Application of IGF-1 replacing insulin in promotion of proliferation of chicken primordial germ cells

Abstract

The invention discloses application of IGF-1 (Insulin-like Growth Factor-1) instead of insulin in promoting proliferation of chicken primordial germ cells. The IGF-1 replaces insulin to play a role in promoting proliferation of the chicken primordial germ cells, it can be determined that a PI3K-AKT signal channel is necessary for proliferation of the PGCs, and a theoretical basis is provided for small molecule research of other signal channels.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of IGF-1 replacing insulin in promoting the proliferation of chicken primordial germ cells. 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 the application of PGCs in genetic breeding.

[0004] Insulin can promote the proliferation of PGCs. Studies have shown that insulin can regulate various biological processes, including metabolism, growth, differentiation and apoptosis, through signaling pathways such as the PI3K / AKT signaling pathway, the MAPK signaling pathway, the FOXO signaling pathway, the mTOR signaling pathway and the RAS signaling pathway. In the culture of PGCs, it is unknown which signaling pathway insulin passes through. Therefore, exploring new culture systems and methods to improve the in vitro proliferation efficiency of PGCs and clarify the signaling pathways has important scientific significance and application value. Summary of the invention

[0005] Technical problems solved: In response to the above technical problems, the present invention provides the use of IGF-1 (insulin-like growth factor-1) instead of insulin in promoting the proliferation of chicken primordial germ cells, which can effectively solve the shortcomings of the above culture method such as low efficiency of in vitro proliferation of PGCs and unclear action signal pathways.

[0006] Technical solution: In the first aspect, the present invention provides the use of IGF-1 instead of insulin in promoting the proliferation of chicken primordial germ cells.

[0007] Preferably, the concentration C of IGF-1 used for in vitro culture of chicken primordial germ cells is 0<C≤100 ng / mL.

[0008] Preferably, the concentration C of IGF-1 used for in vitro culture of chicken primordial germ cells is 25 ng / mL≤C≤100 ng / mL.

[0009] Preferably, the concentration C of IGF-1 used for in vitro culture of chicken primordial germ cells is 100 ng / mL.

[0010] In a second aspect, the present invention provides use of IGF-1 in the preparation of a drug for promoting the proliferation of chicken primordial germ cells.

[0011] Beneficial effects: The present invention promotes the proliferation of chicken primordial germ cells by replacing insulin with IGF-1, which can clarify that the PI3K-AKT signaling pathway is necessary for the proliferation of PGCs and provide a theoretical basis for the study of small molecules in other signaling pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : This is a result diagram of the effect of insulin on the proliferation and biological characteristics of PGCs in the present invention, wherein A is the effect of insulin and no insulin on the morphology of PGCs, B is the statistical result of cell counting after 72 hours of culture with insulin and no insulin, CE is EdU and CCK8 proliferation assay, F is qRT-PCR detection of the effect of insulin and no insulin on PGCs cell adhesion and multipotency, and GH is western blot detection of the effect of insulin and no insulin on PGCs cell adhesion proteins; Figure 2 is the KEGG analysis of insulin and no insulin transcriptomes in the present invention, wherein A is the upregulated signaling pathway in the KEGG analysis, and B is the downregulated signaling pathway in the KEGG analysis; Figure 3 is the effect of different signaling pathway activators in the present invention on the proliferation of PGCs; Figure 4 : It is the result diagram of the effect of different concentrations of IGF-1 on the proliferation of PGCs in the present invention, wherein A is the effect of different concentrations of IGF-1 on the morphology of PGCs, B is the statistical diagram of the cell counting results of culturing PGCs with different concentrations of IGF-1 for 24-72h, and CE is the effect of EdU detection of no insulin, IGF-1 100 ng / mL and insulin on the proliferation of PGCs; Figure 5 The results of the effects of IGF-1 and insulin on the anti-oxidation and apoptosis of PGCs in the present invention are shown in Figure 1, wherein AB is the effects of insulin and IGF-1 on ROS and the statistical graph, CE is the effects of insulin and no insulin on total glutathione, reduced glutathione and oxidized glutathione, and FG is the effects of insulin and no insulin on apoptosis of PGCs; Figure 6 These are the results of the effects of IGF-1 and insulin on the migration ability of PGCs and the establishment of the cell line in the present invention, wherein A is a 7.5 d and 12.5 d fluorescence PGCs migration observation diagram, B is a migration efficiency statistical diagram, and C is a cell line establishment efficiency statistical diagram. DETAILED DESCRIPTION

