Macrophage overexpressing IL-10, preparation method thereof and application of macrophage in intervention of atherosclerosis

Abstract

The embodiment of the invention provides a macrophage overexpressing IL-10, and further provides a preparation method of the macrophage overexpressing IL-10, and application of the macrophage overexpressing IL-10 in preparation of a product for preventing or treating atherosclerosis. According to the invention, RAW264.7 cells are infected by packaging recombinant lentivirus overexpressing mouse source IL-10, the macrophage overexpressing IL-10 is finally prepared, and the macrophage overexpressing IL-10 can reduce the plaque area and the plaque necrosis core and increase the plaque stability when intervening AS. The macrophage overexpressing IL-10 is expected to be applied to clinical prevention and / or treatment of atherosclerosis, reduction of ischemic cardiovascular disease burden and reduction of occurrence of cardiovascular events.

Description

Technical Field

[0001] The embodiments of the present application belong to the field of biomedicine technology, and specifically relate to a macrophage overexpressing IL-10, a method for preparing the macrophage overexpressing IL-10, and an application in intervening in atherosclerosis. Background Art

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide. [1] Atherosclerosis (AS) is one of the main pathological bases of CVD in my country. [2] Despite the existence of mature treatment strategies for AS-related diseases, the risk of recurrent cardiovascular events remains [3] Results of randomized controlled trials show that among patients receiving statins, residual inflammatory risk is more significantly associated with cardiovascular events and all-cause mortality than residual cholesterol risk [4] .

[0003] There is still great controversy about whether anti-inflammatory therapy is suitable for the treatment of human AS-related diseases. Recent clinical trials have provided new evidence for the use of anti-inflammatory drugs. The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) trial demonstrated the benefits of neutralizing antibodies against interleukin (IL)-1β in patients with stable coronary heart disease after myocardial infarction. Despite systemic treatment, these patients had high levels of high-sensitivity C-reactive protein. Anti-inflammatory treatment reduced the relative risk of recurrent myocardial infarction, stroke, or cardiac death by 15%. However, the incidence of infection in patients receiving antibody treatment was slightly increased and statistically significant. Since IL-1β is involved in the host defense mechanism, this side effect is not surprising. [5] . The natural drug colchicine has been used for anti-inflammatory treatment for many years and has become a routine treatment for pericarditis. Two important clinical trials have reported the efficacy of colchicine in reducing the recurrence of cardiovascular events after acute coronary syndrome. The COLCOT (Colchicine Cardiovascular Outcomes Trial) trial showed that the incidence of the primary composite endpoint (including cardiac death, non-fatal myocardial infarction or non-fatal stroke) was reduced by 23%. However, the incidence of pneumonia in patients treated with colchicine more than doubled. [6]The LoDoCo2 (Low Dose Colchicine 2) study gave low-dose colchicine (5 mg / day) to patients with stable coronary artery disease and reported similar results to those in the COLCOT trial. [7] These large-scale clinical trials further consolidate the prospects for clinical transformation of decades of basic research on the inflammatory pathogenesis of AS. Under the premise of limiting the impact on the patient's own defense mechanism, anti-inflammatory therapy is expected to become an effective means of preventing and treating AS-related diseases.

[0004] Interleukin-10 (IL-10) is a soluble cytokine produced by immune cells such as macrophages and regulatory T cells. [8] , which plays an important role in limiting the host's immune response to pathogens, reducing the damage caused by pathogens to the host, and maintaining tissue homeostasis. [9-13] Studies have reported that elevated serum IL-10 levels are associated with a better prognosis in patients with acute coronary syndrome who have elevated C-reactive protein. [9] Elevated baseline IL-10 levels in patients with acute coronary syndrome are an independent predictor of long-term adverse cardiovascular events

[14] Recombinant human IL-10 is currently in clinical trials for the treatment of various inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, chronic hepatitis C, and tumors. [15-17] Currently, there are no clinical trials to verify the efficacy of IL-10 in treating AS. Direct use of IL-10 monomers as therapeutic drugs has significant limitations. Since the half-life of IL-10 in the body is very short (about 3 minutes), it is difficult to reach a sufficient concentration at the site of the disease. Therefore, a larger dose and a higher frequency of medication are required to achieve a therapeutic effect, which brings inconvenience to patients and increases the possibility of side effects such as immunosuppression and infection. [16,17] To make up for this shortcoming, it is necessary to design a strategy that can effectively deliver IL-10 in vivo to better promote the effect of IL-10 in clinical applications.

