Turbot gill tissue cell line and application thereof

Abstract

The present invention provides a turbot gill cell line, which has a deposit number of CCTCCNO: C2022230. The turbot gill tissue cell line SMG provided by the present invention can be continuously passaged. Experimental results show that it can be passaged up to 65 generations and remains stable, and can provide a large number of stable turbot gill cells for the study of immune function genes. The provided cell line has excellent properties and is a fibroblast. The cells are spindle-shaped or irregularly triangular, with several irregular protrusions extending outward from the cell body, and the cells are closely arranged. It is passaged once every 3-4 days, has high recovery efficiency, and can be used for plasmid transfection, siRNA interference and other molecular biology experiments.

Description

Technical Field

[0001] The invention belongs to the technical field of fish cell culture, and particularly relates to a turbot gill tissue cell line and applications thereof. Background Art

[0002] Turbot (Scophthalmus maximus), a member of the Scophthalmidae family and genus Scophthalmus, is an important commercial fish species in my country. However, in recent years, turbot aquaculture has been frequently plagued by bacterial diseases, particularly Aeromonas salmonicida, Edwardsiella tarda, and Vibrio anguillarum, which have severely hampered its development. Therefore, research on the immune system and disease mechanisms of turbot is urgently needed.

[0003] Fish gill tissue, along with the intestine and skin, is a crucial mucosal tissue in fish, playing a crucial role in defending against pathogenic invasion. These three mucosal tissues comprise epithelial cells, which act as a mechanical barrier, and are rich in immune cells that perform various immune functions. These interactions contribute to maintaining the mucosal immune barrier. Therefore, isolating turbot gill tissue cells and culturing them into cell lines will facilitate research into the pathogenic mechanisms of pathogens that use the mucosal tissue as a point of entry.

[0004] Bacterial diseases are among the most devastating to aquatic organisms, severely impacting the development of my country's aquaculture industry. With the advancement of cell culture technology, the role of fish cell culture in fish pathology and immunology research has become increasingly prominent. Although primary fish cell culture is a relatively mature technology, obtaining stable cell lines suitable for experimental model development remains highly haphazard and difficult. Furthermore, reported turbot fin cell lines have not performed very well in various molecular biology experiments, such as plasmid transfection and siRNA interference. Summary of the Invention

[0005] The purpose of the present invention is to provide a turbot gill cell line, which has stable passage and high survival rate; and has a high transfection rate for exogenous nucleic acid substances, thereby providing a basis for the study of turbot gill bacterial diseases and making up for the shortcomings of the existing technology in the study of fish gill diseases.

[0006] The turbot gill cells SMG provided by the present invention were deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on August 24, 2022, with the deposit number CCTCC NO: C2022230.

[0007] The turbot gill cells SMG provided by the present invention are obtained by screening after construction from the gill tissue of turbot juveniles.

[0008] The turbot gill tissue cell line provided by the present invention can be used as an experimental subject for exogenous gene transformation or RNAi;

[0009] The gene, as specifically described in the examples, is an immune function gene or an epithelial adhesion function gene.

[0010] The turbot gill tissue cell line of the present invention can also be used as an experimental subject for pathogenic bacteria in fish mucosal tissues;

[0011] In another aspect, the present invention also provides a method for culturing or passage of the turbot gill cell SMG cell line, which comprises culturing in a culture medium supplemented with 10% fetal bovine serum;

[0012] Preferably, the culture medium is DMEM or L15 culture medium; and the culture temperature is 24°C.

[0013] The turbot gill tissue cell line SMG provided by the present invention can be continuously passaged. Experimental results show that it remains stable up to 65 generations, providing a large number of stable turbot gill cells for the study of immune function genes. The cell line has excellent properties and is composed of fibroblasts with spindle-shaped or irregular triangular cells, several irregular protrusions extending from the cell body, and densely packed cells. It can be passaged every 3-4 days, has high recovery efficiency, and is suitable for a variety of molecular biology experiments such as plasmid transfection and siRNA interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Microscopic photograph of turbot gill cell line primary culture on the 14th day (10×),

[0015] Figure 2 : Microscopic photograph of turbot gill cell line after 24 hours of culture (10×),

[0016] Figure 3 :Chromosome analysis and karyotype distribution of turbot gill cell line,

[0017] Figure 4 :Growth detection chart of turbot gill cell line cultured in different concentrations of fetal bovine serum for 5 days,

[0018] Figure 5 :Graph showing cell growth detection of turbot gill cell lines cultured in different temperature environments for 5 days.

