Separation culture method of dairy cow fat-derived macrophages and application of separation culture method

Abstract

The invention belongs to the technical field of biology, and particularly relates to an isolated culture method of dairy cow fat-derived macrophages and application of the isolated culture method. According to the method for separating and culturing the dairy cow fat-derived macrophages, the dairy cow fat tissue serves as a research object, the ATM is separated through the immunomagnetic bead sorting technology, the treatment mode of the dairy cow fat tissue and the cell separation and culture method are optimized, operability is high, the method is simple, the physiological state is stable, and the ATM is promoted to be differentiated to the functional M1 / M2 phenotype. The invention provides a reliable cell model for research on fat metabolism, inflammation regulation and related diseases of dairy cows. The popularization and application of the invention are expected to provide new tools and ideas for health research and disease prevention and treatment of dairy cows, and the sustainable development of the breeding industry is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a method for separating and culturing adipose tissue macrophages of dairy cows and application thereof. BACKGROUND

[0002] Adipose tissue macrophages (ATM) of dairy cows play an important role in the study of dairy cow health and diseases. These cells not only participate in innate and adaptive immune responses, but also play a key role in fat metabolism, inflammation regulation and tissue repair. ATM regulates the homeostasis and inflammatory response of adipose tissue by secreting various cytokines (such as TNF-α, IL-6 and IL-10) and metabolites. With the large-scale development of dairy farming, the health problems of dairy cows are becoming increasingly prominent, especially diseases related to fat metabolism, such as fatty liver, ketosis and mastitis, which seriously affect the production performance and breeding benefits of dairy cows. Studies have shown that ATM plays a key role in adipose tissue inflammation and insulin resistance, and its dysfunction is closely related to the occurrence of metabolic diseases. Therefore, in-depth study of the biological characteristics of ATM and its mechanism in diseases is of great significance for the development of new prevention and control strategies.

[0003] Currently, the research on ATM mainly relies on in vivo experiments. Although this method can provide a relatively true biological environment, it has problems such as long experimental period, high cost and complex operation. In addition, in vivo experiments are affected by many factors such as individual differences, environmental factors and genetic background, and the repeatability and stability of the experimental results are poor. In vitro culture technology as an alternative method can conduct cell research under controlled conditions, and has the advantages of simple operation, low cost and short experimental period. How to provide a method for constructing ATM that can be stably cultured and functionally differentiated in vitro is a problem that needs to be solved by those skilled in the art. SUMMARY

[0004] The purpose of the present application is to make up for the shortcomings of the prior art, and to provide a method for separating and culturing adipose tissue macrophages of dairy cows and application thereof, so as to solve the technical problem of poor operability of existing ATM in in vitro culture, promote the differentiation of ATM to functional M1 / M2 phenotypes, and lay a foundation for immunological research and pathogenesis research of epidemic diseases.

[0005] The present application provides a method for separating and culturing adipose tissue macrophages of dairy cows, comprising the following steps: The fresh adipose tissue of dairy cows is washed with a first washing solution, a second washing solution, a third washing solution and a fourth washing solution in sequence to obtain washed adipose tissue; the washed adipose tissue is cut into small pieces of 3-5 mm, and collagenase digestion is performed to obtain digested adipose tissue; The digested adipose tissue is subjected to first centrifugation, and after collecting the lower liquid, second centrifugation is performed, and the precipitate is collected; after removing red blood cells in the precipitate, immunomagnetic bead sorting is performed, and macrophages positive for CD14 and CD11b are screened to obtain primary cow fat-derived macrophages; The primary cow fat-derived macrophages are cultured by using RPMI1640 complete medium; The first washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture and 1.0% v / v soluble amphotericin B are added; The second washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture is added; The third washing liquid uses PBS buffer as a solvent, and 1.0% v / v penicillin-streptomycin mixture is added; The fourth washing liquid uses PBS buffer as a solvent, and 0.5% v / v penicillin-streptomycin mixture is added; The RPMI1640 complete medium comprises 84-86% v / v RPMI1640 basic medium, 14-16% v / v fetal bovine serum, and 0.8-1.2% v / v penicillin-streptomycin mixture.

