Chicken serum-free culture medium for promoting proliferation of chicken primordial germ cells and use thereof

Abstract

Disclosed are a chicken serum-free culture medium for promoting the proliferation of chicken primordial germ cells and use thereof. Ovotransferrin is used in the culture medium instead of chicken serum. The effect of using ovotransferrin, instead of chicken serum, in a culture system for PGCs is disclosed. Ovotransferrin can maintain the self-renewal of PGCs, thereby providing a basis for developing a stable culture system. The culture medium can avoid adverse effects caused by the undefined components of chicken serum, and the precise use of various growth factors can provide better culture conditions and reduce culture costs.

Description

A serum-free culture medium for promoting the proliferation of chicken primordial germ cells and its application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a serum-free culture medium for promoting the proliferation of chicken primordial germ cells and its application. Background Technology

[0002] In the field of genetic breeding, primordial germ cells (PGCs) have become important tool cells for gene editing and genetic improvement due to their unique developmental potential and genetic characteristics. Originating from the ectoderm, PGCs migrate to the genital ridge in early embryonic development and differentiate into female or male gametes as the gonads develop. This characteristic makes PGCs a key cell type for altering genotypes at the source and creating gene-edited animals.

[0003] However, the in vitro culture and proliferation of PGCs has always been a technical challenge. Traditional culture methods often suffer from low cell proliferation efficiency and low survival rates, which severely limits their application in genetic breeding. Therefore, exploring new culture systems and methods to improve the in vitro proliferation efficiency of PGCs has significant scientific and practical value. Summary of the Invention

[0004] Technical Problem Solved: To address the aforementioned technical problems, this invention provides a serum-free culture medium for promoting the proliferation of chicken primordial germ cells (PGCs) and its application. Ovotransferrin is a major component of chicken serum and is also a widely distributed iron transporter protein in organisms, playing a crucial role in promoting cell growth and proliferation. In cell culture, ovotransferrin provides the iron required for cell growth and participates in various cellular metabolic processes, thereby contributing to cell proliferation and differentiation. Under specific culture conditions, ovotransferrin can significantly promote the in vitro proliferation of various cell types. When an appropriate amount of ovotransferrin is added to the PGC culture medium, the PGCs exhibit the best condition, a significant increase in PGC numbers, and no obvious cell debris, demonstrating a significant effect on improving the proliferation efficiency of PGCs.

[0005] Technical solution: In a first aspect, the present invention provides a serum-free culture medium for promoting the proliferation of chicken primordial germ cells, wherein ovotransferrin is used in place of chicken serum in the culture medium.

[0006] Preferably, the culture medium further includes DMEM culture medium, ultrafiltered water, and additives, the additives including concentrated calcium chloride stock solution and B-27. TM Additives, GlutaMax additives, MEM non-essential amino acid solution, 2-mercaptoethanol, EmbryoMax ® A mixture of nucleoside, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A, and penicillin-streptomycin.

[0007] Preferably, the concentration of ovotransferrin in the culture medium is 0.1 mg / mL to 1 mg / mL.

[0008] Furthermore, the concentration of ovotransferrin in the culture medium is 0.5 mg / mL.

[0009] Secondly, the present invention provides the application of the culture medium described in the first aspect in promoting the proliferation of chicken primordial germ cells.

[0010] Thirdly, the present invention provides a method for promoting the proliferation of chicken primordial germ cells, comprising the following steps: inoculating chicken primordial germ cells in the culture medium described in the first aspect, and then culturing them at 37 °C for a certain period of time until the desired cell quantity is reached.

[0011] Beneficial effects: This invention discloses a method of using oocyte transferrin to replace chicken serum in a PGC culture system, which can maintain the self-renewal of PGCs and thus provide a basis for exploring a stable culture system; the culture medium of this invention can avoid the adverse effects caused by the unclear composition of chicken serum, provide better culture conditions for the precise use of various growth factors, and reduce culture costs. Attached Figure Description

[0012] Figure 1 shows the morphological observation results of PGCs cultured without chicken serum and with chicken serum, where A is the effect of chicken serum culture and chicken serum-free culture on the morphology of chicken primitive germ cells, and B is the cell number statistics after 3 days of culture.

