Construction method of lateolabrax japonicus kiss cell line and application thereof in toxicity assessment of environmental pollutant microplastics

Abstract

The invention discloses a construction method of a lateolabrax japonicus kiss cell line and application of the lateolabrax japonicus kiss cell line in toxicity assessment of environmental pollutant microplastics. The lateolabrax japonicus kiss cell line is constructed by the following method: under a sterile condition, taking lateolabrax japonicus kiss tissues, cutting the lateolabrax japonicus kiss tissues into pieces, and performing enzyme digestion to obtain primary cells; after the primary cells are subjected to enzyme digestion, complete medium resuspension cells are taken and inoculated into a cell culture bottle, and constant-temperature culture and passage are performed at 28 DEG C. The lateolabrax japonicus kiss cell line can preserve lateolabrax japonicus genetic resources at the cellular level, preservation is simple and convenient, an in-vitro research system is provided, a platform is built for lateolabrax japonicus cell molecular biology research, and the method is suitable for large-scale popularization and application. Meanwhile, the material can also be used as an important material and model for in-vitro researches of molecular genetics, biology, environmental toxicology and the like. According to the method, the cells of the lateolabrax japonicus kiss cell line are cultured in the culture medium containing the environmental pollutant microplastics, and the cell condition is observed, so that the toxicity of the environmental pollutant microplastics can be evaluated more accurately.

Description

Technical Field

[0001] The present invention relates to a Lateolabrax japonicus cell line, and also relates to a method for constructing the Lateolabrax japonicus cell line and an application of the Lateolabrax japonicus cell line in toxicity assessment of environmental pollutant microplastics. Background Art

[0003] In recent years, significant research progress has been made in understanding the toxicity of NPs to fish. These studies have revealed the potential adverse effects of long-term exposure to NPs on organisms. For example, studies have shown that NPs can cause physiological disturbances in fish, including impaired growth and development, behavioral changes, impaired immune function, and reproductive dysfunction. NP exposure has also been associated with oxidative stress, inflammation, and altered gene expression patterns in fish. In addition, in vitro studies using various cell line models have provided valuable insights into the mechanisms of NP toxicity. These studies have shown that NPs can induce significant alterations in the cytoskeleton, cell surface structures, and subcellular structures. For example, NP exposure has been associated with disruption of cytoskeletal organization, leading to changes in cell morphology and impaired cellular function. NPs have also been shown to interact with cell surface receptors and membrane proteins, potentially affecting cell signaling and communication processes. Although the exact mechanisms of NP toxicity in fish and in vitro cells are still being elucidated, new evidence suggests that the small size and high surface area to volume ratio of NPs contribute to their enhanced toxicity. These properties enable NPs to easily interact with cell membranes, enter cells, and induce various cellular responses.

[0004] Despite a growing body of research, there are still major gaps in our understanding of the long-term effects of nanoparticles and their potential impacts on the environment and the health of organisms. Specifically, there is limited research on the long-term toxicity of nanoparticles to organisms and the recovery of organisms after cessation of exposure to nanoparticles. Summary of the invention

[0005] One of the objects of the present invention relates to a Lateolabrax japonicus cell line.

[0006] Specifically, the Lateolabrax rostrums cell line was constructed by the following method: under sterile conditions, the Lateolabrax rostrums tissue was obtained, cut into pieces, and then digested with enzymes to obtain primary cells; the cells were resuspended in complete culture medium after enzymatic digestion of the primary cells, inoculated into a cell culture bottle, and cultured and passaged at a constant temperature of 28°C to obtain the Lateolabrax rostrums cell line.

[0007] The sea bass snout tissue is digested with collagenase and trypsin successively; the primary cells are digested with trypsin.

[0008] The primary cells were passaged when they adhered to and grew to 90-100% confluence.

[0009] The second purpose of the present invention relates to a method for constructing a Lateolabrax japonicus cell line.

[0010] Specifically, the method for constructing the Lateolabrax rostrum cell line is as follows: under sterile conditions, the Lateolabrax rostrum tissue is taken, cut into pieces and digested with enzymes to obtain primary cells; the cells are resuspended in complete culture medium after enzymatic digestion of the primary cells, inoculated into a cell culture bottle, and cultured and passaged at a constant temperature of 28°C to obtain the Lateolabrax rostrum cell line.

[0011] The sea bass snout tissue is digested with collagenase and trypsin successively; the primary cells are digested with trypsin.

[0012] The primary cells were passaged when they adhered to and grew to 90-100% confluence.

[0013] The third object of the present invention relates to the application of Lateolabrax japonicus cell line.

[0014] Specifically, it involves the application of Lateolabrax japonicus cell line in the toxicity assessment of environmental pollutants microplastics.

[0015] A fourth object of the present invention is to provide a method for evaluating the toxicity of environmental pollutants, microplastics.

[0016] Specifically, the method for assessing the toxicity of microplastics, an environmental pollutant, is to take cells from the Lateolabrax japonicus cell line and place them in a culture medium containing microplastics, an environmental pollutant, for cultivation, and observe the cell conditions to assess the toxicity of microplastics, an environmental pollutant.

[0017] Furthermore, the cell conditions including the state of the cell nucleus, survival rate, and cell proliferation time were observed to evaluate the toxicity of environmental pollutants such as microplastics.

[0018] The cells of the Lateolabrax japonicus cell line have undergone at least 20 passages.

[0019] Advantages of the present invention:

[0020] 1. The present invention provides a Lateolabrax japonicus cell line, which can preserve Lateolabrax japonicus genetic resources at the cellular level, and the preservation is relatively simple. It provides an in vitro research system and a platform for molecular biology research on Lateolabrax japonicus cells. It can also be used as an important material and model for in vitro research on molecular genetics, biology, and environmental toxicology.

[0021] 2. The present invention provides a Lateolabrax japonicus cell line, which can be passaged to 40 generations, and can be passaged once every 2-3 days at a ratio of 1:2. The cells can maintain a good growth state and can be cryopreserved. The temperature and serum concentration suitable for its growth are 28°C and 25%, respectively. Through metaphase analysis of LMS cell chromosomes, it was found that 38% of the cells had a normal diploid chromosome number of 2n=48. Through in vitro transfection of pEGFP-N3 plasmids, it was found that the transfection efficiency of LMS cells was about 28%. After transfection of exogenous recombinant plasmids, active proteins can be expressed, which can be used for scientific research.

[0022] 3. The inventors have found through experiments that microplastics can hinder the transcription of nucleoporins, which may affect the integrity and function of the nuclear membrane permeability barrier, significantly inhibit cell proliferation, and cause lasting damage to cells and subcellular structures. The present invention can more accurately assess the toxicity of environmental pollutant microplastics by culturing cells of the Lateolabrax cell line in a culture medium containing environmental pollutant microplastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For primary and subcultured LMS cells;

[0024] A: Day 4 of primary culture; B: Day 10 of primary culture; C: Passage 16; D: Passage 40; Scale bar = 100 μm.

[0025] Figure 2 For LMS cell origin analysis;

[0026] A: Amplification results of 18S rRNA in LMS cells, B: Sequence alignment results of 18S rRNA in LMS cells and AB089346.1.

[0027] Figure 3 Figure 2 shows the growth curves of LMS cells at different temperatures.

[0028] Figure 4 Figure 2 is the growth curve of LMS cells at different serum concentrations.

[0029] Figure 5 This is the chromosome analysis of LMS cells at passage 35;

[0030] A: Normal chromosome metaphase; B: Chromosome number distribution.

[0031] Figure 6 GFP expression in LMS cells;

[0032] A: bright field image; B: fluorescence field image; C: bright field and fluorescence field images; scale bar = 20 μm.

