以上结果显示，IEVs可能是通过调控中性粒细胞从而发挥治疗ALI作用。
实施例4诱导性囊泡(IEVs)的分析
1、NTA检测
PBS重悬实施例2的IEVs沉淀，稀释后用zetaview仪器检测IEVs浓度和粒径。
检测结果如图7所示，IEVs颗粒直径平均为(144.3nm)。
2、电镜检测
用戊二醛磷酸缓冲液固定实施例2的IEVs沉淀，常规脱水、浸透、包埋、染色，制成超薄切片，在电镜下观察并记录IEVs形态和结构。
透射电镜观察，如图8所示，大部分囊泡的直径都在200nm及200nm以下。
3、IEVs的内容物分析
利用蛋白DIA定量技术完成MSCs，MSCs-Exosomes，MSCs-IEVs的蛋白组学定量分析。结果显示，MSCs-Exosomes和MSCs-IEVs的蛋白内容物表达与母细胞具有较高的重叠性，170种蛋白在IEVs中特异性高表达(图9A)。通过生物信息学分析，筛选IEVs 特异性高表达的蛋白，绘制热图(图9B)，进一步结合差异蛋白的GO富集分析结果，明确IEVs能特异性高表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分子(图9C)。与同种MSCs来源的Exosomes相比，IEVs的5种特征性分子的表达量均显著上调，具体为：IEVs中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5和Syntaxin 4相对于Exosomes中相应标记物的表达量分别为1.76倍、2.81倍、2.41倍、3.68倍和4.45倍。最后利用western blot技术再次进行验证，结果与DIA定量分析结果相符(图9D)。
MSCs-Exosomes：指的是来源于MSCs的外泌体。
MSCs-IEVs：指的是来源于MSCs的IEVs，具体获得方法：除了细胞代数为P2，其余步骤同实施例2。
其中，所述的内容物分析中MSCs和与提取Exosomes和IEVs的MSCs为同一细胞株。
MSCs-Exosomes的分离与提取方法：将实施例1中培养至第2代的BMMSCs，用实施例1中的培养基(Dex(+)培养液)继续培养至细胞汇合80％-90％时，用PBS冲洗2遍，加入无血清培养基(α-MEM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。
试验例1
MSCs能够治疗非酒精性脂肪性肝炎(Non-alcoholic steatohepatitis,NASH)(Ezquer et al.,J Hepatol,2011；Winkler et al.,Methods Mol Biol,2014)。
(1)检测步骤或方法：取8周龄雄性C57小鼠，饲喂正常饲料(Normal chow diet,NCD)或蛋氨酸-胆碱缺乏饲料(Methionine and choline deficient diet,MCD)，MCD喂养期间每2周经尾静脉输注IEVs，喂养8周后取材，肝组织石蜡切片、HE染色。
(2)结果：如图10所示，IEVs治疗MCD诱导的非酒精性脂肪性肝炎(Non-alcoholic steatohepatitis,NASH)无效果。肝脏在MCD喂养后出现脂肪化、炎症，IEVs注射未能缓解。
试验例2
MSCs和外泌体能够治疗四氯化碳或硫代乙酰胺(Thioacetamide,TAA)诱导的损伤性肝纤维化(Mehrabani et al.,Arch Razi Inst,2019；Sabry et al.,Int J Stem Cells,2019；Rong et al.,Stem Cell Res Ther,2019)。
(1)检测步骤或方法：取8周龄C57小鼠，腹腔注射100mg/kg的TAA，每周3次，持续8周，造损伤性肝纤维化模型。TAA造模期间每2周经尾静脉输注IEVs，8周后取材，肝组织石蜡切片、HE染色。