[0013] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: Example 1: Verification of the effect of insulin on the proliferation and biological characteristics of PGCs After 70 days of culture, the primary cells were frozen after the system was successfully established and revived when used. The experiment was divided into two groups, the control group was the original culture system (i.e., the insulin group), and the experimental group was the B27 group without insulin. TM The cells were cultured in a 37°C cell culture incubator for 72 h and the cell morphology was observed. Figure 1 As shown in AB: The cells in the no-insulin group were flat and broken, showing an irregular shape; the PGCs in the insulin group were able to proliferate normally.

[0014] Next, the proliferation test was carried out. The first was the CCK-8 proliferation experiment. PGCs were plated and subcultured in a 24-well plate for 72 hours. 100 μL was aspirated from each well and added to 96-well plates. Three parallel wells were set in each group, and 10 μL of CCK-8 solution was added to each well (be careful not to generate bubbles in the wells, as they will affect the OD value reading). The well plate was placed in an incubator at 37°C and 5% CO2; the culture plate was cultured in the incubator for 2-4 hours; the wavelength of the microplate reader was adjusted to 450 nm, the 96-well plate was placed at the plate reader, the instrument was operated, and the absorbance value was read. Figure 1 As shown in C and E: Insulin promotes the proliferation of PGCs.

[0015] Subsequently, EdU was used to detect the proliferation of PGCs. PGCs were plated and passaged in 24-well plates, and no insulin was used as the experimental group. EdU solution was diluted with PGCs complete medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μM EdU medium. 100 μL of 50 μM EdU medium was added to each well and incubated for 2 hours, and the medium was discarded. The cells were washed with PBS 1-2 times, each for 5 minutes. Centrifuged at 1400rpm for 6 min, the supernatant was discarded, and 20 μL of PGCs was mixed by blowing 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 of PBS was added, and after washing for 5 minutes, the PBS was discarded. 20 μL of permeabilization agent (0.5 % TritonX-100 in PBS) for 10 minutes; wash once with PBS for 5 minutes; add 20 μL of 1× Apollo staining reaction solution, incubate at room temperature in the dark for 30 minutes, and then discard the staining reaction solution; add 20 μL of permeabilization agent for 2-3 times, 10 minutes each time, and then discard the permeabilization agent; 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, and then discard the staining solution; add 20 μL of permeabilization agent for 10 minutes, and then 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 the figure. Figure 1 As shown in C and D: Insulin promotes the proliferation of PGCs.

[0016] Subsequent steps: ① Total RNA extraction: PGCs cultured with insulin and without insulin were collected into centrifuge tubes, centrifuged at 1400rpm for 6 min, and the supernatant was discarded; 1 mL of TRIZOL reagent was added, mixed by pipetting, and allowed to stand at 4°C for 5 min; the sample was allowed to stand at room temperature for 5 min to allow the protein to fully dissociate; 0.2 mL of chloroform was added, the lid was tightly closed, and the sample was vigorously shaken for 15 s and allowed to stand at room temperature for 2-3 min; centrifuged at 4°C and 12000×g for 15 min, and the sample was separated into layers after centrifugation. The upper aqueous phase contained RNA, and the lower organic phase contained protein and DNA; 500 μL of supernatant was taken, 0.5 mL of isopropanol was added, and the mixture was gently mixed. After standing at room temperature for 10 min, a gelatinous precipitate appeared at the bottom of the tube, which was RNA; after centrifugation at 4°C and 12000×g for 10 min, the supernatant was discarded; 1 mL of 75% ethanol was added to the precipitate, and the mixture was gently mixed; centrifuged at 4°C and 7500×g for 5 After 10 min, discard the supernatant; dry the RNA sample, add an appropriate amount of enzyme-free water to dissolve (dissolution can be promoted at 55-60 ℃ for 10 min); determine the RNA concentration, detect and calculate the OD with a UV spectrophotometer 260 / OD 280Ratio: 1.9~2.0 indicates good effect.

