Separation and culture method for liver fat storage cells of scatophagus argus

Abstract

The invention discloses a scatophagus argus liver fat storage cell separation and culture method, and belongs to the technical field of cell biology. According to the present invention, the scatophagus argus liver fat storage cells are obtained through separation by using a perfusion method, and then the scatophagus argus liver fat storage cells obtained through separation are cultured by using a DMEM medium sugar culture medium containing 8-12% of FBS, 4-6% of HBS, 0.005-0.015% of HGF, 0.005-0.015% of HGP, 0.12-0.16 nM of vitamin A acid and 2-2.5 mM of L-glutamine; the ITO cells obtained through separation can be normally cultured to five generations, and the cells have good cell viability.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and particularly relates to a method for separating and culturing adipose-storing cells in the liver of Scatophagus argus. Background Art

[0002] In mammals, the liver is an important organ for the body's metabolism, undertaking functions such as detoxification, metabolism, substance synthesis and degradation, and immunity of the body, such as carbohydrate metabolism, urea metabolism, protein and carbohydrate metabolism, lipid metabolism, and the conversion, secretion, and excretion of metabolic wastes. The liver of teleost fish also has similar functions to those of mammals and can provide corresponding material metabolism and material conversion functions for the growth, development, and reproduction of fish. Due to the advantages of economy, stability, and repeatability, fish cell culture has been increasingly valued by researchers and has become a method for studying the functions of fish somatic cells. Currently, researchers have successfully isolated and cultured approximately 150 fish cells from a variety of marine and freshwater fish species, including rainbow trout, Gymnocypris przewalskii, common carp, grass carp, Epinephelus coioides, loach, and other fish species.

[0003] Scatophagus argus is a brackish-water economic fish species commonly cultured in the Zhanjiang area of Guangdong Province, China. The liver of Scatophagus argus participates in regulating the development of germ cells during the breeding period and is also an important site for synthesizing VTG. So far, there has been no report on a cell line of adipose-storing cells in the liver of Scatophagus argus that can be passaged. Therefore, the establishment of a method for separating and culturing adipose-storing cells in the liver of Scatophagus argus is helpful for the establishment of an adipose-storing cell line in the liver of Scatophagus argus and the subsequent research on the regulatory functions of liver cells. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a method for separating adipose-storing cells in the liver of Scatophagus argus, and the separation method includes the following steps:

[0005] (1) Inject the basic medium of Willian’s medium E into the liver blood vessels of Scatophagus argus, wash the blood cells remaining in the liver blood vessels, and then perfuse and wash with sterile PBS until the whole liver shows yellow and the perfused PBS is clear;

[0006] (2) Perfuse a trypsin solution with a concentration of 0.2%-0.3% along the liver blood vessels, start collecting the perfused digestion liquid after 30-40 minutes, and continue until the perfused digestion liquid is no longer turbid;

[0007] (3) After centrifuging the perfused digestion liquid, collect the precipitate to obtain adipose-storing cells in the liver of Scatophagus argus.

[0008] Preferably, the concentration of the trypsin solution in step (2) is 0.25%, manufacturer: Sangon Biotech (Shanghai) Co., Ltd., product number E607003-0500.

[0009] More preferably, in the step (3), the centrifugation speed is 400 rpm / min and the centrifugation time is 15 min.

[0010] More preferably, after perfusion for 35 min in the step (3), the perfusion digestive juice starts to be collected.

[0011] The second object of the present invention is to provide a method for culturing fat-storing cells of golden pompano liver, which uses a DMEM medium containing 8-12% FBS, 4-6% HBS, 0.005-0.015% HGF, 0.005-0.015% HGP, 0.12-0.16 nM retinoic acid, and 2-2.5 mM L-glutamine to culture the fat-storing cells of golden pompano liver.

[0012] Preferably, the culture temperature is 36-38 °C.

[0013] More preferably, the fat-storing cells of golden pompano liver are cultured using a DMEM medium containing 10% FBS, 5% HBS, 0.01% HGF, 0.01% HGP, 0.14 nM retinoic acid, and 2.38 mM L-glutamine.

[0014] The third object of the present invention is to provide the fat-storing cells of golden pompano liver separated by using the above separation method.

[0015] The fourth object of the present invention is to provide the application of the above separation method in separating the fat-storing cells of golden pompano liver.

[0016] The fifth object of the present invention is to provide the application of the above DMEM medium containing 8-12% FBS, 4-6% HBS, 0.005-0.015% HGF, 0.005-0.015% HGP, 0.12-0.16 nM retinoic acid, and 2-2.5 mM L-glutamine in culturing the fat-storing cells of golden pompano liver.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The ITO cells separated by the present invention can be normally cultured to the 5th generation, and the cells have good cell activity. Description of the Drawings

[0019] Figure 1 The ITO cells of golden pompano separated by the trypsin digestion method in Example 1, where A: The ITO cells of golden pompano liver separated by the trypsin digestion method, 20×; B: The ITO cells of golden pompano liver separated by the trypsin digestion method, 40×. a: Single-lipid-drop ITO cells; b: Multi-lipid-drop ITO cells; c: Double-lipid-drop ITO cells; d: Triple-lipid-drop ITO cells; e: The nucleus of ITO cells; f: Lipid-drop-free ITO cells.

