Method for forming macrophages through induced differentiation

Abstract

The invention provides application of a vitamin D receptor agonist in preparation of macrophages. The invention also provides a method for inducing differentiation to form macrophages. The method comprises the step of treating monocytes by adopting a vitamin D receptor agonist. The method can directionally induce the monocyte to differentiate into the M1 type macrophage in vitro, the experimental operation is simple, the consumed time is short, meanwhile, the phagocytic ability of the M1 type macrophage is enhanced, the tumor immune microenvironment is improved, and the anti-tumor immunity is enhanced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method and application of efficiently inducing monocytes to differentiate into macrophages and maintaining anti-tumor activity. Background Art

[0002] Tumor cell immunotherapy technology has been widely used in experimental research and clinical treatment of various malignant tumors, especially chimeric antigen receptor T cells (CAR-T) therapy has achieved good results in the field of hematological tumors, but in solid tumors, its dense cell and matrix barriers first form a physical barrier to lymphocyte infiltration into solid tumors. Even if it enters the solid tumor, the immunosuppression caused by hypoxia and acidic environment, expression of immune checkpoint ligands, and especially a large number of immunosuppressive cells, such as TAM and myeloid-derived suppressor cells (MDSC), is one of the main difficulties in the current immunotherapy of solid tumors. Therefore, it is of great significance to study and explore therapeutic cells that can efficiently infiltrate into solid tumors and continuously exert anti-tumor functions.

[0003] In CAR-based cell therapy, macrophages have great therapeutic potential: strong ability to penetrate solid tumors, strong phagocytosis, rapid killing of tumor cells, prevention of immune escape, and activation of T cells as antigen-presenting cells, etc. However, the collection, culture and maintenance of anti-tumor phenotypes of macrophages are challenging.

[0004] Monocytes are one of the main sources of macrophages. Under different stimuli, monocytes can differentiate into macrophages (Mφ) and dendritic cells (DC). However, existing differentiation methods have limitations such as unstable differentiation efficiency, low cell viability and cell yield, and long cycles. In addition, macrophages are highly plastic and heterogeneous, and can undergo functional transformation in response to different signal stimuli, such as transformation into the classical activated M1 type (ie, M1 polarization), selectively activated M2 type (ie, M2 polarization), etc. M1 macrophages have strong phagocytic ability, can kill tumor cells and resist the invasion of pathogens; while M2 macrophages mainly play the role of immune transplantation to promote tumor growth, invasion and metastasis. Therefore, it is of great significance to develop methods to maintain the M1 phenotype of macrophages and exert anti-tumor functions. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for inducing monocytes to differentiate into M1 macrophages in vitro. The experimental operation is simple and time-saving. At the same time, the phagocytic ability of monocytes is enhanced, the tumor immune microenvironment is improved, and the anti-tumor immunity is enhanced.

[0006] According to one aspect of the present disclosure, there is provided use of a vitamin D receptor agonist (VDR agonist) in preparing macrophages.

[0007] In some embodiments, the vitamin D receptor agonist is used in combination with granulocyte-macrophage colony stimulating factor to prepare macrophages.

[0008] In some embodiments, the vitamin D receptor agonist comprises at least one of vitamin D, a vitamin D precursor, a vitamin D analog, a vitamin D receptor ligand, or a vitamin D receptor agonist precursor.

[0009] In some embodiments, the vitamin D receptor agonist includes at least one of calcitriol, alfacalcidol, paricalcitol, calcipotriol, calcifediol, cholecalciferol, ergocalciferol, tachysterol 3, idecalcitol, secalcitol, maxacalcitol, ciocalcitol, inecalcitol, tacalcitol, or doxorcalcitol.

[0010] In some embodiments, the macrophages are differentiated from monocytes.

[0011] In some embodiments, the vitamin D receptor agonist promotes the differentiation of monocytes into M1 macrophages.

[0012] In some embodiments, the vitamin D receptor agonist inhibits the differentiation of monocytes into M2 macrophages.

