Macrophage as well as hypoxia induction culture method and application thereof

Abstract

The invention relates to the technical field of macrophage differentiation, in particular to a macrophage and a hypoxia induction culture method and application thereof. The macrophages are obtained by inducing and culturing mature macrophages in a low-oxygen environment. According to the macrophage hypoxia induction culture method, bone marrow cells are cultured to be mature by adopting a macrophage culture medium, and are subjected to induction culture for at least 3 days in a hypoxia environment. The macrophage is applied to preparation of a product which is transplanted to the heart to treat myocardial infarction. The low-oxygen pretreated macrophages constructed through low-oxygen treatment effectively avoid the double-cutting-sword characteristic of the macrophages after myocardial infarction, so that the inflammatory response of the macrophages in the early stage is not very strong, and fibrosis transition proliferation can be well balanced in the later-stage repair promoting effect.

Description

Technical Field

[0001] The present invention relates to the technical field of macrophage differentiation, and in particular to a macrophage and a hypoxia-induced culture method and application thereof. Background Art

[0002] Macrophages can be categorized by origin into peripheral monocyte-derived macrophages, embryonic yolk sac-derived macrophages, and embryonic liver-derived macrophages. The latter two are collectively referred to as resident macrophages because they colonize and self-renew within mammalian tissues after birth. Functionally, macrophages can be divided into inflammation-related M1 macrophages and repair-related M2 macrophages.

[0003] M1 macrophages produce a large number of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), as well as chemokines such as monocyte chemoattractant protein-1 (MCP-1) and chemokine (C-C motif) ligand 2 (Ccl2), to recruit more monocytes to eliminate pathogens. M1 macrophages also produce reactive oxygen species (ROS) by activating NADPH oxidase to eliminate microorganisms. M2 macrophages, on the other hand, can produce anti-inflammatory molecules such as IL-10 and transforming growth factor-β (TGF-β).

[0004] It is important to note that the classification of M1 and M2 phenotypes is based on in vitro cell experiments. Macrophage polarization refers to the process by which macrophages develop into specific functional phenotypes through a series of signaling pathways under different microenvironmental stimulation. M1 macrophages are induced by interferon-γ (IFN-γ) and lipopolysaccharide (LPS), while M2 macrophages are induced by IL-4 and IL-13. Neither M1 nor M2 macrophages exist solely in myocardial tissue following myocardial infarction, as macrophages are exposed to a complex microenvironment and express different cell surface markers to exert different functions.

[0005] However, as research progresses, new macrophage phenotypes are constantly being discovered, and cellular RNA transcriptome sequencing technology provides a new and specific method for identifying macrophage transcriptional characteristics.

[0006] Macrophages play a complex and dynamic dual role in myocardial injury and repair after myocardial infarction (MI), with their functional regulation directly influencing the outcome of cardiac remodeling. During the acute injury phase (24-72 hours after MI), macrophages primarily promote inflammation and clear necrosis. During the subacute repair phase (3-7 days), macrophages primarily contribute to inflammation resolution and tissue remodeling. Finally, during the chronic remodeling phase (>1 week), macrophages regulate fibrosis and repair tissue. Macrophages exhibit a double-edged sword nature after MI. Early proinflammatory responses are essential for clearing necrotic tissue, but persistent activation leads to aggressive remodeling. During the repair phase, M2 polarization underlies scar stability, but excessive activation may limit cardiac regenerative potential. Summary of the Invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a macrophage and a hypoxia-induced culture method and application thereof.

[0008] A type of macrophage called M H Type 2 macrophages were obtained by inducing and culturing mature macrophages in a hypoxic environment.

[0009] Preferably, the mature macrophages are derived from bone marrow.

[0010] Preferably, the mature macrophages are prepared by the following steps: bone marrow cells are separated from the body, and cultured in a macrophage culture medium until they mature.

