CN117106700A

CN117106700A - Application of reading protein Prrc2a in differentiation process of female germ stem cells

Info

Publication number: CN117106700A
Application number: CN202311248802.XA
Authority: CN
Inventors: 裴秀英; 俞晓丽; 张彦平; 邱意开; 常青; 刘心蕊; 李进花; 孙伟玮
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2023-09-25
Filing date: 2023-09-25
Publication date: 2023-11-24

Abstract

The invention discloses an application of reading protein Prrc2a in the differentiation process of female germ stem cells, which discovers m through researching the in-vitro differentiation mechanism of female germ stem cells promoted by cistanche deserticola polysaccharide treatment ⁶ The role of A methylation in this process was followed by screening for the key gene Prrc2a (an m ⁶ A reads protein), demonstrating that it can regulate m ⁶ A is modified, so that the in vitro differentiation of female germ stem cells is promoted.

Description

Application of reading protein Prrc2a in differentiation process of female germ stem cells

Technical Field

The invention relates to the field of biotechnology, in particular to application of reading protein Prrc2a in a female germ stem cell differentiation process.

Background

Female germ stem cells (Famale Germline Stem Cells, fgcs) are a class of monoenergetic stem cells that can be directionally differentiated into oocytes by asymmetric division. The traditional concept of reproductive biology considers that female mammals stop the production of ova after birth, can not realize the regeneration and renewal of follicular pools and can only be continuously exhausted until the ovaries age, so that the discovery of FGSCs has important significance for supplementing the number of oocytes in an ovary stock, but under normal conditions, most FGSCs are in a resting stage, and a certain condition of stimulation is required to activate the FGSCs and activate the differentiation ability. Cistanche deserticola polysaccharides (Cistanche Deserticola Polysaccharides, CDPs) have the effect of promoting the in vitro differentiation of FGSCs and indicate that the FGSCs probably act through TGF-beta and BMP signaling pathways, but other mechanisms act synergistically on cell differentiation, regulatory mechanisms have not been clarified yet, and epigenetic modifications may also participate in the process.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an application of the reading protein Prrc2a in the differentiation process of female germ stem cells.

The technical scheme for solving the technical problems is as follows: provides an application of reading protein Prrc2a in the differentiation process of female germ stem cells.

Based on the technical scheme, the invention can also be improved as follows:

further, the nucleotide sequence of the reading protein Prrc2a is shown as SEQ ID No: 1.

Further, in the differentiation process, cistanche deserticola polysaccharide is added into an in-vitro culture system to promote differentiation of female germ stem cells.

Further, the addition amount of cistanche polysaccharide is 0.5 mug/mL.

The invention also provides an in vitro culture system for promoting differentiation of female germ stem cells, which comprises the reading protein Prrc2a.

The invention also provides a preparation for promoting differentiation of female germ stem cells, which comprises the reading protein Prrc2a.

The invention has the following beneficial effects:

1. eukaryote mRNA up-scalingThe most highly posttranscriptional modification is N6-methyladenosine (m ⁶ A) Methylation, which means methylation of the sixth N of adenylate, m ⁶ The A methylation is performed by methylase, wherein methyltransferase METTL3/14/WTAP is responsible for adding modification, demethylase ALKBH5 and FTO are responsible for removing modification, and reading protein can recognize m ⁶ A site and mediates splicing, transport, degradation and translation of mRNA, thereby participating in a variety of biological processes, m ⁶ The A modification plays a key role in various physiological functions such as proliferation and differentiation of stem cells, DNA injury reaction, formation of homeostasis, gametogenesis, embryo development, formation process of biological clock and the like, and comprises the regulation and control of self-renewal and differentiation of spermatogenic stem cells and embryonic stem cells and the generation of sperms and ova.

2. The invention clarifies that m in the process of inducing FGSCs to differentiate in vitro by CDPs ⁶ Important role and m playing the main role of A modification ⁶ A methylase, further elucidate the regulatory mechanism of CDPs inducing FGSCs to differentiate in vitro and is m ⁶ Modification A adds a new function.

3. The method uses FGSCs culture medium of 0.5 mug/mL CDPs to induce FGSCs for 24 hours, and detects obvious differentiation promoting phenotype of cells from the cell morphology, diameter, activity, proliferation, apoptosis and expression of differentiation markers; detection of m in the Induction of differentiation of FGSCs by 0.5 μg/mL CDPs ⁶ Importance of a methylation: detection of m by quantitative kit ⁶ Elevated overall levels of a methylation; there is a significant difference in expression of methylases; knocking down methyltransferase Mettl3, decreasing m ⁶ A modification level, and then induction by CDPs to find that the differentiation capacity of FGSCs is reduced, and overexpressing the demethylase Alkbh5 to obtain the same effect as that of Mettl3 knockdown, thus obtaining m ⁶ Methylation of a is involved in the CDPs promoting FGSCs differentiation process and is necessary; ONT full length transcriptomics and m ⁶ A methylation sequencing is combined for analysis, qRT-PCR and PRM methods are verified, and a key gene Prrc2a (m ⁶ A reading protein), the Prrc2a was knocked down and induced using CDPs, and then the expression of differentiation markers was examined, and a decrease in differentiation ability was found.

Drawings

FIG. 1 is a graph showing the in vitro culture state of FGSCs from an experimental group and statistical analysis of cell diameters from two groups;

FIG. 2 is a normal distribution diagram of cell diameters of a control group and an experimental group;

FIG. 3 shows qRT-PCR detection of female germ cell marker gene and differentiation related gene expression;

FIG. 4 shows qRT-PCR detection of expression of the level of methylase RNA;

FIG. 5 shows the ability of EdU to detect FGSCs cell DNA replication;

FIG. 6 shows two sets of Stra8 and γH by immunofluorescent staining ₂ ax and Sycp3 expression and statistical analysis;

FIG. 7 is a Western blot detection of two sets of Oct4, mvh, stra8 and Sycp3 protein band patterns, and statistical analysis of the grayscale values of the protein bands;

FIG. 8 shows the Western blot detection of expression of protein levels of methylase;

FIG. 9 shows the CCK-8 assay for FGSCs cell viability;

FIG. 10 is a flow chart for detecting FGSCs apoptosis;

FIG. 11 shows the detection of m by the kit method ⁶ ARNA methylation overall level expression;

FIG. 12 shows qRT-PCR assay of si-Mettl3 with CDPs added to induce the expression of FGSCs cell markers and differentiation markers;

FIG. 13 shows FGSCm after Prrc2a knockdown ⁶ A methylation integral level statistics;

FIG. 14 shows qRT-PCR detection of Alkbh5 overexpression;

FIG. 15 shows qRT-PCR detection of sh-Alkbh5 induction of FGSCs cell markers and differentiation markers expression by CDPs;

FIG. 16 is a volcanic diagram of differentially expressed transcripts;

FIG. 17 is a cluster map of differentially expressed transcripts;

