Mouse M2 type macrophage induction method and application

Abstract

The invention discloses a mouse M2 type macrophage induction method and application, and aims to solve the technical problem that mouse tumor cells cannot grow into tumors in a mouse body due to the fact that the polarization degree of current mouse M2 type macrophages is very low in the induction process. S2, after the cells are recovered and cultured, performing planking and virus infection on the recovered and cultured cells; s3, after the cells are subjected to planking and virus infection, protein and medicine configuration is carried out; s4, after the protein and the medicine are prepared, RAW264.7 M2 polarization condition setting is carried out; and S5, after the polarization condition of the RAW264.7 M2 is set. Protein drugs such as IL4, IL13, IL10, M-CSF, AZM, SOD and DEX are exogenously added, and lentivirus is used for mediating macrophages to stably express IL-4R receptors, so that M2 polarization of the macrophages is finally greatly improved, and a foundation is laid for subsequent research on growth and tumor formation of human / mouse tumor cells in mice.

Description

Technical Field

[0001] The present invention relates to the technical field of macrophage induction, and specifically to a method for inducing murine M2 macrophages and its application. Background Art

[0002] Tumors have always been a major factor plaguing human health. Every year, many people die from cancers caused by tumors. Therefore, it is extremely urgent to study the potential mechanisms of tumor occurrence, proliferation, metastasis, etc. Limited by ethical issues, scientific researchers use model animals as a carrier to study various mechanisms of tumors, so as to formulate strategies for controlling and eliminating tumors. We construct a transplanted tumor model in model animals, such as mice (a transplanted tumor model is a common experimental model used in cancer research, which involves transplanting tumor cells or tissues into experimental animals to study the growth, spread, and treatment response of tumors), so as to observe and experiment on the corresponding tumors to explore their occurrence and development laws, and better serve clinical practice. However, due to various reasons, the transplanted tumors often do not grow as expected. Scientific researchers promote the success of the transplanted tumor model by selecting appropriate cell lines or tissues, optimizing transplantation techniques, host environment management, biomechanical and biochemical factors, etc., but the results are often unsatisfactory.

[0003] In the tumor microenvironment, macrophages can be recruited and domesticated by tumor cells and transformed into tumor-associated macrophages, which usually have the characteristics of promoting tumor growth.

[0004] Macrophages play an important role in the interaction between innate and adaptive immune responses. They are clearly divided into classical M1 and M2 types. Generally, interferon-γ (IFN-γ) or lipopolysaccharide (LPS) activates M1 (classically activated) macrophages, which produce pro-inflammatory cytokines, phagocytize microorganisms, and initiate immune responses.

[0005] M2 (alternatively activated) macrophages are activated by cytokines, such as interleukin IL-4, IL-10, and IL-13. M2-polarized macrophages drive an anti-inflammatory environment to inhibit inflammation. M1 macrophages have a pro-inflammatory effect. Under the action of LPS, they are activated by Th1 cytokines such as IFN-γ and GM-CSF, and produce cytokines such as IL-1β, IL-6, IL-12, IL-23, and TNF-α. On the contrary, M2 macrophages have anti-inflammatory and immunomodulatory effects. Under the action of Th2 cytokines such as IL-4 and IL-13, they are polarized and produce cytokines such as IL-10 and TGF-β. These macrophage types have different functions and transcriptional profiles.

[0006] Macrophages exhibit bidirectional inflammatory regulation due to their plasticity, which enables them to undergo reversible polarization between pro-inflammatory M1 and anti-inflammatory M2 phenotypes in response to various challenges. The differentiated M1 and M2 macrophages can transform into each other, M2 into M1 and vice versa, depending on the reversible expression levels of the M1 and M2 subtype marker proteins.

[0007] In the process of realizing the invention, the inventors found that at least the following problems in the prior art have not been solved. The polarization degree of traditional murine M2 macrophages during induction is very low, resulting in the inability to allow murine tumor cells to grow into tumors in mice. Therefore, new technical solutions need to be designed to solve this problem. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for inducing murine M2 macrophages and its application, so as to solve the technical problem that the polarization degree of current murine M2 macrophages during induction is very low, resulting in the inability to allow murine tumor cells to grow into tumors in mice.

[0009] To achieve the above purpose, the present invention provides the following technical solutions: A method for inducing murine M2 macrophages and its application, including the following steps:

[0010] S1. First, resuscitate and culture the cells;

[0011] S2. After the cells are resuscitated and cultured, let the resuscitated and cultured cells be plated and infected with the virus;

[0012] S3. After the cells are plated and infected with the virus, then perform protein and drug preparation;

[0013] S4. After the protein and drug preparation is completed, set the RAW264.7 M2 polarization conditions;

[0014] S5. After the RAW264.7 M2 polarization conditions are set, let the M2 cell polarization supernatant promote tumor cell tumorigenesis;

[0015] S6. Finally, perform data analysis.

[0016] As a preferred embodiment of the present invention, for the cell resuscitation culture: First, take RAW264.7 cells stored at -80°C, resuscitate them at 39°C, then centrifuge them at 1000 rpm for 5 min using a horizontal centrifuge, discard the supernatant, take 1 mL of cell culture medium preheated to 37°C to resuspend the cells, transfer them into a T25 cell flask, supplement the culture medium to 5 mL, place them in a CO2 incubator, and culture them under the conditions of 5% CO2 and 37°C;

[0017] Cell seeding and virus infection: RAW264.7 cells were cultured in 90% DMEM + 10% FBS. The subcultured cells were digested with 0.25% trypsin and seeded into 24-well plates. When the confluence reached 80%, lentivirus overexpressing IL4R purchased from ABM was added for infection. After 24 hours, the cells were changed to normal medium for culture. After 48 hours of infection, positive cells were screened using 1 μg / mL puromycin.

