CN113388614B - Application of RSPO2 gene in porcine ovarian granulosa cells - Google Patents
Application of RSPO2 gene in porcine ovarian granulosa cells Download PDFInfo
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Abstract
The invention discloses an application of RSPO2 gene in swine ovary granular cells, belonging to the technical field of cell engineering and genetic engineering. The invention takes RSPO2 as a research object, and transfects the RSPO2 overexpression vector or the RSPO2-siRNA to the ovarian granulosa cells by constructing the RSPO2 overexpression vector and synthesizing small interfering RNA (RSPO 2-siRNA), then detects the change of the protein level and the mRNA of the signal channel gene related to the apoptosis, proliferation and E2 secretion of the ovarian granulosa cells, and finally detects the phenotypic change of the ovarian granulosa cells. The results show that RSPO2 can inhibit apoptosis of ovarian granulosa cells and promote proliferation and E2 secretion. By researching the application of RSPO2 in the ovarian granulosa cells, the invention has good application value for researching ovarian follicle locking mechanism, incipient situation starting and the like.
Description
Technical Field
The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of an RSPO2 gene in porcine ovarian granulosa cells.
Background
Follicular development is a multicellular process involving oocyte maturation, granulosa cell proliferation, apoptosis and hormone secretion. An increase in granulosa cell death is likely to be a cellular mechanism that directly or indirectly hinders follicular development. For example, disruption of estrogen receptor β in mice results in decreased estrogen secretion by granulosa cells, impeding follicular maturation and ovulation dysfunction. In granulosa cells of mice with premature ovarian failure, the expression of anti-mullerian hormone (anti-mullerian hormone) can be up-regulated to inhibit the apoptosis of the granulosa cells and further promote the development of follicles.
RSPO2 (R-Spondin 2) is a key protein that activates the WNT signaling pathway, which plays a key role in granulosa cell apoptosis, follicular maturation, and primordial events initiation. Meanwhile, in mammals, the RSPO family plays an important role in the proliferation and apoptosis of ovarian cells and the growth and development of follicles. For example, interference with the transcriptional expression of RSPO1 can inhibit the proliferation of human ovarian cancer cells and promote apoptosis of the cells. RSPO2 promotes the development of primary follicles in the mouse ovary into secondary follicles. Compared with wild mice, the knockout of the RSPO2 gene can inhibit the proliferation and differentiation of granulosa cells, thereby hindering the growth of follicles. However, the connection between the RSPO2 gene and the growth and development of the pig ovarian granulosa cells is not reported at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the application of the RSPO2 gene in the porcine ovarian granulosa cells.
It is still another object of the present invention to provide a small interfering RNA fragment (siRNA) that inhibits the expression of RSPO2 gene.
The purpose of the invention is realized by the following technical scheme:
application of RSPO2 gene in swine ovary granular cells.
Under the in vitro environment, the RSPO2 gene can promote the proliferation of the porcine ovarian granulosa cells and can inhibit the apoptosis of the porcine ovarian granulosa cells.
Further, the application of the RSPO2 gene in preparing the medicine for regulating and controlling the proliferation and the apoptosis of the porcine ovarian granulosa cells.
The regulation and control of the proliferation and the apoptosis of the swine ovarian granulosa cells are realized by the following modes: increasing RSPO2 gene to promote proliferation of pig ovary granular cells and inhibit apoptosis of cells; or reducing the RSPO2 gene to inhibit the proliferation of the porcine ovarian granulosa cells and promote the apoptosis of the cells;
the increase of RSPO2 gene promotes the proliferation of pig ovarian granulosa cells and inhibits the apoptosis of cells by a mode of increasing exogenous RSPO2 gene;
the reduction of the RSPO2 gene to inhibit the proliferation of the pig ovarian granulosa cells and the promotion of the apoptosis of the cells are realized in a mode that siRNA for inhibiting the expression of the RSPO2 gene is transfected to the pig ovarian granulosa cells.
The exogenous RSPO2 gene is added by the following method: connecting the RSPO2 gene to a pcDNA3.1 vector to construct a overexpression vector containing the RSPO2 gene; the overexpression vector containing the RSPO2 gene was then transfected into porcine ovarian granulosa cells.
