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

Abstract

The invention discloses a chicken serum-free culture medium for promoting proliferation of chicken primordial germ cells and application of the chicken serum-free culture medium. In the culture medium, ovotransferrin is used for replacing chicken serum. According to the invention, ovotransferrin is adopted to replace chicken serum in a PGCs culture system, and self-renewal of PGCs can be maintained, so that a basis is provided for exploring a stable culture system; according to the culture medium, adverse effects caused by undefined chicken serum components can be avoided, better culture conditions can be provided for accurate use of various growth factors, and the culture cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and in particular relates to a chicken serum-free culture medium for promoting the proliferation of chicken primordial germ cells and an application thereof. Background Art

[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. PGCs originate from the ectoderm, migrate to the genital ridges in the early embryonic development, and differentiate into female or male gametes as the gonads develop. This characteristic makes PGCs a key cell type for changing genotypes from the source and preparing gene-edited animals.

[0003] However, the in vitro culture and proliferation of PGCs has always been a technical challenge. Traditional culture methods often face problems such as low cell proliferation efficiency and low survival rate, which seriously limits its application in genetic breeding. Therefore, exploring new culture systems and methods to improve the in vitro proliferation efficiency of PGCs has important scientific significance and application value. Summary of the invention

[0004] Technical problem to be solved: In view of the above technical problems, the present invention provides a chicken serum-free culture medium for promoting the proliferation of chicken primordial germ cells and its application. Ovotransferrin is the main component of chicken serum. In addition, it is an iron transport protein widely present in organisms and plays an important role in promoting cell growth and proliferation. In cell culture, ovotransferrin can provide the iron required for cell growth and participate in a variety of cell metabolic processes, thereby contributing to cell proliferation and differentiation. Under specific culture conditions, ovotransferrin can significantly promote the in vitro proliferation of various types of cells. In the PGCs culture medium, when an appropriate amount of ovotransferrin is added, the PGCs are in the best state, the number of PGCs increases significantly, and there is no obvious cell debris, which has a significant effect on improving the proliferation efficiency of PGCs.

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

[0006] Preferably, the culture medium further comprises DMEM culture medium, ultrafiltered water and additives, wherein the additives include concentrated calcium chloride solution, B-27 TM Supplements, GlutaMax Supplements, MEM Non-essential Amino Acid Solution, 2-Mercaptoethanol, A mixture of nucleosides, 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-1 mg / mL.

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

[0009] In a second aspect, the present invention provides use of the culture medium described in the first aspect in promoting the proliferation of chicken primordial germ cells.

[0010] In a third aspect, 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 at 37° C. for a certain period of time to reach the required cell amount.

[0011] Beneficial effects: The present invention discloses a method of using ovotransferrin to replace the role of chicken serum in the PGCs culture system, which can maintain the self-renewal of PGCs, thereby providing a basis for exploring a stable culture system; the culture medium of the present invention can avoid the adverse effects caused by the unclear composition of chicken serum, and the precise use of various growth factors can provide better culture conditions and reduce the culture cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The present invention is a graph of the observation results of the cell morphology of PGCs cultured without chicken serum and with chicken serum, wherein A is a graph showing the effect of culture with chicken serum and culture without chicken serum on the morphology of chicken primordial germ cells, and B is a statistical graph of the number of cells after 3 days of culture;

[0013] Figure 2 : It is a result diagram of the effect of ovotransferrin on PGCs cell proliferation in the present invention, wherein A is the effect of different concentrations of estradiol and ovotransferrin on PGCs cell morphology, B is a statistical diagram of the number of cells after 3 days of estradiol culture, C is a statistical diagram of the number of cells after 3 days of culture with different concentrations of ovotransferrin, D is a statistical diagram of PGCs cell viability after 3 days of culture with different concentrations of ovotransferrin detected by CCK-8, E is a statistical diagram of EdU cell proliferation detection, and F is the EdU detection result of PGCs cells cultured with different concentrations of ovotransferrin;

