WO2023019913A1 - 一种诱导剂、巨噬细胞及其应用 - Google Patents

一种诱导剂、巨噬细胞及其应用 Download PDF

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WO2023019913A1
WO2023019913A1 PCT/CN2022/079118 CN2022079118W WO2023019913A1 WO 2023019913 A1 WO2023019913 A1 WO 2023019913A1 CN 2022079118 W CN2022079118 W CN 2022079118W WO 2023019913 A1 WO2023019913 A1 WO 2023019913A1
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inducer
macrophages
repairing
medium
osteopontin
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English (en)
French (fr)
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王春明
母若雨
张哲�
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澳门大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the disclosure relates to the interdisciplinary fields of immunology, cell biology and regenerative medicine, and in particular relates to an inducer, macrophages and applications thereof.
  • the lower extremity ulcer commonly known as “diabetic foot” is a skin wound that is difficult to heal or even does not heal, and has become the leading cause of lower extremity amputation.
  • diabetes foot is a skin wound that is difficult to heal or even does not heal, and has become the leading cause of lower extremity amputation.
  • Routine care and debridement are the main treatments. The treatment effect is poor and the recurrence rate is high.
  • Macrophages and their precursor monocytes, play a key role in wound repair.
  • these cells play roles such as phagocytosis of pathogens, recruitment of cells, and promotion of angiogenesis; in the later stages of repair, these cells promote fibroblast proliferation, vascular maturation, and matrix remodeling.
  • phagocytosis of pathogens in the early stage of trauma, these cells play roles such as phagocytosis of pathogens, recruitment of cells, and promotion of angiogenesis; in the later stages of repair, these cells promote fibroblast proliferation, vascular maturation, and matrix remodeling.
  • the present disclosure provides an inducer comprising: osteopontin and a cytokine selected from the group consisting of MIP-2 (macrophage inflammatory protein 2), CCL8 (monocyte chemoattractant protein 2), VEGF - at least one of B (vascular endothelial growth factor B) and M-CSF (macrophage colony stimulating factor).
  • a cytokine selected from the group consisting of MIP-2 (macrophage inflammatory protein 2), CCL8 (monocyte chemoattractant protein 2), VEGF - at least one of B (vascular endothelial growth factor B) and M-CSF (macrophage colony stimulating factor).
  • the inducer is calculated in parts by weight.
  • the mass ratio of osteopontin to MIP-2 is (80-120): (27-47) ;
  • the mass ratio of osteopontin to CCL8 is (80-120): (9-29);
  • the mass ratio of osteopontin to VEGF-B is (80-120): (5-20);
  • the mass ratio of osteopontin to M-CSF is (80-120): (1-18).
  • the inducer also includes: IL-31 (interleukin 31), IL-10 (interleukin 10), TGF- ⁇ 2 (transforming growth factor-beta 2) and bFGF (basic fibroblast growth factor ) at least one component.
  • IL-31 interleukin 31
  • IL-10 interleukin 10
  • TGF- ⁇ 2 transforming growth factor-beta 2
  • bFGF basic fibroblast growth factor
  • the weight ratio of IL-31 to osteopontin is (0.06-0.10):1.
  • the weight ratio of IL-10 to osteopontin is (0.04-0.08):1.
  • the weight ratio of TGF- ⁇ 2 to osteopontin is (0.02-0.06):1.
  • the weight ratio of bFGF to osteopontin is (0.01-0.05):1.
  • the present disclosure provides a culture medium comprising the inducer as described above.
  • the present disclosure provides a method for preparing repairing macrophages, which includes: using the aforementioned inducer to induce differentiation of monocytes or using the aforementioned medium to differentiate and culture monocytes, To obtain macrophages for tissue wound repair.
  • the final concentration of osteopontin in the medium in the inducer is 100-150 ng/mL;
  • the final concentration of MIP-2 in the medium is 32-52 ng/mL;
  • the final concentration of CCL8 in the medium is 11-31 ng/mL;
  • the final concentration of VEGF-B in the medium is 1-21 ng/mL;
  • the final concentration of M-CSF in the medium is 1-20 ng/mL;
  • the final concentration of IL-31 in the culture medium is 1-20 ng/mL;
  • the final concentration of IL-10 in the medium is 1-15 ng/mL;
  • the final concentration of TGF- ⁇ 2 in the medium is 1-10 ng/mL;
  • the final concentration of bFGF in the culture medium is 1-6 ng/mL.
  • the culture time for inducing differentiation is 1-3 days.
  • the present disclosure provides a repairing macrophage, which is prepared by the above-mentioned preparation method of macrophage.
  • the present disclosure provides an application of the aforementioned inducer, or the aforementioned medium, or the aforementioned repairing macrophages in the preparation of a medicament for tissue wound repair.
  • the repairing of tissue wounds includes: repairing tissue wounds of diabetic patients.
  • the present disclosure provides an application of the above-mentioned inducer, or the above-mentioned medium, or the above-mentioned macrophage in the preparation of a drug for promoting angiogenesis.
  • the present disclosure provides a medicament for repairing tissue wounds, which includes the aforementioned macrophages.
  • the present disclosure provides the use of the repairing macrophages or the drug in repairing tissue wounds.
  • the present disclosure provides the use of the reparative macrophage or the drug in promoting angiogenesis.
  • the present disclosure provides the use of the reparative macrophages, or the medicament, in repairing tissue wounds.
  • the repairing of tissue wounds includes: repairing tissue wounds of diabetic patients.
  • the present disclosure provides the use of the reparative macrophage, or the drug, in promoting angiogenesis.
  • the present disclosure provides a method of tissue wound repair in a subject, comprising:
  • a therapeutically effective amount of the reparative macrophages, or the drug is administered to a subject in need thereof.
  • the tissue wound repair includes: tissue wound repair in diabetic patients.
  • the present disclosure provides a method of promoting angiogenesis in a subject comprising:
  • a therapeutically effective amount of the reparative macrophages, or the drug is administered to a subject in need thereof.
  • Fig. 1 is the concentration ratio of OPN and other cytokines in embodiment 12;
  • Fig. 2 is the morphological diagram (compared with uninduced M0) of 11 kinds of repaired macrophages obtained by inducing human peripheral blood mononuclear cells through the steps of Example 12-14 in Test Example 1;
  • Figure 3 is the test example 2 main gene expression
  • Figure 4 is the test example 4 Can promote angiogenesis in vitro
  • Fig. 5 is in test example 5 Promote fibroblast activation
  • Figure 6 is the test example 6 Promote skin wound healing in diabetic mice in vivo
  • Fig. 7 is test example 6 Can promote the collagen secretion of fibroblasts in wounds of diabetic mice;
  • Fig. 8 is test example 6 It can promote angiogenesis and maturation in wounds of diabetic mice.
  • osteopontin also known as osteopontin, English abbreviation OPN
  • OPN osteopontin
  • Macrophages (English Macrophages, abbreviated as ) is a tissue-located white blood cell derived from monocytes, which in turn are derived from precursor cells in the bone marrow.
  • the term "subject” refers to a vertebrate, optionally a mammal, optionally a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • therapeutically effective amount refers to an amount of an agent sufficient to produce a beneficial or desired result.
  • Therapeutically effective amount may vary according to one or more of the following: the subject being treated and the condition of the disease, the weight and age of the subject, the severity of the condition, the mode of administration, etc., which can be determined by Easily determined by one of ordinary skill in the art. Dosage may vary depending on one or more of: the specific agent chosen, the dosing regimen followed, whether it is co-administered with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the agent .
