Method for in-vitro photon domestication of M2 macrophages and application

Abstract

The invention discloses a method for in-vitro photon domestication of M2 macrophages and application, and belongs to the technical field of biological medicines. The method comprises the following steps: separating a tibia and a femur of a mouse, flushing bone marrow, removing red blood cells and impurities, and obtaining BMDMs of the mouse through a cell seed plate; bMDMs are cultured for 5-10 days, a cell culture medium is used for inducing the cells to be attached to the wall, then the cell culture medium is diluted with a cell culture solution, the cells are put into a cell incubator for incubation, a micro-nano photon material and a light field are used for regulating and inducing the macrophages, and the M2 type function-enhanced macrophages are obtained and can be used for drugs for treating ubiquitous vascular plaques. The M2 type macrophage with specificity and function stability is obtained by inducing and regulating cell differentiation through a micro-nano photon material and a light field regulation technology by utilizing the frequency single characteristic of laser, a rapid, accurate and efficient macrophage photon domestication method is established, and clinical transformation application of engineering transformation of the macrophage is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for in-vitro photon domestication of M2 macrophages and application. BACKGROUND

[0002] In the world heart report released by WHM in 2023, 2050 million people died of cardiovascular diseases in 2021, accounting for about one-third of the total number of global deaths, and cardiovascular and cerebrovascular plaques accounted for 85% of the number of deaths from cardiovascular diseases. Research has found that long-term pro-inflammatory stimulation of blood vessels after injury is a key factor in the formation of vascular plaques. Studies have shown that neutralizing pro-inflammatory factor IL-1β using monoclonal antibodies can reduce the pro-inflammatory response of macrophages, but this reduction in pro-inflammatory response has led to a sharp increase in the number of fatal infections due to a decrease in neutrophils, and in the CIRT trial, the use of long-term low-dose methotrexate (an anti-inflammatory drug) by the test subjects did not reduce the incidence of vascular plaques. Therefore, simple drug molecule therapy is inadequate for the treatment of vascular atherosclerosis.

[0003] The traditional treatment methods mainly include: (1) long-term use of statins to indirectly delay the growth of vascular plaques by reducing blood lipids in the blood, which is a slow process for the treatment of vascular plaques and only has certain indirect effects in the early stage of plaque formation, but for the onset period of plaque occlusion of blood vessels, thrombolytic drugs are needed. (2) Use of thrombolytic drugs in the emergency onset period, which activate the human fibrinolytic system to dissolve fibrin in the thrombus and restore blood vessel patency, but the use of thrombolytic drugs can cause bleeding risk (most common) and problems such as disruption of blood clotting function, anaphylactic shock, and vascular dilation. (3) In the case of vascular embolism caused by the use of statins, surgical vascular stents or vascular bypass surgery are used, but this surgery can only be used for large blood vessels and is ineffective for small blood vessels, especially brain blood vessels. Most importantly, stents can cause the formation of more vascular plaques, and long-term use of anti-allergic drugs is required.

[0004] Currently, the immunotherapy for the control and elimination of vascular plaques and systemic vascular plaques is through drug molecules to regulate metabolic pathways for macrophage polarization. Studies have revealed how metabolic reprogramming affects the immune response of atherosclerosis, including changes in glucose metabolism, glycosaminoglycan metabolism and retinoic acid signaling during the induction of M1 and M2 phenotype polarization. These studies have been provided from proteomics and phosphoproteomics and transcriptomics. Therefore, changing and regulating the metabolic pathways of macrophages is a necessary condition for obtaining and establishing an effective immune response. For example, M1 macrophages rely on aerobic glycolysis to generate ATP, increasing the consumption of glucose and glutamine, but they inhibit oxidative metabolism. In contrast, M2 macrophages maintain an intact tricarboxylic acid (TCA) cycle and tend to oxidative metabolism, especially fatty acid oxidation (FAO), as a model for ATP production. Within atherosclerotic plaques, monocyte-derived M2 macrophages are converted to an anti-inflammatory, fibrogenic phenotype by taking up modified lipoproteins, especially LDL, which can confer protection by promoting tissue repair, relieving inflammation and limiting lesion development. Therefore, it is essential to understand how cellular pathways and metabolites coordinate macrophage activation.