[0014] Figure 3: This is a result diagram of the effect of ovotransferrin on the cell cycle of PGCs in the present invention, wherein A is the proportion of PGCs cell cycle detected by flow cytometry; B is the statistics of the proportion of PGCs cell cycle, C is the relative expression of cell cycle-related genes in PGCs cells, D is a statistical diagram of the effect of different concentrations of ovotransferrin on CCND1 and CCNB protein levels, E is the effect of different concentrations of ovotransferrin on CCND1 and CCNB protein levels, F is a statistical diagram of anti-proliferating cell nuclear antigen antibody-positive cells in different concentrations of ovotransferrin; G is the anti-proliferating cell nuclear antigen antibody detection result of PGCs cells cultured with different concentrations of ovotransferrin;

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

[0016] Figure 5 It is the effect of ovotransferrin on PGCs apoptosis in the present invention, wherein A is the effect of different concentrations of ovotransferrin on the relative expression of BAX gene, B is the effect of different concentrations of ovotransferrin on the relative expression of BCL-2 gene, C is the effect of different concentrations of ovotransferrin on the relative expression of Caspase3 gene, D is the effect of different concentrations of ovotransferrin on the relative expression of Caspase9 gene, E is the effect of different concentrations of ovotransferrin on the relative expression of TP53 gene, F is the effect of different concentrations of ovotransferrin on the relative expression of FAS gene, G is the effect of different concentrations of ovotransferrin on the relative expression of C-myc gene; H is a statistical graph of PGCs apoptosis ratio, and I is a flow cytometric analysis to detect PGCs apoptosis;

[0017] Figure 6It is the effect of ovotransferrin on the PI3K-AKT-mTOR signaling pathway of PGCs cells in the present invention, wherein A is the effect of different concentrations of ovotransferrin on the relative expression of PI3K, AKT and mTOR genes, B is the effect of different concentrations of ovotransferrin on the protein levels of PI3K, AKT and mTOR, and C is a statistical graph of the effect of different concentrations of ovotransferrin on the protein levels of PI3K, AKT and mTOR;

[0018] Figure 7 The invention relates to the effect of ovotransferrin on ferroptosis of PGCs cells, wherein A is the effect of different concentrations of ovotransferrin on the level of total glutathione, B is the effect of different concentrations of ovotransferrin on the level of reduced glutathione, C is the effect of different concentrations of ovotransferrin on the level of reduced glutathione, D is the effect of different concentrations of ovotransferrin on the content of malondialdehyde, E is the effect of different concentrations of ovotransferrin on the activity of superoxide dismutase, F is the effect of different concentrations of ovotransferrin on the content of divalent iron, G is the effect of different concentrations of ovotransferrin on the relative expression of SLA7A11 gene, and H is the effect of different concentrations of ovotransferrin on the relative expression of GPX4 gene. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

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

[0021] After 70 days of culture, the primary cells were frozen after the line was successfully established and revived when used. The specific components and addition amounts of the culture medium are shown in Table 1. Chicken serum was deleted during the PGCs culture process. Figure 1 A- Figure 1 As shown in B: After adding chicken serum, PGCs can proliferate normally, but after lacking chicken serum, PGCs do not proliferate, and the cell morphology is flat and broken, showing irregular shapes. These results show that chicken serum is indispensable in the culture process of PGCs.

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

[0023]

[0024] Example 2: Ovotransferrin promotes the proliferation of PGCs in a dose-dependent manner

[0025] In order to screen the most appropriate amount of ovotransferrin added during the culture of PGCs, different concentrations of estradiol and ovotransferrin were added to the culture medium lacking chicken serum, and then PGCs were cultured and the cell morphology was observed. Figure 2 A- Figure 2As shown in C: estradiol cannot promote the growth of PGCs, but as the concentration of ovotransferrin increases, PGCs proliferate to varying degrees. The number of cells first increases and then decreases with the concentration. PGCs added with medium concentration of ovotransferrin (0.5 mg / mL) show good condition; PGCs exposed to high concentration of ovotransferrin (1 mg / mL) show obvious cell fragments and dead cells; and PGCs added with low concentration of ovotransferrin (0.1 mg / mL) show fewer cell fragments and more cell fragments, and are in poorer condition than those added with 0.5 mg / mL.

