Establishment and application of gobiocypris rarus intestinal cell line
By establishing the rare intestinal cell line Gbr-gut of Crucian carp and developing corresponding chemical acute toxicity evaluation methods, the problem of insufficient development of rare Crucian carp cell lines in the prior art was solved, and the efficiency and accuracy of the evaluation of acute toxicity of chemicals was achieved.
Patent Information
- Application Number
- CN202510132708.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-06
- Publication Date
- 2025-04-25
AI Technical Summary
In the prior art, the number of rare Crucian Crucian cell lines has been developed in a small number and has not been commercialized. No systematic research has been carried out in the field of ecological toxicology, so it is difficult to effectively carry out acute toxicity screening of chemicals based on fish cells.
A rare intestinal cell line Gbr-gut of Crucian carp was established, and the cell line was constructed through primary culture, subculture, frozen storage and resuscitation steps. Based on this cell line, the acute toxicity assessment method of chemicals was developed.
The long-term subculture and low-temperature frozen storage recovery of rare Crucian intestinal cell lines have been achieved, providing a reliable tool for the evaluation of acute toxicity of chemicals, improving the throughput and accuracy of chemical toxicity tests.
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Figure CN119875994A_ABST
Abstract
Description
Technical field:
[0001] The invention relates to the technical field of fish cell culture, and in particular to the establishment and application of a rare Gobius crucianus intestinal cell line. Background technology:
[0002] Gobiocypris rarus is a small carp fish endemic to my country and is also a native organism recommended for use in my country's environmental management of chemicals. Research on the biological characteristics of Gobiocypris rarus involves fields such as ecotoxicology, fish pathology and physiology. Its broodstock has a short sexual maturity period, a long breeding season, a large number of eggs, and can be artificially bred all year round. Therefore, it has the potential to become a model fish species endemic to China. Establishing a cell line of Gobiocypris rarus and conducting ecotoxicological research based on Gobiocypris rarus in the cell field will effectively expand the scope of use of Gobiocypris rarus in toxicity testing, creating conditions for it to become an internationally common model organism and to conduct in vitro alternative tests for fish.
[0003] In order to reduce the use of experimental fish and increase the throughput of chemical toxicity testing, people have proposed the concept of predicting fish acute toxicity based on acute toxicity tests based on fish cells. The number of cell lines developed for rare crucian carp is small and has not been commercialized, and no systematic research has been carried out in the field of ecotoxicology.
[0004] The intestine is the main site for chemical absorption and biotransformation. Developing rare goby intestinal cell lines and establishing acute toxicity screening technology for chemicals based on rare goby intestinal cells are issues that technical personnel in this field urgently need to address. Summary of the invention:
[0005] The purpose of the present invention is to provide the establishment and application of a rare goby intestinal cell line, to develop a rare goby intestinal cell line using the intestine, and to establish a chemical acute toxicity assessment method based on the rare goby intestinal cell line.
[0006] The first object of the present invention is to provide a rare minnow intestinal cell line Gbr-gut, which is preserved on January 3, 2025. The name of the preservation unit is Guangdong Microbial Culture Collection Center, referred to as GDMCC, address: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the preservation number is GDMCC No: 65714.
[0007] The second object of the present invention is to provide a method for constructing the above-mentioned rare minnow intestinal cell line, comprising the following steps:
[0008] (1) Primary culture of rare goby intestinal cells: Take healthy rare goby intestinal tissue, cut it into pieces and digest it, collect the resulting suspended cells and tissue fragments, inoculate them into primary cell culture medium, perform primary culture, and obtain rare goby intestinal primary culture cells;
[0009] (2) Subculture of rare goby intestinal cells: digesting the primary culture cells of step (1), collecting the obtained suspension cells, inoculating them into subculture cell culture medium, and subculture them to obtain a rare goby intestinal cell line;
[0010] (3) Cryopreservation and thawing of cells: When freezing cells, the rare crucian carp intestinal cell line from step (2) is digested, the precipitate obtained after centrifugation is mixed with a cryopreservation protection solution, gradient frozen, and then placed in liquid nitrogen for long-term cryopreservation; when thawing cells, the cell suspension in the cryopreservation tube is first thawed, and then the cell suspension in the cryopreservation tube is transferred to a cell culture medium, centrifuged at 800g for 5 minutes to obtain a precipitate, which is then inoculated into the cell culture medium for culturing.