[0033] Figure 7 The shape and size of NPs.

[0034] A and B depict the morphology of the NPs obtained by SEM, and B shows a magnified view of the yellow box in A. C and D represent the particle size distribution of 500 randomly measured NPs.

[0035] Figure 8 The cytotoxicity of NPs to LMR cells.

[0036] A. LMR cells were treated with different concentrations of NP, i.e., 2, 20, 200, 2000 μg / mL, for 1, 3, 7, and 14 days, showing changes in cell morphology and viability. Different lowercase letters indicate statistical significance (p<0.05). The lower left corner of the image shows the level of toxicity of NP to cells. B. LMR cells cultured for a long time with 20 μg / mL NP were used to measure the doubling time of the LMR cell line at the 10th generation (NP-10, 15 days), the 20th generation (NP-20, 55 days), and the 30th generation (NP-30, 105 days). ** indicates p<0.01.

[0037] Fig. 9 for the accumulation and release of NPs in LMR cells.

[0038] A. Fluorescence microscopy observation of NPs in LMR cells; NPs were observed and photographed at the 1st generation (NP-1st, 1.5 days), 10th generation (NP-10th, 15 days) and 30th generation (NP-30, 105 days) of continuous exposure to 20 μg / mL NPs, as well as 48 hours after stopping exposure to NPs and at the 5th generation (RCY-5, 22 days) and 10th generation (RCY-10, 35 days) of LMR cells; B. The distribution of NPs in cells was observed using a confocal microscope; the yellow arrow indicates NPs around the nucleus, and the red arrow indicates the colocalization of NPs with the nucleus; C and D. Statistical analysis of the average NP fluorescence area per cell (C) and the fluorescence density per cell (D) at different time points; the insets in the upper right corners of C and D show the enlarged views of the statistical analysis of cells in the recovery period.

[0039] Fig.10 To observe F-actin and subcellular morphology in LMR cells in NP-30th and RCY-10th.

[0040] A. Morphology of F-actin in typical cells of categories 1 to 4. B. Statistics of cell classification. Five biological replicates were evaluated for each group, and 200 randomly selected cells were analyzed in each parallel, for a total of 800 cells in each group. The 800 cells were classified, and the percentage of each cell category in each group was calculated. C1 to C4 in the figure represent categories 1 to 4. C. Typical SEM images. The boxed area in the image represents the magnified area, the yellow arrow indicates the cell surface crack, the red arrow indicates the cell surface ulcer, the black arrow indicates the retraction of the filopodia, the green dotted circle indicates the breakage of the filopodia, and the white dotted circle indicates the cell membrane being repaired. D. Typical transmission electron microscopy image. The red boxed area represents the magnified area, and the pink lines and numbers in the magnified image indicate the position and size of the measured perinuclear space. N represents the nucleus, M represents the mitochondria, and RER represents the rough endoplasmic reticulum.

[0041] Fig.11The genotoxicity and oxidative damage of NP-30th and RCY-10th.

[0042] For genotoxicity assay: A. Representative images of EdU+ cells. B. Percentage of EdU+ cells. C. Representative images of γH2AX foci. Cells outlined by white dashed lines are magnified and shown in the lower right corner. D. Quantification of γH2AX foci per cell. E. Transcriptional expression of H2AX. For oxidative damage assay: F. MDA level. G. SOD activity. H. CAT activity. NP represents NP-30th and RCY represents RCY-10th.

[0043] Fig.12 For assessment of nuclear health.

[0044] A. Representative images of PCNA cell immunofluorescence in NP-30th and RCY-10th cells. The cells outlined by the white rectangular box are magnified and shown in the lower right corner. B. RT-PCR detection of transcriptional expression of NUPs. The expression of genes highlighted in red was reduced in NP-30th compared with the control group. GAPDH was used as an internal reference gene. C. Western blog detection of ERK1, ERK2, and PCNA proteins (all with nuclear localization function) in the cytoplasm and total protein. D. MOD ratio of PCNA protein in the nucleus and cytoplasm. E. The relative expression levels of ERK1 and PCNA genes were analyzed using qRT-PCR. GAPDH was used as an internal reference gene. NP represents NP-30th and RCY represents RCY-10th. DETAILED DESCRIPTION

[0045] Part I Experimental Materials

[0046] 1. Experimental Materials

[0047] 1.1.1 Experimental animals

[0048] The Lateolabrax japonicus (500±50 g) was obtained from the Zhuhai Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. Lateolabrax japonicus rostral cell line (named LMS cell).

[0049] 1.1.2 Microplastics

[0050] Polypropylene microplastics (PP-MPs) were purchased from Hengfa Plastics Co., Ltd. with a particle size of 1-6 μm.

[0051] 1.1.3 Experimental reagents

[0052] The experimental reagents are shown in Table 1-1.

[0053] Table 1-1 Experimental reagents

[0054]

[0055] 1.1.4 Reagent preparation

[0056] (1) 2% double antibody DPBS: Add 2% double antibody to DPBS.

[0057] (2) Double Antibody L-15 Medium: Add 2% double antibody to L-15 medium.

[0058] (3) L-15 complete medium: Add 25% FBS and 2% double antibody to L-15 medium.

[0059] (4) Cell freezing solution: Add 10% FBS to DMSO.

[0060] 1.1.5 Experimental equipment

[0061] Experimental equipment: The experimental reagents used in this chapter are shown in Table 1-2.

[0062] Table 1-2 Experimental equipment

[0063]

[0064] Part II Establishment and identification of Lateolabrax japonicus cell line

[0065] 1. Experimental Methods

[0066] 1.1 Primary and secondary cell culture

[0067] After the sea bass was anesthetized, it was disinfected with 75% alcohol. The fish was immediately moved into a sterile environment and the surface liquid of the fish was wiped with sterile gauze. The snout tissue of the fish was cut with sterile scissors and washed three times in double-antibody DPBS; it was transferred to a mixture of 2mL L-15 culture medium and 1mL double-antibody DPBS, and the tissue was cut into pieces with sterile scissors and transferred to a 15mL centrifuge tube; centrifuged at 600×g for 10min, the supernatant was discarded, 5mL collagenase was added for digestion for 1h, centrifuged, the supernatant was discarded, 5mL 0.25% trypsin digestion solution was added, and the centrifuge tube was placed in a 37℃ water bath for digestion for 30min, shaking every 15min, and observing the tissue digestion; after digestion, 5mL L-15 complete culture medium was added to terminate the digestion, sieved (70μm) and centrifuged, and the supernatant was discarded; the cell pellet was suspended with 2-3mL L-15 complete culture medium and inoculated in 25cm 2 The cell culture flask was placed in a constant temperature incubator at 28°C. Half of the culture medium was replaced every 2-3 days, and the cell morphology was observed under a microscope.

[0068] When the primary cells adhere to the wall and grow to 90-100% confluence, they can be subcultured. First, aspirate the culture medium in the culture flask, gently wash the cells with 2mL DPBS and aspirate; then add 2mL 0.25% trypsin digestion solution, fully digest until the cell layer falls off, and add 3mL complete culture medium to stop digestion after about 3 minutes; then transfer to a 15mL centrifuge tube, centrifuge at 600×g for 10 minutes, and discard the supernatant; suspend the cell pellet with L-15 complete culture medium, and inoculate it in a 25cm 2 The cell culture flask was placed in a constant temperature incubator at 28°C. Subculture was performed according to the cell growth conditions, and the FBS concentration was gradually reduced and the addition of dual antibodies was stopped.

