Construction method and application of ovarian particle cell strain capable of being stably subcultured in vitro

Abstract

The invention discloses a construction method and application of an ovarian particle cell strain capable of being stably subcultured in vitro. Belongs to the technical field of cytobiology. The follicular fluid in the ovary of the Shaanbei white cashmere goat is taken out by using an injector, precipitates are collected after centrifugation, and the MEM complete medium containing fetal calf serum is used for culture and passage. Lentiviruses containing SV40T antigens are packaged in vitro and are transduced into the Shaanbei white cashmere goat ovary granular cells, and immortalization is realized after long-term subculture. The construction method disclosed by the invention is high in repeatability, simple and efficient in operation steps, good in ovarian granular cell growth state and stable in physiological state. The ovarian particle cell strain provided by the invention lays a foundation for subsequent research on follicular development of the down producing goat and biological functions of particle cells, and the cell strain provided by the invention can be used for research and creation of follicular development and ovulation regulation methods of the down producing goat.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and more particularly to a method for constructing an ovarian granulosa cell line capable of stable in vitro subculture and its application. Background Art

[0002] Granulosa cells (GC), as an important component of follicles, regulate the development and atresia of follicles through gonadotropin receptors, steroid hormones, various growth factors, etc., and play an important role in the normal development of oocytes and follicles. Therefore, in mammals, ovarian granulosa cells are often used as a model for in vitro research on ovarian function.

[0003] Since primary ovarian granulosa cells have very poor in vitro culture ability and are difficult to be subcultured for more than 10 generations, the existing method for stable cell subculture mainly involves adding cell growth factors. Although this will promote cell proliferation and differentiation, the number of cell subcultures will not change, which severely limits the research on ovarian function. Therefore, there is an urgent need for a cell model that can be stably subcultured in vitro.

[0004] In summary, how to provide a method for constructing ovarian granulosa cells that can be stably subcultured in vitro is an urgent problem that those skilled in the art need to solve. Summary of the invention

[0005] In view of this, the present invention provides a method for constructing an ovarian granulosa cell line that can be stably subcultured in vitro and its application. The purpose of the present invention is to provide a cashmere goat ovarian granulosa cell line and study its biological characteristics to solve the technical problem that existing ovarian granulosa cells cannot be stably subcultured in vitro.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for constructing an ovarian granulosa cell line capable of stable in vitro subculture comprises the following steps:

[0008] 1. Collection of follicular fluid;

[0009] 2. Preparation of ovarian granulosa cells;

[0010] The follicular fluid is centrifuged and the precipitate is obtained, namely, ovarian granulosa cells;

[0011] 3. Primary culture;

[0012] Resuspend the ovarian granulosa cells obtained in step 2 with complete granulosa cell culture medium and adjust the cell concentration to 3×10 5 / ml, inoculated into a culture dish, placed in a 5% CO2, 37℃ constant temperature incubator, and replaced the culture medium every other day;

[0013] 4. Subculture;

[0014] When the density of ovarian granulosa cells reaches 80-90%, subculture is performed;

[0015] 5. Immortalization of ovarian granulosa cells;

[0016] After subculture, the ovarian granulosa cells were transfected with the lentiviral plasmid SV40T to achieve immortalization.

[0017] Furthermore, in step 1, the follicular fluid is collected by suction.

[0018] Furthermore, the centrifugation parameter in step 2 is 1000r / min for 5min.

[0019] Furthermore, the complete granulosa cell culture medium in step three includes the following components in volume: 89 parts of MEM basal culture medium, 10 parts of fetal bovine serum and 1 part of double antibody.

[0020] Further, the specific operation of step 4 is as follows: when the density of ovarian granulosa cells reaches 80-90%, remove the culture medium, wash with PBS, add 0.25% trypsin, and digest at 37°C for 5 minutes; terminate the digestion with an equal volume of granulosa cell complete culture medium, centrifuge at 1000r / min for 5 minutes; discard the supernatant, resuspend the cells with granulosa cell complete culture medium, and centrifuge at 3×10 5 Cells were inoculated into culture dishes, the cell density was observed under a microscope, and the culture medium was replaced every other day.