[0011] Preferably, in step (1), the healthy rare crucian carp is starved for 24 hours in advance.
[0012] Preferably, the primary cell culture medium is an L-15 complete culture medium containing 20% v / v fetal bovine serum and 1% v / v penicillin-streptomycin-amphotericin B triple antibodies, which can provide sufficient nutrients for the culture of primary cells; the passage cell culture medium is an L-15 complete culture medium containing 10% v / v fetal bovine serum and 0-1% v / v penicillin-streptomycin-amphotericin B triple antibodies.
[0013] Preferably, the specific steps of step (1) are as follows: starve 3-5 healthy rare crucian carp for 24 hours in advance, soak them in 75% alcohol for 30-50 seconds, dissect and remove the intestinal tissue along the lateral line under sterile conditions, remove the surface mucus and the internal intestinal material with forceps, and transfer them to PBS buffer containing a final concentration of 2-5% v / v penicillin-streptomycin-amphotericin B; cut the intestinal tissue into 1 mm 2 The tissue block is mixed with the PBS buffer containing the three antibodies, and centrifuged to obtain a precipitate, and the operation of mixing with the PBS buffer containing the three antibodies and centrifuging to obtain a precipitate is repeated 2-3 times to obtain a pretreated tissue block; 5-12mL 0.25% trypsin is added to the pretreated tissue block, digested for 1-2h, centrifuged and the supernatant is discarded, primary cell culture fluid is added, and a cell suspension is obtained after resuspending; the cell suspension is transferred to a T-25 cell culture flask, and cultured at 28°C until the cells cover the bottom of the culture flask, thereby obtaining primary cultured cells of the intestine of the rare minnow.
[0014] Preferably, step (2) specifically comprises the following steps: discarding the solution after primary culture of cells, washing with PBS buffer, adding 0.5-2 mL of 0.25% trypsin for digestion until the cells become round, adding 10-20 mL of subculture cell culture medium to resuspend the cells, and subculture at a ratio of 1:2.
[0015] More preferably, 16 mL of cell culture medium is added to resuspend the cells.
[0016] Preferably, the specific steps of step (3) freezing are: washing the rare crucian carp intestinal cell line with PBS buffer, adding 0.5-2mL 0.25% trypsin for digestion, adding 5-10mL of cell culture medium to terminate digestion after the cells become round, transferring to a centrifuge tube, 800g, centrifuging for 5min to obtain a precipitate, adding a cell freezing solution containing 10% DMSO to resuspend the cells, and transferring the cell suspension to a cryopreservation tube; transferring the cryopreservation tube to a programmed cooling box, placing the programmed cooling box in a -80°C environment for 4-24h, and then transferring the cryopreservation tube to liquid nitrogen for long-term storage.
[0017] Preferably, the specific steps of resuscitation in step (3) are: taking out the frozen rare crucian carp intestinal cells from liquid nitrogen, quickly placing them in a water bath, and quickly shaking the cryotube to melt the liquid in the tube; transferring the cell suspension in the cryotube to 5-9 mL of cell culture medium, centrifuging at 800g for 5 minutes to obtain a precipitate, adding 5-10 mL of cell culture medium to resuspend the cells, transferring them to a T-25 culture flask, culturing at 28°C, and replacing with fresh culture medium after 12-24 hours to continue culturing to obtain revived rare crucian carp intestinal cells.