The invention provides siRNA for inhibiting RSPO2 gene expression, which has the following sequence:
siRNA-RSPO2-1：5′-CCGATGTCAACAGAAGTT-3′；
siRNA-RSPO2-2：5′-GGAACTTGTAGCAGAAATA-3′；
siRNA-RSPO2-3：5′-GGCTGTTTGTCTTGTTCAA-3′。
application of RSPO2 gene in promoting E2 (estradiol) generation in pig ovarian granulosa cells.
After the RSPO2 gene is over-expressed, the generation of E2 can be promoted, and after the RSPO2 gene is interfered, the generation of E2 can be inhibited.
The verification result of the invention is as follows:
1. 3 pairs of small interfering RSPO2 fragments/controls (RSPO 2-siRNA/siRNA-NC) were synthesized, screened and tested for their interference efficiency. As can be seen from the results in FIG. 2, the small gene interference fragment is transfected into the ovarian granule cells, and the small RSPO2-siRNA-3 fragment with better interference effect is finally screened by qRT-PCR means for subsequent experiments.
siRNA-RSPO2-1：5′-CCGATGTCAACAGAAGTT-3′；
siRNA-RSPO2-2：5′-GGAACTTGTAGCAGAAATA-3′；
siRNA-RSPO2-3：5′-GGCTGTTTGTCTTGTTCAA-3′。
2. We transfected pcDNA3.1-RSPO2 or RSPO2-siRNA (siRNA-RSPO 2-3) to ovarian granular cells respectively, and tested the influence of RSPO2 on the expression and proliferation of the gene related to granular cell proliferation by qRT-PCR, WB and Edu methods respectively. The qRT-PCR and WB results showed that pcDNA3.1-RSPO2 promoted the expression level of cell cycle-related genes (PCNA, CDK1, CDK2, CCNA1, CCND1 and CCNE 2). EdU staining showed that the proliferation rate of pcDNA3.1-RSPO2 group was significantly higher than that of pcDNA3.1 group. At the same time, RSPO2-siRNA inhibited the expression levels of PCNA, CDK1, CCNA1 and CCNE 2. The proliferation rate of RSPO2-siRNA group is significantly lower than that of siRNA-NC group. In conclusion, RSPO2 can promote the proliferation of the porcine ovarian granulosa cells.
3. pcDNA3.1-RSPO2 or RSPO2-siRNA (siRNA-RSPO 2-3) is transfected into ovarian granular cells respectively, and the influence of the RSPO2 on the expression and apoptosis of the apoptosis-related genes of the granular cells is detected by utilizing qRT-PCR, WB and Annexin V-FITC methods respectively. The results of qRT-PCR and WB show that pcDNA3.1-RSPO2 inhibits the expression level of apoptosis-promoting related genes (Caspase 3, caspase8, caspase9 and BAX) of cells and promotes the expression level of anti-apoptosis gene BCL2 of cells. Flow cytometry analysis results show that the apoptosis rate (early apoptosis + late apoptosis) of the pcDNA3.1-RSPO2 group is obviously lower than that of the pcDNA3.1 group. Meanwhile, RSPO2-siRNA promotes the expression levels of Caspase3, caspase8, caspase9 and BAX, and inhibits the expression level of BCL 2. The apoptosis rate of the RSPO2-siRNA group is obviously higher than that of the siRNA-NC group. In conclusion, RSPO2 can inhibit the apoptosis of the porcine ovarian granulosa cells.
4. We transfected pcDNA3.1-RSPO2 or RSPO2-siRNA (siRNA-RSPO 2-3) to ovarian granulosa cells respectively, and tested the influence of RSPO2 on the E2 secretion related gene expression and E2 secretion of granulosa cells by qRT-PCR, WB and ELISA methods respectively. The qRT-PCR and WB results show that pcDNA3.1-RSPO2 promotes the expression level of E2 secretion-related genes (CYP 19A1 and HSD17B 1) of the cells. The ELISA results showed that the E2 concentration of pcDNA3.1-RSPO2 group was significantly higher than that of pcDNA3.1 group. Meanwhile, RSPO2-siRNA inhibits the expression level of CYP19A1 and HSD17B 1. The E2 concentration of RSPO2-siRNA group was significantly lower than that of siRNA-NC group. In conclusion, RSPO2 can promote the secretion of E2 of porcine ovarian granulosa cells.
