Bovine primary myocardial fibroblast hypoxia model construction method

Abstract

The invention provides a bovine primary cardiac fibroblast hypoxia model construction method, and belongs to the technical field of cell model construction, trypsin and type II collagenase are adopted for sequentially digesting, differential attachment and the like to obtain bovine primary cardiac fibroblasts, the bovine primary cardiac fibroblasts are treated for 24-72 h under the hypoxia condition of 1% O2, 5% CO2 and 94% N2 at the inoculation cell density of 3.5 * 10 < 5 >-8 * 10 < 5 > / ml, and the bovine primary cardiac fibroblasts are obtained. And constructing the cell hypoxia model. The three-gas incubator hypoxia model based on the primary bovine myocardial fibroblasts can accurately simulate a pathological hypoxia environment, and is suitable for hypoxia stress mechanism research and key molecule screening. The model is high in physiological correlation and simple and convenient to operate, has wide applicability and reference value, and provides a reliable experimental platform for research on the bovine hypoxia myocardial diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of cell model construction, and in particular to a method for constructing a bovine primary myocardial fibroblast hypoxia model. Background Art

[0002] In the high-altitude hypoxic environment, introduced plains cattle suffer from a series of altitude sicknesses, including right ventricular hypertrophy and pulmonary hypertension (PH), due to long-term exposure to hypoxic conditions. Furthermore, plateau hypoxia exacerbates oxidative stress, generating excessive reactive oxygen species (ROS), which further damages myocardial cells.

[0003] Studying the cardiac function of cattle under high-altitude hypoxic environments is of great significance for revealing its pathogenesis and developing targeted intervention measures, and constructing an in vitro hypoxic cell model is an important technical means. By accurately simulating the high-altitude hypoxic environment, the metabolic reprogramming and functional changes of cells under hypoxic conditions can be systematically observed. Myocardial fibroblasts, as the main cellular component of the cardiac interstitium, are not only involved in maintaining the normal structure and function of the heart, but also play a core role in the repair of cardiac damage and pathological remodeling. Compared with cardiomyocytes, fibroblasts are more sensitive to environmental stimuli, can quickly respond to hypoxic stress and activate the corresponding molecular regulatory network.

[0004] Using primary cardiac fibroblasts for hypoxia research offers unique advantages. First, primary cells retain the physiological characteristics of the donor animal to the greatest extent possible, avoiding the phenotypic drift that can occur with passaged cells. Second, through specific isolation and culture techniques, highly pure fibroblast populations can be obtained, effectively eliminating interference from other cell types.

[0005] Currently, the construction of a hypoxic environment in vitro widely uses the method of chemical induction, but this method cannot accurately measure the concentration of oxygen. For example, the Chinese invention patent application with application number 2024104455372 proposes the use of cobalt chloride (CoCl2) to simulate hypoxic conditions. Its mechanism relies on the competitive binding of cobalt ions to oxygen molecules. However, this method cannot achieve dynamic monitoring and precise regulation of oxygen concentration, resulting in uncontrollable experimental conditions. A similar study by Wang Dan et al. (2020) showed that although sodium dithionite (Na2S2O4) can rapidly consume oxygen (2mmol / L to maintain an anaerobic state for 1h), high concentrations of Na2S2O4 can cause cell damage (survival rate drops to 50%-60%). These chemical methods all rely on chemical reaction processes and have defects such as large concentration fluctuations, interference from byproducts, and poor safety, making it difficult to meet the requirements of precise oxygen control and experimental safety. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing a hypoxia model of primary bovine cardiac fibroblasts. By establishing a hypoxia model of primary bovine cardiac fibroblasts, the pathological hypoxic environment can be accurately simulated, which is suitable for the study of hypoxic stress mechanisms and the screening of key molecules. It provides a theoretical basis for the prevention and treatment of heart diseases in cattle herds in plateau areas, and also provides new ideas for the development of prevention and treatment strategies for human plateau heart disease, solving at least one technical problem existing in the above-mentioned background technology.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for constructing a hypoxia model of primary bovine cardiac fibroblasts, comprising the following steps:

[0009] (1) The heart tissue was digested sequentially using two enzyme solutions: trypsin solution digestion once and type II collagenase solution digestion six times to obtain a mixture containing myocardial cells, red blood cells, cell debris, and myocardial fibroblasts;

[0010] (2) The cell debris, red blood cells and myocardial cells in the mixed solution of step (1) were separated and removed by the differential adhesion method. The obtained cells were identified as primary myocardial fibroblasts by Vimentin and α-SMA.

