WO2021147922A1 - 细胞外囊泡及其在皮肤产品的用途 - Google Patents

细胞外囊泡及其在皮肤产品的用途 Download PDF

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WO2021147922A1
WO2021147922A1 PCT/CN2021/072937 CN2021072937W WO2021147922A1 WO 2021147922 A1 WO2021147922 A1 WO 2021147922A1 CN 2021072937 W CN2021072937 W CN 2021072937W WO 2021147922 A1 WO2021147922 A1 WO 2021147922A1
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stem cells
mesenchymal stem
extracellular vesicles
skin
cells
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PCT/CN2021/072937
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French (fr)
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马兰
寇晓星
施松涛
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医微细胞生物技术(广州)有限公司
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Priority to US17/793,833 priority Critical patent/US20230084480A1/en
Priority to JP2022569291A priority patent/JP2023513394A/ja
Publication of WO2021147922A1 publication Critical patent/WO2021147922A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/983—Blood, e.g. plasma
    • 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
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS

Definitions

  • the present disclosure belongs to the field of biomedicine, and relates to extracellular vesicles and their use in skin products.
  • Extracellular vesicles are nano-scale carriers secreted by cells that contain proteins, nucleic acids and various cytokines. Extracellular vesicles can act on target cells through endocrine or paracrine mode, and play an important role in the process of material transfer and information exchange between cells. Studies have found that information exchange mediated by extracellular vesicles plays an important regulatory role in the body's physiological or pathological processes, involving immune regulation, tumor growth, angiogenesis, damage repair, and so on. The current research in this field is mainly focused on exosomes. Exosomes are extracellular vesicles with a diameter of about 30-150nm, which contain components such as RNA, lipids, and proteins.
  • Exosomes are widely involved in various physiological/pathological regulation of the body, and can be used for diagnosis, treatment and prognostic evaluation of various diseases. So far, mesenchymal stem cells (MSCs) are considered to be the cells with the strongest ability to produce exosomes. Numerous studies have found that exosomes derived from MSCs can mimic the biological functions of MSCs, and play an important regulatory role in promoting cell growth and differentiation, repairing tissue defects, and so on. Therefore, in recent years, cell vesicle therapy based on MSCs-derived exosomes has achieved remarkable development.
  • MSCs mesenchymal stem cells
  • Alopecia areata Alopecia areata
  • Ghost shaving is a kind of localized patchy hair loss that occurs suddenly, with normal local skin and no symptoms. At present, the cause is unknown.
  • Pathological examination shows that the hair follicles are reduced and lymphocytes are infiltrated around the hair follicles. The disease is sometimes combined with other autoimmune diseases (Alopecia Areata: Review of Epidemiology, Clinical Features, Pathogenesis, and New Treatment Options. Darwin E, Hirt PA, Fertig R, Doliner B, Delcanto G, Jimenez JJ. Int J Trichology. 2018; 10(2): 51-60).
  • alopecia areata has a natural tendency to heal, and a few cases recur, so treatment is difficult, but there are many treatments that can be combined to treat hair loss. It can occur at any age, but it is more common in young adults, and there is no significant difference in incidence between the sexes.
  • the skin lesions appear as round or oval non-scarring hair loss, and "exclamation mark"-like hairs are often seen on the edge of alopecia areata. All or almost all of the hair falls out, which is called total baldness. All hair (including body hair) of the whole body falls off, which is called general hair loss. Creeping hair loss can also be seen. Except for the lack of hair, the skin of the diseased area has no other abnormalities.
  • a model of alopecia areata can be made by topical application of imiquimod in C3H/HeJ mice. There is currently no effective treatment for alopecia areata.
  • the present disclosure provides the use of inducible vesicles in the preparation or use of skin products or skin accessory products.
  • skin product refers to a product that can act on the skin, for example, a product that treats, improves, repairs, removes wrinkles, or eliminates wounds on the skin, and can act directly or indirectly on the skin .
  • the product is: a drug, a drug carrier, a food, a health care product, a skin care product, or a medical beauty product.
  • the skin is epidermis, dermis, or subcutaneous tissue.
  • the skin appendages are hair, hair, sweat glands, sebaceous glands, nails, or toenails.
  • IEV was not distributed in the heart, kidney, and brain after injection of IEV into nude mice, but it was distributed in the nails, which was speculated to be related to the targeting of IEV.
  • IEV has a good therapeutic effect on hair regeneration and wound healing.
  • IEV has a therapeutic effect on the skin or skin appendages, and on the other hand, it just targets the skin or skin appendages, making it particularly suitable for preparation or application as skin products or skin appendage products.
  • the present disclosure provides a composition including: an inducible vesicle and a skin drug or care product.
  • the skin medication and care products are encapsulated in extracellular vesicles or are present in the composition in separate forms.
  • the dosage form of the composition is preferably selected from freeze-dried powder injection, injection, tablet, capsule or patch; in some embodiments, the patch is selected from microneedle patch; in some In an embodiment, the dosage form is selected from injections or microneedle patches.
  • the inducible vesicles are vesicles produced by external force inducing apoptosis during the normal survival of stem cells; in some embodiments, the inducible vesicles are produced by adding stellaria, Ultraviolet radiation, starvation method, or thermal stress method or a combination of one or more of them induces stem cells or stem cell apoptosis.
  • the present disclosure provides a composition
  • a composition comprising a first agent composed of stem cells or stem cell extracellular vesicles; and, a second agent composed of anti-aging substances or Wnt signaling pathway positive regulators Agent.
  • the extracellular vesicle refers to a type of subcellular product produced during the normal survival of precursor cells (such as stem cells, especially mesenchymal stem cells), which are interfered with or induced to cause apoptosis. , Also known as induced vesicles.
  • precursor cells such as stem cells, especially mesenchymal stem cells
  • induced vesicles also known as induced vesicles.
  • the precursor cell is an early precursor cell.
  • this type of subcellular product has a membrane structure, contains genetic material DNA, cannot divide, can move and migrate. It is a class of substances between cells and extracellular vesicles (apoptotic bodies, exosomes, etc.).
  • the stem cells are selected from induced pluripotent stem cells, blood stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchyme One or more of stem cells, umbilical cord mesenchymal stem cells, periosteal mesenchymal stem cells, and skin mesenchymal stem cells.
  • the stem cells are selected from induced pluripotent stem cells, blood stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, oral mesenchymal stem cells, skin mesenchymal stem cells One or more of.
  • the stem cells are selected from one or more of induced pluripotent stem cells, umbilical cord mesenchymal stem cells, blood stem cells or bone marrow mesenchymal stem cells.
  • the stem cell extracellular vesicles are selected from one or more of exosomes, migrating bodies, microvesicles, apoptotic bodies, and induced extracellular vesicles.
  • the stem cell extracellular vesicles are selected from induced extracellular vesicles.
  • the anti-aging substance is selected from one or more of metformin and resveratrol.
  • the anti-aging substance is selected from metformin.
  • the Wnt signaling pathway is selected from the Wnt/ ⁇ -catenin pathway.
  • the positive modulator is selected from agonists.
  • the agonist is selected from one or more of Licl, CHIR99021, SB-216763, BIO, Wnt Agonist, or WAY 262611.
  • the agonist is selected from Licl.
  • the positive modulator is selected from the group comprising cues to exercise.
  • the prompt is selected from one or more of packaging, labeling, or instructions.
  • the composition contains stem cells or stem cell extracellular vesicles in a ratio of (0.5-5) ⁇ 10 7 : (0.05-10) mg.
  • the composition comprising 1 ⁇ 10 7 -3 ⁇ 10 7 cells or stem cells, stem extracellular vesicles; and, anti-aging substance 0.1-5 mg.
  • 1.8 ⁇ 10 7 -2.7 ⁇ 10 7 stem cells or stem cell extracellular vesicles are included in the composition.
  • 0.2-3 mg of anti-aging substances are included in the composition.
  • the extracellular vesicles can be optionally selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intra-organ infusion.
  • Administer the drug for example, as an example, for intravenous injection, it can be injected through the tail vein.
  • Intra-organ infusion includes infusion into an anatomical space, such as, for example, the gallbladder, gastrointestinal cavity, esophagus, pulmonary system (by inhalation), and/or bladder.
  • intraperitoneal injection in gastrointestinal cavity infusion
  • tail vein injection intraperitoneal injection can also obtain a good therapeutic effect.
  • the safety and operability of intraperitoneal injection are better than those of tail vein injection.
  • the two reagents in the composition are provided in the form of a single pharmaceutical composition, and in some embodiments, it may be expected to be a kit or a combined dispenser package containing each of the two reagents . It should be understood that the content of the present disclosure encompasses the co-administration of any of the two agents to a subject, whether such administration is in a single formulation or a combination of separate formulations, and whether such administration is simultaneous or staggered.
  • the extracellular vesicles are produced by inducing mesenchymal stem cell apoptosis by adding staurosporine, ultraviolet irradiation, starvation, or thermal stress, or a combination thereof.
  • the extracellular vesicles are produced by adding staurosporine to induce apoptosis of mesenchymal stem cells.
  • the generation number of the mesenchymal stem cells may be 2 to 5 generations, but it is not limited thereto.
  • the concentration of the staurosporine is greater than or equal to 1 nM; preferably, 1-15000 nM; preferably, 200-10000 nM; preferably, 250-1000 nM; preferably, 500 -1000nM.
  • the concentration of staurosporine can also be 280-9000nM; it can also be 230-8500nM; it can also be 500-1000nM; it can also be 500-900nM; it can also be 500-800nM.
  • the concentration of the staurosporine is 500 nM.
  • the diameter of the inducible extracellular vesicle is 0.45 ⁇ m or less. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.45 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.1-0.45 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.1-0.35 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.35 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.3 ⁇ m.
  • the diameter of the inducible extracellular vesicle is 0.15-0.2 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.4 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.38 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.35 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.32 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.3 ⁇ m.
  • the diameter of the inducible extracellular vesicle is 0.05-0.25 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.05-0.22 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicle is 0.15-0.22 ⁇ m. In some embodiments, the diameter of the inducible extracellular vesicles can also be 0.15-0.45 ⁇ m, or 0.2-0.3 ⁇ m.
  • the inducible extracellular vesicle has the marker Syntaxin 4. In some embodiments, the inducible extracellular vesicles highly express the marker Syntaxin 4. In some embodiments, the expression of Syntaxin 4, a marker of inducible extracellular 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 marker further includes 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 extracellular vesicles highly express markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5.
  • the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5 of the inducible extracellular vesicles are higher than that of MSC or exosomes. In some embodiments, the expression level of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 in the inducible extracellular vesicles is relative to that of the markers in exosomes derived from mesenchymal stem cells. The expression levels were 1-2 times, 2-3 times, 1-3 times and 3-4 times, respectively.
