Establishment method and application of immortalized cell line of porcine testicular mesenchymal stem cells
Through isolation, enrichment and lentiviral transduction methods, the immortalized cell line of pig testicular interstitial stem cells was established, which solved the problem that pig testicular interstitial stem cells could not proliferate continuously in vitro, achieved efficient testosterone secretion and long-term culture, and provided abundant cellular resources.
Patent Information
- Application Number
- CN202510423731.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-07-08
AI Technical Summary
In the prior art, pig testicular interstitial stem cells are prone to replication and aging during in vitro culture and cannot proliferate continuously. There have been no reports of successful establishment of immortalized cell lines, which limits their application in disease research and regenerative medicine.
Pig testicular interstitial stem cells were separated by type IV collagenase dissociation fluid, and cell sorting was performed using anti-PDGFRα antibody and flow cytometry. Primary cells were inoculated and SV40 large T antigen was transferred into cells using lentivirus to make them immortalized. Pig testicular interstitial stem cells immortalized cell lines were established through lentiviral transduction and subculture.
An immortalized cell line of uniform mass of pig testicular interstitial stem cells can secrete testosterone under the stimulation of luteinizing hormone, pass on for more than 60 generations, and cultured for more than 5 months, providing rich cellular resources for basic research and disease treatment.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for establishing an immortalized cell line of porcine testicular interstitial stem cells and its application. Background Art
[0002] Leydig cells secrete testosterone and other growth factors, which play an important role in the development of the male reproductive system and the lifelong maintenance of male reproductive function. In male animals, persistent low serum testosterone levels can lead to the occurrence of hypogonadism, manifested as decreased libido, low fertility, reduced muscle mass, and decreased bone density. Currently, the most widely used treatment method for patients with primary hypogonadism is testosterone replacement therapy. Although testosterone replacement therapy can effectively alleviate many symptoms of hypogonadism, it can also lead to infertility and increase the risk of diseases such as prostate cancer, cardiovascular problems, and polycythemia. The cell therapy of interstitial stem cells restores the physiological pattern of testosterone production by reconstructing the interstitial cell lineage in vivo. It can avoid most of the problems caused by testosterone replacement therapy and is an ideal treatment method for patients with primary hypogonadism. As an animal model for human disease research, porcine interstitial stem cells have important value in the establishment and research of disease and regenerative medicine models.
[0003] However, cells derived from the interstitial cell lineage, including interstitial stem cells, are prone to replicative senescence during in vitro culture and cannot proliferate continuously in vitro. So far, only an immortalized and continuously proliferating interstitial cell line (TM3) has been successfully established in mice and applied in many studies, while there is no report on the establishment of cell lines (including interstitial stem cells) derived from the testicular interstitial cell lineage in large animals including pigs. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for establishing an immortalized cell line of porcine testicular interstitial stem cells and its application in testosterone production.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] On the one hand, the present invention relates to a method for establishing an immortalized cell line of porcine testicular interstitial stem cells, which includes the following steps:
[0007] (1) Isolation of porcine testicular interstitial stem cells: Take the parenchymal tissue of a piglet's testis, dissociate it with type IV collagenase dissociation solution, and centrifuge and filter to obtain a single-cell suspension of testicular somatic cells including interstitial cells;
[0008] (2) Enrichment of porcine testicular interstitial stem cells: Perform cell sorting with an anti-PDGFRα antibody and a flow cytometer to obtain PDGFRα-positive cells, that is, primary porcine testicular interstitial stem cells;
[0009] (3) Immortalization of porcine testicular interstitial stem cells: Primary porcine testicular interstitial stem cells were inoculated, and SV40 large T antigen was transferred into the cells using lentivirus to immortalize the porcine testicular interstitial stem cells.
[0010] In a preferred embodiment of the present invention, the isolation step of the porcine testicular interstitial stem cells includes: taking the parenchymal tissue of the testes of piglets aged 7 - 30 days, dissociating in a water bath at 36 - 38 °C with a type IV collagenase dissociation solution prepared with DMEM medium at a concentration of 1 - 3 mg / mL for 20 - 40 min; centrifuging, taking the supernatant and filtering it through a 30 - 50 μm sieve to obtain a single - cell suspension of testicular somatic cells including interstitial cells.