FIG. 18 is a volcanic diagram of a differentially expressed gene;

FIG. 19 is a cluster map of differentially expressed genes;

FIG. 20 is a statistical plot of differential methylation sites;

FIG. 21 is a cluster map of differential methylation sites;

FIG. 22 shows the DETs and the difference m ⁶ A comparison analysis chart of the transcript Venn related to the site A;

FIG. 23 is a diagram showing the difference m between DETs and DETs ⁶ A comparison analysis chart of the transcript Venn related to the site A;

FIG. 24 is a graph of the difference m ⁶ Clustering the transcript related to the A site with DETs Complex Heatmap;

FIG. 25 shows the DEGs and the difference m ⁶ A comparison analysis chart of the gene Venn related to the site A;

FIG. 26 shows the DEGs and the difference m ⁶ A comparison analysis chart of the gene Venn related to the site A;

FIG. 27 is a difference m ⁶ Clustering heat map of A site related gene and DEGs Complex Heatmap;

FIG. 28 shows qRT-PCR verification of DEGs and the variance m ⁶ A site-related gene expression;

FIG. 29 is a graph showing the mean value of target protein content for two groups of samples;

FIG. 30 is a qRT-PCR validation of Prrc2a three transcript expression;

FIG. 31 is a qRT-PCR assay for Prrc2a expression;

FIG. 32 shows qRT-PCR detection of FGSCs markers and differentiation-related gene expression;

FIG. 33 is a statistical analysis of Western blot detection of FGCS markers and differentiation-related protein band patterns and grayscale values of protein bands;

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1:

fgcs were induced for 24h using fgcs medium of 0.5 μg/mL CDPs (cistanche polysaccharide), and as an experimental group, a control group (normal cultured female germ stem cells without cistanche polysaccharide) was set, and the results of which were examined from cell morphology and diameter, respectively, are shown in fig. 1-2 (scale: 50 μm and 10 μm in fig. 1, data of each group in fig. 1, left is Ctrl, right is CDPs).

As can be seen from FIGS. 1-2, the in vitro culture of female germ stem cells is interfered by CDPs of 0.5 mu g/mL, and the cell growth state is good, and the cell diameter can be increased by 25 mu m-30 mu m in a certain proportion (FIG. 1); the diameter of the cells of the experimental group (0.5. Mu.g/mL CDPs intervention) and the control group (normally cultured female germ stem cells) were measured, and the results showed that the cell diameters were increased to different extents after the experimental group had treated the cells for 12h,24h,36h and 48h, and that there was a significant difference (P < 0.05) compared with the control group. The cell diameters of the experimental group and the control group were subjected to normal distribution analysis to obtain a normal distribution map (fig. 2). Suggesting that 0.5 mug/mL cistanche deserticola polysaccharide is dry, female germ stem cells have larger diameters and tend to differentiate.

Example 2:

PCR amplification

1.1 extraction of Total RNA from cells

(1) Cells cultured in 35mm dishes were harvested in 1.5mL centrifuge tubes to a quantity of about 10 ⁶ A plurality of;

(2) the pre-chilled milling tube was charged with 0.35. 0.35mL TRK Lysis Buffer and ovarian tissue was milled using a pre-chilled cryomill (20. Mu.L of beta-mercaptoethanol was added per 1mL before TRK Lysis Buffer use). The lysate was transferred to a fresh EP tube, centrifuged at 14000g for 2min at normal temperature, and the supernatant was collected to remove insoluble impurities.

(3) Adding 70% ethanol solution with equal volume into the supernatant, and mixing by vortex;

(4) sleeving the RNA binding column into a collecting pipe, adding the mixed solution obtained in the step (3) into the RNA binding column, centrifuging at 10000rpm for 1min at room temperature, and discarding the filtrate;

(5) repeating step 4 until all the mixed solution is combined to the RNA binding column;

(6) sleeving the RNA binding column into a collecting pipe, adding 500 mu L RNA Wash Buffer I to the binding column, centrifuging at 10000rpm for 30s, and discarding the filtrate;

(7) recovering the RNA binding column sleeve into a collecting pipe, adding 500 mu L RNA Wash Buffer II to the binding column, centrifuging at 10000rpm for 1min, and discarding the filtrate;

(8) repeating step (7);

(9) recovering RNA combined with the column sleeve in a collecting pipe, centrifuging at 10000rpm for 2min, and spin-drying;

the RNA binding column is sleeved into a new 1.5mL centrifuge tube, 30-70 mu L of nucleic-free Water is sucked up to the center of the binding column, the RNA is eluted by centrifugation at 10000rpm for 2min, and after the concentration and purity of the obtained RNA are measured, the RNA is reversed or placed at-80 ℃ for preservation as soon as possible.

(2) The remaining steps are referred to 3.1.

1.2 determination of RNA concentration

(1) The NanoDrop 2000 software was opened and RNA measurements were selected. Lifting a sample arm, sucking 2 mu LDEPC water by using a pipette, correcting the instrument, and repeating the process twice;

(2) after the sample is named, 1 mu L of sample to be detected is dripped on the instrument probe, and a computer is operated for detection;

(3) Wiping off the residual liquid, sucking 1 mu L DEPC water to calibrate the instrument, and measuring the next sample;

(4) repeating the steps (2) and (3) until all samples are measured;

(5) saving the measurement results, recording Nucleic Acid and OD ₂₆₀ /OD ₂₈₀ Numerical value (OD) ₂₆₀ /OD ₂₈₀ The numerical range should be 1.8-2.0, protein contamination is possible when less than 1.8 and DNA contamination is possible when more than 2.0), the instrument is closed, and the remaining RNA sample is inverted or placed at-80 ℃ for preservation as soon as possible.

1.3 preparation of cDNA by reverse transcription

Note that: RNA purity (OD) ₂₆₀ /OD ₂₈₀ ) Subsequent experiments can be carried out between 1.8 and 2.0;

(1) the following mixed solutions were prepared in 200. Mu.L PCR tubes without RNase:

TABLE 1 reverse transcription system (20. Mu.L)

(2) The pipettor was thoroughly mixed with all reaction components and subjected to gentle centrifugation and incubated at 25℃for 10min.