[0005] Based on this, the present invention provides a method for preparing macrophages overexpressing IL-10, using a lentivirus carrying IL-10 mRNA to infect macrophages, constructing macrophages overexpressing IL-10, and synergizing the functions of macrophages and IL-10. On the one hand, the anti-plaque effect of IL-10 is exerted; on the other hand, the macrophage phenotype is regulated to create a microenvironment in the plaque that is more conducive to plaque stabilization and regression. Summary of the invention

[0006] The first purpose of the present invention is to provide a method for preparing macrophages overexpressing IL-10, so as to solve the problem that the existing IL-10 monomer has low stability and limited effect as a therapeutic drug.

[0007] The second object of the present invention is to provide an application of macrophages overexpressing IL-10 in the preparation of products for intervening in atherosclerosis.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for preparing macrophages overexpressing IL-10 comprises the following steps:

[0010] 293FT cells were cultured and transfected with recombinant lentivirus that overexpressed mouse IL-10;

[0011] RAW264.7 cells were infected with a recombinant lentivirus overexpressing mouse IL-10, selected with puromycin, and then expanded and cultured to obtain macrophages overexpressing IL-10.

[0012] In the present invention, the recombinant lentivirus for transfection and packaging overexpression of mouse IL-10 adopts a three-plasmid co-transfection system, including pMD, psPAX and pCDH-GFP-Pure-mIL-10-Myc.

[0013] The present invention also includes the step of isolating and purifying the recombinant lentivirus overexpressing mouse IL-10, which is specifically as follows: culturing the recombinant lentivirus overexpressing mouse IL-10, collecting the supernatant, centrifuging it, and then filtering it to obtain a concentrated recombinant lentivirus sample.

[0014] Furthermore, the centrifugation is performed at 3000-6000 rpm / min and 4° C. for 3-8 min.

[0015] Preferably, the centrifugation is performed at 5000 rpm / min and 4° C. for 5 min.

[0016] Furthermore, the filtration is first performed by filtering with a filter and then by ultrafiltration centrifugation.

[0017] Preferably, the filter is a 0.45 μm filter.

[0018] The ultrafiltration centrifugation is performed at 3000-6000 rpm / min and 4° C. for 15-25 min.

[0019] Furthermore, the ultrafiltration centrifugation is performed at 5000 rpm / min and 4° C. for 20 min.

[0020] A macrophage overexpressing IL-10 is obtained by the above preparation method.

[0021] Application of IL-10-overexpressing macrophages in the preparation of products for intervening in atherosclerosis.

[0022] In the present invention, the product for intervening atherosclerosis includes the above-mentioned macrophages overexpressing IL-10.

[0023] Furthermore, the dosage form of the product for intervening in atherosclerosis is an injection.

[0024] Furthermore, the product for intervening atherosclerosis is administered at an early stage of atherosclerotic plaque formation.

[0025] The present invention has the following beneficial effects:

[0026] (1) The method for preparing macrophages overexpressing IL-10 of the present invention is to infect RAW264.7 cells with a recombinant lentivirus overexpressing mouse IL-10, thereby finally preparing macrophages overexpressing IL-10. The macrophages overexpressing IL-10 can reduce the plaque area and the necrotic core of the plaque and increase the stability of the plaque by intervening in AS.