[0011] In a preferred embodiment of the present invention, the method for immortalizing porcine testicular interstitial stem cells specifically includes the following steps: inoculating the obtained primary porcine testicular interstitial stem cells and placing them in a cell culture incubator at 35 - 37 °C and 5 - 10% CO2 for adherent culture; when the cell confluence reaches 65 - 75%, using lentivirus to transfer SV40 large T antigen into the cells to immortalize the porcine testicular interstitial stem cells; then continuing to place them in a cell culture incubator at 35 - 37 °C and 5 - 10% CO2 for sub - culture, and the obtained cells are the immortalized cell line of porcine testicular interstitial stem cells.
[0012] In a preferred embodiment of the present invention, the lentiviral transduction in the immortalization of porcine testicular interstitial stem cells specifically includes the following steps: co - transfecting HEK293T cells with the transfer vector SV40 large T antigen plasmid and the second - generation packaging vectors pSPAX2 and pMD2.G to produce lentivirus, and then adding a lentivirus concentrate to concentrate the lentivirus; inoculating the isolated and enriched porcine testicular interstitial stem cells (PDGFRα - positive cells) and transducing them with the prepared lentivirus; transducing at a multiplicity of infection (MOI) of 2.5 - 5, and exposing the cells to polybrene and the virus supernatant.
[0013] In a preferred embodiment of the present invention, the immortalized cell line of porcine testicular interstitial stem cells has mRNA / protein expression of the marker genes PDGFRA, LIF, NESTIN, CYP17A1, CYP11A1, HSD3B1 and the immortalization marker SV40.
[0014] On the other hand, the present invention also relates to the immortalized cell line of porcine testicular interstitial stem cells obtained by the above method and its application, and the application refers to being induced to differentiate into functional interstitial cells capable of producing testosterone.
[0015] In a preferred embodiment of the present invention, the induced differentiation means culturing the immortalized cell line of porcine testicular interstitial stem cells in a complete medium supplemented with luteinizing hormone (LH).
[0016] The subculture of the immortalized cell line of porcine testicular interstitial stem cells specifically includes the following steps: The complete culture medium consists of DMEM high-glucose culture solution containing 10% fetal bovine serum (FBS), 1% glutamine, 1% non-essential amino acids (NEAA), and 1% penicillin-streptomycin (P / S). The cells are cultured in a cell incubator at 37°C and 5% CO2, and the culture solution is changed every 48 hours. When the cell confluence reaches 80%, they are passaged into a new culture plate at a ratio of 1:3.
[0017] The cryopreservation and resuscitation methods of the immortalized cell line of porcine testicular interstitial stem cells include the following steps: The cells are resuspended with a cryopreservation solution (the cryopreservation solution is composed of DMEM high-glucose culture medium, fetal bovine serum, and dimethyl sulfoxide, and the volume ratio of DMEM high-glucose culture medium: fetal bovine serum: dimethyl sulfoxide is 6:3:1), inoculated into a cryopreservation tube and placed in a cryopreservation box, frozen at -80°C overnight, and then stored in liquid nitrogen for a long time; The cells stored in liquid nitrogen are rapidly thawed in a water bath at 35 - 39°C for resuscitation.
[0018] The method for stimulating the immortalized cell line of porcine testicular interstitial stem cells to produce testosterone includes the following steps: The immortalized cell line of porcine testicular interstitial stem cells is cultured with a complete culture medium supplemented with 1 IU / mL luteinizing hormone (LH). The supernatant is collected after culturing for 1, 2, 4, and 6 days. The enzyme-linked immunosorbent assay (ELISA) for porcine testosterone is performed using a 96-well kit according to the instructions of the FANKW manufacturer, and the concentration of testosterone is measured using an enzyme-labeling instrument (Shanpu, SuPerMax-3100).
[0019] The beneficial effects of the present invention are reflected in:
[0020] The present invention has established an immortalized cell line of porcine testicular interstitial stem cells for the first time. This cell line has uniform quality, has been passaged more than 60 generations, and cultured for more than 5 months. It has the characteristics of interstitial stem cells and can secrete testosterone under the stimulation of luteinizing hormone (LH), providing rich cell resources for future basic research and disease treatment. Description of the Drawings
[0021] Figure 1 For the isolation, enrichment, and immortalization of porcine testicular interstitial stem cells. A is the bright-field image of the testicular single-cell suspension obtained by dissociation with type IV collagenase; B is the enrichment of PDGFRα-positive cells using a flow cytometer (FACS); C is the live cell staining of PDGFRα before and after FACS enrichment. The scale bar is 50 μm.
[0022] Figure 2 For the plasmid map of the cell immortalization containing SV40 large T antigen.