[0011] Specifically, the following operations are performed: (1) 8-12 week old C57BL / 6 mice were anesthetized and killed by cervical dislocation and then immersed in 75% alcohol for 5 min. (2) Expose the mouse femur, free the surrounding muscles and connective tissues, and completely remove both femurs of the mouse; (3) Soak the separated femur in 4°C PBS buffer and carefully remove the attached muscle and connective tissue; (4) Cut both ends of the femur and repeatedly flush the bone marrow cavity with a 1 mL syringe; (5) Repeatedly blow the bone marrow cavity flushing fluid to disperse the cell clumps; (6) After filtering through a 70 μm cell sieve, centrifuge at 500 g for 5 min at 4°C; (7) Discard the supernatant, add 1 mL of red blood cell lysis buffer, place at room temperature for 5 minutes, and then add 4 mL of PBS buffer to neutralize; (8) Centrifugation at 500 g for 5 min at 4°C; (9) Discard the supernatant and add 5 mL of PBS buffer to resuspend the cell pellet; (10) Centrifuge at 500 g for 5 min at 4°C; (11) Discard the supernatant and resuspend the cell pellet in macrophage culture medium; (12) Place the cells in a cell incubator and replace the culture medium after 3 days; (13) After culturing for 7 days, undifferentiated mature bone marrow-derived macrophages were obtained.

[0012] Preferably, the volume fractions of the gases in the hypoxic environment are as follows: 1% oxygen, 5% carbon dioxide, and the balance nitrogen.

[0013] Preferably, M H M1 macrophages are quasi-round and have fewer pseudopodia than undifferentiated mature macrophages, M1 macrophages, and M2 macrophages.

[0014] Preferably, M H The expression of IL-1α, Nos2, IL-6, IL-1β, and TNF genes in M1 macrophages was lower than that in M1 macrophages and higher than that in M2 macrophages.

[0015] Preferably, M H The Arg-1 gene expression in M1 macrophages is higher than that in M1 macrophages.

[0016] Preferably, M H The expression of Tgtβ1 gene in M2 macrophages is lower than that in M3 macrophages.

[0017] Preferably, M H The expression of Pparg gene in M2 macrophages is lower than that in M2 macrophages and higher than that in M1 macrophages.

[0018] Preferably, M H The IL-10 gene expression of M1 macrophages is higher than that of M1 macrophages and M2 macrophages.

[0019] The above M H A method for preparing macrophages comprises the following steps: culturing bone marrow cells in a macrophage culture medium until they mature, and inducing culture in a hypoxic environment for at least 3 days.

[0020] Preferably, the volume fractions of the gases in the hypoxic environment are as follows: 1% oxygen, 5% carbon dioxide, and the balance nitrogen.

[0021] Specifically, the following specific operation is adopted: undifferentiated mature bone marrow-derived macrophages are placed in a cell culture incubator containing 1% oxygen and 5% carbon dioxide for at least 3 days.

[0022] The above M H The macrophages are used in any of the following (1)-(6): (1) Prepare products that can be transplanted into the heart to improve the long-term survival rate of patients with myocardial infarction; (2) Preparation of products for transplantation into the heart to inhibit or alleviate adverse ventricular remodeling after myocardial infarction; (3) Preparation of products for transplantation into the heart to reduce left ventricular scarring after myocardial infarction; (4) preparing products that can be transplanted into the heart to increase the proportion of type III collagen fibers in myocardial tissue after myocardial infarction; (5) Preparation of products for transplantation into the heart to preserve cardiac function after myocardial infarction; (6) Preparing a product for transplantation into the heart to inhibit the increase in left ventricular internal diameter or / and left ventricular volume after myocardial infarction.

[0023] Preferably, the product is a drug or a reagent.

[0024] Beneficial effects: The hypoxia-pretreated macrophages obtained by the present invention through hypoxia treatment effectively avoid the "double-edged sword" characteristics of macrophages after myocardial infarction, making the early inflammatory response of macrophages less intense, while the fibrosis transition proliferation can be well balanced in the later pro-repair effect.