[0018] Protein and drug preparation: Mouse IL4, IL13, IL10, and M-CSF were prepared into 100 ng / μL stock solutions with ddH2O, aliquoted, and stored at -80°C. AZM and DEX were prepared into 100 mM stock solutions with DMSO, aliquoted, and stored at -80°C. SOD was prepared into 1 μg / μL stock solution with ddH2O, aliquoted, and stored at -80°C.

[0019] Setting of RAW264.7 M2 polarization conditions: RAW264.7 / RAW264.7-IL4R cells were cultured in 90% DMEM + 10% FBS. The subcultured cells were digested with 0.25% trypsin and seeded into 6-well plates. When the confluence reached 80%, IL4, proteins, and AZM drugs were added for grouped treatment. After incubation for 48 hours, the cells were digested with 0.25% trypsin, and the cell pellet was resuspended with PBS and incubated with flow antibodies such as Arg-1-AF700 and CD206-FITC at 4°C for 30 minutes, then washed twice with PBS and detected by flow cytometry.

[0020] Promotion of tumor cell tumorigenesis by M2 cell polarization supernatant: The supernatant of the highest treatment group of M2 polarization cells was collected. Human gastric adenocarcinoma cells were cultured in 79% F12K + 20% FBS + 1% double antibody at 37°C with 5% CO2, expanded and cultured in 10 10-cm cell culture dishes. After digestion with 0.25% trypsin, the cells were resuspended with PBS, counted, and stored at 4°C. 200 μL of the cell suspension was subcutaneously inoculated into the left hind limb of 5-6-week-old female nude mice. After inoculation, the tumorigenesis situation and the size of the tumor mass were observed every 2-3 days, and samples were taken after 15 days for grouping.

[0021] Data analysis: Independent sample T-test was used for comparison between two groups of samples, and one-way ANOVA was used for comparison among multiple groups. The data were analyzed using SPSS, and each group had at least 3 replicates.

[0022] As a preferred embodiment of the present invention, the steps for screening positive cells are as follows:

[0023] M1. First, the RAW264.7 cells infected with the virus were maintained in culture with 1 μg / mL puromycin for 2 days.

[0024] M2. Then, the RAW264.7 cell bank after virus infection was diluted by the infinite dilution method and inoculated into a 96-well cell culture plate (1 cell per well). After the 96-well cell culture plate was fully grown, it was digested with 0.25% trypsin and inoculated into a 48-well plate until it was inoculated into a 6-well plate. After it was fully grown, it was digested with 0.25% trypsin;

[0025] M3. Then, qPCR and WB were detected;

[0026] M4. Finally, 1 / 3 of the cells were taken to extract total RNA for qPCR to verify the overexpression efficiency of IL4R, 1 / 3 of the cells were taken to extract total protein for WB to verify the overexpression efficiency of IL4R, and the other 1 / 3 were inoculated into the original 6-well plate for continuous culture. The operation was repeated for each well until a positive clone cell line was screened out.

[0027] As a preferred embodiment of the present invention, in the qPCR detection in step M3, Trizol was used to extract total RN2A from cells, and reverse transcription cDNA was used for qPCR to detect the transcriptional level of IL4R. The experimental steps were as follows:

[0028] Extracting total RNA from tissues by Trizol method

[0029] ① 1 ml of Trizol was added to the cells, pipetted and mixed evenly, placed at 4°C for 15 min to allow sufficient lysis, and then transferred to a 1.5 ml centrifuge tube;

[0030] ② According to 200 μl of chloroform / ml of Trizol, chloroform was added, shaken vigorously for 30 s, and then left to stand at 4°C for 15 min;

[0031] ③ Centrifuge at 12,000 rpm at 4°C for 15 min on a tabletop centrifuge to allow the solution to be fully stratified;

[0032] ④ Carefully pipette 400 μl of the supernatant and transfer it to a sterile 1.5 RNase-free centrifuge tube, add 400 μl of isopropanol, mix well, and precipitate at room temperature for 40 min;

[0033] ⑤ Centrifuge at 12,000 rpm at 4°C for 15 min on a tabletop centrifuge and discard the supernatant;

[0034] ⑥ Add 1 ml of 75% ethanol to wash twice, centrifuge at 12,000 rpm at 4°C for 5 min, and discard the supernatant;

[0035] ⑦ After the ethanol was evaporated in a laminar flow hood, 30 μl of DEPC water was added to dissolve the RNA and stored at -80°C for standby.

[0036] As a preferred embodiment of the present invention, in step M3, for WB detection, the cell sample is placed in a 1.5 ml EP tube, 1 ml of RIPA, 10 μl of PMSF, and 20 μl of phosphatase inhibitor are added thereto, lysed at 4°C for 1 h, centrifuged at 12000 rpm for 5 min, and the cell sample is quantitatively analyzed by BCA to detect the expression level of IL4R protein. The experimental steps are as follows:

[0037] X1. Take out the gel plate of the gel-making rack, install it on the electrophoresis rack, place it in the electrophoresis tank, and pour 1X Tris-Gly-SDS electrophoresis buffer until the scale line;

[0038] X2. Pull out the electrophoresis comb, load the sample, after electrophoresis at 75 V for 60 min (when the bromophenol blue reaches the separating gel), electrophoresis at 120 V for 80 min, and stop electrophoresis when the bromophenol blue indicating line reaches the bottom;