[0013] In some embodiments, the macrophages are M1 macrophages.

[0014] According to a second aspect of the present disclosure, a method for inducing differentiation to form macrophages is provided, the method comprising the step of treating monocytes with a vitamin D receptor agonist.

[0015] In some embodiments, the vitamin D receptor agonist comprises at least one of vitamin D, a vitamin D precursor, a vitamin D analog, a vitamin D receptor ligand, or a vitamin D receptor agonist precursor.

[0016] In some embodiments, the vitamin D receptor agonist includes at least one of calcitriol, alfacalcidol, paricalcitol, calcipotriol, calcifediol, cholecalciferol, ergocalciferol, tachysterol 3, idecalcitol, secalcitol, maxacalcitol, ciocalcitol, inecalcitol, tacalcitol, or doxorcalcitol.

[0017] In some embodiments, the amount of the vitamin D receptor agonist is 0.1nM-1μM, for example, 0.1nM, 0.5nM, 1nM, 2nM, 4nM, 5nM, 6nM, 8nM, 10nM, 50nM, 100nM, 200nM, 400nM, 500nM, 600nM, 800nM, 1μM or any value therebetween. In some preferred embodiments, the amount of the vitamin D receptor agonist is 1-100nM, preferably 1-50nM, and more preferably 1-20nM.

[0018] In some embodiments, the method further comprises treating the monocytes with granulocyte-macrophage colony stimulating factor.

[0019] In some embodiments, the amount of the granulocyte-macrophage colony stimulating factor is 0.1-1000 ng / mL, for example, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 800 ng / mL, 1000 ng / mL or any value therebetween. In some preferred embodiments, the amount of the granulocyte-macrophage colony stimulating factor is 10-200 ng / mL.

[0020] In some embodiments, the method further comprises inducing culture of monocytes using an immune cell culture medium.

[0021] In some embodiments, the immune cell culture medium includes X-VIVO10 culture medium, X-VIVO15 culture medium or X-VIVO20 culture medium, preferably X-VIVO15 culture medium.

[0022] In some embodiments, the method comprises the steps of:

[0023] (1) first culturing monocytes using an immune cell culture medium;

[0024] (2) adding the vitamin D receptor agonist to the immune cell culture medium after the first culture, and performing a second culture;

[0025] (3) Changing the medium and performing a third culture on the cells after the second culture using an immune cell culture medium.

[0026] In some embodiments, the immune cell culture medium in step (3) contains granulocyte-macrophage colony stimulating factor.

[0027] In some embodiments, the immune cell culture medium in step (1) does not contain granulocyte-macrophage colony stimulating factor.

[0028] In some embodiments, the immune cell culture medium comprises fetal bovine serum.

[0029] In some embodiments, the concentration of the fetal bovine serum is 5-15%, preferably 5-12%. In some specific embodiments, the concentration of the fetal bovine serum is 5%, 6%, 8%, 10%, 12%, 14%, 15% or any value therebetween.

[0030] In some embodiments, the first culture time is 20-26 h, for example, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h or any value therebetween.

[0031] In some embodiments, the second culture time is 6-10 h, for example, 6 h, 7 h, 8 h, 9 h, 10 h or any value therebetween.

[0032] In some embodiments, the third culture time is 14-18 h, for example, 14 h, 15 h, 16 h, 17 h, 18 h or any value therebetween.

[0033] According to yet another aspect of the present disclosure, provided is a macrophage prepared according to the method of the present disclosure.

[0034] In some embodiments, the macrophages have at least one upregulated M1 macrophage marker and / or at least one downregulated M2 macrophage marker.

[0035] In some embodiments, the macrophages have elevated expression of CD80 and / or CD86.

[0036] In some embodiments, the macrophages have reduced expression of CD163 and / or CD206.

[0037] In some embodiments, the macrophages are M1 macrophages.

