Construction of immortalized ovine granulosa cell model and its application in preparation of estradiol in vitro

Abstract

The application discloses a kind of sheep ovary granulosa cell immortalization model construction and its application in preparation of estradiol in vitro, belong to cell engineering technical field. Including the following steps: (1) from sheep ovary follicular fluid primary granulosa cell is separated;(2) using the lentivirus carrying SV40T-GFP transfection the primary granulosa cell;(3) by puromycin screening obtain stable expression SV40T-GFP immortalized granulosa cell line.The application utilizes sheep ovary granulosa cell (GCs) and follicle membrane cell (TCs) co-culture system, successfully establishes and optimizes in vitro estradiol (E2) production platform.Sheep granulosa cell immortalization model construction, the establishment of in vitro co-culture system and bioactive estradiol preparation and mouse verification etc. multiple levels, all verified the possibility of sheep granulosa cell immortalization and in vitro production of estradiol.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, and in particular to the construction of an immortalized sheep ovarian granulosa cell model and its application in the in vitro preparation of estradiol. Background Technology

[0002] Estradiol, as a natural estrogen, has always played a crucial role in the reproductive regulation, bone health, cardiovascular protection, and maintenance of neuroendocrine homeostasis in female animals. Currently, estradiol production mainly relies on traditional chemical synthesis or animal tissue extraction. However, the chemical synthesis route is highly complex, prone to producing inactive isomers, and can also impose significant environmental burdens. On the other hand, animal-derived extraction faces challenges such as yield fluctuations, pathogen contamination, and animal ethics issues. These limitations have prompted researchers to actively seek green and sustainable alternatives for in vitro biosynthesis. Summary of the Invention

[0003] The purpose of this invention is to provide a model for the immortalization of sheep ovarian granulosa cells and its application in the in vitro preparation of estradiol, so as to solve the problems existing in the prior art.

[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for constructing an immortalized sheep ovarian granulosa cell line, comprising the following steps: (1) Isolation of primary granulosa cells from sheep ovarian follicular fluid; (2) The primary granular cells were transfected with a lentivirus carrying SV40T-GFP; (3) Immortalized granular cell lines that stably express SV40T-GFP were obtained by screening with puromycin.

[0005] The second technical solution of the present invention is a method for in vitro synthesis of estradiol, comprising the following steps: (1) The immortalized sheep ovarian granulosa cell line constructed by the above construction method is co-cultured with sheep ovarian theca cells; (2) Add follicle-stimulating hormone and luteinizing hormone to the co-culture system; (3) Collect the co-culture supernatant, extract and concentrate to obtain estradiol.

[0006] The third technical solution of the present invention is an immortalized sheep ovarian granulosa cell line, which is constructed by the aforementioned construction method. This cell line stably expresses SV40T antigen and green fluorescent protein, and retains the activity of CYP19A1, a key enzyme for the synthesis of estradiol.

[0007] The fourth technical solution of the present invention is the application of the immortalized sheep ovarian granulosa cell line in the preparation of estradiol.

[0008] Based on the above technical solution, the present invention has the following technical effects: This invention successfully established and optimized an in vitro estradiol (E2) production platform using a co-culture system of sheep ovarian granulosa cells (GCs) and theca cells (TCs). The construction of an immortalized sheep granulosa cell model, the establishment of the in vitro co-culture system, and the preparation and mouse validation of bioactive estradiol all validated the feasibility of sheep granulosa cell immortalization and in vitro estradiol production. This invention not only provides a new approach for reproductive biology research in sheep and other large livestock, but also opens up an innovative technological path for the large-scale preparation of fully bioactive natural estradiol without relying on chemical synthesis or animal slaughter. This invention is expected to demonstrate certain theoretical value and application potential in basic reproductive endocrinology research, livestock breeding technology improvement, and the biopharmaceutical field. Attached Figure Description

[0009] Figure 1 To isolate and culture sheep granulosa cells in follicular fluid with a diameter of 3-8 mm.

[0010] Figure 2 This is for the determination of primary cell growth density curves.

[0011] Figure 3 Immunofluorescence staining is used for the identification of GCs.

[0012] Figure 4 This section describes the quantification of the fold change in infection (MOI). A represents the results observed under fluorescence microscopy. B represents the quantitative statistics of transfection efficiency. Different MOI concentrations were set at 1, 10, 50, and 100. When MOI = 10, the transfection efficiency reached 90%, which was suitable for experimental use (P < 0.05).

[0013] Figure 5 The images show the fluorescence characteristics of the first generation after transfection with SV40T-GFP lentivirus. In the images, A represents a standard microscope image, and B represents a fluorescence microscope image.

[0014] Figure 6 To verify the transfection status of SV40T-GFP-GCs lentivirus using q-PCR (P<0.05).

[0015] Figure 7 The study included the detection of cell morphology and SV40T-GFP virus fluorescence in generations 1, 10, 25, and 50.

[0016] Figure 8The growth density curves of primary cells and immortalized cells of the 1st and 50th generations were determined every 12 hours.

[0017] Figure 9 The proliferation activity of transfected SV40T-GFP-GCs was detected (P<0.05). In the diagram, (A) represents flow cytometry. (B) indicates that compared to GCs-1, the proportion of S+G1 phase cells in the first and tenth generation SV40T-GFP-GCs after transfection was significantly increased, indicating significantly enhanced proliferation activity. (C) represents the number of cells in the G1 phase.

[0018] Figure 10 The following assays were performed to detect estradiol production in transfected SV40T-GFP-GCs cells (P<0.05). Specifically, (A) compared to GCs-1, estradiol levels were significantly increased in the SV40T-GFP-GCs-10 and SV40T-GFP-GCs-25 groups, while no significant change was observed in the SV40T-GFP-GCs-50 group. (B) The expression level of CYP19A1 at different passages after transfection was detected by q-PCR (P<0.05). (C) The expression level of CYP19A1 at different passages after transfection was detected by Western blotting (P<0.05).

[0019] Figure 11 For cell chromosome karyotype analysis.