[0026] From the morphological observation of PGCs cultured with different concentrations of ovotransferrin, it can be preliminarily known that ovotransferrin can promote the proliferation of PGCs. In order to further explore the proliferation effect of ovotransferrin on PGCs, CCK-8 cell proliferation experiment and EdU cell proliferation experiment were carried out. First, the CCK-8 cell proliferation experiment, PGCs were plated and passaged in 24-well plates, and gradient concentrations of ovotransferrin (0, 0.1mg / mL, 0.5mg / mL and 1mg / mL) were added; the cells were cultured in an incubator for 48h, 100μL was aspirated from each well, added to 96-well plates, 3 parallel wells were set for each group, and 10μL of CCK-8 solution was added to each well (be careful not to generate bubbles in the wells, they will affect the OD value reading). The well plate was placed in an incubator at 37℃ and 5% CO2; the culture plate was cultured in the incubator for 2-4 hours; the wavelength of the microplate reader was adjusted to 450nm, the 96-well plate was placed at the plate reader, the instrument was operated, and the absorbance value was read. The results are as follows Figure 2As shown in D: With the addition of ovotransferrin concentration, the proliferation ability of PGCs under each concentration treatment first increased and then decreased. The difference between the medium concentration ovotransferrin (0.5 mg / mL) and the other concentration groups was significant (P < 0.05), and there was no significant difference between the other concentration groups (P > 0.05). Subsequently, EdU proliferation experiment was performed to detect the proliferation of PGCs. PGCs were plated and passaged in 24-well plates, and gradient concentrations of ovotransferrin (0, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL) were added; the EdU solution was diluted with PGCs complete culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU culture medium; 100 μL of 50 μM EdU culture medium was added to each well and incubated for 2 hours, and the culture medium was discarded; the cells were washed with PBS 1 to 2 times, each for 5 minutes; centrifuged at 1400 rpm for 6 minutes, the supernatant was discarded, and 20 μL of PGCs were blown and mixed 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, and the fixative was discarded; 20 μL of 2 mg / mL glycine was added, and after incubation for 5 minutes, the glycine solution was discarded; 20 μL PBS, wash for 5 minutes, discard PBS; add 20μL permeabilization agent (0.5% TritonX-100 in PBS) and incubate for 10 minutes; wash once with PBS for 5 minutes; add 20μL 1×Apollo staining reaction solution, incubate at room temperature in the dark for 30 minutes, discard the staining reaction solution; add 20μL permeabilization agent to wash 2-3 times, 10 minutes each time, discard the permeabilization agent; wash once with PBS for 5 minutes; add 20μL Hoechst 33342 staining solution, incubate at room temperature in the dark for 30 minutes, discard the staining solution; add 20μL permeabilization agent to incubate for 10 minutes, wash once with PBS for 5 minutes; seal with neutral gum, and observe under a fluorescence microscope using different channels. The results are shown in Figure 2 As shown in E-2F: With the addition of ovotransferrin concentration, the proliferation ability of PGCs under each concentration treatment first increased and then decreased. The difference between the medium concentration ovotransferrin (0.5 mg / mL) and the other concentration groups was significant (P < 0.05), and there was no significant difference between the other concentration groups (P > 0.05). It can be seen that ovotransferrin can promote the proliferation of PGCs, and when the added concentration is 0.5 mg / mL, the proliferation ability is the strongest.

[0027] Example 3: Effects of ovotransferrin on the cell cycle, apoptosis, signaling pathways and cell adhesion-related genes and proteins of PGCs

[0028] In order to further verify the effect of ovotransferrin on PGCs cell cycle, apoptosis, signaling pathways and cell adhesion related genes and proteins, this example detected the expression of cell cycle related genes and proteins by flow cytometry, qRT-PCR and Western blot. Figure 3-Figure 6 shown.