[0018] The third object of the present invention is to provide a method for evaluating the acute toxicity of chemicals based on the rare goby intestinal cell line, comprising the following steps:
[0019] (A) Inoculating rare gouji carp intestinal cells into a well plate: digest the rare gouji carp intestinal cell line described above to obtain a cell suspension, inoculate it into a cell culture plate, and culture it at 28° C. for 24-48 hours to obtain a cell culture plate with rare gouji carp intestinal cells growing all over the bottom of the well plate;
[0020] (B) exposing the intestinal cells of rare goby to the test chemical: dissolving the test chemical in a delivery medium to obtain an exposure solution, taking the cell culture plate with rare goby intestinal cells growing on the bottom of the well plate in step (A), adding the exposure solution, and continuously exposing for 24 hours to obtain a cell culture plate that has completed the exposure test; the delivery medium is L-15 culture medium;
[0021] (C) Detecting the effects of chemicals on intestinal cells of rare goby: Take the cell culture plate that has completed the exposure test in step (B), add working solution A and working solution B in sequence, incubate in the dark, and use a microplate reader to measure and detect cell viability;
[0022] (D) Data analysis: Process the raw data obtained in step (C), draw the concentration-effect curve, and calculate the median effect concentration of the test chemical.
[0023] Preferably, step (A) is specifically as follows: taking the above rare crucian carp intestinal cell line with a confluence of 80-95%, washing with PBS buffer, adding 0.25% trypsin for digestion until the cells become round, adding an appropriate amount of cell culture medium, wherein the cell culture medium is L-15 complete culture medium containing 10% fetal bovine serum and 0-1% penicillin-streptomycin-amphotericin B triple antibody, and adjusting the cell density to 4-6×10 5 / mL to obtain the cell suspension to be inoculated; before inoculation, first add 0.5-2mL of PBS buffer to rinse all the wells in the 24-well cell culture plate, discard the solution, and then transfer the cell suspension to be inoculated to the 24-well cell culture plate at an inoculation volume of 500μL / well according to the plating and dosing diagram, place it at 28°C, and culture it for 24-48h until the bottom of the cell culture plate is covered with rare crucian carp intestinal cells.
[0024] Preferably, step (B) is specifically as follows: dissolving the test chemical in DMSO to obtain a concentrated solution, diluting the concentrated solution in L-15 culture medium at a ratio of 1:200, or directly dissolving the test chemical in L-15 culture medium to obtain an exposure solution; taking the cell culture plate with rare crucian carp intestinal cells growing on the bottom in step (A), gently adding 1-2 mL of PBS buffer to each well, shaking the well plate slightly and discarding it, and then gently adding 2 mL of exposure solution to each well according to the plating and dosing diagram, placing it in an environment of 28°C, and continuously exposing it for 24 hours to obtain a cell culture plate that has completed the exposure test.
[0025] Preferably, step (C) is specifically as follows: taking the cell culture plate on which the exposure test in step (B) is completed, discarding the exposure solution, gently adding 1-2 mL of PBS buffer to each well, slightly shaking the well plate and then discarding it, adding 400 μL of working solution A to each well in a light-proof environment, incubating in the dark for 30-60 min, and measuring the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 590 nm; after measuring the fluorescence intensity of working solution A, discarding working solution A, adding 400 μL of working solution B to each well in a light-proof environment, incubating in the dark for 60-90 min, discarding working solution B, adding 400 μL of fixative to each well for a fixation time of 1-3 min, discarding the fixative, adding 400 μL of extract to each well, gently shaking the plate for 10-15 min, and finally measuring the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 645 nm.
[0026] Preferably, the working solution A comprises the following components in volume percentage: 10% of resazurin solution and 90% of DPBS buffer.
[0027] Preferably, the working solution B comprises the following components in volume percentage: 1-2% neutral red solution and 98-99% DPBS buffer.
[0028] Preferably, the fixing solution is prepared by dissolving 2.5 g of CaCl2 in 200 mL of ultrapure water, adding 3.38 mL of 37% formaldehyde solution, and then making up to 500 mL with ultrapure water.