(2)结果：如图11所示，TAA注射诱导肝脏损伤、肝血窦周围组织破坏、玻璃样变纤维化形成，IEVs注射未有明显治疗效应。
试验例3
(1)检测步骤或方法：取8周龄舍格伦综合征(SS)模型小鼠，经尾静脉系统注射MSCs和IEVs，注射后4周取材，检测唾液流速，收集唾液腺样本行石蜡切片HE染色和B细胞标志物B220染色。
(2)结果：如图12A-图12C，结果显示，对比小鼠骨髓间充质干细胞及其来源的IEVs治疗干燥综合症(舍格伦综合征)唾液流率的影响，间充质干细胞治疗后唾液流率稍有恢复，IEVs治疗后唾液流率未见改善(*p<0.05相较于WT组，###p<0.001相较于MSCs组)。IEVs注射未改变唾液腺的炎症浸润和B细胞堆积情况。
试验例4
利用体外凝血实验检测实施例2获得的IEVs和实施例4提取Exosomes的体外促凝效果。结果如表3显示，IEVs能显著缩短大部分血浆的体外凝固时间，促凝效果好于Exosomes。
但对于因子II，V，X缺乏的血浆，IEVs无法发挥体外促凝作用，说明IEVs的体外促凝作用更多集中于凝血共同途径的上游。
表3
Figure PCTCN2021143113-appb-000003
Figure PCTCN2021143113-appb-000004
利用血友病A小鼠(凝血因子VIII缺乏)为模型，通过尾静脉注射9×10 8个IEVs，观察IEVs的体内促凝作用。结果如图13显示，IEVs治疗后能够显著改善血友病小鼠的出血倾向，治疗作用可以持续稳定地维持14天。
实验结果表明，IEVs能够在体外发挥显著的促凝作用。且体内注射后能够显著改善出血倾向，可用于改善血友病A导致的出血倾向。同时检测小鼠血浆中各种凝血因子的水平，发现凝血因子VIII、vWF因子、组织因子(tissue factor，TF)和凝血酶原(prothrombin)均没有发生显著变化(图14A，图14B，图14C，图14D)。
在血友病A小鼠模型中，分别注射正常IEVs，PS阴性IEVs和TF阴性IEVs，7天后进行剪尾实验，结果如图15A和图15B显示，PS和TF的封闭并没有影响IEVs的体内治疗效果，初步说明IEVs治疗血友病小鼠的机制与PS和TF无关。以往文献报道中，细胞外囊泡发挥促凝作用都高度依赖于其表面的PS和TF，而IEVs的体内实验结果与以往研究不一致，这提示在体内环境下，IEVs可能有新的作用机制发挥促凝作用。
对血友病A小鼠模型，分别进行同种MSCs来源的IEVs(实施例2获得的)和Exosomes(实施例4提取的)的注射治疗(9×10 8个)，结果显示，IEVs能够显著纠正小鼠的出血倾向，而Exosomes没有明显的治疗效果(图16)。

Claims (11)

  1. 诱导性囊泡在制备用于治疗和/或预防肺部疾病或病症药物中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述肺部疾病或病症包括急性炎症引发的肺部疾病；
    优选地，所述肺部疾病或病症为急性呼吸窘迫综合征、急性肺损伤或肺纤维化。
  3. 如权利要求1-2任一所述的应用，其特征在于，所述疾病或病症是由细菌、病毒或真菌感染引起的肺部疾病或病症；
    优选地，所述病毒包括2019冠状病毒；
    优选地，所述细菌包括肺炎链球菌，金黄色葡萄球菌。
  4. 一种组合物，其特征在于，所述组合物包含诱导性囊泡，所述组合物还包含肺部疾病或病症的预防或治疗剂，所述预防或治疗剂选自抗细菌剂、抗病毒剂、抗真菌剂、抗肿瘤剂、抗组胺剂、蛋白质、酶、激素、非甾体抗炎性物质、细胞因子、类固醇、尼古丁和胰岛素中的一种或多种；
    优选地，所述肺部疾病为急性呼吸窘迫综合征、急性肺损伤或肺纤维化；