  • the present disclosure provides an inducer, which includes: osteopontin and cytokines, the cytokines are selected from at least one of MIP-2, CCL8, VEGF-B and M-CSF.
  • the inventors found that the combination of OPN and cytokines can be used to induce differentiation of monocytes, and macrophages (osteopontin-macrophages, osteopontin-macrophages, ). Compared to normal macrophages, It has a strong repair function and can promote wound healing and tissue repair and regeneration.
  • the mass ratio of osteopontin to MIP-2 is (80 ⁇ 120):(27 ⁇ 47), for example, the mass ratio of osteopontin to MIP-2 can be (85 ⁇ 115): (30 ⁇ 45), (90 ⁇ 110): (32 ⁇ 42), (95 ⁇ 105): (35 ⁇ 40), such as (98 ⁇ 102): (35 ⁇ 39), (99 ⁇ 101 ): (36 ⁇ 38);
  • the mass ratio of osteopontin to CCL8 is (80 ⁇ 120):(9 ⁇ 29), for example, the mass ratio of osteopontin to CCL8 can be (85 ⁇ 115):(10 ⁇ 25), (90 ⁇ 110): (12 ⁇ 24), (95 ⁇ 105): (13 ⁇ 23), such as (97 ⁇ 105): (16 ⁇ 23), (99 ⁇ 101): (18 ⁇ 20 );
  • the mass ratio of osteopontin to VEGF-B is (80 ⁇ 120):(5 ⁇ 20), for example, the mass ratio of osteopontin to VEGF-B can be (85 ⁇ 115): (7 ⁇ 15), (90 ⁇ 110): (8 ⁇ 12), (95 ⁇ 105): (8 ⁇ 12), such as (98 ⁇ 102): (8 ⁇ 12), (99 ⁇ 101 ): (9 ⁇ 11);
  • the mass ratio of osteopontin to M-CSF is (80 ⁇ 120):(1 ⁇ 18), for example, the mass ratio of osteopontin to M-CSF can be (85 ⁇ 115): (5 ⁇ 15), (90 ⁇ 110): (6 ⁇ 12), (95 ⁇ 105): (6 ⁇ 12), such as (98 ⁇ 102): (6 ⁇ 10), (99 ⁇ 101 ): (7 ⁇ 9).
  • the inducer includes the following components in parts by weight: 80-120 parts of osteopontin, 27-47 parts of MIP-2, 9-29 parts of CCL8, 5-20 parts of VEGF -B and 1-18 parts of M-CSF. Within this definition, the inducer induces the differentiated It has better repairing effect.
  • the parts by weight of osteopontin can be 80 parts, 85 parts, 90 parts, 95 parts, 100 parts, 105 parts, 110 parts, 115 parts or 120 parts; MIP-2 The number of copies can be 27, 30, 35, 40, 45, or 47; the number of copies of CCL8 can be 9, 10, 15, 20, 25, or 29; VEGF-B The number of parts of M-CSF can be 5 parts, 10 parts, 15 parts or 20 parts; the number of parts of M-CSF can be 1 part, 5 parts, 10 parts, 15 parts or 18 parts.
  • the inducer further includes: at least one component of IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • the weight ratio of IL-31 to osteopontin is (0.06-0.10):1. In some embodiments, the weight ratio of IL-31 to osteopontin may be 0.06:1, 0.08:1 or 0.10:1.
  • the weight ratio of IL-10 to osteopontin is (0.04-0.08):1. In some embodiments, the weight ratio of IL-10 to osteopontin may be 0.04:1, 0.06:1 or 0.08:1.
  • the weight ratio of TGF- ⁇ 2 to osteopontin is (0.02-0.06):1. In some embodiments, the weight ratio of TGF- ⁇ 2 to osteopontin may be 0.02:1, 0.03:1, 0.04:1, 0.05:1 or 0.06:1.
  • the weight ratio of bFGF to osteopontin is (0.01-0.05):1. In some embodiments, the weight ratio of bFGF to osteopontin may be 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1 or 0.06:1.
  • the inducer contains these cytokines and is added according to the above ratio, it can induce differentiation into cells with better repair ability.
  • An embodiment of the present disclosure provides a culture medium, which includes the inducer as described in any of the foregoing embodiments.
  • the medium includes a medium matrix and an inducer.
  • the medium matrix can be selected from existing known medium for culturing cells, such as RPMI 1640, DMEM, IMDM.
  • the culture medium is RPMI-1640 culture fluid.
  • An embodiment of the present disclosure provides a method for preparing repairing macrophages, which includes: using the inducer as described in the foregoing embodiments to induce differentiation of the macrophages to be induced or using the medium as described in the foregoing embodiments
  • the macrophages to be induced were differentiated and cultured to obtain repairing macrophages.
  • the "macrophages to be induced” herein may be peripheral blood macrophages differentiated from monocytes.
  • Monocytes are derived from precursor cells in the bone marrow.
  • the final concentration of osteopontin in the culture medium in the inducer is 100-150 ng/mL. In this concentration range, the induced repairing macrophages have better repairing effect.
  • the final concentration of osteopontin in the culture medium can be 100ng/mL, 105ng/mL, 110ng/mL, 115ng/mL, 120ng/mL, 125ng/mL, 130ng/mL, 135ng/mL, 140ng/mL mL, 145ng/mL or 150ng/mL.
  • the final concentration of MIP-2 in the culture medium is 32-52 ng/mL.
  • the final concentration of MIP-2 in the medium can be 32ng/mL, 34ng/mL, 36ng/mL, 38ng/mL, 40ng/mL, 42ng/mL, 44ng/mL, 46ng/mL, 48ng/mL mL, 50ng/mL or 52ng/mL.
  • the final concentration of CCL8 in the culture medium is 11-31 ng/mL.
  • the final concentration of CCL8 in the medium can be 11 ng/mL, 13 ng/mL, 15 ng/mL, 17 ng/mL, 19 ng/mL, 21 ng/mL, 23 ng/mL, 25 ng/mL, 27 ng/mL, 29ng/mL or 31ng/mL.
  • the final concentration of VEGF-B in the culture medium is 1-21 ng/mL.
  • the final concentration of CCL8 in the medium can be 1 ng/mL, 3 ng/mL, 5 ng/mL, 7 ng/mL, 9 ng/mL, 11 ng/mL, 13 ng/mL, 15 ng/mL, 17 ng/mL, 19ng/mL or 21ng/mL.
  • the final concentration of M-CSF in the medium is 1-20 ng/mL.
  • the final concentration of M-CSF in the medium can be 1 ng/mL, 3 ng/mL, 5 ng/mL, 7 ng/mL, 9 ng/mL, 11 ng/mL, 13 ng/mL, 15 ng/mL, 17 ng/mL mL, 19ng/mL or 20ng/mL.
  • the final concentration of IL-31 in the culture medium is 1-20 ng/mL.
  • the final concentration of IL-31 in the medium can be 1 ng/mL, 3 ng/mL, 5 ng/mL, 7 ng/mL, 9 ng/mL, 11 ng/mL, 13 ng/mL, 15 ng/mL, 17 ng/mL mL, 19ng/mL or 20ng/mL.
  • the final concentration of IL-10 in the culture medium is 1-15 ng/mL.
  • the final concentration of IL-10 in the medium can be 1 ng/mL, 3 ng/mL, 5 ng/mL, 7 ng/mL, 9 ng/mL, 11 ng/mL, 13 ng/mL or 15 ng/mL.
