A turbot intestinal tissue cell line

Abstract

The application provides a scophthalmus maximus intestinal tissue cell line, which has a preservation number of CCTCC NO: C2022231. The scophthalmus maximus intestinal tissue cell line provided by the application can be continuously subcultured, has been subcultured to 60 generations and remains stable, can provide a large number of stable scophthalmus maximus intestinal cells, and is used for researches on immune function genes and epithelial adhesion function genes. The provided cell line has excellent traits, is a fibroblast, the cells are fusiform or irregular triangular, the cell bodies extend several irregular protrusions outward, and the cells are closely arranged. The cell line can be subcultured once every 3-4 days, has high resuscitation efficiency, and can be used for plasmid transfection, siRNA interference and various molecular biology experiments.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fish cell culture, and particularly relates to a Scophthalmus maximus intestinal tissue cell line. BACKGROUND

[0002] Scophthalmus maximus belongs to Scophthalmidae and Scophthalmus, and is an important economic fish species in many countries. However, in recent years, Scophthalmus maximus culture is frequently disturbed by bacterial diseases, especially Aeromonas salmonicida, Edwardsiella tarda and Vibrio anguillarum, which seriously restricts the development of Scophthalmus maximus culture. Therefore, research on the immune system and disease mechanism of Scophthalmus maximus is imminent.

[0003] Cell culture technology is an important technology in biological research, which mainly refers to taking tissues or cells from living organisms, and under appropriate conditions in vitro, the tissues or cells are kept alive, grown and propagated, and their structure and function are maintained. Compared with directly using fish individuals for experiments, using fish cell lines for experiments has the advantages of simple operation, no constraints on site and season, high repeatability, etc. The application range of cell culture in vitro involves fish immunology, virology, molecular genetics, pharmacology, toxicology, cell engineering breeding, germplasm preservation and other disciplines.

[0004] Considering that bacterial diseases are one of the most serious diseases that harm aquatic organisms, and seriously affect the development of China's aquaculture industry. With the development of cell culture technology, the role of fish cell culture in fish disease and immunology research is also becoming more and more obvious. Although fish primary cell culture technology is currently a relatively mature technology, it is still difficult to obtain a stable cell line suitable for experimental model construction. SUMMARY

[0005] The present application aims to provide a Scophthalmus maximus intestinal cell line, its construction method and application. The preserved cell line is stable in passage and has a high survival rate. It provides a research basis for the study of intestinal bacterial diseases of Scophthalmus maximus, thereby making up for the lack of research on fish intestinal diseases.

[0006] The Scophthalmus maximus intestinal cell SMI provided by the present application was preserved in the China Center for Type Culture Collection in Wuhan, Wuhan University on August 24, 2022, and the preservation number is CCTCC NO: C2022231.

[0007] The Scophthalmus maximus intestinal cell SMI provided by the present application is obtained by screening after construction from the intestinal tissue of Scophthalmus maximus juvenile fish.

[0008] The method for establishing the turbot intestinal tissue cell SMI provided by the present invention comprises the following steps:

[0009] 1) Obtaining Turbot Intestinal Tissue:

[0010] The intestinal tissue of 6-month-old turbot juveniles was collected, rinsed with PBS, and then transferred to DMEM basal medium containing penicillin, streptomycin, gentamicin, and amphotericin B;

[0011] 2) Primary culture:

[0012] The intestinal tissue of turbot was soaked in DMEM basal medium containing penicillin, streptomycin, gentamicin and amphotericin B for 2 hours, then moved to 75% ethanol and soaked for 1 minute, and then rinsed with PBS. Finally, the intestinal tissue of turbot was gently scraped to remove mucus and cut into 1mm pieces with scissors. 3 Transfer the tissue block to a T25 culture flask, remove the culture medium, leaving only a small amount to keep it moist, and place the culture flask at 20°C for 2 hours to allow the tissue block to adhere to the bottom of the culture flask. Add complete culture medium and finally place it in a 20°C incubator for culture.

[0013] 3) Subculture: When the primary culture is carried out until the migrated cells cover about 80% of the bottom plate of the culture flask, the culture medium is aspirated and the cells are washed with PBS to remove excess serum. The adherent cells are then digested with 0.25% trypsin and subcultured to obtain a turbot intestinal tissue cell line.

[0014] In a preferred embodiment of the present invention, the complete culture medium is based on DMEM+Hepes, containing final concentrations of 15% fetal bovine serum FBS, 5‰ β-mercaptoethanol, 1× non-essential amino acids, 500U / mL penicillin, 500μg / mL streptomycin, 1.25μg / mL amphotericin B and 250μg / mL gentamicin.