[0006] Preferably, the RPMI1640 complete medium comprises 84% v / v RPMI1640 basic medium, 15% v / v fetal bovine serum, and 1.0% v / v penicillin-streptomycin mixture.

[0007] Preferably, the penicillin-streptomycin mixture contains 10,000 U / mL of penicillin and 10,000 μg / mL of streptomycin.

[0008] Preferably, the collagenase is type II collagenase, and the digestion temperature is 37-38℃, and the digestion time is 1-2 h.

[0009] Preferably, the first centrifugation is performed at a speed of 400-800 rpm for 5-6 min.

[0010] Preferably, the second centrifugation is performed at a speed of 1000-1500 rpm for 8-10 min.

[0011] Preferably, the method for removing red blood cells in the precipitate comprises lysing the precipitate by using a red blood cell lysis solution, and after obtaining a lysis product, solid-liquid separation is performed, and the precipitate is collected; the lysis temperature is 20-25℃, and the lysis time is 5-6 min.

[0012] Preferably, the culture temperature is 37℃.

[0013] Preferably, the fresh bovine fat tissue is fresh fat tissue at the greater omentum and mesentery of the bovine.

[0014] The application further provides application of the bovine fat-derived macrophage obtained by the separation and culture method in preparation of a bovine immunology research model and / or a bovine disease pathogenesis research model.

[0015] Beneficial effects: The application provides a separation and culture method of bovine fat-derived macrophages, taking bovine fat tissue as a research object, separating ATM by an immunomagnetic bead sorting technology, and optimizing the processing mode of the bovine fat tissue and the cell separation and culture conditions, so as to promote differentiation of the ATM into functional M1 / M2 phenotypes. The application provides a reliable cell model for research on bovine fat metabolism, inflammation regulation and related diseases. Popularization and application of the application are expected to provide new tools and ideas for bovine health research and disease prevention and treatment, and promote sustainable development of the breeding industry. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0017] Figure 1 A result of flow cytometry detection of the proportion of bovine fat-derived macrophages in bovine fat tissue stromal cells; Figure 2 A growth morphology diagram of bovine fat-derived macrophages cultured in different culture media for different culture times; Figure 3 A cell identification result of bovine fat-derived macrophages; wherein, the cell nucleus after DAPI staining appears blue, and CD11b and CD14 are green; Figure 4 A flow cytometry detection result of the phenotype of bovine fat-derived macrophages; Figure 5 A result of induction of macrophages into M1 / M2 by adding LPS and IL-4; wherein, A is a Western blot imaging result; B is a statistical result of the expression levels of each protein, wherein, ** represents P <0.01; DETAILED DESCRIPTION

[0018] The application provides a separation and culture method of bovine fat-derived macrophages, including the following steps: The fresh bovine fat tissue is sequentially washed by using a first washing liquid, a second washing liquid, a third washing liquid and a fourth washing liquid to obtain washed fat tissue; the washed fat tissue is cut into small pieces with a size of 3-5 mm, and collagenase digestion is performed to obtain digested fat tissue; The digested adipose tissue is subjected to first centrifugation, and after collecting the lower liquid, subjected to second centrifugation, and the precipitate is collected; after removing the red blood cells in the precipitate, immune magnetic bead sorting is performed to screen CD14 and CD11b positive macrophages, thereby obtaining primary cow fat-derived macrophages; The primary cow fat-derived macrophages are cultured by using RPMI1640 complete medium; The first washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture and 1.0% v / v soluble amphotericin B are added; The second washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture is added; The third washing liquid uses PBS buffer as a solvent, and 1.0% v / v penicillin-streptomycin mixture is added; The fourth washing liquid uses PBS buffer as a solvent, and 0.5% v / v penicillin-streptomycin mixture is added; The RPMI1640 complete medium comprises 84-86% v / v RPMI1640 basic medium, 14-16% v / v fetal bovine serum, and 0.8-1.2% v / v penicillin-streptomycin mixture.