[0023] Figure 3Long-term culture of immortalized cell lines of porcine testicular interstitial stem cells. A is the bright-field images of two immortalized cell lines B1 and B2; B is the cell doubling time of the two cell lines, and the data are expressed as the mean ± standard error of three independent experiments (n = 3), ** indicates P < 0.01; C is the immunofluorescence staining of α-tubulin and phalloidin in the two cell lines. The scale bar is 50 μm.
[0024] Figure 4 Gene expression characteristics of immortalized cell lines of porcine testicular interstitial stem cells. A shows the mRNA expression of PDGFRA, LIF, NESTIN, CYP17A1, CYP11A1, HSD3B1 and SV40 in different passages of cell lines B1 and B2 detected by RT-PCR. ACTB was used as the loading control, RNA from 60-day-old porcine testes was used as the control, and no reverse transcription (-RT) was used as the negative control; B shows the expression of SV40, PDGFRα and STAR at the protein level analyzed by Western blot, and β-ACTIN was used as the loading control; C shows the immunofluorescence staining images of SV40, PDGFRα and STAR in the two cell lines on cell smears. The scale bar is 50 μm.
[0025] Figure 5 Testosterone secretion in primary and immortalized cell lines of porcine testicular interstitial stem cells after stimulation with luteinizing hormone (LH) for different days. The data are expressed as the mean ± standard error of six independent experiments (n = 6), *: P < 0.05, **: P < 0.01. Detailed implementation mode
[0026] The present invention will be further described below in conjunction with the drawings and embodiments, but it does not limit the scope of the rights of the present invention. The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified; the reagents or materials are from commercial channels unless otherwise specified.
[0027] Example 1:
[0028] (1) Isolation, enrichment and immortalization of porcine testicular interstitial stem cells
[0029] 1.1 Primary digestion of porcine testicular tissue
[0030] 1.1.1 Material preparation: Testes of piglets aged 7 - 30 days, 75% alcohol, sterile surgical instruments, penicillin-streptomycin P / S, PBS, DPBS, culture dishes, centrifuge tubes, centrifuge, type IV collagenase, complete medium, shaking water bath.
[0031] 1.1.2 Steps:
[0032] (1) Collect the testes of piglets aged 7 - 30 days, place them in a sampling tank pre-cooled in advance and filled with a sufficient amount of phosphate buffer solution (PBS, added with 3× penicillin-streptomycin P / S to prevent contamination), place the sampling tank on an ice box and quickly transfer it to the laboratory, and place it in a 4℃ refrigerator. Wash the piglet testes 3 times in pre-cooled PBS containing 3×P / S to wash away the blood stains, then briefly soak them in 75% alcohol and shake them from time to time for 3 min, and then transfer them to PBS containing 3×P / S and wash them 3 more times.
[0033] (2) Transfer the testes to a 90 mm Petri dish, cut off the epididymis, use a sterile scalpel to cut open the tunica albuginea of the testes, peel off the tunica albuginea and isolate the interstitial tissue of the testes. Use scissors to cut the testicular parenchymal tissue into tissue blocks about 2 mm 3 in size (note to discard the white testicular mediastinum), collect the tissue into a 50 mL centrifuge tube, at the same time add PBS containing 3×P / S to the Petri dish to rinse it clean, collect it into the same 50 mL centrifuge tube and centrifuge at 50×g for 2 min, discard the supernatant. Repeat once.
[0034] (3) Add 2 mg / mL type IV collagenase dissociation solution to the centrifuge tube containing testicular tissue blocks, seal it with a sealing film to prevent contamination, place it on a shaking water bath at 37℃ and incubate with shaking for 30 min, during which take it out from time to time and invert it up and down to make the tissue fully contact with the collagenase. Stop dissociation when scattered seminiferous tubules can be seen under microscopy. Centrifuge the dissociation product at 50×g for 2 min and collect the supernatant.
[0035] (4) Centrifuge the supernatant at 450×g for 5 min, collect the cell pellet, resuspend it with PBS containing 3×P / S, and filter the cell suspension through a cell sieve with a pore size of 40 μm.
[0036] (5) Add hemocyte lysate, after lysing for 90 s, add Dulbecco's phosphate buffered saline (DPBS) to terminate lysis, centrifuge at 450×g for 5 min and then discard the supernatant. Resuspend the cells with complete medium and evenly inoculate the cells into a culture dish.