  • the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 in the inducible extracellular vesicles are relative to the expression levels of the markers in exosomes derived from mesenchymal stem cells. It is 1.5-2 times, 2.5-3 times, 1.5-2.5 times and 3.5-4 times. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 in the inducible extracellular vesicles are relative to the expression level of the markers in exosomes derived from mesenchymal stem cells They are 1.76 times, 2.81 times, 2.41 times, and 3.68 times respectively.
  • the inducible extracellular vesicles described in the present disclosure are essentially different from exosomes.
  • the inducible extracellular vesicles IEVs described in the present disclosure highly express Syntaxin 4, and its Annexin V, The expression levels of Flotillin-1, Cadherin 11, and Integrin alpha 5 were significantly higher than those of exosomes (see Example 3).
  • the inducible extracellular vesicle IEVs also exhibit characteristics that are different from stem cells and other extracellular vesicles such as exosomes in terms of function or therapeutic effect.
  • the inducible extracellular vesicles also express CD29, CD44, CD73, CD166; and do not express CD34, CD45. In some embodiments, the inducible extracellular vesicles also express one or more of CD9, CD63, CD81, and C1q.
  • the application of the composition in the preparation of a medicament for promoting hair regeneration or promoting wound healing is also provided.
  • the composition further comprises a pharmaceutically acceptable carrier; the dosage form of the composition is preferably selected from lyophilized powder injection, injection, tablet, capsule or patch.
  • the patch is selected from a microneedle patch; in some embodiments, the dosage form is selected from an injection, a microneedle patch, or a tablet.
  • the method for preparing extracellular vesicles includes the following steps:
  • step 2) Add the mesenchymal stem cells prepared in step 1) to a serum-free medium containing 500-1000 nM staurosporine, incubate at 37°C for 16-24 hours, and collect the cell supernatant;
  • step 2) Centrifuge the cell supernatant collected in step 2) at 500-15000g for 5-30 minutes at 4°C to collect the supernatant;
  • step 4) Centrifuge the cell supernatant collected in step 3) at 1500-2500g at 4°C for 5-30 minutes, and collect the supernatant;
  • step 5 Centrifuge the cell supernatant collected in step 4) at 10,000-30,000 g for 15-60 minutes at 4°C, and the resulting precipitate is extracellular vesicles;
  • it also includes a washing step of extracellular vesicles; in some embodiments, the washing step is specifically: 6) Resuspend the extracellular vesicles prepared in step 5) with PBS, and then at 4°C. Centrifuge at 10,000-30,000 g for 15-60 minutes, and the resulting pellet is extracellular vesicles.
  • the present disclosure also provides the use of anti-aging substances in the preparation of concomitant drugs for stem cell medications.
  • stem cell service in the embodiments of the present disclosure includes, but is not limited to, products or methods that enable stem cells or stem cell vesicles to enter an animal's body.
  • the products that enable the stem cell service to enter the animal body include freeze-dried powder injections, injections, tablets, capsules or patches.
  • the methods that can make the stem cell service into the animal body include oral administration, injection, application, smearing, spraying, and drip infusion.
  • the "concomitant drug” in the embodiments of the present disclosure includes a drug that enters the body together with another one or more drugs. Concomitant drugs can enter the body at the same time or almost at the same time.
  • almost simultaneous entry can be one entry after another; or entry into the body at different times on the same day.
  • multiple medicaments or their active ingredients can be partitioned into one formulation, or two or more formulations can enter the body at least substantially simultaneously. For example, within about 1 hour of each other.
  • the stem cell service is stem cells or stem cell extracellular vesicles.
  • the stem cell service is a stem cell or stem cell extracellular vesicle used to ingest an animal body; preferably, the animal is a mammal; preferably, the mammal is a human.
  • the present disclosure also provides a stem cell administration system, which includes: stem cell services, and, concomitant products; the concomitant products are selected from one or two of anti-aging substances or Wnt signaling pathway positive regulators.
  • the stem cell service contains stem cells or stem cell extracellular vesicles; the anti-aging substances or Wnt signaling pathway positive regulators are as described above.
  • the present disclosure also provides the use of stem cells or stem cell extracellular vesicles in the preparation of drugs for the metabolism of stem cells or stem cell extracellular vesicles regulated by the Wnt signaling pathway to promote hair regeneration or wound healing.
  • the drugs that promote hair regeneration or wound healing include stem cells or stem cell extracellular vesicles; and, anti-aging substances.
  • the stem cells or stem cell extracellular vesicles and anti-aging substances are as described above.
  • the present disclosure also provides a method for promoting the discharge of stem cell products from the body of an animal, or hair regeneration, or wound healing.
  • the method includes taking the anti-aging substance at the same time or almost simultaneously during the period of taking the stem cell products, or Exercise.
  • Wnt signaling pathway positive regulators include those agents or methods that can up-regulate and activate Wnt, such as agonists, but are not limited thereto.
  • the realization of the aforementioned "methods" includes prompts, instructions, prescriptions, instructions, etc. to the subject.
  • the present disclosure also provides a treatment method for skin or skin appendage diseases, including the following steps: administering the inducible vesicle, composition, and stem cell administration system to a subject in need.
  • the skin or skin appendage disease is selected from trauma, hair loss, scars, burns and scalds, or ulcers.
  • the wound is selected from a resection wound, a cut, a puncture wound, or a puncture wound.
  • Induced pluripotent stem cells include pluripotent stem cells formed by reprogramming terminally differentiated somatic cells by introducing specific transcription factors.
  • Figure 1 is a flow cytometric detection diagram in Example 1;
  • Figure 2 is a technical roadmap for preparing IEV in Example 2;
  • Figure 3 shows the statistical results of the number of IEVs produced by MSCs (106 MSCs) analyzed by flow cytometry;
  • Figure 4A-4F shows the diameter detection of IEVs particles:
  • Figure 4A shows the particle diameter distribution of IEVs by flow detection;
  • Figure 4B shows the scattered light intensity of IEVs analyzed by side scattered light (SSC), showing the particle diameter distribution of IEVs;
  • Figure 4C In order to analyze the scattered light intensity of IEVs with standardized small particle microspheres produced by Bangs Laboratories, showing the particle diameter distribution of IEVs;
  • Figure 4D shows the IEVs observed by transmission electron microscope (TEM), showing the particle diameter distribution of IEVs;
  • Figure 4E shows the particle diameter distribution of IEVs.
  • Tracking analysis NTA
  • Figure 4F shows the single-vesicle-level particle size detection of IEVs by nanoflow detection technology, showing the particle diameter distribution of IEVs;
  • Figures 5A-5K show the analysis results of the surface membrane proteins of IEVs by flow cytometry
  • Figure 6A- Figure 6D are the content analysis of IEVs:
  • Figure 6A is the proteomic quantitative analysis results of MSCs, MSCs-Exosomes, MSCs-IEVs by DIA quantitative technology;
  • Figure 6B is the heat map drawn by screening IEVs-specific and highly expressed proteins
  • Figure 6C is the GO enrichment analysis of differential proteins and the results of IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules;
  • Figure 6D is the western blot verifying that MSCs, MSCs-Exosomes, and MSCs-IEVs express Annexin The results of V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4;
  • Figure 7A is a schematic diagram of the dynamic metabolism of IEV on the skin surface in Example 3.
  • Figure 7B shows that as time goes by, IEV gradually moves from the subcutaneous tissue to the dermis and epidermis
  • Figure 7C shows that PKH26-IEV is found in the hair follicles in the plucked hairs on the mouse body on day 7;
  • Figure 8A shows the distribution of IEV in the whole body of the mouse on the 1, 3, and 7 days detected by in vivo imaging technology
  • Fig. 8B is a comparison of the distribution of IEV in various organs of mice and the control group on the 7th day;
  • Figure 8C shows a schematic diagram of the metabolism of IEV in mouse nails and incisors on day 3;
  • Fig. 9A is a schematic diagram of back hair regeneration on days 0, 10, and 14 of mice treated with different treatments in Example 4.
  • 9B is a schematic diagram of statistical analysis of the area of back hair regeneration on the 0th day, the 10th day, and the 14th day of the mice with different treatments in Example 4;
  • Fig. 10 is a schematic diagram of back hair regrowth on days 0, 11, 13 and 16 of mice treated with different treatments in Example 6;
  • Figure 11 is a combination of different drugs and IEV in Example 5, and the distribution of IEV on the skin surface after 7 days;
  • Figure 12 is a schematic diagram of the effect of different drugs in combination with IEV on promoting hair regrowth in Example 5;
  • Figure 13A shows the Western Blot results after administration of the Wnt agonist Licl in Example 6;
  • Figure 13B shows the Western Blot results after administration of the Wnt inhibitor Xav939 in Example 6;
  • Figure 13C is the immunofluorescence result under the action of Xav939 in Example 6;
  • Figure 13D shows the results of cell smears under different treatments in Example 6;
  • Figure 13E shows the immunofluorescence results of the excretion of IEV in the epidermis after different treatments in Example 6
  • Figure 14A shows the level of DKK1 in the circulation after exercise
  • Figure 14B shows the number of IEVs in the blood and the results of flow cytometry
  • Figure 14C shows the immunofluorescence results of IEV excretion on the skin surface after exercise
  • Figure 14D shows the distribution of IEV in the whole body of the mouse after 1 or 3 days of exercise detected by in vivo imaging technology
  • Figure 14E shows the distribution of IEV in the skin of mice after 1 or 3 days of exercise detected by in vivo imaging technology
  • Figure 14F shows the result of IEV in the body (blood) after 7 days of exercise by flow cytometry
  • Figure 15A shows the promoting effect of IEV and MSC treatment on wound healing in Example 7.
  • 15B is a schematic diagram of the effect of different treatment groups on wound healing in Example 7.
  • FIG. 15C shows the results of immunofluorescence analysis of different treatment groups in Example 7.
  • 16 is a comparison diagram of wrinkles on the chin area of the subject before and after the introduction of water and light into the IEV in Example 10;
  • Figure 17 shows the IEVs derived from the iPSC under the electron microscope in Example 11.
  • Example 18 is a diagram of the death process of hiPSCs and hUCMSCs taken by the high-content cell imaging analysis system in Example 12;
  • Figure 19 shows the hiPSCs and hUCMSCs induced by flow cytometry in Example 12;
  • Figure 20 shows the positive rate of Annexin5 expression of hiPSCs and hUCMSCs using flow analysis in Example 12;
  • Figures 21-23 show in turn the particle size, yield and potential of hiPSCs-IEVs and hUCMSCs-IEVs characterized by NTA in Example 12;
  • Fig. 24 is a statistical diagram of wound conditions and wound area of different treatment groups at different time points in the skin injury model of Example 12.
  • IEVs in the embodiments of the present disclosure are the abbreviations for induced vesicles, which may be called induced vesicles, and may also be called induced extracellular vesicles (IEVs).
