Method for constructing pig follicular granule cell ferroptosis model

Abstract

The invention discloses a construction method of a pig follicular granule cell ferroptosis model, and relates to the technical field of biology. The construction method comprises the following steps: rinsing a pig ovary with washing liquor until the washing liquor is clear, soaking, putting into the washing liquor for later use, extracting follicular fluid containing granular cells from the pig ovary, and putting into 10% fetal calf serum; filtering, collecting and centrifuging granular cells, removing supernate, re-suspending the cells, repeating centrifugal cleaning, re-suspending the granular cells in 10% fetal calf serum, adjusting the density of the granular cells, and culturing; and treating the granular cells with an ammonium ferric citrate solution, and cleaning to obtain the pig follicle granular cell ferroptosis model. According to the method provided by the invention, the pig follicular granule cell ferroptosis model can be successfully constructed, the standardization degree and repeatability of the model are remarkably improved, and the problems of unstable induction efficiency and limited application range of a traditional method are effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of biological technologies, and particularly to a method for constructing a ferroptosis model of porcine follicular granulosa cells. Background Art

[0002] Ferroptosis is a form of programmed cell death that regulates intracellular lipid metabolism, iron metabolism, and redox levels. It is characterized by the accumulation of ROS, iron overload, and lipid peroxidation, leading to cell rupture and death. Different from other forms of cell death (including autophagy, necrosis, and apoptosis), ferroptosis is accompanied by morphological features such as mitochondrial shrinkage, reduced cristae, and rupture of the outer mitochondrial membrane, as well as biochemical features such as increased intracellular free iron content, ROS accumulation, and lipid peroxidation. Follicular granulosa cells play a key role in the development and maturation of follicles. By screening drugs or nutrients that can inhibit the ferroptosis of granulosa cells, the follicular development and ovulation of sows can be improved, the litter size can be increased, which helps to increase the economic benefits of the pig industry and meet the growing demand of the meat market. In addition, understanding the occurrence mechanism of ferroptosis in granulosa cells is also helpful for the healthy development of the livestock and poultry breeding industry. In view of this, a method for constructing a ferroptosis model of porcine follicular granulosa cells is specifically proposed. Summary of the Invention

[0003] Aiming at the above deficiencies in the prior art, the present invention provides a method for constructing a ferroptosis model of porcine follicular granulosa cells, which significantly improves the standardization degree and repeatability of the model, and effectively solves the problems of unstable induction efficiency and limited applicable range existing in the traditional method.

[0004] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: to provide a method for constructing a ferroptosis model of porcine follicular granulosa cells, including the following steps:

[0005] S1. Rinse the porcine ovaries with a washing solution until the washing solution is clear, then soak them and place them in the washing solution for standby. Then, extract the follicular fluid containing granulosa cells from the porcine ovaries and place it in 10% fetal bovine serum.

[0006] S2. Filter and collect the granulosa cells and centrifuge them. After removing the supernatant, resuspend the cells, repeat centrifugation and washing, then resuspend the granulosa cells in 10% fetal bovine serum, and then adjust the density of the granulosa cells and culture them.

[0007] S3. Treat the granulosa cells with ferric ammonium citrate solution, and after washing, obtain a ferroptosis model of porcine follicular granulosa cells.

[0008] Further, in step S1, the washing solution is a PBS solution containing 1-2% triple antibiotics at 36-38°C.

[0009] Further, in step S1, the washing solution is a PBS solution containing 1% triple antibiotics at 37°C.

[0010] Further, in step S1, soak in an ethanol solution with a concentration of 74 - 76% for 29 - 31 s.

[0011] Further, in step S1, soak in an ethanol solution with a concentration of 75% for 30 s.

[0012] Further, in step S1, place in 10 - 11% fetal bovine serum.

[0013] Further, in step S1, use a 1 mL syringe to aspirate the follicular fluid containing granulosa cells.

[0014] Further, in step S1, place in 10% fetal bovine serum.

[0015] Further, in step S2, filter through a 200 - mesh cell sieve.

[0016] The purpose of adopting the above further scheme is: to remove impurities.

[0017] Further, in step S2, collect into a 15 mL centrifuge tube.

[0018] Further, in step S2, centrifuge at 1400 - 1600 r / min for 9 - 11 min.