[0039] X3. Transfer the membrane: Cut a PVDF membrane according to the PAGE gel electrophoresis area (generally 4×8 cm specification), then activate it with methanol for 1 min, and then equilibrate it in the transfer buffer for 30 s;

[0040] X4. Remove the PAGE gel from the glass plate, place it on the filter paper, mark the membrane, cover it on the gel, drive out the bubbles, cover the filter paper and sponge, close the transfer clip, pour in the pre-cooled transfer solution, and place the transfer tank in an ice-water bath. The transfer electron flow direction is: black plate → sponge → filter paper → gel → membrane → filter paper → sponge → red plate;

[0041] X5. The transfer condition is a constant current of 125 mA (125 mA for one group, 250 mA for two groups, and so on), and the time is the molecular weight plus 10 min;

[0042] X6. Blocking: After the transfer is completed, take out the membrane and block it at room temperature for 1 h with TBST containing 5% skim milk (it can be overnight at 4°C);

[0043] X7. Cut the membrane: Cut the membrane along the Maker;

[0044] X8. Dilute the primary antibody with TBST according to the instruction manual and incubate it at room temperature for 1 h (it can be overnight at 4°C);

[0045] X9. After incubation, wash the membrane twice with 1X TBST, 10 min each time;

[0046] X10. Dilute the secondary antibody with TBST according to the instruction manual and incubate it at room temperature for 1 h;

[0047] X11. After incubation, wash the membrane twice with 1X TBST, 10 min each time;

[0048] X12. Exposure (using ECL chemiluminescence, 2 mL each of Solution A and Solution B, soaking the membrane, and exposing with an instrument).

[0049] As a preferred embodiment of the present invention, the steps for preparing the PAGE gel are as follows:

[0050] XT1. Refer to the instruction manual of the YEASEN gel kit. Generally, the separating gel is 12% and the stacking gel is 5%. Select a 1.0 mm glass plate for preparation;

[0051] XT2. Add 1 mL of isopropanol to seal the glass plate in the gel-making rack, let it stand at room temperature for 35 min, pour out the isopropanol, and blot the residual isopropanol with filter paper;

[0052] XT3. Add the upper-layer stacking gel, insert the electrophoresis comb, and let it stand at room temperature for 35 min.

[0053] As a preferred embodiment of the present invention, the steps for preparing the protein sample are as follows;

[0054] For cell samples, collect cells (such as one dish), centrifuge at 1000 rpm for 5 min, discard the supernatant, add 500 μL of PBS to resuspend and 10 μM PMSF (for phosphorylation, phosphatase inhibitor needs to be added), add 125 μL of 5× Loading Buffer and mix well, boil in a water bath for 10 min, vortex for 30 s, and centrifuge at 12000 rpm for 3 min.

[0055] As a preferred embodiment of the present invention, in step S4, IL4, protein, and AZM drugs are added for grouping treatment, and the drug groups are as follows:

[0056] I. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF;

[0057] II. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 μM AZM, DEX;

[0058] III. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 μM AZM, DEX + 10 μg / mL SOD;

[0059] IV. Normal RAW264.7 + 0.5% DMSO;

[0060] V. RAW264.7-IL4R: 150 ng / mL IL4, IL13, IL10, M-CSF;

[0061] VI.RAW264.7 - IL4R: 150 ng / mL IL4, IL13, IL10, M - CSF + 50 uM AZM, DEX;

[0062] VII.RAW264.7 - IL4R: 150 ng / mL IL4, IL13, IL10, M - CSF + 50 uM AZM, DEX + 10 ug / mL SOD.

[0063] As a preferred embodiment of the present invention, the grouping for sampling after 15 days is as follows;

[0064] (1) 200 uL of AGS cell suspension (1x106 cells) (resuspended in PBS);

[0065] (2) 200 uL of AGS cell suspension (5x106 cells) (resuspended in PBS);

[0066] (3) 200 uL of AGS cell suspension (1x106 cells) (resuspended in M2 polarized cell supernatant);

[0067] (4) 200 uL of AGS cell suspension (5x106 cells) (resuspended in M2 polarized cell supernatant);

[0068] (5) 200 uL of M2 polarized cell supernatant control.

[0069] As a preferred embodiment of the present invention, for the cell resuscitation and culture: First, take RAW264.7 cells stored at -80°C, resuscitate them at 39°C, then centrifuge them at 1000 rpm for 5 min using a horizontal centrifuge, discard the supernatant, take 1 mL of cell culture medium preheated at 37°C to resuspend the cells, transfer them into a T25 cell flask, supplement the culture medium to 5 mL, and place them in a CO2 incubator, and culture them under the conditions of 5% CO2 and 37°C;

[0070] For the cell plating and virus infection: Culture RAW264.7 cells using 90% DMEM + 10% FBS. Digest the sub - cultured cells with 0.25% trypsin and plate them into a 24 - well plate. When the confluence reaches 80%, add the IL4R over - expression lentivirus purchased from ABM to infect for 24 h, then change to normal culture medium. After 48 h of infection, use 1 ug / mL puromycin to screen for positive cells;

[0071] The preparation of the proteins and drugs: Dilute mouse IL4, IL13, IL10, and M-CSF with ddH2O to prepare a 100 ng / uL stock solution, aliquot and store at -80°C; dissolve AZM and DEX with DMSO to prepare a 100 mM stock solution, aliquot and store at -80°C; dissolve SOD with ddH2O to prepare a 1 ug / uL stock solution, aliquot and store at -80°C.