[0027] (2) The macrophages overexpressing IL-10 of the present invention can effectively reduce the overall plaque load and increase plaque stability when intervening in the early stage of plaque progression. Although the effect of reducing the proportion of plaque to arterial lumen when intervening in the late stage of plaque progression is not significant, it can still increase plaque stability. Therefore, macrophages overexpressing IL-10 are expected to be used in the clinical prevention and / or treatment of atherosclerosis, reduce the burden of ischemic cardiovascular disease, and reduce the occurrence of cardiovascular events. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. Some specific embodiments of the present application will be described in detail in an illustrative and non-restrictive manner with reference to the drawings. The same reference numerals in the drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the drawings:

[0029] Figure 1 is the concentration of IL-10 in IL-10M and ConM medium under LPS and oxLDL stimulation conditions;

[0030] Figure 2 is the concentration of IL-6 in IL-10M and ConM medium under LPS stimulation;

[0031] Figure 3 is the concentration change diagram of TNF-α in IL-10M and ConM culture medium under oxLDL stimulation;

[0032] Figure 4The graph is the change of IL-10 secretion concentration in IL-10M and ConM culture medium;

[0033] Figure 5 The graph is the change of TNF-α secretion concentration in IL-10M and ConM culture medium;

[0034] Figure 6 RT-qPCR was used to detect macrophage polarization phenotype markers;

[0035] Figure 7 To detect macrophage polarization phenotype markers for flow cytometry analysis;

[0036] Figure 8 After AS model mice were injected with different numbers of IL-10M via the tail vein, the aorta was isolated for ex vivo fluorescence imaging, and the aorta was placed in a 6-cm culture dish;

[0037] Fig. 9 For tail vein injection, 1 × 10 7 Imaging was performed at different intervals after each IL-10M. Fig.10 After AS model mice were injected with PBS, IL-10M and ConM through the tail vein, blood vessels in the aortic plaque were isolated and stained with DAPI and observed under a fluorescence microscope; scale bar: 50 μm;

[0038] Fig.11 A flowchart for the intervention program;

[0039] Fig.12 The results of aorta oil red O staining after treatment in different subgroups of the previous intervention group;

[0040] Fig.13 The results of Oil Red O staining of aorta after treatment in different subgroups of the late intervention group;

[0041] Fig.14 H&E, Masson staining and immunohistochemistry results of plaques in the early intervention group and the late intervention group after treatment, scale bar: 50μm;

[0042] Figure 15-18 Statistical analysis of each marker. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only embodiments of a part of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0044] The abbreviations and key terms used in the present invention are defined as follows:

[0045] 1. Atherosclerosis (AS): Atherosclerosis is a disease in which lipid plaques (atheromas or atherosclerotic plaques) form on the walls of medium-sized or large arteries, reducing blood flow or blocking blood flow. Atherosclerosis is caused by repeated damage to the arterial walls. Many factors can cause this damage, including high blood pressure, smoking, diabetes, and high blood cholesterol levels. Blockage of blood vessels caused by atherosclerosis is a common cause of heart attacks and strokes. Usually, the first symptom is pain or cramps when blood flow cannot meet the tissue's demand for oxygen. To prevent atherosclerosis, you need to quit smoking, improve your diet, exercise regularly, and control your blood pressure, cholesterol levels, and diabetes. Atherosclerosis progresses to life-threatening complications, such as heart attack or stroke, which requires emergency treatment. Cardiovascular disease, mainly coronary artery disease (atherosclerosis affects the arteries that supply blood to the heart) and stroke (atherosclerosis affects the arteries that supply blood to the brain), is the leading cause of death worldwide. Atherosclerosis is the most important and common type of arteriosclerosis, which can affect the medium-sized and large arteries in the brain, heart, kidneys, other vital organs, and legs.

[0046] 2. IL-10

[0047] Interleukin-10 (IL-10) is a soluble cytokine produced by immune cells such as macrophages and regulatory T cells. It plays an important role in limiting the host's immune response to pathogens, reducing the damage caused by pathogens to the host, and maintaining tissue homeostasis.

[0048] 3. Macrophages

[0049] Macrophages are a type of cell that originates from monocytes and has the function of phagocytosis and digestion. Macrophages are one of the most important members of the mononuclear macrophage system. They can be found in various tissues throughout the body and play important functions such as immune regulation, removal of cell waste and antibacterial.

[0050] 4. Cell therapy

[0051] Cell therapy refers to the transplantation or injection of normal or bioengineered human cells into the patient's body. The newly injected cells can replace damaged cells or have stronger functions, thereby achieving the purpose of treating the disease. Immune cell therapy.