[0019] The first washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture and 1.0% v / v soluble amphotericin B are added.

[0020] As an embodiment, the fresh cow adipose tissue of the present application is fresh adipose tissue at the omental and mesenteric sites of a cow.

[0021] As an embodiment, the penicillin content in the penicillin-streptomycin mixture of the present application is 10,000 u / mL, and the streptomycin content is 10,000 μg / mL. As an embodiment, the number of first washings of the present application is 1.

[0022] After obtaining the first adipose tissue, the second washing liquid is used to wash the first adipose tissue, thereby obtaining the second adipose tissue. The second washing liquid of the present application uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture is added. The components of the penicillin-streptomycin mixture of the present application have been described above, and will not be repeated here. As an embodiment, the number of second washings of the present application is 1-3; as another embodiment, the number of second washings of the present application is 2.

[0023] After obtaining the second adipose tissue, the present invention performs a third washing on the second adipose tissue using a third washing solution to obtain the third adipose tissue. The third washing solution of the present invention uses PBS buffer as a solvent and adds 1.0% v / v penicillin-streptomycin mixture. The composition of the penicillin-streptomycin mixture of the present invention has been described above and will not be repeated here. In one embodiment, the third washing is performed 1 to 3 times; in another embodiment, the third washing is performed 2 times.

[0024] After obtaining the third adipose tissue, the present invention performs a fourth washing on the third adipose tissue using a fourth washing solution to obtain washed adipose tissue. The fourth washing solution of the present invention uses PBS buffer as a solvent and adds 0.5% v / v penicillin-streptomycin mixture. The components of the penicillin-streptomycin mixture of the present invention have been described above and will not be repeated here. In one embodiment, the fourth washing is performed 1 to 3 times; in another embodiment, the fourth washing is performed 2 times.

[0025] This invention uses a first washing solution, a second washing solution, a third washing solution, and a fourth washing solution sequentially to wash fresh bovine adipose tissue, which has the advantage of reducing the risk of adipose tissue contamination, thereby ensuring the success rate of subsequent cell isolation and culture.

[0026] After obtaining the washed adipose tissue, the present invention cuts the washed adipose tissue into small pieces of 3-5 mm and digests them with collagenase to obtain digested adipose tissue.

[0027] In one embodiment, the collagenase described in this invention is type II collagenase. In one embodiment, the digestion temperature is 37-38°C; in another embodiment, the digestion temperature is 37°C. In one embodiment, the digestion time is 1-2 hours; in another embodiment, the digestion time is 1.5 hours.

[0028] After obtaining the digested adipose tissue, the present invention performs a first centrifugation on the digested adipose tissue, collects the lower liquid layer, performs a second centrifugation, and collects the precipitate. After removing red blood cells from the precipitate, immunomagnetic bead sorting is performed to obtain mononuclear cells.

[0029] As an implementation form, the rotating speed of the first centrifugation is 400-800 rpm; as another implementation form, the rotating speed of the first centrifugation is 500 rpm. As an implementation form, the time of the first centrifugation is 5-6 min; as another implementation form, the time of the first centrifugation is 5 min. As an implementation form, the rotating speed of the second centrifugation is 1000-1500 rpm; as another implementation form, the rotating speed of the second centrifugation is 1500 rpm. As an implementation form, the time of the second centrifugation is 8-10 min; as another implementation form, the time of the second centrifugation is 10 min.

[0030] As an implementation form, the method for removing the red blood cells in the precipitate comprises lysing the precipitate by using a red blood cell lysing solution, and then performing solid-liquid separation to obtain a lysate and collecting the precipitate. As an implementation form, the lysing temperature is 20-25℃. As an implementation form, the lysing time is 5-6 min; as another implementation form, the lysing time is 5 min. The composition of the cell lysing solution is not strictly required in the present application, and it can be purchased conventionally. As an implementation form, the immunomagnetic bead sorting uses MicroBeads.