[0025] The present invention induces bone marrow-derived mononuclear macrophages to form undifferentiated macrophages by isolating and culturing them, and constructs hypoxia-pretreated macrophages by hypoxia treatment; at the same time, undifferentiated macrophages are induced to differentiate into M1 / M2 macrophages. Sequencing technology is then used to analyze the different subtypes of macrophages (M N 、M H The transcriptome genes of M1, M2 macrophages were differentially expressed, and GO analysis, KEGG analysis and protein interaction analysis were used to compare M H The differentially expressed genes between MH macrophages and traditional M1 / M2 macrophages and their related functions were analyzed; and qPCR was used to detect the differentially expressed genes between MH macrophages and traditional M1 / M2 macrophages.

[0026] The applicant discovered that M H The up-regulated genes of M-type macrophages were mainly concentrated in energy metabolism and tissue microenvironment changes; the down-regulated genes were concentrated in inflammatory response. H The high expression of IL-10 in M1 macrophages is significantly higher than that in M1 macrophages and M2 macrophages. H This type of macrophage has a strong ability to promote angiogenesis and tissue repair, but a weak ability to induce inflammatory response and cause tissue damage, suggesting that it should be a new macrophage subtype.

[0027] The present invention established a mouse myocardial infarction model and a blank control (sham operation group), and injected different types of macrophages into the myocardium and found that: H MI mice containing type III macrophages have better long-term survival rate, can promote myocardial repair after myocardial infarction in mice, increase the proportion of type III collagen fibers in myocardial tissue after myocardial infarction, reduce left ventricular scar formation after myocardial infarction, inhibit the increase of left ventricular inner diameter and / or left ventricular volume after myocardial infarction, inhibit or reduce ventricular adverse remodeling after myocardial infarction, and preserve cardiac function after myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 M H A is the difference between M1 macrophages and traditional M1 and M2 macrophages. H A is a volcano plot of transcriptome gene changes compared with M1 macrophages or M2 macrophages; B is a volcano plot of transcriptome gene changes compared with M1 macrophages or M2 macrophages; H Venn diagram of upregulated genes in transcriptome of M1 macrophages compared with M1 macrophages and M2 macrophages; C is M H Venn diagram of downregulated genes in the transcriptome of M1 macrophages compared with M1 macrophages and M2 macrophages.

[0029] Figure 2 M H Statistical graphs showing the differential expression of some genes in M1 and M2 macrophages. Figure A shows the differential expression of IL-1α, Nos2, IL-6, IL-1β, and TNF in macrophages of different types; Figure B shows the differential expression of Arg-1, Tgtβ1, and Pparg in macrophages of different types; and Figure C shows the differential expression of IL-10 in macrophages of different types.

[0030] Figure 3 M H Functional analysis of upregulated genes in type 2 macrophages. H Heat map of differentially expressed genes among M1 macrophages, M2 macrophages, and B is the KEGG analysis of M H Function of upregulated genes in macrophages, C is GO analysis M H Type 2 macrophages upregulate gene function, D is M H Protein interaction analysis of upregulated genes in type 2 macrophages.

[0031] Figure 4 M H Functional analysis of down-regulated genes in type 2 macrophages. H Type 2 macrophage down-regulated gene function, B is GO analysis M H Type 2 macrophages downregulate gene function, C is MH Protein interaction analysis of downregulated genes in type 2 macrophages.

[0032] Figure 5 Figure 3 shows the effect of hypoxia on macrophage phenotypic transformation. A shows F4 / 80 immunofluorescence staining of different macrophage subtypes, and B shows the statistical graph of pseudopodia of different macrophage subtypes.

[0033] Figure 6 This is the long-term survival curve of different subtypes of macrophages transplanted into mice after myocardial infarction.

[0034] Figure 7 Figure 1 shows the effects of transplantation of different macrophage subtypes on ventricular remodeling in mice after MI. A shows a comparison of cardiac geometry in each group of mice after MI (n=3); B shows the heart-to-body weight ratio in each group 28 days after MI.

[0035] Figure 8 Figure 1 shows the effect of transplantation of different macrophage subtypes on left ventricular scar formation in mice after MI. A shows Masson-stained paraffin sections of myocardial tissue from mice in each group 28 days after MI (n=3). B shows the statistical analysis of cardiac scar area and left ventricular wall thickness in mice in each group 28 days after MI.

