Trachinotus ovatus spermatogonial stem cell line and establishment, cryopreservation and recovery method and application thereof

Abstract

The invention provides a trachinotus ovatus spermatogonial stem cell line as well as establishment, cryopreservation and recovery methods and application thereof, and relates to the technical field of biologication.The method comprises the steps that tissue cells of trachinotus ovatus testis are placed in a complete culture medium to be cultured, and a fresh culture medium is replaced once every 2-4 days within 15 days before culture; culturing until the bottom of the bottle is full of cells, adding a trypsin solution to digest the cells, sucking out the digested cells every 30 seconds, respectively adding a complete culture medium for culturing for three times, screening out high-proportion spermatogonial stem cells for subsequent continuous culture, subculturing to a new culture bottle according to the proportion of 1: (1-3), and culturing to obtain the trachinotus ovatus spermatogonial stem cell line. The trachinotus ovatus spermatogonial stem cells with relatively high purity are obtained by a differential digestion method, and the trachinotus ovatus spermatogonial stem cell line is obtained by setting a passage proportion for culture and can be stably passed. And gene transfection research is carried out, so that technical support is provided for extensive research on spermatogonial stem cells of trachinotus ovatus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a spermatogonial stem cell line of an oval pomfret, and methods for establishing, freezing, and resuscitating the line and applications thereof. Background Art

[0002] The oval pomfret (Trachinotus ovatus) is a fish of the genus Trachinotus in the family Trachinotidae, order Perciformes, commonly known as the golden pomfret or yellow wax pomfret. The oval pomfret has a long sexual maturation period. Studies have shown that under natural conditions, it takes 7-8 years for the oval pomfret to reach sexual maturity. Under artificial breeding conditions, the sexual maturity period of the oval pomfret is shortened, but it still takes 3-5 years. Fish cell lines have been widely used in gene function analysis, virology, endocrinology, immunology, and toxicology research. Currently, only a few cell lines have been established for the oval pomfret, including the head kidney, mesonephros, brain, tail fin, nose, and nerve. There are no reports on the establishment of an oval pomfret spermatogonial stem cell line.

[0003] Fish spermatogonial stem cells (SSCs) can self-renew and differentiate in vitro to produce sperm. Inducing spermatogonial stem cells to produce sperm in vitro, or transplanting them into recipient fish through germline stem cell transplantation, can significantly shorten the breeding cycle. At present, only spermatogonial stem cells cultured in vitro for about one month in Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss) have been transplanted; in vitro culture and induced differentiation of spermatogonial stem cells have been studied in medaka (Oryzias latipes), zebrafish (Brachydanio rerio var), dusky goby (Gnathopogon caerulescens), eel (Anguilla japonica), catfish (Clarias batrachus), black snakehead fish (Opsariichthys bidens), spotted grouper (Epinephelus coioides) and snakehead fish (Bostrychus sinensis). coioides) has established a long-term spermatogonial stem cell line. Most fish gonadal cell lines cultured in vitro for a long time are usually Sertoli cells, or they differentiate or stop proliferating after a certain number of passages. Therefore, establishing a long-term, stable spermatogonial stem cell line from the oval pomfret is of great significance.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a method for establishing a spermatogonial stem cell line of an oval pomfret, so as to solve the technical problem in the prior art of lacking a method for establishing a spermatogonial stem cell line of an oval pomfret that can be stably propagated for a long time.

[0006] A second object of the present invention is to provide a spermatogonial stem cell line of ovate pomfret.

[0007] A third object of the present invention is to provide a method for cryopreservation of spermatogonial stem cell lines from ovate pomfret.

[0008] A fourth object of the present invention is to provide a method for resuscitating a spermatogonial stem cell line from an oval pomfret.

[0009] A fifth object of the present invention is to provide a method for identifying spermatogonial stem cell lines of the oval pomfret.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0011] In a first aspect, the present invention provides a method for establishing a spermatogonial stem cell line of an oval pomfret, comprising the following steps:

[0012] A. Culture the testicular tissue cells of the oval pomfret in complete culture medium, replacing the culture medium every 2 to 4 days for the first 15 days.

[0013] B. Observe the cell migration. When the cells have grown to the bottom of the flask, add trypsin solution to digest the cells for 1-2 minutes. Aspirate the digested cells every 30 seconds and add complete culture medium to culture them. Repeat three times to select a high proportion of spermatogonial stem cells for subsequent culture.