[0021] Furthermore, the specific operations of step five are:

[0022] ①Virus packaging:

[0023] The 293T cells were washed with serum-free basal medium after discarding the culture medium, starved for 30 minutes, and opti-MEM was pipetted into two sterile centrifuge tubes, which were marked as lipo tubes and plasmid tubes respectively; lipofectamine 2000 was added to the lipo tube, and fully vortexed to mix; SV40T lentiviral plasmid, pSPAX2 and pMD2.G two auxiliary plasmids were added to the plasmid tube at a mass ratio of 10:2:1; the diluted lipofectamine 2000 in the lipo tube was pipetted out and added to the plasmid tube, and gently mixed by pipetting up and down, and allowed to stand at room temperature for 10 minutes to form a lipo-plasmid mixture; the lipo-plasmid mixture was gently dripped into the culture dish; fresh culture medium was replaced after 5 hours of culture, and culture was continued for 24 hours; the supernatant was collected and filtered with a 0.45um filter membrane to obtain the lentiviral venom;

[0024] ②Viral transduction:

[0025] After subculturing, 3 × 10 ovarian granulosa cells were cultured per dish. 6 Cells were inoculated into culture dishes; virus infection was performed after overnight culture, starvation treatment was performed for 30 min, the basal culture medium was discarded and lentiviral venom was added, and complete culture medium containing 2 μM GSK3Inhibitor IX (BIO) was added after culturing for 2 h, and then subculture was continued until the cell proliferation ability was improved and immortalization was achieved.

[0026] Furthermore, the SV40T lentiviral plasmid carries ampicillin resistance.

[0027] Furthermore, the BIO is a specific inhibitor of glycogen synthase kinase 3 (GSK-3) and can promote cell proliferation and differentiation.

[0028] Furthermore, the ovarian granulosa cells are ovarian granulosa cells of Shaanxi white cashmere goats.

[0029] The ovarian granulosa cell line constructed by the above construction method.

[0030] The application of the above-mentioned ovarian granulosa cell line in establishing an in vitro research model of ovarian function.

[0031] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0032] The invention provides a northern Shaanxi white cashmere goat ovarian granulosa cell line, takes the sexually mature northern Shaanxi white cashmere goat ovarian tissue as the research object, establishes a cashmere goat primary ovarian granulosa cell culture system and identifies the separated cells, and can provide a basis for the molecular regulation mechanism of cashmere goat ovarian granulosa cells and the hormone metabolism cell model.

[0033] The present invention provides a method for constructing a granulosa cell line of an ovarian goat in northern Shaanxi, wherein the ovarian tissue of the white cashmere goat in northern Shaanxi is obtained, and after the ovarian tissue is cleaned and disinfected, the follicular fluid is collected using a syringe under sterile conditions, and further centrifuged; the precipitate is resuspended in a MEM complete medium containing fetal bovine serum and double antibodies (penicillin-streptomycin), and primary granulosa cells of the ovarian goat in northern Shaanxi are obtained. The construction method is simple to operate and has strong repeatability. The granulosa cells obtained by separating the ovarian granulosa cells from the ovaries of the white cashmere goats in northern Shaanxi and primary culture in vitro have a stable growth state, can be continuously passaged, and can be obtained in batches by passage. The granulosa cells of the ovarian goat in northern Shaanxi can provide reference materials for studying the molecular regulation mechanism of granulosa cells of the ovarian goats in northern Shaanxi and establishing a hormone metabolism cell model in the future, and provide references for the separation and culture of granulosa cells of other mammals. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0035] Figure 1 The figures are growth morphology diagrams of ovarian granulosa cells of northern Shaanxi white cashmere goats at different stages in Example 1 of the present invention; wherein A is a growth morphology diagram of the cells attached to the wall at one day of primary culture, B is a growth morphology diagram of the cells attached to the wall at five days of primary culture, and C is a growth morphology diagram of immortalized ovarian P0 generation granulosa cells of northern Shaanxi white cashmere goats;

