Inducer, macrophage and use thereof

Abstract

The present invention discloses an inducer, macrophages, and their applications, relating to the interdisciplinary fields of immunology, cell biology, and regenerative medicine. The invention utilizes a composition composed of osteopontin (OPN) and other cytokines to culture and induce monocytes, thereby generating macrophages with strong repair functions. These macrophages can be used to repair tissue wounds, promoting the healing and tissue regeneration of chronic, difficult-to-heal wounds, and exhibiting excellent repair effects, making them suitable for widespread application.

Description

Technical Field

[0001] The present invention relates to the interdisciplinary field of immunology, cell biology and regenerative medicine, and in particular to an inducer, macrophages and applications thereof. Background Art

[0002] Diabetes and its complications pose a serious threat to human health worldwide. Among these, lower limb ulcers, commonly known as "diabetic foot," are difficult-to-heal or even non-healing skin wounds and have become the leading cause of lower limb amputations. However, there are currently no targeted treatment strategies or medications for diabetic foot, primarily relying on routine care and debridement, which are ineffective and have a high recurrence rate.

[0003] In the process of wound repair, macrophages (and their precursor monocytes), as core members of the innate immune system, play a key role. For example, in the early stages of trauma, these cells play roles in phagocytosis of pathogens, cell recruitment, and promotion of angiogenesis; in the later stages of repair, these cells promote fibroblast proliferation, vascular maturation, and matrix remodeling. A large number of pathological studies have shown that in the local area of diabetic wounds, the disorder of macrophage function directly leads to the obstruction of the healing of such wounds. Shaping the function of autologous monocytes / macrophages in vitro and then transplanting them to the wound site is expected to become a therapeutic strategy to promote wound repair.

[0004] At present, there is no method in the world or in China that can effectively repair macrophages on the bed. In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide an inducer, macrophages and applications thereof.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides an inducer comprising: osteopontin and a cytokine, wherein the cytokine is selected from at least one of MIP-2, CCL8, VEGF-B and M-CSF.

[0008] In a second aspect, an embodiment of the present invention provides a culture medium comprising the inducer as described in the above embodiment.

[0009] In a third aspect, an embodiment of the present invention provides a method for preparing macrophages, which comprises: inducing differentiation of monocytes using the inducer described in the preceding embodiment or differentiating and culturing monocytes using the culture medium described in the preceding embodiment to obtain macrophages for tissue wound repair.

[0010] In a fourth aspect, an embodiment of the present invention provides a macrophage, which is prepared by the macrophage preparation method described in the above embodiment.

[0011] In a fifth aspect, embodiments of the present invention provide use of the inducer as described in the preceding embodiments, or the culture medium as described in the preceding embodiments, or the macrophages as described in the preceding embodiments in the preparation of a medicament for tissue wound repair.

[0012] In a sixth aspect, embodiments of the present invention provide use of the inducer as described in the preceding embodiments, or the culture medium as described in the preceding embodiments, or the macrophages as described in the preceding embodiments in the preparation of a drug for promoting angiogenesis.

[0013] In a seventh aspect, an embodiment of the present invention provides a drug for tissue wound repair, comprising the macrophages as described in the above embodiment.

[0014] The present invention has the following beneficial effects:

[0015] The present invention uses a composition composed of osteopontin OPN and other cytokines to culture and induce monocytes, which can form macrophages with strong repair functions. These macrophages can be used to repair tissue wounds, promote the healing of chronic and difficult-to-heal wounds and tissue regeneration, have good repair effects, and are suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The concentration ratio of OPN and other cytokines;

[0018] Figure 2 The morphology of 11 types of repair macrophages obtained by inducing human peripheral blood mononuclear cells through the steps of Examples 12-14 in Experimental Example 1 (compared with uninduced M0);

[0019] Figure 3 For Test Example 1 The main gene expression of

[0020] Figure 4 For Test Example 2 Can promote angiogenesis in vitro;

[0021] Figure 5 For Test Example 3 Promote fibroblast activation;

[0022] Figure 6 For Test Example 4 Promoted skin wound healing in diabetic mice in vivo;

[0023] Figure 7 For Test Example 4 It can promote collagen secretion in fibroblasts in the wounds of diabetic mice;

[0024] Figure 8 For Test Example 4 It can promote angiogenesis and maturation in the wounds of diabetic mice. DETAILED DESCRIPTION

[0025] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0026] The present invention provides an inducer comprising osteopontin and a cytokine, wherein the cytokine is selected from at least one of MIP-2, CCL8, VEGF-B and M-CSF.