[0029] When detecting the cell cycle by flow cytometry, cells are fixed first: the cell pellet is gently mixed with 1 mL of pre-cooled 70% ethanol and fixed at 4°C for more than 2 hours or overnight. Next, the cells are centrifuged at 1000×g for 5 minutes to precipitate the cells, and then resuspended with 1 mL of pre-cooled PBS. Then, the cells are centrifuged again at 1000×g for 5 minutes to precipitate the cells. Add 10μL of propidium iodide stock solution and 10μL of RNase A solution to 0.5mL of staining buffer, mix well and set aside. Add 0.5mL of prepared propidium iodide staining solution to each cell sample, gently mix and resuspend the cells. Incubate at 37°C in the dark for 30 minutes, detect at an excitation wavelength of 488nm, and use flowjo analysis software to analyze cell DNA content and light scattering. The results are as follows Figure 3 A- Figure 3 As shown in B: Compared with the chicken serum-free group (0 mg / mL), the G0 / G1 phase in the 0.5 mg / mL ovotransferrin replacement group increased significantly and the S phase decreased, indicating that 0.5 mg / mL ovotransferrin can significantly accelerate the cell cycle and promote cell proliferation.

[0030] Apoptosis detection was performed by flow cytometry. PGCs were plated and passaged in 24-well plates, and ovotransferrin was added with gradient concentrations and cultured for 72 h. PGCs were detected by flow cytometry. The steps were as follows: 300 × g, 4 ° C centrifugation to collect cells; wash cells twice with pre-cooled PBS, each time centrifugation at 300 × g, 4 ° C for 5 min. Collect 1-5 × 10 5 cells; discard PBS, add 100μL 1×Binding Buffer to resuspend the cells; add 5μL Annexin V-FITC and 10μL PIStaining Solution, mix gently; react at room temperature for 10-15min in the dark; add 400μL 1×Binding Buffer, mix well and place on ice. The samples were detected by flow cytometry within 1 hour. Figure 5 As shown: With the addition of ovotransferrin concentration, the apoptosis of PGCs under each concentration treatment first decreased and then increased. The ovotransferrin concentration of 0.5 mg / mL was significantly downregulated compared with 0 and 1 mg / mL (P < 0.05), and there was no significant difference between the other groups (P > 0.05). The results showed that ovotransferrin can inhibit the apoptosis of PGCs, and the effect was most significant when the ovotransferrin concentration was 0.5 mg / mL.

[0031] PCNA proliferation detection, the method steps are as follows: collect cells into 1.5mL centrifuge tubes, centrifuge at 1400rpm for 6min, discard the supernatant; add PBS to wash once, discard PBS; add 200μL 4% paraformaldehyde to fix for 30min; add PBS to wash once, centrifuge at 1400rpm for 6min, discard the supernatant; add 200μL 0.5% TritonX-100 (diluted with PBS), permeabilize the cell membrane for 20min at room temperature, add PBS to wash once; centrifuge at 1400rpm for 6min, discard the supernatant, and then slowly add 10% FBS-PBS to block at room temperature for 2h; aspirate the blocking solution, wash once with TBST, add 200μL diluted (1:200, using antibody diluent) PCNA to ensure that the surface is fully covered, and incubate at 4℃ overnight; wash with TBST for 5min the next day, then add fluorescent secondary antibody, incubate in a 37℃ incubator in the dark for 2h; use TBST to wash off the secondary antibody, add 200μL DAPI (5 ng / mL) was used for staining for 15 min, and then DAPI was washed off. The coverslips were sealed and observed under an inverted fluorescence microscope in different fluorescence channels and photographed. The results were as follows: Figure 3 F- Figure 3 As shown in G: It shows that 0.5 mg / mL ovotransferrin can significantly increase the rate of anti-proliferating cell nuclear antigen antibody positive cells (PCNA) and promote cell proliferation.