[0036] Figure 9 Figure 1 shows the effect of transplantation of different macrophage subtypes on collagen fiber types in ventricular remodeling in mice after MI. A shows picrosirius red staining (white light) of myocardial tissue from each group of mice 28 days after MI (n=5); B shows picrosirius red staining (polarized light) of myocardial tissue from each group of mice 28 days after MI (n=5); and C shows the ratio of type I to type III collagen in myocardial tissue from each group of mice 28 days after MI.

[0037] Figure 10 Figure 1 shows the effect of transplantation of different macrophage subtypes on left ventricular function in mice after MI. Figures A and B show the EF and FS values ​​of mice in each group assessed by echocardiography 28 days after MI, respectively. Figures C and D show the LVID and LV volume values ​​of mice in each group 28 days after MI, respectively.

[0038] above Figure 2 、 Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 In the middle, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001. DETAILED DESCRIPTION

[0039] The present invention will be further explained below with reference to specific embodiments.

[0040] Example 1M H The difference between M1 macrophages and traditional M1 and M2 macrophages Preparation of undifferentiated mature bone marrow-derived macrophages (M N macrophages): (1) 8-12 week old C57BL / 6 mice were anesthetized and killed by cervical dislocation and then immersed in 75% alcohol for 5 min. (2) Expose the mouse femur, free the surrounding muscles and connective tissues, and completely remove both femurs of the mouse; (3) Soak the separated femur in 4°C PBS buffer and carefully remove the attached muscle and connective tissue; (4) Cut both ends of the femur and repeatedly flush the bone marrow cavity with a 1 mL syringe; (5) Repeatedly blow the bone marrow cavity flushing fluid to disperse the cell clumps; (6) After filtering through a 70 μm cell sieve, centrifuge at 500 g for 5 min at 4°C; (7) Discard the supernatant, add 1 mL of red blood cell lysis buffer, place at room temperature for 5 minutes, and then add 4 mL of PBS buffer to neutralize; (8) Centrifugation at 500 g for 5 min at 4°C; (9) Discard the supernatant and add 5 mL of PBS buffer to resuspend the cell pellet; (10) Centrifuge at 500 g for 5 min at 4°C; (11) Discard the supernatant and resuspend the cell pellet in macrophage culture medium (DMEM medium containing 20 ng / mL M-CSF, 10% FBS, and 1x penicillin-streptomycin); (12) Place the cells in a cell incubator (temperature: 37°C, humidity: 90-95%) and replace the culture medium after 3 days; (13) After culturing for 7 days, undifferentiated mature bone marrow-derived macrophages (M N type macrophages).

[0041] To M N Macrophage polarization: A. Undifferentiated mature bone marrow-derived macrophages were cultured in a cell culture incubator containing 1% oxygen and 5% carbon dioxide (the balance being nitrogen) for at least 3 days to obtain macrophages. H type macrophages; B. Undifferentiated mature bone marrow-derived macrophages were placed in fresh growth medium containing 10% fetal bovine serum (FBS), 100 ng / mL lipopolysaccharide (LPS), and 50 ng / mL interferon-γ (IFN-γ) to obtain M1 macrophages; C. Undifferentiated mature bone marrow-derived macrophages were placed in fresh growth medium containing 10% FBS, 10 ng / mL interleukin-4 (IL-4), and 10 ng / mL interleukin-13 (IL-13) to obtain M2 macrophages.

[0042] The applicant found through transcriptome mRNA sequencing that compared with M1 macrophages, M H There were 583 up-regulated genes and 460 down-regulated genes in M2 macrophages; compared with M3 macrophages, H In type 2 macrophages, 165 genes were upregulated and 248 genes were downregulated (e.g. Figure 1 (as shown in A in the figure).

[0043] But this data can reflect that M H The applicant further found through Venn diagram analysis that: compared with M1 macrophages and M2 macrophages, M H There were 146 up-regulated genes and 199 down-regulated genes in type 2 macrophages (e.g. Figure 1 (As shown in B and C). H Type 2 macrophages have some relatively special molecular biological functions.