  • Inducible extracellular vesicles refer to a type of subcellular product produced when precursor cells (such as stem cells) survive normally and are interfered or induced to cause apoptosis. Usually this type of subcellular product has a membrane structure, expresses apoptotic markers, and partly contains genetic material DNA.
  • inducible extracellular vesicles are a class of substances distinguished from cells and conventional extracellular vesicles (such as exosomes, etc.).
  • the cells that survive normally are, for example, non-apoptotic cells, non-senescent cells, non-aging cells that have stagnated proliferation, cells that are not recovered after cryopreservation, or abnormally proliferating without malignant transformation. Cells or non-damaged cells, etc.
  • the normally viable cells are taken from cells that are in contact with 80-100% of the fusion during the cell culture process. In some embodiments, the cells that survive normally are taken from cells in the log phase. In some embodiments, the cells that survive normally are obtained from primary culture and subculture cells derived from human or murine tissue. In some embodiments, the cells that survive normally are taken from established cell lines or cell lines. In some embodiments, the precursor cells are taken from early cells.
  • IEVs are the same as IEVs.
  • the STS in the present disclosure is staurosporine.
  • the components in the “composition” may exist in a mixed form, or they may be packaged separately. Separately packaged components may also contain their respective adjuvants.
  • the adjuvant refers to a means that can assist the curative effect of drugs in pharmacy.
  • the separately packaged components can be administered simultaneously or in any order, in which the patient is treated with one drug first, and then another drug is administered .
  • the said patient refers to a mammalian subject, especially a human being.
  • the "composition” may also exist in a form in which one component is wrapped by another component.
  • the inducible vesicle in the composition, is used as a drug carrier, and drugs for treating or preventing diseases are encapsulated in the inducible vesicle.
  • compositions e.g., medium
  • method include the listed elements, but do not exclude other elements.
  • Consisting essentially of means excluding other elements that have any significance for the combination for the stated purpose. Therefore, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel features of the claimed disclosure.
  • Consisting of refers to the elimination of trace elements and substantial method steps of other components. The embodiments defined by each of these transition terms are within the scope of this disclosure.
  • the composition may include A alone; B alone; C alone; D alone ; Contains the combination of A and B; Contains the combination of A and C; Contains the combination of A and D; Contains the combination of B and C; Contains the combination of B and D; Contains the combination of C and D; Contains the combination of A, B and C Combination; including A, B and D combination; including A, C and D combination; including B, C and D combination; or A, B, C and D in combination.
  • mice were killed with excessive CO 2 according to the guidance of the animal ethics committee. Under aseptic conditions, the tibia and femur were removed, and the muscle and connective tissue attached to it were peeled off. The metaphysis was further separated and the bone marrow cavity was exposed. 10 mL was used. A sterile syringe draws PBS with a volume fraction of 10% fetal calf serum to repeatedly wash the bone marrow cavity. After filtering with a 70 ⁇ m pore size cell strainer, centrifuge at 500g for 5min, remove the supernatant and collect the cell pellet at the bottom, resuspend in PBS, and centrifuge again at 500g 5min, collect the final cell pellet.
  • BMMSCs bone marrow mesenchymal stem cells
  • the cells were resuspended in Dex(-) medium and seeded in a 10 cm diameter cell culture dish, incubated at 37°C and 5% CO 2 . After 24 hours, the supernatant non-adherent cells were aspirated, washed with PBS, and Dex(-) medium was added to continue the culture. One week later, the same amount of Dex(+) culture medium was added, and one week later, dense primary BMMSCs colonies were seen. The BMMSCs were digested with trypsin at 37°C and subcultured and expanded. Then, the Dex(+) medium was changed every 3 days, and then subcultured after full growth. Use P2 generation BMMSCs for follow-up experiments.
  • composition of Dex(-) culture solution is shown in Table 1
  • composition of Dex(+) culture solution is shown in Table 2:
  • Flow cytometry was used to analyze the surface markers to evaluate the purity of the isolated BMMSCs.
  • surface marker identification after trypsin digestion to collect P2 generation BMMSCs, wash once with PBS, resuspend the cells in 3% FBS-containing PBS at a density of 5 ⁇ 10 5 /mL, and add 1 ⁇ L of PE fluorescence-conjugated CD29, CD44, CD90, CD45 and CD34 antibodies were not added to the blank group. Incubate at 4°C in the dark for 30 minutes, wash with PBS twice, and then test on the machine. The test results are shown in Figure 1.
  • MSCs (MSCs derived from bone marrow) cultured in Example 1 to the second passage were cultured with the medium (Dex(+) medium) in Example 1 until the cells were 80%-90% confluent, then rinsed with PBS 2
  • 500nM STS-containing serum-free medium ( ⁇ -MEM medium) to induce apoptosis, incubate at 37°C for 16-24h, collect the cell supernatant, centrifuge at 800g at 4°C for 10 minutes, and collect the supernatant at 4°C Centrifuge at 2000 g for 10 minutes, and collect the supernatant again at 4° C. and centrifuge at 16000 g for 30 minutes.
  • the resulting precipitate is IEV. Resuspend the pellet in 500 ⁇ l PBS and centrifuge again at 16000g for 30 minutes at 4°C to obtain the washed IEV.
  • the MSCs (MSCs derived from bone marrow, BMMSCs) cultured in Example 1 to the second generation were cultured with the medium in Example 1 until the cells were 80%-90% confluent, rinsed twice with PBS, and added serum-free Incubate the culture medium at 37°C for 48 hours, and collect the cell supernatant for separation and extraction of exosomes.
  • the extraction steps include: 800g centrifugation for 10 minutes-collection of supernatant liquid-2000g centrifugation for 10 minutes-collection of supernatant liquid-16000g centrifugation for 30 minutes-collection of supernatant liquid-120,000g centrifugation for 90 minutes-remove the supernatant, and resuspend in sterile PBS Precipitate-120,000g centrifugation again for 90 minutes, remove the supernatant, collect the bottom exosomes, and resuspend in sterile PBS.
  • the IEVs obtained in Example 2 were quantitatively analyzed by flow cytometry. The measurement time points were 1h, 4h, 8h, 16h and 24h. The results showed that 106 MSCs were induced to the 1h, 4h, and 24h. After 8h, 16h and 24h, 0.76 ⁇ 10 8 , 1.29 ⁇ 10 8 , 1.95 ⁇ 10 8 , 2.48 ⁇ 10 8 , 3.14 ⁇ 10 8 IEVs can be produced respectively. It can be seen from this that the induction to After 24h, a single MSC can produce 300 IEVs (Figure 3).
  • NTA Nanoparticle Tracking Analysis
  • the state-of-the-art nanoflow detection technology was used for particle size detection at the single vesicle level, and the results also showed that the average particle diameter of IEVs was 100.63 nm ( Figure 4F).
  • IEVs The surface membrane proteins of the IEVs extracted in Example 3 were analyzed by flow cytometry. The results showed that IEVs derived from MSCs can express surface proteins similar to MSCs, namely CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. At the same time, IEVs can express the universal surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (Figure 5A-5K).
  • IEVs were screened for specific and highly expressed proteins, and a heat map was drawn (Figure 6B), and further combined with the GO enrichment analysis results of differential proteins, it is clear that IEVs can specifically and highly express Annexin V, Flotillin-1, and Cadherin 11 , Integrin alpha 5 and Syntaxin 4 molecules (Figure 6C).
  • the expression levels of the five characteristic molecules of IEVs are significantly up-regulated, specifically: the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in IEVs
  • the expression levels of corresponding markers in exosomes were 1.76 times, 2.81 times, 2.41 times, 3.68 times and 4.45 times, respectively.
  • the western blot technology was used for verification again, and the results were consistent with the quantitative analysis results of DIA ( Figure 6D).
  • MSCs-Exosomes Refers to exosomes derived from MSCs.
  • MSCs-IEVs Refers to IEVs derived from MSCs.
  • the MSCs in the content analysis and the MSCs from which the exosomes and IEVs are extracted are the same cell line.
  • Example 4 IEV can be discharged through the skin and hair
  • Figure 7A shows that IEV can reach the surface of the skin, the number is the largest on the 3rd day, and basically disappears on the 7th day, showing the dynamic metabolic process of IEV on the skin surface (Figure 7A).
  • Immunofluorescence results showed that PKH26-IEV gradually moved from the subcutaneous tissue to the dermis and epidermis over time after systemic injection of C57 mice. A large amount of IEV was observed in the stratum corneum of the skin on the 7th day, suggesting that the systemic injection of IEV can be excreted with the stratum corneum of the skin ( Figure 7B).
  • IEV can be excreted through the skin and hair, indicating that it is safe to inject or increase the content of IEV in the body.
  • Example 5 IEV can promote hair regeneration
  • mice Female C57 mice (hair quiescent period) of 7 weeks were depilated and injected subcutaneously with PBS, IEVs, MSC and 2% Minoxidil. Comparing the hair regrowth area on the back of mice on day 10 and day 14, the experimental results showed that IEVs and MSC had a significant effect on promoting hair regrowth compared with the control group. Compared with the traditional anti-hair loss drug minoxidil, on day 14 , Both IEVs and MSC have more obvious hair regrowth-promoting effects (Figure 9A-9B).
  • Example 6 IEV introduced with a microneedle patch can promote hair regeneration
  • IEV derived from human umbilical cord mesenchymal stem cells was introduced into the back of C57 mice with a 1300-needle microneedle patch.
  • the cells used are umbilical cord mesenchymal stem cells (UCMSC) provided by Nanjing Taisheng Biotechnology Co., Ltd. After the obtained UCMSC were treated with STS (500nm) for 10 hours to induce apoptosis, the IEV was extracted according to the following procedure:
  • the supernatant collected in the centrifuge tube continues to be centrifuged for 30 minutes, the centrifuge speed is 16000 g, and the temperature is 4°C.
  • the microneedle patch was fixed to the back skin of the shaved C57 mouse with tape, and IEV (4 ⁇ 10 6 mesenchymal stem cells derived from the umbilical cord) was coated on the surface of the tested skin 1 hour later.
  • the introduction of the 1300-needle microneedle patch into the IEV has a good effect of promoting hair regeneration.
  • Example 7 The combined use of rapamycin, metformin, dasatinib and resveratrol with IEV significantly improves the effect and comparison of IEV in skin discharge and hair regeneration speed
  • PKH26-IEV systemically injected C57 mice rapamycin (Rapamycin, 5mg/kg), metformin (Metformin, 100mg/kg), dasatinib (Dadatinib, 5mg/kg) and resveratrol (Resveratrol, 10mg/kg) were injected intraperitoneally for 7 days, immunofluorescence results showed that metformin can significantly increase the excretion of IEV in the skin tissue, followed by resveratrol, and rapamycin and dasatinib did not significantly enhance the effect (Figure 11 ).