[0005] Glutaminolysis-derived α-KG is an anti-inflammatory metabolite that maintains the M2 phenotype and controls the metabolic reprogramming of M2 macrophages through a jmjd3-dependent mechanism. The lipolysis process induced by lysosomal acid lipase (LAL) is important for the increase in oxidative phosphorylation, the enhancement of spare respiratory capacity (SRC), the prolongation of survival time and the expression of genes that collectively determine M2 activation. In a mouse model, mitogen-activated protein kinase (MEK) and histone deacetylase (HDAC) can also effectively promote M2 phenotype macrophage polarization and angiogenesis and wound healing. However, despite the use of specific kinases and inhibitor drugs to coordinate macrophage polarization, M2 macrophages are still prone to functional disorders due to the complex biological crosstalk in the local microenvironment. Therefore, it is very important to develop a fast and effective method to achieve loyal M2 macrophages. SUMMARY

[0006] To achieve the above-mentioned purpose, the main purpose of the present application is to provide a method for in vitro photon domestication of M2 macrophages.

[0007] Another purpose of the present application is to provide an M2 type function-enhanced macrophage prepared by the method for in vitro photon domestication of M2 macrophages.

[0008] Still another purpose of the present application is to provide the use of the M2 type function-enhanced macrophage in the preparation of a drug for treating pan-vascular plaques, and to provide an autoimmune treatment strategy for immune diseases without the action of a non-molecular drug.

[0009] To achieve the above object, the present application adopts the following technical solutions:

[0010] In a first aspect of the present application, a method for in vitro photon domestication of M2 macrophages is provided, comprising the following steps:

[0011] (1) Obtain mouse bone marrow macrophages (BMDMs), which comprises the following steps in sequence: isolate mouse tibia and femur, flush bone marrow, remove red blood cells and impurities, and plate cells to obtain BMDMs;

[0012] (2) Induce differentiation of macrophages, which comprises the following steps: culture BMDMs for 5-10 days, induce with cell culture medium, after the cells adhere, dilute the cell culture medium with cell culture solution, and place in a cell incubator for incubation;

[0013] (3) Use micro-nano photonic materials and light field to control and induce macrophages, which comprises the following steps: after stimulating the cells adhering through micro-nano photonic materials and light field, add carbon quantum dots in the cell culture solution and react for 6-10 hours, irradiate with laser, and then place in a cell incubator for incubation.

[0014] Preferably, in step (1), the process of isolating mouse tibia and femur comprises: taking 4-8 week old C57BL / 6 mice, sacrificing by cervical dislocation, and fully disinfecting with 75% alcohol, then isolating and removing mouse tibia and femur under sterile conditions, and placing them in a cell culture dish containing 75% alcohol for preservation.

[0015] Preferably, in step (1), the process of flushing bone marrow comprises: moving the mouse tibia and femur into a biological safety cabinet, separating and removing the tissue around the mouse tibia and femur, then washing 2-3 times in a cell culture dish containing PBS, and then transferring to a cell culture dish containing complete culture medium, cutting the bone ends of the mouse tibia and femur, then using a 1 mL syringe to suck the complete culture medium, flushing the bone marrow cells from one end of the bone into a 50 mL sterile centrifuge tube, and repeating multiple times until the bone is white; wherein the composition of the complete culture medium is 1% penicillin-streptomycin double antibody + 10% fetal bovine serum + DMEM base medium.

[0016] Preferably, in step (1), the process of removing red blood cells and impurities comprises: adding 5 times the volume of red blood cell lysis solution to the 50 mL sterile centrifuge tube, repeatedly blowing with a Pasteur pipette, and standing for 15 minutes; after standing, centrifuge at 1000 rpm / min for 10 minutes, discard the supernatant; resuspend the cells by adding DMEM cell culture solution, then filter the cells with a 200 mesh screen; centrifuge at 1000 rpm / min for 10 minutes, discard the supernatant, and repeat the cell washing twice.