[0015] The turbot intestinal tissue cell line provided by the present invention is used for the research of immune function genes and epithelial adhesion function genes.

[0016] The cell lines provided by the present invention are also used for functional studies of intestinal flora;

[0017] This cell line can also be used to detect the toxic effects of heavy metal ions and other chemicals in the water environment on intestinal cells.

[0018] In another aspect, the present application provides a method for culturing the enteron tissue cell line SMI of Scophthalmus maximus, which comprises culturing the cell line in DMEM or L15 medium supplemented with 10-15% fetal bovine serum at a temperature of 20-28°C, preferably 24°C.

[0019] The enteron tissue cell line SMI of Scophthalmus maximus provided by the present application can be continuously subcultured, and has been subcultured to the 60th generation and kept stable, and can provide a large amount of stable enteron tissue cells of Scophthalmus maximus for researches on immune function genes and epithelial adhesion function genes, etc. The cell line provided by the present application has excellent properties, is fibroblast, and the cells are spindle-shaped or irregular triangular, with several irregular processes extending out of the cell body, and the cells are closely arranged. The cell line can be subcultured once every 3-4 days, has high resuscitation efficiency, and can be used for plasmid transfection, siRNA interference and various molecular biology experiments. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a microscope photograph of the primary culture of the enteron tissue cell line of Scophthalmus maximus on the 15th day (10x),

[0021] Figure 2 Figure 2 is a microscope photograph of the enteron tissue cell line of Scophthalmus maximus after resuscitation and cultured for 24 hours (10x),

[0022] Figure 3 Figure 3 is a karyotype distribution diagram of the enteron tissue cell line of Scophthalmus maximus,

[0023] Figure 4 Figure 4 is a diagram of cell growth detection of the enteron tissue cell line of Scophthalmus maximus cultured in different concentrations of fetal bovine serum for 5 days,

[0024] Figure 5 Figure 5 is a diagram of cell growth detection of the enteron tissue cell line of Scophthalmus maximus cultured in different temperatures for 5 days,

[0025] Figure 6 Figure 6 is a diagram of cell growth detection of the enteron tissue cell line of Scophthalmus maximus cultured in different types of basic culture medium for 5 days,

[0026] Figure 7 Figure 7 is a microscope photograph of the cell line transfected with GFP-siRNA for 48 hours,

[0027] Figure 8 Figure 8 is a microscope photograph of the cell line transfected with pEGFP-N1 for 48 hours. DETAILED DESCRIPTION

[0028] The complete culture solution used in the application comprises DMEM+Hepes basic culture medium, fetal bovine serum FBS, β-mercaptoethanol, non-essential amino acids (Gibco, 11140050), penicillin, streptomycin, amphotericin B and gentamicin. The DMEM culture medium, fetal bovine serum FBS and non-essential amino acids provide the necessary nutrients for cell growth; Hepes provides a stable growth environment for cells; β-mercaptoethanol stimulates cell proliferation and improves cell activity; penicillin, streptomycin, amphotericin B and gentamicin expand the antibacterial spectrum, especially in primary culture, which can effectively inhibit bacterial growth and prevent contamination.

[0029] The technical solutions of the application will be further described and explained in combination with the drawings and specific embodiments.

[0030] Example 1: Construction of cell line

[0031] The Scophthalmus maximus used for constructing the cell line in the application is from Huanghai Aquatic Products Co., Ltd. of Yantai, Shandong.

[0032] The information related to the solutions used in this example is as follows:

[0033] 1) PBS, a commercial 1x PBS purchased.

[0034] 2) Basic culture medium: DMEM powder 13.5 g (Invitrogen, 12800-058) + Hepes powder 9.528 g + 1 L of triple distilled water, stored at 4℃.

[0035] 3) Complete culture medium: 7.5 mL of fetal bovine serum (FBS, Gibco) + 0.5 mL of 100x penicillin-streptomycin double-antibiotic solution, final concentration of 1.25 μg / mL amphotericin B and 250 μg / mL gentamicin, 50 μL of β-mercaptoethanol, 0.5 mL of 100x non-essential amino acid solution and 40 mL of basic culture medium, stored at 4℃.

[0036] 4) Cell cryopreservation solution: 1 mL of DMSO (purchased from Invitrogen Company) + 2 mL of fetal bovine serum + 7 mL of complete culture medium, prepared fresh.