[0052] The present invention proposes to genetically modify RAW264.7 macrophages to overexpress IL-10, inject the genetically modified macrophages into an AS mouse model via tail vein injection, and observe their effects on AS lesions, thereby verifying the application prospect of a new inflammatory intervention therapy in the treatment of AS.

[0053] Example 1

[0054] 1. Construction of IL-10 overexpressing macrophages

[0055] RAW264.7 and HEK293T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0056] A method for preparing macrophages overexpressing IL-10 comprises the following steps:

[0057] (1) 293FT cells were passaged in 6 cm culture dishes and cultured until their confluence reached 70%.

[0058] (2) ① 0.5 μg of pMD, 1.5 μg of psPAX, 2 μg of pCDH-GFP-Pure and 300 μL Buffer gently mixed. Vortex for 10s and mix thoroughly. This scheme packages the control lentivirus, named LV-GFP. ② pMD 0.5μg, psPAX 1.5μg, pCDH-GFP-Pure-mIL-10-Myc 2μg and 300uL Buffer was gently mixed. Vortexed for 10 seconds to mix thoroughly. This protocol packaged a lentivirus that overexpressed mouse IL-10 and was named LV-IL10.

[0059] (3) Add 8 μL of each of the two mixed solutions in (2). reagent, vortex for 1 second, and incubate at room temperature for 10 minutes.

[0060] (4) Add the mixture from (3) to the cells to complete the transfection process.

[0061] (5) The culture medium was replaced 4 hours after transfection. The supernatant was collected once within 24-36 hours after replacement and supplemented with 5 mL of culture medium. The supernatant was collected again 60 hours after transfection.

[0062] (6) The supernatant collected in step 5 was centrifuged at 5000 rpm / min and 4°C for 5 min to precipitate cells and other impurities. The supernatant was then aspirated and filtered through a 0.45 μm filter, added to a millipore ultrafiltration centrifuge tube, and centrifuged at 5000 rpm / min and 4°C for 20 min to obtain approximately 200 μL of concentrated LV sample, which could be stored at -80°C or used immediately, depending on the situation.

[0063] (7) RAW264.7 cells were plated on a 24-well plate one day before infection, with 3 wells and 10,000 cells per well. Then, 20 μL of concentrated LV was added to two wells of each cell type the next day. Green fluorescence was visible under a fluorescence microscope about 72 hours later. Puromycin selection was performed 5 days after infection, with 5 μg / ml selection for 24 hours. The remaining live cells were expanded and cultured. Cells successfully infected with LV-IL10 were labeled IL-10M, and cells successfully infected with LV-GFP were labeled ConM.

[0064] 2. ELISA determination of cytokines in the supernatant of IL-10M culture medium

[0065] (1) Collect conditioned medium of macrophages stimulated with lipopolysaccharide (LPS), oxLDL, and unstimulated macrophages, centrifuge at 3000 rpm for 15 min at 2-8°C, and take the supernatant. Use the supernatant immediately in the experiment or store it at -20°C or -80°C after aliquoting. Avoid repeated freezing and thawing.

[0066] (2) The experiment was performed according to the instructions of mouse IL-10, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) ELISA kits.

[0067] (3) Determine the secretion trend of IL-10 and TNF-α in the culture medium supernatant over time: After IL-10M and ConM are cultured to the logarithmic growth phase, replace the culture medium with new one, and take the culture medium supernatant 6h, 8h, 12h, 24h, and 36h after the culture medium is replaced, and determine the concentration of IL-10 and TNF-α according to the above steps.

[0068] The effect of lentiviral infection was determined by detecting the concentration of IL-10 in IL-10M and ConM culture media. The results showed that the concentration of IL-10 in IL-10M culture media was significantly higher than that in ConM. Although IL-10 secretion was inhibited after stimulation by LPS and oxLDL, it was still significantly higher than that in ConM ( Figure 1 In addition, when stimulated by LPS, the concentration of IL-6 in IL-10M medium was significantly lower than that in ConM ( Figure 2 ); when stimulated by oxLDL, the concentration of TNF-α in IL-10M medium was significantly lower than that in ConM ( Figure 3 IL-10M continued to secrete IL-10 within 36 hours after the culture medium was replaced, which was always significantly higher than that of ConM, while the secretion trend of TNF-α was similar to that of ConM ( Figure 4 , Figure 5 ).