[0036] Figure 2 The Giemsa staining images of the primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and the immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) in Example 1 of the present invention; wherein A is the Giemsa staining image of the primary Shaanxi white cashmere goat ovarian granulosa cells, and B is the Giemsa staining image of the immortalized Shaanxi white cashmere goat ovarian granulosa cells;

[0037] Figure 3 The immunofluorescence identification result diagram of the primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) in Example 1 of the present invention is of the gene StAR related to granulosa cell differentiation and steroid hormone production, the commonly used marker genes FSHR, CYP19A1 and AMH for granulosa cells, and the gene PCNA that promotes granulosa cell proliferation and differentiation;

[0038] Figure 4 The growth curves of the primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and the immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) in Example 1 of the present invention;

[0039] Figure 5 This is the chromosome karyotype analysis of the immortalized Shaanxi white cashmere goat ovarian granulosa cells in Example 2 of the present invention;

[0040] Figure 6 The figure is a result of using the EdU incorporation method to detect the proliferation of primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) in Example 3 of the present invention; wherein A is the EDU incorporation fluorescence staining result, and B is the EDU positive cell statistical result;

[0041] Figure 7This is a graph showing the results of using CCK-8 to detect the cell proliferation of primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) at 0, 24, and 48 hours in Example 4 of the present invention;

[0042] Figure 8 This is a graph showing the changes in steroid hormone secretion in primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) in Example 5 of the present invention; wherein A is the progesterone PROG content, and B is the estrogen E2 content;

[0043] Fig. 9 This is a graph showing the expression changes of proliferation genes and apoptosis genes related to primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T) in Example 6 of the present invention; wherein A represents proliferation-related genes cyclinD1 and PCNA, and B represents apoptosis-related genes casps3, BCL2 and p21. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0046] Example 1

[0047] Construction of ovarian granulosa cell line of Shaanxi white cashmere goat

[0048] 1. Specimen Source

[0049] It originated from a slaughterhouse in Yuyang District, Yulin City, Shaanxi Province.

[0050] 2. Reagents and Materials

[0051] MEM culture medium (cytiva, China); PBS buffer (cytiva, China); fetal bovine serum (Daxi Biotechnology); penicillin-streptomycin-amphotericin B solution (Beyotime, China); 0.25% trypsin (Beyotime, China); follicle stimulating hormone receptor (FSHR) antibody (Wuhan Boster).

[0052] Preparation of complete culture medium for granulosa cells: 89 mL of MEM culture medium, 10 mL of fetal bovine serum (FBS), and 1 mL of double antibody (penicillin-streptomycin-amphotericin B solution) were mixed evenly.

[0053] The SV40T lentiviral plasmid and the auxiliary plasmids pSPAX2 and pMD2.G were kindly donated by the Shaanxi Stem Cell Engineering Technology Research Center of Northwest Agriculture and Forestry University.

[0054] The EdU (NO.E607204) kit was purchased from Shanghai Shenggong Biotechnology Co., Ltd.

[0055] III. Methods

[0056] 1. Collection of Follicular Fluid

[0057] Use a 2.5mL syringe to collect follicular fluid from Shaanxi white cashmere goats.

[0058] 2. Preparation of Ovarian Granulosa Cells

[0059] The collected follicular fluid of the Shaanxi white cashmere goats was centrifuged at 1000r / min for 5min, and the precipitate was the ovarian granulosa cells of the Shaanxi white cashmere goats.

[0060] 3. Primary Culture

[0061] The ovarian granulosa cells of the Shaanxi white cashmere goat obtained in step (2) were resuspended in complete granulosa cell culture medium (MEM + 10% fetal bovine serum + 1% double antibody), and the cells were counted under a microscope to adjust the cell concentration to 3×10 5 / mL, inoculated into a 60mm culture dish, placed in an incubator at 37°C, 5% CO2, and cultured every other day.

