Application of PLPP3 gene in regulating estradiol production of ovarian granulosa cells

Abstract

The application discloses application of a PLPP3 gene in regulating E2 generation of ovarian granulosa cells and belongs to the technical field of cell engineering and gene engineering. It is found for the first time that overexpression of PLPP3 significantly reduces the E2 level in supernatant of human ovarian granulosa cells, and inhibition of PLPP3 expression significantly increases the E2 level in supernatant of granulosa cells; overexpression of PLPP3 significantly reduces the E2 level in serum of mice, and inhibition of PLPP3 expression significantly increases the E2 level in serum of mice. The application has good application value for studying the influence mechanism of PLPP3 in ovarian follicle development, reproductive capacity and ovarian related diseases. Technical support is provided for improving the reproductive capacity of sows, and the application has important economic value for actual production of the pig industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of a PLPP3 gene in regulating E2 production of ovarian granulosa cells. BACKGROUND

[0002] In mammals, a single follicle is composed of an oocyte and surrounding granulosa cells (GCs) and theca cells. During follicular development, about 99% of follicles will atresia, and only about 1% of follicles can ovulate normally. Estradiol (E2) is produced by granulosa cells (GCs) in the follicle, which controls the development and selection of the pre-ovulatory dominant follicle. E2 is the main hormone affecting the development of mammalian follicles and reproductive capacity, and E2 signal disorder will bring a variety of reproductive diseases, such as polycystic ovary syndrome caused by follicular development arrest.

[0003] Phospholipid phosphatase 3 (PLPP3) belongs to the phospholipid phosphatase family and is a transmembrane enzyme. As a kind of glycoprotein, PLPP3 is located on the cytoplasmic membrane and can hydrolyze lysophosphatidic acid and short-chain phosphatidic acid, converting phosphatidic acid into diacylglycerol, which plays a role in the de novo synthesis of glycerolipids and receptor-activated signaling pathways. In addition, it has been reported that PLPP3 can also hydrolyze ceramide phosphate and phosphatidyl alcohol. Through the production of these products, PLPP3 gene is involved in cell adhesion, intercellular interaction, angiogenesis, and related diseases such as inflammation and cell invasion. Although PLPP3 gene has the above important biological functions, its role in ovarian granulosa cells and follicular development is still unclear. SUMMARY

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide application of a PLPP3 gene in regulating E2 production of ovarian granulosa cells.

[0005] The present application constructs an overexpression plasmid pcDNA3.1-PLPP3 (OE-PLPP3) of the PLPP3 gene, and synthesizes an interference sequence si-PLPP3 (synthesized by Dongze Company) for transfecting human ovarian granulosa cells. Forty 3-week-old C57BL / 6J female mice are fed, and overexpression lentivirus (LV-Plpp3) and interference lentivirus (sh-Plpp3) of PLPP3 (synthesized by Dongze Company) are synthesized, and the lentivirus is injected into the abdominal cavity of the mice every week. The effect of the PLPP3 gene on E2 production of human ovarian granulosa cells and mouse serum is explored by ELISA method; the effect of the PLPP3 gene on mRNA and protein levels of marker genes of the estrogen secretion signaling pathway of humans and mice is detected by qRT-PCR and Western blot (WB) experiments; and finally the effect of PLPP3 on follicular development of mice is verified by HE staining.

[0006] The application is achieved by the following technical scheme: application of the PLPP3 gene in regulating E2 production of ovarian granulosa cells, wherein the application is any one of the following applications:

[0007] I. Application of overexpression of exogenous PLPP3 gene in inhibiting E2 production of ovarian granulosa cells in an in vitro environment.

[0008] II. Application of inhibition of PLPP3 gene expression in promoting E2 production of ovarian granulosa cells in an in vitro environment.

[0009] Further, the ovarian granulosa cells are human ovarian granulosa cells or mouse ovarian granulosa cells.

