Diabetes-based in-vitro macrophage training immune model, construction method and application

Abstract

The invention belongs to the technical field of immunology, and aims to solve the problems of long modeling time, difficulty in exploration of an in-vivo mechanism and the like in research of a mechanism of hyperglycemia-induced macrophage training immunity by using a diabetic animal model in the prior art and the problem that in-vitro hyperglycemia-induced macrophage training immunity phenotype is not obvious. The invention provides a construction method of an in-vitro macrophage training immune model based on diabetes mellitus. The construction method comprises the following steps: S1, acquiring bone marrow cells of a mouse; s2, erythrocyte lysis and cell cleaning; s3, induced differentiation of macrophages; s4, inducing macrophages to train immunity under the stimulation of AGEs. According to the invention, it is determined that 200 [mu] g / mLAGEs stimulate macrophages for 48 hours to effectively induce macrophage training immunophenotype, and the phenotype is consistent with macrophage training phenotype found in a diabetic mouse model, so that the LAGEs can be used for in-vitro exploration of a mechanism of hyperglycemia induced macrophage training immunogenesis.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and specifically relates to an in vitro macrophage training immune model based on diabetes, a construction method and an application thereof. Background Art

[0002] Studies have found that even if blood sugar is under control, short-term hyperglycemia can still cause persistent damage to organs, tissues and cells. This phenomenon is called "hyperglycemia memory," which promotes the occurrence and development of chronic diabetic complications.

[0003] In 1987, Engerman et al. used alloxan monohydrate to induce a canine model of type 1 diabetes mellitus (T1DM) and first discovered the phenomenon of hyperglycemia memory in a study of retinopathy in T1DM dogs. T1DM dogs had poor blood sugar control in the first 2.5 years of onset, and retinopathy was absent or unclear at this time. However, surprisingly, despite good blood sugar control in the following 2.5 years, retinopathy still occurred and progressed. Rapid blood sugar control within the first 2 months could effectively inhibit the occurrence of retinopathy. In 1990, Roy et al. found in a streptozotocin (STZ)-induced T1DM rat model that the level of fibronectin mRNA in the renal cortex and heart tissue of T1DM rats was significantly increased, and after the blood sugar level was restored, the fibronectin mRNA continued to remain at a high level for several weeks. In 1993, Hammes et al. induced a diabetic rat model by sucrose feeding and performed islet transplantation on the diabetic rats. They found that islet transplantation within 6 weeks after the onset of diabetic rats could prevent diabetic retinopathy. However, diabetic retinopathy still occurred when islet transplantation was performed 12 weeks after the onset of the disease.

[0004] In 2003, the Diabetes Control and Complications Trial / Epidemiology of Diabetes Interventions and Complications (DCCT / EDIC) research team explored the long-term impact of previous differences in blood sugar levels on renal disease in patients with T1DM and found that although the HbA1c of T1DM patients in the intensive treatment group and the conventional treatment group remained at similar levels during follow-up, the incidence of renal disease in the conventional treatment group was significantly higher than that in the intensive treatment group, indicating that early hyperglycemia increased the risk of renal disease and confirmed the existence of hyperglycemia memory in diabetic patients for the first time. In 2005, the DCCT / EDIC research team also confirmed that early hyperglycemia increased the risk of cardiovascular disease in patients with T1DM. By lowering blood sugar through early intensive treatment, the risk of cardiovascular events was reduced by 42%, and the risk of serious clinical events including non-fatal myocardial infarction, stroke or death from cardiovascular disease was reduced by 57%. In 2008, the United Kingdom Prospective Diabetes Study (UKPDS) confirmed that early intensive treatment to lower blood sugar can bring long-term benefits (lasting 10 years) to patients with type 2 diabetes mellitus (T2DM), significantly reducing the risk of myocardial infarction and microvascular disease in patients, once again proving the existence of hyperglycemia memory (the phenomenon is called the legacy effect in the article).

[0005] Cardiovascular complications are the main cause of death in diabetic patients. Although many commonly used hypoglycemic therapies have achieved great success in controlling hyperglycemia in diabetic patients, the risk of cardiovascular complications still persists, which may be related to the delayed diagnosis of diabetic patients and the formation of hyperglycemia memory. Recent studies have found that hyperglycemia in the body induces macrophage immune training. Even if blood sugar levels return to normal, macrophages that have trained immunity still drive the occurrence and development of cardiovascular diseases. This better explains the phenomenon of hyperglycemia memory, but the mechanism by which hyperglycemia in the body induces macrophage immune training is still unclear. Exploring the mechanism by which hyperglycemia induces macrophage immune training may provide targets for regulating macrophage immune training to prevent and treat cardiovascular complications of diabetes.