[0069] 1.2 Cryopreservation and thawing of cells

[0070] Cell cryopreservation: Select LMS cells of different generations for cryopreservation. Collect cells by trypsin digestion method of cell subculture mentioned above, discard supernatant and add 1.5mL cell cryopreservation solution, gently blow to fully resuspend cells, transfer into cell cryopreservation tubes and place in program cooling box, place at -80℃ for 24h, and then transfer cells to liquid nitrogen tank for long-term storage.

[0071] Cell recovery: When recovering cells, take out the cells stored in the liquid nitrogen tank and immediately place them in a 37°C water bath to shake to thaw the cryopreservation solution; transfer the thawed cryopreservation solution to a centrifuge tube and add 5 mL of L-15 complete medium. Centrifuge at 600 × g for 10 min and discard the supernatant. Resuspend the cell pellet in complete medium and inoculate it in a 25 cm 2 The cell culture flask was placed in a constant temperature incubator at 28°C for continued culture. After 24 hours, the culture medium was replaced and the morphology and growth of the cells were observed under a microscope.

[0072] 1.3 Molecular identification of cell lines

[0073] 1.3.1 DNA extraction

[0074] The cells in the cell culture flask were digested, centrifuged, and washed to obtain cell pellets, and the total DNA of LMS cells was extracted using a DNA extraction kit.

[0075] Follow the instructions of the Tiangen Marine Animal Tissue Genomic DNA Extraction Kit:

[0076] (1) Take 20 mg of tissue / cells, place them in a centrifuge tube containing 200 μL GA buffer, and vortex for 15 seconds.

[0077] (2) Add 20 μL of Proteinase K (20 mg / mL), mix well, and place in a 56°C water bath until the cells are completely lysed.

[0078] (3) Add 200 μL GB buffer, mix thoroughly, incubate at 70°C for 10 min, and then centrifuge briefly.

[0079] (4) Add 200 μL of anhydrous ethanol, mix thoroughly, and centrifuge briefly.

[0080] (5) Transfer the entire solution obtained in the previous step into the adsorption column CB3 and centrifuge at 12000 rpm for 30 seconds.

[0081] (6) Discard the waste liquid, add 500 μL of GD buffer to the adsorption column, and centrifuge at 12,000 rpm for 30 s.

[0082] (7) Discard the waste liquid, add 600 μL PW rinse solution to the adsorption column, and centrifuge at 12,000 rpm for 30 seconds.

[0083] (8) Repeat step (7).

[0084] (9) Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and let stand at room temperature for 5 min.

[0085] (10) Place the adsorption column in a new centrifuge tube and drop 50 μL of elution solution TE into the middle of the adsorption column.

[0086] (11) Incubate at room temperature for 5 min and centrifuge at 12,000 rpm for 2 min.

[0087] (12) The concentration of the extracted DNA was measured using NanoDrop 2000 and the DNA was stored at -20°C.

[0088] 1.3.2 18S rRNA sequence verification

[0089] (1) According to the sequence of the Lateolabrax japonicus gene (GenBank Accession No. AB089346.1) on NCBI, primers were designed to amplify the 18S rRNA fragment: 18S-F (5'-ACGGCTACCACATCCGAGGA-3') and 18S-R (5'-ACGAATGCCCCCGGCCGTCC-3') for PCR amplification. The PCR reaction is shown in Table 2-1:

[0090] Table 2-1 PCR reaction system

[0091]

[0092] (2) Invert to mix, centrifuge briefly, and set the PCR reaction program according to the primer Tm value, as shown in Table 2-2:

[0093] Table 2-2 PCR amplification program

[0094]

[0095] 1.3.3 PCR product recovery and purification

[0096] Follow the instructions of the Meiji Bio-Agarose Gel DNA Recovery Kit:

[0097] (1) Cut the gel with the target band, weigh it and place it in a centrifuge tube. Calculate that 100 mg of gel block is equivalent to 100 μL volume. Add an equal volume of Buffer GDP and incubate in a 55°C water bath until the gel block is completely dissolved.

[0098] (2) Place the HiPure DNA Mini Column in a 2 mL centrifuge tube, centrifuge briefly, transfer ≤ 700 μL of the sol to the column, and centrifuge at 12,000 × g for 60 s.

[0099] (3) Discard the waste liquid, add 300 μL of Buffer GDP to the column, let it stand at room temperature for 1 min, and then centrifuge at 12,000 × g for 60 s.

[0100] (4) Discard the waste liquid, add 600 μL Buffer DW2 to the column, and centrifuge at 12,000 × g for 60 s.

[0101] (5) Repeat step (4).

[0102] (6) Discard the waste liquid and centrifuge at 12,000 × g for 2 min.

[0103] (7) Open the lid and let stand at room temperature for 5 minutes.

[0104] (8) Place the column in a new 1.5 mL centrifuge tube, vertically drip 30 μL of Elution Butter onto the center of the membrane, let stand at room temperature for 2 min, and centrifuge at 12,000 × g for 60 s.

[0105] (9) The concentration of the extracted DNA was measured using NanoDrop 2000 and the DNA was stored at -20°C.

[0106] 1.3.4 Connection and conversion

[0107] The ligation reaction system is shown in Table 2-3.

[0108] Table 2-3 Ligation reaction system

[0109]

[0110] Conversion:

[0111] (1) Gently add 10 μL of the above gel recovery product to 100 μL of DH5α competent cells and place on ice for 30 min.

[0112] (2) Incubate the mixture in a 42°C water bath for 90 seconds and then place in ice for 2 minutes.

[0113] (3) Add 800 μL LB liquid culture medium and place on a shaker at 37°C, 220 r / min for 1 h.

[0114] (4) Take out 100 μL of bacterial solution and spread it evenly on LA solid culture medium and seal it.

[0115] (5) Invert and culture in a 37°C incubator overnight.

[0116] 1.3.5 Picking single clones

[0117] (1) In an ultra-clean workbench, use a pipette to pick up a single colony and place it in a centrifuge tube containing 1 mL of LA liquid culture medium. Place it on a shaker and culture it at 37°C, 220 r / min for 1 hour. Then perform bacterial liquid PCR verification. The bacterial liquid reaction system PCR is shown in Table 2-4.

[0118] Table 2-4 Bacterial liquid PCR amplification program

[0119]

[0120] (2) Invert to mix, centrifuge briefly, and follow the reaction procedure as shown in Table 2-5.

[0121] Table 2-5 Bacterial liquid PCR reaction procedure

[0122]

[0123] (3) Run the PCR products on gel electrophoresis and send the bacterial solution with the correct band for testing.

[0124] (4) Compare and analyze the sequencing results using the Blast tool.

[0125] 1.4 Effects of different temperatures on cell growth

[0126] LMS cells were digested, centrifuged and resuspended and then inoculated in a 12-well plate. Each well was inoculated with 1 mL of cell suspension, containing approximately 5 × 10 4 cells. After inoculation, the culture plate was placed at different temperatures of 16°C, 22°C, 28°C and 32°C for culture. The experiment lasted for seven days. The number of cells in each well was recorded by a blood cell counting plate under a microscope every day. The culture medium was replaced every 2-3 days, and each group of experiments was repeated three times. The cell growth curve was drawn with the culture time as the horizontal axis and the cell amount as the vertical axis.

[0127] 1.5 Effects of different FBS concentrations on cell growth

[0128] LMS cells were digested, centrifuged and resuspended and then inoculated in a 12-well plate. Each well was inoculated with 1 mL of cell suspension, containing approximately 5 × 10 4 cells. After inoculation, the culture plate was placed in complete culture medium containing different FBS concentrations of 5%, 10%, 20% and 25% for culture. The experiment lasted for seven days. The number of cells in each well was recorded by a blood cell counting plate under a microscope every day. The culture medium was replaced every 2-3 days, and each group of experiments was repeated three times. The cell growth curve was drawn with the culture time as the horizontal axis and the cell amount as the vertical axis.