[0020] Figure 12 For the detection of functional standards for immortalized GCs cells.

[0021] Figure 13 Molecular assays were performed to detect cell function markers (P<0.05). Specifically, (A-B) Western blotting was used to determine whether transfected 50th generation FSHR cells would exhibit a physiological response to different concentrations of E2 (P<0.05). (C) q-PCR was used to determine whether transfected 50th generation FSHR cells would exhibit a physiological response to different concentrations of E2 (P<0.05).

[0022] Figure 14 Mice transfected with SV40T-GFP-GCs were not tumorigenic (P<0.05). (A) Cell morphology and (B) Growth density were also considered. (CE) Mice inoculated with HELA cells showed obvious tumors, while GCs-1 and SV40T-GFP-GCs-50 cells showed no tumors. (FH) MDSC, PDL1, and CD68 cells were significantly increased (P<0.05).

[0023] Figure 15 For the isolation and culture of TC cells. Among them, (A) collection of sheep ovaries. (B) culture of TC cells.

[0024] Figure 16 Identification of TCs and SV40T-GFP-GCs-50 cells.

[0025] Figure 17 Cell co-culture significantly increased estradiol production. Specifically, (A) the culture and identification of TCs and GCs; (BC) cell co-culture significantly enhanced CPY19A1 expression in granulosa cells; and (D) cell co-culture significantly increased estradiol production.

[0026] Figure 18 To improve estradiol concentration through extraction and rotary evaporation. (AB) Screening for maximum FSH and LH concentrations. (CD) Addition of FSH and LH significantly increased CPY19A1 expression. (E) Addition of FSH and LH significantly increased estradiol yield.

[0027] Figure 19 To increase estradiol concentration through extraction and rotary evaporation. Where A represents a standard for industrial-scale E2 production. B represents the concentration after extraction. C represents the concentration after extraction and rotary evaporation. D represents a significant increase in concentration after extraction and rotary evaporation.

[0028] Figure 20 The effects of estradiol concentrations after extraction, concentration, and rotary evaporation on physiological changes in mice were investigated. Specifically, (A) estradiol produced after extraction, concentration, and rotary evaporation significantly increased estradiol levels in mouse blood. (BI) estradiol produced significantly enhanced the expression of CPY19A1 and AR in mouse testes, while attenuating ER expression. Detailed Implementation

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0030] This invention provides a method for constructing an immortalized sheep ovarian granulosa cell line, comprising the following steps: (1) Isolation of primary granulosa cells from sheep ovarian follicular fluid; (2) The primary granular cells were transfected with a lentivirus carrying SV40T-GFP; (3) Immortalized granular cell lines that stably express SV40T-GFP were obtained by screening with puromycin.

[0031] In some specific implementations, the follicular fluid is derived from sheep cavitary follicles with a diameter of 3-8 mm.

[0032] In some specific implementation schemes, the transfection method is as follows: when the granulocyte cell adhesion rate is ≥85%, SV40T-GFP lentivirus solution is transfected into the granulocyte cells at MOI=10, and the infection is carried out for 16 hours.

[0033] This invention also provides a method for in vitro synthesis of estradiol, comprising the following steps: (1) The immortalized sheep ovarian granulosa cell line constructed by the above method is co-cultured with sheep ovarian theca cells; (2) Add follicle-stimulating hormone and luteinizing hormone to the co-culture system; (3) Collect the co-culture supernatant, extract and concentrate to obtain estradiol.

[0034] In some specific implementations, in step (2), the concentration of follicle-stimulating hormone added is 5 IU / mL, and the concentration of luteinizing hormone added is 1 ng / mL.

[0035] In some specific implementations, in step (3), the method for extracting estradiol is to use ether to perform liquid-liquid extraction on the culture supernatant; The method for concentrating estradiol is to perform rotary evaporation on the extract.

[0036] This invention also provides an immortalized sheep ovarian granulosa cell line, constructed by the aforementioned method. This cell line stably expresses SV40T antigen and green fluorescent protein, and retains the activity of CYP19A1, a key enzyme in estradiol synthesis.

[0037] This invention also provides the application of the immortalized sheep ovarian granulosa cell line in the preparation of estradiol.

[0038] Example 1 Establishment of immortalized sheep ovarian granulosa cells (GCs) 1. Laboratory animals In Tianzhu County, Gansu Province, ovarian tissue was collected from adult sheep around two years old at 8:30 AM at the livestock and poultry slaughter and quarantine facility. After opening the abdominal cavity at the slaughterhouse, the ovaries without lesions were immediately removed using sterile scissors. They were sprayed with 37°C alcohol and then rinsed with 37°C sterile PBS to remove surface blood. The intact ovaries were then quickly placed in a constant-temperature container filled with 37°C physiological saline containing 100 IU / mL penicillin and 100 IU / mL streptomycin, and transported back to the laboratory of the School of Life Sciences and Engineering at the Yuzhong Campus of Northwest University for Nationalities within two hours.

[0039] 2. Test Methods 2.1 Pre-test treatment The sheep ovaries were repeatedly washed three times with alcohol and 37°C physiological saline containing 100 IU / mL penicillin and 100 IU / mL streptomycin, for 10 seconds each time. The tissue was then transferred to a sterile culture dish. The mesentery surrounding the ovary was trimmed using autoclaved scissors, and the ovarian tissue was washed three more times with alcohol and 37°C physiological saline containing 100 IU / mL penicillin and 100 IU / mL streptomycin, for 10 seconds each time. The ovarian tissue was then transferred to a beaker, and ovaries with large follicles were identified.

[0040] 2.2 GCs isolation and culture Carefully puncture cavitary follicles with a diameter of 3-8 mm in the ovary using a 0.2 μm needle. Filter the follicular fluid through 70 and 100 mesh cell filters. Add the filtered cells to a suitable amount of DMEM / F12 culture medium containing 5 ml of 15% FBS. Pour the culture medium into a T25 culture flask, shake it evenly, and place it in a cell culture incubator with 5% carbon dioxide.