[0010] When the ovarian granulosa cells are human ovarian granulosa cells, overexpression of exogenous PLPP3 gene significantly reduces the mRNA levels of estrogen signaling pathway marker genes FSHR, ESR2, CYP19A1, HSD17B1, CYP1A1 and ELK1 in the ovarian granulosa cells, and the protein levels of FSHR, CYP19A1 and HSD17B1; inhibition of PLPP3 gene expression significantly increases the mRNA levels of estrogen signaling pathway marker genes FSHR, CYP19A1, HSD17B1 and CYP1A1, and the protein levels of FSHR, CYP19A1 and HSD17B1.

[0011] When the ovarian granulosa cells are mouse ovarian granulosa cells, overexpression of exogenous PLPP3 gene significantly reduces the protein levels of estrogen signaling pathway marker genes FSHR, CYP19A1 and HSD17B1 in the ovarian granulosa cells, and the proportions of antral follicles and corpus luteum in the mouse ovary are significantly reduced, and the proportion of primordial follicles is significantly increased; inhibition of PLPP3 gene expression significantly increases the protein levels of estrogen signaling pathway marker genes FSHR, CYP19A1 and HSD17B1, and the proportions of antral follicles and corpus luteum in the mouse ovary are significantly increased, and the proportion of primordial follicles is significantly reduced.

[0012] Further, the overexpression of exogenous PLPP3 gene is achieved by gene overexpression technology.

[0013] Still further, when the ovarian granulosa cells are human ovarian granulosa cells, the gene overexpression plasmid used in the gene overexpression technology is prepared by the following method:

[0014] (1) cDNA of human ovarian granulosa cells is extracted, and a target fragment is obtained by PCR amplification with the cDNA as a template;

[0015] (2) the target fragment is connected to a pcDNA3.1 vector cut by restriction enzymes XbaI and kpnI to obtain a recombinant plasmid.

[0016] Further, the primers used for PCR amplification in step (1) are as follows:

[0017] Forward: 5'-GG GGTACC CCATGCTGATGGTCCTCCTTGTATC-3';

[0018] Reverse: 5'-GC TCTAGAG CCTACACCATGTTGTGGTGATTGTT-3'.

[0019] Further, the inhibition of PLPP3 gene expression is achieved by RNA interference technology.

[0020] Further, when the ovarian granulosa cells are human ovarian granulosa cells, the RNA interference technology uses a small interfering fragment sequence as follows:

[0021] si-PLPP3: 5'-CTGATGGTCCTCCTTGTAT-3'.

[0022] When the ovarian granulosa cells are mouse ovarian granulosa cells, the reagent for inhibiting PLPP3 gene expression is a small interfering fragment with the sequence as follows:

[0023] sh-Plpp3: 5'-CTGATGGTCCTCCTTGTAT-3'.

[0024] The reagent for inhibiting PLPP3 gene expression in the preparation of a drug for promoting the development of mouse ovarian granulosa cell follicles.

[0025] Further, the reagent for inhibiting PLPP3 gene expression is a small interfering fragment with the sequence as follows:

[0026] sh-Plpp3: 5'-CTGATGGTCCTCCTTGTAT-3'.

[0027] The present application uses cell biology methods to study the influence of PLPP3 on the production of E2 in human ovarian granulosa cells and mouse serum by taking the mutual action between proteins as the breakthrough point.

[0028] The verification results of the present application are as follows:

[0029] Under an in vitro environment, overexpression of PLPP3 (OE-PLPP3) can significantly reduce the level of E2 production in human ovarian granulosa cells, and interference with the expression of PLPP3 (si-PLPP3) gene can significantly improve the level of E2 production in human ovarian granulosa cells.

[0030] In vitro, overexpression of PLPP3 (OE-PLPP3) can significantly reduce the mRNA levels of estrogen signaling pathway marker genes FSHR, ESR2, CYP19A1, HSD17B1, CYP1A1 and ELK1 in human ovarian granulosa cells, and significantly reduce the protein levels of FSHR, CYP19A1 and HSD17B1; and interfering with the expression of PLPP3 (si-PLPP3) gene can significantly increase the mRNA levels of estrogen signaling pathway marker genes FSHR, CYP19A1, HSD17B1 and CYP1A1, and significantly increase the protein levels of FSHR, CYP19A1 and HSD17B1.