[0129] 1.6 Chromosome analysis

[0130] The 32nd generation LMS cells were used for chromosome analysis. 2 In the culture flask, replace the fresh culture medium after the cells are 80% confluent, and rinse with DPBS, then add colchicine with a final concentration of 10μg / mL to the culture medium, and continue to culture for 12h. Aspirate the old culture medium, rinse with DPBS, add trypsin digestion solution, collect the cells into a 15mL centrifuge tube, centrifuge at 1000r / min for 5min, and obtain the cell pellet. Add 2mL of 0.075mol / L KCL solution for hypotonicity at room temperature for 40min; then slowly add 1mL of pre-cooled Carnoy's fixative, pre-fix for 2min, centrifuge at 1000r / min for 10min; discard the supernatant and drop 2mL of pre-cooled Carnoy's fixative, fix at room temperature for 30min, and repeat the fixation 3 times. Centrifuge after fixation, discard the supernatant, add 0.5mL of fixative to resuspend the cells; drip the cell suspension evenly on the pre-cooled slide from a height of 50cm, air dry naturally, stain with the freshly prepared 10% Giemsa for 10min, then rinse with double distilled water and dry naturally. Finally, the number of 100 chromosomes was observed and counted under an inverted microscope, and photos were taken and karyotype analysis was performed.

[0131] 1.7 Cell transfection

[0132] The pEGFP-N3 plasmid of enhanced green fluorescent protein (EGFP) used was stored by our laboratory.

[0133] (1) LMS cells were seeded into 24-well plates and transfection was started when the cells grew to 70-90% confluence.

[0134] (2) Using Opti-MEM TM Dilute Lipofectamine in culture medium TM 3000, as shown in Table 2-6, and mix thoroughly.

[0135] Table 2-6 LipofectamineTM 3000 complex system

[0136]

[0137] (3) Using Opti-MEM TM Dilute pEGFP-N3 plasmid in culture medium and add P3000 TM Reagents, as shown in Table 2-7, and mix thoroughly.

[0138] Table 2-7 DNA complex system

[0139]

[0140] (4) In the diluted Lipofectamine TM Add the diluted pEGFP-N3 plasmid to 3000 μL of reagent (1:1 ratio).

[0141] (5) After incubation at room temperature for 15 min, the DNA-lipid complexes were added to a 24-well plate.

[0142] (6) Place the culture plate in a 28°C incubator and continue culturing. After 48 hours, observe the expression of the gene under a fluorescence microscope.

[0143] 2. Experimental results

[0144] 2.1 Primary culture and subculture

[0145] A cell line was isolated from the snout tissue of the striped sea bass and named LMS cell line. The cells adhered well and showed obvious proliferation on the 4th day, and the cell type tended to be single. On the 10th day, the confluence of primary cells reached 90%. Subsequently, the cells were subcultured at a ratio of 1:2, subcultured every 2-3 days, and the cell line has been subcultured to 40 generations. Figure 1 , observed under a microscope, the LMS cell type is fibroblast-like.

[0146] 2.2 Cell cryopreservation and thawing

[0147] The survival rates of the 15th, 24th and 36th generation frozen cells were all above 80% and 1:2 subculture could be continued after 2-3 days.

[0148] 2.3 18S rRNA analysis

[0149] LMS cell DNA was extracted and 18S rRNA gene was amplified by PCR. The sequence obtained after sequencing the PCR product was compared with the published sequence of Lateolabrax japonicus 18S rRNA (GenBank accession number: AB089346.1). The sequence homology reached 100%, proving that this cell line originated from Lateolabrax japonicus. Figure 2 .

[0150] 2.4 Effects of different temperatures on cell growth

[0151] Different temperatures have different effects on cells. Cells do not proliferate significantly at 16°C. At 22-32°C, the number of cells increases over time, and after 5 days, the cells enter a decline phase. Cells grow best at 28°C, with significant proliferation. Therefore, the optimal growth temperature for LMS cells is 28°C ( Figure 3 ).

[0152] 2.5 Effects of different serum concentrations on cell growth

[0153] As the FBS concentration in the culture medium increases, the number of cells gradually increases. When the FBS concentration is 5%, the cells do not proliferate significantly; when the FBS concentration is increased to 10%, the cells grow significantly; when the FBS concentration is 25%, the cells grow fastest ( Figure 4 ).

[0154] 2.6 Chromosome analysis

[0155] The results of the metaphase statistics of 100 LMS cells at the 32nd generation showed that the number of chromosomes ranged from 30 to 92, showing aneuploidy, indicating that the chromosomes were missing or distorted, but the proportion of normal chromosomes (2n=48) was the highest, accounting for 38% ( Figure 5 ).

[0156] 2.7 Cell transfection

[0157] Lipofectamine TM 3000 pEGFP-N3 plasmid was transferred into LMS cells. Obvious fluorescence signal was observed under fluorescence microscope 48 hours after transfection. The transfection efficiency was about 28% ( Figure 6 ).

[0158] 3. Results

[0159] Using the rostrum tissue of striped seabass as material, an enzyme digestion method was used to establish a rostrum cell line of striped seabass, and it was named LMS cells. The LMS cells have a good growth state and have been passaged to the 40th generation. The analysis of chromosome number and karyotype is an important parameter for identifying cell lines, and the stability of its genetic material can also be determined by analyzing chromosomes. The chromosome analysis results of the present invention showed that the number of diploid chromosomes in LMS cells was 48, of which the number of diploid chromosome cells accounted for 38% of the total number of cells. The 18S rRNA gene is a conservative sequence that evolves slowly and is often used to identify the source of cells. Therefore, this article selects the 18S rRNA gene for identification. The sequence analysis results are 100% consistent with the gene sequence of striped seabass, proving that LMS cells are derived from striped seabass.

[0160] L-15 medium is rich in essential amino acids and is considered the first choice for fish cell culture. The growth rate of LMS cells is also mainly affected by temperature and the concentration of FBS in the culture medium. FBS is an indispensable factor for cell survival and growth. Within a certain range, the growth rate of LMS cells increases with the increase of FBS concentration, and the growth rate is fastest when the FBS concentration is 25%. A high concentration (25%) of FBS was selected during the primary culture process, which is conducive to cell adhesion and growth. Compared with mammalian cells, fish cells have a wider temperature adaptation range. LMS cells can grow at 22-32°C, but the growth rate is fastest at 28°C, which is the optimal growth temperature.

[0161] The present invention successfully established a Lateolabrax rostral cell line (LMS), which has been subcultured to the 40th generation and can be subcultured at a 1:2 ratio in 2-3 days. The growth temperature and serum concentration suitable for LMS cells were determined to be 28°C and 25%, respectively. 18S rRNA was used to identify the cell source, and the results showed that the cell line came from Lateolabrax. By analyzing the metaphase of LMS cell chromosomes, the results showed that 38% of the cells had a normal diploid chromosome number of 2n=48, and LMS cells could be transfected with exogenous genes with a transfection efficiency of about 28%.

[0162] Part III Application of Lateolabrax japonicus cell line

[0163] 1. Materials and Methods

[0164] 1.1 Cells and NPs

[0165] Experiments were performed using a Lateolabrax rostral cell line (LMR), which is characterized by rapid growth. This cell line was isolated and cultured in the laboratory and had undergone more than 30 passages before the start of the experiments. Red carboxylated fluorescent polystyrene microspheres (NPs) composed of polystyrene copolymers with a diameter of 20 nm and a density of 1.05 g / cm were purchased from HuicH (Shanghai, China). 3Due to their small diameter, the morphology of the NPs was examined using scanning electron microscopy (SEM), and the diameters of 500 randomly selected NPs were measured.