[0062] 4. Subculture and Expansion of Ovarian Granulosa Cells

[0063] When the density of ovarian granulosa cells of Shaanxi white cashmere goats reached 80% to 90%, the culture medium was removed, PBS was washed twice, 0.25% trypsin was added, and digestion was carried out at 37°C for 5 minutes. After the digestion was terminated with an equal volume of complete culture medium of granulosa cells, the cells were centrifuged at 1000r / min for 5 minutes. After discarding the supernatant, the cells were resuspended in complete culture medium of granulosa cells and centrifuged at 3×10 5 The cells were inoculated into a 60 mm culture dish, and the cell density was observed under a microscope. The culture medium was replaced every other day, and subculture was performed when the cell confluence reached more than 80%.

[0064] 5. Cell Cryopreservation and Thawing

[0065] The ovarian granulosa cells were placed in liquid nitrogen for long-term storage starting from the third generation. After 30 days of recovery, the growth of the cells after recovery was observed under a microscope, and the cells showed a survival rate of 60% to 65%.

[0066] 6. Immortalization of Ovarian Granulosa Cells

[0067] 6.1 Virus packaging

[0068] When the 293T cell confluency reached 80-90%, the cells were passaged at a rate of 1×10 6 Inoculate cells into 100 mm culture dishes, transfect and package viruses 24 hours later, as follows:

[0069] 293T cells were discarded from the culture medium and washed twice with serum-free basal medium, and starved for 30 minutes with 2 mL of basal medium. 250 μL of opti-MEM was aspirated into two sterile 1.5 mL centrifuge tubes, and they were marked as lipo tubes and plasmid tubes respectively; 20 μL of lipofectamine 2000 was added to the lipo tube, and vortexed thoroughly to mix well; SV40T lentiviral plasmid, pSPAX2 and pMD2.G two auxiliary plasmids were added to the plasmid tube at a mass ratio of 10:2:1; the diluted lipofectamine 2000 in the lipo tube was aspirated and added to the plasmid tube, and the mixture was gently mixed by blowing up and down, and the mixture was allowed to stand at room temperature for 10 minutes to form a lipo-plasmid mixture. The lipo-plasmid mixture was gently dripped into the culture dish; after 5 hours of culture, fresh culture medium was replaced, and culture was continued for 24 hours; the supernatant was collected and filtered with a 0.45 um filter membrane to obtain the lentiviral venom, which can be used.

[0070] 6.2 Viral transduction

[0071] After subculturing, 3 × 10 ovarian granulosa cells were cultured per dish. 6 Cells were inoculated into 60 mm culture dishes; after overnight culture, virus infection was performed, first 2 mL of serum-free and dual-antibody-free basal culture medium was added for starvation treatment for 30 min, the basal culture medium was discarded, 2 mL of filtered lentiviral venom was added to each dish, and after culturing for 2 h, 2 mL of complete culture medium containing 2 μM GSK 3Inhibitor IX (BIO) was added, and then subculture was continued until the cell proliferation ability was improved and immortalization was achieved (about two generations).

[0072] 6.3 Giemsa staining

[0073] SHBGC and SHBGC-SV40T were inoculated in 24-well plates. After culturing for 24 hours, the culture medium was discarded and the plates were washed twice with PBS according to the steps of Beekman Bio-Giemsa staining solution. 1 mL of 4% paraformaldehyde was added to each well for fixation for 3 minutes. Two drops of Giemsa staining solution Ι were added. Two drops of Giemsa staining solution ΙΙ were added and gently shaken to mix. The plates were incubated for 30 minutes. The plates were rinsed with distilled water, dried, and examined under a microscope.