  • the final concentration of TGF- ⁇ 2 in the medium is 1 ⁇ 10 ng/mL.
  • the final concentration of TGF- ⁇ 2 in the culture medium can be 1 ng/mL, 3 ng/mL, 5 ng/mL, 7 ng/mL, 9 ng/mL or 10 ng/mL.
  • the final concentration of bFGF in the culture medium is 1-6 ng/mL.
  • the final concentration of bFGF in the medium can be 1 ng/mL, 2 ng/mL, 4 ng/mL or 6 ng/mL.
  • the culture time for inducing differentiation is 1-3 days, optionally 2 days.
  • the embodiments of the present disclosure also provide a macrophage, which is prepared by the preparation method of the macrophage described in any of the foregoing embodiments.
  • the repairing macrophages prepared by the above preparation method are derived from human peripheral blood mononuclear cells, which highly express CD86, CD163, CD206 and other surface biomarkers after induction, and highly secrete IL-10, IL-16, TGF- ⁇ Cytokines that promote immunosuppression, high secretion of cytokines such as Amphiregulin, IGFBP-3, Ang that promote angiogenesis, high secretion of cytokines such as VEGF-A, PDGF-B, bFGF that promote cell proliferation and matrix secretion, in vitro culture has Proliferation ability (ki67 positive); and the phenotype is relatively stable, partial anti-inflammatory type.
  • the preparation method further includes: inducing and culturing monocytes to obtain macrophages (macrophages to be induced).
  • Embodiments of the present disclosure also provide the inducer as described in any of the foregoing embodiments, or the medium as described in any of the foregoing embodiments, or the preparation of macrophages as described in any of the foregoing embodiments in the preparation of drugs for tissue wound repair in the application.
  • the repairing of tissue wounds includes: repairing tissue wounds of diabetic patients.
  • the macrophages provided by the disclosure can effectively promote the repair of tissue wounds in diabetic patients, and provide a new approach for difficult-to-repair wounds.
  • Embodiments of the present disclosure also provide the inducer as described in any of the foregoing embodiments, or the medium as described in any of the foregoing embodiments, or the preparation of macrophages as described in any of the foregoing embodiments for promoting angiogenesis application in medicine.
  • Embodiments of the present disclosure also provide a medicament for repairing tissue wounds, which includes macrophages as described in any of the foregoing embodiments.
  • the drug may also include other known preparations for tissue wound repair.
  • Embodiments of the present disclosure also provide repairing macrophages or the use of drugs in repairing tissue wounds.
  • Embodiments of the present disclosure also provide repairing macrophages or the use of the drug in promoting angiogenesis.
  • Embodiments of the present disclosure also provide repairing macrophages, or the medicament, for repairing tissue wounds.
  • the repairing of tissue wounds includes: repairing tissue wounds of diabetic patients.
  • the embodiment of the present disclosure also provides the use of the repairing macrophage or the drug for promoting angiogenesis.
  • Embodiments of the present disclosure also provide a method for tissue wound repair in a subject, comprising:
  • a therapeutically effective amount of reparative macrophages, or a drug, is administered to a subject in need thereof.
  • tissue wound repair comprises: tissue wound repair in diabetic patients.
  • Embodiments of the present disclosure also provide a method of promoting angiogenesis in a subject, comprising:
  • a therapeutically effective amount of reparative macrophages, or a drug, is administered to a subject in need thereof.
  • the present disclosure uses a composition composed of osteopontin OPN and other cytokines to culture and induce monocytes, which can form macrophages with a strong repair function. These cells can be applied to tissue wound repair and promote chronic refractory wounds Healing and tissue regeneration, has a good repair effect, and is suitable for popularization and application.
  • a preparation of an inducer which includes: OPN (the name of the gene expressing OPN is SPP1), MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • OPN:MIP-2:CCL8:VEGF-B:M-CSF 1:0.37:0.19:0.1:0.08 (mass ratio).
  • OPN:IL-31:IL-10:TGF- ⁇ 2:bFGF 1:0.08:0.06:0.04:0.03 (mass ratio).
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • an inducer is roughly the same as that in Example 1, except that the inducer does not include: MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, MIP-2, VEGF-B, M-CSF, IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, M-CSF, IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, IL-31, IL-10, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-10, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, TGF- ⁇ 2 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10 and bFGF.
  • the preparation of an inducer is roughly the same as that in Example 1, except that the inducer does not include: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10 and TGF- ⁇ 2.
  • a method for preparing repairing macrophages comprising the following steps.
  • PBMC Human peripheral blood mononuclear cells
  • the monocytes were induced in vitro with RPMI-1640 oxygen solution containing 20ng/ml human M-CSF for 7 days, and the monocytes were differentiated into adult peripheral blood macrophages (uninduced macrophages, denoted as M0).
  • the final concentration of each component in the inducer in RPMI-1640 culture liquid is as follows (with reference to Figure 1) :
  • the final concentration of osteopontin is 113.215ng/mL
  • the final concentration of MIP-2 is 42.513ng/mL
  • the final concentration of CCL8 is 21.894ng/mL
  • the final concentration of VEGF-B is 11.799ng/mL
  • M-CSF The final concentration of IL-31 was 10.157ng/mL
  • the final concentration of IL-31 was 9.102ng/mL
  • the final concentration of IL-10 was 7.372ng/mL
  • the final concentration of TGF- ⁇ 2 was 5.211ng/mL
  • the final concentration of bFGF was 3.522ng/mL.
  • Example 12 Refer to the preparation method of Example 12 to induce the differentiation of human peripheral blood macrophages, with the difference that: human peripheral blood macrophages were stimulated with 40 ng/ml human IL-4 and 20 ng/ml human IL-13 for 48 hours to obtain M2 macrophages (marked as M2).
  • a method for preparing repairing macrophages is roughly the same as that in Example 12, except that the inducer is different, and the difference is as follows.
  • Human peripheral blood macrophages were cultured externally for 2 days with RPMI-1640 medium containing the inducer provided in Example 2. Among them, the final concentration of osteopontin was 113.215ng/mL, the final concentration of MIP-2 was 42.513ng/mL, the final concentration of CCL8 was 21.894ng/mL, the final concentration of VEGF-B was 11.799ng/mL, M- The final concentration of CSF was 10.157 ng/mL.
  • the induced repair macrophages are denoted as The cell morphology is shown in Figure 2. Compared with uninduced macrophages, OPN-induced macrophages have a slightly longer diameter and higher density.
  • the preparation method of other repairing macrophages is roughly the same as that of Example 12, the difference lies in the difference of the inducer, and the differences are as follows.
  • Human peripheral blood macrophages were cultured in vitro for 2 days with RPMI-1640 medium containing the inducers provided in Examples 3-11.
  • the final concentration of osteopontin was 113.215ng/mL
  • the final concentration of MIP-2 was 42.513ng/mL
  • the final concentration of CCL8 was 21.894ng/mL
  • the final concentration of VEGF-B was 11.799ng/mL
  • M- The final concentration of CSF was 10.157 ng/mL
  • the final concentration of IL-31 was 9.102 ng/mL
  • the final concentration of IL-10 was 7.372 ng/mL
  • the final concentration of TGF- ⁇ 2 was 5.211 ng/mL
  • the final concentration of bFGF It was 3.522ng/mL.
