Sexual gland cell line of western hybrid sturgeons and application of sexual gland cell line

Abstract

The invention belongs to the field of prevention and control of aquatic organism cells and aquaculture diseases, and particularly discloses a sexual gland cell line of acipenser sinensis hybrid and application of the sexual gland cell line. The name of the cell line is ZaXFine, the cell line is preserved in the China Center for Type Culture Collection, the preservation date is July 11, 2024, and the preservation number is CCTCC NO: C2024201. The oxtail hybrid sturgeon gonad cell line ZaXFine constructed by the invention can realize stable passage, is sensitive to copper ions, can be applied to heavy metal copper pollution research, lays an important research foundation for healthy culture and disease prevention and control of oxtail hybrid sturgeons, can be applied to stem cell research, and has important significance in promoting sustainable utilization and biodiversity protection of sturgeon resources.

Description

Technical Field

[0001] The present invention belongs to the field of aquatic biological cells and aquaculture disease prevention and control, and specifically relates to a gonad cell line of a western hybrid sturgeon (Acipenser siberiensis♀×Acipenser schrenckii♂ hybrid sturgeon) and an application thereof. Background Art

[0002] As a core tool in the field of biological research, cell lines are used in many key areas, including virology, physiology, toxicology, immunology, cell biology, endocrinology, genetics, and germplasm resource protection. Compared with mammalian cell lines, the research on fish cell lines started later, but they have shown unique advantages in scientific research practice, including but not limited to high cost-effectiveness, good experimental repeatability, strong adaptability to cell environments, long growth cycles, and significantly extended lifespan of cell lines. These advantages make fish cell lines an important resource for in-depth exploration of biological mechanisms and promoting the development of related applications.

[0003] Since the rainbow trout gonad cell line was successfully established as the first fish cell line, the scientific research community has successfully cultivated more than 280 cell lines from different tissue types of freshwater and marine fish. As the core component of the fish reproductive system, the gonads and their somatic cells (such as granulosa cells, theca cells, interstitial cells in the ovaries, and specific cell types in the testes) play a vital role in the proliferation, differentiation and development of germ cells. With the growing popularity of reproductive stem cell research, it is particularly important to establish and optimize fish gonad somatic cell lines, which not only helps to reveal the molecular mechanisms of reproductive development, but also provides a valuable experimental platform for reproductive medicine, genetic improvement, and germplasm resource protection.

[0004] It is worth noting that the long-term culture of reproductive stem cells in vitro (up to several months) has been successfully achieved by using the gonadal somatic cells of model organisms such as zebrafish as feeder layers. This breakthrough not only demonstrates the potential of fish cell lines in supporting stem cell growth, but also opens up new avenues for future research in reproductive biology and regenerative medicine. Therefore, strengthening in-depth research on fish cell lines, especially gonad-derived cell lines, is of immeasurable value in promoting innovation in biological theory and promoting the practical application of related technologies.

[0005] Sturgeons, as a kind of cartilaginous and hard-scaled fish with a long history, high endangerment and great scientific research value, are not only indispensable living fossils in the process of biological evolution, but also deeply reveal the complexity and diversity of fish evolutionary paths. They also play a key role in maintaining the balance of aquatic ecosystems and biodiversity. Their economic value is also not to be ignored. They are favored by the international market for their superior meat quality and rare caviar. There are 8 known species in my country, and more than 20 species in the world. The Chinese sturgeon (Acipenser sinensis), the Yangtze sturgeon (Acipenser dabryanus), and the white sturgeon (Psephuyrus gladius) are sturgeons unique to the Yangtze River Basin in my country. Among them, the white sturgeon has been declared extinct, and the Chinese sturgeon and the Yangtze sturgeon are also endangered to varying degrees. Although my country has made significant progress in the artificial breeding technology of the Chinese sturgeon and the Yangtze sturgeon, due to their long sexual maturity cycle (9-11 years for males and 11-13 years for females) and long spawning interval (4-6 years), the current artificial breeding efficiency is still difficult to meet the needs of species protection. Reproductive stem cell transplantation technology has attracted much attention because it can significantly shorten the breeding cycle and improve breeding efficiency, and is regarded as a potential and efficient means of sturgeon conservation. However, since both the Chinese sturgeon and the Yangtze sturgeon are first-class protected animals, it is extremely difficult and strictly restricted to directly obtain their reproductive cell materials.