[0014] C. Screen out spermatogonial stem cells with a high ratio and transfer them to new culture bottles at a ratio of 1:1 to 3 to obtain spermatogonial stem cell lines of the oval pomfret.

[0015] Furthermore, the mass concentration of trypsin is 0.2% to 3%, preferably 0.25%;

[0016] Furthermore, the complete culture medium comprises the following components at the following concentrations: FBS 20 v / v%, flounder serum 1 v / v%, embryo extract 1 w / v%, penicillin-streptomycin-amphotericin 8 w / v%, bFGF 40 ng / μL and lif 40 ng / μL, and the solvent is L-15 culture medium;

[0017] Preferably, the testicular tissue cells of the oval pomfret are cultured in a complete culture medium at a temperature of 17 to 33° C., preferably 28° C.;

[0018] Preferably, fresh culture medium is replaced every 3 days within the first 15 days of culture.

[0019] Preferably, when the cells have grown to the bottom of the flask, trypsin solution is added to digest the cells for 1 minute.

[0020] Furthermore, the step of preparing the testis tissue cells of the oval pomfret comprises digesting a cleaned tissue block of the testis of a one-month-old oval pomfret with trypsin;

[0021] Preferably, the trypsin digestion treatment time is 30 to 60 minutes, preferably 30 minutes;

[0022] Preferably, the method for preparing a cleaned one-month-old oval pomfret testis tissue block comprises taking a one-month-old oval pomfret testis tissue block and placing it in L-15 culture medium to remove the mucosa on the surface of the tissue; rinsing the tissue block with a buffer solution 4 to 5 times, transferring it to penicillin-streptomycin-amphotericin and soaking it for 3 to 6 minutes;

[0023] Preferably, the buffer is PBS buffer;

[0024] Preferably, transfer to penicillin-streptomycin-amphotericin and soak for 5 minutes;

[0025] Preferably, the concentration of penicillin-streptomycin-amphotericin used for soaking is 100× penicillin-streptomycin-amphotericin;

[0026] Preferably, 5 mM Hepes buffer and 50 μM β-mercaptoethanol are added to the L-15 culture medium.

[0027] Furthermore, the one-month-old oval pomfret weighs 450±20 g and has a body length of 28±2 cm.

[0028] In a second aspect, the present invention provides a spermatogonial stem cell line of an oval pomfret, which is established according to the above-mentioned establishment method.

[0029] In a third aspect, the present invention provides a method for cryopreserving the above-mentioned spermatogonial stem cell line of the oval pomfret, comprising digesting the oval pomfret spermatogonial stem cells from the 3rd day of passage growth to the logarithmic growth phase with trypsin, centrifuging, adding the treated oval pomfret spermatogonial stem cells to a cell freezing solution, and placing them at 4°C for 20 to 40 minutes, at -20°C for 30 to 60 minutes, and at -80°C for at least 12 hours before transferring them to liquid nitrogen for long-term storage;

[0030] Preferably, the mass concentration of trypsin is 0.25%;

[0031] Preferably, complete medium is added after digestion to terminate the digestion;

[0032] Preferably, the centrifugation conditions include centrifugation at 200-400 g for 3-6 min, preferably at 300 g for 5 min;

[0033] Preferably, the cell freezing solution consists of L-15, FBS and DMSO, wherein the ratio of L-15:FBS:DMSO is 7:2:1;

[0034] Preferably, the treated spermatogonial stem cells of the oval pomfret are added to a cell freezing solution and placed at 4°C for 30 minutes, -20°C for 1 hour, and -80°C for at least 12 hours before being transferred to liquid nitrogen for long-term storage;

[0035] Preferably, the concentration of spermatogonial stem cells of the oval pomfret in the cell freezing solution is 2×10 6 cells.

[0036] In a fourth aspect, the present invention provides a method for resuscitating the above-mentioned oval pomfret spermatogonial stem cell line, comprising rapidly placing the frozen oval pomfret spermatogonial stem cells in a water bath, rapidly shaking and thawing for 1 to 2 minutes, centrifuging and discarding the supernatant, adding complete culture medium, and transferring to a culture flask for culture;

[0037] Preferably, the temperature of the water bath is 35-40°C, preferably 37°C;

[0038] Preferably, the centrifugation conditions include centrifugation at 200-400 g for 3-6 min, preferably at 300 g for 5 min;

[0039] Preferably, the culture temperature is 23-33°C, preferably 28°C.