[0166] 1.2 Experimental design

[0167] Experiment 1: To analyze the toxicity of NPs at different concentrations and exposure times to LMR cells, LMR cells were cultured at a rate of 1×10 4 Cells were seeded into 96-well cell culture plates at a density of 10 cells / well. These cells were then placed in L15 medium containing 0 (control), 2, 20, 200 or 2000 μg / mL NPs, supplemented with 5% fetal bovine serum. The morphological changes of the cells were observed, and their survival was evaluated on days 1, 3, 7 and 14. Each experimental group consisted of five biological parallels, and the cell culture medium was changed every 3 days without cell passage.

[0168] Experiment 2: To study the long-term effects of NP exposure on LMR cells and the recovery of cells after cessation of NP exposure, cells were cultured at 7×10 6 The cells were seeded at a density of 100 cells / mL into six T-25 cell culture flasks; three flasks contained complete medium (L15 medium supplemented with 20% fetal bovine serum) supplemented with 20 μg / mL NP (experimental group), and the remaining three flasks contained complete medium without NP (control group). Cells were passaged after the cells had grown to the bottom of the flask. LMR cells were counted during each passage, and approximately 7×10 6 After 30 generations of culture, the NP addition was stopped and the cells were cultured for another 10 generations (recovery group).

[0169] Throughout the experiment, samples were collected and relevant parameters were measured at different time points. For experimental group cell samples, the following naming convention was used: "NP + treatment time or passage number". For recovery group cell samples, "RCY + passage number or time after NP withdrawal" was used. For example, NP-20 th Refers to cell samples treated with NP for 20 passages.

[0170] 2. Experimental Methods

[0171] 2.1 Cytotoxicity assessment

[0172] In Experiment 1, the cell viability was assessed using the methylthiazolyldiphenyltetrazolium bromide (MTT) assay kit (Solarbio, Beijing, China) according to the manufacturer's instructions. The absorbance at 490 nm was measured by ELISA (Leica Biosystems, Wetzlar, Switzerland), and the cell viability of each sample was calculated using the formula [(experimental well-blank well) / (negative control well-blank well)] × 100%. The cell morphology of each sample was also observed under an inverted microscope (Leica, Wetzlar, Germany), and the cytopathic effect (CPE) was recorded.

[0173] For cells treated with 20 μg / mL NPs in T-25 cell culture flasks, the cell doubling time was measured at passages 10, 20, and 30. Cells were cultured at 1×10 4 Cells were seeded into 96-well cell culture plates at a density of 10 cells / well. After 4 hours, non-adherent cells were removed and the remaining cells were subjected to MTT analysis to measure the optical density (OD) at time point N0. After 48 hours of incubation, OD (Nt) was measured again. The cell doubling time was calculated using the following formula: 48×lg2 / (lgNt-lgN0).

[0174] 2.2 Cellular NP observation and immunofluorescence analysis

[0175] In order to study the intracellular distribution of NPs, NP-1 st 、NP-10 th 、NP-30 th 、RCY-48h、RCY-5 th and RCY-10 th Cell samples. Cells were seeded onto round coverslips and fixed with 4% paraformaldehyde for 20 min. Subsequently, cells were washed with phosphate-buffered saline (PBS), stained with 4′,6-diamidino-2-phenylindole (DAPI) solution, and incubated in the dark at room temperature for 10 min. Anti-fluorescence quenching sealant was then added dropwise. Images were captured using a fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan) and a confocal laser scanning microscope (Nikon EclipseTi) at the specified excitation (EX) and emission (EM) wavelengths (DAPI, EX 330-380 nm, EM 420 nm; NP, EX 535 nm; EM 610 nm). Five biological replicates were measured, and 20 fields of view were randomly selected for each replicate. NP fluorescence area, NP fluorescence density, and cell number were calculated using ImageJ software (https: / / imagej.net / ) to determine the average NP fluorescence area and density of cells.

[0176] NP-30 th 、RCY-10 thCell samples of the control group were seeded on round coverslips with a diameter of 20 mm. Cells were fixed with 4% paraformaldehyde and then washed with PBS. They were permeabilized with 0.5% Triton X-100 for 20 min at room temperature and then washed with PBS three times. Cells were blocked with 3% bovine serum albumin for 30 min at room temperature and subsequently incubated with rabbit polyclonal antibody against phosphorylated H2A histone family member X (pH2AX) or mouse polyclonal antibody against proliferating cell nuclear antigen (PCNA) (Servicebio, Wuhan, China) at 4°C overnight. After three PBS washes, cells were incubated with IF488-labeled goat anti-rabbit IgG (Servicebio) for 50 min at room temperature. After three PBS washes, cells were stained with DAPI solution, incubated in the dark at room temperature for 10 min, and treated with dropwise anti-fluorescence quenching sealant. Imaging was performed using a fluorescence microscope at the specified excitation and emission wavelengths (IF488, EX 465-495 nm, EM 515-555 nm). In response to double-strand DNA breaks (DSBs), the histone variant H2AX is precisely phosphorylated at the break sites, resulting in the formation of distinct nuclear structures called γ-H2AX foci. Five biological replicates were evaluated, and 20 cells were randomly selected for γ-H2AX focus counting to determine the average number of pH2AX foci per cell. Similarly, for each experimental group, 50 cells were randomly selected from five biological replicates to measure the PCNA fluorescence intensity in the nuclear and cytoplasmic regions. In order to avoid changes in fluorescence density caused by the adjustment of the brightness level of the cell images, which could affect the accuracy of the experiment, the ratio of the mean optical density (MOD) of the nucleus to the cytoplasm was calculated.

[0177] 2.3 Phalloidin staining

[0178] NP-30 was washed with 4% paraformaldehyde th 、RCY-10 th Cells of the control group were fixed for 20 min and then washed three times with PBS. Membrane permeabilization was achieved by incubating the cells with 0.5% Triton X-100 for 20 min at room temperature and washing them with PBS three times. Then, the cells were stained with IF488-conjugated phalloidin staining solution (Servicebio) for 120 min at room temperature in the dark. After three PBS washes, the nuclei were stained with DAPI for 10 min and then washed three more times with PBS. Finally, anti-fluorescence fading sealant was added dropwise before imaging using the same fluorescence microscope described in Section 2.2.

[0179] Then, images of DAPI (EX 330-380nm, EM 420nm), IF488 (EX 465-495nm, EM 515-555nm), and NP (EX 535nm; EM 610nm) were taken. Five biological replicates were evaluated per group, and 200 randomly selected cells were analyzed per replicate, for a total of 800 cells per group. Phalloidin-stained cells were divided into four categories (see Results section 3.4 for details), and the percentage of each cell type was statistically analyzed.

[0180] 2.4 SEM and Transmission Electron Microscopy (TEM)

[0181] For SEM analysis, NP-30 th 、RCY-10 th and control group cells were seeded onto 20 mm circular coverslips. Each group had three biological replicates. LMR cells adhered to the coverslips were fixed with 2.5% glutaraldehyde for 2 h. After washing three times with 0.1 M phosphate buffer (PB), the cells were fixed with 1% osmium tetroxide in 0.1 M PB at room temperature for 2 h, followed by a series of ethanol dehydrations. Then, the samples were dried and sputter-coated with gold. The cell surface ultrastructure was observed using a SU8100 Hitachi SEM (Tokyo, Japan), and the perinuclear space was measured using ImageJ software.