[0037] The steps of the method for establishing the Scophthalmus maximus intestinal cell line of the application are as follows:

[0038] 1) Put the 6-month-old Scophthalmus maximus juvenile fish (about 10 cm long, about 25 g) into a bucket, then drop 2-3 drops of clove oil until the fish stops swimming, lies on the bottom of the bucket, and the gill cover stops opening and closing. Wipe the surface of the fish with a cotton ball soaked in 75% alcohol, and separate the tissue with sterile surgical scissors. The removed intestinal tissue is placed in a 15 mL centrifuge tube containing a penicillin-streptomycin PBS solution, and then immediately moved to a clean bench for operation.

[0039] 2) In the clean bench, turn on the ultraviolet lamp to sterilize, and take the intestinal tissue. Add PBS to 3 wells, 75% sterile ethanol to 1 well, and DMEM medium containing 4 antibiotics (penicillin, streptomycin, gentamicin, and amphotericin B) to 2 wells in a 6-well plate. The tissue is sequentially passed through PBS, PBS, 75% sterile ethanol (soaked for 2 min), PBS, antibiotic-containing DMEM (1 h), and antibiotic-containing DMEM (0.5-1 h). The tissue block is transferred to a 6 cm dish, and the tissue is cut into small pieces with scissors or a scalpel. The small tissue pieces (1 mm 3 ) are transferred to a T25 culture flask, the tissue is placed, the excess medium is absorbed, and the culture flask is placed horizontally in a 20°C incubator for 2 h and vertically for 0.5 h. Complete medium is added for culture. As shown in Figure 2, 12 days after primary culture of the Scophthalmus maximus intestinal cells, new cells migrate out from the adherent cells and around the tissue block, and small cell clusters appear around the 15th day. Figure 1

[0040] 3) When the coverage of the primary cells on the bottom of the culture flask reaches about 80%, subculture can be performed. The medium is aspirated, and the excess serum is removed by washing with PBS. Then, the adherent cells are digested with 0.25% trypsin for about 3 min without shaking the culture flask. The trypsin is aspirated, complete medium is added and blown, the intestinal cells are suspended, and subculture is performed at a ratio of 1:2. After subculture, the cells are cultured in a 20°C incubator. Subculture is performed every 3-4 days thereafter until after the 50th generation. The cells are stable during culture, have a fast subculture speed, and are suitable as experimental tool cells.

[0041] The final Scophthalmus maximus intestinal tissue cell line obtained after screening is named SMI, and was preserved on August 24, 2022 at the China Center of Type Culture Collection in Wuhan, Wuhan University, with a preservation number of CCTCC NO: C2022231.

[0042] Example 2: Cryopreservation and recovery of Scophthalmus maximus intestinal cells SMI

[0043] 1) Cryopreservation: Take a bottle of T25 culture flask containing Scophthalmus maximus intestinal cell line cells in the logarithmic growth phase, centrifuge the cells after 0.25% trypsin digestion. Use cryopreservation solution to prepare 5×10 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.

[0044] 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 20°C incubator.

[0045] like Figure 2 , the adhesion rate of turbot intestinal cell line reached about 80% 24 hours after recovery, and the cell morphology was similar to that before freezing, and the condition was good.

[0046] Example 3: Chromosome Karyotype Analysis of Turbot Intestinal Cell SMI

[0047] 1) Chromosome karyotype analysis of SMI cells was performed at passage 10. Turbot intestinal 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.

[0048] 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.

[0049] 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 3As shown, the chromosome analysis of the enteron cell line of Scophthalmus maximus showed that the karyotype was approximately normal distribution; 46% of the observed mitotic phase cell chromosome number was 44; the chromosome number was between haploid and tetraploid (22-88). In conclusion, the cell line obtained in the application has the same chromosome number as that of Scophthalmus maximus individuals.

[0050] Example 4: Screening of optimal culture conditions of SMI of Scophthalmus maximus enteron cells

[0051] Firstly, the optimal serum concentration is screened. Prepare complete culture medium with FBS concentrations of 2%, 5%, 10%, 15% and 20%. Digest SMI of passage 52, adjust the cell concentration, and inoculate SMI cells into 48-well plates at 5×10 4 cells per well, 15 wells for each serum concentration of each cell, and place the cells in a 20°C incubator for culture. Then, every 24 hours, collect cells from 3 wells in each serum concentration, and collect cells for 5 days. Count the cells using a cell counter, and calculate the total amount of cells per well. Plot the growth curve of cells under different basal medium conditions with culture time as the horizontal coordinate and cell number as the vertical coordinate, determine the appropriate serum concentration in the cell growth medium, and the results are shown in Figure 4 As the serum concentration increases, the growth rate of SMI increases, and when the serum concentration is 20%, the growth rate of SMI is the fastest, but by the 5th day of growth, the cell number decreases to be basically consistent with that of 10% and 15% serum concentration, and the cell growth reaches the decline phase. To ensure the stable growth of SMI cells, the serum concentration can be maintained at 10% and 15%.