[0031] The RPMI1640 complete culture medium comprises 84-86% v / v RPMI1640 basic culture medium, 14-16% v / v fetal bovine serum, and 0.8-1.2% v / v penicillin-streptomycin mixture. As an implementation form, the RPMI1640 complete culture medium comprises 84% v / v RPMI1640 basic culture medium, 15% v / v fetal bovine serum, and 1.0% v / v penicillin-streptomycin mixture. As an implementation form, the culture temperature is 37℃. As an implementation form, the culture time is 6 days.

[0032] The present application also provides the application of the dairy cow fat-derived macrophage obtained by the separation and culture method in the preparation of a dairy cow immunological research model and / or a dairy cow disease pathogenesis research model.

[0033] In order to further illustrate the present application, the separation and culture method of the dairy cow fat-derived macrophage and the application thereof provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0034] Example 1 1. Reagents and materials Reagents: RPMI-1640 medium (R8758) was purchased from Sigma-Aldrich; PBS buffer, red blood cell lysis solution was purchased from Solabio (Beijing); penicillin-streptomycin mixture was purchased from Beyotime; fetal bovine serum (FBS) was purchased from CellMax (Australia); trypsin was purchased from Gibco (USA); MACS-buffer, MicroBeads were purchased from Miltenyi Biotec (Germany); mouse anti-bovine CD14 monoclonal antibody was purchased from Novus Biologicals (USA).

[0035] Materials: surgical knife, surgical scissors, surgical forceps, shaver, 5 mL syringe, T25 cell culture bottle, six-well plate, 60 mm cell culture dish, etc.

[0036] 2. Collection of adipose tissue Sample source: from a slaughterhouse in Longfeng District, Daqing City, Heilongjiang Province.

[0037] Pre-treatment of reagents and consumables required for sampling: surgical knife, surgical scissors, surgical forceps, conical flask and beaker, and filter screen need to be sterilized by high-pressure steam in advance; 200 mL PBS buffer was placed in No. 1 beaker, 3 mL penicillin-streptomycin mixture (Beyotime, China, penicillin content: 10000 u / mL, streptomycin content: 10000 μg / mL) and 2 mL soluble amphotericin B were added; 200 mL PBS buffer was placed in No. 2 beaker, 3 mL penicillin-streptomycin mixture was added; 200 mL PBS buffer was placed in No. 3 beaker, 2 mL penicillin-streptomycin mixture was added; 200 mL PBS buffer was placed in No. 4 beaker, 1 mL penicillin-streptomycin mixture was added.

[0038] Sample collection method: First, the collection site was sterilized with iodophor alcohol, and the fat on the mesentery of the cow was collected with sterile surgical instruments, and the surrounding excess tissue was cut off and placed in No. 1 beaker to wash off the impurities and blood on the surface of the fat, and then sent into the intercellular space. The fat tissue was washed with No. 2, 3, and 4 beakers in turn for 3 times, and the tissue was placed in a culture dish and cut into small pieces (about 3-5 mm) with scissors. Transfer into a conical flask, add 50 mL collagenase type II (1 g / L), 37°C water bath shaker for 1.5 h, then add 50 mL RPMI1640 medium in the conical flask, place it on a 50 mL conical tube and filter with a 100µm filter.

[0039] 3. Preparation of bovine adipose tissue stromal cells Centrifugation parameters are 500 rpm / min for 5 min, then 1500 rpm / min for 10 min. Remove supernatant, flick the tube to resuspend the pellet in 10 mL of red blood cell lysis solution R1010 (Solarbio). Incubate for 5 min at room temperature. Neutralize the red blood cells by adding an equal volume of PBS buffer, and centrifuge at 1500 rpm / min for 10 min. Resuspend the isolated cells in PBS and centrifuge at 800 rpm / min for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of MACS-buffer to obtain a suspension of bovine adipose tissue stromal cells.