[0020] Figure 2 Results of isolating Scatophagus argus ITO cells by the tissue block migration method in Example 1 (A: 20×; B: 40×).

[0021] Figure 3 Isolation of Scatophagus argus ITO cells by perfusion method in Example 1 (A: 20×; B: 40×).

[0022] Figure 4 ITO cell counting in Example 1 (10×).

[0023] Figure 5 Results of ITO cell diameter measurement in Example 1.

[0024] Figure 6 Results of ITO cell RNA quality and marker gene detection in Example 1, where A: Total RNA quality of ITO cells; B: Detection results of liver tissue marker genes; C: Detection results of ITO cell marker genes.

[0025] Figure 7 Periodic acid-Schiff staining of liver sections of female and male Scatophagus argus in Example 1 (3 mm), where A: Periodic acid-Schiff staining of liver sections of female Scatophagus argus; B: Periodic acid-Schiff staining of liver sections of male Scatophagus argus.

[0026] Figure 8 Periodic acid-Schiff staining of ITO cells in Example 1, where A: Positive control of periodic acid-Schiff staining of liver sections (3 mm); B: Periodic acid-Schiff staining of ITO cells; C: Hematoxylin counterstaining of ITO cell nuclei.

[0027] Figure 9 Coverage of ITO genome on chromosomes of reference genome in Example 1.

[0028] Figure 10 GO enrichment of mutated genes in Example 1.

[0029] Figure 11 Culturing ITO cells with 10% FBS DMEM in Example 1.

[0030] Figure 12 Effect of media supplemented with SOL and media with appropriate cofactors on in vitro proliferation of ITO cells in Example 1, where A: Effect of media supplemented with SOL on proliferation of ITO cells 20×; B: Adhesion process of Scatophagus argus ITO cells 20×; C: Effect of adding cofactors on adhesion and proliferation of ITO cells 20×.

[0031] Figure 13To study the effect of culture temperature conditions on the proliferation of ITO cells in Example 1, where A: Proliferation of ITO cells at 28°C, magnification 20×; B: Proliferation of ITO cells at 37°C, magnification 20×.

[0032] Figure 14 For the in vitro subculture of ITO cells in Example 1, where A: Primary ITO cells; B: ITO cells cultured for 3 passages; C: ITO cells digested with trypsin for 3 passages; D: ITO cells cultured for 5 passages. Detailed implementation methods

[0033] Example 1

[0034] 2.1.1 Experimental materials

[0035] 2.1.1.1 Experimental materials

[0036] Six randomly selected Scatophagus argus (3 females and 3 males, body weight: 176.7 - 350.0 g) were used for the isolation of hepatic stellate cells. The Scatophagus argus were from the Donghai Island Aquaculture Base of Guangdong Ocean University, and were raised under natural photoperiod in an outdoor open concrete pond of 12 m × 5 m. During the breeding process, the water salinity was controlled at 8.0‰, the temperature was 25°C - 34°C, the pH value was 7.5 - 8.5, the dissolved oxygen in the water was about 6.0 - 8.6 mg / L, and oxygen was supplied continuously for 24 hours. Commercial floating bait was used as their food. They were transported by seawater oxygenation and temporarily raised in a disinfected plastic box of 0.6 m × 1 m with artificially prepared 8‰ seawater for 2 - 3 days.

[0037] 2.1.1.2 Sample collection

[0038] 1. Sampling equipment

[0039] Surgical instruments: surgical scissors, surgical forceps, scalpel, tray, etc., sterilized by steam at 121°C for 30 minutes.

[0040] Sampling containers: 5 ml sterile centrifuge tubes containing Bouin's fixative (PH0976, Phygene), 5 ml sterile syringes.

[0041] Disinfection supplies: 75% ethanol solution, alcohol cotton balls containing 75%, potassium permanganate solution, iodophor solution.

[0042] 2. Preparation of experimental solutions

[0043] A. 0.9% sterile normal saline:

[0044] Weigh 4.5 g of analytical pure NaCl (A610476 - 0001, Sangon Biotech, Shanghai) granules and place them in a 500 - ml beaker. Dissolve them with 295.5 g of distilled water. After complete dissolution, transfer the solution to a 500 - ml volumetric flask. Rinse the beaker with distilled water several times and transfer the rinsing solution to the volumetric flask. Add water to the volumetric flask up to the calibration line. The resulting liquid is 0.9% physiological saline. Transfer the liquid to a glass bottle and sterilize it at 121 °C under high - pressure steam for 30 min to obtain 0.9% sterile physiological saline solution.