[0182] For TEM analysis, NP-30 th 、RCY-10 th Cells were collected from the control group and fixed with 2.5% glutaraldehyde for 30 minutes. After fixation, dehydration, resin infiltration and embedding, polymerization, ultrathin sectioning and staining, subcellular structures were observed using HT7800 Hitachi TEM.

[0183] 2.5 5-ethynyl-2'-deoxyuridine (EdU) assay for cell proliferation

[0184] NP-30 th 、RCY-10 th Cells from the control group were seeded onto 20 mm round coverslips and the Click-iT EdU-488 Cell Proliferation Assay Kit (Servicebio) was used according to the instructions to identify proliferating cells. This involved in vitro EdU labeling, fixation, permeabilization, and EdU click reaction to label newly synthesized DNA. Images of DAPI, IF488, and NP were captured using a fluorescence microscope, using the same excitation and emission wavelengths described in section 2.2. Each group had five biological replicates, and 10 random fields of view from each replicate (a total of 50 fields per group) were analyzed to calculate the percentage of EdU-positive (EdU+) cells.

[0185] 2.6 Relative gene expression determination

[0186] From NP-30 th 、RCY-10 th LMR cells were collected from the control group, and their total RNA was harvested using Trizol reagent and PrimeScript TM RT kit (Takara, Japan) was used for reverse transcription to synthesize cDNA. Primer Premier 6 software (http: / / www.premierbiosoft.com / ) was used to design specific primers for H2AX, mitogen-activated protein kinase 1 (ERK1), PCNA and nuclear pore complex proteins NUP43, NUP50, NUP107, NUP133, NUP153, NUP160, NUP85, NUP50, NUP53, NUP54, NUP93, NUP62, NUP88, NUP214, NUP37, NUP214 (Table 3-1). SYBR Green-based fluorescent quantitative real-time PCR (qRT-PCR) was performed according to the reported PCR system and reaction conditions using Roche LightCycler480 instrument (Switzerland) to measure the relative expression levels of H2AX, ERK and PCNA. RT-PCR was performed according to the method described by D'Angelo et al. (D'Angelo MA, Raices M, Panowski SH, Hetzer MWJC, 2009. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. 136 (2): 284-295.) to measure the relative expression levels of NUPs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene. Five biological replicates were performed for each experiment.

[0187] Reaction system: The total reaction system is 12.5 μl, including 6.25 μl Premix Ex TaqTMII (2×), 2 μl cDNA template (<100 ng), 3.25 μl sterile ddH2O, 0.5 μl forward primer (10 μmol / l) and 0.5 μl rear primer (10 μmol / l).

[0188] Reaction conditions: pre-denaturation at 95°C for 30 seconds; denaturation at 94°C for 5 seconds, then at 60°C for 30 seconds, this process was repeated 40 times.

[0189] Table 3-1 Specific primers

[0190]

[0191]

[0192] 2.7 Biochemical assays

[0193] From NP-30 th 、RCY-10 th LMR cells were collected from the control group and malondialdehyde (MDA) content, total superoxide dismutase (T-SOD) activity, and catalase (CAT) activity in the cells were measured using the assay kit provided by Servicebio. The protein concentration of the samples was determined using the BCA protein quantification assay kit, and the results were used to calculate the SOD and CAT activities and MDA content per unit protein. Each group had five biological parallels.

[0194] 2.8 Western blot (WB)

[0195] From NP-30 th 、RCY-10 th LMR cells were collected from the control group and the cytoplasmic protein extraction kit (Beyotim, Shanghai, China) and lysis buffer (Servicebio) were used to extract cytoplasmic proteins and total proteins, respectively. The protein solution was mixed with 5× loading buffer at a ratio of 4:1, denatured in a metal bath at 95°C for 10 min, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The proteins on the membrane were blocked with 5% skim milk at room temperature for 30 min, and then incubated with mouse anti-ERK1 / 2 polyclonal antibody, mouse anti-PCNA polyclonal antibody, and mouse anti-GAPDH polyclonal antibody (Servicebio) at 4°C overnight. After washing the membrane with Post-Tris buffered saline and Tween 20 (TBST), the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (heavy chain and light chain) at ambient temperature for 30 min, followed by TBST washing. Finally, the proteins were visualized on the membrane using an enhanced chemiluminescence reagent, and the image was recorded after 1 min.

[0196] 2.9 Statistical analysis

[0197] All data were graphically represented using GraphPad Prism 8 and Microsoft PowerPoint software. Results are presented as mean ± standard deviation. SPSS (version 19) (IBM Corp., Armonk, NY, USA) software was used to perform Student's t test, one-way ANOVA, followed by Tukey's test to assess statistical differences.

[0198] 3. Results

[0199] 3.1 NP characteristics and distribution

[0200] The purchased NPs were observed by SEM and showed that their surface was regular and smooth and their shape was uniform ( Figure 7 Among the 500 measured NPs, the minimum diameter was 21.601 nm, the maximum diameter was 21.980 nm, and the average and median diameters were 21.755 nm and 21.759 nm, respectively ( Figure 7 ). The size distribution of NPs was narrow and consistent with the technical specifications provided by the manufacturer.

[0201] 3.2NP induces cytotoxicity and prolongs cell doubling time

[0202] In experiment 1, cytotoxicity was divided into six levels according to the degree of cell viability reduction and the presence of CPEs: L0 (no cytotoxicity), cell viability remained unchanged and no CPEs were observed; L1, cell viability decreased by <20% and CPEs were present; L2, cell viability decreased by 20% to 40% and CPE was present; L3, cell viability decreased by 40% to 60% and CPE was present; L4, cell viability decreased by 60% to 90% and CPE was present; L5, cell viability decreased by >90% and CPE was present.

[0203] In the control group and the 2 μg / mL NP-treated group, no CPE was observed in the cells during the first 7 days of the culture period ( Figure 8 A). However, in the 20, 200, and 2000 μg / mLNP-treated groups, CPE began to appear as early as the first day. Specifically, on day 1, the cytotoxicity levels of cells treated with 20, 200, and 2000 μg / mLNP, respectively, were divided into L1, L2, and L4. In the 20 μg / mLNP-treated group, the cytotoxicity level remained at L1 for the first 3 days, but increased to L2 from day 7 to day 14. Throughout the 14-day experiment, even if the culture medium was replaced every 3 days, the cell density increased due to mitosis, resulting in contact inhibition between cells. Therefore, on day 14, CPE was also observed in the control and 2 μg / mLNP-treated groups. ( Figure 8 A)

[0204] The above results indicate that the cytotoxic effect of 20 μg / mL NP treatment on cells gradually increased. Experiment 2 was performed to further evaluate the chronic cytotoxic effect of NP on cells. Cells were cultured for a longer period of time in complete medium supplemented with 20 μg / mL NP or without NP (control group) and passaged. After 10 passages (15 days), NP-treated cells exhibited CPE, but there was no significant change in doubling time. However, at the 20th generation (55 days), CPE was observed in NP-treated cells, and the proliferation time increased significantly. By the 30th generation (105 days), NP-treated cells showed a doubling time of 88.98 hours, which was 56.85 hours longer than the control group ( Figure 8 B) After 30 passages, NP-treated cells showed significant cytotoxicity with a surprising effect on cell proliferation time.