[0074] 7. Plotting the growth curve of ovarian granulosa cells

[0075] Ovarian granulosa cells of Shaanxi white cashmere goats were cultured at 3×10 4 The density of cells per well was uniformly inoculated in a 24-well plate, and the medium was changed every day. Cells were taken from 3 wells at the same time every day, digested with 0.25% trypsin, and counted, and the average value was taken. The growth curve was drawn with the number of culture days as the horizontal axis and the number of cells as the vertical axis, and the population doubling time (PDT) was calculated using the following formula:

[0076] PDT = [lg2 / (lgNt-lgN0)] × t, Nt = number of cells after culture, N0 = number of cells inoculated, t is culture time

[0077] 8. Identification of Ovarian Granulosa Cells

[0078] According to the characteristics of Follicle-Stimulating Hormone Recepor (FSHR) specific expression in ovarian granulosa cells, immunofluorescence identification of ovarian granulosa cells cultured in vitro was performed using commonly used marker genes for ovarian granulosa cells, including FSHR, CYP19A1 and AMH, PCNA, a gene that promotes granulosa cell proliferation and differentiation, and StAR, a gene related to estradiol and progesterone levels and steroid production. The specific steps are as follows:

[0079] a. Remove the culture medium of ovarian granulosa cells after culturing for 24 hours, and wash them with PBS three times, 5 minutes each time;

[0080] b. Fix with 4% paraformaldehyde for 1 hour, wash with PBS 3 times, 5 minutes each time;

[0081] c. Incubate on ice with 0.3% TritionX-100 for 15 min, then wash three times with PBS, 5 min each time;

[0082] d. Incubate in blocking solution (0.5% fetal bovine serum + 0.3% TritionX-100) for 2 h at room temperature, and wash with PBS three times, 5 min each time;

[0083] e. Add primary antibody (anti-FSHR, 1:200), incubate at 4°C overnight, and wash with PBS three times, 5 min each time;

[0084] f. Add secondary antibody (FITC, 1:100) and incubate at 4°C for 1 hour in dark;

[0085] g. Wash with PBS three times, 5 min each time, and add DAPI for nuclear staining for 10 min;

[0086] h. Observe the color development under a fluorescence microscope and take photos for preservation.

[0087] IV. Results

[0088] Figure 1 These are the growth morphology diagrams of ovarian granulosa cells of Shaanxi white cashmere goats at different stages. It can be seen that the proliferation of immortalized cells is significantly higher than that of primary cells.

[0089] Figure 2 This is a Giemsa staining image of primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC) and immortalized Shaanxi white cashmere goat ovarian granulosa cells (SHBGC-SV40T). The results show that after immortalization, the Shaanxi white cashmere goat ovarian granulosa cells have a paving stone-like cell morphology ( Figure 2 B), which is different from the SHBGC before immortalization ( Figure 2 A) is consistent with the shape.

[0090] Ovarian granulosa cell identification results Figure 3 As shown in the figure, the cell nucleus after DAPI staining is blue, and the receptor protein is red and located in the cytoplasm, indicating that the isolated cells are primary Shaanxi white cashmere goat ovarian granulosa cells (SHBGC). AMH, STAR, PCNA, and CYP19A1 of the two groups of cells are all positive, so the cells of the SHBGC group and SHBGC-SV40T group are all ovarian granulosa cells without mutation.

[0091] The growth curve of ovarian granulosa cells is as follows Figure 4 As shown, from 0 to 24 hours of culture, the cells are in the adhesion adaptation period. From 24 to 48 hours, the cell growth is slow and increases slightly. From 48 to 144 hours, the cell division is vigorous and increases rapidly. The cells begin to proliferate in large quantities. At 144 hours, the cell number reaches a peak. After 144 hours, the cell proliferation slows down and shows a downward trend. From 144 to 168 hours, the cell number decreases slightly. The population doubling time of SHBGC is 26.2 hours. The population doubling time of SHBGC-SV40T group cells is 25.4 hours.