[0045] B. 0.1% Sterile Na2EDTA - Physiological Saline Solution:

[0046] Weigh 1 g of Na2EDTA (A500838 - 0500, Sangon Biotech, Shanghai) solid powder and dissolve it in 50 ml of deionized water. After complete dissolution, transfer the solution to a 100 - ml volumetric flask. Rinse the container with distilled water several times and transfer the rinsing solution to the volumetric flask. Add water to the volumetric flask up to the calibration line. The resulting liquid is 1% Na2EDTA solution. Take 10 ml of the 1% Na2EDTA solution and add it to a 100 - ml wide - mouthed glass bottle, then add 90 ml of 0.9% physiological saline. The resulting solution is 0.1% Na2EDTA - physiological saline solution. After preparation, sterilize it at 121 °C under high - pressure for 30 min to obtain 0.1% sterile Na2EDTA - physiological saline solution.

[0047] C. Sterile Phosphate Buffer Solution:

[0048] Weigh 8 g of NaCl (A501218 - 0001, Sangon Biotech, Shanghai), 0.2 g of KCl (A501159 - 0500, Sangon Biotech, Shanghai), 1.44 g of Na2HPO4 (A501727 - 0500, Sangon Biotech, Shanghai) and 0.24 g of KH2PO4 (A501211 - 0500, Sangon Biotech, Shanghai), dissolve them in 800 ml of distilled water, adjust the pH value of the solution to 7.4 with HCl, and finally make up the volume to 1 L with distilled water. Then transfer the liquid to a 1 - L wide - mouthed bottle and sterilize it at 121 °C under high - pressure for 30 min.

[0049] D. Preparation of 0.4% Trypan Blue Working Solution:

[0050] Preparation of 4% Trypan Blue Stock Solution: Weigh 4 g of trypan blue (A601140 - 0010, Sangon Biotech, Shanghai), grind it with a small amount of distilled water, add double - distilled water to 100 mL, and store it at 4 °C. Take 1 ml of the 4% trypan blue stock solution and dilute it 10 - fold with PBS until the trypan blue concentration is 0.4%. Filter it with a 0.22 - μm syringe filter (F513163 - 0001, Sangon Biotech, Shanghai) before use.

[0051] E. Preparation of Glucose - based Medium (MG) in DMEM:

[0052] The high-glucose (HG) DMEM medium (E600003-0500, Sangon Biotech, Shanghai) and the low-glucose (LG) DMEM medium (E600009-0500, Sangon Biotech, Shanghai) were mixed at a ratio of 1:1 for preparation.

[0053] F. Follicular extract (SOL):

[0054] The ovaries of golden pompano with ovarian development at stages 2 to 4 were collected, minced with surgical scissors, and then broken with an ultrasonic crusher. After centrifugation at 1200 r / min for 15 min, the supernatant was filtered through a 0.22-μm syringe filter and stored at -20°C after labeling for later use.

[0055] G. Preparation of media with different serum concentrations:

[0056] Appropriate amounts of DMEM medium (MG), L-15, M199, MEM, HBSS, and Willian’s medium E basal media were taken, and appropriate amounts of fetal bovine serum (FBS), horse serum (HBS), penicillin-streptomycin (100×), and cofactors (0.01% hepatocyte growth factor (HGF), 0.01% hepatocyte promoting factor (HGP), 0.14 nM retinoic acid (VA acid), and 2.38 mM L-glutamine in DMEM medium with medium glucose, 10% SOL) were added according to the proportions to prepare the corresponding media of 5%, 10%, 15%, and 20%. After labeling, they were stored at -20°C for use. The above 5%, 10%, 15%, and 20% refer to the corresponding volume percentages of the mixed solution of fetal bovine serum and horse serum in the medium after mixing at a ratio of 2:1.

[0057] 2.1.2 Experimental methods

[0058] 2.1.2.1 Experimental sample collection

[0059] Approved by the Ethics Committee of Guangdong Ocean University, the experiment was carried out with reference to the experimental animal guide manual. The experimental fish were placed in water containing MS-222 (A5040, Sigma) for anesthesia, the abdomen was removed along the abdominal cavity edge, the liver edge was gently picked up with forceps, and separation was carried out along the ventral surface of the liver. After weighing and photographing the liver under sterile conditions, it was disinfected with iodophor and 75% alcohol for 30 s each. A small piece of liver tissue was cut along the edge and fixed in Bouin's solution overnight. A small piece of liver tissue was placed in a 1.5-ml sterile test tube containing OCT embedding medium and stored at -80°C, and the remaining samples were transferred to a laminar flow hood for cell separation operation.