[0040] Transfer the cell suspension to a 1.5 mL centrifuge tube and centrifuge at 1000 r / min for 6 min. Resuspend in 100 μL of PBS and centrifuge at 1000 r / min for 6 min for 3 times. Dilute the CD11b-PC7-A antibody (0.25 μg / 10 6 ). Incubate at 4°C in the dark for 30 min. After the antibody labeling is complete, add 1 mL of PBS, centrifuge at 1000 r / min for 5 min, and repeat 2-3 times. Discard the supernatant and use a flow cytometer to detect the sample to determine the ratio of bovine adipose tissue stromal cells in the bovine adipose tissue stromal cells. The results show that the proportion of CD11b-positive cells in bovine adipose tissue stromal cells is about 29.7% (n=3). Figure 1 .

[0041] 4. Preparation of primary adipose-derived macrophages Add mouse anti-bovine CD14 monoclonal antibody to the suspension of bovine adipose tissue stromal cells obtained in step 3, mix well, and incubate in a 4°C refrigerator for 30 min, then centrifuge at 250 rpm / min for 5 min. Resuspend in MACS-buffer, add magnetic beads (MicroBeads), mix gently, and incubate in a 4°C refrigerator for 15 min. Place Pre-Separation Filters on MS Columns and place them in a holder. Rinse the MS Columns with MACS-buffer, then add the stromal cells incubated with MicroBeads. After the sample flows into the MS Columns, remove the MS Columns from the holder, add MACS-buffer, collect the magnetically positive cells remaining in the MS Columns, centrifuge at 800 rpm / min for 5 min, and resuspend in RPMI1640 complete medium containing 84% v / v RPMI1640 basal medium, 15% v / v fetal bovine serum, and 1.0% v / v mixture of penicillin-streptomycin to obtain a suspension of primary adipose-derived macrophages.

[0042] 5. Culture Effects of different culture medium on different culture time of bovine adipose-derived macrophages (1) Preparation of culture medium DMEM / F12 complete medium: composed of 84% v / v DMEM / F12 basal medium, 15% v / v fetal bovine serum and 1.0% v / v penicillin-streptomycin mixture; DMEM complete medium: composed of 84% v / v DMEM basal medium, 15% v / v fetal bovine serum and 1.0% v / v penicillin-streptomycin mixture; RPMI-1640 complete medium: composed of 84% v / v RPMI1640 basal medium, 15% v / v fetal bovine serum and 1.0% v / v penicillin-streptomycin mixture.

[0043] (2) The primary adipose-derived macrophages obtained in step 4 were inoculated into different complete culture media for culture. The results showed that on the first day of culture in different complete culture media, the cell density was high, the morphology was consistent, and most of the cells had grown adherently in a circular shape; on the third day, the cell surface area in RPMI-1640 complete medium increased significantly, the morphology and size were different, most of the cells were round or oval, and individual cells had spicules on the surface, while in DMEM / F12 and DMEM complete medium, the cell spacing was larger, the cell morphology was irregular, and individual cells were incomplete; on the sixth day, the cell morphology in RPMI-1640 complete medium was irregular, in a fusiform, triangular and oval shape, and most of the cells had pseudopodia on the surface, while in DMEM / F12 and DMEM complete medium, the cell density continued to decrease, and individual cells had spicules on the edge, and the morphology and number were poorer than those in RPMI-1640 complete medium Figure 2 ). RPMI-1640 complete medium is suitable for in vitro culture of macrophages, and the cell morphology and number are better after 6 days of culture.