[0041] 2.3 Cell counting chamber method Cell proliferation dynamics were systematically monitored over 96 hours using a cell counting chamber method. Specifically, synchronized granulosa cells were cultured in DMEM / F12 complete medium, and samples were taken every 12 hours starting from 0 h. Cells were digested with trypsin and stained with trypan blue. Viable cells were counted using a modified Neubauer counting chamber under an inverted microscope. Three replicates were set up at each time point to reduce error. Finally, cell density was calculated, growth curves were plotted, and population doubling time was calculated.

[0042] 2.4 Immunofluorescence staining After digesting and centrifuging primary GCs, the cells were evenly seeded into 24-well plates. Staining was initiated when cells reached 75%-85% adherence after 3-4 days. The specific steps for immunofluorescence staining were as follows: Step 1: Fixation: Remove the complete cell culture medium and gently wash three times with PBS buffer, 2-3 min each time. Then, add 250 μL of paraformaldehyde and fix the cells for 30 min. Discard the fixative and wash three times with PBS buffer, 2-3 min each time. Step 2: Permeabilization: Add 250 μL of 0.2% Triton X-100 and incubate at room temperature for 15-20 min. Then, remove the liquid and gently wash three times with PBS buffer, 2-3 min each time. Step 3: Blocking and Primary Antibody Incubation: Add diluted primary antibody working solution to each well, and add an equal volume of PBS buffer to the negative control wells. Incubate at 4°C for 12 h, and wash three times with PBS buffer, 5-7 min each time. The fourth step is secondary antibody incubation: Add 500 μL of diluted secondary antibody working solution to each well, then place in a dark room and incubate at room temperature for 3 hours. Wash three times with PBS buffer, 5-7 min each time. The fifth step is DAPI staining: Add 200 μL of DAPI staining solution to each well in a dark room, incubate at room temperature for 1 min, then wash three times with PBS buffer, 5-7 min each time. The sixth step is observation and photography: Observe and photograph using a fluorescence microscope in a dark room.

[0043] 2.5 Validation of GFP-GCs (transfection fold) in sheep granular cells On day 1, SV40T sheep ovarian granulosa cells were seeded into 12-well plates containing 1E5 cells. The following day, the GFP virus stock solution (Cegrogen Shanghai) was removed from the -80°C freezer and thawed on ice before infection. The GFP virus stock solution was diluted with 800 μL of complete culture medium with MOIs of 1, 10, 50, and 100, and the original culture medium for the treatment groups was removed. 500 μL of culture medium containing lentivirus dilution was added to the cells in the treatment groups. On day 3, the culture medium was changed, and 16 h after infection, the lentivirus-containing culture medium was replaced with 1 mL of complete culture medium. On day 5, the infection efficiency was assessed. Fluorescence was observed under an inverted fluorescence microscope, and the effect of lentivirus infection on target cells was calculated based on the fluorescence intensity.

[0044] 2.6 SV40T-GFP-GCs Lentiviral Transfection On day 1, SV40T sheep ovarian granulosa cells were seeded into 12-well plates containing 1E5 cells. When the cells reached 80% saturation, the viral stock solution was removed from the -80°C freezer and thawed in an ice bath. The viral stock solution was diluted with 80 μL of complete culture medium at an MOI of 10, and then the original culture medium in the treatment group was removed. 500 μL of culture medium containing the lentivirus dilution was added to the treatment group cells. On day 3, the culture medium was changed, and 16 h after infection, the culture medium containing lentivirus was replaced with 1 mL of complete culture medium. Then, on day 5, the infection efficiency was assessed, and fluorescence was observed under an inverted fluorescence microscope to determine the effect of lentivirus infection on target cells. Suitable eukaryotic resistance cells were selected for selection. Cells were screened twice with commonly used drugs (once every 2 days, antibiotics determined in preliminary experiments). Cells were stably passaged. After stable passage, cells were maintained using DMEM + 10% FBS + 1% PS + 1.5 μg / mL puromycin complete culture medium.

[0045] 2.7 RNA extraction, reverse transcription, and quantitative RT-PCR RNA extraction: Cells were digested with trypsin and seeded into 6-well plates. The culture medium was discarded, and the cells were washed three times slowly with PBS. Then, 1 mL of TRIzol was added for lysis for 5-7 min. After thorough mixing, the cells were transferred to a 1.5 mL enzyme-free centrifuge tube. 0.2 mL of chloroform was added, and the tube was vigorously vortexed approximately 100 times. The tube was incubated at room temperature for 3 min, then centrifuged at 10000×g for 15 min at 4°C. Approximately 300-400 μL of the supernatant was carefully transferred to a new enzyme-free centrifuge tube. 0.5 mL of isopropanol was added, and the tube was inverted 100 times. The tube was incubated at room temperature for 10 min. The tube was then centrifuged at 10000×g for 10 min at 4°C. The supernatant was discarded, and the milky white precipitate was RNA. 1 mL of 75% ethanol was added, and the tube was centrifuged at 7500×g for 5 min at 4°C. The supernatant was discarded. The tube was dried at room temperature for 5 min, and 25 μL of LEPC water was added. The mixture was gently swirled and incubated at room temperature for 5 min to dissolve the RNA. Total RNA concentration was determined using an ultra-micro spectrophotometer. RNA with an A260 / 280 ratio between 1.9 and 2.2 yielded the best results. RNA with excessively high concentrations was then diluted appropriately to a concentration of 100-200 ng / μL. If not intended for immediate use, the RNA was stored at -80°C for later use.

[0046] cDNA preparation: Following the reverse transcription kit procedure, first remove genomic DNA, then reverse transcribe to prepare cDNA. The genomic DNA removal procedure is shown in Table 1. Place the mixed system into a PCR instrument and react according to the program "42℃ for 2 min, 4℃". The reverse transcription procedure is shown in Table 2. First, place the mixed system into a PCR instrument and react according to the program "37℃ for 15 min, 85℃ for 5 s, 4℃". Finally, store the cDNA prepared by reverse transcription at -20℃ for later use.