[0011] (3) The primary cardiac fibroblasts obtained in step (2) are continued to be adherently cultured.

[0012] (4) After the primary cardiac fibroblasts successfully adhered to the wall, they were randomly divided into a control group and a hypoxia group. The cells in the control group were cultured conventionally. The hypoxia environment parameters of a common three-gas incubator were set to 1% O2, 5% CO2, and 94% N2. The cells in the hypoxia group were placed in the three-gas incubator and cultured for different lengths of time. After joint identification of multiple indicators such as cell morphology, cell viability, and hypoxia stress, an in vitro hypoxia model of bovine primary cardiac fibroblasts was obtained.

[0013] As a further limitation of the first aspect of the present invention, to ensure the stability of the properties of the isolated cells, during the cell subculture process, a representative second generation was selected for immunofluorescence staining identification. The specific experimental process includes:

[0014] (a) When adherent cells reached 50%-70% confluence, they were washed with 1× PBS, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton in ice-cold methanol (pre-chilled at -20°C), and incubated with blocking buffer at room temperature.

[0015] (b) After the blocking solution is discarded, Vimentin and a-SMA primary antibody working solution are added respectively, and the sample is incubated in a wet box at 37℃ for 1.5 hours; after washing with 1xPBS for 3 times, FITC-conjugated Goat anti-Mouse IgG (H+L) is added, and the sample is incubated at 37℃ for 30 minutes in the dark.

[0016] (c) DAPI staining solution is added, and the sample is observed and photographed under a fluorescence microscope.

[0017] As a further limitation of the first aspect of the present application, in order to ensure the successful construction of the bovine primary myocardial fibroblast hypoxic in vitro model, four methods are used for determination, specifically comprising the following steps:

[0018] ①The primary myocardial fibroblasts are randomly divided into normoxic control group and hypoxic treatment group. The control group is cultured under normal conditions, and the hypoxic group is transferred to a hypoxic incubator for culture after adhering to the wall. The hypoxic incubator is set to 1% O2, 5% CO2 and 94% N2;

[0019] ②The cells treated to the desired time are observed for morphology, cell viability is detected, and hypoxic stress level is determined to further determine whether the hypoxic in vitro model of Holstein bovine primary myocardial fibroblasts is successfully constructed;

[0020] As a further limitation of the first aspect of the present application, the calf in step (1) is preferably a 15-day-old Holstein calf;

[0021] As a further limitation of the first aspect of the present application, the specific operation of the trypsin solution and the type II collagenase solution digestion in step (1) is preferably:

[0022] After the heart tissue is washed with pre-cooled D-hanks, a 0.08% trypsin solution is used for preliminary digestion. After removing the digestion solution, a type II collagenase solution with a volume activity unit of 250U / ml is added for digestion 6 times to obtain a mixed solution containing myocardial cells, red blood cells, cell fragments and myocardial fibroblasts;

[0023] As a further limitation of the first aspect of the present application, the specific operation of the differential adhesion method in step (2) is preferably:

[0024] After the mixed solution obtained in step (1) is centrifuged at 800rpm / min, the cells are resuspended in DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody, and are differentially adhered at 37℃ and 5% CO2 for 90 minutes. The cells that have adhered in the culture dish are a mixture containing primary myocardial fibroblasts;

[0025] As a further limitation of the first aspect of the present invention, the vimentin antibody described in step (b) is preferably vimentin (SC-373717, Santa Cruz), and the α-SMA antibody is preferably α-SMA (A5228, Merck), and the dilution ratio used is preferably 1:50 by volume using 1×PBS; the secondary antibody used is preferably FITC-conjugated goat anti-mouse IgG (H+L) (AS001, ABclonal), and the dilution ratio used is preferably 1:200 by volume using 1×PBS;

[0026] As a further limitation of the first aspect of the present invention, the DAPI staining solution used in step (c) is preferably an anti-fluorescence quenching sealing solution (containing DAPI) (P0131, Biyuntian);

[0027] As a further limitation of the first aspect of the present invention, the hypoxic incubator in step ① is preferably a conventional three-gas incubator (HCP-168, Haier Biomedical);

[0028] As a further limitation of the first aspect of the present invention, the cell viability detection method described in step ② is preferably MTT viability detection; the hypoxic stress detection method is preferably a method of measuring reactive oxygen species (ROS) levels and detecting dynamic changes in mitochondrial membrane potential using a JC-1 fluorescent probe method.