[0060] 2.1.2.2 Cell separation and culture

[0061] 1. Trypsin digestion method:

[0062] Place liver tissue blocks sized 2*2 cm into sterilized PBS - 75% alcohol - sterilized PBS - sterilized PBS in sequence for cleaning and sterilization, with the time being 30 s - 15 s - 30 s - 30 s in sequence. Put the disinfected and cleaned liver tissue into a sterile petri dish, trim it into small tissue blocks avoiding visible blood vessels, add sterilized 1×PBS to the petri dish containing the small tissue blocks for cleaning, remove the liquid, and repeat this several times until the small liver tissue blocks are cleaned and no blood red color is seen, and the tissue blocks show a pale yellow color. Transfer the minced liver tissue blocks to a 15 - ml centrifuge tube, add 10 ml of 0.25% trypsin, and digest at 28°C in a water bath for 30 min. Aliquot the digestive solution into 2 - ml enzyme - free centrifuge tubes, centrifuge at 500 r / min, then remove the supernatant, add an appropriate amount of physiological saline to the cell precipitate at the bottom of the tube, resuspend it, and filter it through a 200 - mesh sieve. Take a part of the solution for microscopic observation. When the characteristics that there are obvious fat droplets or fat globules in the middle of the vast majority of cells and the cell nuclei are squeezed to the edge are seen, perform cell counting, and place about 3×10 5 cells into a 25 - cm 2 culture flask, shake well to cover the entire growth area of the culture flask with cells as much as possible, pre - culture in a 28°C cell incubator with a 5% carbon dioxide concentration for 30 min. Subsequently, add 2 ml of 10% FBS DMEM medium with glucose to the culture flask, and place it in a carbon dioxide incubator for culture. Change the medium for the first time after 12 h of culture, and then change half of the medium every 24 h, and culture for 1 - 2 weeks.

[0063] 2. Tissue block migration method:

[0064] Place liver tissue blocks into sterilized PBS - 75% alcohol - sterilized PBS - sterilized PBS in sequence for cleaning and sterilization, with the time being 30 s - 15 s - 30 s - 30 s in sequence. Put the disinfected and cleaned liver tissue into a sterile petri dish, trim it into tissue blocks sized 1*1 mm avoiding visible blood vessels, add sterilized 1×PBS to the petri dish containing the small tissue blocks for cleaning, remove the liquid, and repeat this several times until the small liver tissue blocks are cleaned and no blood color is seen, and the tissue blocks show a pale yellow color. Directly attach the small liver tissue blocks to the inside of the culture flask and place them in a 5% carbon dioxide incubator at 28°C for 10 - 20 min for cell attachment. Then add 2 ml of 10% FBS DMEM medium with glucose to the culture flask and place it back in the incubator for culture. Observe the cell status under the microscope every day, change the liquid every 24 - 48 h, and culture for 1 - 2 weeks, and observe under the microscope whether there are cells migrating out.

[0065] 3. Isolation of ITO cells by perfusion method

[0066] Place the liver in a sterile petri dish. Use a syringe to slowly perfuse the liver with William's medium E basal medium (manufacturer: Gibco, product number: A1217601) through the reserved liver blood vessels with a 5 ml syringe, and try to wash the blood cells remaining in the liver blood vessels as clean as possible. Then, perfuse and wash again with sterile PBS until the whole liver turns yellow and the perfused PBS is clear. Only then can the washed liver be placed in a clean petri dish. Use a syringe to draw 10 ml of 0.25% trypsin and slowly perfuse and separate it along the liver blood vessels. Take a little of the perfusion fluid every 5 minutes for microscopic observation. When the characteristics of obvious fat droplets or fat globules in the middle of the vast majority of cells and the nucleus being squeezed to the edge are seen (the time is about 30 - 40 minutes), start collecting the perfusion digestion fluid until the perfusion fluid is no longer turbid. Collect the perfusion fluid into a 50 ml centrifuge tube and centrifuge it at 400 rpm / min for 15 minutes. Collect the precipitate, resuspend it with 1×PBS and wash it again. After centrifuging at the same speed, resuspend the cells with PBS solution. After shaking well, take a small amount of the liquid and add 0.4% trypan blue dye solution for cell counting and cell viability observation. Subsequently, take 3×10 5 PBS cell dilutions and place them in a 25 cm 2 culture flask, shake well to cover the entire growth area of the culture flask with cells as much as possible, pre-culture in a 28℃ cell incubator with a 5% carbon dioxide concentration for 30 minutes. Subsequently, add 2 ml of 10% FBS DMEM medium with sugar to the culture flask and place it in a carbon dioxide incubator for culture. Change the medium for the first time after 12 hours of culture, and then change half of the medium every 24 hours. Culture for 1 - 2 weeks and observe the cell status every day.