[0006] In view of this, the present invention selected the hybrid offspring of the Siberian sturgeon (Acipenser baerii, ♀) and the Schrenckii (Acipenser schrenckii, ♂) - the western hybrid sturgeon as the research object. This hybrid variety is not only widely cultivated in China and has good economic value, but also provides rich materials for scientific research due to the complexity of its genetic background. The method of constructing the gonadal cell line of the western hybrid sturgeon was explored, and the stability and safety of the cell line were verified through a series of strict identification procedures and preliminary toxicological studies. It is expected to provide a solid experimental foundation and technical platform for the subsequent construction of the somatic gonadal cell line of endangered sturgeons, and then provide important research materials for the subsequent stem cell transplantation of sturgeons. Summary of the invention

[0007] In view of the shortcomings of the prior art, one of the objects of the present invention is to provide a gonad cell line of a hybrid sturgeon, which is a gonad cell line of a hybrid of Acipenser siberianus ♀ and Acipenser schrenckii ♂, named ZaXFine, and preserved in the China Center for Type Culture Collection at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a preservation date of July 11, 2024 and a preservation number of CCTCC NO: C2024201.

[0008] Furthermore, the method for establishing the gonadal cell line ZaXFine of the western hybrid sturgeon is as follows:

[0009] S1. Tissue pretreatment: The gonadal tissue of hybrid sturgeon was taken and immersed in a PBS solution containing penicillin-streptomycin-amphotericin B, and the fat tissue and bacteria were removed by shaking;

[0010] S2. Tissue processing: Filter the tissue with a cell sieve, rinse with PBS solution, and then transfer to a cell culture flask for attachment;

[0011] S3. Primary cell culture: Add DMEM medium containing 20% ​​FBS to a cell culture bottle with good tissue block adhesion, and culture in a 28°C incubator until the cells migrate out from the edge of the tissue to obtain the gonadal cells of the hybrid sturgeon;

[0012] S4. Subculture: When the primary cultured cells cover 90% or more of the bottom of the culture flask, discard the culture medium, wash with PBS, digest with trypsin, and then subculture; the first subculture is 1:2. After the 10th generation of subculture, the fetal bovine serum content in the culture medium is reduced to 10-15%, and subculture is carried out every 48 hours.

[0013] Furthermore, the cell type of the ZaWFine cell line was identified as fibroblasts by immunofluorescence using vimentin and cytokeratin as marker proteins, respectively.

[0014] Furthermore, the cell source of the ZaWFine cell line was identified as a western hybrid sturgeon cell line by extracting total cell DNA, performing PCR amplification using β-actin primers, and sequencing the amplified product. The sequencing results showed 100% similarity with the β-actin sequence of the Siberian sturgeon and the Acipenser schrenckii.

[0015] The second purpose of the present invention is to provide the application of the gonad cell line ZaXFine of the western hybrid sturgeon. The cell line can be used as a cell model for the study of heavy metal copper pollution, and can be used to explore the mechanism of copper ions on cells, monitor the copper ion pollution status in coastal estuaries, etc. The cell line can also be used for stem cell research, as a basic material for reproductive stem cell culture and transplantation, and assist in sturgeon breeding and related molecular mechanism research.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention screened out a hybrid sturgeon gonad cell line that was stable in progeny (has been passed down to the 36th generation) and sensitive to copper ions, thus overcoming the accidental difficulty in cell line screening.