[0017] Preferably, in step (1), the cell plating process comprises: resuspending the cells in a sterile centrifuge tube by adding DMEM complete medium containing 10 ng / mL M-CSF to induce the differentiation of bone marrow cells into macrophages; after cell counting, the cell density is adjusted to 1*10^6 / mL, and the cells are inoculated into a culture dish and cultured in a 37℃, 5% CO2 incubator.

[0018] Preferably, in step (2), for M1 macrophages, 100 ng / mL LPS is used to induce the cell culture medium, and after the cells adhere, 1 mg / mL LPS solution is diluted with cell culture medium and placed in the cell incubator for 12-24 h.

[0019] Preferably, in step (2), for M2 macrophages, 10 ng / mL IL-4 is used to induce the cell culture medium, and after the cells adhere, 0.1 mg / mL IL-4 solution is diluted with cell culture medium and placed in the cell incubator for 12-24 h.

[0020] Preferably, in step (3), the light source wavelength used in the light field regulation is 300-1400 nm, the photon energy regulation range is 1%-99%, the photon frequency regulation is in the visible light band, and the quarter-wave plate regulates the photon phase.

[0021] In a second aspect of the present application, an M2 type functional enhanced macrophage is provided, which is prepared by the method of in vitro photon domestication of M2 macrophages.

[0022] In a third aspect of the present application, the M2 type functional enhanced macrophage is used in the preparation of a drug for treating pan-vascular plaques.

[0023] Preferably, the drug uses the M2 type functional enhanced macrophage as the active ingredient and is administered by intravenous injection.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] I. In view of the problem in the prior art that the use of biological factors to induce macrophage differentiation leads to single signal pathway and incomplete differentiation, the present application uses micro-nano photonic materials and light field regulation technology to induce and regulate cell differentiation by using the frequency single characteristic of laser, domesticates macrophages for pan-vascular plaques, so that they are M2 type macrophages with specificity and stable function, establishes a rapid, accurate and efficient macrophage photon domestication technology, promotes the clinical application of macrophage engineering modification, and has important clinical application value.

[0026] Secondly, the application promotes the change of receptor molecules by using a single frequency of laser without adding any inducing chemical reagent, realizes the change of signal path of immune cells, and realizes the root cell differentiation of reprogramming from the gene, thereby providing a new idea for fast and stable immune cell induced differentiation.

[0027] Thirdly, the application domesticates M2 macrophages in vitro by using photons, and uses the M2 macrophages for treating pan-vascular plaques, so that more than 96% of mouse bone marrow cells or M0 macrophages can be converted into M2 type, 75% of mice with pan-vascular plaques can be cleaned up after being returned through the tail vein, the micro-nano photonic material can be used for accurately targeting macrophages, the light field regulation technology can quickly start the metabolism of macrophages, the macrophages are controlled by using photon frequency, polarization, energy and frequency, and the macrophages have the advantages of high directional polarization efficiency, high purity, fast expansion of the macrophages, and direct use. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A flow chart for the process of using photon domesticated macrophages to clean up pan-vascular plaques in the body in the embodiment.

[0029] Figure 2 Experimental results of micro-nano photonic material and light field regulation for stimulating BMDMs to polarize into M2 type macrophages in vitro in the embodiment; wherein: under the same culture conditions, (a) qPCR analysis of the mRNA expression amount of M1 marker genes of BMDMs stimulated by micro-nano photonic material (PPIX), micro-nano photonic material and light field regulation (PPIX-L), IL-4, LPS and control for 12 hours; (b) qPCR analysis of the mRNA expression of M1 / M2 marker genes of BMDMs stimulated by 100 ng / mL LPS after the PPIX-L step; (c) under the same culture conditions, the macrophage morphology images of BMDMs stimulated by PPIX-L, LPS, PPIX-L+LPS and control for 0h, 6h and 12h, respectively; (d) qPCR analysis of the mRNA expression amount of M1 / M2 marker genes of PPIX-L, LPS, PPIX-L+LPS and control; *p<0.05, **p<0.01, ***p<0.001, the data represent 3 independent experiments, and each group has 5 samples (a-d; mean±s.d); (h) flow cytometry results, APC anti-mouse I-A / I-E antibody and PE anti-mouse CD163 antibody are used for immunofluorescence staining of BMDMs treated by untreated, IL-4, LPS and PPIX-L.