(3) If the synthesized cDNA template is to be used for fluorescent quantitative qPCR detection, please incubate in the PCR instrument according to the following scheme:

42℃15min

85℃5min

stopping the reaction in ice bath

The cDNA thus synthesized can be used for immediate downstream fluorescent quantitative qPCR detection or stored for a long period of time at-20 ℃.1.4 real-time fluorescence quantitative PCR (qRT-PCR)

(1) Using the cDNA obtained in step 1.3 as a template, carrying out qRT-PCR amplification, wherein the reaction system is as follows:

TABLE 2 qRT-PCR System (25. Mu.L)

(2) The primer sequences used for qRT-PCR reactions are shown in the following table:

TABLE 3 qRT-PCR Gene primer sequence Listing

(3) After the eight-connecting tube is centrifuged briefly, amplification is carried out by using a CFX instrument, and the two-step PCR amplification procedure is as follows: stage 1: pre-denaturation

Repeat：1

95 ℃ 30sStage 2: PCR reaction

Repeat：40

95℃5s

60℃30s

Stage 3：Dissociation

(4) Deriving experimental data, results according to 2 ^-ΔΔCt Analytical calculations were performed (delta Ct = delta Ct experimental group-delta Ct control group, delta Ct = Ct gene of interest-Ct β -action) data were analyzed and plotted using Graphpad prism software.

qRT-PCR is carried out on FGSCs marker genes Oct4 and Mvh of an experimental group and a control group; meiosis and differentiation marker genes Stra8 and Sycp3; maintaining multipotency and self-renewal related genes Sox2, tert; differentiation marker Cd117; the zona pellucida marker Zp3 expression was examined and the results are shown in figure 3 (data for each set of data in figure 3, left hand Ctrl, right hand CDPs).

As can be seen from fig. 3, the cells of the experimental group were significantly elevated in the expression of oct4 and Mvh (P < 0.05) by CDPs treatment for 24 hours compared to the control group; meiosis and differentiation marker genes Stra8 and Sycp3 expression were also significantly elevated (P < 0.05); sox2, tert, cd117, zp3 were all significantly elevated (P < 0.05),

detection of m by qRT-PCR ⁶ The results of the expression of A methylase are shown in FIG. 4 (FIG. 4, data for each set of data, left Ctrl, right CDPs).

As can be seen from fig. 4, after CDPs intervened in fgcs for 24 hours, the expression of methyltransferase Mettl3, mettl14 and Wtap were all significantly increased, the expression of methyltransferase Alkbh5 was significantly decreased, and Fto was not significantly different; the reading proteins, ythdf1, ythdf2, ythdf3, were all significantly elevated, P <0.05, P <0.01.

EdU staining

(1) After addition of a cover slip to a 24-well plate, a suitable number of cells were cultured and grown to a density of about 80%;

(2) preparing 2 XEdU working solution: diluting EDU stock solution (10 mM) with cell culture solution preheated at 37 ℃ at a ratio of 1:500 to obtain 2 XEdU working solution with a concentration of 20 mu M, adding the 2 XEdU working solution with the same volume as that of the old culture solution into a cell culture plate to make the final concentration of EdU be 1X, putting the cell culture plate into an incubator, and continuously incubating for 2 hours;

(3) after the EdU labeling of the cells was completed, the culture solution was removed, and 4% paraformaldehyde fixing solution was added thereto, and the mixture was allowed to stand at room temperature for about 15 minutes; removing the fixed liquid, washing the cells with 3% BSA for 3 times, and 3-5 min each time; removing the washing liquid, using 0.3% Triton X-100 penetrating liquid to penetrate the cell membrane, and incubating for 10-15 min under the condition of room temperature; removing the permeation solution, washing the cells with 3% BSA for 1-2 times, each time for 3-5 min;

(4) removing the washing liquid, adding an EdU detection agent (Click Additive Solution) prepared according to the requirements of the kit specification, gently shaking the culture plate to ensure that the detection agent can be uniformly covered on the cell sample, and incubating for about 30min under the condition of being away from light at room temperature; then sucking up the Click reaction liquid, and washing the cells with the washing liquid for 3 times, each time for 3-5 min;

(5) After the washing liquid is sucked off, adding 1X Hoechst 33342 solution, and incubating for 10min at room temperature under the condition of avoiding light;

(6) sucking out the 1 Xhoechst 33342 solution, and washing the cells with the washing solution for 3 times for 3-5 min each time;

(7) taking a concave glass slide, dripping a proper amount of anti-fluorescence quenching agent, reversely buckling the dyed cell cover glass on the glass slide, and dripping oiling liquid sealing sheets around the cover glass;

(8) fluorescence detection and photographing can then be performed. Hoechst 33342 is blue fluorescence with a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460 nm. The results are shown in FIG. 5. (Scale: 100 μm and 20 μm)

As can be seen from fig. 5, the EdU positive cell rate of the experimental group was significantly lower than that of the control group (P < 0.01) by statistical analysis using EdU staining to detect DNA replication in both groups, indicating that CDPs inhibited DNA self-replication of fgcs in a short period. Some of the experimental groups had larger cell diameters and did not express EDU activity, suggesting that CDPs promote the entry of FGSCs from the interval into the meiosis phase.

3. Immunofluorescent staining

(1) Inoculating the cells into a special confocal culture dish, and culturing until the density is about 80%; removing old cell culture medium, adding PBS to rinse cells for 1 time and 5min;

(2) adding 4% paraformaldehyde, and fixing at room temperature for 15min; removing the fixed liquid, adding PBS containing 0.5% Triton to permeate cells for 30min, and preventing membrane protein from permeating (Mvh); removing the permeation solution, and rinsing the cells with PBS for 3 times, each time for 5min;

(3) Blocking for 1h at room temperature with PBS containing 3% BSA;

(4) incubation resistance: discarding the blocking solution, diluting the primary antibody by using an antibody diluent according to the proportion of 1:500, and incubating overnight at 4 ℃; recovering the primary antibody, and rinsing with PBS for 3 times, each time for 5min;

(5) diluting the fluorescent secondary antibody by using PBS according to the proportion of 1:800, and incubating for 1h at room temperature in a dark place; recovering the secondary antibody, and rinsing with PBS for 3 times, each time for 5min;

(6) diluting working solution with DAPI concentration of 10ng/mL by using PBS, and incubating for 5min at room temperature under the condition of light shading; removing DAPI working solution, and rinsing the cells with PBS for 3 times, each time for 5min;

(7) after encapsulation with the anti-fluorescence quencher, images were observed and collected under a Nikon confocal microscope. The results are shown in FIG. 6. (Scale: 50 μm and 10 μm; P < 0.05; P < 0.01)

As can be seen from FIG. 6, immunofluorescent staining detects the meiosis protein Stra8, γH ₂ ax, expression of Sycp3 and statistics of fluorescence intensity, it was found that the expression of the meiosis activating protein Stra8 was significantly increased (P<0.01 First meiosis pre-Congress marker protein gamma H ₂ The expression of the association-marker protein Sycp3 was also significantly increased in ax and even-line phase of the first meiosis prophase (P)<0.01). The cistanche deserticola polysaccharide has the maintenance effect on the characteristics of FGCS cells, and promotes the in vitro differentiation of FGCS.

4. Western immunoblotting technique

4.1 extraction of cell holoprotein

(1) After the cells had sucked the medium, they were operated on a cold table, and cold PBS was added to the dish for 3 washes, shaking several times each time to remove the medium as much as possible, sucking the medium as gently as possible along the side walls, and not touching the bottom cells.

(2) PBS was removed, 100-200. Mu.L of PMSF-containing RIPA protein lysate (volume added determined based on cell density) was added, and the cell membrane was seen to disappear under the microscope leaving only gray nuclei. The pre-chilled EP tube was labeled in detail and the adherent cells were scraped with cells into the labeled pre-chilled EP tube.