[0067] 2.1.2.3 Determination of cell cofactors

[0068] After counting the isolated cells, take similar concentrations and add them to media containing different cofactor formulations for culture. Change the medium for the first time after 12 hours, and then change the medium every 48 hours. Observe the cell adhesion or survival situation under the microscope after culturing for 3 - 5 days. After determining survival, change to the same medium and culture at 28℃.

[0069] 2.1.2.4 Effects of different temperatures on cells

[0070] Take primary cells for trypsin digestion. Digest for 5 - 10 minutes and pipette the bottom of the flask. Wash all the cells with 1×PBS, centrifuge at 500 rmp / min for 15 minutes. Then, disperse the cells with the medium and divide them equally into 4 cell culture flasks for proliferation culture. Place 2 flasks in a 5% carbon dioxide incubator at 28℃ and 37℃ respectively for 48 hours, and observe the cell proliferation situation.

[0071] 2.1.2.5 Determination of the medium

[0072] The primary cells cultured to the 2nd passage were digested, and the digested cells were washed with 1×PBS, resuspended after washing, counted using a hemocytometer, and evenly distributed into a 24-well cell culture plate. The corresponding culture medium was added to each well, and the cells were cultured for 48 h to 96 h. The cells were digested into single cells with 0.25% trypsin, and observed and counted using a hemocytometer.

[0073] 2.1.2.6 RNA Extraction and cDNA Synthesis

[0074] The total RNA of liver tissues and isolated cells was extracted using the Trizol kit, and the RNA integrity number (RIN) was evaluated using a Nanodrop 2100 bioanalyzer; the RNA quality was detected by agarose gel electrophoresis. 1 μg of RNA from each sample was used to synthesize the first strand of cDNA using the Prime ScriptTM RT Reagent Kit with gDNA Eraser kit according to the operating instructions.

[0075] 2.1.2.7 Primer Design

[0076] The liver, kidney, and pancreas marker gene HNF1BA, three liver nuclear factor marker genes HNF1A, HNF4A, and HNF4G, the liver apolipoprotein B gene (APOB), and the liver hepcidin gene (HAMP) were selected. Referring to the CDS sequence of the genome of Scatophagus argus in the laboratory, primers were designed using Oligo primer analysis software version 7.0 (Table 1), and the primers were synthesized by Sangon Biotech Co., Ltd.

[0077] Table 1 Primer sequences of Scatophagus argus ITO cell marker genes

[0078]

[0079]

[0080] 2.1.2.8 ITO Cell Genome Sequencing

[0081] The experimental procedure was carried out according to the standard Protocol provided by Illumina. After the genomic DNA of the sample was detected to be qualified, the DNA was fragmented by mechanical shearing (ultrasonic wave), and then the fragmented DNA was subjected to fragment purification, end repair, addition of A at the 3′ end, ligation of sequencing adapters, followed by fragment size selection using agarose gel electrophoresis, PCR amplification to form a sequencing library. The constructed library was first subjected to library quality inspection, and the library with qualified quality inspection was sequenced using Illumina.

[0082] The main steps of data filtering are as follows:

[0083] (1) Remove the Reads with adapters;

[0084] (2) Filter reads with N content exceeding 10%;

[0085] (3) Reads with bases with quality values ​​below 10 exceeding 50% were removed.

[0086] Clean Reads obtained by sequencing.

[0087] Calculate the Q20, Q30, GC content and sequence duplication level of clean data. Use "bwa-mem2mem-t 4-M" to align Clean Reads with the reference genome, sort the alignment results with Samtools (v1.9) sort, and calculate the sequencing depth, genome coverage and other information of each sample based on the sorted results.

[0088] A graph is drawn based on the coverage depth of each chromosome site. By default, a maximum of 20 chromosome scaffolds are displayed. If the coverage depth is evenly distributed on the chromosome, it can be considered that the sequencing randomness is relatively good.

[0089] The mutation results are strictly filtered to ensure the reliability of the mutation results. The main filtering parameters are as follows:

[0090] (1) SNPs within 5 bp of the InDel and those within 10 bp of the adjacent InDel were filtered out based on the subroutine vcfutils.pl (varFilter-w 5-W 10) in bcftools;

[0091] (2) ClusterSize 2clusterWindowSize 5, indicating that the number of variants within a 5 bp window should not exceed 2;

[0092] (3) QUAL<30, the quality value in Phred format, indicates the possibility of variant variation at the site. Sites with a quality value lower than 30 are filtered out;

[0093] (4) QD < 2.0, the ratio of the variant quality value (Quality) divided by the coverage depth (Depth), the coverage depth is the sum of the coverage depths of all samples containing variant bases at this site. Samples with a QD lower than 2.0 are filtered out;

[0094] (5) MQ<40, the root mean square of the alignment quality values ​​of all reads aligned to this site. Reads with MQ below 40 were filtered out;

[0095] (6) FS > 60.0, a value converted from the P-value by Fisher's test, describes whether there is a significant positive or negative strand specificity for reads containing only variants and reads containing only reference sequence bases during sequencing or alignment. That is to say, if there are no strand-specific alignment results, FS should be close to zero.