[0092] Example 2

[0093] Karyotype Analysis of Chromosomes in Granulosa Cells of Ovaries of Northern Shaanxi White Cashmere Goats

[0094] Take the immortalized Shaanxi white cashmere goat ovarian granulosa cells in Example 1 above and grow them in a 37°C constant temperature incubator for 48h, add colchicine with a final concentration of 10μg / mL for 6h, and centrifuge at 1000rpm for 10min to recover the cells after 0.25% trypsin digestion. 75mM KCl 37°C water bath hypotonic treatment for 30min, centrifuge at 1000rpm for 10min to recover the cell pellet, add 8mL fixative (methanol: glacial acetic acid = 3:1) and fix at room temperature for 30min, and repeat the fixation step twice. For the last fixation, leave about 150μL of fixative and blow gently. Absorb the fixative-cell suspension, drop it on a -20°C pre-cooled slide at a height of about 0.5 meters, quickly blow the droplets away with force, and dry them at room temperature. The dried slides were immersed in 1x Giemsa staining solution (Bioseth, catalog number RS3080) for 10 min, washed with purified water, and dried at room temperature. The cell chromosome morphology was observed under an oil microscope, and the chromosome number was calculated.

[0095] The results are as follows Figure 5 As shown, the cells have diploid chromosomes, the number of chromosomes is 60, and the chromosome morphology is mostly telomeric chromosomes.

[0096] Example 3

[0097] EdU incorporation method was used to detect cell proliferation in ovarian granulosa cells of Shaanxi white cashmere goats

[0098] EdU incorporation method was used to detect cell proliferation, referring to the steps of EdU cell proliferation detection kit (NO.E607204):

[0099] Ovarian granulosa cells were inoculated into 24-well plates. After 24 hours of adherence culture, 300 μL of EdU-containing culture medium was added to each well and the cells were incubated for 2 hours, and the culture medium was discarded; the cells were washed twice with PBS, each time for 5 minutes, 150 μL of 4% paraformaldehyde cell fixative was added to each well, and the cells were incubated at room temperature for 30 minutes, and the fixative was discarded; 150 μL of 2 mg / mL glycine was added to each well, and the cells were incubated on a shaker for 5 minutes to neutralize excess paraformaldehyde; the glycine solution was discarded, and the cells were washed at room temperature for 5 minutes; 300 μL of 0.5% TritonX-100 cell permeabilization solution was added to each well, and the cells were permeabilized at room temperature for 10 minutes, and the cell permeabilization solution was discarded, and the cells were washed at room temperature for 5 minutes with PBS; 100 μL of the prepared detection mixture was added to each well to cover the cells, and the cells were incubated at room temperature for 30 minutes away from light; the detection mixture was discarded, and 300 μL of 0.5% Triton Wash with X-100 cell permeabilization solution 2 to 3 times, each time for 10 minutes; discard the cell permeabilization solution, wash twice with PBS, each time for 5 minutes; add 300μL 1×Hoechst staining solution to each well, incubate at room temperature in the dark for 20 to 30 minutes, and then discard the staining solution; add 300μL PBS to wash the cells twice, and immediately take pictures with a fluorescence microscope after staining.

[0100] The results are as follows Figure 6 As shown, the proliferation rate of immortalized cells was significantly higher than that of primary cells.

[0101] Example 4

[0102] Using CCK-8 to detect cell viability of ovarian granulosa cells of Shaanxi white cashmere goats

[0103] The subcultured SHBGC and SHBGC-SV40T cells were cultured at a rate of 5 × 10 3 Inoculate in 96-well plates to detect cell proliferation at 0, 24, and 48 hours. Aspirate the culture medium after the cells adhere to the wall. Since metabolic waste will be produced during the proliferation of the two groups of cells, which will affect the absorbance value, when performing the test, discard the original culture medium, add 100μL MEM culture medium, add 10μL CCK-8 solvent to each well, put it in a cell culture incubator and incubate for 2 to 3 hours, and measure the absorbance value at OD = 450nm with an enzyme marker at 0h, 24h, and 48h.

[0104] The results are as follows Figure 7 As shown, the proliferation effect of immortalized cells was significantly higher than that of primary cells at 48h.