[0019] Further, in step S2, centrifuge at 1500 r / min for 10 min.

[0020] Further, in step S2, resuspend the cells with a PBS solution containing 1 - 2% triple antibody at 36 - 38°C, repeat centrifugation and washing 2 - 3 times, and then resuspend the granulosa cells in 10 - 11% fetal bovine serum.

[0021] Further, in step S2, resuspend the cells with a PBS solution containing 1% triple antibody at 37°C, repeat centrifugation and washing 2 times, and then resuspend the granulosa cells in 10% fetal bovine serum.

[0022] Further, in step S2, adjust the granulosa cell density to 55 - 65% and culture in an incubator at 36 - 38°C with a carbon dioxide concentration of 4 - 5% until the granulosa cell density reaches 80 - 90%, and change the medium once every 24 - 36 h during this period.

[0023] Further, in step S2, adjust the granulosa cell density to 60% and culture in an incubator at 37°C with a carbon dioxide concentration of 5% until the granulosa cell density reaches 80 - 90%, and change the medium once every 24 - 36 h during this period.

[0024] Further, in step S3, treat the granulosa cells with ammonium ferric citrate at a concentration of 150 - 250 μmol / L for 35 - 37 h.

[0025] Furthermore, in step S3, the granulosa cells were treated with ammonium ferric citrate at a concentration of 200 μmol / L for 36 h.

[0026] Furthermore, in step S3, the cells were washed 2 - 3 times with PBS solution containing 1 - 2% triple antibiotics at 36 - 38°C.

[0027] Furthermore, in step S3, the cells were washed 2 times with PBS solution containing 1% triple antibiotics at 37°C.

[0028] A porcine follicular granulosa cell ferroptosis model constructed by the above - mentioned method for constructing a porcine follicular granulosa cell ferroptosis model.

[0029] The present invention has the following beneficial effects:

[0030] 1. Precision - optimized ferroptosis induction conditions to enhance model stability: By systematically screening the time and dose of different concentrations of FAC (ferric chelate of 1,10 - phenanthroline) for treating porcine follicular granulosa cells, it was determined that the optimal induction condition was treatment with 200 μmol / L for 36 hours (cell viability decreased to about 50%). This solved the ambiguity problem of dose - and - time dependence in the prior art. Compared with the possible unstable induction efficiency or limited applicable range of traditional methods, this scheme significantly improved the standardization and repeatability of the model, providing a reliable basis for subsequent research.

[0031] 2. Multi - dimensional comprehensive verification system to enhance scientific rigor: The present invention integrates multiple detection methods such as Western Blot for detecting ferroptosis - related proteins, transmission electron microscopy (observing mitochondrial morphology), mitochondrial membrane potential (JC - 1 probe), ROS level (immunofluorescence), and iron ion content (FerroOrange probe), comprehensively covering the biochemical, morphological, and functional characteristics of ferroptosis. The prior art mostly relies on a single indicator (such as only detecting lipid peroxidation or ROS), while this method ensures the scientificity and accuracy of model construction through multi - indicator linkage verification, providing multi - dimensional data support for in - depth mechanism research.

[0032] 3. Filling the research gap with species and cell specificity and promoting industrial application: The present invention constructs a ferroptosis model for porcine follicular granulosa cells for the first time, filling the research gap in ferroptosis of the reproductive system of livestock and poultry. Existing research mostly focuses on human or mouse models, while pigs, as important economic animals, their follicular development is directly related to breeding efficiency. This model provides a direct tool for exploring abnormal follicular development in sows and screening nutritional or drug intervention strategies to improve reproductive performance, with both basic research value and industrial transformation potential, helping to improve the quality and efficiency of the livestock and poultry breeding industry. Brief Description of the Drawings

[0033] Figure 1 It is a fluorescence image for immunofluorescence identification of granulosa cells.