[0029] Preferably, the extract is prepared by dissolving 5 mL of acetic acid in 100 mL of ultrapure water, adding 250 mL of anhydrous ethanol, and then making up the volume to 500 mL with ultrapure water.
[0030] Preferably, in step (C), when discarding the exposure solution, working solution A, working solution B, fixative solution, and extracting solution, the liquid is discarded into a waste liquid tank by quickly inverting the cell culture plate.
[0031] Preferably, step (D) is specifically as follows: the value of the well containing cells and exposure solution is subtracted from the value of the blank well without cells to obtain the value J, the value of the control group is subtracted from the value of the blank well without cells to obtain the value P, and then the cell viability at each exposure concentration is calculated using the following formula:
[0032]
[0033] The concentration-effect curve of the test chemical on the intestinal cells of rare goby was drawn according to the set test chemical concentration and the corresponding cell viability, and the half-maximal effect concentration of the test chemical on the intestinal cells of rare goby was calculated using the S-type nonlinear fitting curve.
[0034] The beneficial effects of the present invention are as follows:
[0035] (1) The rare crucian carp intestinal cell line of the present invention can be subcultured for a long time, can be cryopreserved and revived at low temperature; and can be applied to the acute toxicity assessment of chemicals, to be used for in vitro substitution of acute toxicity of fish, to explore the toxic effects of chemicals, and other studies.
[0036] (2) The method for evaluating the acute toxicity of chemicals based on the intestinal cell line of rare crucian carp constructed in the present invention has uniform cell plating, is convenient and quick, and can quickly screen out toxic test chemicals, providing cytotoxicity information and an alternative in vitro alternative method for chemical toxicity evaluation.
[0037] A rare intestinal cell line of Gobiocypris crucianus Gbr-gut, preserved on January 3, 2025, the name of the preservation unit is Guangdong Microbiological Culture Collection Center, abbreviated as GDMCC, address: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the preservation number is GDMCC No: 65714. Description of the drawings:
[0038] Figure 1The morphology of the primary cultured rare minnow intestinal cells under an inverted microscope in Example 1 of the present invention is observed, wherein A: the morphology of the first generation of cells; B: the morphology of the fourth generation of cells.
[0039] Figure 2 This is the morphological observation of the 50th generation of rare minnow intestinal cells subcultured under an inverted microscope in Example 1 of the present invention, wherein C: 100 times magnification; D: 200 times magnification.
[0040] Figure 3 Schematic diagram of the cell plating and dosing regimen in Example 2 of the present invention.
[0041] Figure 4 This is the concentration effect curve of the rare goby intestinal cell line exposed to two test chemicals in Example 2 of the present invention, wherein: (E) tetrabromobisphenol S; (F) 3,4-dichloroaniline. Specific implementation method:
[0042] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention. Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.
[0043] Example 1: Construction of rare gobiid intestinal cell line
[0044] The primary cell culture medium is L-15 complete culture medium containing 20% v / v fetal bovine serum and 1% v / v penicillin-streptomycin-amphotericin B triple antibodies; the passage cell culture medium is L-15 complete culture medium containing 10% v / v fetal bovine serum and 1% v / v penicillin-streptomycin-amphotericin B triple antibodies; the triple antibodies include 100 U / mL penicillin, 100 μg / mL streptomycin, and 0.25 μg / mL amphotericin B.
[0045] (1) Primary culture of intestinal cells of rare crucian carp
[0046] The intestinal tissue of a healthy rare goby is obtained, cut into pieces and digested, and the obtained suspended cells and tissue fragments are collected and inoculated into a primary cell culture medium for primary culture to obtain primary culture cells of the intestinal tract of the rare goby.