(3) The cells were disrupted using a sonicator, the procedure was set and used with ultrasound for 1s, suspended for 9s, and repeated 10 times (the EP tube was inserted into ice and placed into the sonicator to avoid excessive heat generated by cell disruption during sonication, degrading the protein).

(4) Centrifuge at 12000rpm for 20min at 4 ℃.

(5) And slowly transferring the supernatant obtained by centrifugation into a new EP tube to obtain the whole protein extract.

4.2 protein quantification

(1) Preparing BCA working solution with proper volume according to the quantity of standard holes by mixing BCA reagent A and BCA reagent B according to the proportion of 50:1, and fully and uniformly mixing;

(2) 200 mu L of BCA working solution was added to each well;

(3) The ELISA plate was placed on a shaker for 30s shaking, incubated at 37℃for 30min, and then colorimetric assay was performed at 562 nm. Drawing a standard curve by taking the protein content (mug) as an abscissa and the light absorption value as an ordinate;

(4) diluting the sample to be measured to a proper concentration so that the total volume of the sample diluent is 20 mu L (e.g. 18 mu L dd H ₂ O+2 μl of sample to be measured), 3 wells are required for the sample to be measured. Adding 200 mu L of BCA working solution into each hole, fully and uniformly mixing, oscillating for 30s, incubating for 30min at 37 ℃, colorimetrically measuring at 562nm wavelength, and recording the absorbance value;

(5) according to the absorbance value of the measured sample, the corresponding protein content (mug) can be obtained on a standard curve, divided by the total volume of the sample diluent (20 mug), and then multiplied by the dilution factor of the sample to obtain the actual concentration (unit: mug/mug) of the sample to be measured. Taking 30 mug as an example, the total protein amount of each group is adjusted to be consistent according to the protein concentration by using lysate or water.

(6) Denaturation: adding 5×loading Buffer into protein supernatant at ratio of 1:5, mixing, setting metal bath at 100deg.C, decocting for 10min, rapidly inserting into ice, packaging protein, and storing in-80deg.C refrigerator to avoid repeated freeze thawing.

4.3SDS-PAGE electrophoresis

(1) Stacking two cleaned glass plates to align the bottoms of the two cleaned glass plates, and fixing the two cleaned glass plates on a glue making bracket;

(2) after leak detection, 10% SDS-PAGE gel was prepared;

(3) and (3) glue filling: adding separating gel to about 2/3 width of small glass plate, about 1cm of lower edge of comb, immediately adding isopropanol or water to edge of small glass plate for sealing, and standing for 20min to obtain clear boundary; pouring out the water or isopropanol on the gel, and sucking the residual water or isopropanol with filter paper; adding concentrated glue to the upper edge of a small glass plate, immediately inserting a sample feeding comb, inserting the comb in parallel and avoiding generating bubbles when inserting, and standing for 30min at room temperature until the concentrated glue is completely solidified;

(4) taking down the glass plates from the gel making frame, fixing the two small plates in the electrophoresis tank oppositely, filling SDS electrophoresis buffer solution between the two glass plates, and if the liquid level of the electrophoresis tank does not move downwards obviously after 5min, indicating that no liquid leakage exists;

(5) sample adding: slowly pulling out the comb teeth, and using a 10 mu L small-range liquid-transfering device to sample, wherein the Marker is 3-5 mu L, the sample should be carefully added slowly during sample adding, the sample can be punched out of the sample adding hole too quickly, if bubbles exist in the sample adding process, the sample can overflow, and if impurities exist in the lane, the liquid-transfering device can be used for flushing the lane and then adding the sample;

(6) Electrophoresis: and regulating the voltage to 80V to enable the bromophenol blue to run to the boundary of the concentrated gel and the separation gel, regulating the voltage to 120V until the bromophenol blue reaches the bottom to stop electrophoresis, and if the molecular weight of the target protein is large, enabling the bromophenol blue to properly exceed the bottom of electrophoresis.

4.4 film transfer

(1) Taking down the rubber plate, carefully separating the large and small glass plates, avoiding pulling to damage the rubber, taking care to prevent the rubber from drying, and immediately placing the rubber into a culture dish with a film transfer liquid. Gently scrape the concentrated glue portion to avoid scraping the separating glue. Scraping off the unnecessary gel part according to the Marker range;

(2) NC films of appropriate dimensions were cut out and assembled in the following order: white transfer film clamp-sponge-filter paper-NC film-SDS gel-filter paper-sponge-black transfer film clamp.

(3) Closing a film transfer clamp, placing the film transfer clamp in a film transfer groove, placing an ice bag, filling film transfer buffer solution, oppositely connecting two electrodes with the same color, and connecting a power supply to start film transfer;

(4) the film transferring groove is placed in an ice-water mixture and placed in an ice bag (film transferring at low temperature, heating in the film transferring process is avoided), film transferring is carried out by using a constant current of 200mA, and stopping is carried out for about 120 min;

(5) the membrane is taken out and transferred to NC membrane, indirectly proving that the target protein is transferred.

4.5 closure

(1) Weighing 1g of the obtained skimmed milk powder, and dissolving the skimmed milk powder into 20mL of TBST to obtain a 5% skimmed milk sealing solution;

(2) immersing the NC membrane after membrane transfer into a sealing liquid, and incubating for about 1h at room temperature by a shaking table;

(3) the blocking solution was then removed and rinsed 3 times for 5min each with TBS.

4.6 antibody incubation

(1) Incubation resistance: diluting the primary antibody by using an antibody diluent according to the requirements of the specification, adding the antibody to cover an NC membrane, and then placing the incubation box on a shaking table at 4 ℃ for overnight incubation; taking out the sample in the next day, and cleaning the NC membrane for 3 times by TBS after recovering the primary antibody, wherein each time is 5min;

(2) secondary antibody incubation: diluting the Odyssey fluorescent secondary antibody in TBS solution at a ratio of 1:10000, adding the secondary antibody to cover NC membrane, incubating for about 1h in a shaking table under the condition of light-shielding at room temperature, recovering the secondary antibody, and washing the membrane with TBS for 3 times and 5min each time.

4.7 image acquisition

The bands were observed and images were acquired using an Odyssey Sa near infrared two-color fluorescence imaging system.

The results are shown in FIGS. 7-8.

As can be seen from FIG. 7, the Western Blot further examined the expression of FGSCs marker proteins Mvh, oct4, stra8 and Sycp3, and found that 0.5 μg/mL cistanche salsa polysaccharide significantly increased the expression of germ cell marker proteins Mvh, oct4 and meiosis and differentiation marker proteins Stra8, sycp3 (P < 0.01) after in vitro intervention of female germ cells.