Compared with the prior art, the invention has the following advantages and effects:
(1) RSPO2 can be directly or indirectly involved in follicular atresia, follicular development and primordial situation initiation, and the application of the RSPO2 (Gene ID: 100154008) in porcine ovarian granulosa cells is researched by taking the RSPO2 as a research object and adopting a molecular and cell biological method: RSPO2 inhibits apoptosis and promotes proliferation and E2 secretion of ovarian granulosa cells. Has good application value for researching ovarian follicular atresia, incipient situation starting and the like.
(2) The technical scheme of the invention is thoroughly designed and has reliable results. To demonstrate the effect of RSPO2 on ovarian granulosa cell proliferation, apoptosis, and E2 secretion, the present invention was validated at multiple levels, multiple angles, at the levels of the relevant signaling pathway genes, mRNA, and protein, and finally at the phenotype of ovarian granulosa cells.
Drawings
FIG. 1 is a graph showing the overexpression efficiency of pcDNA3.1-RSPO2 detected by qRT-PCR and WB.
FIG. 2 is a graph showing interference efficiency of qRT-PCR and WB detection 3 on RSPO 2-siRNA.
FIG. 3 is a graph of the effect of RSPO2 on granulosa cell proliferation; wherein, the overexpression of RSPO2 promotes the mRNA (A) and protein (B) level expression of the granulosa cell proliferation related gene; c is overexpression RSPO2 for promoting granular cell proliferation; interfering with the mRNA (D) and protein (E) level expression of the RSPO2 inhibition granulosa cell proliferation related gene; f is interference RSPO2 to inhibit the proliferation of granulosa cells.
FIG. 4 is a graph showing the results of apoptosis of granulosa cells by RSPO 2; wherein, the overexpression of RSPO2 inhibits the mRNA (A) and protein (B) level expression of the granular cell apoptosis related gene; c is overexpression RSPO2 for inhibiting granular cell apoptosis; interfering the mRNA (D) and protein (E) level expression of the gene related to the promotion of granular cell apoptosis by RSPO 2; f is interference RSPO2 promoting granular cell apoptosis.
FIG. 5 is a graph of the effect of RSPO2 on E2 secretion from granulocytes; wherein, the overexpression or interference of RSPO2 promotes or inhibits the mRNA (A) and protein (B) level expression of the E2 secretion related gene of the granulosa cells respectively; c is overexpression or interference of RSPO2 to promote or inhibit E2 secretion of granulocytes respectively.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.
In the present invention, the results of 3 independent experiments in each example were analyzed by statistical methods, and the "mean ± standard deviation" was calculated, and the analysis of significance of difference was performed by one-way analysis of variance (in the figure, "+" indicates P <0.05, and "+" indicates P < 0.01).
EXAMPLE 1 construction of overexpression vector of RSPO2
(1) Designing a Primer by using Primer5, and amplifying a CDS (coding sequence) region of RSPO2 by using the extracted cDNA (complementary deoxyribonucleic acid) of the porcine granulosa cell as a template; the amplified fragment is purified, recovered, connected with pMD18T vector (purchased from Takara company), transformed, screened, sequenced and identified correctly, and then common plasmid is extracted.
(2) The CDS region sequence of RSPO2 gene has no Hind III and Kpn I restriction enzyme cutting sites, while pcDNA3.1 vector has Hind III and Kpn I cutting sites. The HindIII and Kpn I restriction site sequences are added to the upstream and downstream primers respectively. PCR amplification is carried out by taking CDS region recombinant pMD18T common plasmid of RSPO2 as a template; the fragment is purified and recovered, subjected to double digestion, connected with a pcDNA3.1 vector, transformed, screened, sequenced and identified to be correct, and then an endotoxin-free plasmid (the endotoxin-free plasmid miniprep kit is purchased from America magenta) is extracted and named as pcDNA3.1-RSPO2.
The RSPO2 gene CDS region primer used by the invention:
RSPO2 Forward：
Reverse：
note: the black bold font is the protecting base, underlined is the restriction site.