[0105] Example 5

[0106] Effects of ovarian granulosa cells on steroid hormone secretion in northern Shaanxi white cashmere goats

[0107] When the secretion of steroid hormones was detected by enzyme-linked immunosorbent assay, the cell culture supernatant was collected after 24 hours of cell culture, and the estrogen E2 and progesterone PROG content of each group of cells was detected with reference to the MLbio Goat ELISA KIT kit.

[0108] The results are as follows Figure 8 As shown, the two groups of cells were cultured for 24 hours to detect the levels of estrogen E2 and progesterone PROG. There was no significant difference in the secretion of estrogen E2 and progesterone PROG between the two groups of cells.

[0109] Example 6

[0110] RT-PCR detection of the expression changes of proliferation genes and apoptosis genes in ovarian granulosa cells of Shaanxi white cashmere goats

[0111] Collect the ovarian granulosa cells of the northern Shaanxi white cashmere goat in the logarithmic growth period, add Trizol lysis solution, let it stand at room temperature for 2-5 minutes, blow and beat to completely lyse the cells, and collect them in EP tubes; let it stand at room temperature for 5 minutes, add -20℃ pre-cooled chloroform according to the volume ratio of Trizol: chloroform = 5:1, shake vigorously for 15 seconds, centrifuge at 4℃, 12000r / min for 15 minutes, and aspirate the upper colorless liquid into a newly prepared 1.5mL EP tube; add an equal volume of -20℃ pre-cooled isopropanol, turn it upside down 5-8 times, let it stand at room temperature for 15 minutes, centrifuge at 4℃, 12000r / min for 10 minutes, and discard the supernatant; add 1mL -20℃ pre-cooled 75% alcohol, centrifuge at 4℃, 8000r / min for 5 minutes, discard the supernatant, and leave a white precipitate. Dissolve the RNA precipitate with an appropriate amount of RNase-free water, measure the OD value, and evaluate the RNA quality by RNA concentration and A260 / 280 value.

[0112] The RNA sample was reverse transcribed into cDNA using the Fermentas reverse transcription kit. According to the kit instructions, 1-2 μg RNA was mixed with RNase-free water and 1 μL Oligo (dT), incubated at 65°C for 5 min, and cooled on ice for 2 min; 4 μL buffer, 2 μL dNTP, 1 μL RNase inhibitor and 1 μL reverse transcriptase were added, reacted at 42°C for 1 h, terminated at 70°C for 5 min, and stored at 4°C. The synthesized cDNA product was stored in a -20°C refrigerator.

[0113] Quantitative primers were designed by searching the CDS region sequence of the goat internal reference β-Actin gene in GenBank, as well as the CDS regions of proliferation-related genes PCNA and CyclinD1 and apoptosis-related genes Bcl-2, caspase3, and P21. Three replicates were obtained for each treatment group, and the relative expression levels of proliferation- and apoptosis-related genes were analyzed with β-Actin as the control. The effects of immortalization gene introduction on the proliferation and apoptosis of ovarian granulosa cells in Shaanxi white cashmere goats were determined.

[0114] The PCR reaction system included 6.8 μL of ddH2O, 10 μL of SYBR, 2 μL of reverse transcription product, 0.8 μL of primers, and 0.4 μL of ROX. The reaction was carried out on a fluorescent quantitative PCR instrument. The reaction conditions were: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 60°C annealing for 1 min, 72°C extension for 1 min, and 40 cycles. The mRNA expression of each gene in each group was statistically analyzed by the 2^-ΔΔT method. The primer sequences are shown in Table 1.

[0115] Table 1 Primer sequence list of proliferation genes and apoptosis genes related to ovarian granulosa cells of Shaanxi white cashmere goats

[0116]

[0117] The results are as follows Fig. 9 As shown, the mRNA expression levels of immortalized cell proliferation-related genes were higher than those of primary granulosa cells; while the mRNA expression levels of apoptosis-related genes were significantly lower than those of primary granulosa cells.

[0118] Comparative Example 1

[0119] Failure of virus packaging: After virus packaging, the cell proliferation rate did not increase, and the SV40T gene could not be expressed by PCR of cell DNA and RNA.