[0019] Figure 6 :The cell growth detection chart of cell line SMG cultured in different types of basal culture medium for 5 days,

[0020] Figure 7 :Microscopic pictures of cell lines transfected with FAM-siRNA for 48 hours,

[0021] Figure 8 : Microscopic photographs of cells transfected with pEGFP-N1 for 48 hours. DETAILED DESCRIPTION

[0022] The complete culture medium used in the present invention comprises DMEM+Hepes basal medium, fetal bovine serum (FBS), β-mercaptoethanol, non-essential amino acids, penicillin, streptomycin, amphotericin B, and gentamicin. The DMEM medium, fetal bovine serum (FBS), and non-essential amino acids provide essential nutrients for cell growth; Hepes provides a stable growth environment for cells; β-mercaptoethanol stimulates cell proliferation and increases cell activity; and penicillin, streptomycin, amphotericin B, and gentamicin expand the antibacterial spectrum, particularly effectively inhibiting bacterial growth and preventing contamination during primary culture.

[0023] The turbot gill cells (SMG) provided by the present invention are obtained through screening and have excellent passage stability, maintaining stable properties even after 65 passages. Passaging is performed every 3-4 days, resulting in high recovery efficiency. Furthermore, the SMG cell line has a strong reproductive capacity, enabling the production of large quantities of stable turbot gill cells for research.

[0024] In addition, the SMG cell line of the present invention can be used for plasmid transfection, siRNA interference and other molecular biology experiments, and the cell state is stable during the experimental operation.

[0025] The technical solution of the present invention will be further illustrated and described below in conjunction with the accompanying drawings and specific implementation methods.

[0026] Example 1: Construction of cell lines

[0027] The turbot used in the present invention comes from Shandong Yantai Haiyang Yellow Sea Aquatic Products Co., Ltd., has a cultivation temperature of 18°C, a salinity of 28-30‰, a neutral pH, sufficient dissolved oxygen, is healthy and energetic, and weighs 25g.

[0028] The names and sources of the solutions used in this example are as follows:

[0029] PBS was purchased from commercial 1× PBS.

[0030] Basal culture medium: DMEM powder 13.5 g (Invitrogen, 12800-058) + Hepes powder 9.528 g + 1 L triple-distilled water, stored at 4°C.

[0031] Complete culture medium: 7.5 mL fetal bovine serum (FBS, Gibco) + 0.5 mL 100× penicillin-streptomycin solution, a final concentration of 1.25 μg / mL amphotericin B and 250 μg / mL gentamicin, 50 μL β-mercaptoethanol, 0.5 mL 100× non-essential amino acid solution and 40 mL basal culture medium, stored at 4°C.

[0032] Cell freezing solution: 1 mL DMSO (purchased from Invitrogen) + 2 mL fetal bovine serum + 7 mL complete culture medium, prepared before use.

[0033] The steps for establishing a turbot gill cell line are as follows:

[0034] 1) Place a 6-month-old turbot juvenile (approximately 10 cm long and 25 g in weight) in a bucket. Add 2-3 drops of eugenol until the fish stops swimming, lies on its side at the bottom of the bucket, and its gill covers stop opening and closing. Wipe the fish's surface with a cotton ball soaked in 75% alcohol and separate the tissue using sterile surgical scissors. Place the excised gill tissue in a 15 mL centrifuge tube containing penicillin-streptomycin in PBS and immediately transfer to a clean bench for manipulation.