Example 2 culture and transfection of ovarian granulosa cells
(1) Collecting ovaries of healthy sows, placing the ovaries in a PBS (ice) buffer solution containing 1% double antibodies, and quickly transporting the ovaries back to a laboratory for treatment;
(2) Firstly, washing ovaries for 3-5 times by using PBS (phosphate buffer solution) containing 2% double antibody outside a cell room, putting the ovaries into a beaker, and putting the beaker into a transfer window;
(3) Wiping a cell room superclean bench with alcohol, clamping an ovary by using forceps, sucking follicular fluid by using an injector, pumping the follicular fluid into a centrifuge tube containing 5mL of DMEM culture fluid, and extracting the follicular fluid to 9mL per tube;
(4) Centrifuging at 1000rpm for 5min, discarding the supernatant, adding 5mL PBS, blowing, mixing, and cleaning twice;
(5) Preparing a complete culture medium: 89% DMEM, 10% serum and 1% double antibody, and mixing up and down;
(6) 5mL of complete medium was added to resuspend the cell pellet;
(7) Adding 10mL of complete culture medium into a 75mL culture bottle, and then adding the resuspension;
(8) Observing with microscope, and standing at 37 deg.C and 5% CO 2 Culturing in an incubator, observing the growth condition of granular cells after 24 hours, pouring out the culture medium when the granular cells grow to about 90%, and washing for 3 times by using preheated PBS containing 1% double antibody;
(9) Adding trypsin for digestion, placing in an incubator for about 3min, observing under a microscope until most cells float, and immediately adding equivalent stopping solution to stop digestion;
(10) Washing with DMEM for 2 times, and centrifuging at 1000rpm for 5min;
(11) Gently resuspending the cell precipitate with complete culture medium, uniformly distributing into each well, supplementing volume with complete culture medium, shaking gently, and culturing in incubator;
(12) Observing the cell state after about 24 hours, and preparing for transfection when the cell confluency reaches about 80%;
(13) Transfection method Invitrogen
3000 kit instructions, each set 3 replicates;
(14) The transfected well plate was placed at 37 ℃ 5% 2 Culturing in an incubator, observing the cell state 24-72 h after transfection, and collecting the cells after good growth.
The double-resistant is penicillin and streptomycin.
Example 3 qRT-PCR
In the present invention, the qRT-PCR detection of the gene was performed using Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific Co.). The content of the sample gene is detected by adopting a Ct value comparison method in the experiment, and the specific calculation formula is as follows:
relative gene expression =2- { (Ct value of target gene in experimental group-) - (inner reference gene Ct value in experimental group-) - (target gene Ct value in control group-) }
GAPDH is used as an internal reference for detecting genes, and qRT-PCR primers used by the invention are as follows:
qRT-PCR-RSPO2 Forward：5′-GATGGAGACGCAGTAAGCGA-3′；
Reverse：5′-CATATCTGGGGCTCGGTGTC-3′；
qRT-PCR-Caspase3 Forward：5′-ACATGGAAGCAAATCAATGGAC-3′；
Reverse：5′-TGCAGCATCCACATCTGTACC-3′；
qRT-PCR-Caspase8 Forward：5′-GAGCCTGGACTACATCCCAC-3′；
Reverse：5′-GTCCTTCAATTCCGACCTGG-3′；
qRT-PCR-Caspase9 Forward：5′-GCTGAACCGTGAGCTTTTCA-3′；
Reverse：5′-CCTGGCCTGTGTCCTCTAAG-3′；
qRT-PCR-BAX Forward：5′-ACTTCCTTCGAGATCGGCTG-3′；
Reverse：5′-AAAGACACAGTCCAAGGCGG-3′；