  • the repair-type macrophages induced by Example 3 are marked as +OPN
  • the repair-type macrophages induced by Example 4 are marked as +MIP-2
  • the repair-type macrophages induced by Example 5 are marked as +CCL8
  • the repair type macrophage induced by Example 6 is marked as +VEGF-B
  • the repair type macrophage induced by Example 7 is marked as +M-CSF
  • the repair type macrophage induced by Example 8 is marked as + IL-31
  • the repair type macrophage induced by Example 9 is recorded as +IL-10
  • the repair type macrophage induced by Example 10 is recorded as +TGF- ⁇
  • the repair type macrophage induced by Example 11 is recorded as +IL-10 Cells were counted as +bFGF.
  • uninduced macrophages were recorded as M0.
  • the morphology of all repaired macrophages obtained is shown in Figure 2.
  • Example 12 Based on the preparation method of Example 12, obtain The mRNA was extracted by Trizol method and analyzed by RT-qPCR method The main gene expression, the results are shown in Figure 3, The surface markers of M1 and M2 were expressed simultaneously (Fig. 3). Moreover, genes related to promoting angiogenesis and promoting stromal cell proliferation were also highly expressed.
  • Test example 3 collection of conditional medium (Conditional medium (CM))
  • Example 12 Prepared by Example 12 respectively M0 macrophages, M1 macrophages and M2 macrophages were cultured in vitro under the same conditions, and then the culture supernatants were collected respectively. The obtained supernatant is The culture supernatants of other control macrophages were respectively marked as M0-CM, M1-CM and M2-CM. Specifically, 10 7 of the above-mentioned four kinds of cells were spread in a 10 cm culture dish, and 10 milliliters of 1640 complete medium were added to culture them in a cell culture incubator for 36-50 hours. 7.2 to 7.4, and 5% carbon dioxide. Then the culture supernatants were collected separately.
  • Test example 4 Induction of angiogenesis in vitro
  • Example 12 Based on the preparation method of Example 12, obtain Spread the Matrigel on the ⁇ -Slide plate.
  • human vascular endothelial cells were planted on Matrigel, and M0-CM, M1-CM, M2-CM and Culture, normal medium (Normal Medium) is used as negative control, and VEGF is positive control, as shown in accompanying drawing 4, with respect to other cells, It has a strong ability to promote angiogenesis.
  • Test example 5 In Vitro Tests for Promoting Fibroblast Activation
  • Test example 6 In vivo promotion of skin wound healing in diabetic mice
  • this test example uses it in the wound model of immunodeficiency diabetic mice.
  • the mouse was anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg), and a full-thickness excision wound with a diameter of 6 mm was created on its back.
  • Will be 3 x 10 5 Plant on the electrospinning of gelatin weaving, then transplant in the wound, transplant other control macrophages with the same method, will be compared with The same number of cells M0, M1 and M2 (obtained in Example 12) were planted on the gelatin-woven electrospinning; the blank control group was: transplanted blank electrospinning was the blank control group.
  • the normal mouse group is: the non-diabetic mice are recorded as the normal mouse group, as the positive control group....
  • the inducer provided by the present disclosure uses a composition composed of osteopontin OPN and other cytokines to culture and induce monocytes, which can form macrophages with strong repair function. These cells can be applied to tissue wound repair and promote The healing and tissue regeneration of chronic refractory wounds has a good repair effect, especially can effectively promote the repair of tissue wounds in diabetic patients, and provides a new way for refractory wounds, which has a wide range of application values.