[0047] Table 1. Genomic DNA Removal Procedure

[0048] Table 2 Reverse transcription operation system

[0049] Primer design: Gene sequences were first queried using the NCBI gene database, and then primer sequences were designed using Primer 6.0 software. The primer sequences are shown in Table 3. The primers used in the experiment were synthesized by Beijing BGI Genomics Co., Ltd.

[0050] Table 3 Primer Sequences

[0051] RT-PCR: Prepare the following reaction system using 0.2 mL of 8-tube PCR apparatus. See Table 4 for the reaction system details. Amplify the reaction using a two-step procedure: "pre-denaturation at 95℃ for 30 s, PCR reaction at 95℃ for 5 s, 60℃ for 20 s, 40 cycles, followed by melting curve analysis at 66℃ for 15 s". Use the GAPHD gene as the internal reference gene for RT-PCR. -△△Ct The method is used to determine the relative expression level of the target gene.

[0052] Table 4. Quantitative Real-Time PCR Operating System

[0053] 2.7 Data Statistics and Analysis All experiments were repeated three times, with three biological replicates per experiment. All data were analyzed using standard one-way ANOVA with GraphPadPrism 8.0.2 software, and are presented as mean ± standard deviation. P A value >0.05 indicates that the difference is not significant. P A value <0.05 indicates a significant difference.

[0054] 3. Experimental Results 3.1 Isolation and culture of GCs like Figure 1 As shown, follicles of approximately 3-8 mm in diameter were selected from ovaries with a diameter of 1-2 cm and punctured with a syringe. After filtration and culture for 96 hours, the morphology was typically spindle-shaped. Subsequent experiments were conducted when the adhesion rate was ≥85%.

[0055] 3.2 Determination of GC growth curves like Figure 2As shown, this study used a cell counting chamber method to systematically monitor cell proliferation dynamics over 96 hours. Sampling was performed every 12 hours, starting from 0 h. Biological and technical replicates were set up at each time point. Finally, cell density was calculated, growth curves were plotted, and population doubling time was calculated.

[0056] 3.3 Identification of GCs like Figure 3 As shown in the immunofluorescence staining image, the FSHR protein of granulocytes was positive, confirming that the isolated cells were GCs, which met the requirements for subsequent experiments.

[0057] 3.4 Validation of GFP-GCs (infection fold) in sheep granular cells like Figure 4 As shown, the GFP virus stock solution (Cegrogen Shanghai) was diluted to four gradients with MOIs of 1, 10, 50, and 100. Fluorescence was observed under an inverted fluorescence microscope; the efficiency reached 90% at MOI=10. This solution can be used for subsequent experiments.

[0058] 3.5 SV40T-GFP-GCs Lentiviral Transfection like Figure 5 As shown, sheep ovarian granulosa cells (GCs) were seeded into 12-well plates with a cell density greater than 70% and transfected with SV40T-GFP (MOI=10) virus. The results showed that the transfected GCs exhibited significant green fluorescence, confirming successful transfection of SV40T-GFP virus into GCs cells.

[0059] 3.6 q-PCR verification of GCs-SV40T-GFP lentiviral transfection like Figure 6 As shown, qRT-PCR was used to further verify the transfection status of SV40T–GFP virus. The results showed that untransfected GCs did not contain SV40T–GFP, while the transfected SV40T-GFP-GCs-1 showed significant expression.

[0060] This embodiment successfully established an immortalized sheep ovarian granulosa cell line GCs-SV40T-GFP using lentiviral transduction of the GFP-SV40T antigen. This cell line exhibits stable proliferation, typical morphology, and continuous green fluorescence expression, overcoming the limitations of primary cell passage and providing a reliable in vitro model for subsequent research.

[0061] Example 2 Biological function verification of immortalized GCs 1. Laboratory animals Nine male nude mice (from Lanzhou Veterinary Research Institute) at 6 weeks of age were divided into three groups: GCs-1, SV40T-GFP-GCs-50, and HELA cells, with three mice in each group.

[0062] 2. Test Methods 2.1 Passaging and Fluorescence Identification of Immortalized GCs When cells reached 80%-90% confluence in 12-well culture dishes, aseptic procedures were performed in a laminar flow hood. The culture medium was discarded, and the cell surface was gently washed three times with PBS buffer (37°C). The PBS was removed, and 0.25% trypsin digestion solution was added. The cells were incubated at 37°C for 2-3 minutes. Observation under an inverted microscope was performed until the cells became rounded and the intercellular spaces increased. Immediately, an equal volume of complete culture medium was added to stop the digestion. The cells were gently agitated with a pipette to completely detach from the bottom of the dish, forming a single-cell suspension. This suspension was transferred to a centrifuge tube and centrifuged at 1000-1200 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in complete culture medium containing 1.5 μg / mL puromycin. The suspension was aliquoted into new culture dishes at a ratio of 1:2 to 1:3, and fresh culture medium was added. The cells were gently shaken and incubated at 37°C, 5% CO2, and saturated humidity. Cell morphology and GFP fluorescence expression were observed after passage.

[0063] 2.2 Cell counting chamber method Primary first-generation immortalized GCs were cultured in vitro at passage 50, and cell proliferation dynamics were systematically monitored over 96 hours using a cell counting chamber. Specifically, synchronized granulocytes were cultured in DMEM / F12 complete medium, and samples were taken every 12 hours starting from 0 h. Trypsin digestion and trypan blue staining were performed, and viable cells were counted using a modified Neubauer counting chamber under an inverted microscope. Triple replicates were set up for each time point. Cell density was calculated, growth curves were plotted, and cell doubling time was calculated.