As can be seen from fig. 8 (×p)<0.05，**P<0.01 Western blot to detect expression of Mettl3, fto and Ythdf1 consistent with gene expression trend, ×p<0.01. Description m ⁶ The methylation of A and CDPs promote the in vitro differentiation process of FGSCs.

5. Cell count

(1) Discarding the culture medium of the cells to be counted, and washing the cells for 3 times by PBS;

(2) adding 0.05% pancreatin into the culture dish, and digesting for 2min; adding culture solution to stop digestion, and blowing the bottom cells into cell suspension;

(3) transferring the cell suspension into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, and sucking and discarding the supernatant;

(4) re-suspending the cells with 1mL of fresh culture solution, taking 100 μl therefrom, adding to 900 μl PBS, and mixing well;

(5) taking a cleaned blood cell counting plate, covering a cover glass, sucking 10 mu L of cell suspension by a liquid transfer device, and slowly dripping from one side of the cover glass; after the cell suspension is scattered under the cover glass, placing a cell counting plate under a microscope for observation and counting;

(6) counting the cell numbers in the upper and lower four corners and the middle 5 grids of the blood cell counting plate respectively, and calculating an average value;

(7) the total number of cells per ml of cell suspension was calculated according to the following formula: 1mL cell number = average value x 10 ⁴ X dilution.

CCK-8 cell viability assay

(1) Inoculating the cells of the experimental group and the control group into a 96-well plate according to 5000 cells per hole, repeating 6 times for each group of cells respectively, and placing the cells into an incubator for pre-culture;

(2) under the dark condition, 110 mu L of diluted CCK8 mixed solution (CCK 8: FGSCs culture solution=1:10) is added into each hole, and the mixture is placed into an incubator for incubation for 2 hours;

(3) the OD of the cells of each well at a wavelength of 450nm was measured with a microplate reader and the results were analyzed by measuring the culture time for 0h,12h,24h,36h and 48h, respectively.

The results are shown in FIG. 9.

As can be seen from fig. 9, the cell viability of the experimental group and the control group was detected by CCK-8 method, and the results showed that the cell viability of the experimental group was increased (P < 0.05) for 12h and 24h, and there was no significant difference between 36h and 48h, indicating that CDPs can maintain the cell viability of fgcs, and is non-toxic to fgcs.

Annexin V-FITC/PI flow apoptosis detection

(1) Inoculating the cells of the control group of the experimental group into 24 pore plates respectively, and carrying out 3 repeated samples on each group;

(2) adding a proper amount of pancreatin cells without EDTA for digestion for 2min, and collecting the cells into a centrifuge tube;

(3) washing cells with PBS, centrifuging at 2000rpm for 5min, discarding supernatant, collecting cells, and repeating twice;

(4) adding 500 mu L of Binding Buffer to lightly suspend the cells;

(5) Under the condition of avoiding light, adding 5 mu L of Annexin V-FITC, and gently mixing;

(6) add 5. Mu. L Propidium Iodide and mix gently;

(7) incubating for 5-15 min under the condition of keeping away from light at room temperature;

(8) two sets of cell suspensions were observed and tested using a Flow Cytometry instrument within 1 h.

The results are shown in FIG. 10. (xp <0.05, < P < 0.01)

As can be seen from FIG. 10, apoptosis was detected by flow through double staining of PI and Annexin V, and when apoptosis occurred in the cells, phosphatidylserine on the cell membrane turned inside out from the membrane, while Annexin V was able to bind specifically thereto, and was detected by FITC fluorescence labeled on Annexin V. PI is a nuclear dye that cannot pass through the cell membrane of living cells and can only enter cells if the cell membrane is broken. Apoptosis flow results are shown in the figure and divided into four quadrants: first quadrant, upper right (Annexin V ⁺ /PI ⁺ ) Is a late apoptotic cell, with membrane eversion and cell breakage; second quadrant, upper left (Annexin V ^- /PI ⁺ ) Necrotic cells and cell debris, no cell membrane and only nuclear signal; third quadrant, lower left (Annexin V ^- /PI ^- ) Is a living cell, both of which have no signal; fourth quadrant, lower right (Annexin V ⁺ /PI ^- ) Is an early apoptotic cell, and membrane eversion has just occurred. And (3) counting the sum of the late apoptosis rate (first quadrant) and the early apoptosis rate (fourth quadrant), and finding that the apoptosis rates of the experimental group and the control group have no significant difference, so that the CDPs cannot induce the FGSCs to undergo apoptosis.

The results show that 0.5 mug/mL CDPs do not affect the cell viability of FGSCs and have good differentiation promoting effect. 8.m ⁶ ARNA methylation global level detection

The experiment uses m provided by Eimer science and technology ⁶ A RNA methylation quantitative detection kit (colorimetric method).

(1) Sample preparation: extracting total RNA of the cells, quantifying to 200ng, and storing the extracted RNA at-20 ℃ or-80 ℃ until use;

(2) buffer and solution preparation: diluting WB, CA, DA, ES, PC solution according to the requirements of the kit instruction;

(3) RNA binding:

a. the number of 8 tubes required for the experiment was calculated in advance. The unwanted 8-up tube was carefully removed from the rack and placed back into the bag (tightly sealed and stored at 4 ℃);

b. add 80. Mu.L of BS (binding solution) to each well;

c. 2. Mu.L NC, 2. Mu.L diluted PC, and 200ng sample RNA were added to each well, gently tilting the plate side-to-side or gently shaking it several times to mix the solution well and ensure that the solution completely covered each well bottom;

d. sealing the experimental pore plate by using a sealing film or a sealing plate strip, and incubating for 90min in a 37 ℃ incubator;

e. the BS solution in each well was removed. Then, 150. Mu.L of diluted WB solution was added to each well for washing, and the diluted WB solution was removed; repeated three times.

④m ⁶ A, RNA capture:

a. 50 μl of diluted Capture Antibody (CA) solution was added to each well, which was then covered with a sealing film and incubated at room temperature for 60min;

b. removing the discarded CA solution;

c. 150. Mu.L of diluted WB solution was added to each well and washed three times;

d. 50. Mu.L of diluted Detection Antibody (DA) solution was added to each well, which was then covered with a sealing film and incubated at room temperature for 30min;

e. removing the discarded DA solution;

f. mu.L of diluted WB solution was added to each well and washed four times;

g. 50. Mu.L of diluted Enhancer (ES) was added, then covered with a sealing film and incubated at room temperature for 30min;

h. removing the ES solution;

i. mu.L of diluted WB solution was added to each well and washed five times.

(5) And (3) signal detection:

a. mu.L of a Developing Solution (DS) was added to each well and incubated at room temperature in the absence of light for 1 to 10min. Observing and monitoring the color change of the sample wells and positive and negative control wells, at m ⁶ When the content of A is enough, the DS solution turns blue;

b. when the color of the positive control well became medium blue, 100. Mu.L of reaction terminator (SS) was added to each well to inhibit the enzyme reaction. After addition of SS, the color turned yellow and absorbance at 450nm was read using a microplate reader within 2-15 min.