[0069] 3. RT-qPCR determination of IL-10M polarization phenotype

[0070] IL-10M and ConM were treated with 100 ng / mL LPS for 12 h and 20 μg / mL oxidized oxLDL for 24 h, respectively. mRNA was extracted using the RNAsimple Total RNA Kit. Reverse transcription was performed according to the instructions of the ReverTra Ace qPCR RT Master Mix Kit. The cDNA obtained by reverse transcription was mixed with SYBR Green Real-Time PCR RTMaster Mix and RT-qPCR was performed according to the instructions. The experiment was repeated at least three times, and β-actin was used as an internal reference. The relative expression of the samples was calculated by using the 2-ΔΔCt method.

[0071] The designed primers are as follows:

[0072] Cd206:forward 5'-CTCTGTTCAGCTATTGGACGC-3',

[0073] reverse 5'-TGGCACTCCCAAACATAATTTGA-3';

[0074] Arg1:forward 5'-CTGGGGATTGGCAAGGTGAT-3',

[0075] reverse 5'-CAGCCCGTCGACATCAAAG-3';

[0076] Tnf-α:forward 5'-AGCCGATGGGTTGTACCTTG-3',

[0077] reverse 5'-ATAGCAAATCGGCTGACGGT-3';

[0078] Inos:forward 5'-AGCTCGGGTTGAAGTGGTATG-3',

[0079] reverse 5'-CACAGCCACATTGATCTCCG-3';

[0080] β-actin:forward 5'-GGCTGTATTCCCCTCCATCG-3',

[0081] reverse 5'-CCAGTTGGTAACAATGCCATGT-3'.

[0082] RT-qPCR results showed that the expression of IL-10M M2 phenotype markers Cd206 and Arg1 increased significantly, and Cd206 mRNA remained highly expressed after LPS stimulation ( Figure 6 Flow cytometry analysis showed similar results, with a higher proportion of IL-10M positive for CD206 than for the M1 phenotype marker CD86 ( Figure 7 ).

[0083] Example 2

[0084] Application of IL-10-overexpressing macrophages in intervention of atherosclerosis

[0085] 1. Construction of AS model mice

[0086] Eight-week-old female ApoE- / - mice were selected and fed with a western diet (WD) containing 21% fat, 50% carbohydrates, 20% protein and 0.21% cholesterol for three months to induce AS.

[0087] 2. Targeting of IL-10M to AS plaques

[0088] In vivo validation of the targeting of IL-10M to AS plaques

[0089] After the ApoE- / - mice were established, IL-10M was injected through the tail vein. According to previous literature, the number of injected cells was set as follows: 1×10 7 , 1×10 6 , 1×10 5 According to the time intervals for macroscopic imaging after injection, 48h, 24h, 12h, and 6h groups were set up, and macroscopic fluorescence imaging of the aorta was performed by an optical imaging system. The aorta was taken for paraffin-embedded pathological sections to detect the green fluorescent protein (GFP) fluorescence signal of IL-10M to confirm the targeting of IL-10M to plaques.

[0090] The results of in vitro aortic imaging showed that the aortic fluorescence signal increased with the increase of cell number ( Figure 8 ). The fluorescence signal began to decay 6 hours after IL-10M injection ( Fig. 9 ). Fluorescence analysis of aortic plaque pathological sections showed that green fluorescence from IL-10M was clearly observed in the plaque area of ​​mouse arteries, further proving that IL-10M can specifically target plaques, and its targeting ability is not inferior to ConM ( Fig.10 ).

[0091] 3. Effect of IL-10M intervention on aortic plaque area

[0092] Intervention process and grouping First, ApoE- / - mice were divided into two groups according to the start time of treatment. The early intervention group (n=45) received tail vein injections from 6 weeks of age and started feeding WD at the same time. The late intervention group (n=45) began to receive injections at 16 weeks of age after feeding WD for 2 months. According to different injection reagents, the mice were further divided into IL-10M, ConM and PBS groups. The early intervention group (early stage of plaque formation) received an injection every 2 weeks, and the late intervention group (late stage of plaque formation) received an injection every 1 week, and the injection was stopped after being fed WD for 5 months. All mice were killed after completing their respective injection schedules, and tissue and blood samples were collected. The weight and survival of the mice were continuously observed and recorded throughout the process.