[0031] In vitro, overexpression of PLPP3 (LV-Plpp3) can significantly inhibit the production of E2 in mouse serum; and interfering with the expression of PLPP3 (sh-Plpp3) gene can significantly promote the production of E2 in mouse serum.

[0032] In vitro, overexpression of PLPP3 (LV-Plpp3) can significantly reduce the protein levels of estrogen signaling pathway marker genes FSHR, CYP19A1 and HSD17B1 in mouse granulosa cells; and interfering with the expression of PLPP3 (sh-Plpp3) gene can significantly increase the protein levels of FSHR, CYP19A1 and HSD17B1.

[0033] The present application has the following advantages and effects relative to the prior art:

[0034] The technical scheme of the present application is designed in detail and the results are reliable. To confirm the influence of PLPP3 on E2 production in humans and mice, the present application verifies from multiple levels and multiple angles. First, the influence of PLPP3 on E2 levels in human ovarian granulosa cells and mouse serum is verified, then the regulation of PLPP3 on the mRNA and protein levels of estrogen signaling pathway marker genes in human and mouse granulosa cells is verified, and finally the influence of PLPP3 on mouse follicle development is verified by HE staining.

[0035] The present application first finds that overexpression of PLPP3 significantly reduces the E2 level in the supernatant of human ovarian granulosa cells, and inhibiting the expression of PLPP3 significantly increases the E2 level in the supernatant of granulosa cells; overexpression of PLPP3 significantly reduces the E2 level in mouse serum, and inhibiting the expression of PLPP3 significantly increases the E2 level in mouse serum.

[0036] The present application has good application value for studying the influence mechanism of PLPP3 on ovarian follicle development, reproductive capacity and ovarian-related diseases, and has important clinical value in ovarian function protection and treatment of ovarian-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Figure 8 is a diagram of the research results of the effect of PLPP3 on estrogen production in human ovarian granulosa cells; wherein a is the effect of OE-PLPP3 and si-PLPP3 on the E2 level in the supernatant of granulosa cells; b is the effect of OE-PLPP3 and si-PLPP3 on the mRNA level of the marker genes FSHR, ESR2, CYP19A1, HSD17B1, CYP1A1 and ELK1 of the estrogen signaling pathway; c is the effect of OE-PLPP3 and si-PLPP3 on the protein level of the key genes FSHR, CYP19A1 and HSD17B1 of the estrogen signaling pathway.

[0038] Figure 2 Figure 9 is a diagram of the research results of the effect of PLPP3 on estrogen production in mouse serum; wherein a is the effect of LV-Plpp3 and sh-Plpp3 on the E2 level in the mouse serum; b is the effect of LV-Plpp3 and sh-Plpp3 on the protein level of the marker genes FSHR, CYP19A1 and HSD17B1 of the estrogen signaling pathway in mouse granulosa cells.

[0039] Figure 3 Figure 10 is a diagram of the research results of the effect of PLPP3 on the development of mouse follicles; wherein a and b are respectively HE staining diagrams of mouse ovaries and statistics of the proportion of follicles at different development stages, wherein "CL" represents corpus luteum, and the arrow represents primordial follicle. DETAILED DESCRIPTION

[0040] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0041] The experimental methods in the following examples not specifically indicated are generally performed according to conventional conditions.

[0042] In the following examples, statistical methods are used to analyze the results of 3 independent experiments in each example, and the "average value ± standard deviation" is calculated, and single factor variance analysis is used for significant difference analysis (in the figure, "*" represents P<0.05, and "**" represents P<0.01).

[0043] Example 1: Culture of human ovarian cancer granulosa cell COV434 cell line

[0044] The application uses COV434 cell line (ATCC) and adopts complete culture medium (containing 10% calf serum and 1% double antibody) and is cultured in a 37°C, 5% CO2 incubator. When the cell confluence reaches 50%-70%, transfection or drug treatment is performed, and the subsequent experiment can be performed after 24 h.