[0027] Osteopontin (OPN) is a glycosylated protein that exists widely in a variety of extracellular matrices as a "non-collagenous" extracellular matrix component.

[0028] After a series of creative work, the inventors found that the combination of OPN and cytokines for the induction of monocyte differentiation can produce macrophages. Compared with normal macrophages, It has strong repair function and can promote wound healing and tissue repair and regeneration.

[0029] When the inducer includes MIP-2, the mass ratio of osteopontin to MIP-2 is (80-120): (27-47);

[0030] When the inducer includes CCL8, the mass ratio of osteopontin to CCL8 is (80-120):(9-29);

[0031] When the inducer includes VEGF-B, the mass ratio of osteopontin to VEGF-B is (80-120): (5-20);

[0032] When the inducer includes M-CSF, the mass ratio of osteopontin to M-CSF is (80-120):(1-18).

[0033] Preferably, the inducing agent comprises the following components by weight: 80 to 120 parts of osteopontin, 27 to 47 parts of MIP-2, 9 to 29 parts of CCL8, 5 to 20 parts of VEGF-B and 1 to 18 parts of M-CSF. Under this definition, the inducing agent induces differentiation Has better repair effect.

[0034] In some embodiments, the number of parts of osteopontin can be 80 parts, 85 parts, 90 parts, 95 parts, 100 parts, 105 parts, 110 parts, 115 parts or 120 parts; the number of parts of MIP-2 can be 27 parts, 30 parts, 35 parts, 40 parts, 45 parts or 47 parts; the number of parts of CCL8 can be 9 parts, 10 parts, 15 parts, 20 parts, 25 parts or 29 parts; the number of parts of VEGF-B 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.

[0035] Preferably, the inducer further comprises at least one component selected from the group consisting of IL-31, IL-10, TGF-β2 and bFGF.

[0036] Preferably, when the inducing agent includes IL-31, 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.

[0037] Preferably, when the inducing agent includes IL-10, 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.

[0038] Preferably, when the inducing agent includes TGF-β2, the weight ratio of TGF-β2 to osteopontin is (0.02-0.06): 1. In some embodiments, the weight ratio of TGF-β2 to osteopontin may be 0.02:1, 0.03:1, 0.04:1, 0.05:1 or 0.06:1.

[0039] Preferably, when the inducing agent includes bFGF, the weight ratio of bFGF to osteopontin is (0.01-0.05): 1. In some embodiments, the weight ratio of bFGF to osteopontin can be 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1 or 0.06:1.

[0040] When the inducer contains these cytokines and is added according to the above ratio, it can induce differentiation of cells with better repair ability.

[0041] An embodiment of the present invention provides a culture medium comprising the inducer as described in any of the aforementioned embodiments.

[0042] Optionally, the culture medium comprises a culture medium matrix and an inducer. The culture medium matrix can be selected from existing culture media known for culturing cells.

[0043] Preferably, the culture medium is RPMI-1640 culture medium.

[0044] An embodiment of the present invention provides a method for preparing repair macrophages, which comprises: using the inducer described in the above embodiment to induce differentiation of the macrophages to be induced or using the culture medium described in the above embodiment to culture the macrophages to be induced to obtain repair macrophages.

[0045] The "macrophages to be induced" herein may be peripheral blood macrophages differentiated from monocytes. Monocytes are derived from precursor cells in the bone marrow.

[0046] Preferably, when the culture medium is used to culture the macrophages to be induced for differentiation, the final concentration of osteopontin in the inducer in the culture medium is 100-150 ng / mL. Within this concentration range, the induced repair macrophages have better repair efficacy.

[0047] Specifically, the final concentration of osteopontin in the culture medium can be 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL or 150 ng / mL.

[0048] When the inducer includes MIP-2, the final concentration of MIP-2 in the culture medium is 32 to 52 ng / mL. Specifically, the final concentration of MIP-2 in the culture medium can be 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL, 40 ng / mL, 42 ng / mL, 44 ng / mL, 46 ng / mL, 48 ng / mL, 50 ng / mL, or 52 ng / mL.