[0032] Follow up with the following steps:

[0033] ① Total RNA extraction: Collect PGCs cultured with ovotransferrin into a centrifuge tube, centrifuge at 1400rpm for 6min, and discard the supernatant; add 1mL TRIZOL reagent, mix by blowing, and let stand at 4℃ for 5min; let the sample stand at room temperature for 5min to allow the protein to fully dissociate; add 0.2mL chloroform, cover the lid tightly, shake vigorously for 15s and let stand at room temperature for 2-3min; centrifuge at 4℃ and 12000×g for 15min, and the sample will be separated after centrifugation. The upper aqueous phase contains RNA, and the lower organic phase contains protein and DNA; take 500μL supernatant, add 0.5mL isopropanol, mix gently, and let stand at room temperature for 10min. A gelatinous precipitate will appear at the bottom of the tube, which is RNA; centrifuge at 4℃ and 12000×g for 10min, and discard the supernatant; add 1mL to the precipitate. Gently mix with 75% ethanol, centrifuge at 4°C, 7500×g for 5 minutes, and discard the supernatant; dry the RNA sample, add an appropriate amount of enzyme-free water to dissolve (dissolution can be promoted at 55-60°C for 10 minutes); determine the RNA concentration, detect and calculate the OD by UV spectrophotometer 260 / OD 280 Ratio: 1.9 to 2.0 indicates good results.

[0034] ②cDNA synthesis: thaw the template RNA on ice, thaw 5×FastKing-RT SuperMix and RNase-FreeddH2O at room temperature, and place on ice immediately after thawing. Vortex each solution before use, centrifuge briefly to collect the liquid remaining on the tube wall; prepare the reverse transcription reaction system: 4μL5×FastKing-RT SuperMix, Total RNA50ng-2μg (depending on RNA concentration), RNase-Free ddH2O to 20μL; reverse transcription reaction: remove the genome and reverse transcription reaction: 42℃, 15min; enzyme inactivation process: 95℃, 3min; add RNase-Free ddH2O to dilute the cDNA concentration according to the RNA content used.

[0035] ③qRT-PCR technology was used to detect the mRNA expression level of each gene in PGCs after treatment with different concentrations of ovotransferrin. 10μL qRT-PCR reaction system consists of 5μL 2×Universal SYBR Green Fast qPCR Mix, 0.4μL upstream primer (10μM), 0.4μL downstream primer (10μM), 2μL cDNA, and RNase-Free ddH2O to 10μL. Reaction process: 95℃, 3 minutes; 95℃ 5 seconds, 60℃ 30 seconds, a total of 40 cycles. PGCs culture was set up with 3 biological replicates, each sample was set up with 3 technical replicates, and 2 -ΔΔCT Methods The expression levels were quantitatively analyzed.