[0205] 3.3NP accumulation and release in cells

[0206] In experiment 2, the accumulation and localization of NPs in cells were analyzed after long-term exposure to a concentration of 20 μg / mL NPs, as well as the elimination of NPs from cells after 30 generations (105 days) of NP treatment when NP addition was stopped ( Figure 8 Laser confocal imaging showed that when the cells underwent 30 generations of proliferation, NPs were dispersed in the cells ( Fig. 9 B). Notably, some NPs were distributed around the nucleus, forming a clean nuclear membrane boundary, and in some cases, they colocalized with the nucleus ( Fig. 9 B) However, further confirmation is needed to determine whether the NPs actually penetrate into the interior of the cell nucleus.

[0207] The intracellular accumulation of NPs was assessed by calculating the NP fluorescence area and density per cell. Fig. 9 C, D). As expected, the accumulation of NPs in cells increased with the increase of NP exposure time, and the fluorescence signal of NPs in cells decreased after NP addition was stopped ( Fig. 9 ). It is noteworthy that after 10 generations of incubation in the absence of NP (RCY-10 th ), the fluorescence signal of NP was no longer detected in the cells. Unexpectedly, NP only accompanied the cell proliferation for one generation (NP-1 st ), the cells have reached NP treatment for 30 generations (NP-30 th ). In addition, after 48 h of incubation in the absence of NPs (RCY-48h), the fluorescence area and density of NPs within cells decreased by about 90%. These results suggest that cells can rapidly take up NPs and then quickly clear them from the cells.

[0208] 3.4 Long-term exposure to NPs has a strong toxic effect on cell morphology and subcellular structure

[0209] Cells stained with phalloidin were divided into four main types ( Fig.10 A): Category 1, cells have thick fibers spanning more than 90% of the central region of the cell, and their fibers cover 50% of the cell width. Category 2, the majority of intracellular F-actin is thin fibers, and at least one thick fiber crosses more than half of the cell width. Category 3, cells have only thin fibers. Category 4, cells have no detectable fibers in the central region and only show diffuse fluorescence. In the two control groups studied (NPs concentration of 0 μg / mL in experiments 1 and 2), category 2 cells dominated, accounting for approximately 50-78% and 49-72% of the total, respectively, while in the NP-30 group, category 3 cells were the most common, accounting for approximately 62-84%, while category 2 cells accounted for only 6-23%. In the RCY-10th group, category 3 cells were still more numerous, ranging from 58-74%, while the proportion of category 2 cells recovered slightly compared to the NP group, ranging from 22-39% ( Fig.10 B).

[0210] SEM showed that cells in the control group displayed uniform features with smooth and intact surface structures. In contrast, cells after long-term NP treatment showed obvious surface cracks, ulcers, and even large cracks, while cell edges shrank and filopodia shortened and retracted. Some filopodia also showed signs of breakage. After stopping NP treatment, cells in the recovery group showed restored elongation, repaired cell membranes, and smoother surfaces, although some local damage remained unrepaired ( Fig.10 C).

[0211] Transmission electron microscopy images showed that the cell membrane of the control group was intact and continuous. The cell organelles were abundant, the nuclear membrane was intact, and there was no obvious abnormality in the perinuclear space. The mitochondria were uniform in size, with intact membranes, regular cristae, and uniform matrix. In the NP-treated group, cell membrane damage, swollen organelles, enlarged perinuclear space, and mitochondrial membrane damage and swelling were observed. In addition, the rough endoplasmic reticulum showed expansion, membrane dissolution, and ribosome separation. In the recovery group, partial cell membrane damage was found, the cytoplasm was sparse and weak, the organelles were slightly swollen, the perinuclear space was slightly widened, the mitochondrial membrane was partially dissolved, and the cristae were reduced and broken ( Fig.10 D).

[0212] 3.5 Long-term exposure to NPs makes it difficult to recover from genotoxicity

[0213] Using the EdU method to detect cell DNA synthesis, it was found that compared with the control group, the DNA synthesis of cells exposed to NP for a long time (30 generations, 105 days) was reduced, resulting in slower cell proliferation. However, after 10 generations without NP exposure, the proportion of cells undergoing DNA synthesis returned to the control group level. Fig.11 A and B)

[0214] Immunofluorescence was used to analyze the formation of γH2AX foci in cells. The average number of spontaneously formed pH2AX foci per cell in the two control groups was 1.28 and 2.16 ( Fig.11 C and D). In cells exposed to NP for a long time (NP-30, 30 generations, 105 days), the average number of pH2AX foci per cell increased to 23.46, which was significantly higher than that in the control group ( Fig.11 C and D). After 10 generations without NP exposure, the average number of pH2AX foci per cell in the recovery group decreased to 18.56, but this value was still significantly higher than that in the control group ( Fig.11 C and D). In addition, NP-30 th and restore RCY-10 th The expression level of H2AX gene in the group was significantly higher than that in the control group ( Fig.11 E) These findings suggest that chronic exposure to NPs leads to severe DNA DSBs that persist even after 10 generations without NP exposure.

[0215] Since oxidative stress is closely related to DNA damage, the key enzymes and products involved in oxidative stress were studied. The MDA content and CAT activity in cells exposed to NP for a long time increased significantly, but the T-SOD activity remained unchanged. After 10 generations without NP exposure, the MDA content and CAT activity in the recovery group returned to the control group level, and the T-SOD activity remained unchanged. ( Fig.11 F, G, and H)

[0216] 3.6 Long-term exposure to NPs inhibits NUP transcription, leading to potential risk of nuclear membrane permeability barrier

[0217] The main function of the cell nucleus is to store and protect DNA while regulating the expression and replication of genetic information. Therefore, based on the pH2AX focus results, the transcription levels of NUPs under NP stress and recovery conditions were preliminarily detected. After 30 generations of NP exposure, the expression of 9 out of 14 NUPs was inhibited ( Fig.12 B). After NP removal, the transcription of NUPs rebounded, and the transcription level in the RCY-10th group was higher than that in the control group ( Fig.12 B).

[0218] Subsequently, the expression of genes and proteins with nuclear translocation functions was evaluated ( Fig.12 ). Compared with the control group, NPS-30 th and RCY-10 thThe protein expression levels of PCNA, ERK1 and ERK2 in the cytoplasm of the NPS-30 and RCY-10 groups were increased. However, compared with the control group, the protein expression levels of PCNA, ERK1 and ERK2 in the total cellular protein (cytoplasm + nucleus) of the NPS-30 and RCY-10 groups were almost unchanged. th Group and RCY-10 th The PCNA level in the total protein of the NP-30 cells even decreased slightly. Similarly, the expression levels of PCNA and ERK1 in NP-30 cells did not change significantly compared with the control group.

[0219] Measurement of MOD of PCNA in nuclear and cytoplasmic regions, NPs-30 th and RCY-10 th The PCNA fluorescence intensity ratio (nucleus / cytoplasm) of the group was also significantly reduced ( Fig.12 A and D).

[0220] 4. Results

[0221] 4.1 Chronic stress can cause severe reactions when it reaches a certain threshold

[0222] Throughout the life cycle of an organism, from differentiation and reproduction to aging and death, cells are constantly battling NPs. While extensive studies have revealed the cytotoxic and genotoxic effects of NPs, few studies have investigated the adverse effects of NPs on cells as they continue to proliferate. The harmful effects of NPs may not be apparent in short- or medium-term experiments. For example, in the presence of NPs at a concentration of 20 μg / mL, the doubling time of LMR cells in the study did not change significantly after 10 generations ( Figure 8 B). However, when reaching the 20th generation, the cell proliferation rate decreased significantly ( Figure 8 B). In addition, by passage 30, the doubling time of LMR cells was approximately three times that of the control group ( Figure 8 B) In addition, compared with the control group, NP-30 th The number of cells undergoing DNA replication (EdU+ cells) in the group also decreased by nearly half ( Fig.11 A). Studies on Daphnia magna have shown that chronic NP or MP stress shortens the lifespan of individuals and prolongs the time to first reproduction, which was not observed under acute stress conditions. These findings suggest that the adverse effects of NP on cell proliferation may only become apparent after reaching a certain threshold or duration, highlighting the importance of long-term evaluation to detect such effects. In addition, studies on human induced pluripotent stem cells have shown that long-term exposure to NP significantly inhibits cell growth and reduces the self-renewal capacity of these cells. It can be seen that the chronic harmful effects of NP on cells deserve careful attention from researchers.