qRT-PCR-BCL2 Forward：5′-GATGCCTTTGTGGAGCTGTATG-3′；
Reverse：5′-CCCGTGGACTTCACTTATGG-3′；
qRT-PCR-PCNA Forward：5′-TCGTTGTGATTCCACCACCAT-3′；
Reverse：5′-TGTCTTCATTGCCAGCACATTT-3′；
qRT-PCR-CDK1 Forward：5′-AGGTCAAGTGGTAGCCATGAA-3′；
Reverse：5′-TCCATGAACTGACCAGGAGG-3′；
qRT-PCR-CDK2 Forward：5′-AAAAGATCGGAGAGGGCACG-3′；
Reverse：5′-GCAGTACTGGGTACACCCTC-3′；
qRT-PCR-CDK4 Forward：5′-CCTCCCGGTATGAACCAGTG-3′；
Reverse：5′-TGCTCAAACACCAGGGTCAC-3′；
qRT-PCR-CCNA1 Forward：5′-GCGCCAAGGCTGGAATCTAT-3′；
Reverse：5′-CCTCAGTCTCCACAGGCTAC-3′；
qRT-PCR-CCNA2 Forward：5′-GTACTGAAGGCCGGGAACTC-3′；
Reverse：5′-AGCTGGCCTCTTTTGAGTCT-3′；
qRT-PCR-CCNB1 Forward：5′-ACGGCTGTTAGCTAGTGGTG-3′；
Reverse：5′-GAGCAGTTCTTGGCCTCAGT-3′；
qRT-PCR-CCNB2 Forward：5′-TGGAAATCGAGTTACAACCAGA-3′；
Reverse：5′-TGGAGCCAACATTTCCATCTGT-3′；
qRT-PCR-CCND1 Forward：5′-CTTCCATGCGGAAGATCGTG-3′；
Reverse：5′-TGGAGTTGTCGGTGTAGATGC-3′；
qRT-PCR-CCND2 Forward：5′-TTCCCCAGTGCTCCTACTTC-3′；
Reverse：5′-CACAACTTCTCAGCCGTCAG-3′；
qRT-PCR-CCNE1 Forward：5′-AGCCTGTGAAAACCCCTGTT-3′；
Reverse：5′-TCCAGAAGAATCGCTCGCAT-3′；
qRT-PCR-CCNE2 Forward：5′-GGGGGATCAGTCCTTGCATT-3′；
Reverse：5′-AGCCAAACATCCTGTGAGCA-3′；
qRT-PCR-CYP19A1 Forward：5′-CTGAAGTTGTGCCTTTTGCCA-3′；
Reverse：5′-CTGAGGTAGGAAATTAGGGGC-3′；
qRT-PCR-STAR Forward：5′-CGACGTTTAAGCTGTGTGCT-3′；
Reverse：5′-ATCCATGACCCTGAGGTTGGA-3′；
qRT-PCR-HSD17B1 Forward：5′-GTCTGGCATCTGACCCATCTC-3′；
Reverse：5′-CGGGCATCCGCTATTGAATC-3′；
qRT-PCR-HSD3B1 Forward：5′-ATCTGCAGGAGATCCGGGTA-3′；
Reverse：5′-CCTTCATGACGGTCTCTCGC-3′；
qRT-PCR-GAPDH Forward：5′-TCACCAGGGCTGCTTTTAACT-3′；
Reverse：5′-CTTGACTGTGCCGTGGAACT-3′；
total RNA extraction of cells was performed according to the instructions of TRIzol of Takara, and the following steps were performed:
(1) Adding the granular cells into TRIzol directly;
(2) Standing at room temperature for 10min to fully lyse cells, centrifuging at 12000g for 5min, discarding the precipitate, and taking the supernatant in a new RNase-free tube;
(3) Adding 0.2mL of chloroform (1 mL of TRIzol) and shaking vigorously for 15-30 s, standing at room temperature for 5min, and centrifuging at 4 ℃ and 12000g for 15min;
(4) Absorbing the upper aqueous phase and placing the upper aqueous phase in a new RNase-free EP tube;
(5) Adding 0.5mL of isopropanol (1 mL of TRIzol), gently turning upside down, mixing, standing at room temperature for 10min, and centrifuging at 4 deg.C for 12000g for 10min;
(6) Discarding the supernatant, placing at room temperature, adding 1mL of 75% ethanol-DEPC (per 1mL of TRIzol) along the tube wall to wash RNA, centrifuging at 4 ℃ at 12000g for 5min, and discarding the supernatant as much as possible;
(7) Vacuum drying for 5-10 min, and taking care to avoid excessive drying of RNA precipitate;
(8) DEPC water was added to dissolve the RNA pellet.
PrimeScript from TaKaRa was used TM The RT Master Mix (Perfect Real Time) cDNA reverse transcription kit reverse transcribes total RNA.
Example 4 Western Blot
(1) Extraction and quantification of total protein from adherent cell monolayer (ovarian granulosa cells in example 2): the cell culture was decanted and the cells were washed three times with an appropriate amount of pre-cooled PBS to wash out the culture. 100-200. Mu.L of protein lysate and 10. Mu.L of 100mM PMSF are added into each well of 6-well plate cells, and the cells are lysed for 30min. The cell lysate was collected and transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm at 4 ℃ for 5min. Protein sample concentrations were determined using the BCA method.