⑥m ⁶ And A, calculating:

the following formula was used to calculate m in total RNA of the sample ⁶ Percentage of a: m is m ⁶ A％＝(Sample OD－NC OD)÷S/(PC OD－NC OD)÷P×100％

S is the input quantity of the RNA sample, and the unit is ng; p is the Positive Control (PC) input in ng.

The results are shown in FIG. 11. (in FIG. 11, for each set of data, ctrl on the left and CDPs on the right, < 0.01. Times. P)

As can be seen from FIG. 11, female germ stem cells m before and after cistanche polysaccharide intervention were detected by methylation quantitative detection kit (colorimetric method) ⁶ The result shows that after CDPs intervene in FGSCs for 24 hours in vitro culture, m ⁶ Overall a methylation levels were significantly elevated (P<0.01 With an upward trend after 12h and 36h of intervention, but without significant differences.

9. Small interfering RNA transfection

Querying target gene sequence, sending to biological company to design and synthesize siRNA.

(1) The day before transfection, 4-5×10 ⁴ The cells are inoculated on a 24-hole plate, and the cell confluence rate reaches 70-90% after 24 hours of control;

(2) 20pmol of siRNA is added into 50 mu L of DMEM serum-free medium, and the mixture is gently blown and mixed uniformly;

(3) the GP-transmission-Mate transfection reagent is subjected to centrifugation and gentle mixing before use, then 1 mu LGP-transmission-Mate reagent is added into 50 mu L of serum-free DMEM culture medium for dilution, gently blown and mixed evenly, and the mixture is placed for 5min at room temperature;

(4) Mixing the diluted GP-transmit-Mate transfection reagent with the diluted siRNA; standing at room temperature for 15-20 min, forming a complex, and immediately starting transfection;

(5) adding 100 mu L of siRNA/GP-transmission-Mate compound into a culture plate hole containing cells and 400 mu L of culture medium, and gently shaking the cell culture plate back and forth to ensure that liquid uniformly covers the bottom of the culture plate;

(6) placing the cell culture plate at 37deg.C CO ₂ In the incubator, the culture medium is changed into a complete culture medium for 4-6 hours, and after 24-48 hours, other detection steps after transfection are carried out.

The results are shown in FIG. 12. (xp <0.05, < P < 0.01)

As can be seen from FIG. 12, CDPs dry prognosis, m ⁶ Significant differences in modification level of A and methylase indicate m ⁶ Methylation of A involved in CDPs to promote in vitro differentiation of FGSCs in order to explore m ⁶ Whether A modification is necessary in this process, we knockdown the methyltransferase Mettl3 to reduce m ⁶ A modification level, CDPs are used for intervention after knocking down, and related expression is detected.

The results of testing the expression of fgcs marker gene Mvh and Oct4, and differentiation marker gene Stra8 and Sycp3, as shown in fig. 12, showed that the maintenance and differentiation ability of fgcs stem cells knocked down with Mettl3 were significantly reduced (P <0.05, < 0.01), indicating that Mettl3 was deleted, inhibiting CDPs from promoting fgcs differentiation in vitro.

To determine whether Prrc2a can regulate m ⁶ A modification, using negative control small interfering RNA (Si-NC) as negative control group, by m ⁶ Quantitative detection kit A for determining m before and after Prrc2a knockdown in FGSCs ⁶ A is horizontal overall. The results are shown in FIG. 13. (XP)<0.01)

As can be seen from FIG. 13, the m of FGSCs was knocked down after Prrc2a, compared with the negative control group ⁶ Overall a level was significantly reduced (P<0.01 Indicating Prrc2a as m ⁶ A reading protein regulatable m ⁶ And A modification.

10. Lentivirus overexpression

10.1 Pre-experiments for target cell infection

(1) Inoculating 3-5×10 respectively one day before experiment ³ The method comprises the steps that target cells are placed in each hole of a 96-hole culture plate, the volume of a culture medium is 100 mu L, the growth speed of different types of cells is different, and in order to ensure a better experimental result, the fusion rate of the cells during virus infection is 40% -60%, and the cell inoculation is not too dense during infection because the expression time of Lentivirus is longer;

(2) the interference pre-test was divided into two groups, each group having a different gradient of MOI values. The first group is normally infected, i.e. the virus is added directly to the complete medium. The second group is Polybrene with 5 mug/mL added during infection, and Polybrene can effectively improve the infection efficiency in most cells;

(3) Cell replacement is carried out before infection, cell supernatant is sucked, 90 mu L of required culture medium is added according to different grouping conditions, and each group comprises three holes;

(4) prepare 2 sterile EP tubes and aspirate 10. Mu.L of 1X 10 ⁸ TU/mL of virus (previously removed from-80 ℃ C., dissolved in ice bath) was added to the first tube and gently mixed without foaming. Also, 10. Mu.L of virus was pipetted from the first tube into the second tube and mixed well. Three different gradients of virus were thus obtained: 10 times of dilution and 100 times of dilution are carried out on the stock solution;

(5) three different gradients of virus solution were added to each 10 μl of each of the three wells, and calculated to give MOI of 100, 10,1 for each well. If the virus titer used does not reach 1X 10 ⁸ TU/mL, then correspondingly increasing the virus volume so that a different MOI can be obtained;

(6) placing the cells back into an incubator for incubation;

(7) after 8-12 h, please observe the cell state. If the cell state has no obvious difference from the uninfected group, the lentivirus has no obvious toxic effect on the cells, the liquid is not required to be replaced, the culture is continued, and the fresh culture medium is replaced after 24 hours;

(8) after 72-96 hours of infection, the fluorescent expression was observed. For cells with slow growth and metabolism, the observation time can be prolonged appropriately, and the liquid can be changed in the middle to keep the good state of the cells;

(9) The above procedure was designed for adherent cells. The differentiation of suspension cells is mainly on the cell separation disc, which does not need to be separated one day in advance. During operation, the cells are directly centrifuged and then suspended in different culture mediums, and viruses can be added after counting and dividing;

and (3) confirming the infection method and the infection parameters of the target cells through a first experiment and a repeated experiment.

10.2 minimum lethal concentration screening

The working concentration of puromycin hydrochloride for screening stable transformants is required to vary depending on the cell type, medium, growth conditions and cell metabolic rate, and a concentration of 0.1-10. Mu.g/mL is recommended. It is recommended for the first-time experimental system to screen out the appropriate concentration by a minimum lethal concentration experiment.

(1) When the cells were cultured in 35mm dishes to 80-90% confluency, the cells were washed twice with 1mL of PBS, 1mL of 10% FBS-containing medium was added, and the cells were blown to form a single cell suspension.

(2) According to 2X 10 ⁴ Inoculating 12-well plate with cell/well concentration, mixing, and adding 5% CO at 37deg.C ₂ Culturing for 24h, and adding culture medium +10% FBS with final concentration of Puromycin of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5, 2.0, 2.5, 3.0 μg/ml in the preliminary experiment.