[0040] In a fifth aspect, the present invention provides a method for identifying the above-mentioned oval pomfret spermatogonial stem cell line, comprising staining the 20th generation oval pomfret spermatogonial stem cells with alkaline phosphatase until the oval pomfret spermatogonial stem cells appear blue-purple, and identifying the stemness of the established oval pomfret spermatogonial stem cells through marker gene identification.

[0041] In a sixth aspect, the present invention provides the use of the above-mentioned oval pomfret spermatogonial stem cell line in expressing exogenous genes.

[0042] The present invention provides a method for establishing a spermatogonial stem cell line from an oval pomfret. Based on the characteristics of oval pomfret spermatogonial stem cells, a differential digestion method is used to obtain high-purity oval pomfret spermatogonial stem cells. By setting an appropriate passage ratio, an oval pomfret spermatogonial stem cell line is cultured and can be stably passaged. Based on the obtained oval pomfret spermatogonial stem cells, gene transfection research was conducted, providing technical support for extensive research on oval pomfret spermatogonial stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 This is a diagram showing the cell morphology of different generations in the passage culture of spermatogonial stem cells of the oval pomfret provided in Example 1 of the present invention;

[0045] Figure 2 This is a cell morphology diagram of spermatogonial stem cells of the oval pomfret recovered after cryopreservation provided in Example 2 of the present invention;

[0046] Figure 3 This is a growth curve of the 20th generation of oval pomfret spermatogonial stem cells at different temperatures provided in Example 3 of the present invention;

[0047] Figure 4 This is a growth curve of the 20th generation of oval pomfret spermatogonial stem cells in different culture media provided in Example 4 of the present invention;

[0048] Figure 5 This is an alkaline phosphatase staining identification image of the 15th generation of oval pomfret spermatogonial stem cells provided in Example 5 of the present invention;

[0049] Figure 6 This is a diagram of the origin and stemness gene detection of spermatogonial stem cells of the 22nd generation of oval pomfret provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0050] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0051] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0052] In one aspect, the present invention provides a method for establishing a spermatogonial stem cell line of an oval pomfret, comprising the following steps:

[0053] A. Culture the testicular tissue cells of the oval pomfret in complete culture medium, replacing the culture medium every 2 to 4 days for the first 15 days.

[0054] B. Observe the cell migration. When the cells have grown to the bottom of the flask, add trypsin solution to digest the cells for 1-2 minutes. Aspirate the digested cells every 30 seconds and add complete culture medium to culture them. Repeat three times to select a high proportion of spermatogonial stem cells for subsequent culture.

[0055] C. After screening out a high proportion of spermatogonial stem cells, the cells were subcultured into new culture flasks at a ratio of 1:1 to 3 to obtain an oval pomfret spermatogonial stem cell line.

[0056] According to the characteristics of spermatogonial stem cells of the oval pomfret, the present invention obtains spermatogonial stem cells of the oval pomfret with high purity by a differential digestion method, and by setting a suitable passage ratio, a spermatogonial stem cell line of the oval pomfret is obtained and can be stably passaged.

[0057] Among them, aspirating the digested cells every 30 seconds means that the digestion time of different cells is different, such as aspirating the cells digested for 30 seconds, 60 seconds, 1 minute, 1.5 minutes and 2 minutes respectively (the specific time can be determined according to the total digestion time), and adding complete culture medium to the cells aspirated at each time for culture.

[0058] Spermatogonial stem cells usually have 1-2 obvious nucleoli and are stained blue-purple by alkaline phosphatase. Based on cell morphology observation and alkaline phosphatase staining, the proportion of spermatogonial stem cells after culture of cells aspirated at each time is compared, and the cells with the highest proportion of spermatogonial stem cells are screened as high-proportion spermatogonial stem cells.

[0059] In some specific embodiments, the mass concentration of trypsin is 0.2% to 3%, preferably 0.25%.

[0060] In some specific embodiments, the step of preparing the testis tissue cells of the oval pomfret comprises digesting a cleaned tissue block of the testis of a one-month-old oval pomfret with trypsin.

[0061] In some specific embodiments, the trypsin digestion treatment lasts for 30 to 60 minutes, preferably 30 minutes.