(2) SDS-PAGE electrophoresis: mu.g of total protein per group was mixed with 5 Xloading buffer at 5. Performing SDS-PAGE electrophoresis until bromophenol blue just comes out of the bottom of the gel;
(3) Film transfer: pretreating PVDF membrane with methanol for 3-5 s, and soaking in transfer printing solution for 30min. Taking out the gel, and placing the gel on filter paper to form a sandwich structure of a gel transfer accumulation layer. This operation must remove the bubbles completely. Constant pressure of 100V for 60-120 min;
(4) Immunoblotting: the hybridization membrane was removed, rinsed for 5min in TBST and repeated three times. 5% skimmed milk powder solution was sealed at room temperature for 90min, and rinsed with TBST for 5min, repeated three times. Membranes were incubated overnight at 4 ℃ with primary antibodies diluted as follows: RSPO2 (17781-1-AP, proteintech, 1. After washing the membranes 3 times with TBST, they were incubated with secondary goat-anti-rabbit (ab 205718, abcam,1 10000) or goat-anti-mouse (ab 6789, abcam,1 5000) antibodies for 2h at room temperature. And (3) after ECL luminous liquid treatment, visualizing the protein band by using an Odyssey Fc image system, and finally analyzing the protein band by using ImageJ software.
Example 6 granular cell apoptosis assay
The Annexin V-FITC technology for detecting the Apoptosis refers to the Annexin V-FITC Apoptosis Detection Kit operating instruction of BioVision, and comprises the following specific operating steps:
(1) Placing the cell culture plate at room temperature, gently rinsing cells in the culture plate by using 2mL of PBS solution, and removing the PBS solution;
(2) Adding pancreatin-digested cells without EDTA, and gently resuspending the cells in the medium of step (1) to a density of about 1X 10 6 cell/mL;
(3) 0.5mL of cell suspension was removed from the cell culture plate (approximately 5X 10) 5 Individual cell) toIn a clean centrifugal tube, 500. Mu.L of 1 × Binding Buffer is added;
(4) Adding 5 mu.L of Annexin V-FITC and 5 mu.L of propidium iodide at room temperature;
(5) Reacting at room temperature in dark for 5min;
(6) Analysis was immediately performed using a FACSCalibur flow cytometer (triplicates per group).
Example 7 granulosa cell proliferation assay
The present invention uses the EdU method to detect Cell proliferation, refer to Cell-Light of Ruibo corporation TM The EdU Apollo 567 In vitro Kit detection Kit comprises the following specific operation steps:
(1) Preparation of 50 μ M EdU medium: culturing the cells in a cell culture medium at a temperature of 1: diluting the EdU solution at a ratio of 1000;
(2) Discarding the culture solution when the cell fusion degree is 50-80%, and adding 100 mu L of 50 mu M EdU culture medium to incubate for 2h;
(3) Fixing the cells: discarding the culture solution, adding 100 μ L of cell fixing solution (4% paraformaldehyde PBS) into each well, and incubating at room temperature for 15-30 min;
(4) Incubation with 2mg/mL glycine for 10min, washing with PBS for 2 times;
(5) Discarding the supernatant, adding 100 μ L of penetrant (0.5% (v/v) TritonX-100 PBS) to permeabilize the cells, and washing with PBS for 1 time;
(6) EdU detection: adding 100 μ L of
Staining reaction solution, incubating at room temperature in dark for 30min, washing with PBS for 1 time, precipitating cells, and removing supernatant;
(7) DNA staining: adding 100 mu L of DAPI reaction solution into each hole, and incubating for 30min at room temperature in a dark place;
(8) Adding 100 μ L of penetrating agent (0.5% (v/v) TritonX-100 PBS) to wash for 3 times, eluting DAPI reaction solution;
(9) Fluorescence microscopy (triplicates in each group).
Example 8 measurement of E2 content in porcine ovarian granulosa cell supernatant samples by ELISA
E2 concentration detection, referring to a porcine E2 ELISA kit of Jiangsu Jingmei biological company, the specific operation steps are as follows: respectively adding 50 mu L of standard substances with different concentrations into the standard hole, and adding 50 mu L of samples to be detected into the sample hole. Then 100. Mu.L of horseradish peroxidase (HRP) was added and incubated at 37 ℃ for 60min. After washing, 50. Mu.L of each of the substrates A and B was added and incubated at 37 ℃ for 15min. Finally, a stop solution was added and the OD was measured at 450 nm.