[0035] 2) In the clean bench, turn on the UV lamp for sterilization and take the gill tissue. In a 6-well plate, add PBS to 3 wells, 75% sterile ethanol to 1 well, and DMEM culture medium containing 4 antibiotics (penicillin, streptomycin, gentamicin and amphotericin B) to 2 wells. Pass the tissue through PBS, PBS, 75% sterile ethanol (soak for 2 minutes), PBS, DMEM containing antibiotics (1 hour), and DMEM containing antibiotics (0.5-1 hour). Transfer the tissue block to a 6cm dish and cut the tissue into small pieces with scissors or a scalpel. Cut the small pieces of tissue (1mm 3 ) Transfer the tissue to a T25 culture flask, place the tissue, absorb the excess culture medium, place the culture flask flat in a 24℃ incubator for 2 hours, place the culture flask upright for 0.5 hours, and add complete culture medium for culture. Figure 1 As shown in the figure, 12 days after the primary culture of turbot gill cells, new cells migrated out from around the adherent cells and tissue blocks, and small cell clusters appeared around the 15th day.

[0036] 3) When the coverage of primary cells at the bottom of the culture flask reaches about 80%, subculture can be performed. Aspirate the culture medium and wash with PBS to remove excess serum. Then use 0.25% trypsin to digest the adherent cells for about 3 minutes. Do not shake the culture flask. Aspirate the trypsin, add complete culture medium and pipette to suspend the gill cells. Subculture at a ratio of 1:2. After subculture, place in a 24°C incubator for culture. Thereafter, subculture every 3-4 days until after the 50th generation. During the culture period, the cells are stable and the subculture speed is fast, making them suitable as experimental tool cells.

[0037] The turbot gill tissue cell line finally obtained by screening was named SMG and deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on August 24, 2022, with the deposit number CCTCC NO: C2022230.

[0038] Example 2: Cryopreservation and thawing of turbot gill cells SMG

[0039] 1) Cryopreservation: Take a bottle of turbot gill cell line cells in the logarithmic growth phase in a T75 culture flask, digest them with 0.25% trypsin and collect the cells by centrifugation. 5 Resuspend the cells in 100 cells / mL freezing buffer and gently pipette the cell pellet to evenly disperse it in the freezing buffer. Aliquot the cell suspension into cryovials, 1.5-1.8 mL per tube. Follow the slow freezing principle for cell freezing: place at 4°C for 10 minutes, -20°C for 30 minutes, and -80°C for 1 day before transferring to liquid nitrogen for storage.

[0040] 2) Thawing: Cells should be thawed quickly. Remove the cryovial from liquid nitrogen and quickly place it in a 37°C water bath. Gently shake the cryovial to allow for rapid and even thawing. Thawing should take no longer than 1 minute. Centrifuge the thawed cell suspension at 1,200 rpm for 5 minutes to remove DMSO. Resuspend the centrifuged cells in 5 mL of complete culture medium. Transfer the cell suspension to a T25 cell culture flask and incubate in a 24°C incubator.

[0041] like Figure 2 As shown in the figure, the attachment rate of turbot gill cell line reached about 80% 24 hours after recovery, and the cell morphology was similar to that before freezing, indicating that the cell line was in good condition.

[0042] Example 3: Chromosome Karyotype Analysis of Turbot Gill Cells SMG

[0043] 1) Chromosome karyotype analysis of SMG cells was performed at passage 12. Turbot gill cells in the logarithmic growth phase were cultured with colchicine at a final concentration of 10 μg / mL for 12 h, and then trypsinized to obtain a cell suspension.

[0044] 2) Centrifuge the collected cell suspension at 1,200 rpm for 10 minutes and gently aspirate the supernatant. Resuspend the cells in 2 mL of 1× PBS in a 15 mL centrifuge tube. Add 10 mL of ice water to allow the cells to hypotonically swell. Transfer the cells to a T75 culture flask and incubate at room temperature for 10 minutes. Slowly add 1 mL of fixative (1:3 glacial acetic acid:methanol). Transfer the cells to a 15 mL centrifuge tube and centrifuge at 1,200 rpm for 7 minutes. Carefully remove the supernatant and slowly add 5 mL of fixative dropwise. Resuspend the cells by gently pipetting. Incubate at room temperature for 10 minutes and centrifuge at 1,200 rpm for 7 minutes. Repeat this step three times and resuspend the cells in 0.5 mL of fixative. Drop the cell suspension onto a pre-chilled glass slide. Allow to dry naturally and then stain with 1× Giemsa for 10 minutes at room temperature. Gently rinse the slide with enzyme-free sterile water. After drying, mount the slide with neutral resin.