[0045] Example 2: Construction of overexpression plasmid and interference fragment of PLPP3 gene

[0046] (1) Primers were designed using the NCBI website to amplify the CDS region of the PLPP3 gene (Gene ID: 8613) using the extracted cDNA from human ovarian granulosa cells as a template. The amplified fragment was purified, recovered, ligated into the pMD18T vector (Takara Corporation, Japan), transformed, screened, and sequenced to confirm its correctness before extracting ordinary plasmids.

[0047] (2) Xba I and Kpn I restriction enzyme sites were added to the upstream and downstream primers of the PLPP3 gene, respectively. PCR amplification was performed using the recombinant pMD18T plasmid of the CDS region of the PLPP3 gene as a template. After purification, double digestion, ligation into the pcDNA3.1 vector, transformation, screening, and sequencing to confirm correctness, an endotoxin-free plasmid was extracted using an endotoxin-free plasmid rapid extraction kit (Magen, USA) and named OE-PLPP3.

[0048] Primers for the CDS region of the PLPP3 gene used in this invention:

[0049] Forward: 5'-GG GGTACC CCATGCTGATGGTCCTCCTTGTATC-3′;

[0050] Reverse: 5'-GC TCTAGAG CCTACACCATGTTGTGGTGATTGTT-3′.

[0051] (3) Design and commission Dongze Company to synthesize small interfering fragment sequences of the PLPP3 gene:

[0052] si-PLPP3: 5′-CTGATGGGTCCTCCTTGTAT-3′.

[0053] (4) Observe the cell state. When the cell fusion reaches about 50%-70%, transfect the cells with 1000ng / mL PLPP3 overexpression vector (OE-PLPP3) and overexpression control (OE-NC), and 100nmol / L PLPP3 interference fragment (si-PLPP3) and interference control (si-NC). After 24 hours, collect the cell supernatant and use an ELISA kit to detect the E2 concentration. At the same time, collect the cells to extract RNA and protein, and use qRT-PCR and WB to detect the mRNA and protein levels of estrogen signaling pathway marker genes.

[0054] Example 3: Synthesis of mouse overexpression plasmid and interfering fragment lentivirus of PLPP3 gene

[0055] (1) Primers were designed using the NCBI website to amplify the CDS region of the Plpp3 gene (Gene ID: 67916) using the extracted cDNA from mouse granulocytes as a template. The amplified fragment was purified, recovered, ligated into the pcDNA3.1 vector (Dongze Biotechnology Co., Ltd., China), transformed, screened, and sequenced to confirm its correctness before extracting ordinary plasmids.

[0056] (2) Xba I and Kpn I restriction site sequences were added to the upstream and downstream primers of the Plpp3 gene, respectively. PCR amplification was performed using the recombinant pMD18T plasmid of the CDS region of the PLPP3 gene as a template. After purification, double digestion, ligation into the pcDNA3.1 vector, transformation, screening, and sequencing to confirm correctness, an endotoxin-free plasmid was extracted using an endotoxin-free plasmid rapid extraction kit (Magen, USA) and named OE-Plpp3.

[0057] (3) Design and commission Dongze Company to synthesize small interfering fragment sequences of the Plpp3 gene:

[0058] sh-Plpp3: 5′-CTGATGGGTCCTCCTTGTAT-3′

[0059] (4) The OE-Plpp3 overexpression plasmid and the interfering fragment sh-Plpp3 synthesized in (2) and (3) were packaged into lentivirus with a viral titer of 1*10^8 TU / ml. The lentivirus was injected intraperitoneally into C57BL / 6J (purchased from Guangdong Provincial Experimental Animal Center) female mice weekly. When the mice were 7 weeks old, their serum was collected and the E2 level in the mouse serum was detected by ELISA. Mouse ovaries were collected and stained with hematoxylin-eosin (HE) to detect the development of mouse follicles. Mouse ovaries were collected and proteins were extracted and detected by Western blotting to detect the level of estrogen signaling pathway marker gene proteins.