[0038] According to another aspect of the present disclosure, provided is a macrophage prepared according to the method of the present disclosure or a use of the macrophage described in the present disclosure in preparing a drug for preventing, treating or alleviating inflammatory diseases and / or tumors.

[0039] In some embodiments, the tumor is a solid tumor.

[0040] In some embodiments, the medicament further comprises other anti-tumor therapeutic agents.

[0041] In some embodiments, the drug is used to inhibit the growth of tumor cells and / or induce apoptosis of tumor cells.

[0042] The disclosed in vitro method for inducing monocytes to differentiate into M1 macrophages is not only simple in experimental operation and time-saving, but also can improve the killing and phagocytic ability of macrophages, improve the tumor immune microenvironment, and enhance anti-tumor immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The results of identification of M1 and M2 macrophages under different differentiation induction conditions are shown.

[0044] Figure 2 The viability of macrophages under different differentiation induction conditions is shown.

[0045] Figure 3 Shown are the results of identification of M1 / M2 phenotypes after co-culture of macrophages and tumor cells under different differentiation induction conditions.

[0046] Figure 4 The killing effect of macrophages on target cells under different differentiation induction conditions is shown.

[0047] Figure 5 The phagocytosis of target cells by macrophages under different differentiation-inducing conditions is shown.

[0048] Figure 6 The figure shows the differentiation identification of macrophages under different differentiation induction conditions in vivo (Day 3 of intratumoral injection), where A is the statistical graph of immune cell counts; and B is the statistical graph of dendritic cell percentages.

[0049] Figure 7 The results of identification of M1 and M2 macrophages under different differentiation induction conditions in vivo are shown.

[0050] Figure 8 The results of identification of M1 and M2 macrophages after induction with different VDR agonists are shown. DETAILED DESCRIPTION

[0051] Macrophages are phenotypically heterogeneous immune cells that play an important role during inflammation (both initiation and resolution). Macrophages can be polarized into two phenotypes upon stimulation: (1) one is the classically activated M1 type, which can be induced by lipopolysaccharide (LPS) or interferon γ (IFN-γ) to produce proinflammatory cytokines such as TNFα and IL-1β; (2) the other is the selectively activated M2 type, which can be induced by IL-4, IL-13, etc. to produce anti-inflammatory cytokines such as IL-10, IL-13, Arg1, etc. The balance of M1 / M2 macrophage polarization determines the fate of an organ in inflammation or injury. M1 plays a proinflammatory role in counteracting stimulation in the early stages of inflammation, but continued stimulation will cause tissue damage; M2 plays an anti-inflammatory role, promoting tissue repair and angiogenesis.

[0052] Vitamin D is a class of fat-soluble steroid prohormones and hormones. Its two main forms are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), which can be converted into 1α,25-dihydroxyvitamin D3, also known as calcitriol, which is the physiologically active form of vitamin D.

[0053] Vitamin D receptor (VDR) agonists refer to any compound that binds to and activates VDR. VDR agonists may be naturally occurring or synthetic, including but not limited to: vitamin D, vitamin D precursors, vitamin D analogs, vitamin D receptor ligands, vitamin D receptor agonist precursors, or combinations thereof. Specific examples of VDR agonists include but are not limited to alfacalcidol, paricalcitol, calcipotriol, 25-hydroxy-D3 (25-OH-D3) (calcidiol), vitamin D3 (cholecalciferol), vitamin D2 (ergocalciferol), 1α,25-dihydroxyvitamin D3 (calcitriol), tachysterol 3, idecalcitol, sircalcitol, maxacalcitol, seocalcitol, inecalcitol, tacalcitol, doxorcalcitol, or combinations thereof.

[0054] The present disclosure provides a method for inducing monocytes to differentiate into M1 macrophages in vitro, wherein monocytes are treated with a VDR agonist or a combination of a VDR agonist and GM-CSF during the induction process, differentiation-related indicators are identified in vitro by FACS, and the killing and phagocytic effects of macrophages differentiated from monocytes on tumor cells are verified by killing experiments and phagocytic experiments; in vivo, a SKOV-3 subcutaneous tumor model is constructed in immunodeficient mice to explore its differentiation in tumors and the maintenance of the M1 phenotype.