[0029] In a second aspect, the present invention provides a bovine primary cardiac fibroblast hypoxia model constructed by the method described in the first aspect.

[0030] The beneficial effects of the present invention are as follows: a hypoxia model of primary bovine myocardial fibroblasts was successfully established, providing a corresponding hypoxia model of bovine myocardial fibroblasts for the study of hypoxia problems in livestock myocardial fibroblasts; the hypoxia incubator used is a three-gas incubator, which solves the problem that chemical substances such as cobalt chloride (CoCl2) and sodium dithionite (Na2S2O4) cannot accurately measure oxygen concentration; by optimizing parameters such as morphological observation, cell viability detection and oxidative stress level determination, the obtained in vitro hypoxia model of primary Holstein bovine myocardial fibroblasts shows morphological changes compared with the control, cell activity first increases and then decreases, ROS content significantly increases, mitochondrial damage significantly increases, and has typical clinical characteristics.

[0031] Additional advantages of the present invention will be more clearly given in the following description or learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. The drawings only illustrate some embodiments of the present invention.

[0033] Figure 1 This is an example flow chart for isolating and extracting primary cardiac fibroblasts and establishing a hypoxic cell model as described in an embodiment of the present invention.

[0034] Figure 2 This is a schematic diagram of immunofluorescence identification (20×) of second generation cells using Vimentin and α-SMA as described in an embodiment of the present invention.

[0035] Figure 3 Schematic diagram of the comparison of cell morphology (10×) between the cells described in the examples of the present invention at 24h, 48h, and 72h of hypoxia and the control.

[0036] Figure 4 This is a schematic diagram of the results of detecting cell viability at 24 h, 48 h, and 72 h using the MTT method in 1% O2 as described in an embodiment of the present invention.

[0037] Figure 5 Schematic diagram of ROS detection results (4×) of cells described in an embodiment of the present invention at 1% O2 for 24h, 48h, and 72h.

[0038] Figure 6 This is a schematic diagram (4×) of the results of detecting changes in mitochondrial membrane potential of cells described in an example of the present invention using a JC-1 kit at 1% O2 for 24 hours, 48 ​​hours, and 72 hours. DETAILED DESCRIPTION

[0039] The following describes embodiments of the present invention in detail, with examples of the embodiments illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative and intended only to illustrate the present invention, and are not to be construed as limiting the present invention. The accompanying drawings are merely schematic illustrations of the embodiments, and the components shown in the drawings are not necessarily required to practice the present invention.

[0040] Example 1

[0041] This Example 1 provides a protocol for the isolation and identification of primary cardiac fibroblasts. Figure 1 As shown, an efficient and convenient method for isolating primary Holstein bovine cardiac fibroblasts is provided, which specifically comprises the following steps:

[0042] (1) Heart tissue of a 15-day-old Holstein calf was obtained, and approximately 300 mg of heart tissue was cut using ophthalmic scissors. After washing with pre-cooled D-hanks solution, the tissue was fully minced and then digested with a 0.08% trypsin solution once and a type II collagenase solution with a volume activity unit of 250 U / ml six times to obtain a mixture containing cardiomyocytes, red blood cells, cell fragments, and cardiac fibroblasts.

[0043] (2) The mixed solution obtained in step (1) was centrifuged at 800 rpm / min for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody by volume, and the cells were inoculated into a cell culture dish with a diameter of 10 cm. After culturing in a cell culture incubator with a volume fraction of 5% CO2 and 37°C for 90 min, the cell suspension was aspirated and discarded. The cells attached to the dish were myocardial fibroblasts. Among them, myocardial cells and myocardial fibroblasts have different attachment speeds. Myocardial cells need 2-24 h to attach to the wall, but myocardial fibroblasts can attach to the wall in about 10-30 min. By utilizing the different attachment speeds of these two types of cells, myocardial cells can be effectively removed.

[0044] (3) The primary cardiac fibroblasts obtained in step (2) are continued to be adherently cultured.

[0045] (4) After the primary cardiac fibroblasts successfully adhered to the wall, they were randomly divided into a control group and a hypoxia group. The cells in the control group were cultured conventionally. The hypoxia environment parameters of a common three-gas incubator were set to 1% O2, 5% CO2, and 94% N2. The cells in the hypoxia group were placed in the three-gas incubator and cultured for different lengths of time. After joint identification of multiple indicators such as cell morphology, cell viability, and hypoxia stress, an in vitro hypoxia model of bovine primary cardiac fibroblasts was obtained.