[0062] In some specific embodiments, the cleaning step of the cleaned testis tissue block of one-month-old oval pomfret includes taking the testis tissue block of one-month-old oval pomfret and placing it in L-15 culture medium to remove the mucosa on the surface of the tissue; rinsing the tissue block with buffer 4 to 5 times, transferring it to penicillin-streptomycin-amphotericin and soaking it for 3 to 6 minutes; in some specific embodiments, the buffer is PBS buffer; in some specific embodiments, transferring it to penicillin-streptomycin-amphotericin and soaking it for 5 minutes; in some specific embodiments, the concentration of penicillin-streptomycin-amphotericin used for soaking is 100× penicillin-streptomycin-amphotericin.

[0063] On the basis of obtaining spermatogonial stem cells of oval pomfret, through exploration and optimization of different culture conditions, a culture medium system and culture conditions suitable for the stable passage of spermatogonial stem cells of oval pomfret were established. In some specific embodiments, the complete culture medium includes the following components at the following concentrations: FBS 20v / v%, flatfish serum 1v / v%, embryo extract 1w / v%, penicillin-streptomycin-amphotericin 8w / v%, bFGF 40ng / μL and lif 40ng / μL, and the solvent is L-15 culture medium. In some specific embodiments, the digested tissue cells are transferred to the complete culture medium and cultured at a temperature of 17 to 33°C, preferably 28°C.

[0064] In some specific embodiments, the L-15 medium is supplemented with 5 mM Hepes buffer and 50 μM β-mercaptoethanol.

[0065] In some specific embodiments, when the cells are cultured until they have grown to the bottom of the flask, trypsin solution is added to digest the cells for 1 minute.

[0066] In some specific embodiments, the one-month-old oval pomfret weighs 450±20 g and has a body length of 28±2 cm.

[0067] According to another aspect of the present invention, a spermatogonial stem cell line of an oval pomfret is provided. The spermatogonial stem cell line of an oval pomfret is established according to the above-mentioned establishment method.

[0068] According to another aspect of the present invention, a method for freezing the above-mentioned oval pomfret spermatogonial stem cell line is also provided, comprising using trypsin to digest the oval pomfret spermatogonial stem cells between 3 days of passage growth and the logarithmic growth phase, centrifuging, taking the treated oval pomfret spermatogonial stem cells, adding them to a cell freezing solution, placing them at 4°C for 20 to 40 minutes, -20°C for 30 to 60 minutes, and -80°C for at least 12 hours, and then transferring them to liquid nitrogen for long-term storage.

[0069] In some specific embodiments, the mass concentration of the trypsin is 0.25%; in some specific embodiments, complete culture medium is added after digestion to terminate digestion; in some specific embodiments, the centrifugation conditions include centrifugation at 200-400g for 3-6 minutes, preferably centrifugation at 300g for 5 minutes; in some specific embodiments, the cell freezing solution consists of L-15, FBS and DMSO, and the L-15:FBS:DMSO = 7:2:1; in some specific embodiments, the treated oval pomfret spermatogonial stem cells are added to the cell freezing solution and placed at 4°C for 30 minutes, -20°C for 1 hour, and -80°C for at least 12 hours before being transferred to liquid nitrogen for long-term storage.

[0070] In some specific embodiments, the concentration of spermatogonial stem cells of the pomfret in the cell freezing solution is 2×10 6 cells.

[0071] According to another aspect of the present invention, a method for reviving the above-mentioned oval pomfret spermatogonial stem cell line is also provided, comprising rapidly placing the frozen oval pomfret spermatogonial stem cells in a water bath, rapidly shaking and thawing for 1 to 2 minutes, centrifuging and discarding the supernatant, adding complete culture medium and transferring to a culture flask for culture.

[0072] In some specific embodiments, the temperature of the water bath is 35-40°C, preferably 37°C; in some specific embodiments, the centrifugation conditions include centrifugation at 200-400g for 3-6 min, preferably centrifugation at 300g for 5 min; in some specific embodiments, the culture temperature is 23-33°C, preferably 28°C.

[0073] According to another aspect of the present invention, a method for identifying the above-mentioned oval pomfret spermatogonial stem cell line is also provided, comprising staining the 20th generation oval pomfret spermatogonial stem cells with alkaline phosphatase until the oval pomfret spermatogonial stem cells appear blue-purple, and identifying the stemness of the established oval pomfret spermatogonial stem cells through marker gene identification.