[0060] Example 4: qRT-PCR

[0061] In this invention, the qRT-PCR detection of genes was performed using YEASEN's technology. SYBR Green qPCRMaster Mix (2X) kit.

[0062] The experiment used the comparative Ct value method to detect the gene content in the samples. The specific calculation formula is as follows:

[0063] Relative gene expression level = 2 - {<(Ct value of target gene in experimental group) - (Ct value of internal reference gene in experimental group)> - <(Ct value of target gene in control group) - (Ct value of internal reference gene in control group)>}

[0064] GAPDH was used as an internal control for gene detection. The qRT-PCR primers used in this invention are:

[0065] qFSHR Forward: 5′-GGTTTGTCCTCACCAAGCTTCG-3′;

[0066] Reverse: 5′-GGTTGGAGAACACATCTGCCTC-3′;

[0067] qESR2 Forward: 5′-ATGGAGTCTGGTCGTGTGAAGG-3′;

[0068] Reverse: 5′-TAACACTTCCGAAGTCGGCAGG-3′;

[0069] qCYP19A1 Forward: 5′-GACGCAGGATTTCCACAGAAGAG-3′;

[0070] Reverse: 5′-ATGGTGTCAGGAGCTGCGATCA-3′;

[0071] qELK1 Forward: 5′-GCTGCCTCCTAGCATTCACTTC-3′;

[0072] Reverse: 5′-CCACGCTGATAGAAGGGATGTG-3′;

[0073] qCYP17A1 Forward: 5′-GCACACCAACTATCAGTGACCG-3′;

[0074] Reverse: 5′-CCTTGTCCACAGCAAACTCACC-3′;

[0075] qHSD17B1 Forward: 5′-TTCCTGCCAGACATGAAGAGGC-3′;

[0076] Reverse: 5′-AGAACCGCCAGACTCTCGCATA-3′;

[0077] qCYP1A1 Forward: 5′-GATTGAGCACTGTCAGGAGAAGC-3′;

[0078] Reverse: 5′-ATGAGGCTCCAGGAGATAGCAG-3′;

[0079] qGAPDH Forward: 5′-TCACCAGGGCTGCTTTTAACT-3′;

[0080] Reverse: 5′-CTTGACTGTGCCGTGGAACT-3′;

[0081] Total RNA extraction from cells was performed according to the instruction manual for the RNA fast 200 Total RNA Rapid Extraction Kit from Shanghai Feijie Technology Co., Ltd., and the specific steps are as follows:

[0082] (1) Add 500 μl of RA2 solution to the granule cells digested with trypsin and mix thoroughly by inverting for 1 min.

[0083] (2) Aspirate or pour the sample lysate into the inner tube and centrifuge for 1 min.

[0084] (3) Discard the liquid in the outer tube, add 500 μl of washing solution to the inner tube, and centrifuge for 1 min. Repeat this process once more.

[0085] (4) Remove the inner tube, discard the liquid in the outer tube, put the inner tube back on, and centrifuge for 1 minute without adding washing liquid.

[0086] (5) Transfer the inner tube into a new eppendorf tube, add 25-50 μl of elution buffer (or DEPC-treated water with pH > 7.0) to the center of the membrane, let stand at room temperature for 1 min, centrifuge for 1 min, and obtain total RNA.

[0087] Using PrimeScript from TaKaRa TM RT Master Mix (Perfect Real Time) cDNA Reverse Transcription Kit for Reverse Transcription of Total RNA.

[0088] Example 5: Western Blot

[0089] (1) Extraction of total cell protein

[0090] ① Prepare protein lysis buffer: Add 1% proteasome inhibitor to RIPA protein lysis buffer (White Shark Company) and mix by inverting the container.

[0091] ② Add 120 μl of protein lysis buffer to each well (six-well plate), place on ice for 10-15 min for lysis, and gently shake occasionally to promote cell detachment.

[0092] ③ Collect the lysed blood cells, centrifuge at 12,000g at 4℃ for 10min, carefully aspirate the supernatant and remove the lower precipitate.