[0064] 2.3 Flow cytometry GCs-1 and various passages of SV40T-GFP-GCs cells were seeded at appropriate densities in 6-well plates. 2 mL of complete culture medium containing 10% fetal bovine serum was added to each well. Cells were incubated at 37°C with 5% CO2 for 48 hours until 90% confluence was achieved. The culture medium was then aspirated, and the cells were washed twice with pre-chilled PBS. Simultaneously, 0.25% trypsin was added for digestion, and the cell suspension was collected. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in pre-chilled PBS and counted, yielding approximately 1 × 10⁶ cells. 6Centrifuge the cells again and discard the supernatant. Add 1 mL of pre-chilled 70% ethanol and fix overnight (at least 2 hours) at 4°C. After fixation, centrifuge to remove ethanol, wash the cells twice with PBS, and resuspend the cells in 500 μL of PBS. Add 50 μL of RNase A (final concentration 50 μg / mL) and 50 μL of propidium iodide (PI) staining solution (final concentration 50 μg / mL) sequentially, gently pipette to mix, and then incubate at 37°C in the dark for 30 minutes. Filter the stained cell suspension through a 300-mesh nylon mesh into a dedicated flow cytometry tube for analysis. Detect the PI fluorescence signal (excitation wavelength 488 nm, emission wavelength 617 nm) using flow cytometry, and analyze the distribution ratio of each cell cycle phase (G0 / G1, S, G2 / M) using ModFit or FlowJo software. Collect at least 1 × 10⁶ cells per sample. 4 One cell, the experiment was repeated three times.

[0065] 2.4 Immunoblotting detection First, protein samples fshr, AR, er, and CYP19A1 were prepared, and then gel electrophoresis, membrane transfer, blocking, antibody incubation, and chemiluminescence detection were performed sequentially.

[0066] 2.5 RNA extraction, reverse transcription, and quantitative RT-PCR RNA extraction: Cells were digested with trypsin and seeded into 6-well plates. The culture medium was discarded, and the cells were washed three times slowly with PBS. Then, 1 mL of TRIzol was added for lysis for 5-7 min. After thorough mixing, the cells were transferred to a 1.5 mL enzyme-free centrifuge tube. 0.2 mL of chloroform was added, and the tube was vigorously vortexed approximately 100 times. The tube was incubated at room temperature for 3 min, then centrifuged at 10000×g for 15 min at 4°C. Approximately 300-400 μL of the supernatant was carefully transferred to a new enzyme-free centrifuge tube. 0.5 mL of isopropanol was added, and the tube was inverted 100 times. The tube was incubated at room temperature for 10 min. The tube was then centrifuged at 10000×g for 10 min at 4°C. The supernatant was discarded, and the milky white precipitate was RNA. 1 mL of 75% ethanol was added, and the tube was centrifuged at 7500×g for 5 min at 4°C. The supernatant was discarded. The tube was dried at room temperature for 5 min, and 25 μL of DEPC water was added. The mixture was gently swirled and incubated at room temperature for 5 min to dissolve the RNA. Total RNA concentration was determined using an ultra-micro spectrophotometer; RNA with an A260 / 280 ratio between 1.9 and 2.2 yielded the best results. RNA with excessively high concentrations was then diluted appropriately to a concentration of 100-200 ng / μL. If not intended for immediate use, the RNA was stored at -80°C for later use.

[0067] cDNA preparation: Following the reverse transcription kit procedure, first remove genomic DNA, then reverse transcribe to prepare cDNA. The genomic DNA removal procedure is shown in Table 5. Place the mixed system into a PCR instrument and react according to the program "42℃ for 2 min, 4℃". The reverse transcription procedure is shown in Table 6. First, place the mixed system into a PCR instrument and react according to the program "37℃ for 15 min, 85℃ for 5 s, 4℃". Finally, store the cDNA prepared by reverse transcription at -20℃ for later use.

[0068] Table 5. Genomic DNA Removal Procedure

[0069] Table 6 Reverse transcription operation system

[0070] Primer design: Gene sequences were retrieved from the NCBI gene database, and primer sequences were designed using Primer 6.0 software. The primer sequences are shown in Table 7. The primers used in the experiment were synthesized by Beijing BGI Genomics Co., Ltd.

[0071] Table 7 Primer Sequences

[0072] RT-PCR: Prepare the following reaction system using 0.2 mL of 8-tube PCR apparatus. See Table 8 for the reaction system details. Amplify the reaction using a two-step procedure: "pre-denaturation at 95℃ for 30 s, PCR reaction at 95℃ for 5 s, 60℃ for 20 s, 40 cycles, followed by melting curve analysis at 66℃ for 15 s". Use the GAPHD gene as the internal reference gene for RT-PCR. -△△Ct The method is used to determine the relative expression level of the target gene.

[0073] Table 8. Quantitative Real-Time PCR Operating System

[0074] 2.6 Karyotype Analysis Primary, 10th, 25th, and 50th generation cells were selected for karyotype analysis. Cells in logarithmic growth phase were seeded in 6-well plates and treated with 1 μg / mL colchicine for 4–6 h to arrest cells in metaphase by inhibiting spindle formation. Cells were collected after trypsin digestion and centrifuged at 1000 rpm for 8 min. The pellet was resuspended in pre-warmed 0.075 M KCl hypotonic solution and incubated at 37°C for 20 min. After centrifugation, the supernatant was discarded, and cells were fixed in freshly prepared methanol:glacial acetic acid (3:1) fixative at room temperature for 30 min. The fixation process was repeated twice, and cells were then resuspended in fresh fixative. The cell suspension was dropped onto pre-chilled slides and air-dried. Sections were stained with Giemsa solution for 10–15 min, rinsed with distilled water, and air-dried. Chromosome morphology was observed under an optical microscope (100 × oil-immersion eyepiece). At least 50 complete metaphase diffusions were analyzed for each sample, and the chromosome number was determined.