And (4) analyzing results:
1. in order to study the effect of RSPO2 gene on the function of ovarian granulosa cells, we transfected RSPO2 overexpression vector (pcDNA3.1-RSPO 2) or small interfering RNA (RSPO 2-siRNA) into ovarian granulosa cells to explore the effect of RSPO2 on the proliferation, apoptosis and E2 secretion of ovarian granulosa cells; the construction method of the RSPO2 overexpression vector comprises the following steps: firstly, CDS region (protein coding region) of RSPO2 is amplified by PCR, after the sequencing verification is correct, common plasmid is extracted, and then endotoxin-free plasmid is extracted after the double enzyme digestion, connection, transformation and single clone sequencing verification of the common plasmid and pcDNA3.1 vector are carried out. And carrying out double enzyme digestion identification on the extracted endotoxin-free plasmid, and verifying whether the RSPO2 overexpression vector is successfully constructed or not. Finally, the successfully constructed pcDNA3.1-RSPO2 is transfected into ovarian particle cells, and verified by qRT-PCR and WB methods, as shown in FIG. 1, as the concentration of the pcDNA3.1-RSPO2 vector increases, the mRNA and protein expression level of the RSPO2 also increases.
2. 3 pairs of small interfering RSPO2 fragments/controls (RSPO 2-siRNA/siRNA-NC) were synthesized, screened and tested for their interference efficiency. The result is shown in figure 2, the gene interference small fragment is transfected into the ovary granular cell, and the RSPO2-siRNA-3 fragment with better interference effect is finally screened by qRT-PCR and WB means for subsequent experiments.
siRNA-RSPO2-1：5′-CCGATGTCAACAGAAGTT-3′；
siRNA-RSPO2-2：5′-GGAACTTGTAGCAGAAATA-3′；
siRNA-RSPO2-3：5′-GGCTGTTTGTCTTGTTCAA-3′。
The small interference fragments are synthesized by Ribo Biotech, inc., guangzhou; control siRNA-NC was from Ribo Biotech, inc., guangzhou.
3. pcDNA3.1-RSPO2 or RSPO2-siRNA (siRNA-RSPO 2-3) is transfected into ovarian granular cells respectively, and the influence of the RSPO2 on the expression and proliferation of genes related to the proliferation of the granular cells is detected by qRT-PCR, WB and Edu methods respectively. As a result, as shown in FIG. 3, the results of qRT-PCR and WB showed that pcDNA3.1-RSPO2 promoted the expression level of cell cycle-related genes (PCNA, CDK1, CDK2, CCNA1, CCND1 and CCNE 2). EdU staining showed that the proliferation rate of pcDNA3.1-RSPO2 group was significantly higher than that of pcDNA3.1 group. At the same time, RSPO2-siRNA inhibited the expression levels of PCNA, CDK1, CCNA1 and CCNE 2. The proliferation rate of the RSPO2-siRNA group is obviously lower than that of the siRNA-NC group. In conclusion, RSPO2 can promote the proliferation of the porcine ovarian granulosa cells.
4. We transfect pcDNA3.1-RSPO2 or RSPO2-siRNA (siRNA-RSPO 2-3) to ovary granular cells respectively, and utilize qRT-PCR, WB and Annexin V-FITC methods to detect the influence of the RSPO2 on the expression and apoptosis of granular cell apoptosis-related genes respectively. The results are shown in FIG. 4, and the results of qRT-PCR and WB show that pcDNA3.1-RSPO2 inhibits the expression level of the apoptosis-promoting related genes (Caspase 3, caspase8, caspase9 and BAX) of the cell and promotes the expression level of the apoptosis-resisting gene BCL2 of the cell. Flow cytometry analysis shows that the apoptosis rate (early apoptosis + late apoptosis) of the pcDNA3.1-RSPO2 group is obviously lower than that of the pcDNA3.1 group. Meanwhile, RSPO2-siRNA promotes the expression levels of Caspase3, caspase8, caspase9 and BAX, and inhibits the expression level of BCL 2. The apoptosis rate of the RSPO2-siRNA group is obviously higher than that of the siRNA-NC group. In conclusion, RSPO2 can inhibit the apoptosis of the porcine ovarian granulosa cells.