[0013] Figure 2 shows the effect of ovotransferrin on PGC cell proliferation in this invention. In the figure, A shows the effect of different concentrations of estradiol and ovotransferrin on PGC cell morphology, B shows the cell number statistics after 3 days of estradiol culture, C shows the cell number statistics after 3 days of ovotransferrin culture at different concentrations, D shows the PGC cell viability statistics after 3 days of culture with different concentrations of ovotransferrin detected by CCK-8, E shows the EdU cell proliferation detection statistics, and F shows the EdU detection results of PGC cells cultured with different concentrations of ovotransferrin.

[0014] Figure 3 shows the effect of ovotransferrin on the cell cycle of PGCs in this invention. A represents the proportion of PGCs cells in the cell cycle as detected by flow cytometry; B represents the statistical distribution of PGCs cell cycle proportions; C represents the relative expression levels of cell cycle-related genes in PGCs cells; D represents the statistical distribution of the effect of different concentrations of ovotransferrin on the levels of CCND1 and CCNB proteins; E represents the effect of different concentrations of ovotransferrin on the levels of CCND1 and CCNB proteins; F represents the statistical distribution of cells expressing positive anti-proliferating cell nuclear antigen antibodies in PGCs cultured with different concentrations of ovotransferrin; and G represents the detection results of anti-proliferating cell nuclear antigen antibodies in PGCs cultured with different concentrations of ovotransferrin.

[0015] Figure 4 illustrates the effects of ovotransferrin on PGC cell adhesion and pluripotency in this invention. A represents the effect of different concentrations of ovotransferrin on the relative expression level of the DAZL gene; B represents the effect of different concentrations of ovotransferrin on the relative expression level of the NANOG gene; C represents the effect of different concentrations of ovotransferrin on the relative expression level of the POUV gene; D represents the effect of different concentrations of ovotransferrin on the relative expression level of the SOX2 gene; E represents the effect of different concentrations of ovotransferrin on the relative expression level of the ZO-1 gene; F represents the effect of different concentrations of ovotransferrin on the relative expression level of the Occludin gene; G represents the effect of different concentrations of ovotransferrin on the relative expression level of the JAM-A gene; H represents the effect of different concentrations of ovotransferrin on the relative expression level of the Claudin-1 gene; I represents the effect of different concentrations of ovotransferrin on the protein levels of ZO-1, Occludin, JAM-A, and Claudin-1; and J is a statistical graph showing the effects of different concentrations of ovotransferrin on the protein levels of ZO-1, Occludin, JAM-A, and Claudin-1.

[0016] Figure 5 illustrates the effect of ovotransferrin on PGC cell apoptosis in this invention. A represents the effect of different concentrations of ovotransferrin on the relative expression level of the BAX gene; B represents the effect of different concentrations of ovotransferrin on the relative expression level of the BCL-2 gene; C represents the effect of different concentrations of ovotransferrin on the relative expression level of the Caspase3 gene; D represents the effect of different concentrations of ovotransferrin on the relative expression level of the Caspase9 gene; E represents the effect of different concentrations of ovotransferrin on the relative expression level of the TP53 gene; F represents the effect of different concentrations of ovotransferrin on the relative expression level of the FAS gene; G represents the effect of different concentrations of ovotransferrin on the relative expression level of the C-myc gene; H is a statistical graph of the apoptosis rate of PGC cells; and I represents the flow cytometry analysis of PGC cell apoptosis.