  • mice showed that IEV can significantly promote hair regeneration, and the combined application of anti-aging substances, such as metformin or resveratrol, can enhance the effect of IEV on hair regeneration (Figure 12).
  • This example shows that the combination of metformin or resveratrol and IEV can effectively promote the excretion of IEV in the skin, shorten the time of IEV in the body, and improve the safety of IEV.
  • This example also shows that the combination of metformin or resveratrol and IEV can promote hair regrowth, suggesting that the combination of anti-aging substances and IEV may be used as a medicine for promoting hair regrowth.
  • Example 8 Wnt signaling pathway regulates the functional metabolism of IEV in the skin
  • Wnt pathway is a classic signaling pathway in cell biology. It keeps cells in a state similar to stem cells, helping them to continuously produce new cells. The Wnt signaling pathway plays an important role in the self-renewal of the skin.
  • the Wnt agonist Licl was dissolved in PBS at 10mg/kg and injected into the abdominal cavity for 3 consecutive days; the Wnt inhibitor Xav939 was dissolved in 10% DMSO saline at 1mg/kg and injected into the abdominal cavity for 3 consecutive days.
  • Example 9 IEVs can promote wound healing
  • the method of obtaining IEV is the same as in Example 6.
  • the IEV was introduced into the IEV by injection using a hydrometer.
  • the principle of water-light injection using negative pressure technology, it can accurately replenish the required substances at different depths of the skin. Before the microneedles enter the skin, the skin is lifted by the negative pressure meter, and then multiple needles are accurately entered into the deep epidermis. The injection dosage and injection frequency can be adjusted. In addition, the pressure of the syringe is released before the needle comes out, so there is no loss of nutrients.
  • step 9 Place the UCMSC-derived IEV prepared in step 9 in the syringe, place it in the card slot of the introduction instrument, and adjust the injection depth to 0.3 mm.
  • the subject was informed of the entire process and confirmed informed consent. Disinfect the tested part, direct the water light into the instrument and aim the needle at the tested part (chin area), turn on the negative pressure, follow the operating procedure, and inject the IEV into the tested part.
  • Example 11 Induced pluripotent stem cells (induced pluripotent stem cells, iPS cells, iPSC) cell culture and preparation of IEV derived from it
  • 5 ⁇ 10 4 induced cells were seeded into 10 cm petri dishes with feeder cells (mEFs). The next day, the medium was changed to Es medium containing bFGF (4ng/ml), and changed every other day. After 5 days, the cells began to clone. If there were no Es-like clones after 40 days, it was considered a failure.
  • IEVs derived from iPSCs are shown in Figure 17.
  • Example 12 IEV derived from stem cells with different differentiation potential
  • Flow cytometry detection of cell apoptosis use astrocyticin (500nM) to induce hiPSCs (human induced pluripotent stem cells (hiPSCs)) passaged to P26-P29 generations and hUCMSCs P7-P9 generations (Human umbilical cord mesenchymal stem cells (hUCMSCs)) Approximately 9 hours to cause cell apoptosis, stain AnnexinV (15mins) and 7AAD (3mins), flow cytometric detection of cell apoptosis rate. In this example, hiPSCs passaged to the P26 generation and hUCMSCs passaged to the P7 generation were used.
  • hiPSCs passaged to the P26 generation and hUCMSCs passaged to the P7 generation were used.
  • IEV and use flow cytometry to detect AnnexinV expression rate prepare IEV by differential centrifugation, the steps include: 800g centrifugation for 10 minutes, then 2000g centrifugation for 5 minutes, then 16000g centrifugation for 30 minutes, and then 16000g centrifugation for 30 minutes to obtain IEV, AnnexinV staining for 15 minutes, flow cytometry machine.
  • Modeling of skin injury model A 1.7cm square skin wound was made on the back of the mouse. On Days 1, 4, 8, and 12, the IEV was injected into the tail vein (the injection volume of hiPSCs-IEVs and hUCMSCs were both 6 ⁇ 10 7 /time), and the wound size was recorded by taking pictures every day. Day14 collection.
  • hiPSCs and hUCMSCs were photographed using a high-content cell imaging analysis system. As shown in Figure 18, it is found that there is a difference in the death process between the two.
  • hiPSCs contract with multiple centers of nucleus and cytoplasm, and then emit dendritic branches with bubbles; while hUCMSCs are single-center contractions with nuclei as the center, accompanied by branching. And bubble and other activities.
  • NTA Nanoparticle Tracking Analysis
  • the particle size of hiPSCs-IEVs is about 100nm, and the particle size of hUCMSCs-IEVs is about 180nm;
  • the potential of hiPSC-IEVs is about -12mV, and the potential of hUCMSC-IEVs is about -45mV.
  • hiPSCs-IEVs can significantly promote wound healing. Compared with hUCMSCs-IEVs, hiPSCs-IEVs have stronger tissue repair and regeneration functions.

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Abstract

诱导性细胞外囊泡作为用于皮肤的药物、保健品或护肤品方面的用途。一种组合物包括诱导性囊泡和皮肤药物或护养物。

Description

细胞外囊泡及其在皮肤产品的用途 技术领域
本公开属于生物医药领域，涉及细胞外囊泡及其在皮肤产品的用途。
发明背景