[0018] 2. The present invention provides a method for constructing a stably propagated western hybrid sturgeon gonad cell line, which provides a solid experimental basis and technical platform for the subsequent culture of germ cells of endangered sturgeons such as the Chinese sturgeon and the subsequent further culture of stem cells, and is of great significance for promoting the sustainable utilization of sturgeon resources and the protection of biodiversity.

[0019] 3. The copper-sensitive western hybrid sturgeon gonad cell line provided by the present invention can provide a cell model for subsequent studies on the mechanism of action of copper ions on cells, monitoring of copper ion pollution in coastal estuaries, and other heavy metal copper pollution studies, laying an important research foundation for the healthy breeding and disease prevention and control of western hybrid sturgeon. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a morphological picture of the hybrid sturgeon gonad cell line ZAXFine under an inverted microscope in Example 1.

[0021] In the figure: A is the primary gonadal tissue cell of the western hybrid sturgeon; B is the gonadal tissue cell of the 10th generation of the western hybrid sturgeon; C is the gonadal tissue cell of the 25th generation of the western hybrid sturgeon; D is the gonadal tissue cell of the 35th generation of the western hybrid sturgeon.

[0022] Figure 2 This is a diagram of the cell source identification experiment results of Example 2. In the diagram: M is Maker; the amplified product of Siberian sturgeon β-actin is xbβ-actin; the amplified product of Acipenser schrenckii β-actin is ssβ-actin.

[0023] Figure 3 1 is a diagram showing the results of the cell type identification experiment in Example 3. In the diagram: A is the staining result of cytokeratin, and B is the immunofluorescence result of vimentin.

[0024] Figure 4 The copper sulfate poisoning test results of the cells in Example 4. In the figure: A is a control group with a copper sulfate concentration of 0 μmol / L, B is a test group 1 with a copper sulfate concentration of 20 μmol / L, C is a test group 2 with a copper sulfate concentration of 100 μmol / L, D is a test group 3 with a copper sulfate concentration of 200 μmol / L, and E is a test group 4 with a copper sulfate concentration of 300 μmol / L. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] Example 1: Establishment of the gonadal cell line ZaXFine of hybrid sturgeon

[0027] (1) Tissue pretreatment

[0028] After anesthetizing healthy second-generation hybrid sturgeons (male juveniles), rinse and soak them in 75% alcohol for 1 to 3 minutes. In the clean bench of the laboratory, sterilized scalpels and scissors were used to cut open the abdominal cavity of the hybrid sturgeon, and the gonadal tissue was carefully removed and soaked in a PBS solution containing 5v / v% penicillin-streptomycin-amphotericin B for 30 minutes. During this period, the tissue block was shaken up and down to peel off the fat tissue and bacteria.

[0029] (2) Tissue processing

[0030] After filtering with a sterile 100-mesh cell strainer, the tissue fragments were continuously rinsed with a sterile Pasteur pipette containing 5 v / v% penicillin-streptomycin-amphotericin B. 2 The cell culture flask was allowed to adhere to the wall for 30 minutes so that the tissue blocks could adhere to the wall and would not fall off easily during the subsequent culture treatment.

[0031] (3) Primary cell culture

[0032] 25cm in tissue block with good adhesion 2 Add culture medium (DMEM containing 20% ​​FBS) to the cell culture bottle and culture in a 28°C incubator. After the cells migrate out from the edge of the gonadal tissue of the hybrid sturgeon, replace the culture medium at regular intervals. If mycoplasma contamination is encountered, add mycoplasma scavenger in time for timely control.

[0033] (4) Subculture

[0034] When the primary culture cells cover 90% or more of the bottom of the culture flask, discard the culture medium, wash once with PBS, add 1 mL of trypsin solution for 30 seconds, quickly remove the trypsin, place on a clean bench or a 28°C constant temperature incubator, observe under a microscope every 2 minutes, and when the cells are dispersed into single cells, add culture medium to blow the cells down and mix them; the first subculture uses a 1:2 subculture (the first generation of cell types is complex and some may not be able to be subcultured). The first time the hybrid sturgeon gonad cells fill the culture flask is 5 days, and the subculture is carried out at a 1:2 ratio, and the subculture is performed every 2 days. After the 10th generation of subculture, the fetal bovine serum content in the culture medium is reduced to 10-15v / v%, and subsequent subculture is carried out. During the subculture period, the growth state is stable and the proliferation rate is fast. It can be subcultured every 48 hours. It has been subcultured to the 36th generation. The cell shape is as follows Figure 1 Thus, the present invention obtains a stably propagated western hybrid sturgeon gonad cell line, and the cell line is named ZaXFine.