[0017] 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 and centrifuge briefly to collect the 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), RNase-Free ddH2O to 20 μL; reverse transcription reaction: remove the genome and reverse transcription reaction: 42 ℃, 15 min; enzyme inactivation process: 95 ℃, 3 min; add RNase-FreeddH2O to dilute the cDNA concentration according to the RNA content used.

[0018] ③qRT-PCR technology was used to detect the mRNA expression level of each gene in PGCs after treatment with different concentrations of ovotransferrin: 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 supplemented to 10 μL. 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, and each sample was set up with 3 technical replicates. 2 -ΔΔCT The expression level was quantitatively analyzed by the method. Figure 1 As shown in F, cell adhesion genes ZO-1, Occludin, Claudin1, β-catenin, and JAM are downregulated, and pluripotency genes LIN28, NANOG, POUV, and SOX2 are downregulated. The above results indicate that insulin deficiency can reduce cell adhesion and pluripotency.

[0019] Next, we performed Western Blot: 1) Extraction of total protein from PGCs: PGCs were plated and passaged in 24-well plates, and cultured with or without insulin for 72 h. The cells were centrifuged at 1400 rpm for 6 min, the supernatant was discarded, 1 mL of RIPA lysis buffer was added, and the cells were lysed on ice for 40 min. The cells were then centrifuged at 12,000 × g for 10 min at 4 °C, the supernatant was aspirated into a 1.5 mL centrifuge tube, and stored at -80 °C. 2) Determination of protein concentration by BCA method: Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and mix thoroughly. The BCA working solution can be placed at room temperature for 24 hours. Microwell detection of protein concentration (20-2000 μg / mL): Prepare a 96-well plate, first add 25 μL of BSA standard diluted at each concentration to each of the 7 wells, and 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, mix well, and each well has a total of 225 μL of mixed solution; cover the well plate, place it in a 37 ℃ incubator for 30 minutes, and cool to room temperature after the reaction is activated; detect the absorbance value at 562 nm on an enzyme reader within 3-5 minutes; 3) Preparation of WB reagents: Prepare electrophoresis solution: add one pack of SDS powder to 1000 mL of ultrapure water and stir for 10 minutes; prepare transfer solution: add one pack of Transfer powder to 900 mL of ultrapure water, stir for 10 minutes and then add 100 mL of methanol; prepare 1×TBST buffer: prepare 500 mL of 1×TBST buffer, mix 25 mL of 20×TBST buffer with 475 mL of ultrapure water; 4) SDS-PAGE gel electrophoresis, use a pipette to absorb the electrophoresis solution, gently blow the sample hole of 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 the constant voltage of 80V to run for about 40 minutes, and then switch the voltage to 110V constant voltage electrophoresis until it reaches the bottom of the separation gel; 5) Transfer, PVDF membrane is completely immersed in methanol for activation, and then transferred into the transfer solution for 10 minutes. Filter paper and black sponge are also placed in the transfer solution to soak. Cut the gel: Cut the gel at the position corresponding to the size of the target protein, remember the front and back sides, and place 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 place in the transfer device. Note that black corresponds to the black transfer wall. The direction of the current flows from the negative electrode to the positive electrode. The current is 230mA. The transfer time depends on the size of the protein. 6) Blocking: After the transfer, cut the target protein to an appropriate size, add the freshly prepared blocking solution (50ml blocking solution: 2.5g skim milk powder + 50ml 1×TBST, mix well, and shake for 10 minutes) and incubate on a shaker for two hours. After the blocking is completed, wash three times with 1×TBST, 10 minutes each time; 7) Incubate with primary antibody. After washing, add 1×TBST to dilute the primary antibody according to the ratio (1:1000), soak the PVDF membrane, incubate on a shaker for 10 minutes, and then 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 with secondary antibody. After washing, add 1×TBST to dilute the secondary antibody according to the ratio (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 the ratio of A solution to B solution = 1:1, use it immediately after preparation, evenly drip about 100μl 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 showed that after removing insulin, the adhesion of PGCs cells decreased and their pluripotency decreased; the results are as follows Figure 1 As shown in GH: cell adhesion proteins ZO-1, Occludin and JAM are downregulated.