细胞外囊泡(extracellular vesicles,EVs)是细胞分泌的含有蛋白质、核酸及各种细胞因子的纳米级载体。细胞外囊泡可以通过内分泌或旁分泌的方式作用于靶细胞，在细胞间物质传递和信息交流过程中发挥了重要作用。研究发现，细胞外囊泡所介导的信息交流在机体生理或病理过程中发挥了重要的调控作用，涉及到免疫调节、肿瘤生长、血管生成、损伤修复等。目前该领域的研究主要集中在外泌体(exosomes)方向。外泌体是直径在30-150nm左右的细胞外囊泡，其内含有RNA、脂质和蛋白质等成分。外泌体广泛参与了机体的各种生理/病理性调控，能够用作多种疾病的诊断、治疗和预后评估。迄今为止，间充质干细胞(mesenchymal stem cells，MSCs)被认为是产生外泌体能力最强的细胞。众多的研究发现MSCs来源的外泌体能模拟MSCs的生物学功能，在促进细胞生长和分化，修复组织缺损等方面发挥了重要的调控作用。因此，近年来以MSCs来源的外泌体为基础的细胞囊泡疗法取得了显著的发展。然而，目前以外泌体为基础的细胞囊泡治疗仍然存在诸多问题，主要表现在外泌体的提取和纯化过程复杂，耗时长，对设备和试剂的要求较高，生理性外泌体产量较低等等，这些缺陷都限制了外泌体治疗的临床转化和应用。
导致毛发脱落的疾病有多种，其中最常见的是斑秃。斑秃(Alopecia areata)俗称“鬼剃头”，是一种突然发生的局限性斑片状的脱发，局部皮肤正常，无自觉症状。目前病因不明，病理检查可见毛囊减少，在毛囊周围有淋巴细胞浸润，且本病有时合并其他自身免疫性疾病(Alopecia Areata:Review of Epidemiology,Clinical Features,Pathogenesis,and New Treatment Options.Darwin E,Hirt PA,Fertig R,Doliner B,Delcanto G,Jimenez JJ.Int J Trichology.2018；10(2):51-60)。大多数普通斑秃有自然痊愈倾向，少数病例反复发生，所以治疗困难，但有很多疗法可以联合治疗脱发。可发生于任何年龄，但以青壮年多见，两性发病率无明显差异。皮损表现为圆形或卵圆形非瘢痕性脱发，在斑秃边缘常可见“感叹号”样毛发。头发全部或几乎全部脱落，称为全秃。全身所有的毛发(包括体毛)都脱落，称为普脱，还可见匍行性脱发。病区皮肤除无毛发外，不存在其他异常。目前有文献报道在C3H/HeJ小鼠中可通过局部涂抹咪喹莫特的方法制作斑秃模型，目前对于斑秃尚无有效的治疗方法。
发明内容
一方面，本公开提供了诱导性囊泡在制备或作为皮肤产品或皮肤附属器产品方面的用途。
在本公开中，“皮肤产品”是指，可以对皮肤起到作用的产品，例如，对皮肤起到治疗、改善、修复、除皱或消除伤口等作用的产品，可以直接或间接作用于皮肤，可以是药物、药物载体、食品、保健品、护肤品或医美用品。
在一些实施方案中，所述产品为：药物、药物载体、食品、保健品、护肤品、或医美用品。
在一些实施方案中，所述皮肤为表皮、真皮、或皮下组织。
在一些实施方案中，所述皮肤附属器为毛、头发、汗腺、皮脂腺、指甲、或趾甲。
发明人在研究过程中，偶然发现，向裸鼠注射IEV，IEV在心、肾、脑中无分布，但在指甲中分布，推测与IEV的靶向性有关。
经过进一步研究，发明人发现IEV对毛发再生，伤口愈合有很好的治疗效果。一方面是IEV对皮肤或皮肤附属器具有治疗作用，一方面是其刚好靶向于皮肤或皮肤附属器，使得其尤其适合制备或作为皮肤产品或皮肤附属器产品进行应用。
一方面，本公开提供了一种组合物，包括：诱导性囊泡和皮肤药物或护养物。
在一些实施方案中，所述皮肤药物与护养物被包裹在细胞外囊泡中，或以分开的形式存在于组合物中。
在一些实施方案中，所述组合物的剂型优选地选自冻干粉针、注射剂、片剂、胶囊或贴剂；在一些实施方案中，所述贴剂选自微针贴剂；在一些实施方案中，所述剂型选自注射剂或微针贴剂。
在一些实施方案中，所述诱导性囊泡是在干细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡；在一些实施方案中，所述诱导性囊泡是通过添加星形孢菌、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合诱导干细胞或干细胞凋亡产生。
一方面，本公开提供了一种组合物，所述的组合物包括由干细胞或干细胞胞外囊泡组成的第一剂；和，由抗衰老物质或Wnt信号通路正向调节剂组成的第二剂。
在一些实施方案中，所述细胞外囊泡指的是在前体细胞(例如干细胞、尤其例如间充质干细胞)正常存活期间，被干预或诱导，使其凋亡产生的一类亚细胞产物，也称之为诱导性囊泡。优选地，前体细胞是早期的前体细胞。
通常这一类亚细胞产物：其有膜结构，包含有遗传物质DNA，不能分裂的、可运动的和迁移的。是介于细胞和细胞外囊泡(凋亡小体、外泌体等)之间的一类物质。
在一些实施方案中，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、尿液间充质干细胞、口腔间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带间充质干细胞、骨膜间充质干细胞、皮肤间充质干细胞中的一种或多种。
在一些实施方案中，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、口腔间充质干细胞、皮肤间充质干细胞中的一种或多种。
在一些实施方案中，所述的干细胞选自诱导性多能干细胞、脐带间充质干细胞、血液干细胞或骨髓间充质干细胞中的一种或多种。
在一些实施方案中，所述的干细胞胞外囊泡选自外泌体、迁移体、微泡、凋亡小体、诱导性细胞外囊泡中的一种或多种。
在一些实施方案中，所述的干细胞胞外囊泡选自诱导性细胞外囊泡。
在一些实施方案中，所述抗衰老物质选自二甲双胍，白黎芦醇中的一种或多种。
在一些实施方案中，所述抗衰老物质选自二甲双胍。
在一些实施方案中，所述Wnt信号通路选自Wnt/β-catenin通路。
在一些实施方案中，所述正向调节剂选自激动剂。
在一些实施方案中，所述激动剂选自Licl、CHIR99021、SB-216763、BIO、Wnt Agonist或WAY 262611中的一种或多种。
在一些实施方案中，所述激动剂选自Licl。
在一些实施方案中，所述正向调节剂选自包含进行运动的提示。
在一些实施方案中，所述提示选自包装，标签或说明书中的一种或多种。
在一些实施方案中，所述组合物中，包含干细胞或干细胞胞外囊泡与抗衰老物质的比例为(0.5-5)×10 7个：(0.05-10)mg。
在一些实施方案中，所述组合物中，包含1×10 7-3×10 7个干细胞或干细胞胞外囊泡；和，0.1-5mg抗衰老物质。
在一些实施方案中，所述组合物中包含1.8×10 7-2.7×10 7个干细胞或干细胞胞外囊泡。
在一些实施方案中，所述组合物中包含0.2-3mg抗衰老物质。
将所述组合物应用时，可以将所述细胞外囊泡任选地通过选自由静脉内注射、肌肉注射、皮下注射、鞘内注射或输注以及器官内输注所组成的组中的途径进行给药。例如，作为例子，对于静脉注射，可以通过尾静脉注射。器官内输注包括输注至解剖学空间中，例如，作为例子，胆囊、胃肠腔、食道、肺系统(通过吸入)和/或膀胱。
作为例子，对于胃肠腔输注中腹腔注射，与尾静脉注射注射相比，腹腔注射也可以获得良好的治疗效果。腹腔注射的安全性和可操作性要优于尾静脉注射。
在一些实施方案中，组合物中的两种试剂以单一药物组合物的形式提供，并且在一些实施方案中，可以预期是包含两种试剂中的每一种的试剂盒或组合的分配器包装。应当理解，本公开的内容涵盖两种试剂中的任一种对受试者的共同施用，无论这种施用是以单一制剂还是以分开的制剂组合，以及这种施用是同时的还是交错的。
在一些实施方案中，所述细胞外囊泡是通过添加星形孢菌素、紫外线照射、饥饿法、或热应力法或其组合诱导间充质干细胞凋亡产生。
在一些实施方案中，所述细胞外囊泡是通过添加星形孢菌素诱导间充质干细胞凋亡产生。
一些实施方案中，所述间充质干细胞的代数可以为第2～5代，但又不限于此。
在一些实施方案中，所述星形孢菌素的浓度为大于或等于1nM；优选地，为1-15000nM；优选地，为200-10000nM；优选地，为250-1000nM；优选地，为500-1000nM。除此之外，星形孢菌素的浓度还可以是280-9000nM；还可以是230-8500nM；还可以是500-1000nM；还可以是500-900nM；还可以是500-800nM。在一些实施方案中，所述星形孢菌素的浓度为500nM。
在一些实施方案中，所述诱导性细胞外囊泡的直径为0.45μm或以下。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.45μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.1-0.45μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.1-0.35μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.15-0.35μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.15-0.3μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.15-0.2μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.4μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.38μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.35μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.32μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.3μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.25μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.05-0.22μm。一些实施方案中，所述诱导性细胞外囊泡的直径为0.15-0.22μm。一些实施方案中，所述诱导性细胞外囊泡的直径还可以是0.15-0.45μm，还可以是0.2-0.3μm。
在一些实施方案中，所述诱导性细胞外囊泡具有标志物Syntaxin 4。在一些实施方案中，所述诱导性细胞外囊泡高表达标志物Syntaxin 4。在一些实施方案中，所述诱导性细胞外囊泡的标志物Syntaxin 4的表达高于MSC或外泌体。在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，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。在一些实施方案中，所述标志物为Syntaxin 4、Annexin V、Flotillin-1、Cadherin 11和Integrin alpha 5的组合。在一些实施方案中，所述诱导性细胞外囊泡高表达标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5。在一些实施方案中，所述诱导性细胞外囊泡的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5的表达量高于MSC或外泌体。在一些实施方案中，所述诱导性细胞外囊泡中的标志物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。在一些实施方案中，所述诱导性细胞外囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5相对于来源于间充质干细胞的外泌体中标记物的表达量分别为1.5-2倍、2.5-3倍、1.5-2.5倍和3.5-4倍。在一些实施方案中，所述诱导性细胞外囊泡中的标志物Annexin V、Flotillin-1、Cadherin 11、Integrin alpha 5相对于来源于间充质干细胞的外泌体中标记物的的表达量分别为1.76倍、2.81倍、2.41倍、3.68倍。本公开中所述诱导性细胞外囊泡与外泌体有本质的不同，例如与外泌体相比，本公开所述的诱导性细胞外囊泡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3)。除了标志物表达有差别之外，所述诱导性细胞外囊泡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。在一些实施方案中，所述诱导性细胞外囊泡还表达CD29、CD44、CD73、CD166；且不表达CD34、CD45。在一些实施方案中，所述诱导性细胞外囊泡还表达CD9、CD63、CD81和C1q中的一个或多个。