[0045] The number and karyotype of chromosomes are the basis of cytogenetics and are important indicators for identifying the species and sex of organisms. In cell culture, chromosomes are important indicators for identifying the origin of cells and whether they have undergone transformation during the culture process. Figure 3 As shown, chromosome analysis of turbot gill cell lines showed that their karyotypes were approximately normally distributed; 48% of the observed mitotic cells had 44 chromosomes; and the chromosome numbers were between haploid and tetraploid (22-88).

[0046] In summary, it is proved that the cell line obtained in the present invention has the same chromosome number as that of individual turbot.

[0047] Example 4: Screening for the Optimal Fetal Bovine Serum Concentration for Turbot Gill Cells SMG

[0048] Prepare complete culture medium with 2%, 5%, 10%, 15% and 20% FBS concentration. Take 53 passage SMG for digestion and adjust the cell concentration to 5×10 cells per well. 4 SMG cells were seeded into 48-well plates, and 15 wells of each serum concentration for each cell type were seeded. The cells were cultured in a 24°C incubator. After that, cells were digested and collected from 3 wells of each serum concentration every 24 hours for a total of 5 days. Cells were counted using a cell counter to calculate the total number of cells in each well. The growth curve of cells under different basal culture medium conditions was drawn with culture time as the horizontal axis and cell number as the vertical axis to determine the appropriate serum concentration in the cell growth medium. The results are shown in the figure. Figure 4As shown in the figure, SMG cells grow rapidly in 20% and 15% serum concentrations, exhibiting exponential growth on the second day after plating, but the growth rate decreases with increasing culture time. SMG cells maintain rapid growth in 10% serum and also maintain good growth in 2%-5% serum. Therefore, these cells can maintain a certain growth state in a wide range of serum concentrations. However, to maintain optimal growth of SMG cells and consider culture costs, the optimal culture of these cells can be selected in 10% serum concentration.

[0049] Example 5: Screening for the Optimal Growth Temperature of Turbot Gill Cells SMG

[0050] The optimal growth temperature of 53 passage SMG cells was determined. SMG cells were plated at 5×10 4 The cells were inoculated into 6 48-well plates, with 15 wells in each 48-well plate. The 48-well plates were placed in a 28°C incubator for 3 hours. After the cells adhered to the wall, the 48-well plates were transferred to incubators at 5 different temperatures: 16°C, 20°C, 24°C, 28°C and 32°C. After that, the cells were collected by digesting 3 wells every 24 hours for a total of 5 days. The collected cells were counted using a cell counter to calculate the total number of cells in each well. With the culture time as the horizontal axis and the number of cells as the vertical axis, the growth curve of the cells under different temperature conditions was drawn to determine the optimal growth temperature of SMG cells. The results are shown as follows: Figure 5 As shown in the figure, SMG can grow rapidly in culture medium at 24°C, 20°C, and 28°C, with the fastest growth at 24°C. SMG can also grow slowly at 16°C, but its growth is inhibited at 32°C.

[0051] Example 6: Screening of the Optimal Basic Culture Medium for Turbot Gill Cells SMG

[0052] Prepare complete culture medium based on DMEM, DMEM:F12, M-199, RPMI-1640, and L15, respectively. Take 53 passage SMG for digestion and adjust the cell concentration to 5 × 10 cells per well. 4 SMG cells were seeded into 48-well plates, and 15 wells of each basal culture medium for each cell type were inoculated. The cells were cultured in a 24°C incubator. After that, 3 wells of each cell type were digested and collected every 24 hours for a total of 5 days. Cells were counted using a cell counter to calculate the total number of cells in each well. With the culture time as the horizontal axis and the number of cells as the vertical axis, the growth curve of the cells under different basal culture medium conditions was drawn to determine the optimal basal culture medium for cell growth. The results are shown in the figure. Figure 6The cells can continue to grow in DMEM, L15, DMEM:F12 and 1640 medium, but the growth rate in DMEM and L15 is the fastest, while the growth rate in DMEM:F12 and 1640 is slightly slower. In M199 medium, the growth of SMG is inhibited.