[0030] Figure 3To show the effect of BMDMs on the clearance of inducers of pan-atherosclerotic plaque formation by apoptotic red blood cells, high-density lipoprotein and low-density lipoprotein under different stimulations in the examples; 20 pg / mL ox-LDL: green treated macrophages; 20 pg / mL LDL: pink; 10 4 / mL RBC: red; observed by laser confocal scanning microscopy after 5h incubation; n.s.: no significant difference; *P<0.05, **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test, data represent 3 independent experiments or 2 independent experiments with 3 samples per group.

[0031] Figure 4 To show the results of animal experiments with macrophages obtained from BMDMs under different stimulations in the examples; Apoe- / - mice were imaged on a 9.4T Bruker BioSpec MR scanner using a 3D spoiled gradient echo sequence with an isotropic field of view of 24 mm 3 ; TR 20.8 ms; TE 2.6 ms; flip angle 20°, resulting in a voxel size of 47 x 63 x 94 pm 3; c, d are 3D MR images of brain arteries of 60-day and 68-day mice, respectively; f: MR transverse image of carotid artery before injection of photon-tamed M2 macrophages; g, h are 3D MR images of carotid artery of 60-day and 68-day mice, respectively; j: 3D MR image of brain artery of mice 1 day after injection of photon-tamed M2 macrophages; e, i, k are statistical analysis of MR images of aortic plaques of Apoe- / - mice in untreated group, LPS group, IL-4 group and photon group (n = 10 biological independent mice; see Figs. S27-S28); e) brain artery occlusion rate; i) carotid artery occlusion rate; k) brain artery occlusion rate at different times after injection of photon-tamed M2 macrophages; l: representative images of ORO staining of facial aorta after different treatments (n = 5 biological independent mice; see Fig. S29); n: cross-sectional images of Masson's staining of aortic root (scale bar, 400 pm) and H&E staining of aortic root (scale bar, 400 pm); m: quantification of ORO-positive lesion area; o and q: ratio of ORO-positive lesion area to whole aorta (o) and aortic root (q) (n = 5 biological independent mice); p, r and s: representative microscope images of heart sections stained with Masson's trichrome (p); H&E (r); scale bar, 400 pm; blue in Masson's trichrome staining is the collagen cap region; red 5-pointed star in H&E staining indicates the necrotic core region and a large number of acellular cholesterol crystals; s: quantitative analysis of fibrosis area of whole aortic root plaque (n = 5 biological independent mice); data analysis was performed by way of ANOVA and Dunnett's T3 post-hoc test, and represented as mean ± s.d.; ***P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DETAILED DESCRIPTION

[0032] In order to more fully understand the technical solutions, objectives and advantages of the present application, the technical effects produced by the present application will be further described in detail below in combination with the drawings and specific examples. Obviously, the described examples are only a part of the examples of the present application, but not all. It should be noted that other examples obtained by those skilled in the art without departing from the concept of the present application all belong to the protection scope of the present application.

[0033] Example 1

[0034] According to the technical route shown in Figure 1 Photon-tamed macrophages were prepared in vitro as follows:

[0035] Step 1, obtain mouse bone marrow macrophages (BMDMs)

[0036] (1) Isolation of mouse tibia and femur: Take 4-8 weeks old C57BL / 6 mice, sacrifice by cervical dislocation, and sterilize with 75% alcohol. Isolate and remove the tibia and femur of the mouse under sterile conditions without breaking the bone, and then put it into a cell culture dish containing 75% alcohol.

[0037] (2) Flush the bone marrow: The isolated mouse tibia and femur are transferred to a biological safety cabinet, the remaining tissue around the tibia and femur is removed, washed 2-3 times in a cell culture dish containing PBS, and transferred to a cell culture dish containing complete medium (1% penicillin-streptomycin double antibody + 10% fetal bovine serum + DMEM base medium). The bone ends of the tibia and femur are cut with ophthalmic scissors, 1 mL syringe is used to suck complete medium, and the medium is flushed from one end of the bone to a 50 mL sterile centrifuge tube to flush bone marrow cells, and repeated several times until the bone is white.