[0055] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0056] The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0057] Example 1: Isolation and differentiation of monocytes

[0058] The single blood sample obtained from the commercial company (the single blood sample has passed the relevant ethical approval) was centrifuged at 200g for 10 minutes to remove platelets and some red blood cells, and the supernatant was discarded. The cell pellet was mixed with PBS, added to Ficoll (lymphocyte separation fluid, purchased from Sigma), and centrifuged at 1200rpm for 30 minutes. After centrifugation, the mononuclear cells (PBMC) were recovered and washed once with phosphate buffered saline (PBS). After PBMC was co-incubated with CD14+ magnetic beads in the CD14+ cell sorting kit (purchased from Miltenyi) for 15 minutes, CD14+ cells were separated using LS sorting columns (purchased from Miltenyi), and CD14+ monocytes were finally obtained.

[0059] According to the experimental groups shown in Table 1, on day 0, the cells were fed with X-VIVO containing 10% FBS. TM 15 medium, without or with different concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF); on the first day (24h), without or with VDR agonist (cholecalciferol VD3) after incubation for 8h, the medium was changed (replaced with X-VIVO containing 10% FBS) TM 15 culture medium), aspirate the cell culture supernatant, centrifuge at 400g for 5 min, and then wash with X-VIVO containing 10% FBS. TM The cells were resuspended in 15% culture medium, and the induction differentiation culture was continued for 16 h (24 h for induction differentiation culture from the beginning of the addition of VDR agonist) without or with different concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), for a total of 48 h.

[0060] Table 1

[0061]

[0062] Example 2: In vitro macrophage differentiation and phenotypic identification

[0063] The cells of each group prepared in Example 1 were collected, mixed with PBS, centrifuged at 400 g for 5 min, 2 μL FcR Blocking (Miltenyi Biotec, 130-059-901) was added, incubated at room temperature in the dark for 10 min, anti-human-CD68 antibody, anti-human-CD11b antibody, anti-human-CD80 antibody, anti-human-CD86 antibody, anti-human-CD163 antibody and anti-human-CD206 (all purchased from BD) were added, incubated at 4° C. in the dark for 30 min, centrifuged at 400 g for 5 min, washed once with PBS and then subjected to flow cytometry detection.

[0064] The results showed that compared with the untreated group G1, the positive rates of macrophage surface markers CD11b and CD68 in the different differentiation induction condition groups exceeded 95%, and both groups showed high expression rates and intensities of M1-related proteins CD80 and CD86, and CD163 was downregulated to varying degrees (see Figure 1 ), indicating that the above-mentioned induction differentiation systems (G2-G6) can induce monocytes into macrophages, the macrophage differentiation ratio is higher than 95%, and has an M1 macrophage phenotype.

[0065] At the same time, through statistical analysis of the viability of each group of cells prepared in Example 1, it was found that adding induction factors under different conditions to culture cells would basically not affect the viability of the cells (see Figure 2 ).

[0066] After esophageal cancer cells OE19 and each group of macrophages prepared in Example 1 were co-cultured for 2 days, the macrophages were collected, and the M1 and M2 surface markers (CD86, CD163) of the macrophages were detected by flow cytometry.

[0067] The results showed that compared with the cells before culture (G1-day 0), after co-culture with tumor cells, the expression of CD86 in the G1 cells without any treatment was significantly reduced, while the expression of CD163 was significantly increased, indicating that the G1 cells without any treatment showed an M2 phenotype, while the cells in different treatment groups (G2-G6) still expressed high CD86 and low CD163, indicating that the cells in the treated groups could resist the transformation of the M2 phenotype and maintain the M1 phenotype (see Figure 3 ).