[0044] The applicant further verifies M H The similarities and differences between M1 and M2 macrophages were analyzed, and the marker genes of M1 or M2 macrophages were detected by qPCR.

[0045] The applicant found that: (1) Compared with M2 macrophages, M H The expression of IL-1α, Nos2, IL-6, IL-1β, and TNF (marker genes of M1 macrophages) in M1 macrophages showed a significant increase, but was still lower than that in M1 macrophages (such as Figure 2 (as shown in A); (2) M H The expression of Arg-1, Pparg and other genes in M2 macrophages was significantly lower than that in M2 macrophages but higher than that in M1 macrophages (e.g. Figure 2 (as shown in B); (3) It is worth noting that M H M1 macrophages show their uniqueness in the expression of IL-10. Compared with M1 and M2 macrophages, IL-10 is expressed in M H There was a significant increase in the number of macrophages (eg Figure 2 (as shown in C in the figure).

[0046] Example 2 MH Unique functions of macrophages The applicant conducted an in-depth analysis of transcriptome mRNA sequencing data, focusing on the study of M H The functional uniqueness of type I macrophages can further explain their role in tissue repair.

[0047] To M H GO analysis and KEGG analysis of 146 genes upregulated in M1 macrophages (compared with traditional M1 and M2 types) revealed that: H The genes upregulated in macrophages were mainly concentrated in pathways related to cellular energy metabolism. The protein interaction analysis showed that H Type 2 macrophages have a strong role in metabolic regulation, paracrine secretion and angiogenesis (mainly VEGF) (such as Figure 3 shown).

[0048] And for M H An analysis of 199 genes down-regulated in M1 macrophages (compared to traditional M1 and M2 types) revealed that their effects on inducing inflammatory responses and apoptosis were relatively weak (e.g. Figure 4 shown).

[0049] Based on the above analysis, M H The role of M1 macrophages in tissue repair is significantly stronger than that of traditional M1 and M2 macrophages, but they are weaker than M1 and / or M2 macrophages in mediating inflammatory responses and inducing cell apoptosis.

[0050] Example 3 Effect of hypoxia on macrophage phenotypic conversion In order to further study the relationship between macrophage phenotypic conversion and hypoxia stimulation, the applicant further conducted in vitro experiments.

[0051] The applicant found that the M H M1 macrophages are different from traditional M1 macrophages and M2 macrophages.

[0052] From the cell appearance, we can find that: N In comparison, M H Type 2 macrophages are round in shape and have few pseudopodia (e.g. Figure 5 A and B in the figure).

[0053] To M N 、M H After comparing M1 and M2 macrophages, it was found that M N M macrophages are long spindle-shaped with few pseudopodia; HM1 macrophages are spherical and have almost no pseudopodia; M2 macrophages are spherical and have many pseudopodia (such as Figure 5 A and B in the figure).

[0054] Therefore, it is believed that hypoxia preconditioning macrophages (M H M1-type macrophages are different from traditional M1 and M2 macrophages in appearance.