[0074] The present invention conducts gene transfection research based on the aforementioned spermatogonial stem cell line, providing technical support for extensive research on spermatogonial stem cells of the same species. According to another aspect of the present invention, the use of the aforementioned spermatogonial stem cell line of the same species in expressing exogenous genes is also provided.

[0075] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0076] Example 1 Primary Culture of Spermatogonial Stem Cells of Pomfret ovata

[0077] 1. Material Acquisition and Primary Culture

[0078] One-month-old oval pomfret (weight 450 g ± 20 g, body length 28 ± 2 cm) was anesthetized with MS-222 and placed in 75% alcohol for 5 minutes for preliminary disinfection. Then, the oval pomfret testis tissue was placed in a small culture dish containing 1 mL of PBS and the tissue mucosa was removed. The tissue was washed 5 to 6 times in PBS with sterile forceps and then transferred to 1 mL of 0.25% trypsin (Gibco, 25200072). The tissue was cut into 1 mm pieces with sterile surgical ophthalmic scissors. 3 Digest the tissue pieces at room temperature for 30 minutes, shaking the culture dish to remove the cells. Transfer the digested tissue and cell suspension to a 25cm 2 Add 5 mL of complete culture medium to the culture flask and incubate at 28°C. Observe cell migration and observe cell morphology and photograph using a Leica inverted microscope. Change the culture medium every 3 days for the first 15 days, replacing the old medium while preserving the tissue fragments whenever possible. Once the tissue fragments have fully adhered and cells have migrated, discard any remaining unattached fragments.

[0079] 2. Culture medium preparation

[0080] Complete medium: L-15 medium containing 20% FBS (Shanghai Jianglin Biotechnology Co., Ltd.), 8% 100× penicillin-streptomycin-amphotericin B (Beijing Solebao Technology Co., Ltd., referred to as Solebao), 40 ng / μL lif (Yishen Biotechnology (Shanghai) Co., Ltd., referred to as Yisheng), 40 ng / μL bFGF (Yishen), 1% flounder serum and embryo extract. Subculture medium: L-15 medium containing 15% FBS (Shanghai Jianglin Biotechnology Co., Ltd., referred to as Shanghai Jianglin), 1% 100× penicillin-streptomycin-amphotericin B (Solebao), 40 ng / μL lif (Yishen), 40 ng / μL bFGF (Yishen), 1% flounder serum and embryo extract.

[0081] The washing solution was prepared by adding 8% 100× penicillin-streptomycin-amphotericin B to L-15 medium.

[0082] 3. Cell Subculture

[0083] When the spermatogonial stem cells of the oval pomfret fill the bottom of the monolayer flask (cell confluence >80%), or when cells aggregate and grow in piles around the tissue mass, they are passaged. First, remove the old culture medium with a pipette, rinse twice with 1 mL of rinse buffer, and add 1 mL of 0.25% trypsin containing EDTA to digest the cells for 1 minute. Aspirate the cells after digestion for 30 seconds, 60 seconds, and 1 minute, respectively. Add complete culture medium to the cells aspirated at each time and culture them. Repeat three times. The cells aspirated after 60 seconds are selected as having a high proportion of spermatogonial stem cells and are immediately passaged. The passage ratio is 1:2. Then, the culture medium is filled to 5 mL and placed in culture at 28°C. Cells in the logarithmic growth phase are passaged every 3 to 4 days.

[0084] Figure 1 Middle A shows primary cultured cells migrating from around the testicular tissue mass of the oval pomfret; Figure 1 Middle B is the morphology of spermatogonia of the third generation of oval pomfret; Figure 1 Middle C is the morphology of spermatogonia of the fourth generation of oval pomfret; Figure 1 Middle D is the morphology of spermatogonia of the 5th generation of oval pomfret; Figure 1 Middle E is the morphology of spermatogonia of the 15th generation of oval pomfret; Figure 1 Middle F is the morphology of spermatogonia of the 25th generation of oval pomfret.