[0017] Figure 6 shows the effect of ovotransferrin on the PI3K-AKT-mTOR signaling pathway in PGCs cells in this invention. In this figure, A represents the effect of different concentrations of ovotransferrin on the relative expression levels of PI3K, AKT, and mTOR genes; B represents the effect of different concentrations of ovotransferrin on the protein levels of PI3K, AKT, and mTOR; and C is a statistical graph showing the effect of different concentrations of ovotransferrin on the protein levels of PI3K, AKT, and mTOR.

[0018] Figure 7 illustrates the effect of ovotransferrin on ferroptosis in PGCs cells in this invention. A represents the effect of different concentrations of ovotransferrin on total glutathione levels; B represents the effect of different concentrations of ovotransferrin on reduced glutathione levels; C represents the effect of different concentrations of ovotransferrin on reduced glutathione levels; D represents the effect of different concentrations of ovotransferrin on malondialdehyde (MDA) content; E represents the effect of different concentrations of ovotransferrin on superoxide dismutase (SOD) activity; F represents the effect of different concentrations of ovotransferrin on ferrous iron (Fe2+) content; G represents the effect of different concentrations of ovotransferrin on the relative expression level of the SLA7A11 gene; and H represents the effect of different concentrations of ovotransferrin on the relative expression level of the GPX4 gene. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments:

[0020] Example 1: Verification of the effect of chicken serum on the morphology of PGCs

[0021] Primary cells were cultured for 70 days and successfully established before freezing. They were then thawed before use. The specific components and amounts of the culture medium are shown in Table 1. Chicken serum was substituted during PGC culture, and the results are shown in Figures 1A-1B: PGCs proliferated normally after the addition of chicken serum, but did not proliferate after the absence of chicken serum, and the cells were flattened, fragmented, and irregular in shape. These results indicate that chicken serum is indispensable in the culture of PGCs.

[0022] Table 1. Specific components of the culture medium used for 70-day culture.

[0023]

[0024] Example 2: Ovotransferrin promotes PGC proliferation in a dose-dependent manner.

[0025] To screen the optimal amount of ovotransferrin added during PGC culture, PGCs were cultured in serum-deficient medium with different concentrations of estradiol and ovotransferrin, and cell morphology was observed. The results are shown in Figures 2A-2C: estradiol did not promote PGC growth, but with increasing ovotransferrin concentration, PGCs showed varying degrees of proliferation. The cell number initially increased and then decreased with increasing concentration. PGCs with a medium concentration of ovotransferrin (0.5 mg / mL) exhibited good cell condition; PGCs exposed to a high concentration of ovotransferrin (1 mg / mL) showed obvious cell debris and dead cells; while PGCs with a low concentration of ovotransferrin (0.1 mg / mL) showed fewer cells, more cell debris, and poorer cell condition compared to those with a concentration of 0.5 mg / mL.