[0096] Those with FS higher than 60 are filtered out;

[0097] (7) Other variant filtering parameters are processed using the default values specified by GATK official.

[0098] The filtered Clean reads are used to detect SNP and InDel variants using the Haplotype caller (local haplotype assembly) algorithm of GATK (v3.8). First, each sample generates its own gVCF, and then population joint-genotyping is performed. The variant genes are aligned with functional databases such as NR, SwissProt, GO, COG, and KEGG through Diamond to obtain the annotations of these genes for analyzing gene functions.

[0099] 2.1.2.9 Marker gene amplification

[0100] Add 1 μl of cDNA, 8 μl of enzyme-free water, 0.5 μl of each upstream and downstream primer, and 10 μl of 2× Taq enzyme to the cDNA according to a 20 μl system. React in a PCR instrument for 35 cycles, and the program is as follows: 95°C for 5 min, 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and 72°C for 5 min. Electrophorese the amplified product on a 1.2% agarose gel and observe and photograph it with a gel imaging system.

[0101] 2.1.2.10 PAS staining of ITO cells

[0102] Treat the cells in a 24-well plate with 70% ethanol for 10 min, and wash the cells 2 times with sterile PBS after filtration. Drop 100 μl of periodic acid solution balanced at room temperature for each sample, react in the dark in a 37°C incubator for 10 min, remove the periodic acid solution, soak in distilled water and place on a shaker for washing for 5 min. Drop 100 μl of Schiff reagent balanced at room temperature for each sample, place in a 37°C incubator for dark staining for 30 min - 1 h, remove the staining solution, soak in distilled water and place on a shaker for washing for 5 min, and observe under an inverted microscope.

[0103] 2.1.3 Statistical analysis

[0104] The data is analyzed by independent samples t-test using Spass software V13.0 and plotted using GraphPad prism 9.0 software.

[0105] 2.2 Results

[0106] 2.2.1 Isolation and Identification of Ito Cells in Grass Carp Liver

[0107] (1) Liver cells were isolated with 0.25% trypsin. Under an inverted microscope at 20× magnification, clear cell outlines and visible lipid droplets were observed ( Figure 1 A). Under a 40× microscope, it was found that the cells isolated by the trypsin digestion method had various lipid droplet structures, including single lipid droplets, double lipid droplets, triple lipid droplets, and multi-lipid droplet structures, a small number of cells without lipid droplet structures, and a small number of giant fat cell structures. Under the microscope, single and double lipid droplet cells were mainly observed, and the nucleus and lipid droplets were pushed to one side, with a shift in position ( Figure 1 B).

[0108] (2) The hepatocytes migrated out by the tissue block method were small in volume, and the cells were prone to form cell island structures by proliferation from the bottom layer cells. Under a 40× microscope, clear cell outlines were visible, the cell size was about 5 μm, the nucleus was clearly visible, and it was located in the middle or off-center position of the cell under a light microscope. The cells were mainly mononuclear, and binuclear cells were occasionally seen. No lipid droplet structure characteristics of Ito cells were found in the migrated cells ( Figure 2 ).

[0109] (3) The perfusate of the cells isolated by perfusion with trypsin for about 30 min was collected for observation. The observation results showed that the isolated cells had the lipid droplet characteristics of Ito cells. The cells were intact, and their morphologies were all close to spherical. The cytoplasm was transparent, the intracellular lipid droplet structure was clear, and there was no cell fusion phenomenon. In the isolated cells, single, double, and multi-lipid droplets (lipid droplets ≥ 3) and other unique lipid droplet structures of Ito cells could be seen, and the lipid droplets almost occupied the entire cell, and the nucleus was squeezed to the edge ( Figure 3 ).

[0110] The isolated cells with Ito cell characteristics (cells obtained by the trypsin digestion method and the perfusion method) were diluted 10 times with 1×PBS, and cell counting was performed using a hemocytometer. The results showed that the cell counts in the four counting grids were 52, 55, 35, and 52 cells in sequence. After conversion, the cell concentration was approximately 3.55×10 7 cells / ml, and no cells stained with trypan blue were seen ( Figure 4 ).

[0111] Randomly select three fields of view to count the cell diameters. The results showed that the diameters of the newly isolated cells ranged from 7 to 24 μm, with cells mainly in the range of 14 to 15 μm, and the main diameter range was between 12 and 17 μm ( Figure 5 ).