[0036] Western Blot was then performed: 1) Extraction of total protein from PGCs: PGCs were plated and passaged in 24-well plates, and gradient concentrations of ovotransferrin were added for 72 h, centrifuged at 1400 rpm for 6 min, the supernatant was discarded, 1 mL of RIPA lysis buffer was added, and the cells were lysed at low temperature on ice for 40 min, then centrifuged at 12000 g for 10 min in a 4 °C centrifuge, the supernatant was aspirated into a 1.5 mL centrifuge tube, and stored at -80 °C. 2) BCA method was used to determine protein concentration, and BCA working solution was prepared: according to the number of samples, 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1) were used to prepare an appropriate amount of BCA working solution, and the mixture was thoroughly mixed. The BCA working solution can be left at room temperature for 24 h. Protein concentration microwell detection (20-2000μg / mL): Prepare a 96-well plate, first add 25μL of each diluted BSA standard into each of the 7 wells, then add 25μL of the sample to be tested into the corresponding well according to the number of samples; add 200μL of BCA working solution into each well, mix well, a total of 225μL of mixed solution in each well; cover the well plate, place in a 37℃ incubator for 30min, cool to room temperature after the reaction is activated; detect the absorbance at 562nm on a microplate reader within 3-5 minutes. 3) Preparation of WB reagents: Prepare electrophoresis solution: add 1000mL ultrapure water to a pack of SDS powder, stir for 10 minutes; prepare transfer solution: add 900mL ultrapure water to a pack of Transfer powder, stir for 10 minutes and then add 100mL methanol; prepare 1×TBST buffer: prepare 500mL 1×TBST buffer, mix 25mL 20×TBST buffer with 475mL ultrapure water. 4) For SDS-PAGE gel electrophoresis, use a pipette to absorb the electrophoresis solution, gently blow the sample well on the gel plate, and start loading the sample after it is unblocked. Add the marker and the protein sample to be tested from left to right, and try to shorten the loading time to avoid sample diffusion; connect the electrophoresis tank, set a constant voltage of 80V and run for about 40 minutes, then switch the voltage to 110V constant voltage electrophoresis until it reaches the bottom of the separation gel. 5) Transfer, PVDF membrane is completely soaked in methanol for activation, transferred to transfer solution for 10 minutes, filter paper and black sponge are also placed in transfer solution for soaking. Cut gel: Cut the gel at the position corresponding to the size of the target protein, remember the front and back, and put the gel into the transfer solution. Install from bottom to top in the order of black sponge, 3 layers of filter paper, PVDF membrane, gel, 3 layers of filter paper, and black sponge, use a roller to press out bubbles, and put it into the transfer device. Note that black corresponds to the black transfer membrane wall, the current direction flows from the negative electrode to the positive electrode, the current is 230mA, and the transfer time depends on the size of the protein. 6) Blocking: After the transfer is completed, cut the target protein of appropriate size, add the freshly prepared blocking solution (50mL blocking solution: 2.5g skim milk powder plus 50mL1×TBST, mix well, shake for 10 minutes, incubate on a shaker for two hours, after the blocking is completed, wash 3 times with 1×TBST, 10 minutes each time.7) Incubate the primary antibody. After washing, add 1×TBST to dilute the primary antibody in a ratio of (1:1000), soak the PVDF membrane, incubate on a shaker for 10 minutes, and place at 4°C overnight. The next day, recover the primary antibody (can be reused 3 times), and wash 3 times with 1×TBST, 10 minutes each time. 8) Incubate the secondary antibody. After washing, add 1×TBST to dilute the secondary antibody in a ratio of (1:5000), soak the PVDF membrane, incubate on a shaker for 2 hours, recover the secondary antibody (can be reused 3 times), and wash 3 times with 1×TBST, 10 minutes each time. 9) ECL color development and luminescence, prepare ECL color development solution according to A solution: B solution = 1:1, prepare and use it now, evenly drop about 100μL of color development solution on each PVDF membrane, place it in the gel imaging system for development and observation, and calculate the gray value using Image J software. The results are as follows. Figure 3 D- Figure 3 E. Figure 4 A- Figure 4 I and Figure 6 A- Figure 6 As shown in C: 0.5 mg / mL ovotransferrin can significantly improve cell adhesion, cyclin expression, activate the PI3K-AKT-mTOR signaling pathway, and promote cell proliferation.

[0037] The above results show that ovotransferrin can promote the proliferation of PGCs. When the concentration of ovotransferrin is 0.5 mg / mL, the proliferation ability is the strongest. It can also inhibit the apoptosis of PGCs, reduce cell adhesion, and promote the activation of the PI3K-AKT-mTOR signaling pathway.

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

[0039] PGCs ferrous ion detection: collect the cells and collect them at a rate of about 1×10 6 Add 0.2mL of reagent 1 to each cell, mix well, place on an ice box for lysis for 10 minutes, then centrifuge at 15000×g for 10 minutes, and take the supernatant for later use; standard wells: take 80μL of standards of different concentrations and add them to the corresponding wells of the ELISA plate; assay wells: take 80μL of the sample to be tested and add them to the corresponding wells of the ELISA plate; control wells: take 80μL of the sample to be tested and add them to the corresponding wells of the ELISA plate; add 80μL of reagent 2 to the control wells; add 80μL of reagent 3 to the assay wells and standard wells; mix well, incubate at 37℃ for 10 minutes; measure the OD value of each well at 593nm on an ELISA instrument.