[0026] Preliminary morphological observations of PGCs cultured with different concentrations of ovotransferrin indicate that ovotransferrin can promote PGC proliferation. To further investigate the effect of ovotransferrin on PGC proliferation, CCK-8 cell proliferation experiments and EdU cell proliferation experiments were conducted. First, in the CCK-8 cell proliferation experiment, PGCs were seeded and passaged in 24-well plates with gradient concentrations of ovotransferrin (0, 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL). Cells were cultured for 48 h, and 100 μL was aspirated from each well and added to three parallel wells in a 96-well plate. 10 μL of CCK-8 solution was added to each well (avoiding air bubbles, as they would affect OD readings). The plates were placed in a 37 ℃, 5% CO2 incubator for 2-4 hours. The absorbance was then measured using a microplate reader set to 450 nm and the 96-well plate was placed at the reading position. The results are shown in Figure 2D: With the increase of ovotransferrin concentration, the proliferation capacity of PGCs under each concentration treatment first increased and then decreased. The medium concentration of ovotransferrin (0.5 mg / mL) was significantly different from the other concentration groups (P < 0.05), while there was no significant difference among the other concentration groups (P > 0.05). Subsequently, an EdU proliferation assay was performed to detect PGC proliferation. PGCs were seeded and passaged in 24-well plates with gradient concentrations of ovotransferrin (0, 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL). EdU solution was diluted 1000:1 with complete PGC culture medium to prepare 50 μM EdU medium. 100 μL of 50 μM EdU medium was added to each well and incubated for 2 hours, then the medium was discarded. Cells were washed 1-2 times with PBS for 5 minutes each time. After centrifugation at 1400 rpm for 6 minutes, the supernatant was discarded, and 20 μL of PGCs was mixed by pipetting and then dropped onto a glass slide. 20 μL of cell fixative (PBS containing 4% paraformaldehyde) was added and incubated at room temperature for 30 minutes, then the fixative was discarded. 20 μL of 2 mg / mL glycine was added and incubated for 5 minutes, then the glycine solution was discarded. 20 μL of PBS was added and washed for 5 minutes, then the PBS was discarded. 20 μL of osmotic buffer (0.5 mg / mL) was added. Incubate with % Triton X-100 in PBS for 10 minutes; wash once with PBS for 5 minutes; add 20 μL of 1× Apollo staining solution, incubate at room temperature in the dark for 30 minutes, then discard the staining solution; add 20 μL of penetrant and wash 2-3 times for 10 minutes each time, then discard the penetrant; wash once with PBS for 5 minutes; add 20 μL of Hoechst 33342 staining solution, incubate at room temperature in the dark for 30 minutes, then discard the staining solution; add 20 μL of penetrant and incubate for 10 minutes, then wash once with PBS for 5 minutes; mount with neutral resin and observe under a fluorescence microscope using different channels.The results are shown in Figures 2E-2F: With the increase of ovotransferrin concentration, the proliferation capacity of PGCs under each concentration treatment first increased and then decreased. The medium concentration of ovotransferrin (0.5 mg / mL) showed a significant difference compared with the other concentration groups (P < 0.05), while there were no significant differences among the other concentration groups (P > 0.05). Therefore, ovotransferrin can promote PGC proliferation, and the proliferation capacity is strongest when the concentration is 0.5 mg / mL.

[0027] Example 3: Changes in PGC cell cycle, apoptosis, signaling pathways, and cell adhesion-related genes and proteins by ovotransferrin

[0028] To further verify the effects of ovotransferrin on genes and proteins related to cell cycle, apoptosis, signaling pathways, and cell adhesion in PGCs, this embodiment used flow cytometry, qRT-PCR, and Western blot to detect the expression of cell cycle-related genes and proteins. The results are shown in Figures 3-6.

[0029] For flow cytometry analysis of the cell cycle, cell fixation was performed first: the cell pellet was gently mixed with 1 mL of pre-chilled 70% ethanol and fixed at 4°C for at least 2 hours or overnight. Next, the cells were centrifuged at 1000×g for 5 min to pellet the cells, and then resuspended in 1 mL of pre-chilled PBS. This was followed by another centrifugation at 1000×g for 5 min to pellet the cells again. 10 μL of propidium iodide stock solution and 10 μL of RNase A solution were added to 0.5 mL of staining buffer and mixed well. 0.5 mL of the prepared propidium iodide staining solution was added to each cell sample, and the cells were gently mixed and resuspended. The cells were incubated at 37°C in the dark for 30 min, and then detected at an excitation wavelength of 488 nm. FlowJO analysis software was used for cell DNA content analysis and light scattering analysis. The results are shown in Figures 3A-3B: Compared with the group without chicken serum (0 mg / mL), the 0.5 mg / mL oocyte transferrin replacement group showed a significant increase in the G0 / G1 phase and a decrease in the S phase, indicating that 0.5 mg / mL oocyte transferrin can significantly promote cell cycle acceleration and cell proliferation.