[0037] The results showed that after enzymatic dissociation, a single-cell suspension of testicular somatic cell population including interstitial stem cells was obtained ( Figure 1 A), most of the cells were oval or round, with granular protrusions on the surface, and the cell surface was not smooth.
[0038] 1.2 Enrich pig testicular interstitial stem cells, namely platelet-derived growth factor receptor α (PDGFRα)-positive cells, using a flow cytometer (FACS)
[0039] 1.2.1 Material preparation: Accutase enzyme, DPBS, complete medium, DNase Ι, 40μm cell strainer, FACS buffer, PDGFRα antibody (product number ab203491, Abcam, UK), FITC-labeled goat anti-rabbit IgG (H+L) green fluorescent antibody (Yeasen Biotech Co., Ltd., China), hemocytometer.
[0040] 1.2.2 Steps:
[0041] (1) Cells growing adherently were digested step by step with Accutase enzyme, and the digestion was terminated with PBS. The cells were collected, centrifuged at 450×g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 5 mL of complete medium. 100 μL of DNase Ι was added to prevent cell aggregation. After mixing, the suspension was filtered through a 40μm cell strainer, counted, and then centrifuged at 450×g for 5 min. The supernatant was discarded.
[0042] (2) The cells were resuspended with pre-cooled FACS buffer. One ten million cells were resuspended in 5 mL of buffer, allowed to stand at room temperature for 3 - 5 min, and then centrifuged at 450×g for 5 min. The supernatant was discarded.
[0043] (3) The antibody was diluted with pre-cooled FACS buffer at a dilution volume ratio of 1:50. 100 μL of the diluted antibody was used to incubate every one million cells, incubated on ice for 40 min, and then incubated at room temperature for 10 min. Centrifuged at 450×g for 5 min, the supernatant was discarded. Then, it was washed once with FACS buffer.
[0044] (4) The green fluorescent-labeled antibody was diluted with pre-cooled FACS buffer at a dilution volume ratio of 1:400. 100 μL of the diluted antibody was used to incubate every one million cells, incubated in the dark on ice for 40 min, and then incubated in the dark at room temperature for 10 min. Centrifuged at 450×g for 5 min, the supernatant was discarded. Then, it was washed once with FACS buffer.
[0045] (5) 3 million cells were resuspended in 100 μL of FACS buffer, placed on ice and in the dark, and then sorted on the machine. The software collected data on cell size, structure, and fluorescence intensity, excluded cell debris, and framed positive cells for sorting.
[0046] (6) The sorted and collected PDGFRα-positive cells (i.e., porcine testicular interstitial stem cells) were resuspended in complete medium, and every 30,000 cells were inoculated into one well of a 96-well plate.
[0047] Flow cytometry analysis results showed that PDGFRα-positive cells, namely porcine testicular interstitial stem cells, existed in the isolated testicular somatic cell population, with a proportion of 15.00% ( Figure 1 B). After sorting, almost all cells were PDGFRα-positive ( Figure 1 C).
[0048] 1.3 Packaging of SV40 large T antigen-immortalized lentivirus and transduction of porcine testicular interstitial stem cells
[0049] 1.3.1 Material preparation: HEK293T cell line, liposome transfection reagent, SV40 large T antigen transfer plasmid ( Figure 2 , catalog number 12246, Addgene), lentivirus packaging plasmids pSPAX2 and pMD2.G, DPBS, culture dishes, centrifuge tubes, centrifuge, complete medium, polybrene. Among them, SV40 large T antigen is derived from simian vacuolating virus, and its core sequence (SEQ ID No.1) of the plasmid can encode SV40 large T antigen protein. The large T antigen can bind to the tumor suppressor proteins of host cells (such as p53 and Rb proteins), inhibit the normal functions of these proteins, thus breaking the normal regulation of the cell cycle and promoting continuous cell division.
[0050] 1.3.2 Steps:
[0051] (1) Inoculate 800,000 HEK293T cells per well into a 6-well plate, and perform cell transfection when the cell confluence reaches 85%.
[0052] (2) Refer to the liposome transfection reagent instruction manual, add the SV40 large T antigen transfer plasmid, lentivirus packaging plasmids pSPAX2 and pMD2.G in proportion to prepare the transfection system. According to the steps in the instruction manual, mix the prepared transfection system well and let it stand at room temperature for 30 min. Add the transfection system dropwise to the cultured cells, mix gently, and then put it into the incubator for further culture.