[0112] Total RNA was extracted from Scatophagus argus ITO cells (cells obtained by perfusion method). The results of 1.2% agarose gel electrophoresis showed clear 28s, 18s, and 5s bands of total RNA. The liver tissue cDNA and cell cDNA were used to amplify the liver marker genes APOB, HNF1A, HNF4A, HNF1BA, HNF4G, and HAMP, respectively. The results of 1.2% agarose gel electrophoresis showed that the target fragment bands of the marker genes were present in both liver tissue and cells, and were consistent with the designed size of the target fragment. The amplification results of HNF1A showed that the isolated cells were liver cells or pancreatic cells. The amplification results of HAMP, HNF4A, HNF1BA, and HNF4G showed that the isolated cells were liver cells. The amplification results of APOB showed that the isolated cells were adipocytes. Combining the marker genes, it was determined that the isolated cells were ITO cells( Figure 6 B,C).

[0113] Liver frozen sections and ITO cells obtained by perfusion method were simultaneously stained with periodic acid-Schiff (PAS) to observe the glycogen staining in the liver sections of male and female Scatophagus argus. The results showed that a large amount of glycogen was present in the liver tissue sections of male and female Scatophagus argus, and could be stained red by PAS, and there was no significant difference between male and female individuals( Figure 7 ).

[0114] Observation of ITO staining found that ITO cells could be stained purple-red by periodic acid-Schiff (PAS) reagent, and its color was close to the same staining effect as liver frozen sections( Figure 14 A,B). Through hematoxylin nuclear counterstaining, it was found that ITO cells had multinucleated phenomena( Figure 8 C “↑”).

[0115] Whole-genome resequencing was performed on the second-generation Scatophagus argus ITO cells isolated and cultured by perfusion method. A total of 6,201,569,956 Raw reads were obtained. After data filtering, 41,519,528 Clean reads were obtained, and the total data volume was 6.2 Gbp. Q20 and Q30 reached 97.59% and 93.90% respectively, and the CG content accounted for 41.21% of the total bases (Table 2).

[0116] Table 2 Genome data analysis

[0117]

[0118] The 41,519,528 Clean Reads were re-aligned to the reference genome. The alignment found that the coincidence rate of Clean reads and the reference genome reached 97.01%, the percentage of paired-end sequencing mapped to the reference genome was 92.64%, and the coverage depth was 99.1% (Table 3).

[0119] Table 3 Comparison Results of ITO Cell Genome and Reference Genes

[0120]

[0121] Chromosome coverage depth analysis found that the distribution of Clean reads on chromosomes was evenly colored, indicating that the randomness of sequencing was very good. The first 24 chromosomes were selected for plotting ([[]] Figure 9 ).

[0122] A total of 2,111,175 SNP variations were found in the SNP variation analysis. Among them, the number of SNP variations of the transition type was 1,349,257, and the number of SNP variations of the transversion type was 761,918. The ratio of transition to transversion type SNPs was 1.77. By analyzing heterozygous type SNPs, 1,471,560 were found, and 639,615 were homozygous type SNPs. The ratio of heterozygous type SNPs was 69.7% (Table 4).

[0123] Table 4 Statistical Table of SNP Variation Analysis

[0124]

[0125] The GO functional classification enrichment of the mutated genes showed that the mutations mainly occurred in the processes of cell metabolism, biological regulation, multicellular organism process, cell composition and tissue or biogenesis process, biological adhesion, reproduction, reproductive process, cell proliferation process, etc. in biological processes. Mutations occurred in cell components such as cell part, membrane, organelle part, and cell junction in cell components. In terms of molecular function, mutations mainly occurred in functions such as catalytic activity, transport activity, molecular transducer activity, molecular function regulatory activity, and transcriptional regulatory activity ([[]] Figure 10 ).

[0126] KEGG analysis found that mutations mainly occurred in glycolysis / gluconeogenesis, TCA cycle, pentose phosphate cycle, pentose and gluconate interconversion pathway, fructose and mannose metabolism pathway, galactose metabolism, ascorbate and aldarate metabolism pathway, fatty acid biosynthesis pathway, fatty acid elongation pathway, and fatty acid degradation pathway.

[0127] 2.2.2 Screening of Culture Conditions for Scatophagus argus ITO Cells

[0128] Approximately 3×10 5 ITO cells obtained by the perfusion method were inoculated into 25 cm 2In the cell culture flask, add 10% FBSDMEM medium and place it in a 5% carbon dioxide environment for culture. The culture flask was placed under a microscope to observe the proliferation of ITO cells. The results showed that the newly inoculated cells showed a discrete morphology, with more single cells and fewer cell aggregates. The cells were round in morphology, transparent in cytoplasm, with clear fat droplet structure in the cells and clear cell boundaries ( Figure 11 A).

[0129] Observation under the microscope 3 hours after inoculation showed that most cells in the culture flask aggregated to form cell clusters with transparent cytoplasm, clear fat droplets and distinct boundaries ( Figure 11 B).