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Abstract

提供了一种诱导剂、巨噬细胞及其应用。该诱导剂使用骨桥素OPN及其他细胞因子组成的组合物对单核细胞进行培养诱导，能够形成具有较强修复功能的巨噬细胞，用于组织创面修复、创伤愈合和组织再生。

Description

一种诱导剂、巨噬细胞及其应用
相关申请的交叉引用
本公开要求于2021年08月18日提交中国专利局的申请号为“202110947887.5”名称为“一种诱导剂、巨噬细胞及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及免疫学、细胞生物学和再生医学交叉领域，具体而言，涉及一种诱导剂、巨噬细胞及其应用。
背景技术
糖尿病及其并发症严重危害全球人类健康。其中，俗称“糖尿病足”的下肢溃疡，是一种难愈合甚至不愈合的皮肤创伤，已成为下肢截肢的首要原因。然而，对于糖尿病足目前尚无针对性治疗策略或药物，以常规护理和清创为主，治疗效果不佳，复发率高。
在创伤修复过程中，作为固有免疫系统核心成员的巨噬细胞(及其前体单核细胞)，发挥着关键作用。例如，在创伤早期，这类细胞发挥吞噬病原体、招募细胞、促进血管新生等作用；在修复后期，这类细胞促进成纤维细胞增殖、血管成熟、基质重塑。大量病理学研究表明，在糖尿病创伤局部，巨噬细胞功能的紊乱直接导致该类创伤愈合受阻。在体外对自体来源单核/巨噬细胞的功能进行塑造后移植至创面部位，有望成为促进创面修复的治疗策略。
目前，国际国内尚无能够有效修复临床上的巨噬细胞。
发明内容
本公开提供了一种诱导剂，其包括：骨桥素和细胞因子，所述细胞因子选自MIP-2(巨噬细胞炎性蛋白2)、CCL8(单核细胞趋化蛋白2)、VEGF-B(血管内皮细胞生长因子B)和M-CSF(巨噬细胞集落刺激因子)中的至少一种。
在一些实施方式中，所述诱导剂，按重量份数计，当所述诱导剂包括MIP-2时，骨桥素与MIP-2的质量比为(80～120)：(27～47)；
当所述诱导剂包括CCL8时，骨桥素与CCL8的质量比为(80～120)：(9～29)；
当所述诱导剂包括VEGF-B时，骨桥素与VEGF-B的质量比为(80～120)：(5～20)；
当所述诱导剂包括M-CSF时，骨桥素与M-CSF的质量比为(80～120)：(1～18)。
在一些实施方式中，所述诱导剂还包括：IL-31(白介素31)、IL-10(白介素10)、TGF-β2(转化生长因子-beta 2)和bFGF(碱性成纤维细胞生长因子)中的至少一种组分。
在一些实施方式中，当所述诱导剂包括IL-31时，IL-31与骨桥素的重量比为(0.06～0.10)：1。
在一些实施方式中，当所述诱导剂包括IL-10时，IL-10与骨桥素的重量比为(0.04～0.08)：1。
在一些实施方式中，当所述诱导剂包括TGF-β2时，TGF-β2与骨桥素的重量比为(0.02～0.06)：1。
在一些实施方式中，当所述诱导剂包括bFGF时，bFGF与骨桥素的重量比为(0.01～0.05)：1。
本公开提供了一种培养基，其包括如前所述的诱导剂。
本公开提供了一种修复型巨噬细胞的制备方法，其包括：采用如前述所述的诱导剂对单核细胞进行诱导分化或采用如前述所述的培养基对单核细胞进行分化培养，以获得用于组织创伤修复的巨噬细胞。
在一些实施方式中，当采用所述培养基对单核细胞进行分化培养时，所述诱导剂中骨桥素在所述培养基中的终浓度为100～150ng/mL；
当所述诱导剂包括MIP-2时，MIP-2在所述培养基中的终浓度为32～52ng/mL；
当所述诱导剂包括CCL8时，CCL8在所述培养基中的终浓度为11～31ng/mL；
当所述诱导剂包括VEGF-B时，VEGF-B在所述培养基中的终浓度为1～21ng/mL；
当所述诱导剂包括M-CSF时，M-CSF在所述培养基中的终浓度为1～20ng/mL；
当所述诱导剂包括IL-31时，IL-31在所述培养基中的终浓度为1～20ng/mL；
当所述诱导剂包括IL-10时，IL-10在所述培养基中的终浓度为1～15ng/mL；
当所述诱导剂包括TGF-β2时，TGF-β2在所述培养基中的终浓度为1～10ng/mL；
当所述诱导剂包括bFGF时，bFGF在所述培养基中的终浓度为1～6ng/mL。
在一些实施方式中，所述诱导分化的培养时间为1～3天。
本公开提供了一种修复型巨噬细胞，其由前述所述的巨噬细胞的制备方法制备获得。
本公开提供了如前述所述的诱导剂、或如前述所述的培养基，或如前述所述的修复 型巨噬细胞在制备用于组织创伤修复的药物中的应用。
在一些实施方式中，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
本公开提供了如前述所述的诱导剂、或如前述所述的培养基，或如前述所述的巨噬细胞在制备用于促血管新生的药物中的应用。
本公开提供了一种用于组织创伤修复的药物，其包括如前述所述的巨噬细胞。
本公开提供所述修复型巨噬细胞、或所述药物在用于组织创伤修复中的用途。
本公开提供所述修复型巨噬细胞、或所述药物在用于促血管新生中的用途。
本公开提供所述修复型巨噬细胞、或所述药物，用于组织创伤修复中的用途。
在一些实施方式中，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
本公开提供所述修复型巨噬细胞、或所述药物，用于促血管新生中的用途。
本公开提供一种受试者中组织创伤修复的方法，包括：
向有此需要的受试者给药治疗有效量的所述修复型巨噬细胞、或所述药物。
在一些实施方式中，所述组织创伤修复包括：糖尿病患者的组织创伤修复。
本公开提供一种促进受试者中血管新生的方法，包括：
向有此需要的受试者给药治疗有效量的所述修复型巨噬细胞、或所述药物。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例12中OPN及其他细胞因子的浓度配比；
图2为试验例1中通过实施例12-14步骤诱导人外周血单核细胞所得的11种修复型巨噬细胞的形态图(与未诱导M0对比)；
图3为试验例2中
Figure PCTCN2022079118-appb-000001
的主要基因表达；
图4为试验例4中
Figure PCTCN2022079118-appb-000002
可体外促进血管新生；
图5为试验例5中
Figure PCTCN2022079118-appb-000003
促进成纤维细胞活化；
图6为试验例6中
Figure PCTCN2022079118-appb-000004
在体内促进糖尿病小鼠皮肤创面愈合；
图7为试验例6中
Figure PCTCN2022079118-appb-000005
可促进糖尿病小鼠伤口处成纤维细胞的胶原分泌；
图8为试验例6中
Figure PCTCN2022079118-appb-000006
可促进糖尿病小鼠伤口处血管新生及成熟。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
术语定义：
如本文所用，术语“骨桥素”，(又称骨桥蛋白，英文osteopontin，英文缩写为OPN)是一种糖基化蛋白，作为一种“非胶原”的细胞外基质成分，广泛存在与多种细胞外基质中。
如本文所用，术语“巨噬细胞”(英文Macrophages，缩写为
Figure PCTCN2022079118-appb-000007
)是一种位于组织内的白血球，源自单核细胞，而单核细胞又来源于骨髓中的前体细胞。
如本文所用，术语“受试者”是指脊椎动物，可选地是哺乳动物，可选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
如本文所用，术语“治疗有效量”是指足以产生有益的或期望的结果的药剂的量。治疗有效量可能会根据：接受治疗的受试者和疾病状况、受试者的体重和年龄、疾病状况的严重程度、给药的方式等中的一个或多个因素的不同而变化，其可由本领域普通技术人员容易地确定。剂量可能会根据：所选择的特定药剂、遵循的剂量方案、是否与其他化合物联合给药、给药时间、待成像的组织以及携带药剂的物理递送系统中的一个或多个因素的不同而变化。
本公开提供了一种诱导剂，其包括：骨桥素和细胞因子，所述细胞因子选自MIP-2、CCL8、VEGF-B和M-CSF中的至少一种。
经一系列创造性劳动，发明人发现将OPN和细胞因子组合后用于单核细胞的诱导分化，能获得巨噬细胞(骨桥素-巨噬细胞，
Figure PCTCN2022079118-appb-000008
)。相对于通常的巨噬细胞而言，
Figure PCTCN2022079118-appb-000009
具有较强的修复功能，能够促进创面愈合和组织修复再生。