[0047] The specific steps are as follows: starve 3-5 healthy rare goby crucians for 24 hours in advance, soak them in 75% alcohol for 30-50 seconds, dissect and remove the intestinal tissues of 3-5 rare goby crucians along the lateral line under sterile conditions, remove the surface mucus and intestinal internal materials with tweezers, and transfer the tissues to PBS buffer containing a final concentration of 5% v / v penicillin-streptomycin-amphotericin B; take the intestinal tissue and cut it into 1 mm 2 The method comprises the steps of: mixing the tissue block with the PBS buffer solution containing the three antibodies, centrifuging to obtain a precipitate, repeating the operation of mixing with the PBS buffer solution containing the three antibodies, centrifuging to obtain a precipitate 2-3 times, and obtaining a pretreated tissue block; adding 5-12 mL of 0.25% trypsin to the pretreated tissue block, digesting for 1-2 hours, centrifuging and discarding the supernatant, adding 5 mL of primary cell culture fluid, and resuspending to obtain a cell suspension; transferring the cell suspension to a T-25 cell culture flask, culturing at 28° C. until the cells cover the bottom of the culture flask, and obtaining primary cultured cells of the intestine of the rare goby.
[0048] (2) Subculture of intestinal cells of rare crucian carp
[0049] The solution after the primary culture of the intestinal cells of rare goby crucian carp was discarded, and the cells were washed with PBS buffer. 1 mL of 0.25% trypsin was added for digestion until the cells became round, and 12 mL of subculture cell culture medium was added to resuspend the cells. The cells were subcultured at a ratio of 1:2 to obtain a rare goby crucian carp intestinal cell line.
[0050] (3) Cryopreservation and recovery of cells
[0051] The rare crucian carp intestinal cell line in step (2) was washed with PBS buffer, and 1 mL of 0.25% trypsin was added for digestion. After the cells became round, 5 mL of cell culture medium was added to terminate the digestion, and the cells were transferred to a centrifuge tube, centrifuged at 800 g for 5 minutes to obtain a precipitate, and cell freezing solution (60% cell culture medium, 30% fetal bovine serum, 10% DMSO, volume ratio) was added to resuspend the cells, and the cell suspension was transferred to a cryopreservation tube; the cryopreservation tube was transferred to a programmed cooling box, and the programmed cooling box was placed in a -80°C environment for 24 hours, and then the cryopreservation tube was transferred to liquid nitrogen for long-term storage.
[0052] When performing cell recovery, the frozen rare goby intestinal cells are taken out from liquid nitrogen, quickly placed in a 28°C water bath, and the cryovial is quickly shaken to melt the liquid in the tube; the cell suspension in the cryovial is transferred to 8 mL of cell culture medium, centrifuged at 800g for 5 minutes, the precipitate obtained is added with 7 mL of cell culture medium to resuspend the cells, and the precipitate is transferred to a T-25 culture flask, cultured at 28°C, and replaced with fresh culture medium after 24 hours to continue culturing, thereby obtaining recovered rare goby intestinal cells.
[0053] The above-mentioned rare minnow intestinal cell line Gbr-gut was preserved on January 3, 2025. The name of the preservation unit is Guangdong Microbial Culture Collection Center, abbreviated as GDMCC, address: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and the preservation number is GDMCC No: 65714.
[0054] Example 2
[0055] A method for evaluating the acute toxicity of chemicals based on a rare goby intestinal cell line comprises the following steps:
[0056] (A) Inoculation of rare carp intestinal cells in 24-well plates
[0057] The rare crucian carp intestinal cell line described in step (2) of Example 1 was taken, and the confluence was 80-95%. It was washed with PBS buffer, and digested with 0.25% trypsin until the cells became round. An appropriate amount of the cell culture medium in Example 1 was added to adjust the cell density to 4×10 5 / mL, and obtain the cell suspension to be inoculated;
[0058] Before inoculation, add 1 mL of PBS buffer to rinse all wells in the 24-well cell culture plate. After discarding the solution, place the cell suspension to be inoculated according to the plating and dosing diagram ( Figure 3 ) were transferred to a 24-well cell culture plate at an inoculum volume of 500 μL / well and cultured at 28°C for 24 h until the bottom of the cell culture plate was covered with rare goby intestinal cells.