[0072] The setting of RAW264.7 M2 polarization conditions: Culture RAW264.7 / RAW264.7-IL4R cells with 90% DMEM + 10% FBS. Digest the subcultured cells with 0.25% trypsin and plate them into 6-well plates. When the confluence reaches 80%, add IL4, proteins, and AZM drugs for grouped treatment. After incubation for 48 h, digest with 0.25% trypsin, resuspend the cell pellet with PBS, incubate with flow antibodies such as Arg-1-AF700 and CD206-FITC at 4°C for 30 min, wash twice with PBS, and detect by flow cytometry.

[0073] The promotion of tumor cell tumorigenesis by the supernatant of M2 cell polarization: Collect the cell supernatant of the highest M2 polarization treatment group. Culture human gastric adenocarcinoma cells with 79% F12K + 20% FBS + 1% double antibody at 37°C and 5% CO2, expand the culture to 10 10-cm cell culture dishes. After digestion with 0.25% trypsin, resuspend with PBS, count, and store at 4°C. Inject 200 uL of the cell suspension subcutaneously into the left hind limb of 5-6-week-old female nude mice. Observe the tumorigenesis situation and measure the tumor size every 2-3 days after injection. After 15 days, group and collect samples.

[0074] Data analysis: Independent sample T-test is used for comparison between two groups of samples, and one-way ANOVA is used for comparison among multiple groups. The data is analyzed using SPSS, and each group has at least 3 replicates.

[0075] As a preferred embodiment of the present invention, the method for inducing murine M2 macrophages is applied to promoting tumor cell tumorigenesis.

[0076] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0077] 1. The present invention uses exogenous addition of protein drugs such as IL4, IL13, IL10, M-CSF, AZM, SOD, and DEX, and uses lentivirus-mediated stable expression of IL-4R receptor in macrophages, thereby ultimately greatly enhancing macrophage M2 polarization, laying a foundation for subsequent research on the growth and tumorigenesis of human / murine tumor cells in mice.

[0078] 2. The present invention provides a supernatant of M2 polarized macrophages for promoting tumor growth, which is obtained based on a series of induction systems. The supernatant of M2 polarized macrophages expresses a large number of factors required for promoting the growth of tumor cells. When applied to the construction of a transplanted tumor model, it is convenient for subsequent research on the potential mechanisms of tumor occurrence and development. It can be generally applied to the growth of tumor cells, with high and stable expression efficiency of cytokines, greatly improving the tumor formation effect. The operation of applying the supernatant of M2 polarized macrophages is simple and easy, and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent:

[0080] Figure 1 It is the bright field image (40x) of RAW264.7 cells of the present invention and the result images of WB and qPCR;

[0081] Figure 2 It is the flow cytometry result image of M2 polarization of cells in different treatment groups of the present invention;

[0082] Figure 3 It is the tumor formation photo and tumor volume statistical chart of AGS of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0083] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0084] Example 1: A method for inducing murine M2 macrophages and its application, see Figures 1 to 3 , including the following steps:

[0085] S1. Cell resuscitation and culture: First, take RAW264.7 cells stored at -80°C, resuscitate them at 39°C, then centrifuge them at 1000 rpm for 5 min using a horizontal centrifuge, discard the supernatant, take 1 mL of cell culture medium preheated at 37°C to resuspend the cells, transfer them into a T25 cell flask, supplement the culture medium to 5 mL, and place it in a CO2 incubator, and culture it under the conditions of 5% CO2 and 37°C;

[0086] S2. Cell seeding and virus infection: RAW264.7 cells were cultured in 90% DMEM + 10% FBS. The passaged cells were digested with 0.25% trypsin and seeded into 24-well plates (1x105 cells). When the confluence reached 80%, lentivirus overexpressing IL4R purchased from ABM (400 uL medium + 100 uL virus solution + 8 ug / mL Polybrene) was added for infection. After 24 h, the medium was changed to normal medium for culture. After 48 h of infection, positive cells were screened with 1 ug / mL puromycin;

[0087] S3. Protein and drug preparation: Mouse IL4, IL13, IL10, and M-CSF were prepared into 100 ng / uL stock solutions with ddH2O and stored in aliquots at -80 °C. AZM and DEX were prepared into 100 mM stock solutions with DMSO and stored in aliquots at -80 °C. SOD was prepared into 1 ug / uL stock solution with ddH2O and stored in aliquots at -80 °C;

[0088] S4. Setting of RAW264.7 M2 polarization conditions: RAW264.7 / RAW264.7-IL4R cells were cultured in 90% DMEM + 10% FBS. The passaged cells were digested with 0.25% trypsin and seeded into 6-well plates (5x105 cells). When the confluence reached 80%, IL4, proteins, and AZM drugs were added for grouped treatment;

[0089] The grouping is as follows:

[0090] I. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF;

[0091] II. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 uM AZM, DEX;

[0092] III. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 uM AZM, DEX + 10 ug / mL SOD;

[0093] IV. Normal RAW264.7 + 0.5% DMSO;

[0094] V. RAW264.7-IL4R: 150 ng / mL IL4, IL13, IL10, M-CSF;

[0095] VI. RAW264.7-IL4R: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 uM AZM, DEX;

[0096] VII. RAW264.7 - IL4R: 150 ng / mL IL4, IL13, IL10, M - CSF + 50 uM AZM, DEX + 10 ug / mL SOD;

[0097] After treatment and incubation for 48 h, after digestion with 0.25% trypsin, the cell pellet was resuspended with PBS and incubated with flow antibodies such as Arg - 1 - AF700 and CD206 - FITC at 4°C for 30 min (100 ul cell suspension + 2 ul flow antibody), washed twice with PBS, and detected by flow cytometry;