[0093] (2) SDS-PAGE:

[0094] ① Protein quantification (Beyotime BCA protein quantification kit): A protein was measured using a multi-functional microplate reader. 562 The OD value of the wavelength was used to plot a standard curve. The concentration of each protein sample was calculated using the regression equation and the average OD value of each protein standard.

[0095] ② Sample preparation: Mix 20 μg of total protein with 5× loading buffer at a ratio of 5:1 and boil for 10 min;

[0096] ③ Gel preparation and sample application: Add 20ug of protein to each well, run the gel at 120-150V for about 40 minutes (stop electrophoresis when the bromofenblue runs to about 1-2cm from the bottom of the gel; or adjust the running voltage and time according to the specific situation).

[0097] ④ Cut the gel strip containing the target protein according to the molecular weight standard of the protein marker, select a transfer current of 300mA, and transfer for 20min;

[0098] ⑤ After the transfer is complete, gently rinse the membrane with TBST for 5 minutes, and seal it with 5% skim milk powder at room temperature for 2 hours;

[0099] ⑥ Gently rinse the membrane twice with TBST for 5 minutes each time. Dilute the primary antibody with TBST according to the antibody instructions and incubate overnight at 4°C.

[0100] ⑦ Gently rinse the membrane 3 times with TBST for 10 min each time, dilute the secondary antibody Goat anti-rabbit IgG (purchased from SAB) 1:10000, and incubate at room temperature for 2 h;

[0101] ⑧ Gently rinse the membrane three times with TBST, 10 minutes each time;

[0102] ⑨ The protein bands were developed using the ultrasensitive ECL chemiluminescence reagent kit (Beyotime International Co., Ltd., China), observed and photographed using the Tianneng gelation system (Tianneng Co., Ltd., China), and analyzed using ImageJ software.

[0103] Example 6: ELISA detection of E2 levels in cell supernatant

[0104] This invention uses ELISA technology to detect the E2 content in cell supernatant. Referring to the operating instructions of the porcine estradiol (E2) enzyme-linked immunosorbent assay kit from Shanghai Jianglai Biotechnology Co., Ltd., the specific operating steps are as follows:

[0105] (1) Set up standard wells and sample wells. Add 50 μL of standard at different concentrations to each of the 96-well plates.

[0106] (2) Seed cells into 6-well plates and culture the cells until the confluence is 50% to 80%. Centrifuge the cell supernatant at 1000g for 20 min and take 50 μL into a 96-well plate to obtain the sample wells. Do not add any to the blank wells.

[0107] (3) Seal the reaction wells with sealing film, incubate at 37°C for 30 min, discard the liquid, and pat dry on absorbent paper;

[0108] (4) Add 350 μL of washing solution to each well, let stand for 1 min, discard the liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0109] (5) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, incubate at 37°C for 30 min, discard the liquid, and pat dry on absorbent paper.

[0110] (6) Add 350 μL of washing solution to each well, let stand for 1 min, discard the liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0111] (7) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0112] (8) Add 50 μL of stop solution to each well and use an ELISA reader within 15 min. 450 Measure the OD value.

[0113] Example 7: ELISA detection of E2 levels in mouse serum

[0114] This invention uses ELISA technology to detect the E2 content in cell supernatant. Referring to the instruction manual of the porcine estradiol (E2) enzyme-linked immunosorbent assay kit (Shanghai Jianglai Biotechnology Co., Ltd., China), the specific operating steps are as follows:

[0115] (1) Serum: Collect whole blood samples in serum separation tubes and incubate overnight at 4°C. Then centrifuge at 1000×g for 20 min and collect the supernatant.

[0116] (2) Set up standard wells and sample wells. Add 50 μL of standard at different concentrations to each of the 96-well plates.

[0117] (3) Seal the reaction wells with sealing film, incubate at 37°C for 30 min, discard the liquid, and pat dry on absorbent paper;

[0118] (4) Add 350 μL of washing solution to each well, let stand for 1 min, discard the liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0119] (5) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, incubate at 37°C for 30 min, discard the liquid, and pat dry on absorbent paper.