[0020] Conclusion: Insulin deficiency can lead to downregulation of PGCs cell adhesion.

[0021] Example 2: Exploring the molecular mechanism of insulin promoting PGCs proliferation through transcriptome sequencing In order to systematically analyze the molecular mechanism of insulin in regulating the formation of chicken PGCs, the samples were sent to a sequencing company for transcriptome sequencing and KEGG analysis. The results are as follows Figure 2 As shown in the figure: After the lack of insulin, the PI3K-Akt signaling pathway, ECM receptor interaction pathway, RAS signaling pathway and mTOR signaling pathway are downregulated. In the absence of insulin, activators of these four signaling pathways (i.e., IGF-1, Pyrintegrin, ML-099 and MHY1485) were used to replace the effects of insulin, and DMSO was used to dissolve the activators. The results are shown in the figure. Figure 3 As shown: IGF-1 can significantly promote the proliferation of PGCs, and other signal pathway activators added alone or in combination cannot achieve the effect of IGF-1 replacement, and the combined addition of IGF-1 and other groups of signal pathway activators did not improve the proliferation efficiency of PGCs.

[0022] Example 3: Optimal concentration of IGF-1 to promote proliferation of PGCs In order to determine the most suitable concentration of IGF-1, B27 without insulin was used. TMAs a control, each group was then added with 0, 25, 50, 100, 200 and 300 ng / mL of insulin, and cultured in a 37°C cell culture incubator for 72 h. The cell growth morphology was observed every day, and cell proliferation and cell activity were detected. Figure 4 As shown: After removing insulin, PGCs hardly proliferated. With the addition of IGF-1 concentration, the state of PGCs improved and began to proliferate. The results of CCK-8 and EdU detection showed that when the concentration of IGF-1 reached 100 ng / mL, its proliferation rate no longer increased with the increase of concentration, that is, 100 ng / mL was its most suitable concentration.

[0023] Example 4: Effects of IGF-1 and insulin on PGCs anti-oxidation and apoptosis In order to explore the effect of IGF-1 on the cellular antioxidant level of PGCs, the following experiments were performed: GSH and GSSG detection: Collect cells by centrifugation at 1400 rpm for 6 min, use a 96-well plate, add samples or standards in sequence, and mix well. Add 15 μL of total glutathione detection working solution, mix well, and incubate at 25 ℃ 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 enzyme reader.

[0024] ROS detection: The collected cells were suspended in an appropriate amount of diluted drugs and incubated in a 37 °C cell culture incubator in the dark. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. The cells were collected by centrifugation and the appropriately diluted probe was added to a cell density of 1.0×10 6 ~2.0×10 7 Incubate in a 37 ℃ cell culture incubator for 20-60 min, mix by inversion every 3-5 min, wash cells 1-2 times with serum-free cell culture medium to fully remove DCFH-DA that has not entered the cells, and detect by flow cytometry.

[0025] Flow cytometry was used to detect cell apoptosis: PGCs were plated and passaged in 24-well plates, and ovotransferrin was added with gradient concentrations and cultured for 72 h. Flow cytometry was used to detect PGCs. The steps were as follows: cells were collected by centrifugation at 300×g and 4°C; cells were washed twice with pre-cooled PBS, each time centrifuged at 300×g and 4°C for 5 min. 1-5×10 5Cells; discard PBS, add 100 μL 1×Binding Buffer to resuspend cells; add 5 μL Annexin V-FITC and 10 μL PI Staining Solution, mix gently; react at room temperature for 10-15 min in the dark; add 400 μL 1×Binding Buffer, mix well and place on ice. Samples were detected by flow cytometry within 1 hour. ( Figure 5 ).

[0026] The results are as follows Figure 5 The results showed that there was no significant difference in the total GSH content in PGCs (P>0.05); compared with the control group without insulin, the reduced GSH in the IGF-1 and insulin groups was upregulated; the oxidized GSSG was downregulated; and the ROS was downregulated. The results showed that the effects of IGF-1 and insulin on the anti-oxidation and apoptosis of PGCs were consistent, and IGF-1 could replace the effects of insulin.