[0068] Example 3: In vitro killing experiment of macrophages

[0069] OE19 cells (human esophageal cancer cells, purchased from COBIOER BIOSCIENCES) were plated in 6-well plates, and the target cells were transformed using commercial luciferase lentivirus (purchased from GeneCare Biotechnology) at an MOI of 10. Puromycin was used to screen the transformed cells, and the OE19 transformed cell line stably expressing luciferase (hereinafter referred to as OE19-luc) was obtained for in vitro killing experiments.

[0070] Luciferase-overexpressing esophageal cancer OE19 (OE19-luc) target cells and effector cells (macrophages prepared in Example 1) were incubated at 37° C. for 48 h at effector-target ratios of 5:1, 2:1, 1:1, 1:5 and 1:10, respectively, and then Steady-Glo Luciferase Assay reagent (Promega) was added, and the plate reader was used to read the result after 5 min of reaction.

[0071] The results showed that compared with the untreated group (G1) and the group treated with GM-CSF only on day 0 (G2), the macrophages in the treated groups (G3-G6) had a significant killing effect on OE19-luc cells, and the higher the effector-target ratio, the more obvious the killing effect on target cells (see Figure 4 ).

[0072] Example 4: In vitro phagocytosis experiment

[0073] OE19 cells (human esophageal cancer cells, purchased from COBIOER BIOSCIENCES) target cells were suspended in PBS, DiR dye (5 μM) was added to the cell suspension, and the cells were incubated at 37°C for 20 min. The cells were centrifuged at 400 g for 5 min, the supernatant was aspirated, and pre-warmed X-VIVO15 medium (purchased from Lonza, hereinafter referred to as "complete medium") containing 10% FBS (purchased from Gibco) was added to resuspend the cells to a density of 1×10 6 Cells / mL. Each group of macrophages (effector cells) prepared in Example 1 was harvested and centrifuged at 400g for 5 minutes, the supernatant was discarded, and preheated complete medium was added to the cell pellet to resuspend the cells. Effector cells and target cells were added to the culture dish in a 1:1 ratio. After co-culture for 2 hours, the cells were collected, anti-human-CD11b-FITC (purchased from eBioscience) was added for staining, and cells containing DiR markers in the CD11b+ group were analyzed by flow cytometry, and these cells were considered to be macrophages that phagocytized OE19.

[0074] The results showed that compared with the untreated group (G1) and the group treated with GM-CSF only on day 0 (G2), the effector cells in each treatment group (G3-G6) had a significant phagocytic effect on target cells, and the macrophages in the G4 group had the strongest phagocytic ability on target cells (see Figure 5 ).

[0075] Example 5: SKOV3 transplanted tumor mouse model

[0076] The mononuclear cells were prepared in the same manner as in Example 1 and incubated in X-VIVO medium containing 10% FBS. TM 15 culture medium. On the first day (24h), the cells were cultured without or with 4nM VDR agonist (cholecalciferol VD3) for 8h, and then the medium was changed (replaced with X-VIVO containing 10% FBS). TM 15 culture medium), and the differentiation induction culture was continued for 16 h (24 h from the addition of VDR agonist), and macrophages were harvested after a total of 48 h of differentiation induction.

[0077] The subcutaneous tumor model was constructed using 6-8 week-old immunodeficient NCG mice (purchased from Jicui Yaokang). When the tumor volume grew to 200 mm 3 The macrophages of different treatment groups were treated by intratumoral injection. At the same time, on the second day after intratumoral injection, mice in groups G2 and G4 were injected with 50ng / mL GM-CSF by subcutaneous injection. The specific experimental groups are shown in Table 2. Ten days after treatment, the mice were euthanized, the tumors of the mice were removed and the tumor cells were digested. The number of monocytes, macrophages, dendritic cells (DC cells) and granulocytes in the tumor tissues and the expression intensity of polarization markers of macrophages were detected by flow cytometry.