[0046] To ensure the stability of the properties of the isolated cells, in this example, a representative second generation of cells was selected for immunofluorescence staining during the cell subculture process. The specific experimental process includes:

[0047] (a) The cells from step (3) were seeded into 12-well or 24-well plates, and when the cells reached a confluence of 50% to 70%, they were washed with 1× PBS, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton in ice-cold methanol (pre-chilled at -20°C), and incubated at room temperature with blocking solution;

[0048] (b) After discarding the blocking solution, vimentin (SC-373717, Santa Cruz) and α-SMA (A5228, Merck) antibody working solutions diluted 1:50 in 1× PBS were added and incubated in a humidified chamber at 37°C for 1.5 h. After washing three times with 1× PBS, FITC-conjugated goat anti-mouse IgG (H+L) (AS001, ABclonal) diluted 1:200 in 1× PBS was added and incubated at 37°C in the dark for 30 min.

[0049] (c) Anti-fluorescence quenching mounting solution (containing DAPI) (P0131, Biyuntian) was added and the sections were observed and photographed under a fluorescence microscope.

[0050] Experimental results

[0051] The successfully isolated primary cells can be successfully propagated to the 9th generation through continuous subculture. At the 7th generation, the growth rate is reduced by half. After the 9th generation, the cell growth rate slows down after attachment, and the cells die after one day.

[0052] The second generation cells were identified using Vimentin and α-SMA, and it was found that both Vimentin and α-SMA were positive. Figure 2 As shown, primary cardiac fibroblasts from Holstein cattle were successfully isolated in this example.

[0053] Example 2

[0054] In Example 2, a hypoxia model was constructed for primary bovine cardiac fibroblasts. To address the difficulty in accurately controlling O2 concentrations when using chemical drugs, as well as the difficulty in purchasing precursor drugs and their inherent toxicity, this example employed a three-gas incubator with adjustable O2 concentration, resulting in a precise and simple experiment. The specific process is as follows:

[0055] ① The primary cardiac fibroblasts obtained in Example 1 were seeded into a cell culture dish with a diameter of 10 cm, and when cultured to a confluence of 80%-90%, digested and centrifuged; the cells were resuspended in DMEM / F12 medium containing 10% fetal bovine serum and 1% double-antibody;

[0056] ② The cell suspension was seeded at a density of 8×10 at 24h, 48h, and 72h. 5 , 5×10 5 , 3.5×10 5 / ml, and seeded into 6-well plates, the cells were randomly divided into a control group (Control) and a hypoxia group (Hypoxia); the cells in the control group were placed in a cell culture incubator with a volume fraction of 5% CO2 and 37°C for conventional culture, while the cells in the hypoxia group were placed in a three-gas incubator with a volume fraction of 1% O2, 5% CO2, and 94% N2 at 37°C for hypoxia culture.

[0057] Experimental results:

[0058] Comparison of cell morphology between the three hypoxia-treated groups at 24h, 48h, and 72h and the control group revealed that at 24h, the cell density of the two groups was similar, the morphology was uniform, and there was no significant difference; at 48h, the cells in the control group were dense and well-spread, while the hypoxia group showed cell vacuoles (red circles), and the morphological integrity was affected; at 72h, the cells in the control group filled the well plate and were arranged regularly, while the hypoxia group showed cell detachment and death areas (red circles), and the density was lower than that of the control group, reflecting the inhibition and damage of the hypoxic environment (48h, 72h) on the cell growth state. Figure 3 shown.

[0059] Example 3

[0060] This Example 3 provides an MTT assay for detecting the viability of cardiac fibroblasts. The experimental procedures for cells with a 1% O2 concentration and a control group are as follows:

[0061] ①Same as Example 2①

[0062] ② The cell suspension was seeded at a density of 8×10 at 24h, 48h, and 72h. 3 , 5×10 3 , 3.5×10 3 / ml, and then seeded into 96-well plates, the cells were randomly divided into a control group (Control) and a hypoxia group (Hypoxia), with 10 replicates in each group; the cells in the control group were placed in a cell culture incubator with a volume fraction of 5% CO2 and 37°C for conventional culture, while the cells in the hypoxia group were placed in a three-gas incubator with a volume fraction of 1% O2, 5% CO2, and 94% N2 at 37°C for hypoxia culture;

[0063] ③ After 24h-72h, remove the cells, add 10ul of MTT solution to each well, and incubate in a 5% CO2, 37℃ cell culture incubator for 4h. Aspirate the liquid and add 100ul of DMSO solution. After incubation for 10min, detect the OD value (492nm) in a microplate reader.