[0034] Figure 2 Cell viability graphs of porcine granulosa cells treated with different concentrations of FAC;

[0035] Figure 3 Protein band graphs of ferroptosis-related proteins;

[0036] Figure 4 Quantification graphs of ferroptosis-related protein expression;

[0037] Figure 5 Fluorescence intensity graphs of ROS in granulosa cells;

[0038] Figure 6 Quantification graphs of ROS levels in granulosa cells;

[0039] Figure 7 Mitochondrial morphology graphs of granulosa cells;

[0040] Figure 8 Mitochondrial fluorescence graphs of granulosa cells;

[0041] Figure 9 Quantification graphs of mitochondrial numbers in granulosa cells under different treatments;

[0042] Figure 10 Mitochondrial membrane potential fluorescence graphs of granulosa cells;

[0043] Figure 11 Quantification graphs of mitochondrial membrane potential in granulosa cells;

[0044] Figure 12 Fluorescence intensity graphs of iron ions in granulosa cells;

[0045] Figure 13 Quantification graphs of iron ion content in granulosa cells;

[0046] Figure 14 Quantification graphs of LPO and GSH / GSSH content in granulosa cells. Detailed implementation manners

[0047] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0048] The main reagents include: FBS (Vivacell, 2250340); DMEM / F12 1:1 basic (Hyclone, SH30023.01); Enhanced CCK-8 kit (Beyotime, C0042); PBS phosphate buffer powder (Solarbio, P1010); Penicillin-streptomycin-gentamicin mixed solution (Solarbio, P1410); Rapid membrane transfer solution (Beyotime, P0575-10L); Electrophoresis solution (Beyotime, P0014B); Precision Plus Protein TM Dual Color Standards (Bio-Rad, 1610374); 30% gel preparation solution (Solarbio, A1010); Ultra-sensitive ECL chemiluminescence kit (Beyotime, P0018AM); Wb primary antibody dilution solution (Beyotime, P0023A); Wb secondary antibody dilution solution (Beyotime, P0023D); β-actin (abclonal, AC026, 1 / 1000); Horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1 / 1000) FDX1 (Abmart, T510671, 1 / 2500); FHC (Abcam, ab81444, 1 / 1000); GPX4 (Abcam, ab125066, 1 / 1000); SLC7A11 (Proteintech, 26864-1-AP, 1 / 1000); ACSL4 (Abcam, ab155282, 1 / 1000); HO-1 (Proteintech, 10701-1-AP, 1 / 1000); QuickBlock TM Immunofluorescence staining primary antibody dilution solution (Beyotime, P0262); QuickBlock TM Immunofluorescence staining secondary antibody dilution solution (Beyotime, P0265); QuickBlock TM Immunofluorescence staining blocking solution (Beyotime, P0620); DAPI staining solution (Beyotime, C1005); FerroOrange (Dojindo, F374); Enhanced mitochondrial membrane potential detection kit (Beyotime, C2003S); Reactive oxygen species detection kit (Beyotime, S0033S); LPO detection kit (Nanjing Jiancheng, A106-1-2); GSH / GSSG assay kit (Nanjing Jiancheng, S0053).

[0049] The main instruments include

[0050]

[0051] Example 1 Isolation, identification and culture of porcine primary granulosa cells

[0052] The freshly collected porcine ovaries were placed in PBS solution containing 1% triple antibiotics at 37°C. The collected ovaries were rinsed in a beaker with PBS until the washing solution was clear. The washed ovaries were soaked in 75% alcohol solution for 30 s, and then the ovaries were placed in PBS solution containing 1% triple antibiotics at 37°C for standby; in the laminar flow hood, a 1 mL syringe was used to aspirate the follicular fluid containing granulosa cells from the ovaries, and placed in 10% FBS. After the collection of granulosa cells, impurities were removed using a 200-mesh cell filter sieve and collected into a 15 mL centrifuge tube; centrifuged at 1500 r / min at room temperature for 10 min. After pouring out the supernatant, PBS containing 1% triple antibiotics at 37°C was added again to resuspend the cells, and the centrifugation and washing were repeated 2 times; the washed granulosa cells were resuspended in 10% FBS, and the density of granulosa cells was adjusted to about 60%. The cell culture plate was placed in an incubator at 37°C and 5% CO2. Then, the medium was changed once every 24 - 36 h. After the cell density reached 80 - 90%, the granulosa cells were processed; after the separation and culture of granulosa cells, they were washed 3 times with PBS at 37°C for 30 s each time, fixed with 4% paraformaldehyde for 20 min, then washed 3 times with PBS for 10 s each time, permeabilized with 0.5% TritonX100 for 10 min, then washed 3 times with PBS for 10 s each time, blocked with immunostaining blocking solution at room temperature for 60 min, incubated with primary antibody FSHR overnight in a 4°C refrigerator, then washed 3 times with PBS for 3 min each time, incubated with fluorescent secondary antibody Cy3 in the dark at room temperature for 1 h, then washed 3 times with PBS for 3 min each time, incubated with DAPI in the dark for 5 min, then washed 3 times with PBS for 3 min each time, and images were collected under a fluorescence inverted microscope. The results are as Figure 1 shown, Figure 1 Among them, from left to right are the immunofluorescence image of FSHR protein (FSHR), the nuclear fluorescence image (DAPI), and the merged image (Merge) of the two. Scale bar = 100 μm.