    优选地，所述治疗剂为抗病毒剂；
    更为优选地，所述病毒包括2019冠状病毒。
  5. 一种药品套装，其特征在于，包含：(a)诱导性囊泡；(b)肺部疾病或病症预防或治疗剂；在所述的药品套装中诱导性囊泡和所述肺部疾病或病症预防或治疗剂被分开包装；
    优选地，所述预防或治疗剂选自抗细菌剂、抗病毒剂、抗真菌剂、抗肿瘤剂、抗组胺剂、蛋白质、酶、激素、非甾体抗炎性物质、细胞因子、类固醇、尼古丁和胰岛素中的一种或多种；
    优选地，所述肺部疾病为急性呼吸窘迫综合征、急性肺损伤或肺纤维化。
    优选地，所述治疗剂为抗病毒剂；
    更为优选地，所述病毒包括2019冠状病毒。
  6. 一种药物组合物在制备用于治疗或预防肺部疾病或病症的药物中的应用，所述药物组合物包含诱导性囊泡；
    优选地，所述组合物还包含治疗剂，所述治疗剂选自抗细菌剂、抗病毒剂、抗真菌剂、抗肿瘤剂、抗组胺剂、蛋白质、酶、激素、非甾体抗炎性物质、细胞因子、类固醇、尼古丁和胰岛素中的一种或多种；
    优选地，所述肺部疾病为急性呼吸窘迫综合征、急性肺损伤或肺纤维化。
    优选地，所述治疗剂为抗病毒剂；
    更为优选地，所述病毒包括2019冠状病毒。
  7. 一种治疗和/或预防肺部疾病或病症的方法，其特征在于，包括：给予患者诱导性囊泡；
    优选地，所述肺部疾病或病症包括急性炎症引发的肺部疾病；
    优选地，所述肺部疾病或病症为急性呼吸窘迫综合征、急性肺损伤或肺纤维化；
    优选地，所述疾病或病症是由细菌、病毒或真菌感染引起的肺部疾病或病症；
    优选地，所述病毒包括2019冠状病毒；
    优选地，所述细菌包括肺炎链球菌，金黄色葡萄球菌。
  8. 如权利要求1-3任一所述的应用或权利要求4所述的药物组合物或权利要求5所述的药品套装或权利要求6所述的应用或权利要求7所述的方法，其特征在于，所述诱导性囊泡是在干细胞处于正常存活时通过外加因素诱导凋亡而产生的囊泡；
    优选地，所述诱导性囊泡是诱导干细胞凋亡产生的，所述诱导方法包括添加星形孢菌、紫外线照射、饥饿法、或热应力法；
    更为优选地，所述干细胞为间充质干细胞；
    更为优选地，所述间充质干细胞来源包括骨髓、牙髓、尿液、口腔、脂肪、胎盘、脐带、骨膜、肌腱或外周血；
    优选地，所述间充质干细胞为骨髓间充质干细胞；
    优选地，所述间充质干细胞来源于哺乳动物；
    优选地，所述哺乳动物选自灵长类动物或鼠；
    优选地，所述灵长类动物为人。
  9. 如权利要求1-3任一所述的应用或权利要求4所述的药物组合物或权利要求5所述的药品套装或权利要求6所述的应用或权利要求7所述的方法，其特征在于，所述诱导性囊泡的直径为0.03-10μM；
    优选地，所述诱导性囊泡的直径为0.03-6μM；
    更为优选地，所述诱导性囊泡的直径为0.03-4.5μM；
    更为优选地，所述诱导性囊泡的直径为0.03-1μM。
  10. 如权利要求1-3任一所述的应用或权利要求4所述的药物组合物或权利要求5所述的药品套装或权利要求6所述的应用，其特征在于，所述的药物选自注射剂、雾化吸入剂、喷雾剂、口服制剂或外用制剂。
  11. 如权利要求1-3任一所述的应用或权利要求4所述的药物组合物或权利要求5所述的药品套装或权利要求6所述的应用，其特征在于，所述药物为注射剂；
    优选地，所述药物选自静脉注射剂、肌肉注射剂、皮下注射剂或气管滴注或鞘内注射剂；
    或优选地，所述药物还包括药学上可接受的药用载体；
    更为优选地，所述药用载体包括稀释剂、赋形剂、填充剂、粘合剂、崩解剂、表面活性剂和润滑剂中的一种或几种。
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