当所述诱导剂包括MIP-2时，骨桥素与MIP-2的质量比为(80～120)：(27～47)，例如骨桥素与MIP-2的质量比可以为(85～115)：(30～45)、(90～110)：(32～42)、(95～105)：(35～40)，诸如(98～102)：(35～39)、(99～101)：(36～38)；
当所述诱导剂包括CCL8时，骨桥素与CCL8的质量比为(80～120)：(9～29)，例如骨桥素与CCL8的质量比可以为(85～115)：(10～25)、(90～110)：(12～24)、(95～105)：(13～23)，诸如(97～105)：(16～23)、(99～101)：(18～20)；
当所述诱导剂包括VEGF-B时，骨桥素与VEGF-B的质量比为(80～120)：(5～20)，例如骨桥素与VEGF-B的质量比可以为(85～115)：(7～15)、(90～110)：(8～12)、(95～105)：(8～12)，诸如(98～102)：(8～12)、(99～101)：(9～11)；
当所述诱导剂包括M-CSF时，骨桥素与M-CSF的质量比为(80～120)：(1～18)，例如骨桥素与M-CSF的质量比可以为(85～115)：(5～15)、(90～110)：(6～12)、(95～105)：(6～12)，诸如(98～102)：(6～10)、(99～101)：(7～9)。
在一些典型的实施方式中，按重量份数计，所述诱导剂包括以下组分：80～120份骨桥素，27～47份MIP-2，9～29份CCL8，5～20份VEGF-B以及1～18份M-CSF。在该限定下，诱导剂诱导分化的
Figure PCTCN2022079118-appb-000010
具有更好的修复功效。
在一些实施方式中，按重量份数计，骨桥素的份数可以为80份、85份、90份、95份、100份、105份、110份、115份或120份；MIP-2的份数可以为27份、30份、35份、40份、45份或47份；CCL8的份数可以为9份、10份、15份、20份、25份或29份；VEGF-B的份数可以为5份、10份、15份或20份；M-CSF的份数可以为1份、5份、10份、15份或18份。
在一些典型的实施方式中，所述诱导剂还包括：IL-31、IL-10、TGF-β2和bFGF中的至少一种组分。
在一些典型的实施方式中，当所述诱导剂包括IL-31时，IL-31与骨桥素的重量比为(0.06～0.10)：1。在一些实施方式中，IL-31与骨桥素的重量比可以为0.06：1、0.08：1或0.10：1。
在一些典型的实施方式中，当所述诱导剂包括IL-10时，IL-10与骨桥素的重量比为(0.04～0.08)：1。在一些实施方式中，IL-10与骨桥素的重量比可以为0.04：1、0.06：1或0.08：1。
在一些典型的实施方式中，当所述诱导剂包括TGF-β2时，TGF-β2与骨桥素的重量比为(0.02～0.06)：1。在一些实施方式中，TGF-β2与骨桥素的重量比可以为0.02：1、0.03：1、0.04：1、0.05：1或0.06：1。
在一些典型的实施方式中，当所述诱导剂包括bFGF时，bFGF与骨桥素的重量比为(0.01～0.05)：1。在一些实施方式中，bFGF与骨桥素的重量比可以为0.01：1、0.02： 1、0.03：1、0.04：1、0.05：1或0.06：1。
当诱导剂包含由这些细胞因子且按照上述配比进行添加时，能够诱导分化出修复能力更好的
Figure PCTCN2022079118-appb-000011
本公开实施方式提供了一种培养基，其包括如前述任意实施方式所述的诱导剂。
可选地，所述培养基包括培养基基质以及诱导剂。培养基基质可以选自现有已知用于培养细胞的培养基，诸如RPMI 1640、DMEM、IMDM。
在一些典型的实施方式中，所述培养基基质为RPMI-1640培养液。
本公开实施方式提供了一种修复型巨噬细胞的制备方法，其包括：采用如前述实施方式所述的诱导剂对待诱导的巨噬细胞进行诱导分化或采用如前述实施方式所述的培养基对待诱导的巨噬细胞进行分化培养，以获得修复型巨噬细胞。
本文中的“待诱导的巨噬细胞”可以为由单核细胞分化的外周血巨噬细胞。单核细胞来源于骨髓中的前体细胞。
在一些典型的实施方式中，当采用所述培养基对待诱导的巨噬细胞进行分化培养时，所述诱导剂中骨桥素在所述培养基中的终浓度为100～150ng/mL。该浓度范围内，诱导的修复型巨噬细胞具有更好的修复功效。
例如，骨桥素在所述培养基中的终浓度可以为100ng/mL、105ng/mL、110ng/mL、115ng/mL、120ng/mL、125ng/mL、130ng/mL、135ng/mL、140ng/mL、145ng/mL或150ng/mL。
当所述诱导剂包括MIP-2时，MIP-2在所述培养基中的终浓度为32～52ng/mL。例如，MIP-2在所述培养基中的终浓度可以为32ng/mL、34ng/mL、36ng/mL、38ng/mL、40ng/mL、42ng/mL、44ng/mL、46ng/mL、48ng/mL、50ng/mL或52ng/mL。
当所述诱导剂包括CCL8时，CCL8在所述培养基中的终浓度为11～31ng/mL。例如，CCL8在所述培养基中的终浓度可以为11ng/mL、13ng/mL、15ng/mL、17ng/mL、19ng/mL、21ng/mL、23ng/mL、25ng/mL、27ng/mL、29ng/mL或31ng/mL。
当所述诱导剂包括VEGF-B时，VEGF-B在所述培养基中的终浓度为1～21ng/mL。例如，CCL8在所述培养基中的终浓度可以为1ng/mL、3ng/mL、5ng/mL、7ng/mL、9ng/mL、11ng/mL、13ng/mL、15ng/mL、17ng/mL、19ng/mL或21ng/mL。
当所述诱导剂包括M-CSF时，M-CSF在所述培养基中的终浓度为1～20ng/mL。例如，M-CSF在所述培养基中的终浓度可以为1ng/mL、3ng/mL、5ng/mL、7ng/mL、9ng/mL、11ng/mL、13ng/mL、15ng/mL、17ng/mL、19ng/mL或20ng/mL。
当所述诱导剂包括IL-31时，IL-31在所述培养基中的终浓度为1～20ng/mL。例如，IL-31在所述培养基中的终浓度可以为1ng/mL、3ng/mL、5ng/mL、7ng/mL、9ng/mL、11ng/mL、13ng/mL、15ng/mL、17ng/mL、19ng/mL或20ng/mL。
当所述诱导剂包括IL-10时，IL-10在所述培养基中的终浓度为1～15ng/mL。例如，IL-10在所述培养基中的终浓度可以为1ng/mL、3ng/mL、5ng/mL、7ng/mL、9ng/mL、11ng/mL、13ng/mL或15ng/mL。
当所述诱导剂包括TGF-β2时，TGF-β2在所述培养基中的终浓度为1～10ng/mL。例如，TGF-β2在所述培养基中的终浓度可以为1ng/mL、3ng/mL、5ng/mL、7ng/mL、9ng/mL或10ng/mL。
当所述诱导剂包括bFGF时，bFGF在所述培养基中的终浓度为1～6ng/mL。例如，bFGF在所述培养基中的终浓度可以为1ng/mL、2ng/mL、4ng/mL或6ng/mL。
在一些典型的实施方式中，所述诱导分化的培养时间为1～3天，可选地为2天。
本公开实施方式还提供了一种巨噬细胞，其由前述任意实施方式所述的巨噬细胞的制备方法制备获得。
由上述制备方法制备获的修复型巨噬细胞来源于人外周血单核细胞，经诱导后高表达CD86，CD163,CD206等表面生物标志物，高分泌IL-10，IL-16，TGF-β等促进免疫抑制的细胞因子，高分泌Amphiregulin，IGFBP-3，Ang等促进血管新生的细胞因子，高分泌VEGF-A，PDGF-B，bFGF等促进细胞增殖和基质分泌的细胞因子，体外培养具有增殖能力(ki67阳性)；且表型较为稳定，偏抑炎型。
可选地，所述制备方法还包括：对单核细胞进行诱导培养，以获得巨噬细胞(待诱导的巨噬细胞)。
本公开实施方式还提供如前述任意实施方式所述的诱导剂、或如前述任意实施方式所述的培养基，或如前述任意实施方式所述的巨噬细胞在制备用于组织创伤修复的药物中的应用。
在一些典型的实施方式中，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
糖尿病患者由于体内胰岛素分泌调节功能不正常，影响了机体的糖代谢，使糖的利用率降低，组织的再生修复功能受累，从而造成伤口延迟愈合。本公开提供的巨噬细胞能够有效促进糖尿病患者组织创伤的修复，为难修复的创伤提供了一种新的途径。
本公开实施方式还提供了如前述任意实施方式所述的诱导剂、或如前述任意实施方 式所述的培养基，或如前述任意实施方式所述的巨噬细胞在制备用于促血管新生的药物中的应用。
本公开实施方式还提供了一种用于组织创伤修复的药物，其包括如前述任意实施方式所述的巨噬细胞。
可选地，所述药物还可以包括其他已知用于组织创伤修复的制剂。
本公开实施方式还提供修复型巨噬细胞、或药物在用于组织创伤修复中的用途。