[0085] Example 2 Cell cryopreservation and thawing

[0086] The spermatogonial stem cell line of the oval pomfret prepared in Example 1 was selected as a sample, and the operation was carried out according to the following steps:

[0087] The oval pomfret spermatogonial stem cells in the logarithmic growth phase were cryopreserved. First, the cells were rinsed once with rinsing solution, and then 1 mL of 0.25% trypsin digestion solution containing EDTA was added for digestion. When the cells became round and fell off, 1 mL of complete culture medium was added to terminate the digestion. The cells were collected into a centrifuge tube and centrifuged at 300 g for 5 minutes. The supernatant was removed and 2 mL of cell freezing solution was added to the cell pellet. The cell freezing solution contained L-15:FBS:DMSO = 7:2:1. The cells were mixed by pipetting and then placed at 4°C for 30 minutes, -20°C for 1 hour, and -80°C overnight. Finally, the cells were transferred to a liquid nitrogen tank for long-term storage.

[0088] Cell recovery adheres to the principle of rapid dissolution. The spermatogonial stem cells of the oval pomfret were taken out of the liquid nitrogen tank and immediately placed in a preheated 37℃ water bath. They were shaken to thaw quickly for about 1 minute, then centrifuged at 300g for 5 minutes, the supernatant was removed, 1mL of complete culture medium was added to blow the cell pellet, and the cell pellet was transferred to a culture flask and complete culture medium was added to 5mL. Figure 2 Middle A shows the state of spermatogonial stem cells of the 15th generation of oval pomfret after 1 day of recovery. Figure 2Middle B shows the state of spermatogonial stem cells of the 15th passage of the oval pomfret after 3 days of recovery. The cell confluence is 90%, and the cell morphology is similar to that before cryopreservation.

[0089] Example 3 Comparison of the growth of spermatogonial stem cells of ovate pomfret at different temperatures

[0090] The growth rate of spermatogonial stem cells of the 20th generation of oval pomfret at different temperatures was detected. First, the cells passaged to the 20th generation were digested into a cell suspension, and then counted with a hemocytometer to calculate the cell concentration. 4000 cells were added to each well and placed in an incubator at different temperatures (17°C, 23°C, 28°C and 33°C) for culture. The CCK-8 detection kit (Yisheng, 40203ES88) was used to detect the cells at 1d, 2d, 3d, 4d and 5d of culture according to the instructions. Finally, a cell growth curve was drawn. Figure 3 As shown in the growth curves of spermatogonial stem cells from the 20th generation of the oval pomfret at different temperatures, cells barely proliferated at 17°C, maintaining only viability. At 23°C, cells proliferated, but at a slower rate than at 28 and 33°C. At 28°C, cells grew fastest, exhibiting exponential growth, peaking between days 3 and 5. At 33°C, cells proliferated most rapidly during the first day 1 and 2, but the rate of proliferation decreased over time. Therefore, 28°C is the optimal growth temperature for spermatogonial stem cells of the oval pomfret.

[0091] Example 4 Comparison of the growth of spermatogonial stem cells of Pomfret ovata in different culture media

[0092] The growth rate of spermatogonial stem cells of the 20th generation of oval pomfret under different culture media was detected. First, the cells passaged to the 20th generation were digested into a cell suspension, and then counted with a hemocytometer to calculate the cell concentration. 4000 cells were added to each well, and different culture media (DMEM / F12 (1:1), L-15 (Business), M199, DMEM, MEM and L-15 (self-made)) were added and cultured at 28°C. Then, the CCK-8 detection kit was used to detect the cells at 1d, 2d, 3d, 4d and 5d according to the instructions. Finally, a cell growth curve was drawn. Figure 4 As shown, at passage 20, spermatogonial stem cells of elliptical pomfret grew fastest in DMEM / F12 culture at 28°C, while the growth rates of M199, L-15 (self-made) and L-15 (Business) cells were almost the same, but higher than those of DMEM and MEM cells.

[0093] Among them, DMEM / F12 (1:1) (Gibco, C1330500BT), L-15 (Business) (Gibco, 41300070), M199 (Gibco, 11150059), DMEM (Gibco, C1995500BT) and MEM (Gibco, 10370-021) were purchased from commercial sources; L-15 (self-made) (the configuration of the complete medium is the same as in Example 1).