[0006] At present, the research establishes diabetic animal models, and then detects the macrophage training immune phenotype and explores its formation mechanism after several months. However, there are problems such as long modeling time, difficulty in exploring the in vivo mechanism, and cumbersome later mechanism verification. Researchers tried to directly stimulate macrophages with high sugar in vitro, but there were problems such as unclear training immune phenotype, which could not be used to simulate the exploration of macrophage training immune mechanism induced by high blood sugar in vivo. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an in vitro macrophage training immune model based on diabetes, a construction method and an application thereof.

[0008] Previous studies have explored the short-term effects of AGEs stimulation on macrophages. AGEs can activate macrophages, enhance macrophage autophagy, promote macrophage polarization to M1 type, affect diabetic wound healing, participate in diabetic endothelial damage, etc. This is the mechanism of diabetes pathogenesis under hyperglycemia, but the in vitro research model of the long-term effects of AGEs on macrophages has not been established, and it cannot be used to study the mechanism of the "hyperglycemia memory" phenomenon, that is, it cannot be used to explain why cardiovascular system complications and other diseases still occur for a long period of time after blood sugar returns to normal. The concentration and time of pathogen-associated molecular patterns / damage-associated molecular patterns stimulating macrophages are crucial to the formation of macrophage training immunity. The establishment of macrophage training immunity formation under different stimulation conditions has greatly promoted the study of the "memory effect" mechanism in the corresponding disease model. The present invention establishes a stable in vitro macrophage training immunity model based on diabetes by studying the effects of diabetes damage-associated molecular patterns AGEs of different stimulation concentrations and times on the phenotype of macrophage training immunity, which lays a good foundation for studying the mechanism of the "hyperglycemia memory" phenomenon in diabetes.

[0009] The first object of the present invention is to provide a method for constructing an in vitro macrophage training immune model based on diabetes, comprising the following steps:

[0010] S1. Obtain bone marrow cells from mice;

[0011] S2. Red blood cell lysis and cell washing

[0012] Add the red blood cell lysate to the bone marrow cells obtained in step S1, mix and keep for 3 to 5 minutes, then add an equal volume of DMEM complete medium, centrifuge, retain the cell pellet, resuspend the cell pellet, centrifuge again, remove the supernatant, and obtain lysed cells;

[0013] S3, resuspending the lysed cells obtained in step S2 with the modified culture medium, and culturing them. After 3 days of culture, changing the culture medium and continuing the culture, and changing the modified culture medium once every 2 days until the 7th day, to obtain mature macrophages;

[0014] S4. Continue to culture the mature macrophages obtained in step S3 with the modified medium, and add 200 μg / mL of AGEs to stimulate the mature macrophages for 48 hours, replace the modified medium, and continue to culture with the modified medium for 6 days, changing the medium once every 2 days to obtain an in vitro macrophage training immune model.

[0015] Preferably, step S1 comprises the following steps:

[0016] S11, select 6-8 week old mice, kill them after anesthesia, disinfect the body surface of the mice, and separate the femur and tibia;

[0017] S12, placing the femur and tibia separated in step S11 in a sterile phosphate buffer precooled at 4°C, and then flushing the bone marrow cavity with a sterile phosphate buffer precooled at 4°C to flush the bone marrow cells out of the bone marrow cavity to obtain bone marrow cells.

[0018] Preferably, in step S2, the cell pellet is resuspended in sterile phosphate buffer precooled at 4°C.

[0019] Preferably, in step S3 and step S4, the modified culture medium is a DMEM complete culture medium containing 20 ng / mL M-CSF and 5 mM glucose.

[0020] Preferably, in step S3, before culturing, the lysed cells need to be resuspended in the modified medium to count the lysed cells obtained in step S2, and the lysed cells are divided into 3*10 6 cells / mL were inoculated in 6-well plates and then cultured.

[0021] Preferably, in step S3, the culture conditions are: 37° C. and 5% carbon dioxide.

[0022] The second object of the present invention is to provide an in vitro macrophage training immune model based on diabetes mellitus obtained by constructing the above-mentioned method for constructing an in vitro macrophage training immune model based on diabetes mellitus.