[0064] Experimental results:

[0065] The cell viability was detected by MTT assay at 24 h, 48 h, and 72 h. Figure 4As shown in the results, 1% O2 conditions can promote cell proliferation for 24 hours; while the low oxygen environment at 48 hours and 72 hours has a significant inhibitory effect on cell proliferation, reflecting the temporal regulatory effect of low oxygen intervention on cell proliferation.

[0066] Example 4

[0067] In this example, an immunofluorescence assay is provided to detect the reactive oxygen species (ROS) content in primary bovine cardiac fibroblasts. To further detect the content of reactive oxygen species (ROS) produced by cardiac fibroblasts under hypoxic conditions, this example employs an immunofluorescence assay using a ROS kit from YEASEN to detect the ROS content of cardiac fibroblasts at 1% O₂ for 24, 48, and 72 hours. The specific procedure is as follows:

[0068] ①Same as Example 2①

[0069] ② The cell suspension was seeded at a density of 8×10 at 24h, 48h, and 72h. 5 , 4×10 5 , 3.5×10 5 Cells were seeded at a concentration of 1 μg / ml into a 3.5 cm diameter cell culture dish and randomly divided into a control group (Control) and a hypoxia group (Hypoxia), with two replicates per group. The cells in the control group were placed in a 5% CO2, 37°C cell culture incubator for conventional culture, while the cells in the hypoxia group were placed in a 37°C, 1% O2, 5% CO2, 94% N2, three-gas incubator for hypoxia culture.

[0070] ③ After 24 h, 48 h, and 72 h, remove the cells, wash them with 1× PBS, add 1 ml of DCFH-DA working solution diluted with serum-free DMEM / F12 medium to a final concentration of 10 μM, and incubate in a cell culture incubator at 37°C in the dark for 30 min;

[0071] ④ Wash the cells with serum-free DMEM / F12 medium and observe and photograph them under a fluorescence microscope.

[0072] Experimental results:

[0073] ROS detection at 24h, 48h, and 72h Figure 5As shown, the ROS production level in the control group was low at the three time periods, and the fluorescence signal was almost invisible. The hypoxic intervention group showed a time-dependent change: the ROS level was low at 24 h, and the fluorescence intensity was the lowest in the group; at 48 h, ROS production increased significantly, and the fluorescence signal also increased, with the fluorescence intensity being the highest in the group; at 72 h, the ROS level decreased slightly compared with that at 48 h, and the fluorescence intensity was slightly weaker than that at 48 h, but was still significantly higher than that at 24 h, indicating that the hypoxic environment could induce dynamic time-dependent changes in ROS production in cells.

[0074] Example 5

[0075] In this example, the JC-1 immunofluorescence method was used to detect the changes in mitochondrial membrane potential of cardiac fibroblasts. To further detect whether the mitochondria of cardiac fibroblasts were damaged under hypoxic conditions and the extent of the damage, the JC-1 immunofluorescence method was used to detect the changes in mitochondrial membrane potential of cardiac fibroblasts under 1% O2 for 24 h, 48 h, and 72 h. The mitochondrial membrane potential can represent whether the mitochondria are damaged, and the specific process is as follows:

[0076] ① Same as Example 1 ①

[0077] ② Same as Example 4 ②

[0078] ③ At 24 h, 48 h, and 72 h, the cells were washed with 1xPBS, and then 1 ml of JC-1 staining working solution prepared according to the instructions was added. The cells were incubated in a 37°C cell incubator for 20 min in the dark;

[0079] ④ During the incubation period, the JC-1 staining buffer (1X) was prepared according to the instructions and ice-bathed. After incubation at 37°C, the supernatant was aspirated, and the cells were washed with the JC-1 staining buffer (1X);

[0080] ⑤ 2 ml of cell culture medium was added, and the cells were observed and photographed under a fluorescence microscope.