[0053] It can be Figure 1 seen that FSHR (granulosa cell Maker protein) is highly expressed in the separated and cultured granulosa cells, indicating that the separation purity of granulosa cells is high and can be used for subsequent experiments.

[0054] Example 2 CCK8 assay for cell viability

[0055] Porcine granulosa cells were treated with ammonium ferric citrate (FAC) at 0 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, and 800 μmol / L for 24 h and 36 h respectively. The cells were washed twice with PBS solution containing 1% triple antibiotics at 37°C. 10% CCK8 solution was added to each well, and the cells were cultured in an incubator at 37°C and 5% CO2 for 2 h. The absorbance at 450 nm was measured using a microplate reader, and then the cell viability was calculated. The cell viability results are asFigure 2 As shown Figure 2 In it, the left figure is the result after 24 h of treatment, and the right figure is the result after 36 h of treatment. ** indicates (P<0.01).

[0056] It can be seen from Figure 2 that treating porcine granulosa cells with different concentrations of FAC for 24 h has no significant effect on the activity of granulosa cells. As the treatment time increases to 36 h, the cell activities in the different concentration FAC treatment groups are significantly decreased compared with the control group. In summary, treating granulosa cells with FAC for 36 h will significantly affect their activity.

[0057] Example 3 WB detection of the expression of ferroptosis-related proteins FHC, GPX4, SLC7A11, ACSL4 and HO-1

[0058] Porcine granulosa cells were treated with 0 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, and 800 μmol / L of FAC for 36 h, and then the culture medium was discarded. The cells were washed 5 times with PBS at 4°C for 30 s each time. 60 μL of RIPA was evenly added to each 6-well cell culture plate and then placed in a refrigerator at 4°C for 15 min. The lysed granulosa cells were scraped off with a cell scraper and then placed in a 1.5 mL EP tube. The cells were fully lysed using a cell disruptor, and centrifuged at 15000 r / min for 10 min at 4°C using a high-speed refrigerated centrifuge. The supernatant was taken and placed in a new EP tube. After detecting the protein concentration by the BCA method, the protein concentration was normalized. The proteins were separated by a 12% SDS-PAGE separating gel and a 5% SDS-PAGE stacking gel and then transferred to a PVDF membrane. The membrane was transferred at a constant current of 400 mA for 25 min; the PVDF membrane was blocked with 5% skim milk for 2 h. The PVDF membrane was incubated with primary antibodies against β-actin, FHC, GPX4, SLC7A11, ACSL4 and HO-1 overnight; the PVDF membrane was washed 3 times with TBST for 10 min each time; the PVDF membrane was incubated with a secondary antibody (goat anti-rabbit) for 2 h. After washing 3 times with TBST, the PVDF membrane was soaked with a hypersensitive ECL chemiluminescent solution and then placed in the sample placement area of a chemiluminescent imaging system for development and imaging. The protein band diagram is as Figure 3 shown, and the quantification results are as Figure 4 shown. Figure 4 In it, A is the result of ACSL4 / β-actin, B is the result of SLC7A11 / β-actin, C is the result of HO-1 / β-actin, D is the result of FHC / β-actin, and E is the result of GPX4 / β-actin. The same lowercase letters indicate no significant difference between the two groups (P>0.05), and different lowercase letters indicate a significant difference between the two groups (P<0.05).

[0059] It can be seen fromFigure 3 and Figure 4 It can be seen that compared with the control group, 200 μmol / L FAC significantly down-regulated the levels of the key ferroptosis proteins FHC, GPX4, SLC7A11, and ACSL4 in granulosa cells (P<0.05), and significantly up-regulated the level of HO-1 (P<0.05). Therefore, the ferroptosis inducer FAC at 200 μmol / L was selected for treatment for 36 h for subsequent experiments.