[0055] Example 4 Transplantation of M H Type 2 macrophages promote myocardial repair after myocardial infarction (MI) in mice A myocardial infarction model was established in 120 mice and a sham operation group (Sham group) was established in 10 mice as a blank control, as follows: (1) Anesthetize mice with isoflurane (3-4%) in an anesthesia induction chamber; (2) The mouse is fixed in a supine position on a constant temperature blanket and placed under a stereomicroscope. A ventilation mask is buckled over the nose and mouth of the mouse, with an oxygen flow rate of 300-500 mL / min and isoflurane 1-1.5% to maintain anesthesia; (3) Clean the hair on the neck and left side of the chest of the mouse and disinfect the skin with iodine disinfectant. Check the mouse's pain reflex to ensure that the mouse is fully anesthetized; (4) Under a stereomicroscope, the skin of the mouse neck was cut open through a midline incision, and the subcutaneous fat, neck glands and muscles were separated in sequence to expose the trachea; (5) The mouse trachea was cut under the cricoid cartilage and endotracheal intubation was performed; (6) Use a ventilator to assist breathing, with a respiratory rate (RR) of approximately 110-120 breaths / min and a pressure of approximately 17 cmH2O. Observe whether the frequency of the mouse's chest rise and fall is consistent with the ventilator frequency to ensure that the ventilator is effectively assisting breathing; (7) Change the mouse's body position to the right lateral decubitus position, make an incision in the skin between the 4th and 5th intercostal spaces on the left side, separate the intercostal muscles in sequence, and stop the bleeding with a cauterizer; (8) Open the chest cavity and avoid touching the lungs with sharp instruments. Carefully remove the pericardium and expose the heart; (9) Carefully observe the beating heart. The left anterior descending coronary artery of the mouse heart originates between the pulmonary artery and the left atrium and runs on the surface of the heart. Determine the ligation point and ligate the left anterior descending coronary artery with 7-0 prolene suture (for mice in the sham group, the needle was inserted and the suture was passed over the surface of the heart, but the blood vessels were not ligated, and damage to the coronary artery should be avoided); (10) Observe the color change of the heart surface distal to the ligation point of the left anterior descending artery (red-light red-red and white) to ensure the successful construction of the myocardial infarction model.

[0056] The mice model of myocardial infarction was randomly divided into two groups, and different types of macrophages or PBS were injected into the myocardium. The mice were divided into blank control PBS group (MI+PBS inj group), normoxic undifferentiated macrophage transplantation group (MI+M N inj group) and hypoxia preconditioned macrophage transplantation group (MI+M H inj group), as follows: (11) Enrichment of macrophages to be transplanted (M N macrophages or M H macrophages) 1×10 6 The cell pellet was resuspended in 30 μL PBS buffer; (12) A 50 μL microinjector was used to select three injection points in the myocardial infarction margin area of ​​each group of mice, and 10 μL of cell suspension or 10 μL of PBS buffer was injected into each point; (13) After the catheter is placed, the chest cavity is closed, the muscles are sutured with 7-0 prolene sutures, and the drainage tube is aspirated with a syringe to maintain negative chest pressure to prevent pneumothorax or pericardial tamponade; (14) Suture the chest skin with a continuous suture of 5-0 prolene; (15) Stop anesthesia and gradually transition from ventilator-assisted breathing to spontaneous breathing. Turn off the ventilator, remove the endotracheal tube, suture the tracheal incision with 7-0 prolene sutures, and suture the neck skin with 5-0 prolene sutures; (16) After disinfecting the skin incision again, place the mouse in an incubator until it recovers its autonomous activity.

[0057] 4.1 Porting M H Type 2 macrophages improve long-term survival in mice after MI The applicant found that compared with transplantation of undifferentiated mature macrophages (MI+M N inj group) and MI mice injected with PBS intramyocardially (MI+PBS inj group), MI mice transplanted with hypoxia-preconditioned macrophages (MI+M H inj group) had a significantly improved long-term survival rate (e.g. Figure 6 shown).

[0058] 4.2 Transplantation M H Type 2 macrophages alleviate left ventricular scar formation and inhibit adverse ventricular remodeling after MI in mice The applicant further discovered that transplantation of M H MI mice with type 2 macrophages (MI+M H inj group) with intact heart geometry (e.g. Figure 7 As shown in A), the heart-body ratio was significantly lower than that of the normoxic transplantation group (MI+M Ninj group) and PBS control group (MI+PBS inj group) (e.g. Figure 7 (shown in B).

[0059] By analyzing the scar area of ​​left ventricle after MI in mice, the applicant found that: MI+M H The left ventricular scar area of ​​mice in the inj group after MI was significantly smaller than that in the MI+M group. N inj group and MI+PBSinj group) (e.g. Figure 8 ), and the left ventricular wall thickness did not decrease significantly compared with the sham operation group (as shown in Figure 8 shown).