[0049] When the inducer includes CCL8, the final concentration of CCL8 in the culture medium is 11 to 31 ng / mL. Specifically, the final concentration of CCL8 in the culture 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, 29 ng / mL, or 31 ng / mL.

[0050] When the inducer includes VEGF-B, the final concentration of VEGF-B in the culture medium is 1 to 21 ng / mL. Specifically, the final concentration of CCL8 in the culture 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, 19 ng / mL, or 21 ng / mL.

[0051] When the inducer includes M-CSF, the final concentration of M-CSF in the culture medium is 1 to 20 ng / mL. Specifically, the final concentration of M-CSF in the culture 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, 19 ng / mL, or 20 ng / mL.

[0052] When the inducer includes IL-31, the final concentration of IL-31 in the culture medium is 1 to 20 ng / mL. Specifically, the final concentration of IL-31 in the culture 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, 19 ng / mL, or 20 ng / mL.

[0053] When the inducer includes IL-10, the final concentration of IL-10 in the culture medium is 1 to 15 ng / mL. Specifically, the final concentration of IL-10 in the culture 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.

[0054] When the inducer includes TGF-β2, the final concentration of TGF-β2 in the culture medium is 1 to 10 ng / mL. Specifically, the final concentration of TGF-β2 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.

[0055] When the inducer includes bFGF, the final concentration of bFGF in the culture medium is 1-6 ng / mL. Specifically, the final concentration of bFGF in the culture medium can be 1 ng / mL, 2 ng / mL, 4 ng / mL or 6 ng / mL.

[0056] Preferably, the culture time for inducing differentiation is 1 to 3 days, preferably 2 days.

[0057] An embodiment of the present invention further provides a macrophage, which is prepared by the macrophage preparation method described in any of the above embodiments.

[0058] The repair macrophages prepared by the above preparation method are derived from human peripheral blood monocytes. After induction, they highly express surface biomarkers such as CD86, CD163, and CD206, highly secrete cytokines that promote immunosuppression such as IL-10, IL-16, and TGF-β, highly secrete cytokines that promote angiogenesis such as Amphiregulin, IGFBP-3, and Ang, and highly secrete cytokines that promote cell proliferation and matrix secretion such as VEGF-A, PDGF-B, and bFGF. They have proliferation capacity (ki67 positive) in vitro culture; and the phenotype is relatively stable, tending to be anti-inflammatory.

[0059] Optionally, the preparation method further comprises: inducing and culturing monocytes to obtain macrophages (macrophages to be induced).

[0060] The present invention also provides use of the inducer as described in any of the foregoing embodiments, or the culture medium as described in any of the foregoing embodiments, or the macrophages as described in any of the foregoing embodiments in the preparation of a medicament for tissue wound repair.

[0061] Preferably, the method for repairing tissue wounds includes repairing tissue wounds of diabetic patients.

[0062] Diabetic patients experience abnormal insulin secretion and regulation, which affects their glucose metabolism, reduces glucose utilization, and impairs tissue regeneration and repair, leading to delayed wound healing. The macrophages provided by this invention can effectively promote the repair of tissue wounds in diabetic patients, providing a new approach for treating difficult-to-repair wounds.

[0063] The present invention also provides the use of the inducer as described in any of the foregoing embodiments, or the culture medium as described in any of the foregoing embodiments, or the macrophages as described in any of the foregoing embodiments in the preparation of a drug for promoting angiogenesis.

[0064] An embodiment of the present invention further provides a drug for tissue wound repair, comprising the macrophages as described in any of the aforementioned embodiments.

[0065] Optionally, the drug may also include other agents known to be used for tissue wound repair.

[0066] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0067] Example 1

[0068] The invention discloses a preparation of an inducer, which comprises: OPN (SPP1), MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF-β2 and bFGF.

[0069] Among them, OPN:MIP-2:CCL8:VEGF-B:M-CSF=1:0.37:0.19:0.1:0.08 (mass ratio).

[0070] OPN:IL-31:IL-10:TGF-β2:bFGF=1:0.08:0.06:0.04:0.03 (mass ratio).

[0071] Example 2

[0072] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain IL-31, IL-10, TGF-β2 and bFGF.

[0073] Example 3

[0074] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF-β2 and bFGF.

[0075] Example 4

[0076] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF-β2 and bFGF.

[0077] Example 5

[0078] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, VEGF-B, M-CSF, IL-31, IL-10, TGF-β2 and bFGF.