[0060] Example 5 Immunofluorescence detection of ROS levels in granulosa cells

[0061] After granulosa cells were treated with 200 μmol / L FAC for 36 h, the culture medium was discarded, and the cells were washed 3 times with PBS at 37 °C for 30 s each time. An appropriate volume of diluted DCFH-DA was added, and the cells were incubated in a cell culture incubator at 37 °C for 20 min. The cells were washed 3 times with serum-free cell culture medium for 10 s each time, and images were collected under a fluorescence inverted microscope. The results are as Figure 5 shown, Figure 5 in, scale bar = 200 μm. The quantification graph is as Figure 6 shown.

[0062] From Figure 5 and Figure 6 it can be seen that by detecting with the cell-permeable probe DCFH-DA, compared with the control group, the ROS level in granulosa cells treated with FAC was significantly increased (P<0.05).

[0063] Example 6 Transmission electron microscopy observation of mitochondrial morphology

[0064] The treated porcine granulosa cells were digested with trypsin and collected by centrifugation (2000 r, 10 min), and then fixed with 3% glutaraldehyde at 4 °C for 10 h. Since a certain number of cells will inevitably be lost during the preparation of electron microscopy samples, the cells must reach a certain quantity, that is, the cell quantity after centrifugation is at least the size of a soybean. Image observation and collection were carried out by transmission electron microscopy. The results are as Figure 7 shown, scale bar = 500 nm.

[0065] From Figure 7 it can be seen that after FAC treatment, the mitochondria in granulosa cells shrank, the mitochondrial membrane density increased, the cristae became thicker, the cristae decreased or disappeared, and the outer membrane ruptured.

[0066] Example 7 Mito-Tracker Red staining to observe the number of mitochondria

[0067] The mitochondrial morphology of granulosa cells was detected using a mitochondrial red fluorescent probe. When the cells reached 50% confluence, the Mito-Tracker Red CMXRos working solution was added and incubated at 37 °C for 20 min. The working solution was removed, and pre-warmed cell culture medium was added. Observation was performed under a fluorescence microscope. The results are as Figure 8 shown, Figure 8 in which the scale bar = 200 μm. The quantification results are as Figure 9 shown.

[0068] From Figure 8 and Figure 9 it can be seen that after FAC treatment, the number of mitochondria in granulosa cells decreased significantly (P < 0.05).

[0069] Example 8 Detection of mitochondrial membrane potential using JC-1 mitochondrial membrane potential fluorescent probe

[0070] After granulosa cells were treated with 200 μmol / L FAC for 36 h, the culture medium was discarded, and the cells were washed 3 times with PBS at 37 °C for 30 s each time. The JC-1 staining working solution was added and incubated in a 37 °C cell culture incubator for 20 min. After incubation at 37 °C, the cells were washed 2 times with JC-1 staining buffer for 10 s each time. Images were collected under a fluorescence inverted microscope. The fluorescence images of mitochondrial membrane potential in granulosa cells after treatment of porcine granulosa cells with the control group and FAC group for 36 h are as Figure 10 shown, and the quantification results are as Figure 11 shown.

[0071] From Figure 10 and Figure 11 it can be seen that the ratio of JC-1 polymers / JC-1 monomers in the FAC treatment group decreased, indicating mitochondrial depolarization and damage.

[0072] Example 9 Detection of iron ion content in granulosa cells using FerroOrange probe

[0073] 35 μL of DMSO was added to a tube containing 24 μg of FerroOrange and pipetted to mix well to prepare a 1 mmol / L FerroOrange solution. The 1 mmol / L FerroOrange solution was diluted with HBSS solution to prepare a 1 μmol / L FerroOrange working solution. After granulosa cells were treated with 200 μmol / L FAC for 36 h, the culture medium was discarded, and the cells were washed 3 times with serum-free medium. The 1 μmol / L FerroOrange working solution was added, and the cells were cultured in a 37 °C, 5% CO2 incubator for 30 min. The cells were observed under a fluorescence microscope (without washing, directly observed after culture). The fluorescence intensity results of iron ions in granulosa cells after treatment of porcine granulosa cells with the control group and FAC group for 36 h are as Figure 12 shown, and its quantification diagram is asFigure 13 as shown

[0074] As can be seen from Figure 12 and Figure 13 it can be seen that after treating granulosa cells with FAC for 36 h, the intracellular iron ion content was significantly increased as observed by FerroOrange probe (P < 0.05).