[0038] (3) Remove red blood cells and other impurities: Add 5 times the volume of red blood cell lysis solution to the 50 mL sterile centrifuge tube, repeatedly blow with a Pasteur pipette, and after standing for 15 min, centrifuge at 1000 rpm / min for 10 min, discard the supernatant, resuspend the cells with an appropriate amount of DMEM cell culture solution, and filter the cells with a 200 mesh filter. Centrifuge at 1000 rpm / min for 10 min again, discard the supernatant, and repeat twice to wash the cells.

[0039] (4) Cell plating: After the last centrifugation, discard the supernatant, resuspend the cells in DMEM complete medium containing 10 ng / mL M-CSF in a sterile centrifuge tube, and induce the differentiation of bone marrow cells into macrophages. After cell counting, adjust the cell density to 1 x 10^6 / mL, inoculate the cells in a culture dish, and incubate in a 37°C, 5% CO2 incubator.

[0040] Step 2, micro-nano photonic material and light field regulation induced macrophages

[0041] (1) After about 7 days of BMDM culture, macrophage induction and differentiation:

[0042] For M1, use 100 ng / mL LPS medium for induction. After the cells adhere, take 1 mg / mL LPS solution from -20°C refrigerator, dilute the LPS solution with culture medium, use it to culture cells, and put it in an incubator for 12-24 h.

[0043] For M2, use 10 ng / mL IL-4 medium for induction. After the cells adhere, dilute the 0.1 mg / mL IL-4 solution with culture medium, use it to culture cells, and put it in an incubator for 12-24 h.

[0044] (2) After the micro-nano photonic material and the light field regulation, a proper amount of carbon quantum dots is added to the cell culture solution, and after 8 hours of reaction, the cells are irradiated with a laser and incubated in a cell incubator to obtain M2 type functional enhanced macrophages.

[0045] The above-mentioned micro-nano photonic material and the light field regulation system for inducing macrophages have the functions of photon energy regulation, photon polarization regulation, photon frequency regulation and photon phase regulation, and include:

[0046] A light source module for emitting photons, including a tunable laser and a light beam regulation component; the wavelength of the light source module is 300-1400 nm, the photon energy regulation range is 1%-99%, the photon frequency regulation range is the visible light band, and a quarter wave plate regulates the photon phase;

[0047] A light path adjustment module for regulating the light source emitted photons and the light beam processing component, and adjusting the incident light irradiation range;

[0048] A cell and sample input module for culturing and domesticating the macrophages output from the photon domestication system and the domestication transmission module; including a fast and accurate microsyringe with an injection speed in the range of nl / s-mL / s, the syringe tip is connected with a microchannel to directly enter the system cabin, vertically injects into the system culture dish, the rate is controllable, the position is adjustable, and it is ensured that the number of injected samples and cells is within the controllable range;

[0049] A cell domestication culture module for irradiating and testing the polarization of the macrophages after the regulation of the photons; the culture dish in the sample injection system cabin is conveyed to the irradiation under the expanded light beam by a conveyor belt.

[0050] In particular, the system for inducing macrophages by micro-nano photonic material and light field regulation includes a laser, a broadband beam splitter, a polarizer, a quarter wave plate, an energy regulation, a light beam expander, a temperature and CO2 controller, a stage (culture dish and conveyor belt) in the system cabin, a light beam expander, a microsyringe, and a sample input pipeline (plastic and stainless steel).

[0051] Example 2: Animal experiment

[0052] (I) Establishment of mouse myocardial infarction model: anterior descending artery permanent ligation model, steps as follows:

[0053] The neck and left chest are shaved and disinfected with 80% ethanol.

[0054] Place the mouse flat and place a mask on its nose and mouth to maintain anesthesia.

[0055] Pinch the mouse tail and hind foot to check the reflex to determine that the mouse has been fully anesthetized.

[0056] A midline incision of the neck was performed under a microscope to separate the skin, muscle and tissue covering the trachea.

[0057] When the trachea was exposed, a small hole was cut in the tissue between the two rings below the glottis and a tracheal tube was inserted. The tracheal tube was inserted using a microsurgical probe to support the cranial part of the trachea. The chest movement was checked to ensure that both lungs were well ventilated.