[0053] (3) 16 h after adding the transfection system, change the medium to complete medium supplemented with 20 mmol / L HEPES to maintain the stability of the pH value. After culturing for 24 h, collect the cell culture supernatant and store it at 4 °C. Add fresh complete medium containing 20 mmol / L HEPES to the cells and continue to culture for 24 h, and then collect the cell culture supernatant and store it at 4 °C.
[0054] (4) Mix the cell culture supernatants collected twice, centrifuge at 4 °C and 500 × g for 10 min. After collecting the supernatant, filter the collected supernatant with a 0.45 μm sterile filter. Add the lentivirus concentrate, and the volume ratio of the supernatant to the lentivirus concentrate is 4:1, and then let it stand at 4 °C for 2 h to concentrate the lentivirus particles containing SV40 large T antigen.
[0055] (5) Centrifuge at 4°C and 4000×g for 30 min. Mark the position of the lentivirus particle precipitate with a marker pen. Discard the supernatant, and then dissolve the lentivirus particles in 500 μL of high-glucose DMEM medium for aliquot storage. Pipette the virus solution to mix well, and use a lentivirus titer detection card to measure the lentivirus titer according to the instruction manual. Aliquot the dissolved and concentrated lentivirus into 1.5 mL EP tubes and store them in an -80°C refrigerator to avoid repeated freezing and thawing, which may reduce the virus titer.
[0056] (6) Porcine testicular interstitial stem cells are cultured in 24-well plates. When the confluence reaches 70%, lentiviral transduction is carried out. Calculate the added volume of the virus solution according to the lentivirus titer and MOI (multiplicity of infection). Prepare a complete medium containing virus particles and 1× polybrene (the role is to enhance the contact between the virus and the cell membrane to improve the transduction efficiency), mix well and add it to the cell culture plate. Two MOI values are designed in the experiment to optimize the transduction efficiency and reduce cytotoxicity, and two batches of immortalized porcine testicular interstitial stem cell lines (B1, B2) are established accordingly. Among them, MOI of B1 = 2.5 and MOI of B2 = 5.
[0057] (7) Seal the cell culture plate with parafilm. Use a plate centrifuge to centrifuge at 32°C and 3000×g for 1 h to evenly attach the virus particles to the cell surface. After centrifugation, remove the parafilm, wipe the periphery of the plate with alcohol, and then put it back into the incubator for continuous culture.
[0058] (8) After 16 h of lentiviral transduction, replace it with fresh complete medium for continuous culture.
[0059] (II) Subculture of immortalized porcine testicular interstitial stem cell lines
[0060] (1) The cells are changed with fresh medium every 48 h.
[0061] (2) When the cell confluence reaches about 80%, discard the culture medium, rinse once with DPBS, add 0.125% trypsin digestion solution (the added amount should cover the cells) and digest at room temperature for 2 - 3 min. When the cells shrink and become round, immediately add fetal bovine serum (about 1 / 3 of the volume of the trypsin digestion solution) to terminate the digestion, and then centrifuge at 450×g for 5 min.
[0062] (3) After discarding the supernatant, resuspend the cell pellet with complete medium, inoculate the cell suspension into a new plate, and place it in a 37°C, 5% CO2 incubator for continuous culture. The cells are changed with fresh medium every 48 h, and the next subculture is carried out when the confluence reaches 80%.
[0063] (4) Cryopreservation and resuscitation
[0064] Cryopreservation: Resuspend the cells in cryopreservation medium, inoculate them into cryotubes, and place them in a cryobox. Freeze at -80°C overnight, and then store them in liquid nitrogen for long-term preservation. The cryopreservation medium consists of high-glucose DMEM medium, fetal bovine serum, and dimethyl sulfoxide. The volume ratio of high-glucose DMEM medium: fetal bovine serum: dimethyl sulfoxide = 6:3:1.
[0065] Recovery: Rapidly thaw the cells stored in liquid nitrogen in a water bath at 35 - 39°C. Then transfer the cell suspension to a centrifuge tube containing complete medium, centrifuge at 450×g for 5 min, and discard the supernatant. Resuspend the cells in complete medium and inoculate them into a culture plate, and continue culturing using the same culture methods and conditions as for subculture.
[0066] The experimental results show that the immortalized cell line of porcine testicular interstitial stem cells can be subcultured and cryopreserved normally. So far, it has been stably subcultured more than 60 times and cultured for more than 5 months.