[0079] Example 6

[0080] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, M-CSF, IL-31, IL-10, TGF-β2 and bFGF.

[0081] Example 7

[0082] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, IL-31, IL-10, TGF-β2 and bFGF.

[0083] Example 8

[0084] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-10, TGF-β2 and bFGF.

[0085] Example 9

[0086] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, TGF-β2 and bFGF.

[0087] Example 10

[0088] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10 and bFGF.

[0089] Example 11

[0090] The preparation of an inducer is substantially the same as that of Example 1, except that the inducer does not contain: OPN, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10 and TGF-β2.

[0091] Example 12

[0092] A method for preparing repair macrophages comprises the following steps.

[0093] (1) Preparation of peripheral blood macrophages:

[0094] Human peripheral blood mononuclear cells were obtained by density gradient centrifugation, and then purified by magnetic bead sorting using CD14 magnetic beads.

[0095] Monocytes were differentiated into human peripheral blood macrophages (macrophages to be induced) by induction in vitro for 7 days using RPMI-1640 culture medium containing 20 ng / ml human M-CSF.

[0096] (2) Preparation of repair macrophages:

[0097] Human peripheral blood macrophages were cultured in vitro for 2 days using RPMI-1640 culture medium containing the inducer provided in Example 1. During the culture, the final concentrations of the components of the inducer in the RPMI-1640 culture medium were as follows (refer to Figure 1 ): The final concentration of osteopontin was 113.215 ng / mL, the final concentration of MIP-2 was 42.513 ng / mL, the final concentration of CCL8 was 21.894 ng / mL, the final concentration of VEGF-B was 11.799 ng / mL, the final concentration of M-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-β2 was 5.211 ng / mL, and the final concentration of bFGF was 3.522 ng / mL.

[0098] 2 days later, I got The cell morphology is as follows Figure 2 As shown, the cells have a longer diameter, which is similar to fibroblasts.

[0099] Example 13

[0100] A method for preparing repair macrophages is substantially the same as that of Example 12, except that the inducer is different, as follows.

[0101] Human peripheral blood macrophages were cultured in vitro for 2 days using RPMI-1640 medium containing the inducer provided in Example 2. The final concentrations of osteopontin, MIP-2, CCL8, VEGF-B, and M-CSF were 113.215 ng / mL, 42.513 ng / mL, 21.894 ng / mL, and 11.799 ng / mL, respectively.

[0102] The induced repair macrophages are recorded as The cell morphology is shown in the attached figure Figure 2 As shown, OPN-induced macrophages were slightly longer in diameter and more densely packed than uninduced macrophages.

[0103] Example 14

[0104] The preparation methods of other repair macrophages are roughly the same as those in Example 12, except for the different inducers, as follows.

[0105] Human peripheral blood macrophages were cultured in vitro for 2 days using RPMI-1640 culture medium containing the inducer provided in Example 3-11. The final concentrations of osteopontin, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF-β2, and bFGF were 113.215 ng / mL, 42.513 ng / mL, 21.894 ng / mL, 11.799 ng / mL, 10.157 ng / mL, 9.102 ng / mL, 7.372 ng / mL, 5.211 ng / mL, and 3.522 ng / mL, respectively. The repair macrophages induced by Example 3 were recorded as +OPN, the repair macrophages induced by Example 4 were recorded as +MIP-2, the repair macrophages induced by Example 5 were recorded as +CCL8, the repair macrophages induced by Example 6 were recorded as +VEGF-B, the repair macrophages induced by Example 7 were recorded as +M-CSF, the repair macrophages induced by Example 8 were recorded as +IL-31, the repair macrophages induced by Example 9 were recorded as +IL-10, the repair macrophages induced by Example 10 were recorded as +TGF-β, and the repair macrophages induced by Example 11 were recorded as +bFGF. The morphology of all the repair macrophages obtained was as follows: Figure 2 shown.

[0106] Test Example 1

[0107] Based on the preparation methods of Examples 12-14, 11 types of repair macrophages were obtained and photographed under a microscope. The results are as follows: Figure 2 As shown, compared with other types of macrophages, The cells show a longer diameter, which is similar to fibroblasts. M0 is an unpolarized macrophage. So the next two experiments are mainly studied.

[0108] Test Example 2

[0109] Detection The main expressed genes.