[0130] After culturing in 10% FBSDMEM medium for 7 days, it was observed that the transparent characteristics of the cytoplasm disappeared, the cells collapsed, the cell morphology integrity was lost, the cells died, and a white oily film appeared on the surface of the cell culture medium ( Figure 11 C). Therefore, 10% sugar medium in FBSDMEM is not suitable for adherent culture of ITO.

[0131] A variety of auxiliary factors were selected to screen for auxiliary factors suitable for the growth of ITO cells (Table 5). The results showed that ovarian extract (SOL) had no effect on the adhesion ability of ITO cells, and 5% HBS was one of the necessary conditions for the survival of ITO, but its addition alone or in combination with human recombinant epidermal growth factor (EGF) could not make ITO cells adhere to and proliferate normally. When appropriate amounts of vitamin A acid and L-glutamine were added to the culture medium, the cells were able to adhere to and proliferate. On this basis, the addition of 0.01% hepatocyte growth factor (HGF) and 0.01% hepatocyte repair factor (HGP) can accelerate the rate of ITO cell adhesion growth (Table 5).

[0132] Therefore, DMEM medium supplemented with 10% FBS, 5% HBS, 0.01% HGF, 0.01% HGP, 0.14nM vitamin A acid (VA acid) and 2.38mM L-glutamine can effectively improve the survival, adhesion and proliferation ability of cells ( Figure 12 C).

[0133] Table 5 Effects of different auxiliary factors on the adhesion and proliferation of golden croaker ITO cells

[0134]

[0135] Note: “*” indicates added, “ / ” indicates not added.

[0136] By observing the changes in the morphology of ITO cells attached to the wall obtained by the perfusion method, it was found that the morphology of the separated cells began to change after 24 hours of culture, from a spherical shape to a flat spherical shape, and then a pseudopodia-like structure extended from the cell boundary (Figure 12 B), and finally transform into cells in the shape of long strips, triangles, and polygons Figure 12 C). By subculturing ITO cells through passage of the second-generation cells, it was found that the growth rate of ITO cells was higher at 37 °C than at 28 °C Figure 13 ).

[0137] 2.2.3 Culture of Scatophagus argus ITO cells

[0138] The morphological structure of ITO cells during subculture obtained by perfusion method was observed under an inverted microscope. The results showed that when subcultured to the 3rd generation, ITO cells still had lipid droplet structures and the cell proliferation was good Figure 14 B). When the 3rd-generation cells were treated with 0.25% trypsin for 3 minutes, plasmodesmata between ITO cells could be seen Figure 14 C "↑"), and the cells could be normally cultured to the 5th generation, and the cells had good cell viability Figure 14 D).

[0139] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for isolating fat-storing cells from the liver of a golden pompano, characterized in that, The separation method includes the following steps: (1) Inject Willian’s medium E basal medium into the liver blood vessels of Epinephelus coioides, wash the blood cells remaining in the liver blood vessels, and then perfuse and wash with sterile PBS until the whole liver turns yellow and the perfused PBS is clear; (2) Perfuse a trypsin solution with a concentration of 0.2%-0.3% along the liver blood vessels, start collecting the perfused digestive fluid 30-40 minutes later, until the perfused digestive fluid is no longer turbid; (3) After centrifuging the perfused digestive fluid, collect the precipitate to obtain the hepatic stellate cells of Epinephelus coioides.

2. The separation method according to claim 1, wherein In step (2), the concentration of the trypsin solution is 0.25%.

3. The separation method according to claim 2, characterized in that, In step (3), the centrifugation speed is 400 rpm / min and the centrifugation time is 15 minutes.

4. The separation method according to claim 3, characterized in that, In step (3), start collecting the perfused digestive fluid 35 minutes after perfusion.

5. A method for culturing fat-storing cells of miiuy croaker liver, characterized in that, Culture the hepatic stellate cells of Epinephelus coioides in a DMEM medium containing 8-12% FBS, 4-6% HBS, 0.005-0.015% HGF, 0.005-0.015% HGP, 0.12-0.16 nM retinoic acid, and 2-2.5 mM L-glutamine.

6. The culturing method according to claim 5, characterized in that, The culture temperature is 36-38 °C.

7. The culturing method according to claim 6, wherein, Culture the hepatic stellate cells of Epinephelus coioides in a DMEM medium containing 10% FBS, 5% HBS, 0.01% HGF, 0.01% HGP, 0.14 nM retinoic acid, and 2.38 mM L-glutamine.

8. Hepatic stellate cells of Epinephelus coioides separated by the separation method according to any one of claims 1-4.

9. Use of the separation method according to any one of claims 1-4 in separating hepatic stellate cells of Epinephelus coioides.

10. Use of the DMEM medium in claim 5 in culturing hepatic stellate cells of Epinephelus coioides.

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