[0067] (III) Characteristics of the Immortalized Cell Line of Porcine Testicular Interstitial Stem Cells
[0068] 3.1 Cell Morphology
[0069] During the subculture of the immortalized cell lines B1 and B2 of porcine testicular interstitial stem cells, when the cell confluence reached 70 - 80%, observe the cell morphology under an inverted microscope and take pictures. The results show that during long-term culture, the B1 and B2 cell lines are similar in morphology, both showing a spindle-shaped or relatively round cell morphology with short extensions ( Figure 3 A). The culture time of both the B1 and B2 cell lines has exceeded 5 months, and they have been subcultured more than 60 times. Their morphology is stable, without obvious changes or abnormalities.
[0070] 3.2 Detection of Cell Doubling Time
[0071] During the subculture of the immortalized cell line of porcine testicular interstitial stem cells, calculate the cell doubling time (PDT) according to the following formula: PDT = [lg 2 / lg(Nt / No)] × t, where Nt is the number of cells after culturing for t h, and No is the number of cells initially inoculated. Repeat this three times (n = 3) to calculate the cell doubling time.
[0072] The results show that the proliferation activity of the B1 cell line is lower than that of the B2 cell line. The average doubling time of the B1 cell line is 38.66 h, while that of the B2 cell line is 32.51 h, and there is a significant difference between the two (P < 0.01, Figure 3 B).
[0073] 3.3 Detection of the Cytoskeleton
[0074] (1) Seed the immortalized porcine testicular interstitial stem cell line in a 48-well plate. When the cell confluence reaches approximately 50%, discard the culture medium, wash twice with DPBS, and then fix, permeabilize, and block.
[0075] (2) Incubate with the primary antibody. Dilute the rabbit anti-α-tubulin antibody at a ratio of 1:200. Take 100 μL of the diluted antibody and add it to the culture plate. Incubate overnight at 4°C, and then wash 3 times with DPBS for 3 minutes each time.
[0076] (3) Incubate with the secondary antibody in the dark. Dilute the donkey anti-rabbit green fluorescent antibody at a ratio of 1:400. Take 100 μL of the diluted antibody and add it to the culture plate. Incubate at 4°C for 2 hours, and then wash 3 times with DPBS for 3 minutes each time.
[0077] (4) Dilute the rhodamine-labeled phalloidin dye at a ratio of 1:200. Take 100 μL of the diluted dye and add it to the culture plate. Incubate in the dark at room temperature for 30 minutes. Then, examine under a fluorescence microscope. Stop the staining according to the coloring situation, and wash 3 times with DPBS for 3 minutes each time.
[0078] (5) Stain the cell nuclei. Dilute DAPI at a ratio of 1:1000. Take 100 μL of the diluted solution and add it to the culture plate. Incubate at room temperature for 5 - 10 minutes, and then wash 3 times with DPBS for 3 minutes each time.
[0079] (6) Observe and take pictures under an inverted fluorescence microscope.
[0080] The results showed that multinucleated cells were not observed in the obtained immortalized porcine testicular interstitial stem cell lines B1 and B2, and the cytoskeleton was normal ( Figure 3 C).
[0081] (IV) Identification of the immortalized porcine testicular interstitial stem cell line
[0082] 4.1 Detection of mRNA expression of interstitial cell markers by RT-PCR
[0083] Use RT-PCR to detect the mRNA expression of the interstitial cell lineage marker genes PDGFRA, LIF, NESTIN, CYP17A1, CYP11A1, HSD3B1 and the immortalization marker SV40 in different passages of the B1 and B2 cell lines of the immortalized porcine testicular interstitial stem cell line during subculture (the primer sequences and PCR product length information are shown in Table 1). Use the total RNA without reverse transcription (-RT) but with polymerase chain reaction as the negative control, and the total RNA of 60-day-old porcine testicular tissue as the positive control. Use ACTB as the loading control.
[0084] Table 1
[0085]
[0086] (1) The total RNA of the 9th, 12th, and 15th passages of B1 and B2 cell lines was extracted separately using the Trizol method. Genomic DNA was removed by treatment with DNase. mRNA was reverse transcribed into cDNA using a cDNA synthesis kit.
[0087] (2) Using the synthesized cDNA as a template, polymerase chain reaction (PCR) was carried out. The primer sequences used are shown in Table 1.
[0088] (3) The PCR amplification products were separated by 2% agarose gel electrophoresis, stained with a nucleic acid dye, visualized and photographed under ultraviolet light.