[0059] (B) Exposure of rare goby intestinal cells to the test chemicals
[0060] The test chemicals (tetrabromobisphenol S, 3,4-dichloroaniline) were dissolved in DMSO to obtain a concentrated solution, and the concentrated solution was diluted in L-15 culture medium at a ratio of 1:200 to obtain an exposure solution; the concentration settings of the test chemicals (concentration setting interval ≤ 2.5) were: tetrabromobisphenol S, 200, 153.8, 102.6, 51.3, 25.6, 12.8 mg / L, 3,4-dichloroaniline, 250, 156.3, 65.1, 32.6, 13, 5.2 mg / L;
[0061] Take the cell culture plate with rare crucian carp intestinal cells growing on the bottom in step (A), gently add 1 mL of PBS buffer to each well, shake the well plate slightly and discard it, and then according to the plating and dosing diagram (L-15 culture medium containing 0.5% DMSO as the control group), gently add 2 mL of exposure solution to each well, place it at 28°C, and continue to expose it for 24 hours to obtain a cell culture plate that has completed the exposure test.
[0062] (C) Detecting the effects of chemicals on intestinal cells of rare goby
[0063] Take the cell culture plate that has completed the exposure test in step (B), gently add 1-2 mL of PBS buffer to each well, shake the well plate slightly and discard, add 400 μL of working solution A to each well in a light-proof environment, incubate in the dark for 30-60 minutes, and measure the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 590 nm; working solution A includes the following components in volume percentage: 10% resazurin solution and 90% DPBS buffer.
[0064] After the fluorescence intensity of working solution A is measured, working solution A is discarded, and 400 μL working solution B is added to each well in a light-proof environment. After incubation in the dark for 60-90 min, working solution B is discarded, 400 μL fixative is added to each well for a fixation time of 1-3 min. After the fixative is discarded, 400 μL extract is added to each well, the plate is gently shaken for 10-15 min, and finally, the fluorescence intensity is measured using an excitation wavelength of 530 nm and an emission wavelength of 645 nm; the working solution B comprises the following components in volume percentage: 1-2% neutral red solution and 98-99% DPBS buffer; the fixing solution is prepared by: 2.5 g of CaCl2 is dissolved in 200 mL of ultrapure water, 3.38 mL of 37% formaldehyde solution is added, and the volume is then diluted to 500 mL with ultrapure water; the extract is prepared by: 5 mL of acetic acid is dissolved in 100 mL of ultrapure water, 250 mL of anhydrous ethanol is added, and the volume is then diluted to 500 mL with ultrapure water.
[0065] (D) Data analysis
[0066] The value J is obtained by subtracting the blank wells without cells from the wells containing cells and exposure solution, and the value P is obtained by subtracting the blank wells without cells from the control group. The cell viability at each exposure concentration is calculated using the following formula:
[0067]
[0068] The concentration-effect curve of the test chemical on the intestinal cells of rare goby was drawn according to the set test chemical concentration and the corresponding cell viability, and the half-maximal effect concentration of the test chemical on the intestinal cells of rare goby was calculated using the nonlinear fitting curve.
[0069] Experimental results:
[0070] The concentration-effect curves of tetrabromobisphenol S and 3,4-dichloroaniline on the intestinal cells of rare goby Figure 4When working solution A was used to quantify the cytotoxic effects of the test chemicals on the intestinal cells of rare goby, the half-maximum effect concentrations of tetrabromobisphenol S and 3,4-dichloroaniline were 44.65 mg / L and 72.84 mg / L, respectively.
[0071] When working solution B was used to quantify the cytotoxic effects of the test chemicals on the intestinal cells of rare goby, the half-maximal effect concentrations of tetrabromobisphenol S and 3,4-dichloroaniline were 45.94 mg / L and 85.12 mg / L, respectively.