[0098] S5. M2 cell polarization supernatant promotes tumor cell tumorigenesis: Collect the cell supernatant of the highest M2 polarization treatment group, culture human gastric adenocarcinoma cells (AGS) with 79% F12K + 20% FBS + 1% double antibody at 37°C in 5% CO2, expand the culture to 10 10 - cm cell culture dishes, after digestion with 0.25% trypsin, resuspend with PBS, count, and store at 4°C. Inject 200 uL of the cell suspension subcutaneously into the left hind limb of 5 - 6 - week - old female nude mice. Observe the tumorigenesis situation and measure the tumor size every 2 - 3 d after injection. After 15 d, group and collect samples;

[0099] The grouping is as follows;

[0100] (1) 200 uL of AGS cell suspension (1x106 cells) (resuspended with PBS);

[0101] (2) 200 uL of AGS cell suspension (5x106 cells) (resuspended with PBS);

[0102] (3) 200 uL of AGS cell suspension (1x106 cells) (resuspended with M2 polarization cell supernatant);

[0103] (4) 200 uL of AGS cell suspension (5x106 cells) (resuspended with M2 polarization cell supernatant);

[0104] (5) 200 uL of M2 polarization cell supernatant control;

[0105] S6. Data analysis: Independent - sample T - test was used for comparison between two groups of samples, and one - way ANOVA was used for comparison among multiple groups. The data was analyzed using SPSS, with at least 3 replicates for each group. The graphing software was Graphpad prism5.0 (*p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference).

[0106] Specifically, the steps for screening positive cells in step S2 are:

[0107] M1. First, maintain the RAW264.7 cells infected with the virus in culture with 1 μg / mL puromycin for 2 days;

[0108] M2. Then, dilute the RAW264.7 cell bank infected with the virus by the limiting dilution method, inoculate it into a 96-well cell culture plate (1 cell per well), and when the 96-well cell culture plate is full, digest it with 0.25% trypsin and inoculate it into a 48-well plate, and continue until inoculating into a 6-well plate. After it is full, digest it with 0.25% trypsin;

[0109] M3. Then, perform qPCR and WB detections;

[0110] M4. Finally, take 1 / 3 of the cells to extract total RNA for qPCR verification of the IL4R overexpression efficiency, take 1 / 3 of the cells to extract total protein for WB verification of the IL4R overexpression efficiency, and inoculate the other 1 / 3 into the original 6-well plate for continuous culture. Repeat the operation for each well until a positive clone cell line is screened out.

[0111] Furthermore, in step M3, for the qPCR detection, use Trizol to extract total RN2A from the cells, reverse transcribe cDNA for qPCR detection of the IL4R transcription level. The experimental steps are as follows:

[0112] Extract total RNA from tissues by the Trizol method

[0113] ① Add 1 ml of Trizol to the cells, pipette and mix well, place at 4°C for 15 min to allow sufficient lysis, and then transfer to a 1.5 ml centrifuge tube;

[0114] ② Add chloroform at 200 μl chloroform / ml Trizol, shake vigorously for 30 s, and then let stand at 4°C for 15 min;

[0115] ③ Centrifuge at 12,000 rpm at 4°C for 15 min on a tabletop centrifuge to allow the solution to separate fully;

[0116] ④ Carefully pipette 400 μl of the supernatant and transfer it to a sterile 1.5 RNase-free centrifuge tube, add 400 μl of isopropanol, mix well, and precipitate at room temperature for 40 min;

[0117] ⑤ Centrifuge at 12,000 rpm at 4°C for 15 min on a tabletop centrifuge and discard the supernatant;

[0118] ⑥ Wash twice with 1 ml of 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 min, and discard the supernatant;

[0119] ⑦ After the ethanol has evaporated in the laminar flow hood, add 30 μl of DEPC water to dissolve the RNA and store it at -80°C for later use.

[0120] Reverse transcription cDNA (total volume of reaction system preparation: 20 μl)

[0121]

[0122] Fluorescent quantitative PCR (total volume of reaction system preparation: 20 μl

[0123]

[0124] Furthermore, in step M3, for WB detection, the cell sample is placed in a 1.5 ml EP tube, 1 ml of RIPA, 10 μl of PMSF, and 20 μl of phosphatase inhibitor are added thereto, lysed at 4°C for 1 h, centrifuged at 12000 rpm for 5 min, and the cell sample is quantitatively analyzed by BCA to detect the expression level of IL4R protein. The experimental steps are as follows:

[0125] X1. Take out the gel plate of the gel-making rack, install it on the electrophoresis rack, and place it in the electrophoresis tank. Pour 1X Tris-Gly-SDS electrophoresis buffer until the scale line;

[0126] X2. Pull out the electrophoresis comb and load the sample. After electrophoresis at 75 V for 60 min (when the bromophenol blue reaches the separating gel), electrophoresis at 120 V for 80 min. Stop electrophoresis when the bromophenol blue indicator line reaches the bottom;

[0127] X3. Transfer the membrane. Cut the PVDF membrane according to the PAGE gel electrophoresis area (usually 4×8 cm specification), then activate it with methanol for 1 min, and then place it in the transfer buffer for equilibration for 30 s;

[0128] X4. Remove the PAGE gel from the glass plate, place it on the filter paper, mark the membrane, cover it on the gel, drive out the air bubbles, cover the filter paper and sponge, close the transfer clip, pour in the pre-cooled transfer solution, and place the transfer tank in an ice-water bath. The electron flow direction of membrane transfer is: black plate → sponge → filter paper → gel → membrane → filter paper → sponge → red plate;

[0129] X5. The membrane transfer condition is a constant current of 125 mA (125 mA for one group, 250 mA for two groups, and so on), and the time is the molecular weight plus 10 min;