[0027] Example 5: Effects of IGF-1 and insulin on PGCs migration and lineage establishment In order to clarify the effects of IGF-1 and insulin on the migration ability of PGCs and the establishment of the cell line, migration observation was carried out. Fluorescent PGCs were cultured with IGF-1 and insulin, and the cells were injected into chicken embryos after 72 h of culture. The specific process is as follows: (1) Collection and incubation of fertilized eggs (1-1) Disinfect the freshly obtained eggs, wipe the eggshells with a chlorhexidine solution (1:1000 1.0% chlorhexidine solution), and then place the eggs in an incubator for incubation. Record the incubation time and mark the eggs; (1-2) Collect the eggs at 48-60 hours of incubation (HH stage 16-18). At this time, you can take out an egg and open it from the large end (blunt end) to observe whether the chicken embryo has reached the corresponding period of incubation. It is necessary to focus on observing whether the embryo has developed visible blood vessels and formed a heart and other body parts. The eggs that have reached the period can be taken out for injection.

[0028] (2) Opening the egg window (2-1) Use a cotton ball soaked in 75% alcohol to gently wipe the surface of the eggshell, especially the open end of the egg; (2-2) Use forceps to create a small window (<1 cm in diameter) in the eggshell surface to expose the embryo and remove the shell membrane. Place the egg on a holder under a dissecting microscope and prepare for injection.

[0029] (3) Chicken embryo vascular injection (3-1) Find the embryo under a stereomicroscope and use ophthalmic forceps to gently open the embryo membrane to expose the embryo and its blood vessels; (3-2) Use a micropipette to fill the fluorescent PGCs into the glass needle through the pipette tip (Note: the filling process should be slowly sucked and filled to avoid bubbles rushing into the glass needle), connect the injection needle to the handheld syringe, adjust the dosage, and slowly insert the injection needle along the dorsal aorta of the embryo under a stereo microscope. Slowly fill the exogenous solution (containing fluorescent cells) into the glass needle, and avoid bubbles in the injection needle; (3-3) When using a pneumatic syringe to push the fluorescent cells in, it is important to note that the voltage or air pressure should be low enough to reduce damage to the embryonic blood vessels. At the same time, the injection volume is usually 1-5 μL. Too much can easily cause embryonic death. Once the exogenous solution is injected into the embryonic blood vessels, the color of the embryonic blood vessels will quickly change to the color of the solution and then gradually return to red, indicating that the exogenous solution has been successfully delivered to the embryo; (3-4) After the injection is completed, gently pull out the injection needle, gently drip 20 μL of penicillin-streptomycin to the injection site, and then seal it with medical tape. The sealed eggs are placed in the incubator for incubation, and the migration efficiency is observed at 7.5 days, 12.5 days, and 18.5 days.

[0030] The results are as follows Figure 6 As shown: At 7.5d and 12.5d, IGF-1 and insulin had the same effect on migration ability. Then the ability to establish a cell line was tested. The results showed that both cells were able to establish a cell line and there was no difference. The above results show that IGF-1 can replace the effect of insulin.

[0031] 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. Application of IGF-1 as an alternative to insulin in promoting the proliferation of chicken primordial germ cells.

2. The use of IGF-1 as a substitute for insulin in promoting the proliferation of chicken primordial germ cells according to claim 1, characterized in that: The concentration of IGF-1 used in in vitro culture of chicken primordial germ cells is 0<C≤100 ng / mL.

3. The use of IGF-1 as a substitute for insulin in promoting the proliferation of chicken primordial germ cells according to claim 1, characterized in that: The concentration of IGF-1 used for in vitro culture of chicken primordial germ cells is C25 ng / mL≤C≤100 ng / mL.

4. The use of IGF-1 as a substitute for insulin in promoting the proliferation of chicken primordial germ cells according to claim 1, characterized in that: The concentration of IGF-1 used in in vitro culture of chicken primordial germ cells was 100 ng / mL.

5. Application of IGF-1 in the preparation of drugs for promoting the proliferation of chicken primordial germ cells.

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