[0089] The results showed that the immortalized cell lines B1 and B2 of porcine testicular interstitial stem cells had mRNA expression of the interstitial cell lineage marker genes PDGFRA, LIF, NESTIN, CYP17A1, CYP11A1, HSD3B1, and the immortalization marker SV40 in different passages ( Figure 4 A), confirming that the cell lines B1 and B2 had interstitial cell characteristics and that cell immortalization was successful (able to continuously proliferate in vitro and stably express SV40 large T antigen), thus confirming the successful establishment of the immortalized cell line of porcine testicular interstitial stem cells.
[0090] 4.2 Western blot detection of the expression of interstitial cell lineage marker proteins
[0091] Western blot was used to detect the protein expression of the interstitial cell lineage markers PDGFRα, STAR, and the immortalization marker SV40 in different passages of the immortalized cell line of porcine testicular interstitial stem cells in B1 and B2 cell lines during subculture.
[0092] (1) The total proteins of the 9th, 12th, and 15th passages of B1 and B2 cell lines were extracted separately using RIPA lysis buffer (containing 1% PMSF). The protein concentration was detected using a BCA kit.
[0093] (2) According to the molecular weight of the target protein, an SDS-PAGE gel with the corresponding concentration was selected, and 30 μg of protein was loaded for gel electrophoresis. The electrophoresis conditions were 110 V for about 90 min, and the electrophoresis time was adjusted according to the size of the target protein. After electrophoresis, the gel was taken out, peeled off, and placed in the transfer buffer.
[0094] (3) Immerse the PVDF membrane in methanol for 1 min for activation, and then wash it in the transfer buffer for 5 - 10 min. Transfer the membrane using a wet transfer apparatus under the conditions of 300 mA and 120 min. After the transfer, take out the PVDF membrane and place it in 5% non-fat milk powder, and block it on a shaker at room temperature for 2 h. Then wash it 3 times with a TBST shaker, 10 min each time.
[0095] (4) Incubate with the primary antibody. Dilute the primary antibody according to the antibody instruction manual, immerse the PVDF membrane in the diluted primary antibody, and incubate it on a shaker at 4 °C overnight. Then wash it 3 times with a TBST shaker, 10 min each time.
[0096] (5) Incubate with the secondary antibody. Dilute the secondary antibody according to the antibody instruction manual, immerse the PVDF membrane in the diluted secondary antibody, and incubate it on a shaker at room temperature for 2 h. Then wash it 3 times with a TBST shaker, 10 min each time.
[0097] (6) Prepare the developing solution, develop and take pictures under the developer.
[0098] The results showed that the obtained immortalized cell lines B1 and B2 of porcine testicular interstitial stem cells had the protein expressions of the interstitial cell lineage markers PDGFα, STAR and the immortalization marker SV40 in different passages ( Figure 4 B), further corroborating that the cell lines B1 and B2 had the characteristics of interstitial cells, and the cells were successfully immortalized (able to continuously proliferate in vitro and stably express SV40 large T antigen), thus confirming the successful establishment of the immortalized cell line of porcine testicular interstitial stem cells.
[0099] 4.3 Immunofluorescence detection of the expression of interstitial cell lineage marker proteins
[0100] Detect the expressions of the interstitial cell lineage markers PDGFRα, STAR and the immortalization marker SV40 of the immortalized cell line of porcine testicular interstitial stem cells by cell immunofluorescence.
[0101] Inoculate the immortalized cell line of porcine testicular interstitial stem cells in a 96-well plate. When the cell confluence reaches 60%, discard the culture medium and wash it twice with DPBS. The steps of fixation, permeabilization, blocking, incubation with the primary antibody, incubation with the secondary antibody in the dark, nuclear staining, observation and photography under an inverted microscope are the same as those in "3.3 Detection of the cytoskeleton".
[0102] The results showed that the obtained immortalized cell lines B1 and B2 of porcine testicular interstitial stem cells both expressed the interstitial cell lineage markers PDGFRα, STAR and the immortalization marker SV40 ( Figure 4 C), further corroborating that the cell lines B1 and B2 had the characteristics of interstitial cells, and the cells were successfully immortalized (able to continuously proliferate in vitro and stably express SV40 large T antigen), thus confirming the successful establishment of the immortalized cell line of porcine testicular interstitial stem cells.
[0103] (V) ELISA detection of testosterone secretion in immortalized cell lines of porcine testicular interstitial stem cells
[0104] The primary porcine testicular interstitial stem cells, immortalized cell lines B1 and B2 of porcine testicular interstitial stem cells were cultured respectively in complete medium supplemented with 1 IU / mL luteinizing hormone (LH). The supernatants were collected after 1, 2, 4, and 6 days of culture. Enzyme-linked immunosorbent assay (ELISA) for porcine testosterone was performed using a 96-well kit according to the instructions of the manufacturer FANKW, and the concentration of testosterone was measured using a microplate reader (Shanpu, SuPerMax-3100).