本公开实施方式还提供修复型巨噬细胞、或所述药物在用于促血管新生中的用途。
本公开实施方式还提供修复型巨噬细胞、或所述药物，用于组织创伤修复中的用途。
在一些实施方式中，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
本公开实施方式还提供所述修复型巨噬细胞、或所述药物，用于促血管新生中的用途。
本公开实施方式还提供一种受试者中组织创伤修复的方法，包括：
向有此需要的受试者给药治疗有效量的修复型巨噬细胞、或药物。
在一些实施方式中，组织创伤修复包括：糖尿病患者的组织创伤修复。
本公开实施方式还提供一种促进受试者中血管新生的方法，包括：
向有此需要的受试者给药治疗有效量的修复型巨噬细胞、或药物。
本公开使用骨桥素OPN及其他细胞因子组成的组合物对单核细胞进行培养诱导，能够形成具有较强修复功能的巨噬细胞，这些局势细胞能够应用于组织创面修复，促进慢性难愈合创伤的愈合和组织再生，具有较好的修复效果，适用于推广应用。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
一种诱导剂的制备，其包括：OPN(表达OPN的基因名称为SPP1)、MIP-2、CCL8、VEGF-B、M-CSF、IL-31、IL-10、TGF-β2和bFGF。
其中，OPN:MIP-2:CCL8:VEGF-B:M-CSF＝1:0.37:0.19:0.1:0.08(质量比)。
OPN：IL-31：IL-10：TGF-β2：bFGF＝1：0.08：0.06：0.04：0.03(质量比)。
实施例2
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：IL-31、IL-10、TGF-β2和bFGF。
实施例3
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：MIP-2、CCL8、VEGF-B、M-CSF、IL-31、IL-10、TGF-β2和bFGF。
实施例4
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、CCL8、VEGF-B、M-CSF、IL-31、IL-10、TGF-β2和bFGF。
实施例5
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、VEGF-B、M-CSF、IL-31、IL-10、TGF-β2和bFGF。
实施例6
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、CCL8、M-CSF、IL-31、IL-10、TGF-β2和bFGF。
实施例7
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、CCL8、VEGF-B、IL-31、IL-10、TGF-β2和bFGF。
实施例8
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、CCL8、VEGF-B、M-CSF、IL-10、TGF-β2和bFGF。
实施例9
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、CCL8、VEGF-B、M-CSF、IL-31、TGF-β2和bFGF。
实施例10
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、CCL8、VEGF-B、M-CSF、IL-31、IL-10和bFGF。
实施例11
一种诱导剂的制备，大致与实施例1相同，区别在于，诱导剂不包含：OPN、MIP-2、CCL8、VEGF-B、M-CSF、IL-31、IL-10和TGF-β2。
实施例12
一种修复型巨噬细胞的制备方法，其包括以下步骤。
(1)外周血巨噬细胞的制备：
人外周血单个核细胞(PBMC)购于上海儒百生物科技有限公司(美国
Figure PCTCN2022079118-appb-000012
中国区代理))，通过磁珠分选方法，用CD14磁珠将人外周血单核细胞纯化出来。
用含有20ng/ml人M-CSF的RPMI-1640培氧液体外诱导7天，单核细胞分化成人外周血巨噬细胞(未经诱导的巨噬细胞，记为M0)。
(2)修复型巨噬细胞的制备：
用含有实施例1提供诱导剂的RPMI-1640培养液体外培养人外周血巨噬细胞2天，培养时，诱导剂中各组分在RPMI-1640培养液中的终浓度如下(参照图1)：骨桥素的终浓度为113.215ng/mL，MIP-2的终浓度为42.513ng/mL，CCL8的终浓度为21.894ng/mL，VEGF-B的终浓度为11.799ng/mL，M-CSF的终浓度为10.157ng/mL，IL-31的终浓度为9.102ng/mL，IL-10的终浓度为7.372ng/mL，TGF-β2的终浓度为5.211ng/mL，bFGF的终浓度为3.522ng/mL。
2天后，得到
Figure PCTCN2022079118-appb-000013
的细胞形态如附图2所示，呈现出更长的细胞直径，于成纤维细胞有一定的相似。
(3)对照组巨噬细胞的制备：
参照实施例12的制备方法诱导人外周血巨噬细胞分化，区别在于：用100ng/ml LPS和40ng/ml人源IFN-γ刺激人外周血巨噬细胞48h，得到M1巨噬细胞(记为M1)。
参照实施例12的制备方法诱导人外周血巨噬细胞分化，区别在于：用40ng/ml人源IL-4和20ng/ml人源IL-13刺激人外周血巨噬细胞48h得到M2巨噬细胞(记为M2)。
实施例13
一种修复型巨噬细胞的制备方法，大致与实施例12相同，区别在于诱导剂的不同，区别如下。
用含有实施例2提供诱导剂的RPMI-1640培养液体外培养人外周血巨噬细胞2天。其中，骨桥素的终浓度为113.215ng/mL，MIP-2的终浓度为42.513ng/mL，CCL8的终浓度为21.894ng/mL，VEGF-B的终浓度为11.799ng/mL，M-CSF的终浓度为10.157ng/mL。
诱导出的修复型巨噬细，记为
Figure PCTCN2022079118-appb-000014
的胞形态图如附图2所示，较未诱导的巨噬细胞相比，OPN诱导的巨噬细胞直径稍长，且密度更多。
实施例14
其他修复型巨噬细胞的制备方法，大致与实施例12相同，区别在于诱导剂的不同， 区别如下。
分别用含有实施例3-11提供诱导剂的RPMI-1640培养液体外培养人外周血巨噬细胞2天。其中，骨桥素的终浓度为113.215ng/mL，MIP-2的终浓度为42.513ng/mL，CCL8的终浓度为21.894ng/mL，VEGF-B的终浓度为11.799ng/mL，M-CSF的终浓度为10.157ng/mL，IL-31的终浓度为9.102ng/mL，IL-10的终浓度为7.372ng/mL，TGF-β2的终浓度为5.211ng/mL，bFGF的终浓度为3.522ng/mL。用实施例3诱导的修复型巨噬细胞记为+OPN，用实施例4诱导的修复型巨噬细胞记为+MIP-2，用实施例5诱导的修复型巨噬细胞记为+CCL8，用实施例6诱导的修复型巨噬细胞记为+VEGF-B，用实施例7诱导的修复型巨噬细胞记为+M-CSF，用实施例8诱导的修复型巨噬细胞记为+IL-31，用实施例9诱导的修复型巨噬细胞记为+IL-10，用实施例10诱导的修复型巨噬细胞记为+TGF-β，用实施例11诱导的修复型巨噬细胞记为+bFGF。其中，未经诱导的巨噬细胞记为M0。得到的所有的修复型巨噬细胞形态如图2所示。
试验例1 修复型巨噬细胞的形态表征
基于实施例12-14的制备方法，得到的11种修复型巨噬细胞，用显微镜拍照，结果如图2所示，与其他种类的巨噬细胞比，
Figure PCTCN2022079118-appb-000015
呈现出更长的细胞直径，于成纤维细胞有一定的相似。M0为未极化的巨噬细胞。所以接下来的试验例主要研究
Figure PCTCN2022079118-appb-000016
试验例2 检测
Figure PCTCN2022079118-appb-000017
的主要表达基因
基于实施例12的制备方法，获取
Figure PCTCN2022079118-appb-000018
通过Trizol法提取其mRNA，通过RT-qPCR方法分析
Figure PCTCN2022079118-appb-000019
的主要基因表达，结果如附图3所示，
Figure PCTCN2022079118-appb-000020
同时表达M1和M2的表面标志物(附图3)。而且还高表达促血管新生和促基质细胞增殖的相关基因。
试验例3 条件培养基(Conditional medium(CM))的收集
分别将实施例12制备得到的
Figure PCTCN2022079118-appb-000021
M0巨噬细胞、M1巨噬细胞和M2巨噬细胞在相同条件下进行体外培养，而后分别收集培养上清液，其中，培养