[0094] Example 5 Alkaline phosphatase staining of spermatogonial stem cells of ovate pomfret

[0095] Alkaline phosphatase staining was used to detect whether the spermatogonial stem cells of the 15th generation of oval pomfret have stemness. Alkaline phosphatase is expressed at high levels in primordial germ cells (PGC), pluripotent stem cells and embryonic stem cells, and is often used as a marker to indicate cell stemness. After 1 day of cell passage, the old culture medium was aspirated, and the cells were rinsed twice with PBS. Then 3 mL of 4% paraformaldehyde fixative (Solabo, P1110) was added, and the cells were fixed at 4°C for 30 minutes. The cells were rinsed twice with PBS, and the alkaline phosphatase detection kit (Shanghai Biotech Co., Ltd., P0321S) was used for detection. Finally, the cells were photographed under an inverted microscope. Figure 5 As shown, spermatogonial stem cells of the 15th passage of the oval pomfret appeared purple after alkaline phosphatase staining for 24 hours.

[0096] Example 6 Identification of the Origin and Stemness Gene Expression of Spermatogonial Stem Cell Lines in Oval Pomfret

[0097] RNA was extracted from spermatogonial stem cells of the oval pomfret using the RNAsimple Total RNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP419) according to the manufacturer's instructions. The RNA was reverse-transcribed into cDNA, and a partial sequence of the oval pomfret 18S rRNA was amplified as a template to verify the origin of the cell line. Sequence analysis of GPSSCs revealed 100% similarity to the oval pomfret 18S rRNA sequence (Genbank accession number: AJ421455). A conventional PCR reaction was performed using 2× Hieff PCR Master Mix. The reaction volume (20 μL) consisted of 10 μL 2× Hieff PCR Master Mix, 7 μL ddH₂O, 1 μL forward primer, 1 μL reverse primer, and 1 μL template cDNA. PCR reaction conditions included initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s, followed by a complete extension at 72°C for 10 min. The primer sequences used for cell origin identification and stemness gene expression detection (the nucleotide sequence numbers of each primer are SEQ ID NO. 1 to 6, respectively) are shown in Table 1.

[0098] Table 1

[0099]

[0100]

[0101] The results of cell line origin verification are as follows Figure 6 As shown, spermatogonial stem cells of the oval pomfret can express the germinal stem cell-specific marker gene pou5f1 and the sex-related gene dmrt1.

[0102] In order to further study the application of the spermatogonial stem cell line of the oval pomfret, the present inventors carried out gene transfection research on the basis of obtaining spermatogonial stem cells of the oval pomfret to express exogenous genes.

[0103] The 18th generation of the spermatogonial stem cells of the oval pomfret of the present invention were inoculated into a 24-well plate and cultured in a 28°C incubator overnight for 24 hours before being transfected with the plasmid pEGFP-N1. The transfection method was carried out according to the operating instructions of the lipo3000 transfection kit (Invitrogen Corporation). The specific operation was to dilute the lipo3000 reagent with Opti-MEM culture medium and mix it thoroughly, and name it as mixed solution 1; dilute the plasmid DNA (0.5 μg / well) with Opti-MEM and mix it thoroughly, and name it as mixed solution 2; mix the mixed solution 1 and the mixed solution 2 thoroughly and incubate at room temperature for 15 minutes, add them to the cells, and observe the fluorescence expression after 24 hours. The inventors found that the fluorescence signal was observed 48 hours after transfection, and the transfection efficiency was about 60%, which proved that the spermatogonial stem cell line of the oval pomfret of the present invention has the ability to express exogenous genes.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a spermatogonial stem cell line from an oval pomfret, characterized in that: The following steps are involved: A. Culture the testicular tissue cells of the oval pomfret in complete culture medium, replacing the culture medium every 2 to 4 days for the first 15 days. B. Observe the cell migration. When the cells have grown to the bottom of the flask, add trypsin solution to digest the cells for 1-2 minutes. Aspirate the digested cells every 30 seconds and add complete culture medium to culture them. Repeat three times to select a high proportion of spermatogonial stem cells for subsequent culture. C. Screen out spermatogonial stem cells with a high ratio and transfer them to new culture bottles at a ratio of 1:1 to 3 to obtain spermatogonial stem cell lines of the oval pomfret.

2. The establishment method according to claim 1, characterized in that The mass concentration of the trypsin is 0.2% to 3%, preferably 0.25%.