[0075] 2.7 Immunofluorescence staining After digesting and centrifuging primary GCs, the cells were evenly seeded into 24-well plates. Staining was initiated when cells reached 75%-85% adherence after 3-4 days. The specific steps for immunofluorescence staining were as follows: Step 1: Fixation: Remove the complete cell culture medium and gently wash three times with PBS buffer, 2-3 min each time. Then, add 250 μL of paraformaldehyde and fix the cells for 30 min. Discard the fixative and wash three times with PBS buffer, 2-3 min each time. Step 2: Permeabilization: Add 250 μL of 0.2% Triton X-100 and incubate at room temperature for 15-20 min. Then, remove the liquid and gently wash three times with PBS buffer, 2-3 min each time. Step 3: Blocking and Primary Antibody Incubation: Add diluted primary antibody working solution to each well, and add an equal volume of PBS buffer to the negative control wells. Incubate at 4°C for 12 h, and wash three times with PBS buffer, 5-7 min each time. The fourth step is secondary antibody incubation: Add 500 μL of diluted secondary antibody working solution to each well, then place in a dark room and incubate at room temperature for 3 hours. Wash three times with PBS buffer, 5-7 min each time. The fifth step is DAPI staining: Add 200 μL of DAPI staining solution to each well in a dark room, incubate at room temperature for 1 min, then wash three times with PBS buffer, 5-7 min each time. The sixth step is observation and photography: Observe and photograph using a fluorescence microscope in a dark room.

[0076] 2.8 HALA cell culture Frozen HALA cells were rapidly thawed at 37°C, and 5 ml of DMEM high-glucose medium (fetal bovine serum (FBS): penicillin-streptomycin = 89:10:1) was added. The cells were then placed in a cell culture incubator (37°C, 5% CO2, saturated humidity). Experiments were conducted when the cells reached 80% adherence in the culture dish.

[0077] 2.9 Mouse tumorigenesis experiment Nine 5-week-old male BALB / c nude mice were purchased from the Lanzhou Veterinary Research Institute and randomly divided into three groups of three. After one week of acclimatization culture, they were inoculated with primary granulosa cells, 50th generation immortalized granulosa cells, and HeLa cells, respectively. The cells were placed in two T25 culture flasks and cultured in DMEM medium containing 20% ​​fetal bovine serum. The incubator was set at 37°C with 5% carbon dioxide. Cell growth continued until it exceeded 98%. After trypsin digestion, the cell density was adjusted to 1 × 7 × 10⁶ cells per passage. 6 Tumor size was measured at 10 μL per 10 mL cell count and injected subcutaneously into the left back of mice. Tumor size was monitored using a ruler after 4 weeks of continuous observation.

[0078] 2.10 Data Statistics and Analysis All experiments were repeated three times, with three biological replicates per experiment. All data were analyzed using standard one-way ANOVA with GraphPadPrism 8.0.2 software. Data are expressed as mean ± standard deviation. P > 0.05 indicated no significant difference, and P < 0.05 indicated a significant difference.

[0079] 3 Results 3.1 Successful isolation and identification of GCs cells like Figure 7 As shown, immortalized cells were passaged, and morphological examination and SV40T-GFP virus fluorescence content were detected at passages 1, 10, 25, and 50. Fluorescence was still observed to be well-expressed at passage 50.

[0080] 3.2 Determination of growth curves of immortalized GCs like Figure 8 As shown, cell proliferation dynamics were systematically monitored over 96 hours using a cell counting chamber. Cell density was calculated and growth curves were plotted. It was observed that the growth rate of transfected cells was significantly faster than that of primary cells.

[0081] 3.3 The viability of SV40T-GFP-GCs cells was significantly enhanced after transfection. like Figure 9As shown, cell cycle was detected using flow cytometry. From passage 1 onwards, the S and G1 phases of SV40T-GFP-GCs cells significantly increased in passage 10. The S and G1 phases of cells in passages 25 and 50 also showed a slight increase relative to GCs (P<0.05).

[0082] 3.4 Detection of estradiol production in transfected SV40T-GFP-GCs cells like Figure 10 As shown, the estradiol content in SV40T-GFP-GCs-10 and SV40T-GFP-GCs-25 was significantly higher than that in GCs-1, as detected by ELISA. The CYP19A1 expression levels in SV40T-GFP-GCs-10 and SV40T-GFP-GCs-25 were also significantly higher than those in GCs-1, as detected by Western blotting and q-PCR.

[0083] 3.5 Karyotype analysis of immortalized cells like Figure 11 As shown, chromosome karyotype analysis was performed on primary cells and at passages 10, 25, and 50 after transfection. The results showed that cells at passages 10, 25, and 50 maintained normal chromosome morphology compared to the primary cells.

[0084] 3.6 Immunofluorescence detection of cell functional markers like Figure 12 As shown in the immunofluorescence imaging results, the 50th generation immortalized GCs cells continued to show high expression of key granulocyte cell functional markers FSHR and CYP19a1.

[0085] 3.7 Molecular experimental detection of cell functional markers like Figure 13 As shown, Western blot and quantitative PCR analyses confirmed that FSHR protein expression was significantly upregulated with increasing exogenous estradiol concentration.

[0086] 3.8 Culture of HELA cells HELA cells were passaged and cultured, and their morphology was typically oval.

[0087] 3.9 Tumorigenicity test in mice like Figure 14As shown, GCs-1, SV40T-GFP-GCs-50, and HELA cells were used as controls. After 4 weeks of culture, HeLa cells showed obvious tumors. GCs-1 and SV40T-GFP-GCs-50 cells did not show tumors. Flow cytometry analysis of mouse blood revealed no significant changes in the levels of MDSC, PDL1, and CD68 cells in GCs-1 and SV40T-GFP-GCs-50 cells, but a significant increase in HELA levels.

[0088] This embodiment demonstrates that the sheep ovarian granulosa cells prepared by the present invention retain the morphology of the primary cells, and the immortalized cells retain endocrine function and nuclear shape, and do not have tumorigenicity.

[0089] Example 3 Study on estradiol synthesis using an in vitro co-culture system of immortalized sheep follicular granulosa cells and theca cells. 1. Laboratory animals Fifteen male BALB / c mice were used.