[0120] (6) Add 350 μL of washing solution to each well, let stand for 1 min, discard the liquid, pat dry on absorbent paper, and repeat the washing process 5 times.

[0121] (7) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0122] (8) Add 50 μL of stop solution to each well and use an ELISA reader within 15 min. 450 Measure OD value

[0123] Example 8: Ematoxylin-eosin staining (HE) of mouse ovaries

[0124] (1) Paraffin sections were prepared from mouse ovaries soaked in 4% paraformaldehyde solution;

[0125] (2) Dewaxing paraffin sections to water: The sections were placed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water.

[0126] (3) Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0127] (4) Eosin staining of cytoplasm: Immerse the section in eosin staining solution for 1-3 min.

[0128] (5) Dehydration and mounting: Place the sections in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and make them transparent. Remove the sections from the xylene and let them dry slightly. Then mount them with neutral resin.

[0129] (6) Microscopic examination, image acquisition and analysis, and statistical analysis of the proportion of follicles at different developmental stages.

[0130] result:

[0131] 1. The PLPP3 gene inhibits the production of E2 in human ovarian granulosa cells.

[0132] The effect of PLPP3 on E2 levels in the supernatant of human ovarian granulosa cells was detected using ELISA. The effect of PLPP3 on the mRNA and protein levels of estrogen signaling pathway marker genes in granulosa cells was detected using qRT-PCR and Western Blot experiments.

[0133] ELISA experiments showed that OE-PLPP3 significantly reduced E2 levels in the supernatant of human ovarian granulosa cells. Figure 1 (a) of the above, while si-PLPP3 significantly increased the E2 level in granulocyte supernatant (a). Figure 1 (a) in the middle.

[0134] qRT-PCR results showed that OE-PLPP3 significantly reduced the mRNA levels of estrogen signaling pathway marker genes FSHR, ESR2, CYP19A1, HSD17B1, CYP1A1, and ELK1 in granulosa cells; while si-PLPP3 significantly increased the mRNA levels of these same genes. Figure 1 (b)

[0135] Western blot results showed that OE-PLPP3 significantly reduced the protein levels of the estrogen signaling pathways FSHR, CYP19A1, and HSD17B1, while si-PLPP3 significantly increased the protein levels of FSHR, CYP19A1, and HSD17B1. Figure 1 (c)

[0136] 2. The PLPP3 gene inhibited the production of E2 in mouse serum.

[0137] The effect of PLPP3 on E2 levels in mouse serum was detected using ELISA. The effect of PLPP3 on the mRNA and protein levels of estrogen signaling pathway marker genes in mouse granulosa cells was detected using qRT-PCR and Western Blot experiments.

[0138] ELISA results showed that LV-Plpp3 significantly reduced E2 levels in mouse serum, while sh-Plpp3 significantly increased E2 levels in mouse serum. Figure 2 a)

[0139] Western blot results showed that LV-Plpp3 significantly reduced the protein levels of the estrogen signaling pathways FSHR, CYP19A1, and HSD17B1, while sh-Plpp3 significantly increased the protein levels of FSHR, CYP19A1, and HSD17B1. Figure 2 (b)

[0140] 3. The PLPP3 gene inhibits mouse follicle development.

[0141] HE staining was used to detect the effect of the PLPP3 gene on mouse follicle development.

[0142] HE staining results showed that LV-Plpp3 significantly reduced the proportion of cavitary follicles and corpora lutea in mice, and increased the proportion of primordial follicles. Figure 3 (a, b) ; sh-Plpp3 significantly increased the proportion of cavitary follicles and corpora lutea in mice, and decreased the proportion of primordial follicles (a, b). Figure 3 (a, b) In the diagram, “PF” represents primordial follicle, “AF” represents antral follicle, and “CL” represents corpus luteum.

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Inhibition PLPP3 The application of gene expression reagents in the preparation of drugs that promote mouse follicle development, characterized by: The suppression PLPP3 The reagent for gene expression is a small interfering fragment, the sequence of which is as follows: sh-Plpp3:5′-CTGATGGTCCTCCTTGTAT-3′。

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