[0023] The third object of the present invention is to provide an application of the above-mentioned diabetes-based in vitro macrophage training immune model in screening targeted macrophage training immune prevention and treatment drugs.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention optimizes the stimulation time and concentration of AGEs and determines that stimulating macrophages with 200 μg / mL AGEs for 48 hours can effectively induce the macrophage training immune phenotype, and this phenotype is consistent with the macrophage training phenotype found in the diabetic mouse model, which can be used to explore the mechanism of hyperglycemia-induced macrophage training immune formation in vitro;

[0026] (2) The method for constructing an in vitro macrophage training immune model provided by the present invention has the advantages of high controllability of in vitro experimental conditions, good repeatability, shortened experimental time, and low cost of phenotypic detection, and can accelerate the exploration of diabetes-related macrophage training immune mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A flowchart of a method for constructing an in vitro macrophage training immune model provided by an embodiment of the present invention;

[0028] Figure 2 The expression levels of Tnfa, Il6 and Il1b genes in macrophages in the in vitro macrophage training immune model provided by the embodiment of the present invention;

[0029] Figure 3 The levels of cytokines such as TNF-α and IL-6 in the macrophage culture supernatant in the in vitro macrophage training immune model provided in the embodiment of the present invention;

[0030] Figure 4 The graph shows the detection results of the transcription and secretion levels of training immune-related cytokines after macrophages were stimulated with 30 μg AGEs for 24 hours;

[0031] in, Figure 4 A is the transcription level; Figure 4 B is the secretion level;

[0032] Figure 5 The graphs show the results of the detection of the transcriptional levels of training immune-related cytokines after macrophages were stimulated with 0.01 μg / mL, 1 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL AGEs for 48 hours;

[0033] Figure 6 The graph shows the growth of tumor cells cultured with macrophage supernatants from different groups. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] like Figure 1 As shown, the embodiment of the present invention provides a method for constructing an in vitro macrophage training immune model based on diabetes, which specifically includes the following steps:

[0037] S1. Obtaining bone marrow cells from mice, specifically comprising the following steps:

[0038] S11. Select 6- to 8-week-old C57BL / 6 mice, kill them after anesthesia, place them on a sterile operating table, and disinfect the body surface with 70% ethanol to reduce surface microbial contamination; use sterile surgical instruments to carefully separate the femur and tibia; during the operation, try to maintain the integrity of the bones to avoid bone marrow loss and contamination caused by fractures.

[0039] S12, placing the femur and tibia separated in step S11 in a culture dish containing 4°C precooled sterile phosphate buffer (PBS) to maintain the activity of bone marrow cells; then a syringe draws an appropriate amount of precooled PBS, inserts the needle into one end of the femur and tibia, and uses 4°C precooled sterile phosphate buffer to flush the bone marrow cavity, flushing the bone marrow cells out of the bone marrow cavity into a new sterile centrifuge tube to obtain bone marrow cells. During the flushing process, attention should be paid to gentle movements to ensure that the bone marrow cells can be fully flushed out, while avoiding mechanical damage to the cells.

[0040] S2. Red blood cell lysis and cell washing

[0041] Add an appropriate amount of red blood cell lysis solution to the centrifuge tube containing the bone marrow cells obtained in step S1, gently invert and mix, so that the red blood cells are fully in contact with the lysis solution. The action time of the red blood cell lysis solution is usually carried out according to the instructions, generally 3 to 5 minutes. After the red blood cells are lysed, the solution will appear transparent red, and then an equal volume of DMEM complete medium is added to terminate the red blood cell lysis reaction, and then the centrifuge tube is placed in a centrifuge, centrifuged at 300g for 5 minutes, and the supernatant is discarded, leaving the cell pellet at the bottom; resuspend the cell pellet with pre-cooled PBS and centrifuge again (centrifugation conditions are the same as before) to remove the residual red blood cell lysis solution and other impurities to obtain lysed cells;

[0042] S3, resuspend the lysed cells obtained in step S2 using the modified culture medium, count the lysed cells, and divide the lysed cells into 3*106 cells / mL were inoculated in a 6-well plate and cultured in an incubator at 37°C and 5% carbon dioxide. After 3 days of culture, the modified medium was replaced with fresh one and continued to be cultured. The modified medium was replaced with fresh one every 2 days until the 7th day to obtain mature macrophages. The purity of macrophages (CD11b+F4 / 80+) can be detected by flow cytometry, and the purity is usually above 90%.

[0043] S4. The mature macrophages obtained in step S3 are further cultured with the modified medium, and 200 μg / mL of AGEs are added to stimulate the mature macrophages for 48 hours. 200 μg / mL of BSA is added to the control group. After the stimulation is completed, fresh modified medium is replaced, and the culture is continued for 6 days with the modified medium, and fresh modified medium is replaced once every 2 days to obtain an in vitro macrophage training immune model.