[0090] 2. Test Methods 2.1 Isolation and Culture of Theca Cells (TCs) Ovaries from healthy sheep around 2 years old were collected from a slaughterhouse in Tianzhu County, Gansu Province. The ovaries were then placed in a 37°C incubator and transported to the laboratory within 2 hours. The preservation solution consisted of 500 mL of physiological saline with 0.48 g of penicillin-streptomycin. The outer membrane of the follicle was carefully dissected with forceps, and the follicle was cut open. The inner follicle wall was then gently scraped away to remove granulosa cells. The remaining tissue was fragmented as much as possible and digested with collagenase at 37°C for 20 min. The mixture was filtered through 100 μm and 70 μm cell filters, and the filtrate was centrifuged at 1500 rpm for 10 min. 10 mL of DMEM medium containing 10% fetal bovine serum was added, and the mixture was incubated at 37°C with 5% CO2 saturated humidity. Cell morphology was monitored periodically under a microscope.

[0091] Three days after culturing theca cells, immortalized sheep granulosa cells were added and cultured together for 24 hours in a 37°C, saturated humidity, 5% CO2 incubator.

[0092] 2.2 Immunofluorescence staining After digesting and centrifuging primary GCs, the cells were evenly seeded into 24-well plates. Staining was initiated when cells reached 75%-85% adherence after 3-4 days. The specific steps for immunofluorescence staining were as follows: Step 1: Fixation: Remove the complete cell culture medium and gently wash three times with PBS buffer, 2-3 min each time. Then, add 250 μL of paraformaldehyde and fix the cells for 30 min. Discard the fixative and wash three times with PBS buffer, 2-3 min each time. Step 2: Permeabilization: Add 250 μL of 0.2% Triton X-100 and incubate at room temperature for 15-20 min. Then, remove the liquid and gently wash three times with PBS buffer, 2-3 min each time. Step 3: Blocking and Primary Antibody Incubation: Add diluted primary antibody working solution to each well, and add an equal volume of PBS buffer to the negative control wells. Incubate at 4°C for 12 h, and wash three times with PBS buffer, 5-7 min each time. The fourth step is secondary antibody incubation: Add 500 μL of diluted secondary antibody working solution to each well, then place in a dark room and incubate at room temperature for 3 hours. Wash three times with PBS buffer, 5-7 min each time. The fifth step is DAPI staining: Add 200 μL of DAPI staining solution to each well in a dark room, incubate at room temperature for 1 min, then wash three times with PBS buffer, 5-7 min each time. The sixth step is observation and photography: Observe and photograph using a fluorescence microscope in a dark room.

[0093] 2.3 Immunoblotting assay First, samples of proteins AR, er, FSHR, and CYP19A1 were prepared, and then gel electrophoresis, membrane transfer, blocking, antibody incubation, and chemiluminescence detection were performed in sequence.

[0094] 2.4 RNA extraction, reverse transcription, and quantitative RT-PCR RNA extraction: Cells were digested with trypsin and seeded into 6-well plates. The culture medium was discarded, and the cells were washed three times slowly with PBS. Then, 1 mL of TRIzol was added for lysis for 5-7 min. After thorough mixing, the cells were transferred to a 1.5 mL enzyme-free centrifuge tube. 0.2 mL of chloroform was added, and the tube was vigorously vortexed approximately 100 times. The tube was incubated at room temperature for 3 min, then centrifuged at 10000×g for 15 min at 4°C. Approximately 300-400 μL of the supernatant was carefully transferred to a new enzyme-free centrifuge tube. 0.5 mL of isopropanol was added, and the tube was inverted 100 times. The tube was incubated at room temperature for 10 min. The tube was then centrifuged at 10000×g for 10 min at 4°C. The supernatant was discarded, and the milky white precipitate was RNA. 1 mL of 75% ethanol was added, and the tube was centrifuged at 7500×g for 5 min at 4°C. The supernatant was discarded. The tube was dried at room temperature for 5 min, and 25 μL of DEPC water was added. The mixture was gently swirled and incubated at room temperature for 5 min to dissolve the RNA. Total RNA concentration was determined using an ultra-micro spectrophotometer. RNA with an A260 / 280 ratio between 1.9 and 2.2 yielded the best results. RNA with excessively high concentrations was then diluted appropriately to a concentration of 100-200 ng / μL. If not intended for immediate use, the RNA was stored at -80°C for later use.

[0095] cDNA preparation: Following the reverse transcription kit procedure, first remove genomic DNA, then reverse transcribe to prepare cDNA. The genomic DNA removal procedure is shown in Table 9. Place the mixed system into a PCR instrument and react according to the program "42℃ for 2 min, 4℃". The reverse transcription procedure is shown in Table 10. First, place the mixed system into a PCR instrument and react according to the program "37℃ for 15 min, 85℃ for 5 s, 4℃". Finally, store the cDNA prepared by reverse transcription at -20℃ for later use.

[0096] Table 9. Genomic DNA Removal Procedures

[0097] Table 10 Reverse transcription operation system

[0098] Primer design: Gene sequences were queried using the NCBI gene database, and primer sequences were designed using Primer 6.0 software. The primer sequences are shown in Table 11. The primers used in the experiment were synthesized by Beijing BGI Genomics Co., Ltd.

[0099] Table 11 Primer Sequences

[0100] RT-PCR: Prepare the following reaction system using 0.2 mL of 8-tube PCR apparatus. See Table 12 for the reaction system details. Amplify the reaction using a two-step procedure: "pre-denaturation at 95℃ for 30 s, PCR reaction at 95℃ for 5 s, 60℃ for 20 s, 40 cycles, followed by melting curve analysis at 66℃ for 15 s". Use the GAPHD gene as the internal reference gene for RT-PCR. -△△Ct The method is used to determine the relative expression level of the target gene.

[0101] Table 12 Quantitative Real-Time PCR Operation System

[0102] 2.5 ELISA Detection After the culture was completed, the culture supernatant was collected. After centrifugation at 3000 r / min for 15 min, the supernatant was used to detect the estradiol concentration using a kit from Mlbio (Shanghai, China).

[0103] 2.6 High-performance liquid chromatography-tandem mass spectrometry detection The concentrated sample was filtered through a 0.22 μm PTFE membrane and analyzed using the method model (Agilent 1290 InfinityII / 6470 QQQ). Chromatographic separation was performed using a ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm) at 40 °C, with gradient elution using 0.1% formic acid and methanol as the mobile phase at a flow rate of 0.3 mL / min. Mass spectrometry was performed in ESI + MRM mode, monitoring estradiol * m / z * 273.2→23.1. Quantification was performed using Agilent Mass Hunter software (B.01.01) via an external standard curve.