[0058] The respiratory rate (RR) was approximately 110 / min and the inspiratory pressure was 17-18 cm H20. The mouse was carefully turned on its right side, facing the left side. A thoracotomy was performed between the third and fourth ribs on the left side, and the tissue and muscle were dissected carefully using a cautery to prevent bleeding.

[0059] The thoracic cavity was carefully opened and the heart was found without touching the lungs with any sharp objects. The pericardium covering the heart was removed.

[0060] The LAD was located between the pulmonary artery and the left auricle, and was ligated using an 8-0 Prolene suture (Ethicon, Norderstedt, Germany) with a suture at the proximal end of the LAD.

[0061] A chest drain (28G, venous catheter) was placed between the 4th and 5th ribs.

[0062] The thoracic incision was layered and sutured with a 6-0 Prolene continuous suture (Ethicon, Norderstedt, Germany) and the ribs were sutured with a 4-0 Prolene running suture (Ethicon, Norderstedt, Germany) to close the skin.

[0063] The remaining gas in the thoracic cavity was carefully aspirated with a 2 mL syringe. The mouse was placed flat and the tracheal tube was removed and sutured with a 7-0 Prolene suture (Ethicon, Norderstedt, Germany) using a one-needle method.

[0064] The skin was sutured with a 4-0 Prolene continuous suture (Ethicon, Norderstedt, Germany).

[0065] (ii) Intravenous injection of macrophages into the tail vein of mice

[0066] (1) Preparation:

[0067] 1. The animals were placed on a heating plate at 37°C for about 10 minutes, and observed during heating to identify signs of overheating.

[0068] 2. Resuspend isolated monocytes in 150 μΐ of NaCl and load cells into a 1 mL insulin syringe using a 30G needle. Gently vortex the solution before loading into the syringe to ensure all monocytes are injected and handle cells on ice to avoid heat-mediated adhesion and activation of monocytes.

[0069] (2) Mouse tail vein injection and fixation:

[0070] 1. When selecting a mouse for intravenous injection, avoid disturbing other mice in the cage.

[0071] 2. Place the mouse into the restrainer without too much stress and be careful of the hind legs. Note: Alternatively, use general anesthesia to ensure the procedure.

[0072] 3. Make sure the mouse has room to breathe before closing the restrainer.

[0073] (3) Injection of the transfusion:

[0074] 1. Disinfect the injection site with disinfectant and spray on the mouse tail, let stand for at least 1 minute.

[0075] 2. Limit the venous blood flow of the tail by applying gentle pressure on the lateral tail above the injection site for better visualization of the vein.

[0076] 3. Turn the tail 90 degrees before injection.

[0077] 4. Inject at a 45-degree angle, inject slowly, no more than 5 μΐ per gram, and avoid harming the animal.

[0078] 5. If edema appears, which is a sign of a failed injection, stop immediately and repeat the injection at a closer location.

[0079] 6. Press the injection side gently for about 1 minute to stop the bleeding.

[0080] 7. After the procedure, open the restrainer and place the mouse in the cage.

[0081] 8. Observe the mouse for 20 minutes to make sure the animal is not injured from the injection and is awake from the anesthesia.

[0082] Isolation, formation, and stimulation of mouse BMDMs and results characterization as shown in Figure 2 Tibiae and femurs of 4-week-old mice were flushed with PBS supplemented with 2% heat-inactivated fetal bovine serum to collect bone marrow cells, which were then cultured in BMDM growth medium for 7 days before maturation and analysis of the purity of the cell population. For further analysis of macrophage polarization, cells were stimulated with LPS to activate M1 or with IL-4 to activate M2, and analyzed for phagocytosis of Figure 2a) Polarized macrophages were evaluated for changes in cell morphology, surface marker expression, cytokine production, and cell signaling pathway activation: bone marrow mononuclear cells, M0 macrophages, and induced M2 macrophages under 40x microscopy Figure 2 b0-b2) Surface markers associated with activation were analyzed by flow cytometry after il-4, LPS, and photon stimulation. M2 response markers CD163 and CD206 were evaluated after stimulation, and CD68 and MCH II markers of Ml phenotype were analyzed after stimulation. APC anti-mouse I-A / I-E antibody was used to identify MCH II of Ml macrophages, and PE anti-mouse CD163 antibody was used to identify M2 macrophages Figure 2 c-k), n.s.: not significant; *P<0.05, **P<0.01, ***P<0.001 unpaired two-tailed Student’s t test, data are 3 independent experiments, 10 samples per group.