[0044] In step S3 and step S4, the modified culture medium is a DMEM complete culture medium containing 20 ng / mL M-CSF and 5 mM glucose.

[0045] Comparative Example 1

[0046] This comparative example provides a method for constructing an in vitro macrophage training immune model based on diabetes. The construction method is the same as that of Example 1, except that step S4 of this comparative example is:

[0047] The mature macrophages obtained in step S3 were further cultured with the modified medium, and 30 μg / mL of AGEs were added to stimulate the mature macrophages for 24 hours. 30 μg / mL of BSA was added to the control group. After the stimulation, fresh modified medium was replaced, and the culture was continued for 6 days with the modified medium, with the medium changed once every 2 days, to obtain an in vitro macrophage training immune model.

[0048] Comparative Example 2

[0049] This comparative example provides a method for constructing an in vitro macrophage training immune model based on diabetes. The construction method is the same as that of Example 1, except that step S4 of this comparative example is:

[0050] The mature macrophages obtained in step S3 were further cultured with the modified medium, and 0.01 μg / mL of AGEs was added to stimulate the mature macrophages for 48 hours. 0.01 μg / mL of BSA was added to the control group. After the stimulation, fresh modified medium was replaced, and the culture was continued for 6 days with the modified medium, with the medium changed once every 2 days, to obtain an in vitro macrophage training immune model.

[0051] Comparative Example 3

[0052] This comparative example provides a method for constructing an in vitro macrophage training immune model based on diabetes. The construction method is the same as that of Example 1, except that step S4 of this comparative example is:

[0053] The mature macrophages obtained in step S3 were further cultured with the modified medium, and 1 μg / mL of AGEs was added to stimulate the mature macrophages for 48 hours. 1 μg / mL of BSA was added to the control group. After the stimulation, fresh modified medium was replaced, and the culture was continued for 6 days with the modified medium, with the medium changed once every 2 days, to obtain an in vitro macrophage training immune model.

[0054] Comparative Example 4

[0055] This comparative example provides a method for constructing an in vitro macrophage training immune model based on diabetes. The construction method is the same as that of Example 1, except that step S4 of this comparative example is:

[0056] The mature macrophages obtained in step S3 were further cultured with the modified medium, and 50 μg / mL of AGEs were added to stimulate the mature macrophages for 48 hours. 50 μg / mL of BSA was added to the control group. After the stimulation, fresh modified medium was replaced, and the culture was continued for 6 days with the modified medium, with the medium changed once every 2 days, to obtain an in vitro macrophage training immune model.

[0057] Comparative Example 5

[0058] This comparative example provides a method for constructing an in vitro macrophage training immune model based on diabetes. The construction method is the same as that of Example 1, except that step S4 of this comparative example is:

[0059] The mature macrophages obtained in step S3 were further cultured with the modified medium, and 100 μg / mL of AGEs were added to stimulate the mature macrophages for 48 hours. 100 μg / mL of BSA was added to the control group. After the stimulation, fresh modified medium was replaced, and the culture was continued for 6 days with the modified medium, with the medium changed once every 2 days, to obtain an in vitro macrophage training immune model.

[0060] The present invention uses 20 ng / mL of LPS to stimulate the macrophages in each group of in vitro macrophage training immune models obtained in Example 1 for 12 hours, and uses PCR technology to detect the expression levels of proinflammatory cytokines such as Tnfa, Il6 and Il1b genes in macrophages. And use 20 ng / mL of LPS to stimulate each group of macrophages for 24 hours, and use ELISA or multi-factor detection technology to detect the protein levels of corresponding cytokines in the supernatant. The test results are as follows: Figure 2 and Figure 3 As shown, through Figure 2 and Figure 3 It can be seen that the levels of expression and secretion of proinflammatory cytokines such as TNF-α and IL-6 by macrophages after AGEs training were significantly increased, and they possessed the characteristics of the macrophage training immune phenotype induced by hyperglycemia in vivo.

[0061] The present invention uses 20 ng / mL of LPS to stimulate the macrophages in each group of in vitro macrophage training immune models obtained in Comparative Example 1 for 24 hours. After the stimulation is completed, the transcription level and secretion level of training immune-related cytokines are detected. The results are as follows: Figure 4 A and Figure 4 As shown in B; Figure 4 A and Figure 4 As can be seen from B, under the stimulation condition of 30 μg / mL AGEs, the trained immune phenotype could not be induced.