[0104] 2.7 Estradiol Extraction All extraction processes were performed under ventilated conditions, away from ignition sources. For each sample, 200 mL of the thawed supernatant was added to a 5 L test tube, followed by 400 mL of chromatographic grade diethyl ether, and vortexed vigorously for 3 min. The mixture was incubated at -20 °C for 10 min to allow the aqueous phase to solidify, after which the upper ether phase was discarded. The extraction was repeated once, and the ether fractions were combined.

[0105] 2.8 Rotary evaporation of extract The combined ether extracts (~400 mL) were concentrated using an IKA RV 10 rotary evaporator equipped with 500 mL pear-shaped flasks. Evaporation was carried out at 35 °C (water bath), 80 rpm, and approximately 0.08 MPa, with the condenser cooling to 4 °C. The process was carefully monitored, and the vacuum and heat were immediately shut off once the volume decreased to approximately 100 mL. After evacuation, the concentrate was collected from the flasks.

[0106] 2.9 Mouse Experiment Nine 8-week-old male BALB / c mice were randomly divided into five experimental groups (n = 3 per group). The experimental groups were as follows: primary treatment group, extraction treatment group, extraction control group, rotary evaporation treatment group, and rotary evaporation control group. Testicular tissue and blood samples were collected 12 hours after injection for subsequent analysis.

[0107] 2.10 Statistical Analysis All values ​​are expressed as mean ± standard deviation of at least three independent trials. One-way ANOVA was used for comparisons between groups, and LSD or Tukey post-hoc tests were performed in Graph Pad Prism 8. P < 0.05 was considered statistically significant.

[0108] 3 Results 3.1 Cell isolation and culture like Figure 15 As shown, follicles of approximately 3-8 mm in diameter were selected from ovaries with a diameter of 1-2 cm and dissected using forceps. The follicles were then minced, trypsin-digested, and cultured as TCs. After 96 hours of culture, the adherent cells of the TCs exhibited a typical villous appearance.

[0109] 3.2 Identification of GCs and TCs like Figure 16 As shown, immunofluorescence staining was used to stain the marker proteins Vimentin of TCs and the FSHR protein receptor of SV40T-GFP-GCs-50 to verify that the TCs cells were the desired cells.

[0110] 3.3 Co-culturing of TCs and GCs increased estradiol synthesis. like Figure 17 As shown, the co-culture of TCs and SV40T-GFP-GCs-50 was identified by immunofluorescence. Western blotting and q-PCR confirmed that the expression level of CYP19A1 in co-cultured TCs and SV40T-GFP-GCs-50 was significantly higher than that in granulocytes cultured alone. ELISAJ showed that the E2 content in co-cultured TCs and SV40T-GFP-GCs-50 was significantly higher than that in granulocytes cultured alone.

[0111] 3.4 FSH and LH promote E2 synthesis like Figure 18 As shown, CCK8 analysis revealed that when TCs and SV40T-GFP-GCs-50 were co-cultured, the highest estradiol level in the cell supernatant was observed at FSH concentrations of 5 IU / mL and LH concentrations of 1 ng / mL. Furthermore, Western blotting and q-PCR demonstrated that FSH and LH significantly increased the expression of TCs and CYP19A1.

[0112] 3.5 E2 in the supernatant was extracted using extraction and rotary evaporation methods. like Figure 19 As shown, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) analysis confirmed that liquid-liquid extraction with ether followed by rotary evaporation significantly increases the concentration of estradiol (E2).

[0113] 3.6 Physiological changes in mice caused by estradiol concentrations after extraction, concentration, and rotary evaporation. like Figure 20 As shown, the levels of estradiol in the blood of mice in both the extraction and concentration group and the rotary evaporation group were significantly increased. Western blot and quantitative PCR analysis further showed that the expression of androgen receptor (AR) and aromatase (CPY19A1) was significantly upregulated in the testes, while the expression of estrogen receptor (ER) was significantly downregulated.

[0114] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing an immortalized sheep ovarian granulosa cell line, characterized in that, Includes the following steps: (1) Isolation of primary granulosa cells from sheep ovarian follicular fluid; (2) The primary granular cells were transfected with a lentivirus carrying SV40T-GFP; (3) Immortalized granular cell lines that stably express SV40T-GFP were obtained by screening with puromycin.

2. The construction method according to claim 1, characterized in that, The follicular fluid was derived from sheep cavitary follicles with a diameter of 3-8 mm.

3. The construction method according to claim 1, characterized in that, The transfection method is as follows: when the granulocyte adhesion rate is ≥85%, SV40T-GFP lentivirus solution is transfected into granulocytes at MOI=10 and infected for 16 hours.

4. A method for in vitro synthesis of estradiol, characterized in that, Includes the following steps: (1) The immortalized sheep ovarian granulosa cell line constructed by any one of claims 1-3 is co-cultured with sheep ovarian theca cells; (2) Add follicle-stimulating hormone and luteinizing hormone to the co-culture system; (3) Collect the co-culture supernatant, extract and concentrate to obtain estradiol.

5. The method according to claim 4, characterized in that, In step (2), the concentration of follicle-stimulating hormone added is 5 IU / mL, and the concentration of luteinizing hormone added is 1 ng / mL.

6. The method according to claim 4 or 5, characterized in that, In step (3), the method for extracting estradiol is to use ether to perform liquid-liquid extraction on the culture supernatant; The method for concentrating estradiol is to perform rotary evaporation on the extract.

7. An immortalized sheep ovarian granulosa cell line, characterized in that, The cell line is constructed by the method described in any one of claims 1-3. It stably expresses SV40T antigen and green fluorescent protein and retains the activity of CYP19A1, a key enzyme in the synthesis of estradiol.

8. The use of the immortalized sheep ovarian granulosa cell line as described in claim 7 in the preparation of estradiol.

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