5. We transfected pcDNA3.1-RSPO2 or RSPO2-siRNA (siRNA-RSPO 2-3) to ovarian granulosa cells respectively, and tested the influence of RSPO2 on the E2 secretion related gene expression and E2 secretion of granulosa cells by qRT-PCR, WB and ELISA methods respectively. The results are shown in FIG. 5, and qRT-PCR and WB results show that pcDNA3.1-RSPO2 promotes the expression level of E2 secretion-related genes (CYP 19A1 and HSD17B 1) of cells. The ELISA results showed that the E2 concentration of pcDNA3.1-RSPO2 group was significantly higher than that of pcDNA3.1 group. Meanwhile, RSPO2-siRNA inhibits the expression level of CYP19A1 and HSD17B 1. The E2 concentration of RSPO2-siRNA group was significantly lower than that of siRNA-NC group. In conclusion, RSPO2 can promote the secretion of E2 of porcine ovarian granulosa cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> southern China university of agriculture
Application of <120> RSPO2 gene in porcine ovarian granulosa cells
<160> 51
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<220>
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<223> qRT-PCR-Caspase8 Reverse
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<220>
<223> qRT-PCR-Caspase9 Reverse
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-BCL2 Reverse
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-PCNA Forward
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<220>
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<213> Artificial Sequence (Artificial Sequence)
<220>
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<220>
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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<213> Artificial Sequence (Artificial Sequence)
<220>
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNB2 Forward
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCND1 Forward
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCND1 Reverse
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<220>
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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<400> 41
<210> 42
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CYP19A1 Forward
<400> 42
ctgaagttgt gccttttgcc a 21
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CYP19A1 Reverse
<400> 43
ctgaggtagg aaattagggg c 21
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-STAR Forward
<400> 44
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-STAR Reverse
<400> 45
atccatgacc ctgaggttgg a 21
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD17B1 Forward
<400> 46
gtctggcatc tgacccatct c 21
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD17B1 Reverse
<400> 47
cgggcatccg ctattgaatc 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD3B1 Forward
<400> 48
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD3B1 Reverse
<400> 49
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Forward
<400> 50
tcaccagggc tgcttttaac t 21
<210> 51
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
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<400> 51
Claims (4)
- The application of the RSPO2 overexpression vector in the preparation of a reagent for promoting the generation of E2 in porcine ovarian granulosa cells is characterized in that: after the RSPO2 gene is over-expressed, the generation of E2 can be promoted; e2 refers to estradiol.
- 2. The application of siRNA interfering RSPO2 gene expression in preparing a reagent for inhibiting E2 generation in swine ovary granular cells is characterized in that: inhibiting the generation of E2 after interfering RSPO2 gene; e2 refers to estradiol;wherein, the inhibition of the generation of E2 after the interference of the RSPO2 gene is realized by a mode that siRNA for inhibiting the expression of the RSPO2 gene is transfected into the pig ovary granular cells;the siRNA interfering RSPO2 gene expression is siRNA-RSPO2-3, and the sequence is as follows:siRNA-RSPO2-3：5′-GGCTGTTTGTCTTGTTCAA-3′。
- 3. use according to claim 1, characterized in that:the method for promoting the generation of E2 after over-expression of RSPO2 gene is realized by the following steps: the overexpression vector containing the RSPO2 gene is transfected into the porcine ovarian granulosa cells.
- 4. Use according to claim 3, characterized in that:the method for promoting the generation of E2 after over-expressing RSPO2 gene is realized by the following steps: connecting the RSPO2 gene to a pcDNA3.1 vector to construct a overexpression vector containing the RSPO2 gene; then, the overexpression vector containing the RSPO2 gene is transfected into the pig ovarian granulosa cells.
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| R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth;Marie-Cécile De Cian et al.;《Cell Death & Differentiation》;20200427;第27卷(第10期);摘要，第2857页右栏最后一段-第2858页左栏第1段，第2859页左栏最后一段-第2860页左栏第2段，第2860页右栏最后一段-第2863页左栏第2段 * |
| 肠癌中RSPO2抑瘤的分子机制研究;武长杰;《万方中国学位论文全文数据库》;20131231;doi：10.7666/d.Y2404917 * |
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