[0078] Table 2

[0079] Group In vitro culture In vivo injection G1 N / A N / A G2 N / A One subcutaneous injection of GM-CSF G3 Add VDR agonist for 8 hours at 24 hours, change the medium, and harvest at 48 hours N / A G4 Add VDR agonist for 8 hours at 24 hours, change the medium, and harvest at 48 hours One subcutaneous injection of GM-CSF

[0080] The results showed that monocytes in all groups differentiated into macrophages to varying degrees, and the degree of differentiation and the number of differentiated cells in groups G3 and G4 increased significantly ( Figure 6 A). Meanwhile, 10-20% of the cells in the G1 group differentiated into DCs, and fewer cells in the G3 and G4 groups differentiated into DCs compared with the control group G1 ( Figure 6 B).

[0081] According to the method of Example 2, the macrophage polarization markers were analyzed and it was found that compared with the G1 group, more than 80% of the cells in the G2-G4 groups were differentiated into macrophages, and maintained high expression of CD86 and low expression of CD206 ( Figure 7 ).

[0082] The above data indicate that the treatment methods of monocytes in the G2-G4 group, especially the G3-G4 group, can efficiently induce differentiation into macrophages and maintain the M1 phenotype for a long time.

[0083] Example 6: Identification of macrophage polarization phenotypes after induction with different VDR agonists

[0084] In the same manner as in Example 1, monocytes were prepared.

[0085] The sorted monocytes were induced and cultured with different VDR agonists (calcitriol, alfacalcidol and paricalcitol) and optional GM-CSF. Specifically, the monocytes were cultured in X-VIVO containing 10% FBS. TM 15 culture medium. On the first day (24h), different VDR agonists were added or not added for 8h, and then the medium was changed (replaced with X-VIVO containing 10% FBS). TM15 culture medium), with or without GM-CSF, the induction differentiation culture was continued for 16 h (24 h from the start of the addition of VDR agonist), and macrophages were harvested after a total of 48 h of induction differentiation. The specific grouping is shown in Table 3.

[0086] The cells from each group were collected, mixed with PBS, and centrifuged at 400 g for 5 min. 2 μL FcR Blocking (Miltenyi Biotec, 130-059-901) was added. After incubation at room temperature in the dark for 10 min, anti-human-CD80 antibody, anti-human-CD86 antibody, and anti-human-CD163 antibody (purchased from BD) were added. The cells were incubated at 4°C in the dark for 30 min, centrifuged at 400 g for 5 min, washed once with PBS, and then subjected to flow cytometry detection.

[0087] Table 3

[0088]

[0089]

[0090] The results showed that compared with the G1 group without any treatment, the GM-CSF group alone and the different VDR agonist groups alone could increase the expression of macrophage M1 phenotype markers CD80 and CD86 proteins, and there were significant differences. However, the GM-CSF and different VDR agonist co-induced group, compared with the single treatment group, the expression of CD80 and CD86 proteins increased exponentially. At the same time, the expression of CD163 protein, a surface marker of M2 macrophages, showed a decrease to varying degrees, and the decrease in CD163 protein expression was more obvious in the different VDR and GM-CSF co-treatment groups.

[0091] The above results showed that the cells in the G1 group without any treatment showed an M2 phenotype, while the cells in the different treatment groups showed high expression of CD80 and CD86, low expression of CD163, and maintained the M1 macrophage phenotype. In addition, the difference in the induction of macrophage differentiation into M1 type in the group treated with GM-CSF and different VDR agonists was more obvious, indicating that GM-CSF and different VDR agonists were more able to resist the conversion of macrophages to M2 type and maintain the M1 phenotype compared with the single treatment group (see Figure 8 ).

[0092] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of vitamin D receptor agonists in the preparation of macrophages.