在一些实施方案中，还提供了所述的组合物在制备促毛发再生或促伤口愈合药物中的应用。
在一些实施方案中，所述组合物还包含药学上可接受的载体；所述组合物的剂型优选地选自冻干粉针、注射剂、片剂、胶囊或贴剂。
在一些实施方案中，所述贴剂选自微针贴剂；在一些实施方案中，所述剂型选自注射剂、微针贴剂或片剂。
在一些实施方案中，还提供了所述的细胞外囊泡的制备方法，包括如下步骤：
1)体外培养间充质干细胞，细胞汇合80％-90％时，PBS冲洗2-5遍；
2)将步骤1)制得的间充质干细胞加入含有500-1000nM星形孢菌素的无血清培养基，37℃孵育16-24h，收集细胞上清液；
3)将步骤2)收集的细胞上清液在4℃下500-15000g离心5-30分钟，收集上清液；
4)将步骤3)收集的细胞上清液在4℃下1500-2500g离心5-30分钟，收集上清液；
5)将步骤4)收集的细胞上清液在4℃下10000-30000g离心15-60分钟，所得沉淀物为细胞外囊泡；
在一些实施方案中，还包括细胞外囊泡的清洗步骤；在一些实施方案中，所述清洗步骤具体为：6)将步骤5)制得的细胞外囊泡用PBS重悬，在4℃下10000-30000g离心15-60分钟，所得沉淀物为细胞外囊泡。
一方面，本公开还提供了抗衰老物质在制备干细胞服用品的伴随药物方面的用途。
本公开实施方案中的“干细胞服用品”包括但不限于能使干细胞或干细胞囊泡进入动物体内的产品或方法。
作为示范性的例子，能使干细胞服用品进入动物体内的产品包括冻干粉针、注射剂、片剂、胶囊或贴剂。
作为示范性的例子，能使干细胞服用品进入动物体内的方法包括口服、注射、敷贴、涂抹、喷撒、滴注。
本公开实施方案中的“伴随药物”包括随另外一种或多种药物一同进入体内的药物。伴随药物可以同时或几乎同时进入体内。
作为示范性的例子，几乎同时进入可以是一个接一个地进入；或在同一天的不同时刻进入体内。例如，可以将多个药剂或其活性成分配制成一种制剂，或者两种或多种制剂至少基本上同时进入体内。例如，相互在约1小时内。
在一些实施方案中，所述的干细胞服用品为干细胞或干细胞胞外囊泡。
在一些实施方案中，所述的干细胞服用品为用以摄入动物体的干细胞或干细胞胞外囊泡；优选地，所述动物为哺乳动物；优选地，所述哺乳动物为人。
一方面，本公开还提供了一种干细胞服用系统，其包括：干细胞服用品，和，伴随品；所述的伴随品选自抗衰老物质或Wnt信号通路正向调节剂中的一种或两种；所述的干细胞服用品包含干细胞或干细胞胞外囊泡；所述抗衰老物质或Wnt信号通路正向调节剂如前所述。
一方面，本公开还提供了干细胞或干细胞胞外囊泡在制备Wnt信号通路调控的干细胞或干细胞胞外囊泡代谢而促进毛发再生或伤口愈合的药物方面的用途。
在一些实施方案中，所述促进毛发再生或伤口愈合的药物包括干细胞或干细胞胞外囊泡；和，抗衰老物质。所述干细胞或干细胞胞外囊泡及抗衰老物质如前所述。
一方面，本公开还提供了一种促进干细胞服用品排出动物体外、或毛发再生、或伤口愈合的方法，所述方法包括，在服用干细胞服用品期间，同时或几乎同时服用抗衰老物质，或进行运动。
本公开的一些实施方案中，“Wnt信号通路正向调节剂”包括那些可以上调、激活Wnt的试剂或方法，如激动剂，但并不限于此。前述的“方法”的实现包括对受试者的提示，说明，处方，嘱咐等。
一方面，本公开还提供一种皮肤或皮肤附属器疾病的治疗方法，包括以下步骤：向有需要的受试者施用所述的诱导性囊泡，组合物，干细胞服用系统。
在一些实施方案中，所述皮肤或皮肤附属器疾病选自创伤、脱发、瘢痕、烧烫伤或溃疡。在一些实施方案中，所述创伤选自切除伤口、切割、刺伤或穿刺伤口。
诱导性多能干细胞包括通过导入特定的转录因子将终末分化的体细胞重编程而形成的多能性干细胞。
附图说明
图1为实施例1中流式细胞检测图；
图2为实施例2制备IEV的技术路线图；
图3为用流式细胞分析技术分析的MSCs(106个MSCs)产生的IEVs数量统计结果；
图4A-图4F为IEVs颗粒的直径检测：图4A为流式检测IEVs的颗粒直径分布图；图4B为侧向散射光(SSC)分析IEVs的散射光强，显示IEVs颗粒直径分布；图4C为以Bangs Laboratories公司生产的标准化小颗粒微球分析IEVs的散射光强，显示IEVs的颗粒直径分布；图4D为透射电镜(TEM)观察的IEVs，显示IEVs的颗粒直径分布；图4E为纳米粒子跟踪分析(NTA)，显示IEVs颗粒直径分布；图4F为纳米流式检测技术对IEVs进行单囊泡水平的粒径检测，显示IEVs的颗粒直径分布；
图5A-图5K为流式细胞技术IEVs的表面膜蛋白进行分析结果；
图6A-图6D为IEVs的内容物分析：图6A为DIA定量技术对MSCs、MSCs-Exosomes、MSCs-IEVs蛋白组学定量分析结果；图6B为筛选IEVs特异性高表达的蛋白绘制的热图；图6C为差异蛋白的GO富集分析IEVs表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分子的结果；图6D为western blot验证MSCs、MSCs-Exosomes、MSCs-IEVs表达Annexin V,Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4的结果；
图7A为实施例3中IEV在皮肤表面的动态代谢示意图；
图7B显示随着时间推移，IEV逐渐从皮下组织向真皮层和表皮移动；
图7C显示第7天时在小鼠体表拔下的毛发中发现毛囊中存在PKH26-IEV；
图8A显示活体成像技术检测的第1，3，7天IEV在小鼠全身的分布情况；
图8B为第7天时IEV在小鼠各器官中的分布与对照组的对比；
图8C表示第3天时，IEV在小鼠指甲和门齿中的代谢示意图；
图9A为实施例4中不同处理的小鼠在第0天，第10天，第14天的背部毛发再生情况示意图；
图9B为实施例4中不同处理的小鼠在第0天，第10天，第14天的背部毛发再生面积统计分析示意图；
图10为实施例6中不同处理的小鼠在在第0天，第11天，第13天，第16天的背部毛发再生情况示意图；
图11为实施例5中不同药物与IEV联用，7天后IEV在皮肤表面的分布图；
图12为实施例5中不同药物与IEV联用对毛发再生促进效果示意图；
图13A为实施例6中施用Wnt激动剂Licl后的Western Blot结果；
图13B为实施例6中施用Wnt抑制剂Xav939后的Western Blot结果；
图13C为实施例6中在Xav939作用下的免疫荧光结果；
图13D为实施例6不同处理下的细胞涂片结果；
图13E为实施例6中不同处理后，IEV在表皮的排出情况免疫荧光结果
图14A显示运动后循环中DKK1的水平；
图14B显示运动与否血液中IEV的数量及流式结果；
图14C为运动后IEV在皮肤表面排出量的免疫荧光结果；
图14D为活体成像技术检测的运动1或3天后IEV在小鼠全身的分布情况；
图14E为活体成像技术检测的运动1或3天后IEV在小鼠皮肤的分布情况；
图14F为流式检测运动7天后IEV在体内(血液)中的结果；
图15A显示实施例7中IEV和MSC处理对伤口愈合的促进作用；
图15B为实施例7中不同处理组对伤口愈合的作用示意图；
图15C为实施例7中不同处理组的免疫荧光分析结果；
图16为实施例10中水光导入IEV前后，受试者下巴部位皱纹对比图；
图17为实施例11中电镜下iPSC来源的IEVs；
图18为实施例12中高内涵细胞成像分析系统拍摄的hiPSCs和hUCMSCs死亡过程图；
图19为实施例12中使用流式分析诱导凋亡的hiPSCs和hUCMSCs；
图20为实施例12中使用流式分析的hiPSCs和hUCMSCs的Annexin5表达阳性率；
图21-23依次为实施例12中，NTA表征的hiPSCs-IEVs和hUCMSCs-IEVs的粒径、产量和电位；
图24为实施例12皮肤损伤模型中，不同处理组在不同时间点的伤口情况及伤口面积统计图。
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，IEV同IEVs。
本公开中的STS为星形孢菌素。
在本公开中，“组合物”中的组分可以是混合的形式存在，也可以被分开包装。分开的包装的组分也可以含有其各自的佐剂。所述的佐剂是指在药学中，可辅助药物疗效的手段。对于组合物中的组分在分开包装的情况下，各个分开包装的组分可以是同时施用或者是以任意的前后顺序施用，其中患者先用一种药物治疗，然后再给以另一种药物。所述的患者是指哺乳动物受治疗者，尤其是人类。
在本公开中，所述的“组合物”还可以是一种组分被另外一种组分包裹的形式存在。在一些实施方式中，在组合物中，所述的诱导性囊泡作为药物载体，将治疗或预防疾病的药物包裹在所述诱导性囊泡中。
“包含”或“包括”旨在表示组合物(例如介质)和方法包括所列举的要素，但不排除其他要素。当用于定义组合物和方法时，“基本上由……组成”意味着排除对于所述目的的组合具有任何重要意义的其他要素。因此，基本上由本文定义的元素组成的组合物不排除不会实质上影响要求保护的本公开的基本和新颖特征的其他材料或步骤。“由……组成”是指排除其他组成部分的微量元素和实质性的方法步骤。由这些过渡术语中的每一个定义的实施方案都在本公开的范围内。
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。
实施例1 MSCs的分离培养
依据动物伦理委员会的指导用过量CO 2处死小鼠，在无菌条件下，取下胫骨和股骨，剥净附着在其上的肌肉和结缔组织，进一步分离干骺端、暴露骨髓腔，用10mL无菌注射器抽取喊体积分数为10％胎牛血清的PBS反复冲洗骨髓腔，70μm孔径细胞滤网过滤后，500g离心5min，去除上清液后收集底部的细胞沉淀，PBS重悬，再次500g离心5min，收集最终的细胞沉淀。而后对细胞进行流式分选，以CD34-和CD90+为分选标准，分选出骨髓间充质干细胞(BMMSCs)。最后以Dex(-)培养液重悬细胞并接种于10cm直径细胞培养皿，37℃、5％CO 2培养。24h后，吸除上清液未贴壁细胞，PBS清洗后，加入Dex(-)培养液继续培养。1周后加入等量Dex(+)培养液，再1周后可见密集的原代BMMSCs集落。采用胰蛋白酶37℃孵育消化BMMSCs并传代扩增，之后每3日换Dex(+)培养液，长满后传代。使用P2代BMMSCs进行后续实验。
其中，Dex(-)培养液成分如表1所示，Dex(+)培养液成分如表2所示：
表1 Dex(-)培养液成分表
Figure PCTCN2021072937-appb-000001
表2 Dex(+)培养液配方表
Figure PCTCN2021072937-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。检测结果如图1所示。
实施例2 BMMSC来源的IEV的制备
将实施例1中培养至第2代的MSCs(骨髓来源的MSCs)，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，PBS冲洗2遍，加入含500nM STS的无血清培养基(α-MEM培养基)诱导凋亡，37℃孵育16-24h，收集细胞上清液，4℃下800g离心10分钟，收取上清液4℃下2000g离心10分钟，再次收集上清液4℃下16000g离心30分钟，所得沉淀物为IEV。500μl PBS重悬沉淀，4℃下16000g再次离心30分钟，即得到清洗后的IEV。
制备路线如图2所示。
对比例1 同种MSC来源的外泌体分离和提取
将实施例1中培养至第2代的MSCs(骨髓来源的MSCs,BMMSCs)，用实施例1中的培养基继续培养至细胞汇合80％-90％时，用PBS冲洗2遍，加入无血清培养基，37℃孵育48h，收集细胞上清液，用于分离和提取外泌体。
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，收集底部的外泌体，无菌PBS重悬。
实施例3 IEVs的分析
(1)IEVs的定量和膜蛋白的分析