[0083] Functional characterization of macrophages as shown in Figure 3 20 pg / mL ox-LDL: green treated macrophages; 20 pg / mL LDL: pink; 104 / mL RBC: red. After 5 h incubation, observed by laser confocal scanning microscopy, n.s.: not significant; *P<0.05, **P<0.01, ***P<0.001 unpaired two-tailed Student’s t test. Data represent 3 independent experiments or 2 independent experiments, 3 samples per group.

[0084] The anti-atherosclerotic efficacy of photo-tamed M2 macrophages in Apoe- / - mice was evaluated by quantifying lesion area and assessing plaque stability features as shown in Figure 4 Carrying plaque Apoe- / - mice were administered intravenously (i.v.) twice a week for 1 week with saline, LPS-stimulated macrophages, IL-4-stimulated macrophages, and photo-tamed M2 macrophages, respectively. a, Timeline and treatment regimen as shown in Figure 4 a, Schematic and anatomical structure of mouse brain arteries and aorta as shown in Figure 4 b-b1.

[0085] Carrying plaque Apoe- / - mice were imaged using a 9.4T Bruker BioSpec MR scanner with a 3D fast gradient echo sequence with an isotropic field of view of 24 mm 3 a repetition time of 20.8 milliseconds, an echo time of 2.6 milliseconds, a flip angle of 20°, and a resulting voxel size of 47 x 63 x 94 micrometers 3 3D NMR images of mouse brain arteries at 60 days and 68 days as shown in Figure 4c and 4d, cross-sectional NMR images of the mouse carotid artery before injection of light-tamed M2 macrophages are shown in Figure 4 f, 3D NMR images of the mouse carotid artery at 60 days and 68 days are shown in Figure 4 g and 4h, 3D NMR images of the mouse brain artery one day after injection of light-tamed M2 macrophages are shown in Figure 4 j.

[0086] Statistical analysis of magnetic resonance imaging of aortic plaques of Apoe- / - mice in different groups, including untreated group, LPS group, IL-4 group and light group (n=10 biologically independent mice). Among them, the cerebral artery occlusion rate is shown in Figure 4 e, the carotid artery occlusion rate is shown in Figure 4 i, the cerebral artery occlusion rate at different times after injection of light-tamed M2 macrophages is shown in Figure 4 k, the representative images of the front of the aorta stained with ORO after different treatments (n=5 biologically independent mice) are shown in Figure 4 l. Cross-sectional images of the aortic root stained with Masson (scale, 400 microns) and cross-sectional images of the aortic root stained with H&E (scale, 400 microns) are shown in Figure 4 n, the quantification of the area of ORO-positive lesions in the aorta is shown in Figure 4 m, the ratio of the area of ORO-positive lesions to the whole aortic area in the aorta Figure 4 o) and the aortic root Figure 4 q) (n=5 biologically independent mice).

[0087] Representative microscope images of heart sections stained with Masson trichrome Figure 4 p) and H&E Figure 4 r), scale bar: 400 microns, blue in Masson trichrome represents collagen cap area, red pentagram in H&E represents necrotic core area and a large number of acellular cholesterol crystals. Quantitative analysis of the fibrotic area of the aortic root plaque to the whole aortic root area (n=5 biologically independent mice) is shown in Figure 4 s, the data was analyzed by one-way ANOVA and Dunnett's T3 post-hoc test, the results were expressed as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0088] To sum up, the application domesticates M2 macrophages in vitro by photons, and uses them to treat pan-vascular plaques, so that more than 96% of mouse bone marrow cells or M0 macrophages can be M2 type, 75% of mice pan-vascular plaques can be cleaned up after being returned through the tail vein, the micro-nano photonic material can accurately target macrophages, the light field regulation technology can quickly start the metabolism of macrophages, and the macrophages are controlled by the photon frequency, polarization, energy and frequency, so that the application has the advantages of high macrophage directional polarization efficiency, high purity, fast expansion of the macrophages and direct use.