[0035] (5) Cryopreservation of cell lines

[0036] The gonadal cells of hybrid sturgeon grow to 25cm 2After the cells were cultured in the flask, the cells were collected after trypsin digestion for 1 min and centrifuged at 2000×g for 5 min. The supernatant was removed and 1 mL of cell freezing solution was added for freezing. The cell freezing solution consisted of 10% DMSO, 40% FBS and 50% M199.

[0037] Example 2: Identification of the cell source of the gonadal cell line ZaXFine of the hybrid sturgeon

[0038] The Tiangen Rapid DNA Extraction Detection Kit (KG203) was used to extract total DNA from the gonadal cells of the 30th generation of western hybrid sturgeon. The specific operation method is shown in the DNA extraction section in the kit manual. The extracted DNA was amplified by PCR, and the amplification primers are as follows:

[0039] The Siberian sturgeon primers xbβ-actinF: 5′-ATGGTTGGTATGGGACAGAAAGA-3′ and xbβ-actinR: 5′-ATGGCTGGGGTGTTGAAGG-3′ were used for amplification, and the target band was 266 bp;

[0040] The primers ssβ-actinF: 5′-ATCGCCGCACTGGTTGTTGA-3′ and ssβ-actinR: 5′-ATGCCGTGCTCGATGGGATA-3′ were used for amplification, and the target band was 212 bp;

[0041] The obtained PCR products were detected by agarose gel electrophoresis. Figure 2 As shown, two target bands were amplified. At the same time, the PCR product was sent to Sangon for sequencing. The sequencing results were compared with the sequences on NCBI. The results showed that it had 100% similarity with the β-actin reference gene sequences of Siberian sturgeon and Acipenser schrenckii. Therefore, it was proved that the ZaXFine cell line was a cell of western hybrid sturgeon.

[0042] Example 3: Cell type identification of the gonadal cell line ZaXFine of western hybrid sturgeon

[0043] (1) The gonadal cells of hybrid sturgeon were inoculated into a 6-well plate, the culture medium was removed the next day, and the plate was washed once with PBS to remove the residual culture medium;

[0044] (2) fixing the washed western sturgeon gonad cells obtained in step (1) with 4% paraformaldehyde at room temperature, and washing them twice with PBS after 10 minutes to remove the 4% paraformaldehyde remaining on the hybrid sturgeon gonad cells;

[0045] (3) adding Triton X-100 to the fixed hybrid sturgeon gonadal cells obtained in step (2) and permeabilizing them at room temperature for 20 min, and then washing them with PBS three times;

[0046] (4) adding 5% bovine serum albumin (BSA) to the permeabilized gonadal cells obtained in step (3) and blocking the cells at room temperature for 1 hour;

[0047] (5) Adding a primary antibody diluent to the hybrid sturgeon gonad cells blocked in step (4), incubating at 4°C overnight, recovering the primary antibody, and washing with PBS three times, each time for 3 minutes; using vimentin antibody (ab8069, abcam) and cytokeratin antibody (ab7753, abcam) as primary antibodies for the experiment, respectively, diluting at a ratio of 1:800, and preparing the primary antibody diluent with 5% BSA;

[0048] (6) adding 200 μL of fluorescently labeled secondary antibody (i.e., FITC-labeled goat anti-mouse IgG antibody, the secondary antibody was diluted at a ratio of 1:2000 and prepared in 5% BSA) to the hybrid sturgeon gonad cells treated in step (5), incubating at room temperature for 1 h, removing the secondary antibody, and washing the cells with PBS for 3 times, each time for 3 min, to obtain fluorescently labeled hybrid sturgeon gonad cells;