[0030] Apoptosis was detected by flow cytometry. PGCs were seeded and passaged in 24-well plates and cultured for 72 h with gradient concentrations of ovotransferrin. The following steps were performed: cells were collected by centrifugation at 300×g, 4℃; cells were washed twice with pre-chilled PBS, each time by centrifugation at 300×g, 4℃ for 5 min. Cells ranging from 1 to 5 × 10⁶ cells were collected. 5Cells were resuspended in 100 μL of 1×Binding Buffer after PBS was removed. 5 μL of Annexin V-FITC and 10 μL of PI Staining Solution were added and gently mixed. The mixture was incubated in the dark at room temperature for 10-15 min. 400 μL of 1×Binding Buffer was added, mixed, and the mixture was placed on ice. Samples were analyzed by flow cytometry within 1 hour. The results are shown in Figure 5: With increasing ovotransferrin concentration, apoptosis in PGCs initially decreased and then increased at each concentration. The apoptosis was significantly downregulated at 0.5 mg / mL compared to 0 and 1 mg / mL (P < 0.05), while no significant differences were observed among the other groups (P > 0.05). The results indicate that ovotransferrin can inhibit PGC apoptosis, with the most significant effect observed at a concentration of 0.5 mg / mL.

[0031] The PCNA proliferation assay was performed as follows: Cells were collected into 1.5 mL centrifuge tubes and centrifuged at 1400 rpm for 6 min, then the supernatant was discarded. Cells were washed once with PBS and then discarded. 200 μL of 4% paraformaldehyde was added for fixation for 30 min. Cells were washed once with PBS, centrifuged at 1400 rpm for 6 min, and the supernatant was discarded. 200 μL of 0.5% Triton X-100 (diluted with PBS) was added, and the cells were permeabilized at room temperature for 20 min, then washed once with PBS. Cells were centrifuged at 1400 rpm for 6 min, the supernatant was discarded, and then 10% FBS-PBS was slowly added for blocking at room temperature for 2 h. The blocking solution was removed, and the cells were washed once with TBST. 200 μL of diluted (1:200, using antibody dilution) PCNA was added, ensuring complete surface coverage, and incubated overnight at 4 ℃. The next day, cells were washed with TBST for 5 min, then fluorescent secondary antibody was added, and the cells were incubated at 37 ℃ in the dark for 2 h. The secondary antibody was washed away with TBST, and 200 μL of DAPI (5% ... Stain with DAPI (0.5 mg / mL) for 15 min, then wash off; mount the slide and observe different fluorescence channels under a fluorescence inverted microscope and take pictures to record the results, as shown in Figures 3F-3G: indicating that 0.5 mg / mL oocyte transferrin can significantly increase the positive rate of anti-proliferating cell nuclear antigen antibody (PCNA) cells and promote cell proliferation.

[0032] The following steps will be taken next:

[0033] ① Total RNA extraction: Collect PGCs cultured from oocyte transferrin into centrifuge tubes, centrifuge at 1400 rpm for 6 min, and discard the supernatant; add 1 mL TRIZOL reagent, mix well by pipetting, and incubate at 4 ℃ for 5 min; incubate the sample at room temperature for 5 min to allow for complete protein dissociation; add 0.2 mL chloroform, tighten the cap, shake vigorously for 15 s, and incubate at room temperature for 2-3 min; centrifuge at 4 ℃ and 12000×g for 15 min. After centrifugation, the sample separates into layers, with the upper aqueous phase containing RNA and the lower organic phase containing protein and DNA; take 500 μL of supernatant, add 0.5 mL isopropanol, mix gently, and incubate at room temperature for 10 min. A gel-like precipitate will appear at the bottom of the tube, which is the RNA; centrifuge at 4 ℃ and 12000×g for 10 min, and discard the supernatant; add 1 mL 75% ethanol to the precipitate, mix gently, and centrifuge at 4 ℃ and 7500×g for 5 min. After 10 min, discard the supernatant; air-dry the RNA sample, add an appropriate amount of enzyme-free water to dissolve it (dissolution can be accelerated at 55-60 ℃ for 10 min); determine the RNA concentration, detect and calculate the OD using a UV spectrophotometer. 260 / OD 280 A ratio of 1.9 to 2.0 indicates good results.