Figure PCTCN2022079118-appb-000022
得到的上清液，即为
Figure PCTCN2022079118-appb-000023
其他对照巨噬细胞的培养上清液分别依次记为M0-CM、M1-CM和M2-CM。具体地，10 7个上述得到的四种细胞铺在10cm培养皿中，加入10毫升1640完全培养基在细胞培养箱中培养36-50小时，培养条件包括：、温度为37～38℃、 酸碱度为7.2～7.4、二氧化碳为5％。而后分别收集培养上清液。
试验例4 
Figure PCTCN2022079118-appb-000024
在体外诱导血管新生的测试
基于实施例12的制备方法，获取
Figure PCTCN2022079118-appb-000025
将基质胶Matrigel铺在μ-Slide板上。
之后将人血管内皮细胞(HUVECs)种在基质胶上，分别加入M0-CM、M1-CM、M2-CM和
Figure PCTCN2022079118-appb-000026
培养，正常培养液(Normal Medium)作为阴性对照，VEGF为阳性对照，如附图4所示，相对于其他细胞，
Figure PCTCN2022079118-appb-000027
有很强的促血管新生的能力。
试验例5 
Figure PCTCN2022079118-appb-000028
在体外促进成纤维细胞活化的测试
基于实施例12的制备方法，获取
Figure PCTCN2022079118-appb-000029
用收集的四种细胞的培养上清(试验例2中的M0-CM、M1-CM、M2-CM和
Figure PCTCN2022079118-appb-000030
)分别去培养小鼠成纤维细胞(L929)，通过免疫荧光观察其平滑肌动蛋白(α-SMA)和胶原的表达情况。
如附图5所示，与其他细胞相比，
Figure PCTCN2022079118-appb-000031
能更好地活化成纤维细胞。
试验例6 
Figure PCTCN2022079118-appb-000032
在体内促进糖尿病小鼠皮肤创面愈合
为了验证
Figure PCTCN2022079118-appb-000033
的促再生能力，本试验例将其运用在免疫缺陷的糖尿病小鼠伤口模型中。
腹腔注射戊巴比妥钠(50mg/kg)麻醉小鼠，在其背部造出一个直径为6mm大小的全层切除伤口。将3×10 5个
Figure PCTCN2022079118-appb-000034
(实施例12)种植在明胶纺织的静电纺丝上，之后移植在伤口处，用同样的方法移植其他对照巨噬细胞，将与
Figure PCTCN2022079118-appb-000035
相同数量的细胞M0、M1和M2(实施例12获得)种植在明胶纺织的静电纺丝上；空白对照组为：移植空白电纺丝为空白对照组。正常小鼠组为：非糖尿病小鼠记为正常小鼠组，作为阳性对照组……。分别在0，3，7，11，15，21天观察其伤口大小，结果如附图6所示，相较于其他巨噬细胞，在促伤口愈合方面，
Figure PCTCN2022079118-appb-000036
展现出更强的能力。附图7同时证明了
Figure PCTCN2022079118-appb-000037
可以促进伤口处成纤维细胞的胶原的沉积。以及附图8展示了
Figure PCTCN2022079118-appb-000038
促进伤口处血管的新生以及成熟。
以上所述仅为本公开的典型实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何 修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供的诱导剂使用骨桥素OPN及其他细胞因子组成的组合物对单核细胞进行培养诱导，能够形成具有较强修复功能的巨噬细胞，这些局势细胞能够应用于组织创面修复，促进慢性难愈合创伤的愈合和组织再生，具有较好的修复效果，特别是可以有效促进糖尿病患者组织创伤的修复，为难修复的创伤提供了一种新的途径，具有广泛的应用价值。

Claims (16)

  1. 一种诱导剂，其特征在于，其包括：骨桥素和细胞因子，所述细胞因子选自MIP-2、CCL8、VEGF-B和M-CSF中的至少一种。
  2. 根据权利要求1所述的诱导剂，其特征在于，按重量份数计，当所述诱导剂包括MIP-2时，骨桥素与MIP-2的质量比为(80～120)：(27～47)；
    当所述诱导剂包括CCL8时，骨桥素与CCL8的质量比为(80～120)：(9～29)；
    当所述诱导剂包括VEGF-B时，骨桥素与VEGF-B的质量比为(80～120)：(5～20)；
    当所述诱导剂包括M-CSF时，骨桥素与M-CSF的质量比为(80～120)：(1～18)。
  3. 根据权利要求1所述的诱导剂，其特征在于，所述诱导剂还包括：IL-31、IL-10、TGF-β2和bFGF中的至少一种组分；
    优选地，当所述诱导剂包括IL-31时，IL-31与骨桥素的重量比为(0.06～0.10)：1；
    优选地，当所述诱导剂包括IL-10时，IL-10与骨桥素的重量比为(0.04～0.08)：1；
    优选地，当所述诱导剂包括TGF-β2时，TGF-β2与骨桥素的重量比为(0.02～0.06)：1；
    优选地，当所述诱导剂包括bFGF时，bFGF与骨桥素的重量比为(0.01～0.05)：1。
  4. 一种培养基，其特征在于，其包括如权利要求1～3任一项所述的诱导剂。
  5. 一种修复型巨噬细胞的制备方法，其特征在于，其包括：采用如权利要求1～3任一项所述的诱导剂对待诱导的巨噬细胞进行诱导分化或采用如权利要求4所述的培养基对待诱导的巨噬细胞进行培养，以获得修复型巨噬细胞。
  6. 根据权利要求5所述的修复型巨噬细胞的制备方法，其特征在于，当采用所述培养基对单核细胞进行分化培养时，所述诱导剂中骨桥素在所述培养基中的终浓度为100～150ng/mL；
    当所述诱导剂包括MIP-2时，MIP-2在所述培养基中的终浓度为32～52ng/mL；
    当所述诱导剂包括CCL8时，CCL8在所述培养基中的终浓度为11～31ng/mL；
    当所述诱导剂包括VEGF-B时，VEGF-B在所述培养基中的终浓度为1～21ng/mL；
    当所述诱导剂包括M-CSF时，M-CSF在所述培养基中的终浓度为1～20ng/mL；
    当所述诱导剂包括IL-31时，IL-31在所述培养基中的终浓度为1～20ng/mL；
    当所述诱导剂包括IL-10时，IL-10在所述培养基中的终浓度为1～15ng/mL；
    当所述诱导剂包括TGF-β2时，TGF-β2在所述培养基中的终浓度为1～10ng/mL；
    当所述诱导剂包括bFGF时，bFGF在所述培养基中的终浓度为1～6ng/mL；
    优选地，所述诱导分化的培养时间为1～3天。
  7. 一种修复型巨噬细胞，其特征在于，其由权利要求5或6所述的修复型巨噬细胞的制备方法制备获得。
  8. 如权利要求1～3任一项所述的诱导剂、或如权利要求4所述的培养基，或如权利要求7所述的修复型巨噬细胞在制备用于组织创伤修复的药物中的应用；
    优选地，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
  9. 如权利要求1～3任一项所述的诱导剂、或如权利要求4所述的培养基，或如权利要求7所述的修复型巨噬细胞在制备用于促血管新生的药物中的应用。
  10. 一种用于组织创伤修复的药物，其特征在于，其包括如权利要求7所述的巨噬细胞。
  11. 如权利要求7所述的修复型巨噬细胞、或权要要求10所述的药物在用于组织创伤修复中的用途；
    优选地，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
  12. 如权利要求7所述的修复型巨噬细胞、或权要要求10所述的药物在用于促血管新生中的用途。
  13. 如权利要求7所述的修复型巨噬细胞、或如权要要求10所述的药物，用于组织创伤修复中的用途；
    优选地，所述用于组织创伤修复包括：糖尿病患者组织创伤的修复。
  14. 如权利要求7所述的修复型巨噬细胞、或权要要求10所述的药物，用于促血管新生中的用途。
  15. 一种受试者中组织创伤修复的方法，包括：
    向有此需要的受试者给药治疗有效量的权利要求7所述的修复型巨噬细胞、或权要要求10所述的药物；
    优选地，所述组织创伤修复包括：糖尿病患者的组织创伤修复。
  16. 一种促进受试者中血管新生的方法，包括：
    向有此需要的受试者给药治疗有效量的权利要求7所述的修复型巨噬细胞、或权要要求10所述的药物。
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