[0089] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of in vitro photon domestication of M2 macrophages, characterized in that, It comprises the following steps: (1) Obtain mouse bone marrow macrophages BMDMs, including sequentially isolating mouse tibia and femur, flushing bone marrow, removing red blood cells and impurities, and cell plating, to obtain BMDMs; (2) Macrophage induction and differentiation, including: BMDMs are cultured for 5-10 days, induced with cell culture medium, and after cell adhesion, the cell culture medium is diluted with cell culture fluid, and then placed in a cell incubator for incubation; (3) Using micro-nano photonic materials and light field to induce macrophages, including: After stimulating the cells to adhere by micro-nano photonic materials and light field, carbon quantum dots are added to the cell culture fluid and reacted for 6-10 hours, and then placed in a cell incubator for incubation after laser irradiation.

2. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (1), the process of isolating mouse tibia and femur includes: taking 4-8 week old C57BL / 6 mice, sacrificing by cervical dislocation, and disinfecting with 75% alcohol, and then isolating and removing the mouse tibia and femur under sterile conditions, and placing them in a cell culture dish containing 75% alcohol for storage.

3. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (1), the process of flushing bone marrow includes: the mouse tibia and femur are moved into a biological safety cabinet, the surrounding tissue of the mouse tibia and femur is removed, and then placed in a cell culture dish containing PBS for washing 2-3 times, and then transferred to a cell culture dish containing complete culture medium, the both ends of the mouse tibia and femur are cut, and then 1 mL of a syringe is used to suck the complete culture medium, the complete culture medium is used to flush the bone marrow cells from one end of the bone to a 50 mL sterile centrifuge tube, and the process is repeated multiple times until the bone is white; wherein the complete culture medium comprises 1% penicillin-streptomycin double antibody + 10% fetal bovine serum + DMEM base medium.

4. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (1), the process of removing red blood cells and impurities includes: adding 5 times the volume of red blood cell lysis solution to the 50 mL sterile centrifuge tube, repeatedly blowing with a Pasteur pipette, and standing for 15 min; after standing, centrifuge at 1000 rpm / min for 10 min, discard the supernatant; add DMEM cell culture fluid to resuspend the cells, and then filter the cells with a 200 mesh filter; centrifuge at 1000 rpm / min for 10 min, discard the supernatant, and repeat the washing of the cells twice.

5. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (1), the process of cell plating includes: adding DMEM complete culture medium containing 10 ng / mL M-CSF to the sterile centrifuge tube to resuspend the cells, and inducing the bone marrow cells to differentiate into macrophages; after cell counting, the cell density is adjusted to 1×10^6 / mL, the cells are inoculated in a culture dish, and placed in a 37°C, 5% CO2 incubator for culture.

6. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (2), for M1 macrophages, 100 ng / mL LPS cell culture medium is used for induction, after cell adhesion, 1 mg / mL LPS solution is diluted with cell culture fluid, and then placed in a cell incubator for incubation for 12-24 h.

7. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (2), for M2 macrophages, 10 ng / mL IL-4 cell culture medium is used for induction, and after the cells are attached, 0.1 mg / mL IL-4 solution is diluted with cell culture solution and placed in a cell incubator for 12-24 h.

8. The method of in vitro photonically domesticating M2 macrophages of claim 1, wherein, In step (3), the light source wavelength used in the light field regulation is 300-1400 nm, the photon energy regulation range is 1%-99%, the photon frequency regulation is the visible light band, and the quarter-wave plate regulates the photon phase.

9. A function-enhanced macrophage of M2 type, characterized by, The M2 macrophages are prepared by the method of any one of claims 1 to 8.

10. The use of the M2 type functional enhanced macrophages of claim 9 in the preparation of a drug for treating pan-vascular plaques, wherein the drug takes the M2 type functional enhanced macrophages as an active ingredient and is administered by intravenous injection.

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