[0130] X6. Blocking: After membrane transfer, take out the membrane and block it at room temperature for 1 h with TBST containing 5% skim milk (it can be overnight at 4°C);

[0131] X7. Cut the membrane: Cut the membrane along the Maker;

[0132] X8. Dilute the primary antibody with TBST according to the instruction manual and incubate at room temperature for 1 h (it can be overnight at 4°C);

[0133] X9. After incubation, wash the membrane twice with 1X TBST, 10 min each time;

[0134] X10. Dilute the secondary antibody according to the instruction manual with TBST and incubate at room temperature for 1 h;

[0135] X11. After incubation, wash the membrane twice with 1XTBST for 10 min each time;

[0136] X12. Expose the film (using ECL chemiluminescence, 2 mL each of Solution A and Solution B, soak the membrane, and expose the film using the instrument).

[0137] It should be noted that the steps for preparing the PAGE gel are as follows:

[0138] XT1. Refer to the instruction manual of the YEASEN gel kit. Generally, the separating gel is 12% and the stacking gel is 5%. Select a 1.0 mm glass plate for preparation;

[0139] XT2. Add 1 mL of isopropanol to seal the glass plate in the gel-making rack, let it stand at room temperature for 35 min, pour out the isopropanol, and blot the residual isopropanol with filter paper;

[0140] XT3. Add the upper stacking gel, insert the electrophoresis comb, and let it stand at room temperature for 35 min.

[0141] It should be noted that the steps for preparing the protein sample are as follows;

[0142] For cell samples, collect the cells (such as one dish), centrifuge at 1000 rpm for 5 min, discard the supernatant, add 500 μL of PBS to resuspend and 10 μM PMSF (for phosphorylation, phosphatase inhibitor needs to be added), add 125 μL of 5×Loading Buffer and mix well, boil in a water bath for 10 min, vortex for 30 s, and centrifuge at 12000 rpm for 3 min.

[0143] It is worth introducing the construction and verification of the RAW264.7-IL-4R overexpression cell line

[0144] The positive monoclonal cell lines screened were verified for IL-4R expression by WB and qPCR. The results are shown in the figure. Compared with the control group, the IL-4R expression in the positive monoclonal group was extremely significantly increased (p < 0.01), indicating that the construction of the IL-4R-RAW264.7 overexpression cell line was successful, as Figure 1 shown.

[0145] It should be emphasized that the M2 polarization of WT and IL-4R overexpressing RAW264.7 cells after treatment with different protein drugs

[0146] After treating WT and IL-4R overexpressing RAW264.7 cells with different protein and drug combination formulations for 48 h, antibodies such as ARG-1 and CD206 were used to detect the proportion of M2 polarization of the cells. The results are shown in the figure. Compared with the control group, adding IL-4 protein could increase the M2 polarization ratio of the cells, and adding drugs could further promote the M2 polarization of the cells. After overexpressing IL-4R, the M2 polarization ratio of the cells was further increased. The results showed that adding proteins such as IL-4 and drugs such as AZM could significantly increase the M2 polarization ratio of the cells (p < 0.01); compared with this group, after overexpressing IL-4R, adding proteins such as IL-4 and drugs such as AZM could significantly increase the M2 polarization ratio of the cells (p < 0.01), as Figure 2 shown.

[0147] Promotion of tumor cell tumorigenesis by M2 cell polarization supernatant

[0148] The AGS cells were resuspended with the supernatant of the treatment group with the highest M2 cell polarization ratio as the solvent, and a control group was set up. 200 μL of the cell suspension was subcutaneously inoculated into 5-6-week-old female nude mice in the left hind limb. After inoculation, the tumorigenesis was observed every 2-3 days and the tumor size was measured. The samples were taken 15 days after inoculation. The results are shown in the figure. The M2 polarization supernatant could significantly promote the subcutaneous tumorigenesis of AGS cells (p < 0.01) and significantly increase the tumorigenesis rate of AGS cells, as Figure 3 shown.

[0149] It should be noted that the method for inducing murine M2 macrophages was applied to the experimental study of tumor cell tumorigenesis.

[0150] In the tumor microenvironment, macrophages can be recruited and tamed by tumor cells and transformed into tumor-associated macrophages, which usually have the characteristics of promoting tumor growth. Macrophages can secrete vascular endothelial growth factor (VEGF) and other pro-angiogenic factors to promote tumor angiogenesis, providing nutrients and oxygen for the tumor. Macrophages can secrete matrix metalloproteinases and other proteolytic enzymes to help tumor cells degrade the basement membrane and extracellular matrix, promoting tumor invasion and metastasis. Macrophages can produce immunosuppressive cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), inhibiting the activity of T cells and the anti-tumor functions of other immune cells. M2-polarized macrophages are a phenotypic and functional state of macrophages, which mainly play an anti-inflammatory and tissue repair-promoting role in the immune response. M2 macrophages are opposite to M1 macrophages, the latter mainly participating in pro-inflammatory reactions and anti-pathogen defenses. In the tumor microenvironment, M2 macrophages promote tumor growth and spread by promoting angiogenesis, invasion and metastasis, and inhibiting the immune response. The innovation in this study lies in using the culture supernatant of M2-polarized macrophages (with more than 90% M2-polarized cells, which contains a large number of anti-inflammatory factors), which can provide the microenvironment required for tumor growth, thus greatly promoting tumor growth. We promoted the M2 polarization of tumor cells by optimizing a series of conditions, increasing the number of M2-polarized macrophages by 50-60% compared with other studies, and transforming it into actual scientific research results.