[0062] The present invention uses 20 ng / mL of LPS to stimulate the macrophages in each group of in vitro macrophage training immune models obtained in Example 1 and Comparative Examples 2-5 for 12 hours. After the stimulation is completed, the transcription levels of training immune-related cytokines are detected. The results are as follows: Figure 5 As shown, through Figure 5 It can be seen that Example 1 of the present invention can induce macrophage trained immune phenotype, that is, 200 μg / mL AGEs stimulation of macrophages for 48 hours can induce macrophage trained immune phenotype.

[0063] The present invention cultured tumor cells (HL-1) with supernatants of different groups of macrophages for 24 hours and observed the growth of tumor cells. The results are as follows: Figure 6 shown.

[0064] The specific settings of different groups of macrophages are as follows:

[0065] BSA-NO group: initially stimulated by 200 μg / mL BSA, without restimulation by LPS;

[0066] BSA-LPS group: initially stimulated with 200 μg / mL BSA, followed by LPS stimulation for 24 hours;

[0067] AGEs-NO group: initially stimulated by 200 μg / mL AGEs, without restimulation by LPS;

[0068] AGEs-LPS group: initially stimulated with 200 μg / mL AGEs, and then stimulated with LPS for 24 h;

[0069] pass Figure 6It can be seen that the tumor cells cultured in the supernatants of the BSA-NO group, BSA-LPS group, and AGEs-NO group grew well, while a large number of tumor cells cultured in the supernatant of the AGEs-LPS group died. The above results further confirmed that the AGEs-induced macrophage training immune model was successfully established. The supernatant of the macrophages induced by the AGEs group after LPS re-stimulation had a killing effect on the tumor.

[0070] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for constructing an in vitro macrophage training immune model based on diabetes, characterized in that: The following steps are involved: S1. Obtain bone marrow cells from mice; S2. Red blood cell lysis and cell washing Add the red blood cell lysate to the bone marrow cells obtained in step S1, mix and keep for 3 to 5 minutes, then add an equal volume of DMEM complete medium, centrifuge, retain the cell pellet, resuspend the cell pellet, centrifuge again, remove the supernatant, and obtain lysed cells; S3. Macrophage differentiation induction The lysed cells obtained in step S2 are resuspended in the modified medium and cultured. After 3 days of culture, the medium is changed and culture is continued. The modified medium is changed once every 2 days until the 7th day, and mature macrophages are obtained. S4. AGEs stimulate macrophages to train immunity The mature macrophages obtained in step S3 were further cultured with the modified medium, and 200 μg / mL of AGEs were added to stimulate the mature macrophages for 48 hours. The modified medium was replaced, and the culture was continued for 6 days with the modified medium, with the medium being changed every 2 days to obtain an in vitro macrophage training immune model.

2. The method for constructing an in vitro macrophage training immune model based on diabetes according to claim 1, characterized in that: Step S1 comprises the following steps: S11, select 6-8 week old mice, kill them after anesthesia, disinfect the body surface of the mice, and separate the femur and tibia; S12, placing the femur and tibia separated in step S11 in a sterile phosphate buffer precooled at 4°C, and then flushing the bone marrow cavity with a sterile phosphate buffer precooled at 4°C to flush the bone marrow cells out of the bone marrow cavity to obtain bone marrow cells.

3. The method for constructing an in vitro macrophage training immune model based on diabetes according to claim 1, characterized in that: In step S2, the cell pellet is resuspended in sterile phosphate buffer precooled at 4°C.

4. The method for constructing an in vitro macrophage training immune model based on diabetes according to claim 1, characterized in that: In step S3 and step S4, the modified culture medium is a DMEM complete culture medium containing 20 ng / mL M-CSF and 5 mM glucose.

5. The method for constructing an in vitro macrophage training immune model based on diabetes according to claim 1, characterized in that: In step S3, before culturing, the lysed cells need to be resuspended in the modified medium to count the lysed cells, and the lysed cells are divided into 3*10 6 cells / mL were inoculated in 6-well plates and then cultured.

6. The method for constructing an in vitro macrophage training immune model based on diabetes according to claim 1, characterized in that: In step S3, the culture conditions are: 37° C. and 5% carbon dioxide.

7. An in vitro macrophage training immune model based on diabetes mellitus constructed according to the method for constructing an in vitro macrophage training immune model based on diabetes mellitus according to any one of claims 1 to 6.

8. Use of the diabetes-based in vitro macrophage training immune model according to claim 7 in screening targeted macrophage training immune prevention and treatment drugs.

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