3. The establishment method according to claim 2, characterized in that The complete culture medium comprises the following components: FBS 20 v / v%, flounder serum 1 v / v%, embryo extract 1 w / v%, penicillin-streptomycin-amphotericin 8 w / v%, bFGF 40 ng / μL and lif 40 ng / μL, and the solvent is L-15 culture medium; Preferably, the testicular tissue cells of the oval pomfret are cultured in a complete culture medium at a temperature of 17 to 33° C., preferably 28° C.; Preferably, fresh culture medium is replaced every 3 days within the first 15 days of culture; Preferably, when the cells have grown to the bottom of the flask, add 0.25% trypsin solution to digest the cells for 1 minute; Preferably, after three repetitions, the cells are passaged into new culture flasks at a ratio of 1:

2.

4. The establishment method according to claim 3, characterized in that: The step of preparing the testis tissue cells of the oval pomfret comprises digesting and treating a cleaned tissue block of the testis of the oval pomfret of one month old with trypsin; Preferably, the trypsin digestion treatment time is 30 to 60 minutes, preferably 30 minutes; Preferably, the method for preparing a cleaned one-month-old oval pomfret testis tissue block comprises taking a one-month-old oval pomfret testis tissue block and placing it in L-15 culture medium to remove the mucosa on the surface of the tissue; rinsing the tissue block with a buffer solution 4 to 5 times, transferring it to penicillin-streptomycin-amphotericin and soaking it for 3 to 6 minutes; Preferably, the buffer is PBS buffer; Preferably, transfer to penicillin-streptomycin-amphotericin and soak for 5 minutes; Preferably, the concentration of penicillin-streptomycin-amphotericin used for soaking is 100× penicillin-streptomycin-amphotericin; Preferably, 5 mM Hepes buffer and 50 μM β-mercaptoethanol are added to the L-15 culture medium.

5. The establishment method according to claim 4, characterized in that: The one-month-old oval pomfret weighs 450±20 g and has a body length of 28±2 cm.

6. A spermatogonial stem cell line of Pomfret ovata, characterized in that: The spermatogonial stem cell line of the oval pomfret is established according to the establishment method according to any one of claims 1 to 5.

7. The method for cryopreservation of the spermatogonial stem cell line of Pomfret ovata according to claim 6, characterized in that: The method comprises using trypsin to digest spermatogonial stem cells of the oval pomfret between 3 days of passage growth and the logarithmic growth phase, centrifuging, adding the treated spermatogonial stem cells of the oval pomfret to a cell freezing solution, placing the cells at 4°C for 20 to 40 minutes, storing the cells at -20°C for 30 to 60 minutes, storing the cells at -80°C for at least 12 hours, and then transferring the cells to liquid nitrogen for long-term storage; Preferably, the mass concentration of trypsin is 0.25%; Preferably, complete medium is added after digestion to terminate the digestion; Preferably, the centrifugation conditions include centrifugation at 200-400 g for 3-6 min, preferably at 300 g for 5 min; Preferably, the cell freezing solution consists of L-15, FBS and DMSO, wherein the ratio of L-15:FBS:DMSO is 7:2:1; Preferably, the treated spermatogonial stem cells of the oval pomfret are added to a cell freezing solution and placed at 4°C for 30 minutes, -20°C for 1 hour, and -80°C for at least 12 hours before being transferred to liquid nitrogen for long-term storage; Preferably, the concentration of spermatogonial stem cells of the oval pomfret in the cell freezing solution is 2×10 6 cells.

8. The method for resuscitating the spermatogonial stem cell line of Pomfret ovata according to claim 6, characterized in that: The method includes rapidly placing the frozen oval pomfret spermatogonial stem cells in a water bath, rapidly shaking and thawing for 1 to 2 minutes, centrifuging and discarding the supernatant, adding complete culture medium, and transferring to a culture bottle for culture; Preferably, the temperature of the water bath is 35-40°C, preferably 37°C; Preferably, the centrifugation conditions include centrifugation at 200-400 g for 3-6 min, preferably at 300 g for 5 min; Preferably, the culture temperature is 23-33°C, preferably 28°C.

9. The method for identifying the spermatogonial stem cell line of the oval pomfret according to claim 6, characterized in that: The method includes staining the 15th generation oval pomfret spermatogonial stem cells with alkaline phosphatase until the oval pomfret spermatogonial stem cells appear blue-purple, and identifying the stemness of the oval pomfret spermatogonial stem cells through marker gene identification.

10. Use of the oval pomfret spermatogonial stem cell line according to claim 6 in expressing exogenous genes.

Images