[0034] ②cDNA synthesis: Thaw the template RNA on ice, and thaw 5×FastKing-RT SuperMix and RNase-Free ddH2O at room temperature, immediately placing them on ice after thawing. Before use, vortex each solution to mix thoroughly and briefly centrifuge to collect any liquid remaining on the tube wall; prepare the reverse transcription reaction system: 4 μL 5×FastKing-RT SuperMix, Total RNA 50 ng - 2 μg (depending on RNA concentration), and RNase-Free ddH2O to a final volume of 20 μL; reverse transcription reaction: genomic removal and reverse transcription reaction: 42 ℃, 15 min; enzyme inactivation process: 95 ℃, 3 min; dilute the cDNA concentration with RNase-Free ddH2O according to the amount of RNA used.

[0035] ③ The mRNA expression levels of various genes in PGCs treated with different concentrations of oocyte transferrin were detected using qRT-PCR. A 10 μL qRT-PCR reaction system consisted of 5 μL 2×Universal SYBR Green Fast qPCR Mix, 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 2 μL cDNA, and RNase-free ddH2O, brought to a final volume of 10 μL. The reaction procedure was: 95 °C for 3 minutes; 95 °C for 5 seconds, 60 °C for 30 seconds, for a total of 40 cycles. PGCs were cultured in 3 biological replicates, and each sample was tested in 3 technical replicates. -ΔΔCT The method quantifies the expression level.

[0036] Next, Western blotting was performed: 1) Extraction of total protein from PGCs: PGCs were seeded and passaged in 24-well plates, and cultured for 72 h with gradient concentrations of ovotransferrin. After centrifugation at 1400 rpm for 6 min, the supernatant was discarded, 1 mL of RIPA lysis buffer was added, and lysis was performed at ice for 40 min. Subsequently, the plates were centrifuged at 12000 g for 10 min at 4 °C, and the supernatant was transferred to 1.5 mL centrifuge tubes and stored at -80 °C. 2) Determination of protein concentration by BCA method: Preparation of BCA working solution: According to the number of samples, an appropriate amount of BCA working solution was prepared by adding 1 volume of BCA reagent B (50:1) to 50 volumes of BCA reagent A and mixing thoroughly. The BCA working solution can be stored at room temperature for 24 h. Protein concentration microplate assay (20-2000 μg / mL): Prepare a 96-well plate. First, add 25 μL of BSA standard diluted at various concentrations to each of the 7 wells. Then, add 25 μL of the sample to be tested to the corresponding well according to the number of samples. Add 200 μL of BCA working solution to each well and mix well, for a total of 225 μL of mixture per well. Cover the plate and incubate at 37 ℃ for 30 min. After the reaction is activated, cool to room temperature. Detect the absorbance at 562 nm using a microplate reader within 3-5 minutes. 3) Preparation of WB reagents: Electrophoresis buffer preparation: Add one packet of SDS powder to 1000 mL of ultrapure water and stir for 10 minutes; Transfer buffer preparation: Add one packet of Transfer powder to 900 mL of ultrapure water and stir for 10 minutes, then add 100 mL of methanol; 1×TBST buffer preparation: Prepare 500 mL of 1×TBST buffer by mixing 25 mL of 20×TBST buffer with 475 mL of ultrapure water. 4) SDS-PAGE gel electrophoresis: Use a pipette to draw up the electrophoresis buffer and gently blow into the sample wells on the gel plate to clear the blockage. Add the marker and the protein sample to be tested sequentially from left to right, minimizing the loading time and avoiding sample diffusion; connect the electrophoresis tank and set a constant voltage of 80 V for approximately 40 minutes. Then, adjust the voltage to a constant voltage of 110 V until the sample reaches the bottom of the separating gel. 5) Transfer: Activate the PVDF membrane by completely soaking it in methanol, then transfer it to the transfer buffer for 10 minutes. Also soak the filter paper and black sponge in the transfer buffer. Cut the gel: Cut the gel at the position corresponding to the size of the target protein, remembering the front and back sides. Place the gel in the transfer buffer. Assemble the membrane from bottom to top in the following order: black sponge, 3 layers of filter paper, PVDF membrane, gel, 3 layers of filter paper, black sponge. Use a roller to remove air bubbles. Place the membrane in the transfer apparatus, ensuring the black part aligns with the black membrane wall. The current direction is from the negative electrode to the positive electrode, and the current is 230 mA. The transfer time depends on the protein size.6) Blocking: After transfer, cut the target protein to a suitable size, add freshly prepared blocking buffer (50 mL blocking buffer: 2.5 g skim milk powder plus 50 mL 1×TBST, mix thoroughly, shake for 10 minutes, incubate on a shaker for 2 hours, after blocking, wash 3 times with 1×TBST, 10 minutes each time). 7) Incubation with primary antibody: After washing, dilute the primary antibody with 1×TBST at a ratio of 1:1000, saturate the PVDF membrane, incubate on a shaker for 10 minutes, then place at 4 ℃ overnight. The next day, recover the primary antibody (can be reused 3 times), wash 3 times with 1×TBST, 10 minutes each time. 8) Incubation with secondary antibody: After washing, dilute the secondary antibody with 1×TBST at a ratio of 1:5000, saturate the PVDF membrane, incubate on a shaker for 2 hours, recover the secondary antibody (can be reused 3 times), wash 3 times with 1×TBST, 10 minutes each time. 9) ECL color development and luminescence: Prepare the ECL color development solution using a 1:1 ratio of solution A to solution B, and use immediately. Add approximately 100 μL of the solution evenly to each PVDF membrane, place it in a gel imaging system for development and observation, and calculate the grayscale value using ImageJ software. The results are shown in Figures 3D-3E, 4A-4I, and 6A-6C: 0.5 mg / mL ovalbumin significantly improved cell adhesion, cyclin expression, activated the PI3K-AKT-mTOR signaling pathway, and promoted cell proliferation.