[0105] The results showed that LH stimulated testosterone secretion in three cell populations (primary porcine testicular interstitial stem cells, immortalized cell lines B1 and B2 of porcine testicular interstitial stem cells) to a certain extent ( Figure 5 ), indicating that the immortalized cell lines of porcine testicular interstitial stem cells are similar to primary porcine testicular interstitial stem cells and can produce testosterone after stimulation with LH. The established immortalized cell lines of porcine testicular interstitial stem cells can be induced to differentiate into functional interstitial cells that can produce testosterone.
[0106] Based on the above experimental results of the present invention, it can be confirmed that the established immortalized cell lines of porcine testicular interstitial stem cells have the characteristics of interstitial stem cells and can be induced to differentiate into functional interstitial cells that can produce testosterone. The present invention can provide rich cell resources for future basic research and the treatment of primary hypogonadism, and lay a foundation for studying the characteristics of the interstitial cell lineage.
Claims
1. A method for establishing an immortalized cell line of porcine testicular interstitial stem cells, which comprises the following steps: (1) Isolation of porcine testicular interstitial stem cells: Take the parenchymal tissue of a piglet's testis, dissociate it with a type IV collagenase dissociation solution, and centrifuge and filter to obtain a single-cell suspension of testicular somatic cells including interstitial cells; (2) Enrichment of porcine testicular interstitial stem cells: Use an anti-PDGFRα antibody and a flow cytometer for cell sorting to obtain PDGFRα-positive cells, which are the primary porcine testicular interstitial stem cells; (3) Immortalization of porcine testicular interstitial stem cells: Inoculate the primary porcine testicular interstitial stem cells, and use lentivirus to transfer the SV40 large T antigen into the cells to immortalize the porcine testicular interstitial stem cells.
2. According to the establishment method described in claim 1, the isolation step of the porcine testicular interstitial stem cells includes: Take the parenchymal tissue of a piglet's testis at 7-30 days old, and in a water bath at 36-38 °C, dissociate it with a type IV collagenase dissociation solution of 1-3 mg / mL in DMEM medium for 20-40 min; centrifuge, take the supernatant and filter it through a 30-50 μm sieve to obtain a single-cell suspension of testicular somatic cells including interstitial cells.
3. According to the establishment method described in claim 1, the method for immortalizing porcine testicular interstitial stem cells specifically includes the following steps: Inoculate the obtained primary porcine testicular interstitial stem cells and place them in a cell culture incubator at 35-37 °C and 5-10% CO2 for adherent culture; when the cell confluence reaches 65-75%, use lentivirus to transfer the SV40 large T antigen into the cells to immortalize the porcine testicular interstitial stem cells; then continue to place them in a cell culture incubator at 35-37 °C and 5-10% CO2 for passage culture, and the obtained is the immortalized cell line of porcine testicular interstitial stem cells.
4. The lentiviral transduction in the immortalization of porcine testicular interstitial stem cells in the establishment method according to claim 3 specifically includes the following steps: Co-transfect HEK293T cells with the transfer vector SV40 large T antigen plasmid and the second-generation packaging vectors pSPAX2 and pMD2.G to produce lentivirus, and then add lentivirus concentrate to concentrate the lentivirus; inoculate the isolated and enriched porcine testicular interstitial stem cells (PDGFRα-positive cells), and transduce them with the prepared lentivirus; transduce at a virus infection index MOI of 2.5-5, and expose the cells to polypropylene and virus supernatant.
5. According to the establishment method described in any one of claims 1-4, the immortalized cell line of porcine testicular interstitial stem cells has mRNA / protein expression of the marker genes PDGFRA, LIF, NESTIN, CYP17A1, CYP11A1, HSD3B1 and the immortalization marker SV40.
6. An immortalized cell line of porcine testicular interstitial stem cells obtained according to the establishment method described in any one of claims 1-4.
7. Use of the immortalized cell line of porcine testicular interstitial stem cells described in claim 6, wherein the use refers to being induced to differentiate into functional interstitial cells capable of producing testosterone.
8. According to the use described in claim 7, the induction of differentiation refers to culturing the immortalized cell line of porcine testicular interstitial stem cells in a complete medium supplemented with luteinizing hormone (LH).