[0151] In summary, the present invention uses exogenous addition of protein drugs such as IL4, IL13, IL10, M-CSF, AZM, SOD, and DEX, and uses lentivirus-mediated stable expression of the IL-4R receptor in macrophages, thereby ultimately greatly enhancing the M2 polarization of macrophages, laying a foundation for subsequent studies on the growth and tumorigenesis of human / mouse tumor cells in mice.

[0152] The present invention provides M2-polarized macrophage supernatant for promoting tumor growth, which is obtained based on a series of improvements in the induction system. The M2-polarized macrophage supernatant expresses a large number of factors required for promoting tumor cell growth. Applied to the construction of xenograft tumor models, it is convenient for subsequent studies on the potential mechanisms of tumor occurrence and development, can be generally applicable to the growth of tumor cells, has high and stable cytokine expression efficiency, greatly improves the tumorigenesis effect, and the operation of using M2-polarized macrophage supernatant is simple and easy to implement, and is easy to promote.

[0153] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For a person skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention. Any reference signs in the claims should not be construed as limiting the claims concerned.

[0154] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for inducing murine M2 macrophages, characterized in that: It includes the following steps: S1. First, resuscitate and culture the cells; S2. After the cells are resuscitated and cultured well, let the resuscitated and cultured cells be plated and infected with the virus; S3. After the cells are plated and infected with the virus well, then perform protein and drug preparation; S4. After the protein and drug preparation is completed, set the RAW264.7 M2 polarization conditions; S5. After the RAW264.7 M2 polarization conditions are set, let the M2 cell polarization supernatant promote tumor cell tumorigenesis; S6. Finally, perform data analysis.

2. A method for inducing murine M2 macrophages according to claim 1, characterized in that: For the cell resuscitation culture: First, take RAW264.7 cells stored at -80°C, resuscitate at 39°C, then centrifuge at 1000 rpm for 5 min using a horizontal centrifuge, discard the supernatant, take 1 mL of cell culture medium preheated at 37°C to resuspend the cells, transfer them into a T25 cell flask, supplement the culture medium to 5 mL, and place it in a CO2 incubator, and culture under the conditions of 5% CO2 and 37°C; For the cell plating and virus infection: Culture RAW264.7 cells using 90% DMEM + 10% FBS, digest the subcultured cells with 0.25% trypsin and plate them into a 24-well plate. When the confluence reaches 80%, add the lentivirus overexpressing IL4R purchased from ABM and infect for 24 h, then change to normal culture medium. After 48 h of infection, use 1 μg / mL puromycin to screen for positive cells; For the protein and drug preparation: Prepare mouse IL4, IL13, IL10, and M-CSF into a 100 ng / μL stock solution using ddH2O, aliquot and store at -80°C. Prepare AZM and DEX into a 100 mM stock solution using DMSO, aliquot and store at -80°C. Prepare SOD into a 1 μg / μL stock solution using ddH2O, aliquot and store at -80°C; For the setting of RAW264.7 M2 polarization conditions: Culture RAW264.7 / RAW264.7-IL4R cells using 90% DMEM + 10% FBS, digest the subcultured cells with 0.25% trypsin and plate them into a 6-well plate. When the confluence reaches 80%, add IL4, protein, and AZM drugs for grouped treatment. After treatment, incubate for 48 h. After digestion with 0.25% trypsin, resuspend the cell pellet with PBS and incubate with flow antibodies such as Arg-1-AF700 and CD206-FITC at 4°C for 30 min, then wash twice with PBS and detect by flow cytometry; For the promotion of tumor cell tumorigenesis by the M2 cell polarization supernatant: Collect the cell supernatant of the highest treatment group of M2 polarization, culture human gastric adenocarcinoma cells at 37°C and 5% CO2 using 79% F12K + 20% FBS + 1% double antibody, expand the culture to 10 10-cm cell culture dishes. After digestion with 0.25% trypsin, resuspend with PBS, count, and store at 4°C. Subcutaneously inoculate 200 μL of the cell suspension into the left hind limb of 5-6-week-old female nude mice. After inoculation, observe the tumorigenesis situation and measure the tumor size every 2-3 days. After 15 days, group and collect samples; The data analysis: Independent sample T-test was used for comparison between two groups of samples, and one-way ANOVA was used for comparison among multiple groups. The data was analyzed using SPSS, with at least 3 replicates in each group.

3. The method for inducing murine M2 macrophages according to claim 2, characterized in that: The steps for screening positive cells are as follows: M1. First, the RAW264.7 cells infected with the virus were maintained in culture with 1 μg / mL puromycin for 2 days. M2. Then, the RAW264.7 cell library infected with the virus was diluted by the limiting dilution method and inoculated into a 96-well cell culture plate. After the 96-well cell culture plate was full, it was digested with 0.25% trypsin and inoculated into a 48-well plate until it was inoculated into a 6-well plate. After it was full, it was digested with 0.25% trypsin. M3. Then, qPCR and WB were detected. M4. Finally, 1 / 3 of the cells were taken to extract total RNA for qPCR to verify the overexpression efficiency of IL4R, 1 / 3 of the cells were taken to extract total protein for WB to verify the overexpression efficiency of IL4R, and the other 1 / 3 were inoculated into the original 6-well plate for continued culture. The operation was repeated for each well until a positive clone cell line was screened out.