[0052] Secondly, SMI of passage 52 is used to determine the optimal growth temperature. SMI cells are inoculated into 6 48-well plates at 5×10 4 cells per well, and 15 wells are inoculated in each 48-well plate. Place the 48-well plates in a 28°C incubator for 3 hours, and then transfer the 48-well plates to incubators at 5 different temperatures of 16°C, 20°C, 24°C, 28°C and 32°C for culture. Then, every 24 hours, collect cells from 3 wells, and collect cells for 5 days. Count the collected cells using a cell counter, and calculate the total amount of cells per well. Plot the growth curve of cells under different temperature conditions with culture time as the horizontal coordinate and cell number as the vertical coordinate, determine the optimal growth temperature of SMI cells, and the results are shown in Figure 5 The cells grow most rapidly at 24°C, followed by 20°C and 28°C, grow slowly at 16°C, and are inhibited at 32°C.

[0053] The best base medium for SMI growth was detected by preparing the whole medium based on DMEM, DMEM:F12, M-199, RPMI-1640, and L15, respectively. The 52th generation SMI was digested, and the cell concentration was adjusted. The SMI cells were inoculated into a 48-well plate at 5x10 4 cells per well, and 15 wells were inoculated for each cell in each base medium. The cells were placed in a 20°C incubator for culture. Then, every 24 hours, 3 wells of each cell were digested to collect cells, and the cells were counted by a cell counter. The total number of cells per well was calculated. The growth curve of the cells in different base medium conditions was plotted with the culture time as the horizontal coordinate and the cell number as the vertical coordinate to determine the best base medium for cell growth. The results are shown in Figure 6 . SMI grew fastest in DMEM and L15 base medium, grew slowly in DMEM:F12 and 1640 base medium, and was inhibited in M-199 medium.

[0054] Example 5: Detection of siRNA transfection effect of SMI of Scophthalmus maximus intestinal cells

[0055] The Scophthalmus maximus intestinal cells were inoculated in 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. The Xfect Transfection Reagent kit of Takara was used for transfection. First, 25 pmol of GFP-siRNA was mixed in 30 μL of Xfect Reaction Buffer, and after standing for 5 s, 2.5 μL of Xfect RNA Polymer was mixed and incubated at room temperature for 10 min. 0.2 mL of culture medium was aspirated from the 24-well plate, leaving 0.3 mL / well, and then the transfection reagent was added to the 24-well plate. After 6 h of transfection, 0.5 mL of fresh culture medium was replaced for continued culture for 48 h. After 48 h of transfection, the cell transfection was observed under an inverted fluorescence microscope, and photographs were taken for record.

[0056] RNA interference technology prevents 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. After 48 h of transfection, the green fluorescence (GFP) was observed under a fluorescence microscope, as shown in Figure 7 . The results showed that the established Scophthalmus maximus intestinal tissue cell line could be used for small RNA interference experiments.

[0057] Example 6: Detection of plasmid transfection effect of SMI of Scophthalmus maximus intestinal cells

[0058] Turbot intestinal 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 Takara's Xfect Transfection Reagent transfection kit, first take 1μg pEGFP-N1 and add it to 30μL Xfect Reaction Buffer and mix it evenly. After standing for 5s, add 0.3μL XfectPolymer 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. Observe the cell transfection status under an inverted fluorescence microscope 48h after transfection, and take pictures and record them. The results are as follows Figure 8 As shown, reporter green fluorescence (EGFP) was observed.

[0059] The experimental results demonstrated that the established turbot intestinal tissue cell line is suitable for plasmid transfection experiments. Fluorescence microscopy revealed that SMI transfection efficiencies for both siRNA and plasmids exceeded 30%, exceeding those of other commonly used intestinal tissue-derived fish cells, such as EPCs and ZF4s.

[0060] 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 cell line, characterized in that: The deposit number of the cell line is CCTCC NO: C2022231.

2. Use of the cell line according to claim 1 in detecting the toxic effects of heavy metal ions and other chemicals in water environments on intestinal cells.

3. A method for culturing the cell line according to claim 1, characterized in that: The method is to culture in a culture medium supplemented with 10-15% fetal bovine serum.

4. The method according to claim 3, wherein The culture medium is DMEM or L15 culture medium.

5. The method according to claim 3, wherein The method, wherein the culture temperature is 20-28°C.

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