[0037] The above results indicate that ovotransferrin can promote the proliferation of PGCs, with the strongest proliferative capacity observed at a concentration of 0.5 mg / mL. It can also inhibit PGC apoptosis, reduce cell adhesion, and promote the activation of the PI3K-AKT-mTOR signaling pathway.

[0038] Example 4: Effect of ovotransferrin on ferroptosis in PGCs cells

[0039] Ferrous ion detection in PGCs: Collected cells were processed at approximately 1 × 10⁻⁶ cells per cell. 6 Add 0.2 mL of Reagent I to each cell, mix well, and place on an ice box for lysis for 10 min, then centrifuge at 15000×g for 10 min. Collect the supernatant for later use. Standard wells: Add 80 μL of different concentrations of standard to the corresponding wells of the microplate. Assay wells: Add 80 μL of the sample to be tested to the corresponding wells of the microplate. Control wells: Add 80 μL of the sample to be tested to the corresponding wells of the microplate. Add 80 μL of Reagent II to the control wells. Add 80 μL of Reagent III to the assay wells and standard wells. Mix well and incubate at 37 ℃ for 10 min. Measure the OD value of each well at 593 nm using a microplate reader.

[0040] GSH and GSSG assays: Collect cells by centrifugation at 1400 rpm for 6 min. Using a 96-well plate, add samples or standards sequentially and mix well. Add 150 μL of total glutathione assay working solution, mix well, and incubate at 25 ºC or room temperature for 5 min. Add 50 μL of 0.5 mg / mL NADPH solution and mix well. Immediately measure the absorbance at 405 nm using a microplate reader.