(3) Puromycin concentration was maintained, and the solution was changed every other day and observed. For 4 consecutive days, the lowest Puromycin concentration that completely killed the cells was selected.

10.3 formal experiments

Pre-experiments can help determine optimal conditions for infecting cells of interest with recombinant Lentivirus particles, such as cell seeding density, whether Polybrene is required to be added, appropriate MOI values, and the like. Before formal experiments, it is important to adjust and maintain the cells in good condition. Some are Polybrene sensitive, and Polybrene cannot be added at this time to enhance infection efficiency.

Lentivirus expression time is longer, but GFP fluorescence can be observed 24 hours after virus infection on cells with general vigorous metabolism (such as 293T, BHK21, etc.); cells with slower metabolism (such as primary cultured cells, neural stem cells, embryonic stem cells, etc.) have longer GFP protein expression time, and GFP fluorescence can be observed even longer 72-96 hours after infection. The infected cells can be cultured continuously for one week, and the infection of the target cells by the Lentivirus can be determined by observing the expression time and the expression intensity of GFP. In the later period of infection, the cells are timely replaced and passaged according to the growth condition of the cells so as to ensure the good growth state of the cells. Screening of stable strains is performed by adding a minimum lethal concentration of Puromycin for at least 4 days on the basis of transient infection.

The expression of Alkbh5 was detected by qRT-PCR using lentiviral infection to establish a stable transgenic cell line, harvesting the cells, and the results are shown in FIG. 14. P <0.01

As can be seen from fig. 14, the overexpression of Alkbh5 (sh-Alkbh 5) modification significantly increased the expression level (P < 0.01), about 6 times that of the overexpression NC (sh-NC) group, and the subsequent experiments were possible.

The demethylase Alkbh5 was overexpressed using lentiviruses and then induced using CDPs. The results of detecting the expression of fgcs marker gene Mvh and Oct4, and differentiation marker gene Stra8 and Sycp3 are shown in fig. 15 (P <0.05, P < 0.01), and the maintenance and differentiation ability of fgcs stem cells knocked down with Mettl3 is significantly reduced (P <0.05, P < 0.01), consistent with the results of knockdown with Mettl3, and the CDPs are inhibited to promote the in vitro differentiation of fgcs.

It can be concluded that: m is m ⁶ The A modification is involved in the process of CDPs to promote in vitro differentiation of FGSCs and is essential.

ONT full length transcriptomic sequencing

The experimental procedure operates according to the standard protocol provided by Oxford Nanopore Technologies (ONT) company, and mainly comprises the following steps:

(1) extracting RNA, and detecting the purity, concentration and integrity of the RNA sample to ensure that the qualified sample is used for subsequent transcriptome sequencing;

(2) library construction: a. annealing the primer, reversely transcribing RNA into cDNA, and adding a switch oligo; b. synthesizing a complementary strand; DNA damage repair and end repair, purification using magnetic beads;

(3) And (5) connecting a sequencing joint, and performing on-machine sequencing.

To go deep into m ⁶ A participates in the molecular mechanism of CDPs in promoting the in vitro differentiation process of FGSCs. We performed ONT full length transcriptomic sequencing and m on cells before and after CDPs treatment ⁶ A methylation sequencing, carrying out joint analysis on sequencing results, and screening m-bearing ⁶ A methylation regulated differential genes, and screening key genes through experimental verification.

The results are shown in FIGS. 16-19.

Using DESeq2 software, fold Change was > 1.5 and P value <0.05 was used as screening conditions. 187 differential transcripts were screened, 141 of which were up-regulated and 46 of which were down-regulated, and the expression levels of the differential transcripts in the two groups of samples and the statistical significance of the differences were represented by volcanic images (FIG. 16), and transcripts with greater differences were selected for labeling according to Fold Change values. Hierarchical clustering analysis was performed on the screened differentially expressed transcripts, and transcripts with the same or similar expression patterns were clustered and represented by a cluster map (FIG. 17).

Using DESeq2 software, fold Change was ≡1.5 and Pvalue <0.05 as screening conditions. 93 differentially expressed genes were screened, 75 up-regulated genes and 18 down-regulated genes, the expression levels of the differentially expressed genes in the two groups of samples and the statistical significance of the differences were represented by volcanic images (FIG. 18), and the genes with larger differences were selected for labeling according to the Fold Change values. Hierarchical clustering analysis was performed on the screened differentially expressed genes, and genes with the same or similar expression patterns were clustered and represented by a cluster map (FIG. 19).

12.m ⁶ aRNA methylation sequencing

The experimental procedure is operated according to the standard protocol provided by ONT company, and mainly comprises the following steps:

(1) extracting high-quality total RNA, and performing RNA concentration, purity and integrity quality inspection by using Nanodrop and Agilent 2100 software;

(2) enrichment of mRNA from total RNA using oligo (dT) beads;

(3) library construction (SQK-RNA 002 direct RNA sequencing kit): a. reverse transcription linker ligation; b. reverse transcription synthesizes a cDNA strand (the cDNA strand produced in this step is not used for sequencing, but this step can significantly increase sequencing throughput); c. purifying magnetic beads; d, connecting RNA sequencing joints; e. purifying magnetic beads; quantification of qubit library;

(4) Sequencing on a machine.

Differential methylation site (Differential methylation loci, DML) analysis was performed using the DMC module of SMART2 software, controlling methylation site specificity to 0.3 or greater, significance p-value to less than 0.05, screening out 3765 differential methylation sites, 1886 of which were hypermethylation sites, 1879 of which were hypomethylation sites (fig. 20). For the screened differential methylation sites, the top 100 differential sites of hypermethylation and hypomethylation were selected for cluster analysis, respectively, in order of differential significance (fig. 21).

Difference m ⁶ Methylation level of A-site related Gene all m on all transcripts corresponding to the Gene ⁶ The methylation level average value of the A site is calculated, and the methylation state of each gene is obtained by subtracting the methylation level average value of the gene in the treatment group and the control group. DEG, DET, DML the differential expression genes, differential expression transcripts, and differential m ⁶ The A site is related. up and Hyper represent DEG up and difference m, respectively, in the treatment group ⁶ The hypermethylation state of the A locus related gene, down and Hypo respectively represent DEG downregulation and difference m ⁶ Hypomethylation of a site-related gene.

(1) Differential transcript and differential m ⁶ A site-related transcript association analysis

Differential expression transcripts and differential m ⁶ The A site-related transcripts were compared and mapped by means of a venn comparison. The combined analysis resulted in 25 intersection transcripts, 9 of which were hypermethylated and hypoexpressed, 2 of which were hypermethylated and 7 of which were hypomethylated and hypoexpressed, 7 of which were hypomethylated and hypoexpressed (FIGS. 22-23, FIG. 22 did not distinguish between up-down regulation, and FIG. 23 did distinguish between up-down regulation). Differential transcripts and differential m ⁶ The intersection of the related transcripts of site A is subjected to combined display of a cluster heat map, the cluster modes of the intersection transcripts and the change of the expression level of the transcripts corresponding to each cluster mode are represented, the cluster map (figure 24) is drawn, and the corresponding transcript m is displayed ⁶ Double changes in a methylation level and expression level.