[0120] Comparative Example 2

[0121] Failure of virus attack: The cell proliferation rate of cells that were not starved did not change significantly after virus attack, and the SV40T gene could not be expressed by PCR of cell DNA and RNA. The cell proliferation rate increased after starvation treatment and addition of BIO.

[0122] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0123] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing an ovarian granulosa cell line capable of stable in vitro subculture, characterized in that: The steps include:

1. Collection of follicular fluid; 2. Preparation of ovarian granulosa cells; The follicular fluid is centrifuged and the precipitate is obtained, namely, ovarian granulosa cells; 3. Primary culture; Resuspend the ovarian granulosa cells obtained in step 2 with complete granulosa cell culture medium and adjust the cell concentration to 3×10 5 / ml, inoculated into a culture dish, placed in a 5% CO2, 37℃ constant temperature incubator, and replaced the culture medium every other day; 4. Subculture; When the density of ovarian granulosa cells reaches 80-90%, subculture is performed; 5. Immortalization of ovarian granulosa cells; After subculture, the ovarian granulosa cells were transfected with the lentiviral plasmid SV40T to achieve immortalization.

2. The construction method according to claim 1, characterized in that: In the step 1, the follicular fluid is collected by suction.

3. The construction method according to claim 1, characterized in that: The centrifugation parameter in step 2 is 1000 r / min for 5 min.

4. The construction method according to claim 1, characterized in that: The complete culture medium of granulosa cells in step 3 includes the following components in volume: 89 parts of MEM basal medium, 10 parts of fetal bovine serum and 1 part of double antibody.

5. The construction method according to claim 1, characterized in that: The specific operation of step 4 is as follows: when the density of ovarian granulosa cells reaches 80-90%, remove the culture medium, wash with PBS, add 0.25% trypsin, and digest at 37°C for 5 minutes; terminate the digestion with an equal volume of granulosa cell complete culture medium, centrifuge at 1000r / min for 5 minutes; discard the supernatant, resuspend the cells with granulosa cell complete culture medium, and centrifuge at 3×10 5 Cells were inoculated into culture dishes, the cell density was observed under a microscope, and the culture medium was replaced every other day.

6. The construction method according to claim 1, characterized in that: The specific operations of step five are: ①Virus packaging: The culture medium of 293T cells was discarded and washed with serum-free basal medium, starved for 30 minutes, and opti-MEM was pipetted into 2 sterile centrifuge tubes, which were marked as lipo tube and plasmid tube respectively; lipofectamine 2000 was added to the lipo tube and vortexed thoroughly to mix, and SV40T lentiviral plasmid, pSPAX2 and pMD2.G two auxiliary plasmids were added to the plasmid tube at a mass ratio of 10:2:1; the diluted lipofectamine 2000 in the lipo tube was pipetted out and added to the plasmid tube, and gently mixed by pipetting up and down, and allowed to stand at room temperature for 10 minutes to form a lipo-plasmid mixture; the lipo-plasmid mixture was gently dripped into the culture dish; fresh culture medium was replaced after 5 hours of culture, and culture was continued for 24 hours; The supernatant was collected and filtered with a 0.45 μm filter membrane to obtain the lentiviral venom; ②Viral transduction: After subculturing, 3 × 10 ovarian granulosa cells were cultured per dish. 6 Cells were inoculated into culture dishes; virus infection was performed after overnight culture, starvation treatment was performed for 30 min, the basal culture medium was discarded and lentiviral venom was added, and complete culture medium containing 2 μM GSK 3Inhibitor IX (BIO) was added after culturing for 2 h, and then subculture was continued until the cell proliferation ability was improved and immortalization was achieved.

7. The construction method according to claim 1, characterized in that: The ovarian granulosa cells are ovarian granulosa cells of Shaanxi white cashmere goats.

8. The ovarian granulosa cell line constructed by the construction method according to any one of claims 1 to 7.

9. Use of the ovarian granulosa cell line according to claim 8 in establishing an in vitro research model of ovarian function.

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