[0093] Intervention process diagram Fig.11 As shown in Figure 2, the intervention results showed that the AS plaque area in the aorta of mice injected with IL-10M was significantly reduced compared with mice injected with ConM or PBS in both the early and late intervention groups ( Fig.12 and Fig.13 ).

[0094] Effect of IL-10M intervention on aortic plaque stability

[0095] To evaluate the pathological characteristics of aortic plaques after treatment in different groups, histopathological sections of aortic arch plaques were stained.

[0096] H&E staining results showed that in the early intervention group, the ratio of plaque area to lumen area in mice treated with IL-10M was significantly reduced, however, no significant statistical difference was observed in the late intervention group ( Figures 14 to 18 Notably, the area of ​​necrotic core within aortic plaques was significantly smaller in mice treated with IL-10M compared with mice treated with ConM or PBS in both the early and late intervention groups ( Figures 14 to 18). Molecular marker immunohistochemical staining was used to analyze the changes in plaque composition. The results showed that although no statistically significant difference was observed in the pre-intervention group, mice treated with IL-10M showed higher levels of IL-10 expression in their plaques ( Figures 14 to 18 In the late intervention group, the expression of IL-10 in the plaques of mice treated with IL-10M was significantly higher than that of mice treated with PBS ( Figures 14 to 18 There were no significant differences in CD68-positive macrophages, α-SMA-positive smooth muscle cells, or collagen content as indicated by Masson staining among the different treatment groups. However, a significant decrease in the content of matrix metalloproteinase 9 (MMP9), a biomarker associated with plaque rupture, was observed in the plaques of mice treated with IL-10M in both the early and late intervention groups ( Figures 14 to 18 ).

[0097] References

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[0114]

[17] WANG

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing macrophages overexpressing IL-10, characterized in that: The following steps are involved: 293FT cells were cultured and transfected with recombinant lentivirus that overexpressed mouse IL-10; RAW264.7 cells were infected with a recombinant lentivirus overexpressing mouse IL-10, selected with puromycin, and then expanded and cultured to obtain macrophages overexpressing IL-10.

2. The method for preparing macrophages overexpressing IL-10 according to claim 1, characterized in that: The recombinant lentivirus that overexpressed mouse IL-10 was transfected and packaged using a three-plasmid co-transfection system, including pMD, psPAX, and pCDH-GFP-Pure-mIL-10-Myc.

3. The method for preparing macrophages overexpressing IL-10 according to claim 1, characterized in that: The method also includes the steps of isolating and purifying the recombinant lentivirus overexpressing mouse IL-10, which is specifically as follows: culturing the recombinant lentivirus overexpressing mouse IL-10, collecting the supernatant, centrifuging it, and then filtering it to obtain a concentrated recombinant lentivirus sample.

4. The method for preparing macrophages overexpressing IL-10 according to claim 3, characterized in that: Centrifugation was performed at 3000-6000 rpm / min and 4°C for 3-8 min.

5. The method for preparing macrophages overexpressing IL-10 according to claim 3, characterized in that: The filtration is first performed by filtering with a filter and then by ultrafiltration centrifugation.

6. The method for preparing macrophages overexpressing IL-10 according to claim 5, characterized in that: The ultrafiltration centrifugation is performed at 3000-6000 rpm / min and 4° C. for 15-25 min.

7. A macrophage overexpressing IL-10, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the IL-10 overexpressing macrophages according to claim 7 in preparing a product for intervening in atherosclerosis.

9. The use of the IL-10 overexpressing macrophages according to claim 8 in preparing a product for intervention of atherosclerosis, characterized in that: The product for intervention of atherosclerosis includes the macrophages overexpressing IL-10.

10. The use of the IL-10 overexpressing macrophages according to claim 9 in preparing a product for intervention of atherosclerosis, characterized in that: The dosage form of the product for intervening in atherosclerosis is injection.

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