[0041] MDA assay: Centrifuge at 1400 rpm / min for 6 min, collect approximately 1×10⁻⁶ samples. 6 Cells were placed in 1.5 mL centrifuge tubes, and 1 mL of pre-chilled cell lysis buffer was added. The tubes were shaken vigorously for 30 s every 5 min, and lysed on ice for 20 min. The tubes were centrifuged at 12000×g for 5 min. The supernatant was collected and 1 mL of double-distilled water was added to each tube. The mixture was quickly mixed and placed in a boiling water bath for 50 min (a small hole was punched in the cap before boiling to prevent the tubes from bursting). The tubes were then quickly placed in ice water to cool and centrifuged at 3000 r / min and 4 ℃ for 15 min. The supernatant was taken and the reading was taken at 532 nm.

[0042] SOD assay: Isolated PGCs were passaged in 24-well plates with gradient concentrations of ovotransferrin (0, 0.1, 0.5, and 1 mg / mL). After culturing for 72 h, the cells were centrifuged at 1400 rpm for 6 minutes, and the supernatant was discarded, leaving the cell pellet. 0.3-0.5 mL of buffer (PBS or physiological saline) was added to the cell pellet, and the cells were sonicated at 300 W in an ice-water bath, sonicating once every 3-5 seconds with a 30-second interval, for a total of 4 times. Subsequently, enzyme working solution and enzyme dilution buffer were added, and the assay was performed using a microplate reader.

[0043] The results are shown in Figure 7: After culturing PGCs with different concentrations of ovotransferrin for 3 days, the results (Figure 7A) showed no significant difference in total GSH content (P > 0.05); reduced GSH first increased and then decreased (Figure 7B); oxidized GSH first decreased and then increased (Figure 7C); malondialdehyde (MDA) content first decreased and then increased (Figure 7D); superoxide dismutase (SOD) activity first increased and then decreased (Figure 7E); and ferrous ion content first decreased and then increased. The results indicate that appropriate concentrations of ovotransferrin can improve the antioxidant capacity of PGCs, reduce the degree of PGC cell damage, and thus affect the intracellular ferrous ion content of PGCs (Figure 7F). As is well known, GPX4 and SLC7A11 are negative regulatory genes for ferroptosis. As shown in Figures 7G-7H, ovotransferrin can inhibit ferroptosis in PGCs. When the ovotransferrin concentration was 0.5 mg / mL, the antioxidant capacity was strongest, the MDA level was lowest, and the SOD level was highest. Therefore, it can be concluded that ovotransferrin can inhibit ferroptosis in PGCs and improve cell viability.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A serum-free culture medium for promoting the proliferation of chicken primordial germ cells, characterized in that: Ovotransferrin was used in the culture medium instead of chicken serum.

2. The serum-free culture medium for promoting the proliferation of chicken primordial germ cells according to claim 1, characterized in that: The culture medium also includes DMEM medium, ultrafiltered water, and additives, including concentrated calcium chloride stock solution and B-27. TM Additives, GlutaMax additives, MEM non-essential amino acid solution, 2-mercaptoethanol, EmbryoMax ® A mixture of nucleoside, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A, and penicillin-streptomycin.

3. The serum-free culture medium for promoting the proliferation of chicken primordial germ cells according to claim 1, characterized in that: The concentration of ovotransferrin in the culture medium is 0.1 mg / mL to 1 mg / mL.

4. A serum-free culture medium for promoting the proliferation of chicken primordial germ cells according to claim 3, characterized in that: The concentration of ovotransferrin in the culture medium was 0.5 mg / mL.

5. The use of the culture medium according to any one of claims 1-4 in promoting the proliferation of chicken primordial germ cells.

6. A method for promoting the proliferation of chicken primordial germ cells, characterized in that, The method includes the following steps: inoculating chicken primordial germ cells into the culture medium described in any one of claims 1-4, and then culturing them at 37 °C for a certain period of time until the desired cell volume is reached.

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