利用流式细胞技术对实施例2获得的IEVs进行定量分析，测量时间点为第1h、第4h、第8h、第16h和第24h，结果显示106个MSCs在诱导至第1h、第4h、第8h、第16h和第24h后分别可以产出0.76×10 8个、1.29×10 8个、1.95×10 8个、2.48×10 8个、3.14×10 8个IEVs，从中可以看出，诱导至24h后，单个MSC可以产出300个IEVs(图3)。
此外，流式检测发现IEVs的颗粒直径分布都集中在1μm以下，占94.97％(图4A)。
侧向散射光(SSC)分析结果同样显示IEVs散射光强集中在1μm以下范围(图4B)。
进一步的，通过Bangs Laboratories公司生产的标准化小颗粒微球(0.2μm，0.5μm，1μm)分析IEVs的散射光强，结果显示IEVs的颗粒直径都在0.2μm以下(图4C)。
透射电镜(TEM)观察的结果与流式检测结果相似，大部分囊泡的直径都在200nm及200nm以下(图4D)。
纳米粒子跟踪分析(NTA)结果与透射电镜观察结果相符，IEVs颗粒直径平均为169nm(图4E)。
利用最先进的纳米流式检测技术进行单囊泡水平的粒径检测，结果也显示IEVs的平均颗粒直径在100.63nm(图4F)。
利用流式细胞技术对实施例3提取的IEVs的表面膜蛋白进行分析，结果显示，MSCs来源的IEVs能够表达和MSCs相似的表面蛋白，即CD29，CD44，CD73，CD166阳性，CD34，CD45阴性。同时，IEVs能够表达细胞外囊泡的普遍性表面蛋白CD9，CD63，CD81和C1q(如图5A-5K)。
(2)IEVs的内容物分析
利用蛋白DIA定量技术完成MSCs，MSCs-Exosomes(对比例1提取的)，MSCs-IEVs(实施例2获得的)的蛋白组学定量分析。结果显示，MSCs-Exosomes和MSCs-IEVs的蛋白内容物表达与母细胞具有较高的重叠性，170种蛋白在IEVs中特异性高表达(图6A)。通过生物信息学分析，筛选IEVs特异性高表达的蛋白，绘制热图(图6B)，进一步结合差异蛋白的GO富集分析结果，明确IEVs能特异性高表达Annexin V，Flotillin-1，Cadherin 11，Integrin alpha 5和Syntaxin 4分子(图6C)。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相对于外泌体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6D)。
MSCs-Exosomes：指的是来源于MSCs的外泌体。
MSCs-IEVs：指的是来源于MSCs的IEVs。
其中所述的内容物分析中的MSCs和与提取外泌体和IEVs的MSCs为同一细胞株。
实施例4 IEV可经皮肤和毛发排出
取4×10 6的实施例2制备的IEV用DIR标记，200微升PBS重悬，通过尾静脉系统性注射裸鼠BALB/c-nu/nu体内，观察1，3，7天后用活体成像仪器检测IEV在皮肤表面的分布，结果如图7A-7C所示。
图7A显示IEV可到达皮肤表面，在第3天时数量最多，第7天基本消失，显示IEV在皮肤表面的动态代谢过程(图7A)。免疫荧光结果显示PKH26-IEV系统性注射C57小鼠后，随着时间推移，逐渐从皮下组织向真皮层和表皮移动。第7天在皮肤表面角质层观测到IEV大量存在，提示系统性注射的IEV可以随着皮肤角质层的脱落而排泄出去(图7B)。同时，第7天时在小鼠体表拔下的毛发中发现毛囊中存在PKH26-IEV，说明系统性注射的IEV还可随着毛发的脱落而代谢出去(图7C)。
此外，活体成像数据和免疫荧光结果均显示IEV在心、肾、脑中无分布，但在指甲中分布，进一步证明IEV可随着代谢排出体外(图8A-图8C)。
本实施例表明IEV可以通过皮肤和毛发排出，说明注射或增加IEV在体内的含量具有安全性。
实施例5 IEV能够促进毛发再生
7周的雌性C57小鼠(毛发静止期)进行脱毛处理，分别皮下注射PBS、IEVs、MSC和2％米诺地尔(Minoxidil)。比较第10天和第14天小鼠背部的毛发再生面积，实验结果显示IEVs和MSC较对照组有明显的促毛发再生作用，与传统的防脱发药物米诺地尔相比，在第14天时，IEVs和MSC都具有更明显的促毛发再生效果(图9A-9B)。
实施例6 用微针贴片导入的IEV可促进毛发再生
用1300针微针贴片向C57小鼠背部导入人脐带间充质干细胞(UCMSC)来源的IEV。
UCMSC来源的IEV的获取方式：
采用的细胞是南京泰盛生物科技有限公司提供的脐带间充质干细胞(UCMSC)。获得的UCMSC使用STS(500nm)处理10小时诱导凋亡后，按以下流程提取IEV：
1.诱导完毕,自培养箱取出诱导完毕的细胞，用移液器吹打涮洗皿底及皿壁。混匀后，吸出混悬液,分别收集在对应编号的无菌离心管中，离心，离心时间10min,离心转速：800g，温度4℃。
2.离心完毕，将上述离心管中上清液吸出，收集于对应编号的新准备的无菌离心管中待二次离心提纯。管中沉淀用于凋亡率检测。
3.同步将上述离心后收集的上清液继续离心，离心时间10min，离心转速2000g，温度4℃。
4.离心完毕，收集上清液，弃去沉淀，过程进行观察。
5.将上清液分别吸入对应新编号无菌管中，待用。
6.将上述二次离心后，离心管中收集的上清液继续离心，离心时间30min，离心转速16000g，温度4℃。
7.离心完毕，收集沉淀，弃去上清液，过程进行观察。
8.准备好生理盐水，待离心完毕，弃去上清液，往离心管中分别加入适量生理盐水，吹打混悬洗涤，肉眼观察液相均匀为止。继续离心，离心时间30min，离心转速16000g，温度4℃。
9.离心完毕，弃去上清液，往离心管中加入适量生理盐水，与沉淀物混悬。
无菌操作，利用胶布将微针贴片固定与剃毛后的C57小鼠背部皮肤处，1小时后受试皮肤表面涂布IEV(4×10 6个来源于脐带间充质干细胞)。
各处理组的设计如表3所示。
表3
组别 载体
1 对照组
2 皮下注射IEV
3 1300针微导入IEV
如图10所示，1300针微针贴片导入IEV具有良好的促进毛发再生效果。
实施例7 雷帕霉素、二甲双胍、达沙替尼和白黎芦醇与IEV联用显著提升IEV在皮肤的排出及提升毛发再生速度等方面的作用及对比
PKH26-IEV系统性注射C57小鼠，雷帕霉素(Rapamycin，5mg/kg)、二甲双胍(Metformin，100mg/kg)、达沙替尼(Dadatinib，5mg/kg)和白黎芦醇(Resveratrol，10mg/kg)分别腹腔连续注射7天后，免疫荧光结果显示二甲双胍能够显著增加IEV在皮肤组织的排出量，白黎芦醇次之，雷帕霉素和达沙替尼无明显增强作用(图11)。
小鼠毛发再生结果显示IEV能显著促进毛发再生，而联合应用抗衰老物质，如二甲双胍或白黎芦醇，都能提升IEV对毛发再生的促进作用(图12)。
本实施例表明，二甲双胍或白黎芦醇与IEV联用，能有效促进IEV在皮肤的排出量，缩短了IEV在体内的时间，提升了IEV的安全性。
本实施例还表明，二甲双胍或白黎芦醇与IEV联用，能够促进毛发再生，提示了抗衰老物质和IEV的组合物在作为促毛发再生的药物应用的可能性。
实施例8 Wnt信号通路调控IEV在皮肤的功能性代谢
Wnt通路是细胞生物学中的一个经典信号通路。它能让细胞保持在类似于干细胞的状态中，帮助它们不断生出新的细胞。Wnt信号通路在皮肤的自我更新中发挥重要作用。
Wnt激动剂Licl以10mg/kg溶于PBS中，腹腔连续注射3天；Wnt抑制剂Xav939以1mg/kg溶于10％DMSO的生理盐水中，腹腔连续注射3天。
Western Blot结果显示使用Wnt激动剂Licl后IEV在皮肤的排泄显著加强，使用Wnt抑制剂Xav939后IEV在皮肤的排泄明显降低(图13A-B)。免疫荧光结果也同样证明在Xav939影响下皮下注射的PKH26-IEV在皮肤表面的排出大大减少(图13C)。同时细胞涂片结果显示皮肤间充质干细胞对IEV的摄入能力受Licl和Xav939的影响(图13D)。更重要的是，免疫荧光结果证明IEV注射后大大增强了皮肤中active-β-catenin的强度，提示IEV能刺激皮肤中Wnt信号通路的活化，Xav939抑制剂使这一活化过程被减弱，且active-β-catenin强度的变化与IEV在表皮层的排出数量变化相一致(图13E)。
运动已被证实对Wnt信号通路具有调控作用。ELISA结果显示运动后循环中DKK1水平显著降低(图14A)，说明运动可以激活体内Wnt信号通路。
流式结果显示运动后小鼠体内生理性IEV大量减少(图14B)，免疫荧光结果显示运动后PKH26-IEV在皮肤表面的排出增加(图14C)，活体成像结果显示运动1天后IEV在体内和皮肤的分布大大加强，运动3天后较对照组开始减弱(图14D-E)。利用流式检测PKH-67标记的IEV运动后在体内的数目，结果显示运动7天后PKH67-IEV在血液中较对照组大量减少(图14F)。以上数据提示运动可能激活Wnt信号通路，促进IEV从皮肤排泄。
实施例9 IEVs可促进伤口愈合
在小鼠身上制作1cm×1cm的全皮层创口，检测系统性注射MSCs和IEVs对伤口愈合的促进作用。结果显示IEVs和MSCs均对伤口愈合有促进作用(图15A)，无显著差异。Wnt激动剂Licl能加速IEVs对伤口愈合的促进作用，而抑制剂Xav939能显著减缓IEVs对伤口愈合的促进作用。免疫荧光结果显示Licl组IEVs在伤口处的聚集明显增强，Xav939组IEVs在伤口处的聚集显著减少，说明Wnt信号通路可能通过调控IEVs的排泄来影响伤口愈合(图15B-C)。
实施例10 局部注射IEV的除皱效果
IEV的获取方式同实施例6。
使用水光仪注射导入IEV。水光注射的原理：采用负压技术，可以准确地在皮肤不同深度下补充所需物质，微针进入皮肤前利用负压仪将皮肤提起，然后多针头再准确的进入到表皮深层。注入剂量、注射频率可调节。另外，针头在出来前就已经放开了注射器的压力，因此不会有营养物的损耗。
导入步骤：
将步骤9制备的UCMSC来源的IEV置于注射器内，放置于导入仪卡槽中、调节注射深度至0.3mm。
受试者被告知整个过程，并确认知情同意。受试部位消毒、将水光导入仪针头对准受试部位(下巴部位)，开启负压，按照操作程序进行，将IEV注入 受试部位。
如图16所示，水光导入注射IEV50天后(2020年1月16日至2020年3月8日)，注射部位的皱纹有显著改善，说明IEV有良好的除皱效果，有望应用于医美行业。
实施例11 诱导性多能干细胞(induced pluripotent stem cells，iPS细胞，iPSC)细胞培养及其来源的IEV制备
(1)慢病毒制备：
将1mL DMEM移入EP管，加入5μg基因表达质粒和5μg vsvg质粒加入25微升的脂质体，轻轻搅拌，室温20分钟。将混合物逐滴加入培养的GP2-293细胞(95％混匀)中，旋转使混合物均匀分布。12小时后更换培养基(DMEM+10％FBS(热灭活)+谷氨酰胺)。更换培养基24小时后，收集含有病毒的培养基，48小时后再次收集培养基。
(2)诱导细胞重编程：
每孔(12孔板)接种步骤(1)中培养的GP2-293细胞5×10 5个，80％汇合在500-1000微升/孔的培养基(DMEM+10％FBS(热灭活)+谷氨酰胺)中加入病毒100ng，加入4微克/毫升聚布伦，孵育12h，换新培养基，重复此步骤。在7天内，5×10 4个经诱导的细胞接种到带有饲养细胞的10cm培养皿中(mEFs)。第二天，将培养基改为Es培养基，含bFGF(4ng/ml)，每隔一天换一次，5天后，细胞开始出现克隆，如果40天后没有Es样克隆，则认为失败。
(3)细胞传代：
60％汇合后，每皿添加0.5毫升的accutase，在室温下静置1分钟。将分离的细胞聚集物转移到15mL离心管中，并用另外的2mL mTeSR1以收集任何剩余的聚集物。将冲洗液加入15毫升试管中。在室温下离心含有200×g细胞聚集物的15mL试管5分钟。吸出上清液。使细胞再悬浮并确保细胞保持聚集状态。将人iPS细胞与mTeSR1聚集在涂有基质凝胶的新板上。将培养皿放入37℃培养箱中，快速左右移动培养皿使细胞聚集物均匀分布团块的运动。在37℃下用5％的二氧化碳和95％湿度培养。每天换液。
(4)iPS来源的IEV制备
同实施例2的制备方法。
400倍光镜下，iPS细胞(iPSC)来源的IEVs如图17所示。
实施例12 不同分化潜能干细胞来源的IEV
一般实验方法：