4. A method for inducing murine M2 macrophages according to claim 3, characterized in that: The qPCR detection in step M3 is to use Trizol to extract total RN2A from cells, reverse transcribe cDNA for qPCR to detect the transcriptional level of IL4R. The experimental steps are as follows: Extraction of total RNA from tissues by Trizol method ① Add 1 ml of Trizol to the cells, pipette and mix well, place at 4 °C for 15 min to allow sufficient lysis, and then transfer to a 1.5 ml centrifuge tube. ② Add chloroform at 200 μl of chloroform / ml of Trizol, shake vigorously for 30 s, and then let stand at 4 °C for 15 min. ③ Centrifuge at 12,000 rpm at 4 °C for 15 min on a tabletop centrifuge to allow the solution to separate fully. ④ Carefully pipette 400 μl of the supernatant and transfer it to a sterile 1.5 RNase-free centrifuge tube, add 400 μl of isopropanol, mix well, and precipitate at room temperature for 40 min. ⑤ Centrifuge at 12,000 rpm at 4 °C for 15 min on a tabletop centrifuge and discard the supernatant. ⑥ Wash twice with 1 ml of 75% ethanol, centrifuge at 12,000 rpm at 4 °C for 5 min, and discard the supernatant. ⑦ After the ethanol has evaporated in the laminar flow hood, add 30 μl of DEPC water to dissolve the RNA and store it at -80 °C for later use.

5. The method for inducing murine M2 macrophages according to claim 3, characterized in that: The WB detection in step M3 is to place the cell sample in a 1.5 ml EP tube, add 1 ml of RIPA, 10 μl of PMSF, and 20 μl of phosphatase inhibitor, lyse at 4 °C for 1 h, centrifuge at 12,000 rpm for 5 min, and perform BCA quantification on the cell sample to detect the protein expression level of IL4R. The experimental steps are as follows: X1. Take out the gel plate of the gel-making rack, install it on the electrophoresis rack, place it in the electrophoresis tank, and pour 1X Tris-Gly-SDS electrophoresis buffer until the scale line. X2. Pull out the electrophoresis comb, load the samples, perform electrophoresis at 75 V for 60 min, then at 120 V for 80 min. Stop electrophoresis when the bromophenol blue indicator line reaches the bottom. X3. Transfer the membrane. Cut the PVDF membrane according to the PAGE gel electrophoresis area, then activate it with methanol for 1 min, and then place it in the transfer buffer to equilibrate for 30 s. X4. Remove the PAGE gel from the glass plate, place it on the filter paper, mark the membrane, cover it on the gel, expel the air bubbles, cover with filter paper and sponge, close the transfer cassette, pour in the pre-cooled transfer buffer, and place the transfer tank in an ice-water bath. The electron flow direction for transfer is: black plate → sponge → filter paper → gel → membrane → filter paper → sponge → red plate; X5. The transfer conditions are a constant current of 125 mA and the time is the molecular weight plus 10 min; X6. Blocking: After the transfer is completed, take out the membrane and block it at room temperature for 1 h with TBST containing 5% skim milk; X7. Cut the membrane: Cut the membrane along the Maker; X8. Dilute the primary antibody with TBST according to the instruction manual and incubate at room temperature for 1 h; X9. After incubation, wash the membrane twice with 1X TBST for 10 min each time; X10. Dilute the secondary antibody with TBST according to the instruction manual and incubate at room temperature for 1 h; X11. After incubation, wash the membrane twice with 1X TBST for 10 min each time; X12. Expose to light.

6. The method for inducing murine M2 macrophages according to claim 5, wherein: The steps for preparing the PAGE gel are as follows: XT1. Refer to the instruction manual of the YEASEN gel kit. Generally, the separating gel is 12% and the stacking gel is 5%. Select 1.0 mm glass plates for preparation; XT2. Add 1 ml of isopropanol to seal the glass plates on the gel-making rack, let it stand at room temperature for 35 min, pour out the isopropanol, and blot the residual isopropanol with filter paper; XT3. Add the upper-layer stacking gel, insert the electrophoresis comb, and let it stand at room temperature for 35 min.

7. A method for inducing murine M2 macrophages according to claim 5, characterized in that: The steps for preparing the protein sample are as follows; For cell samples, collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 500 μl of PBS to resuspend and 10 μM PMSF, add 125 μl of 5× Loading Buffer and mix well, boil in a water bath for 10 min, vortex for 30 s, and centrifuge at 12000 rpm for 3 min.

8. A method for inducing murine M2 macrophages according to claim 2, characterized in that: In step S4, IL4, protein, and AZM drugs are added for grouping treatment. The drug grouping is as follows: I. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF; II. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 μM AZM, DEX; III. Normal RAW264.7: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 μM AZM, DEX + 10 μg / mL SOD; IV. Normal RAW264.7 + 0.5% DMSO; V. RAW264.7-IL4R: 150 ng / mL IL4, IL13, IL10, M-CSF; VI. RAW264.7-IL4R: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 μM AZM, DEX; VII. RAW264.7-IL4R: 150 ng / mL IL4, IL13, IL10, M-CSF + 50 μM AZM, DEX + 10 μg / mL SOD.

9. A method for inducing murine M2 macrophages according to claim 2, characterized in that: The grouping for sample collection after 15 days is as follows; (1) 200 μL of AGS cell suspension (1x106 cells), resuspended in PBS; (2) 200 μL of AGS cell suspension (5x106 cells), resuspended in PBS; (3) 200 μL of AGS cell suspension (1x106 cells), resuspended in M2 polarized cell supernatant; (4) 200 μL of AGS cell suspension (5x106 cells), resuspended in M2 polarized cell supernatant; (5) 200 μL of M2 polarized cell supernatant control.

10. Use of the method for inducing murine M2 macrophages according to any one of claims 1 - 9 in promoting tumor cell tumorigenesis.

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