[0075] Example 10 Detection of antioxidant capacity of granulosa cells by LPO and GSH / GSSH kits

[0076] ① Culture and treat granulosa cells in 60 mm dishes, wash twice with PBS, digest the cells with trypsin, transfer to 1.5 mL EP tubes, centrifuge at 1000 r / min at room temperature for 10 min, discard the supernatant, add 0.5 mL PBS, centrifuge at 1000 r / min for 10 min, wash twice with PBS, discard the supernatant, and freeze the cell pellet at -80 °C for detection the next day. Thaw on ice the next day, add as little lysis buffer as possible (50 μL / time), lyse for 40 min, centrifuge to collect the supernatant, measure BCA, and then detect LPO according to the manufacturer's instructions. ② Culture and treat granulosa cells in 60 mm dishes, collect the cells, and detect GSH / GSSG of granulosa cells according to the manufacturer's instructions. Prepare a standard curve with different concentrations of GSSG solution, freeze-thaw the cells 3 times to repeat lysis, add the cell samples and standards in the order of the instructions, then add 300 μL of total GSH detection working solution, mix well, incubate at 25 °C for 5 min, add 100 μL of 0.5 mg / ml NADPH, mix well, immediately detect with an enzyme-linked immunosorbent assay (ELISA) reader, measure the OD value once every 5 min for 5 times, and obtain the total GSH and GSSG contents according to the standard curve, and calculate the GSH content. The results are as Figure 14 shown Figure 14 In the figure, the left figure is the result of LPO, and the right figure is the result of GSH / GSSG.

[0077] As can be seen from Figure 14 it can be seen that after treating granulosa cells with FAC for 36 h, lipid peroxides were significantly accumulated (P < 0.05), and GSH / GSSG was significantly decreased (P < 0.05).

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a ferroptosis model of porcine follicular granulosa cells, characterized in that, It includes the following steps: S1. Rinse the porcine ovary with a washing solution until the washing solution is clear, then soak it and place it in the washing solution for standby. Then, extract the follicular fluid containing granulosa cells from the porcine ovary and place it in fetal bovine serum. S2. Filter and collect the granulosa cells and centrifuge them. After removing the supernatant, resuspend the cells, repeat centrifugation and washing, then resuspend the granulosa cells in fetal bovine serum, and then adjust the density of the granulosa cells and culture them. S3. Treat the granulosa cells with ammonium ferric citrate solution, and after washing, obtain a ferroptosis model of porcine follicular granulosa cells.

2. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, characterized in that In step S1, the washing solution is a PBS solution containing 1-2% triple antibiotics at 36-38°C.

3. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, characterized in that, In step S1, soak with a 74-76% ethanol solution for 29-31 s.

4. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, wherein, In step S1, place it in 10-11% fetal bovine serum.

5. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, wherein In step S2, centrifuge at 1400-1600 r / min for 9-11 min.

6. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, wherein In step S2, resuspend the cells with a PBS solution containing 1-2% triple antibiotics at 36-38°C, repeat centrifugation and washing 2-3 times, and then resuspend the granulosa cells in 10-11% fetal bovine serum.

7. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, wherein In step S2, adjust the density of the granulosa cells to 55-65% and culture them in an incubator at 36-38°C with a carbon dioxide concentration of 4-5% until the density of the granulosa cells reaches 80-90%, and change the medium once every 24-36 h during this period.

8. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, characterized in that, In step S3, treat the granulosa cells with ammonium ferric citrate at a concentration of 150-250 μmol / L for 35-37 h.

9. The method for constructing a ferroptosis model of porcine follicular granulosa cells according to claim 1, wherein In step S3, wash with a PBS solution containing 1-2% triple antibiotics at 36-38°C for 2-3 times.

10. A ferroptosis model of porcine follicular granulosa cells constructed by the method for constructing a ferroptosis model of porcine follicular granulosa cells according to any one of claims 1-9.

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