[0060] Further analysis of the collagen fiber types in the scar area revealed that: MI+M H After MI, the proportion of type III collagen fibers in myocardial tissue increased in the inj group mice, which was significantly higher than that in MI+M N inj group and MI+PBSinj group (e.g. Figure 9 shown).

[0061] 4.3 Transplantation M H Type 2 macrophages can preserve left ventricular function and alleviate adverse ventricular remodeling in mice after MI The applicant further measured cardiac function through small animal ultrasound and found that the cardiac function of mice in the MI+PBSinj group declined significantly after MI, which was about 50% lower than that of normal mice. At the same time, the left ventricular internal diameter and left ventricular volume measured by ultrasound in the MI+PBSinj group also increased significantly, which was 100% higher than that of normal mice.

[0062] MI+M H The cardiac function of mice in the inj group was only slightly decreased; and the increase in left ventricular internal diameter and left ventricular volume was not significant, and their cardiac function was significantly higher than that of mice in the MI+M group. N In the inj group, the left ventricular internal diameter and left ventricular volume were significantly lower than those in the MI+M group. N Mice in the inj group (e.g. Figure 10 shown).

[0063] The above results showed that: cardiac function of mice declined significantly after MI, and severe adverse remodeling occurred in the left ventricle; intramyocardial injection of PBS aggravated the deterioration of cardiac function and accelerated heart failure after MI; while transplantation of M H Type 2 macrophages can preserve cardiac function after MI and reduce the occurrence of adverse left ventricular remodeling.

[0064] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A macrophage, characterized in that: Its name is M H Type 2 macrophages were obtained by inducing and culturing mature macrophages in a hypoxic environment.

2. The macrophage according to claim 1, characterized in that The mature macrophages are derived from the bone marrow.

3. The macrophage according to claim 1, characterized in that The mature macrophages are prepared by the following steps: bone marrow cells are separated from the body and cultured in a macrophage culture medium until they mature.

4. The macrophage according to claim 1, characterized in that The volume fractions of each gas in a hypoxic environment are as follows: oxygen 1%, carbon dioxide 5%, and the balance nitrogen.

5. The macrophage according to claim 1, characterized in that The macrophages are quasi-round and have fewer pseudopodia than undifferentiated mature macrophages, M1 macrophages and M2 macrophages.

6. The macrophage according to claim 1, characterized in that The expression of IL-1α, Nos2, IL-6, IL-1β, and TNF genes in the macrophages is lower than that in M1 macrophages and higher than that in M2 macrophages; The Arg-1 gene expression of the macrophages is higher than that of M1 macrophages; The Tgtβ1 gene expression of the macrophages is lower than that of M2 macrophages; The Pparg gene expression of the macrophages is lower than that of M2 macrophages and higher than that of M1 macrophages; The IL-10 gene expression of the macrophages is higher than that of M1 macrophages and M2 macrophages.

7. A method for culturing macrophages induced by hypoxia according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: culturing bone marrow cells with macrophage culture medium until they mature, and placing them in a low oxygen environment for induction culture for at least 3 days.

8. The method for culturing macrophages by hypoxia induction according to claim 7, characterized in that: The volume fractions of each gas in a hypoxic environment are as follows: oxygen 1%, carbon dioxide 5%, and the balance nitrogen.

9. A macrophage according to any one of claims 1 to 6, for use in any one of the following (1) to (6): (1) Prepare products that can be transplanted into the heart to improve the long-term survival rate of patients with myocardial infarction; (2) Preparation of products for transplantation into the heart to inhibit or alleviate adverse ventricular remodeling after myocardial infarction; (3) Preparation of products for transplantation into the heart to reduce left ventricular scarring after myocardial infarction; (4) preparing products that can be transplanted into the heart to increase the proportion of type III collagen fibers in myocardial tissue after myocardial infarction; (5) Preparation of products for transplantation into the heart to preserve cardiac function after myocardial infarction; (6) Preparing a product for transplantation into the heart to inhibit the increase in left ventricular internal diameter or / and left ventricular volume after myocardial infarction.

10. The use according to claim 9, characterized in that: The product is a drug or reagent.

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