流式检测细胞凋亡：使用星形环孢菌素(500nM)诱导传代至P26-P29代的hiPSCs(人诱导性多能干细胞(human induced pluripotent stem cells,hiPSCs))和P7-P9代的hUCMSCs(人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs))约9h使细胞发生凋亡，染AnnexinV(15mins)和7AAD(3mins)，流式检测细胞凋亡率。在本实施例中，使用传代至P26代的hiPSCs和传代至P7代的hUCMSCs。
分离IEV并用流式检测AnnexinV表达率：使用差数离心法制备IEV，步骤包括：800g离心10mins，之后2000g离心5mins，之后16000g离心30mins，之后16000g离心30mins，获取IEV，AnnexinV染色15mins，流式上机。
皮肤损伤模型造模：在小鼠背部造边长1.7cm的正方形皮肤创口。在Day1，4，8，12分别尾静脉给IEV(hiPSCs-IEVs，hUCMSCs的注射量均为6×10 7个/次)，并且每天拍照记录伤口大小。Day14取材。
结果：
1、使用高内涵细胞成像分析系统拍摄了hiPSCs和hUCMSCs死亡过程。如图18所示，发现两者死亡过程存在差异，hiPSCs以核和质多个中心进行收缩，继而发出树枝状分支伴随出泡；而hUCMSCs是以核为中心的单中心收缩，同时伴随发出分支和出泡等活动。
2、如图19所示，对诱导凋亡的hiPSCs和hUCMSCs使用流式分析凋亡率，证明绝大部分细胞发生了凋亡。
3、如图20所示，上述使用差数离心法获取的IEV，使用流式分析Annexin5表达的阳性率hiPSCs和hUCMSCs两者表达均在80％以上。
4、使用Nanoparticle Tracking Analysis(NTA)对两种IEVs进行表征：
4.1如图21所示，hiPSCs-IEVs的粒径约100nm，hUCMSCs-IEVs的粒径约180nm；
4.2如图22所示，IEVs的产量：21971particles/hiPSCs，886particles/hUCMSCs；
4.3如图23所示，电位hiPSC-IEVs电位约-12mV，hUCMSC-IEVs电位约-45mV。
5、使用皮肤损伤模型对两种细胞来源的IEVs进行体内修复再生功能的比较。
如图24所示，结果显示hiPSCs-IEVs能明显促进伤口的愈合，hiPSCs-IEVs相比hUCMSCs-IEVs有更强的组织修复再生功能。
鉴于可以应用所公开的发明的原理的许多可能的实施例，应当认识到，所示的实施例仅是本公开的优选示例，而不应视为限制本公开的范围。相反，本公开的范围由所附权利要求书限定。因此，我们要求保护所有落入这些权利要求的范围和精神内的发明。

Claims (15)

  1. 诱导性囊泡在制备或作为皮肤产品或皮肤附属器产品方面的用途。
  2. 如权利要求1所述的用途，其特征在于，所述产品为：药物、药物载体、食品、保健品、护肤品、或医美用品；
    优选地，所述皮肤为表皮、真皮、或皮下组织；
    优选地，所述皮肤附属器为毛、头发、汗腺、皮脂腺、指甲、或趾甲。
  3. 一种组合物，其特征在于，包括：
    诱导性囊泡和皮肤药物或护养物。
  4. 如权利要求3所述的组合物，其特征在于，所述皮肤药物与护养物被包裹在细胞外囊泡中，或
    以分开的形式存在于组合物中；
    优选地，所述组合物的剂型优选地选自冻干粉针、注射剂、片剂、胶囊或贴剂；
    优选地，所述贴剂选自微针贴剂；
    优选地，所述剂型选自注射剂或微针贴剂。
  5. 如权利要求1-4任一所述的用途/组合物，其特征在于，所述诱导性囊泡是在干细胞处于正常存活期间通过外力诱导凋亡而产生的囊泡；
    优选地，所述诱导性囊泡是通过添加星形孢菌、紫外线照射、饥饿法、或热应力法或其中一种或多种的组合诱导干细胞或干细胞凋亡产生；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、尿液间充质干细胞、口腔间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带间充质干细胞、骨膜间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、口腔间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、脐带间充质干细胞、血液干细胞或骨髓间充质干细胞中的一种或多种。
  6. 一种组合物，其特征在于，包括：
    由干细胞或干细胞胞外囊泡组成的第一剂；和，
    由抗衰老物质或Wnt信号通路正向调节剂组成的第二剂。
  7. 如权利要求6所述的组合物，其特征在于，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、尿液间充质干细胞、口腔间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带间充质干细胞、骨膜间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、口腔间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、脐带间充质干细胞、血液干细胞或骨髓间充质干细胞中的一种或多种；
    优选地，所述的干细胞胞外囊泡选自外泌体、迁移体、微泡、凋亡小体、诱导性细胞外囊泡中的一种或多种；
    优选地，所述的干细胞胞外囊泡选自诱导性细胞外囊泡；
    优选地，所述诱导性囊泡是在干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，所述抗衰老物质选自二甲双胍，白黎芦醇中的一种或多种；
    优选地，所述抗衰老物质选自二甲双胍；
    优选地，所述Wnt信号通路选自Wnt/β-catenin通路；
    优选地，所述正向调节剂选自激动剂；
    优选地，所述激动剂选自Licl、CHIR99021、SB-216763、BIO、Wnt Agonist或WAY 262611中的一种或多种；更优选Licl；
    优选地，所述正向调节剂选自包含进行运动的提示；
    优选地，所述提示选自包装，标签或说明书中的一种或多种。
  8. 如权利要求6所述的组合物，其特征在于，所述组合物中，包含干细胞或干细胞胞外囊泡与抗衰老物质的比例为(0.5-5)×10 7个：(0.05-10)mg；
    优选地，包含1×10 7-3×10 7个干细胞或干细胞胞外囊泡；和，0.1-5mg抗衰老物质；
    优选地，包含1.8×10 7-2.7×10 7个干细胞或干细胞胞外囊泡；
    优选地，包含0.2-3mg抗衰老物质。
  9. 如权利要求6所述的组合物，其特征在于，所述干细胞胞外囊泡是通过添加星形孢菌素、紫外线照射、饥饿法、或热应力法或其组合诱导间充质干细胞凋亡产生；
    优选地，所述干细胞胞外囊泡是通过添加星形孢菌素诱导间充质干细胞凋亡产生；
    优选地，所述星形孢菌素的浓度为500-1000nM；
    优选地，所述星形孢菌素的浓度为500-900nM；
    优选地，所述星形孢菌素的浓度为500nM-800nM；
    优选地，所述星形孢菌素的浓度为500nM；
    优选地，所述组合物还包含药学上可接受的载体；
    优选地，所述组合物的剂型优选地选自冻干粉针、注射剂、片剂、胶囊或贴剂；
    优选地，所述贴剂选自微针贴剂；
    优选地，所述剂型选自注射剂、微针贴剂或片剂。
  10. 如权利要求6-9任一所述的组合物在制备促毛发再生或促伤口愈合药物中的应用。
  11. 如权利要求6-9任一所述的组合物，其特征在于，所述干细胞胞外囊泡的制备方法包括如下步骤：
    1)体外培养间充质干细胞，细胞汇合80％-90％时，PBS冲洗2-5遍；
    2)将步骤1)制得的间充质干细胞加入含有500-1000nM星形孢菌素的无血清培养基，37℃孵育16-24h，收集细胞上清液；
    3)将步骤2)收集的细胞上清液在4℃下500-15000g离心5-30分钟，收集上清液；
    4)将步骤3)收集的细胞上清液在4℃下1500-2500g离心5-30分钟，收集上清液；
    5)将步骤4)收集的细胞上清液在4℃下10000-30000g离心15-60分钟，所得沉淀物为细胞外囊泡；
    优选地，还包括细胞外囊泡的清洗步骤；
    优选地，所述清洗步骤具体为：6)将步骤5)制得的细胞外囊泡用PBS重悬，在4℃下10000-30000g离心15-60分钟，所得沉淀物为细胞外囊泡。
  12. 抗衰老物质在制备干细胞服用品的伴随药物方面的用途；
    优选地，所述的干细胞服用品为干细胞或干细胞胞外囊泡；
    优选地，所述的干细胞服用品为用以摄入动物体的干细胞或干细胞胞外囊泡；优选地，所述动物为哺乳动物；优选地，所述哺乳动物为人；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、尿液间充质干细胞、口腔间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带间充质干细胞、骨膜间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、口腔间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、脐带间充质干细胞、血液干细胞或骨髓间充质干细胞中的一种或多种；
    优选地，所述的干细胞胞外囊泡选自外泌体、迁移体、微泡、凋亡小体、诱导性细胞外囊泡中的一种或多种；
    优选地，所述的干细胞胞外囊泡选自诱导性细胞外囊泡；
    优选地，所述抗衰老物质选自二甲双胍，白黎芦醇中的一种或多种；
    优选地，所述抗衰老物质选自二甲双胍。
  13. 一种干细胞服用系统，其包括：
    如权利要求12所述的干细胞服用品，和，
    伴随品；所述的伴随品选自抗衰老物质或Wnt信号通路正向调节剂中的一种或两种；
    所述的干细胞服用品包含干细胞或干细胞胞外囊泡；
    优选地，所述抗衰老物质选自二甲双胍，白黎芦醇中的一种或多种；
    优选地，所述抗衰老物质选自二甲双胍；
    优选地，所述Wnt信号通路为Wnt/β-catenin通路；
    优选地，所述正向调节剂选自激动剂；
    优选地，所述激动剂选自Licl、CHIR99021、SB-216763、BIO、Wnt Agonist或WAY 262611中的一种或多种；更优选Licl；
    优选地，所述正向调节剂选自包含进行运动的提示；
    优选地，所述提示选自包装，标签或说明书中的一种或多种。
  14. 干细胞或干细胞胞外囊泡在制备Wnt信号通路调控的干细胞或干细胞胞外囊泡代谢而促进毛发再生或伤口愈合的药物方面的用途；
    优选地，所述的药物包括干细胞或干细胞胞外囊泡；和，抗衰老物质；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、尿液间充质干细胞、口腔间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带间充质干细胞、骨膜间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、血液干细胞、骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、口腔间充质干细胞、皮肤间充质干细胞中的一种或多种；
    优选地，所述的干细胞选自诱导性多能干细胞、脐带间充质干细胞、血液干细胞或骨髓间充质干细胞中的一种或多种；
    优选地，所述的干细胞胞外囊泡选自外泌体、迁移体、微泡、凋亡小体、诱导性细胞外囊泡中的一种或多种；
    优选地，所述的干细胞胞外囊泡选自诱导性细胞外囊泡；
    优选地，所述抗衰老物质选自二甲双胍，白黎芦醇中的一种或多种；
    优选地，所述抗衰老物质选自二甲双胍。
  15. 一种皮肤或皮肤附属器疾病的治疗方法，其特征在于，包括以下步骤：
    向有需要的受试者施用如权利要求1-14任一所述的诱导性囊泡，组合物，干细胞服用系统；
    优选地，所述皮肤或皮肤附属器疾病选自创伤、脱发、瘢痕、烧烫伤或溃疡
    优选地，所述创伤选自切除伤口、切割、刺伤或穿刺伤口；
    优选地，所述皮肤为表皮、真皮、或皮下组织；
    优选地，所述皮肤附属器为毛、头发、汗腺、皮脂腺、指甲、或趾甲。
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