[0081] Experimental results:

[0082] JC-1 staining was used to detect the mitochondrial membrane potential at 24 h, 48 h, and 72 h, and the results are shown in Figure 6 The results showed that under hypoxic treatment, the JC-1 monomer green fluorescence increased with time, and the polymer red fluorescence decreased in a time-dependent manner, indicating that the longer the hypoxic treatment time, the more significant the decline in mitochondrial membrane potential and the more severe the functional damage.

[0083] In summary, this invention provides a method for constructing a hypoxia model with high physiological relevance, ease of operation, broad applicability, and valuable reference. This method allows cells to be cultured in hypoxia for up to 72 hours. This method not only addresses the difficulty of precisely controlling oxygen concentration with chemical agents but also accurately simulates pathological hypoxic environments, providing a reliable experimental platform for studying hypoxic stress mechanisms and screening key molecules.

[0084] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solutions disclosed in the present invention without the need for creative work should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a hypoxia model of primary bovine cardiac fibroblasts, characterized in that: include: The heart tissue was digested with an enzyme solution to obtain a mixture containing cardiomyocytes, red blood cells, cell debris, and cardiac fibroblasts; The cell debris, red blood cells and cardiomyocytes in the mixed solution were separated and removed by differential adhesion method to obtain primary cardiac fibroblasts; The obtained primary cardiac fibroblasts were continued to be cultured adherently; After the primary cardiac fibroblasts successfully adhered to the wall, they were randomly divided into a control group and a hypoxia group. The cells in the control group were cultured in a conventional manner. The hypoxia environment parameters of the common three-gas incubator were set to 1% O2, 5% CO2 and 94% N2. The cells in the hypoxia group were placed in a three-gas incubator and cultured for different lengths of time. After joint identification of multiple indicators such as cell morphology, cell viability, and hypoxia stress, an in vitro hypoxia model of bovine primary cardiac fibroblasts was obtained.

2. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 1, characterized in that: Representative second-generation cells cultured by the differential adhesion method were selected for immunofluorescence staining identification, including: When adherent cells were cultured to 50%-70% confluence, they were washed with 1× PBS, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton in ice-cold methanol, and incubated with blocking solution at room temperature. After discarding the blocking solution, add vimentin and α-SMA primary antibody working solutions respectively, and incubate in a humidified chamber at 37°C for 1.5 h. After washing three times with 1× PBS, add FITC-conjugated goat anti-mouse IgG (H+L) and incubate at 37°C in the dark for 30 min. Add DAPI staining solution and observe and take pictures under a fluorescence microscope.

3. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 1, characterized in that: Digest the heart tissue using trypsin solution and collagenase type II solution, including: After washing the heart tissue with pre-cooled D-hanks, it was initially digested with a 0.08% trypsin solution. After removing the digestion solution, a type II collagenase solution with a volume activity unit of 250 U / ml was added and digested six times to obtain a mixture containing cardiomyocytes, red blood cells, cell fragments and cardiac fibroblasts.

4. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 1, characterized in that: The specific operation of the differential adhesion method is as follows: the obtained mixed solution is centrifuged at 800 rpm / min, and the cells are resuspended in DMEM / F12 culture medium containing 10% fetal bovine serum and 1% double-antibody. The cells are differentially adhered to the culture dish for 90 minutes at 37°C and 5% CO2. The cells that have adhered to the culture dish are a mixture containing primary cardiac fibroblasts.

5. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 1, characterized in that: The hypoxic incubator is a conventional three-gas incubator HCP-168, Haier Biomedical.

6. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 1, characterized in that: The cell viability detection method is MTT viability detection; the hypoxia stress detection method is to measure the level of reactive oxygen species and detect the dynamic changes of mitochondrial membrane potential using the JC-1 fluorescent probe method.

7. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 2, characterized in that: The second generation was subjected to an immunofluorescence staining identification experimental process, including: the vimentin antibody was SC-373717, Santa Cruz; the α-SMA antibody was A5228, Merck, and the dilution ratio used was 1:50 by volume using 1×PBS.

8. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 2, characterized in that: The secondary antibody used was FITC-conjugated Goat anti-Mouse IgG (H+L), and the dilution ratio was 1:200 in 1× PBS.

9. The method for constructing a hypoxia model of primary bovine cardiac fibroblasts according to claim 2, characterized in that: The DAPI staining solution used is an anti-fluorescence quenching sealing solution.

10. A bovine primary cardiac fibroblast hypoxia model, characterized in that: It is constructed by the method according to any one of claims 1 to 10.

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