2. The use according to claim 1, characterized in that The vitamin D receptor agonist is used in combination with granulocyte-macrophage colony stimulating factor to prepare macrophages; and / or The vitamin D receptor agonist comprises at least one of vitamin D, a vitamin D precursor, a vitamin D analog, a vitamin D receptor ligand or a vitamin D receptor agonist precursor; Preferably, the vitamin D receptor agonist includes at least one of calcitriol, alfacalcidol, paricalcitol, calcipotriol, calcifediol, cholecalciferol, ergocalciferol, tachysterol 3, idecalcitol, secalcitol, maxacalcitol, ciocalcitol, inacalcitol, tacalcitol or doxorcalcitol.

3. The use according to claim 1 or 2, characterized in that The macrophages are obtained by inducing differentiation of monocytes; Preferably, the vitamin D receptor agonist promotes the differentiation of monocytes into M1 macrophages; Preferably, the vitamin D receptor agonist inhibits the differentiation of monocytes into M2 macrophages; Preferably, the macrophages are M1 macrophages.

4. A method for inducing differentiation into macrophages, comprising the step of treating monocytes with a vitamin D receptor agonist.

5. The method according to claim 4, characterized in that The vitamin D receptor agonist comprises at least one of vitamin D, a vitamin D precursor, a vitamin D analog, a vitamin D receptor ligand or a vitamin D receptor agonist precursor; Preferably, the vitamin D receptor agonist comprises at least one of calcitriol, alfacalcidol, paricalcitol, calcipotriol, calcifediol, cholecalciferol, ergocalciferol, tachysterol 3, idecalcitol, sircalcitol, maxacalcitol, siocalcitol, inacalcitol, tacalcitol or doxorcalcitol; Preferably, the dosage of the vitamin D receptor agonist is 0.1 nM-1 μM, preferably 1-100 nM.

6. The method according to claim 4 or 5, characterized in that: The method further comprises treating the monocytes with granulocyte-macrophage colony stimulating factor; and / or The method further comprises inducing monocytes to be cultured using immune cell culture medium; Preferably, the dosage of the granulocyte-macrophage colony stimulating factor is 0.1-1000 ng / mL, preferably 10-200 ng / mL; Preferably, the immune cell culture medium includes X-VIVO10 culture medium, X-VIVO15 culture medium or X-VIVO20 culture medium, more preferably X-VIVO15 culture medium.

7. The method according to any one of claims 4 to 6, characterized in that: The method comprises the following steps: (1) first culturing monocytes using an immune cell culture medium; (2) adding the vitamin D receptor agonist to the immune cell culture medium after the first culture, and performing a second culture; (3) changing the medium and performing a third culture on the cells after the second culture using an immune cell culture medium; Preferably, the immune cell culture medium in step (3) contains granulocyte-macrophage colony stimulating factor; Preferably, the immune cell culture medium in step (1) does not contain granulocyte-macrophage colony stimulating factor; Preferably, the immune cell culture medium comprises X-VIVO10 culture medium, X-VIVO15 culture medium or X-VIVO20 culture medium, preferably X-VIVO15 culture medium; Preferably, the immune cell culture medium comprises fetal bovine serum, preferably 5-15% fetal bovine serum; Preferably, the first culture time is 20-26h; Preferably, the second culture time is 6-10h; Preferably, the third culture time is 14-18 hours.

8. Macrophages prepared according to the method of any one of claims 4 to 7; Preferably, the macrophages have at least one upregulated M1 macrophage marker and / or at least one downregulated M2 macrophage marker; More preferably, the macrophages have elevated expression of CD80 and / or CD86; More preferably, the macrophages have reduced expression of CD163 and / or CD206; More preferably, the macrophages are M1 macrophages.

9. Use of the macrophage prepared by the method of any one of claims 4 to 7 or the macrophage according to claim 8 in the preparation of a drug for preventing, treating or alleviating inflammatory diseases and / or tumors.

10. The use according to claim 9, characterized in that: The tumor is a solid tumor; Preferably, the drug also includes other anti-tumor therapeutic agents; Preferably, the drug is used to inhibit the growth of tumor cells and / or induce apoptosis of tumor cells.

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