[0044] Example 2 Identification of bovine adipose-derived macrophages by immunofluorescence According to the specific expression of CD11b and CD14 in bovine adipose-derived macrophages, the expression of CD11b and CD14 in the extracted cells was detected by immunofluorescence method to identify the phenotype of macrophages. The specific operation steps are as follows: (1) Cell preparation: the bovine adipose-derived macrophages obtained in Example 1 were inoculated into six-well plates containing cell slides at a concentration of 1×10 5 cells / mL, and washed with PBS buffer for 3 times, 5 min each time when the cell confluence reached 80%~90%; (2) Fixation: fixed with 4% paraformaldehyde for 30 min, and washed with PBS buffer for 3 times, 5 min each time; (3) Permeation: the fixed cells were permeated, 0.3% Trition X-100 was incubated on ice for 15 min, so that the antibody could reach the epitope of the antigen, and then PBS buffer was washed for 3 times, 5 min each time; (4) Blocking: the blocking solution (0.5% fetal bovine serum + 0.3% Trition X-100) was incubated at room temperature for 1 h, and PBS buffer was washed for 3 times, 5 min each time; (5) Antibody binding: CD14 (dilution ratio 1:500) and CD11b antibody (dilution ratio 1:1000) were added, and incubated at 4℃ overnight, and PBS buffer was washed for 3 times, 5 min each time; (6) PBS buffer was washed for 3 times, 5 min each time, and DAPI was added for nuclear staining for 10 min; (7) Fluorescence microscope observation of specific fluorescence signal, and taking pictures.

[0045] The results show that the bovine fat-derived mononuclear macrophages prepared by the preparation method have surface specific markers (M0 markers: CD11b, CD14) Figure 3 ).

[0046] Example 3 Identification of the phenotype of bovine fat-derived macrophages by flow cytometry M0 macrophages are characterized by high expression of CD11b and CD14. The macrophage markers were detected by flow cytometry, and the specific operation steps were as follows: The bovine fat-derived macrophage suspension obtained in Example 1 was transferred to a 1.5 mL centrifuge tube, and centrifuged at 1000 r / min for 6 min. The supernatant was discarded, and 100 μl of PBS was resuspended, centrifuged at 1000 r / min for 6 min, and repeated for 3 times. CD14-PC5.5-A and CD11b-PC7-A antibodies (0.25 μg / 10 6 ). After 30 min of incubation at 4℃ in the dark, 1 mL of PBS was added, and centrifuged at 1000 r / min for 5 min, repeated for 2-3 times. The supernatant was discarded, and the sample was detected by flow cytometry to determine the ratio of positive cells. The results show that the bovine fat-derived macrophages separated and cultured highly express CD14 and CD11b Figure 4 ).

[0047] Example 4 Identification of bovine fat-derived macrophage polarization by Western blot method Based on the characteristic that iNOS and CD206 are specifically expressed in macrophages M1 and M2, macrophages were induced to polarize towards M1 and M2 by LPS and IL-4 for 24 hours. Macrophage M1 and M2 markers were then detected by Western blot. The specific procedures were as follows: The bovine adipose-derived macrophages isolated in Example 1 were divided into 1×10 5 Cells were seeded at 84% v / v RPMI 1640 basal medium, 15% v / v fetal bovine serum, and 1.0% v / v penicillin-streptomycin in 60 mm culture dishes and cultured at 37°C and 5% CO2 until 80%–90% confluence. The medium was then replaced with 2% BSA (Biofroxx) RPMI-1640 and treated with 100 ng / mL LPS (Sigma-Aldrich) or 10 ng / mL IL-4 (R&D) for 24 hours. Cells were then divided into Con, LPS, and IL-4 groups. Total protein was extracted from cells cultured under different stimuli, and protein concentration was determined using the BCA method (BCA kit purchased from Beyotime). Protein samples were dropped onto SDS-PAGE gels for electrophoresis and transferred to PVDF membranes (total protein concentration: 20 μg). Blocking with 5% skim emulsion at room temperature for 1.5 h was followed by overnight incubation with the following primary antibodies at 4°C: iNOS (1:2000; Proteintech), CD206 (1:2000; Proteintech), and GAPDH (1:500000; Proteintech). Primary antibodies were washed 5 times with TBST, 6 min each time. Incubation with HRP-labeled secondary antibody (1:10000; Proteintech) at room temperature for 1 h was performed, followed by TBST washing 5 times, 6 min each time. Bands were visualized using an enhanced chemiluminescent substrate (ThermoFischer Scientific) and imaged on a multifunctional imaging system. Image analysis was performed using Image Pro Plus 5.0 software. The above results indicate that the protein expression level of the M1 marker iNOS in macrophages increased while the protein expression level of the M2 marker CD206 decreased in the LPS group; and the protein expression level of the M1 marker iNOS in macrophages decreased while the protein expression level of the M2 marker CD206 increased in the IL-4 group. P <0.01, Figure 5 ).