(2) Differentially expressed genes and differences m ⁶ A site-related gene association analysis

Differentially expressed genes and differences m ⁶ The A site-related genes were compared and mapped by Venn comparison. Combined analysis gave 19 intersection genes, possibly subject to m ⁶ The fgcs differentiation key genes regulated by a, 7 of which were hypermethylated and highly expressed, 1 of which were hypermethylated and highly expressed, 9 of which were hypomethylated and highly expressed, and 2 of which were hypomethylated and lowexpressed (fig. 25-26, fig. 25 did not distinguish between up-down regulation, and fig. 26 distinguished between up-down regulation). Differential Gene and differential m ⁶ The intersection of the related genes of the site A is subjected to combined display of a cluster heat map, the cluster modes of the intersection genes and the gene expression level change corresponding to each cluster mode are represented, the cluster map (figure 27) is drawn, and the transcript m corresponding to the gene is displayed ⁶ Double changes in a methylation level and expression level.

The trend of the intersection genes was verified using qRT-PCR, the results are shown in fig. 28. (in FIG. 28, for each set of data, ctrl on the left and CDPs on the right, P <0.05, P < 0.01)

As can be seen from fig. 28, the expression of 8 genes was significantly different (P < 0.05) and the trend was consistent with the sequencing results, respectively: arrb2, cebpd, fscn1, galnt11, gga1, lasp1, nr1h2, prrc2a.

PRM targeted proteomic analysis

(1) Sample preparation: a. extracting protein; b. carrying out proteolysis;

(2) LC-PRM/MS detection analysis: a. high performance liquid chromatography; b. high resolution mass spectrometry PRM/MS analysis.

PRM detection is carried out on samples to be detected respectively, and Skyline 3.5.0 software is adopted for raw data analysis.

Statistical analysis was performed using SPSS software, experimental data were expressed as Mean ± standard deviation (Mean ± SEM), comparisons between two groups were performed using t-test, and comparisons between groups were performed using one-way analysis of variance; the Graph was drawn by Graph Prism software. All data were repeated at least three times; p <0.05 indicates significant inter-group differences, statistically significant, and P <0.01 indicates significant inter-group differences; in the experimental results, P <0.05 and P <0.01 are indicated.

Through sequencing result analysis and experimental verification, we focused on part of the key genes, and in order to determine whether the gene and protein level expression trend are consistent, PRM (Parallel Reaction Monitoring) targeted proteome was used for verification.

And (3) analyzing the expression quantity of the peptide fragments corresponding to each target protein in different samples, so as to obtain the relative expression quantity of the target protein in the samples, and carrying out statistical analysis. The results show (table 4) that the expression of Cyp51 is significantly increased (P < 0.05) and Fscn1 expression is significantly decreased (P < 0.05) compared to the control group; histogram analysis was performed on the average of the protein expression levels of the two groups of samples (fig. 29, data of each group in fig. 29, left is Ctrl, right is CDPs), and the results show that the expression of arb 2, lasp1 and Cdk4 has an upward trend, and the expression of Prrc2a has a downward trend, but no significant difference.

TABLE 4 results of relative quantitative analysis of target protein PRM

By the above combined transcriptome sequencing and methylation sequencing analysis, expression at gene and protein levels was verified using qRT-PCR and PRM, respectively, and m was found ⁶ Expression of the a reading protein Prrc2a is significantly elevated (P<0.05 And methylation occurs on transcripts without significant differences in protein expression. Three transcripts were detected in Prrc2a by sequencing (see Table 5), with one transcript having significantly elevated expression and methylation levels.

TABLE 5Prrc2a three transcript expression profiles

Primers of three transcripts of Prrc2a were designed, and qRT-PCR experiments demonstrated that experimental group ensmsust 00000025253 expression was significantly elevated (P < 0.05) compared to control group, consistent with sequencing results (fig. 30) (×p < 0.05)

Expression of Prrc2a and fgcs marker genes Oct4 and Mvh, in vitro differentiation genes Stra8 and Sycp3 was detected by qRT-PCR. The results are shown in FIGS. 31-32. (in FIG. 31, A is, and B is)

31-32, the addition of CDPs after Prrc2a knockdown showed that Prrc2a expressed significantly higher than that of the knockdown group (P < 0.01), suggesting that CDPs could regulate Prrc2a expression (FIG. 31).

After Prrc2a is knocked down, the expressions of Oct4, mvh, stra8 and Sycp3 are obviously reduced, which indicates that the knockdown Prrc2a reduces the in vitro differentiation capacity of FGSCs; CDPs were added after Prrc2a knockdown, oct4, mvh, stra8 and Sycp3 expression were significantly elevated (P < 0.05), but still lower than the normal addition CDPs group. It was suggested that CDPs could compensate for the decrease in differentiation ability caused by knock-down Prrc2a, and Prrc2a was involved in regulating CDPs to promote in vitro differentiation of FGSCs (FIG. 32).

Western blot results (fig. 33, P <0.05, P < 0.01) consistent with qRT-PCR trends, demonstrating that knock-down Prrc2a reduces fgs cs differentiation capacity in vitro; CDPs can promote fgcs differentiation in vitro by modulating Prrc2 a.

Research on cistanche deserticola polysaccharide treatment to promote in vitro differentiation mechanism of female germ stem cells has found thatM is m ⁶ The role of a methylation in this process. M of FGSCs treated by CDPs ⁶ Elevated a methylation levels, significantly elevated expression of the reading protein Prrc2a in the experimental group (P<0.05 And through preliminary verification, the expression of the knockdown Prrc2a is found, so that the promotion effect of CDPs on the in vitro differentiation of FGSCs can be obviously inhibited. The research results help us to further understand the epigenetic characteristics of the FGSCs, help us to more deeply and comprehensively disclose the in vitro differentiation mechanism of the FGSCs, and promote the application of the Chinese herbal medicine compound in the aspects of female fertility maintenance, female infertility treatment and the like.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. Use of the reading protein Prrc2a in the differentiation process of female germ stem cells.

2. The use of the reading protein Prrc2a according to claim 1 in the differentiation of female germ stem cells, wherein the differentiation is to add cistanche deserticola polysaccharide to the in vitro culture system to promote the differentiation of female germ stem cells.

3. The use of the reading protein Prrc2a according to claim 2 in the differentiation of female germ stem cells, wherein the amount of cistanche deserticola polysaccharide added is 0.5 μg/mL.

4. An in vitro culture system for promoting differentiation of female germ stem cells, comprising the reading protein Prrc2a of any of claims 1-3.

5. A formulation for promoting differentiation of female germ stem cells comprising the reading protein Prrc2a of any of claims 1-3.