[0110] 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 results are shown in the attached Figure 4 As shown, Simultaneous expression of surface markers of M1 and M2 (Appendix Figure 3 ). It also highly expresses genes related to promoting angiogenesis and stromal cell proliferation.

[0111] Test Example 3

[0112] In vitro angiogenesis induction assay.

[0113] Based on the preparation method of Example 12, obtain Matrigel was spread on a μ-Slide plate.

[0114] Then human vascular endothelial cells (HUVECs) were seeded on the matrigel and M0-CM, M1-CM, M2-CM and Normal medium was used as negative control and VEGF as positive control. Figure 4 As shown, compared with other cells, It has a strong ability to promote angiogenesis.

[0115] Test Example 4

[0116] In vitro assay for promoting fibroblast activation.

[0117] Based on the preparation method of Example 12, obtain

[0118] The culture supernatants of the four cells (M0-CM, M1-CM, M2-CM and ) were used to culture mouse fibroblasts (L929), and the expressions of smooth actin (α-SMA) and collagen were observed by immunofluorescence.

[0119] As attached Figure 5 As shown, compared with other cells, Can better activate fibroblasts.

[0120] Test Example 5

[0121] Promotes skin wound healing in diabetic mice in vivo.

[0122] To verify The pro-regenerative ability of the drug was applied in the wound model of immunodeficient diabetic mice in this study.

[0123] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and a full-thickness excision wound with a diameter of 6 mm was made on the back. 5 indivual (Example 12) The cells were grown on electrospun gelatin and then transplanted into the wound. Other control macrophages were transplanted in the same way. The wound size was observed at 0, 3, 7, 11, 15, and 21 days. The results are shown in the attached figure. Figure 6 As shown, compared with other macrophages, Show greater ability. Figure 7 It also proved It can promote the deposition of collagen in fibroblasts at the wound. Figure 8 Shown Promote the regeneration and maturation of blood vessels in the wound.

[0124] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An inducer, characterized in that It is composed of osteopontin, MIP-2, CCL8, VEGF-B, M-CSF, IL-31, IL-10, TGF-β2, and bFGF; The mass ratios of osteopontin, MIP-2, CCL8, VEGF-B, and M-CSF were (80-120):(27-47):(9-29):(5-20):(1-18); The mass ratio of osteopontin, IL-31, IL-10, TGF-β2 and bFGF is 1:(0.06~0.10):(0.04~0.08):(0.02~0.06):(0.01~0.05).

2. A culture medium, characterized in that It includes: The inducer according to claim 1.

3. A method for preparing repair macrophages, characterized in that: The method comprises: using the inducer according to claim 1 to induce differentiation of the macrophages to be induced or using the culture medium according to claim 2 to culture the macrophages to be induced, so as to obtain repair macrophages.

4. The method for preparing repair macrophages according to claim 3, characterized in that: When the culture medium is used to culture monocytes for differentiation, the final concentration of osteopontin in the inducer in the culture medium is 100 to 150 ng / mL; The final concentration of MIP-2 in the culture medium was 32 to 52 ng / mL; The final concentration of CCL8 in the culture medium was 11 to 31 ng / mL; The final concentration of VEGF-B in the culture medium was 1 to 21 ng / mL; The final concentration of M-CSF in the culture medium was 1 to 20 ng / mL; The final concentration of IL-31 in the culture medium was 1 to 20 ng / mL; The final concentration of IL-10 in the culture medium was 1 to 15 ng / mL; The final concentration of TGF-β2 in the culture medium was 1 to 10 ng / mL; The final concentration of bFGF in the culture medium is 1-6 ng / mL.

5. The method for preparing repair macrophages according to claim 3 or 4, characterized in that: The culture time for inducing differentiation is 1 to 3 days.

6. A repair macrophage, characterized in that: The repair macrophage is prepared by the preparation method of any one of claims 3 to 5.

7. Use of the repair macrophages according to claim 6 in the preparation of a medicament for tissue wound repair; The tissue is skin tissue.

8. The use according to claim 7, characterized in that The method for repairing tissue wounds includes repairing tissue wounds of diabetic patients.

9. Use of the repair macrophages according to claim 6 in the preparation of a medicament for promoting angiogenesis in wounds.

10. A drug for repairing skin tissue trauma, characterized in that: It includes the macrophage according to claim 6.

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