[0048] According to the above, the present application takes cow adipose tissue as a research object, successfully isolates and cultures cow adipose-derived macrophages, provides a reliable cell model for the research of cow fat metabolism, inflammation regulation and related diseases, is expected to provide a new tool and thought for cow health research and disease prevention, and promotes the sustainable development of the breeding industry.

[0049] Although the above embodiment describes the present application in detail, it is only a part of the embodiment of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which belongs to the protection scope of the present application.

Claims

1. A method for isolating and culturing bovine adipose-derived macrophages, characterized by, The method comprises the following steps: Fresh cow adipose tissue is sequentially washed by a first washing liquid, a second washing liquid, a third washing liquid and a fourth washing liquid to obtain washed adipose tissue; The washed adipose tissue is cut into small pieces of 3-5 mm, and is digested by collagenase to obtain digested adipose tissue; The digested adipose tissue is subjected to first centrifugation, and the lower liquid is collected and subjected to second centrifugation, and the precipitate is collected; the red blood cells in the precipitate are removed, and the positive macrophages of CD14 and CD11b are screened by immunomagnetic bead sorting to obtain primary cow adipose-derived macrophages; The primary cow adipose-derived macrophages are cultured by using RPMI1640 complete medium; The first washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture and 1.0% v / v soluble amphotericin B are added; The second washing liquid uses PBS buffer as a solvent, and 1.5% v / v penicillin-streptomycin mixture is added; The third washing liquid uses PBS buffer as a solvent, and 1.0% v / v penicillin-streptomycin mixture is added; The fourth washing liquid uses PBS buffer as a solvent, and 0.5% v / v penicillin-streptomycin mixture is added; The RPMI1640 complete medium comprises 84-86% v / v RPMI1640 basic medium, 14-16% v / v fetal bovine serum, and 0.8-1.2% v / v penicillin-streptomycin mixture.

2. The separation culture method according to claim 1, characterized by, The RPMI1640 complete medium comprises 84% v / v RPMI1640 basic medium, 15% v / v fetal bovine serum, and 1.0% v / v penicillin-streptomycin mixture.

3. The separation culture method according to claim 1, characterized by, The penicillin content in the penicillin-streptomycin mixture is 10,000 u / mL, and the streptomycin content is 10,000 μg / mL.

4. The separation culture method according to claim 1, characterized by, The collagenase is type II collagenase; the digestion temperature is 37-38℃, and the time is 1-2 h.

5. The separation and culture method according to claim 1, wherein The first centrifugation is performed at a speed of 400-800 rpm for 5-6 min.

6. The separation and culture method according to claim 1, wherein The second centrifugation is performed at a speed of 1000-1500 rpm for 8-10 min.

7. The separation and culture method according to claim 1, wherein The method for removing the red blood cells in the precipitate comprises lysing the precipitate by using a red blood cell lysis solution, performing solid-liquid separation on the lysis product to collect the precipitate; the lysis temperature is 20-25℃, and the time is 5-6 min.

8. The separation and culture method according to claim 1, wherein The culture temperature is 37℃.

9. The separation culture method according to any one of claims 1 to 8, characterized by, The fresh cow adipose tissue is fresh adipose tissue at the greater omentum and mesenteric membrane of a cow.

10. The application of cow adipose-derived macrophages obtained by the separation and culture method in claims 1-9 in the preparation of a cow immunology research model and / or a cow disease pathogenesis research model.

Images