Claims
1. A rare intestinal cell line Gbr-gut, characterized in that: The deposit number is GDMCC No:65714.
2. The method for constructing the rare goby intestinal cell line according to claim 1, characterized in that: The following steps are involved: (1) Primary culture: The intestinal tissue of healthy rare goby was obtained, cut into pieces and digested, and the suspended cells and tissue fragments were collected and inoculated into primary cell culture medium to obtain primary culture cells; (2) Subculture: digesting the primary cultured cells in step (1), collecting the suspension cells, inoculating them into subculture cell culture medium, and culturing them to obtain a rare goby intestinal cell line; (3) Cryopreservation and thawing of cells: When freezing cells, the rare crucian carp intestinal cell line of step (2) is digested, the precipitate obtained after centrifugation is mixed with a cryopreservation protection solution, and after gradient freezing, it is placed in liquid nitrogen for long-term cryopreservation; when thawing cells, the cell suspension in the cryopreservation tube is first thawed, and then the cell suspension in the cryopreservation tube is transferred to a cell culture medium, and the precipitate obtained after centrifugation is inoculated in the cell culture medium for culture.
3. The method according to claim 2, characterized in that The primary cell culture medium is an L-15 complete culture medium containing 20% v / v fetal bovine serum and 1% v / v penicillin-streptomycin-amphotericin B triple antibodies, and the passage cell culture medium is an L-15 complete culture medium containing 10% v / v fetal bovine serum and 0-1% v / v penicillin-streptomycin-amphotericin B triple antibodies.
4. The method according to claim 2 or 3, characterized in that: Step (1) is as follows: starve healthy rare crucian carp for 24 hours, soak in 75% alcohol for 30-50 seconds, dissect and remove intestinal tissue along the lateral line under sterile conditions, remove surface mucus and intestinal internal materials with forceps; cut the intestinal tissue into 1 mm 2 The tissue blocks were mixed with PBS buffer containing 2-5% penicillin-streptomycin-amphotericin B triple antibodies, and the precipitate was obtained by centrifugation. The mixing and centrifugation operations were repeated 2-3 times to obtain pretreated tissue blocks, 5-12 mL of 0.25% trypsin was added for digestion for 1-2 h, the supernatant was discarded after centrifugation, primary cell culture medium was added for resuspending to obtain a cell suspension, which was transferred to a T-25 cell culture flask and cultured at 28°C. When the cells covered the bottom of the culture flask, the primary cultured cells of the intestine of the rare goby were obtained.
5. The method according to claim 2 or 3, characterized in that: Step (2) specifically includes: discarding the culture solution, washing with PBS buffer, adding 0.5-2 mL of 0.25% trypsin for digestion, and adding 10-20 mL of cell culture medium to resuspend the cells after the cells become round, and performing subculture at a ratio of 1:
2.
6. The method according to claim 2 or 3, characterized in that: In step (3), the specific steps of freezing are: washing the rare crucian carp intestinal cell line with PBS buffer, adding 0.5-2mL 0.25% trypsin for digestion, adding 5-10mL of cell culture medium to terminate digestion after the cells become round, centrifuging to obtain a precipitate, adding a cell freezing solution containing 10% DMSO to resuspend the cells, and transferring them to a cryopreservation tube; transferring the cryopreservation tube to a program cooling box, placing it in a -80°C environment for 4-24 hours, and then transferring the cryopreservation tube to liquid nitrogen for long-term storage; the specific steps of revival are: taking out the frozen rare crucian carp intestinal cell line from liquid nitrogen, placing it in a 28°C water bath to melt the liquid in the cryopreservation tube, transferring the cell suspension in the tube to 5-9mL of cell culture medium, centrifuging to obtain a precipitate, adding 5-10mL of cell culture medium to resuspend the cells, transferring to a T-25 culture flask for culturing at 28°C, replacing it with fresh culture medium after 12-24 hours to continue culturing, and obtaining revived rare crucian carp intestinal cells.