[0040] GSH and GSSG detection: Collect cells by centrifugation at 1400 rpm for 6 minutes, use a 96-well plate, add samples or standards in sequence, and mix well. Add 150 μl of total glutathione detection working solution, mix well, and incubate at 25°C or room temperature for 5 minutes; add 50 μl of 0.5 mg / mL NADPH solution, mix well; immediately measure the absorbance value at 405 nm using an ELISA reader.

[0041] MDA detection: 1400 rpm / min, centrifuge for 6 min, collect about 1×10 6 Place the cells in a 1.5 mL centrifuge tube, add 1 mL of pre-cooled cell lysis buffer, shake vigorously for 30 seconds every 5 minutes, lyse on ice for 20 minutes, centrifuge at 12000×g for 5 minutes, take the supernatant and add 1 mL of double distilled water to each tube, mix quickly, boil in a boiling water bath for 50 minutes (pierce a small hole in the lid before boiling to prevent the tube from bursting), quickly put into ice water to cool, centrifuge at 3000r / min, 4℃ for 15 minutes, take the supernatant and read at 532nm.

[0042] SOD detection: The isolated and cultured PGCs were plated and passaged in 24-well plates, and gradient concentrations of ovotransferrin (0, 0.1, 0.5 and 1 mg / mL) were added. After 72 hours of culture, the plates were centrifuged at 1400 rpm for 6 minutes, and the supernatant was discarded to keep the cell pellet. 0.3-0.5 mL of buffer (PBS or normal saline) was added to the cell pellet, and ultrasonically disrupted at a power of 300 W in an ice water bath, with an ultrasonic wave every 3-5 seconds, 30 seconds between each wave, and 4 times in interval. Subsequently, enzyme working solution and enzyme diluent were added, and the cells were measured using a microplate reader.

[0043] The results are as follows Figure 7 As shown: After 3 days of culture with different concentrations of ovotransferrin, the results are as follows Figure 7 The total GSH content in PGCs shown in A had no significant difference (P>0.05); the reduced GSH first increased and then decreased ( Figure 7 B); Oxidized GSSG first decreases and then increases ( Figure 7 C), the MDA content first decreased and then increased ( Figure 7 D); Superoxide dismutase activity first increased and then decreased ( Figure 7 E), the content of divalent iron ions first decreased and then increased. The results showed that appropriate concentrations of ovotransferrin can improve the antioxidant capacity of PGCs, reduce the degree of PGCs cell damage, and thus affect the content of divalent iron ions in PGCs cells ( Figure 7 F) As we all know, GPX4 and SLC7A11 are negative regulatory genes of ferroptosis. Figure 7 G- Figure 7As shown in Figure H, ovotransferrin can inhibit PGCs cell ferroptosis. When the concentration of ovotransferrin is 0.5 mg / mL, the antioxidant capacity is the strongest, the MDA level is the lowest, and the SOD level is the highest. Therefore, it can be seen that ovotransferrin can inhibit PGCs cell ferroptosis and improve cell viability.

[0044] The above description is only 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 in the protection scope of the present invention.

Claims

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

2. The chicken 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 culture medium, ultrafiltered water and additives, wherein the additives include concentrated calcium chloride solution, B-27 TM Supplements, GlutaMax Supplements, MEM Non-Essential Amino Acid Solution, 2-Mercaptoethanol, EmbryoMax ® A mixture of nucleosides, sodium pyruvate, albumin, sodium heparin, basic fibroblast growth factor, activin A and penicillin-streptomycin.

3. The chicken 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-1 mg / mL.

4. The chicken 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 is 0.5 mg / mL.

5. Use of the culture medium according to any one of claims 1 to 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 comprises the following steps: inoculating chicken primordial germ cells in the culture medium according to any one of claims 1 to 4, and then culturing at 37° C. for a certain period of time to reach a required cell amount.

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