[0053] Example 7: Detection of siRNA transfection effect in turbot gill cells SMG

[0054] Turbot gill cells were seeded into 24-well plates (3 wells) one day in advance, ideally at the time of transfection, when the cells were in the logarithmic phase and the cell density was approximately 60%. Using the Xfect Transfection Reagent transfection kit (TAKARA, 631450), 25 pmol of FAM-siRNA (GenePharma, China) (a fluorescent siRNA mimic) was first added to 30 μL of Xfect Reaction Buffer and mixed thoroughly. After standing for 5 seconds, 2.5 μL of Xfect RNA Polymer was added and mixed thoroughly. The mixture was then incubated at room temperature for 10 minutes. 0.2 mL of culture medium was aspirated from the 24-well plate, leaving 0.3 mL per well. The transfection reagent was then added to the 24-well plate. Six hours after transfection, the cells were replaced with 0.5 mL of fresh culture medium and cultured for a further 48 hours. 48 hours after transfection, the transfected cells were observed under an inverted fluorescence microscope and photographed.

[0055] RNA interference technology inhibits the expression of a specific gene by inhibiting transcription and translation, and has become an important tool for verifying gene function and drug targeting in the post-genomic era. 48 hours after transfection, the cells were examined under a fluorescence microscope. Figure 7 The green fluorescence of the reporter was observed. The experimental results showed that the established turbot gill tissue cell line SMG can be used for RNA interference experiments.

[0056] Example 8: Detection of plasmid transfection effect in turbot gill cells SMG

[0057] Turbot gill cells were inoculated into a 24-well plate (3 wells) one day in advance, so that the cells were in the logarithmic phase and the cell density was about 60% at the time of transfection. Using the Xfect Transfection Reagent transfection kit (TaKaRa, 631317), first take 1μg pEGFP-N1 and add it to 30μL Xfect Reaction Buffer and mix it evenly. After standing for 5s, add 0.3μL Xfect Polymer and mix it evenly. Let it stand at room temperature for 10min. Aspirate 0.2mL of culture medium in the 24-well plate, leaving 0.3mL / well, and then add the transfection reagent to the 24-well plate. After 6h of transfection, replace it with 0.5mL of fresh culture medium and continue to culture for 48h. After 48h of transfection, observe the cell transfection status under an inverted fluorescence microscope and take pictures to record. The results are as follows Figure 8 As shown in the figure, green fluorescence (EGFP) was observed. The experimental results showed that the established turbot gill tissue cell line is suitable for plasmid transfection experiments.

[0058] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of implementation of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A turbot gill tissue cell line, characterized in that: The deposit number of the cell line is CCTCC NO: C2022230.

2. Use of the turbot gill tissue cell line according to claim 1 as a host for exogenous gene transformation or RNAi experiments.

3. The use according to claim 2, characterized in that The exogenous gene is an immune function gene or an epithelial adhesion function gene.

4. Use of the turbot gill tissue cell line according to claim 1 as an experimental subject for pathogens of fish mucosal tissues.

5. A method for culturing the turbot gill tissue cell line according to claim 1, characterized in that: The method is to culture in a culture medium supplemented with 2-20% fetal bovine serum.

6. The method according to claim 5, wherein The method is to culture in a culture medium supplemented with 10% fetal bovine serum.

7. The method according to claim 5 or 6, wherein: The culture medium is DMEM or L15 culture medium.

8. The method according to claim 5, wherein The culture temperature is 20-28°C.

9. The method according to claim 8, wherein The culture temperature is 24°C.

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