7. A method for evaluating the acute toxicity of chemicals based on the rare goby intestinal cell line according to claim 1, characterized in that: The steps include: (A) Inoculating cells; digesting the rare goby intestinal cell line of claim 1 to obtain a cell suspension, inoculating it into a cell culture plate, and culturing it at 28° C. for 24-48 hours to obtain a cell culture plate with the bottom of the well plate full of rare goby intestinal cells; (B) Exposure to the test chemical: dissolve the test chemical in a delivery medium to obtain an exposure solution, take the cell culture plate with rare goby intestinal cells growing on the bottom of the well plate in step (A), add the exposure solution, and continue to expose for 24 hours to obtain a cell culture plate that has completed the exposure test; (C) Detection: Take the cell culture plate that has completed the exposure test in step (B), add working solution A and working solution B in sequence, incubate in the dark, and measure using an ELISA reader to detect cell viability; (D) Data analysis: Process the raw data obtained in step (C), draw a concentration-effect curve, and calculate the median effect concentration of the test chemical.
8. The method according to claim 7, characterized in that The specific steps are: (A) The rare goby intestinal cell line of claim 1 was taken to a confluence of 80-95%, washed with PBS buffer, digested with 0.25% trypsin until the cells became round, and then added with cell culture medium to adjust the cell density to 4-6×10 5 / mL, to obtain a cell suspension to be inoculated; before inoculation, first add 0.5-2mL of PBS buffer to rinse all wells in the 24-well cell culture plate, discard the solution, and then transfer the cell suspension to be inoculated to the 24-well cell culture plate at an inoculation volume of 500μL / well, place it at 28°C, and culture it for 24-48h until the bottom of the cell culture plate is covered with rare crucian carp intestinal cells; (B) dissolving the test chemical in DMSO to obtain a concentrated solution, diluting it in L-15 culture medium in proportion, or directly dissolving the test chemical in L-15 culture medium to obtain an exposure solution; taking the cell culture plate with rare crucian carp intestinal cells growing on the bottom in step (A), gently adding 1-2 mL of PBS buffer to each well, shaking the well plate slightly and discarding it, adding 2 mL of exposure solution to each well, placing it in an environment of 28°C, and continuously exposing it for 24 hours to obtain a cell culture plate that has completed the exposure test; (C) Take the cell culture plate that has completed the exposure test in step (B), discard the exposure solution, add 1-2 mL of PBS buffer to each well, shake the plate slightly and discard, add 400 μL of working solution A to each well in a light-proof environment, incubate in the dark for 30-60 min, and measure the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 590 nm; Discard working solution A, add 400 μL working solution B to each well in a light-proof environment, incubate in the dark for 60-90 min, discard working solution B, add 400 μL fixative to each well, fix for 1-3 min, discard the fixative, add 400 μL extraction solution to each well, gently shake the plate for 10-15 min, and measure the fluorescence intensity using an excitation wavelength of 530 nm and an emission wavelength of 645 nm; (D) The value J is obtained by subtracting the blank wells without cells from the wells containing cells and exposure solution, and the value P is obtained by subtracting the blank wells without cells from the control group. The cell viability at each exposure concentration is then calculated using the formula: The concentration-effect curve of the test chemical on the intestinal cells of rare goby was drawn according to the set test chemical concentration and the corresponding cell viability, and the half-maximal effect concentration of the test chemical on the intestinal cells of rare goby was calculated using the S-type nonlinear fitting curve.
9. The method according to claim 7 or 8, characterized in that: The working solution A comprises the following components in volume percentage: 10% of resazurin solution and 90% of DPBS buffer solution, and the working solution B comprises the following components in volume percentage: 1-2% of neutral red solution and 98-99% of DPBS buffer solution.
10. The method according to claim 8, characterized in that In step (C), the fixing solution is prepared by dissolving 2.5 g of CaCl2 in 200 mL of ultrapure water, adding 3.38 mL of 37% formaldehyde solution, and then making the volume to 500 mL with ultrapure water; the extract is prepared by dissolving 5 mL of acetic acid in 100 mL of ultrapure water, adding 250 mL of anhydrous ethanol, and then making the volume to 500 mL with ultrapure water.