[0049] (7) Nuclear staining: Add nuclear staining solution DAPI (diluted at a ratio of 1:2000, prepared in PBS) to the fluorescently labeled western sturgeon gonad cells obtained in step (6), incubate at room temperature for 5 min, and wash with PBS three times, each time for 3 min, to obtain the western sturgeon gonad cells after nuclear staining;

[0050] (8) Observe and collect images of the hybrid sturgeon gonad cells after the cell nucleus staining obtained in step (7) under a fluorescence microscope.

[0051] The results are as follows Figure 3 As shown in the figure, cytokeratin, which marks epithelial cells, is negative, while vimentin, which marks fibroblasts, is positive. Thus, the cell type of the ZaXFine cell line is identified as fibroblasts rather than epithelial cells.

[0052] Example 4: Experiment on the poisoning of the gonadal cell line ZaXFine of western hybrid sturgeon with copper ions

[0053] The gonadal cell line of the 33rd generation hybrid sturgeon with good growth condition was digested with 0.25% trypsin and then passaged. The cell density was adjusted to 2×10 5 / ml, inoculated into a 25mL cell culture flask, the inoculation volume of the hybrid sturgeon gonadal cells was 5mL. After culturing for 24 hours, the old culture medium was discarded, and the same volume of new culture medium with copper sulfate concentrations of 0, 20, 100, 200 and 300μmol / L was added, and cultured in a CO2 incubator (5%, 25℃) for 24 hours, and then the changes in the cells after poisoning were observed under an inverted microscope and images were collected. The results are shown in Figure 4 As shown, it can be seen that with the increase of copper sulfate concentration, the number of cell deaths increased significantly, proving that the ZaXFine cell line is sensitive to copper ions.

[0054] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A gonadal cell line of a hybrid sturgeon, characterized in that: The name is ZaXFine, deposited in the China Center for Type Culture Collection, the preservation date is July 11, 2024, and the preservation number is CCTCC NO: C2024201.

2. The gonadal cell line of western hybrid sturgeon according to claim 1, characterized in that: The cell line was established as follows: S1. Tissue pretreatment: The gonadal tissue of hybrid sturgeon was taken and immersed in a PBS solution containing penicillin-streptomycin-amphotericin B, and then shaken to remove the fat tissue and bacteria; S2. Tissue processing: Filter the tissue with a cell sieve, rinse with PBS solution, and then transfer to a cell culture flask for attachment; S3. Primary cell culture: Add DMEM medium containing 20% ​​FBS to a cell culture bottle with good tissue block adhesion, and culture in a 28°C incubator until the cells migrate out from the edge of the tissue to obtain the gonadal cells of the hybrid sturgeon; S4. Subculture: When the primary cultured cells cover 90% or more of the bottom of the culture flask, discard the culture medium, wash with PBS, digest with trypsin, and then subculture; the first subculture is 1:

2. After the 10th generation of subculture, the fetal bovine serum content in the culture medium is reduced to 10-15%, and subculture is carried out every 48 hours.

3. The gonadal cell line of western hybrid sturgeon according to claim 1, characterized in that: The cell type of the cell line was identified as fibroblasts by immunofluorescence using vimentin and cytokeratin as marker proteins, respectively.

4. The gonadal cell line of western hybrid sturgeon according to claim 1, characterized in that: The cell source of the cell line was identified as a western hybrid sturgeon cell line by extracting total cell DNA, performing PCR amplification using β-actin primers, and sequencing the amplified product. The sequencing results showed that the β-actin sequence had 100% similarity with that of the Siberian sturgeon and the Schrenckii sturgeon.

5. Use of the gonadal cell line of hybrid sturgeon described in claim 1 as a cell model in the study of heavy metal copper pollution.

6. Use of the gonadal cell line of hybrid sturgeon of claim 1 in stem cell research.

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