[0223] 4.2 LMR cells did not fully recover after rapid release of all NPs

[0224] NPs enter cells through passive diffusion and active endocytosis, and internalized NPs have extremely harmful effects on cells. In the inventors' experiments, as the number of cell passages increased, up to 30 passages, the accumulation of NPs in cells also increased ( Fig. 9 ), which may be due to the enhanced uptake of particles by cell proliferation. Notably, the intracellular NP levels decreased by about 90% within 48 hours after NP removal ( Fig. 9 C, D). This rapid release is unexpected. Studies have shown that with each cell division, the concentration of internalized particles in the daughter cells is halved. However, for most cells that have just entered the recovery phase, 48 hours is not enough to complete the division. Therefore, the inventors speculate that the internalized NPs may also be released outside the cell through exocytosis, and cells that complete mitosis may also reduce the number of NPs in a single cell.

[0225] By the 10th generation without NP exposure, no fluorescent NP signal was observed in the cells, and the cell proliferation rate and antioxidant level returned to the level of the control group ( Fig.11 A, F, G, H). However, after complete elimination of NP, cells did not fully recover, especially the enlarged perinuclear space, F-actin disorder and genotoxicity (H2AX foci number) in subcellular structures ( Fig.10 , 11 ).

[0226] The nuclear envelope separates the nucleus from the cytoplasm and consists of an outer membrane and an inner membrane. The perinuclear space is usually between 30 and 50 nm. The mechanical connection between the nuclear envelope and the cytoskeleton is mediated by the adaptor protein F-actin. After 30 generations (105 days) of NP exposure, the inner and outer membranes were disrupted, resulting in an enlargement of the perinuclear space up to 324 nm ( Fig.10 D), which may be related to the impaired F-actin morphology. Even after a recovery period of 10 generations (35 days), the perinuclear space was still larger than 83 nm. In addition, the main cell types based on F-actin morphology were still 3 categories ( Fig.10 B), indicating that the function of F-actin as a molecular adaptor is severely impaired, which affects the spatial and structural integrity of the nucleus. Abnormal nuclear membrane structure may disrupt gene expression and lead to severe phenotypes.

[0227] A key response of cellular systems to DSBs involves precise phosphorylation of the histone variant H2AX at the site of lesions, generating discrete nuclear foci called γ-H2AX foci. In the inventors' study, the number of γ-H2AX foci per cell was significantly increased in the NPS and RCY groups compared with the control group. In other words, long-term exposure to NPs (30 generations, 105 days) leads to DSBs that remain unrepaired or repaired incorrectly even after the cells no longer contain NPs. Although researchers have reported that DNA toxicity of NPs to mouse intestinal and mesenchymal stromal cells includes an increase in γ-H2AX foci, DSBs in cells during the recovery period after long-term exposure to NPs have not been evaluated. In fact, such recovery assessments are very important because they can provide a more accurate understanding of the level and duration of toxicity of pollutants to organisms or cells.

[0228] After a DSB occurs in DNA, the aggregation of F-actin helps to form the necessary nuclear structures, promote the recognition of damaged sites and the recruitment of repair factors. In heterochromatin regions, F-actin and related myosins may be involved in the process of relocating DSBs to the nuclear periphery for safe homologous recombination repair. Therefore, when NP induces DSBs in LMR cells, the aggregation of F-actin is required to assist in DSB repair.

[0229] F-actin forms a special structure around the cell nucleus, called the perinuclear actin cap. The cap is connected to the cell nucleus through the nuclear membrane and proteins on the nuclear membrane, forming a linker between the nucleoskeleton and cytoskeleton complexes. Inhibition of myosin polymerization or myosin contraction will disrupt the structure of the F-actin cap, thereby affecting the nuclear shape. The inventors speculate that NP may affect the spatial structure of the perinuclear space by disrupting the formation of the F-actin cap. The expansion of the perinuclear space and the occurrence of DSBs will in turn affect the synthesis and secretion of F-actin, which may be the challenge that makes it difficult for LMR cells to fully recover during the repair process.

[0230] This severe effect on F-actin under long-term NP stress may be related to the physical damage caused by NP to F-actn. F-actin shows high sensitivity to mechanical stimulation, and this sensitivity may be related to changes in cell morphology, the stability of intercellular connections, and the activation of signaling pathways. Therefore, it is believed that continuous NP stress will lead to the accumulation of a large amount of NP in cells, and free particles that are not wrapped by lysosomes may cause mechanical damage to F-actin, ultimately leading to the clearance and decomposition of F-actin.

[0231] 4.3NP inhibition of NUP transcription leads to damage of nuclear membrane permeability barrier

[0232] The nuclear pore complex (NPC) consists of water channels composed of more than 30 different NUPs. It spans the two lipid bilayers of the nuclear membrane. During cell proliferation, the NPC disassembles during mitosis and reassembles to form new NPCs. The formation of new pores occurs during mitosis and interphase and requires the expression of NUPs. The inventors observed that under NP stress, the expression levels of 9 out of the 14 NUP genes tested were suppressed ( Fig.12 B) This inhibition leads to insufficient NUP proteins involved in the reestablishment of the NPC in newly dividing cells, which severely affects the integrity and function of the nuclear membrane permeability barrier. This disruption may lead to various problems, such as leakage of nuclear contents, changes in nucleocytoplasmic transport, loss of nuclear compartmentalization, and increased susceptibility to stress and injury.

[0233] The expression levels of nuclear localization proteins ERK1, ERK2, and PCNA were measured, and their fluorescence intensity ratios between the nucleus and cytoplasm were analyzed. th and RCY-10 th In the 24-hour group, the expression of ERK1, ERK2, and PCNA proteins in cells (cytoplasm and nucleus) remained unchanged, but their expression in the cytoplasm increased. Therefore, it is speculated that ERK1, ERK2, and PCNA may have leaked from the nucleus to the cytoplasm ( Fig.12 C, E). In addition, compared with the control group, the ratio of PCNA in the nucleus / cytoplasm in the NPS and RCY groups was significantly decreased ( Fig.12 A, D). This effect may be related to nuclear protein leakage caused by damage to the nuclear membrane permeability barrier, but it cannot be ruled out that it is related to protein expression.

[0234] Although further experiments are needed to prove the functionality of the nuclear membrane permeability barrier, our results suggest that long-term exposure to NPs can affect the expression of NUPs and hinder the formation of new pores in dividing cells, indicating the nuclear damage that may be caused by long-term exposure to NPs.

[0235] The results show profound and lasting effects of NPs on cellular health, particularly at the nuclear level. The results clearly demonstrate that chronic exposure to NPs leads to a significant suppression of cell proliferation and lasting damage to cellular and subcellular structures. Notably, the nuclear abnormalities observed after exposure, such as enlarged perinuclear space and disruption of F-actin in the nuclear envelope, highlight the profound vulnerability of the nucleus to NP toxicity. The observed disturbances in nucleoporin transcription further suggest the possibility of compromised nuclear envelope integrity and function.

Claims

1. A method for constructing a Lateolabrax japonicus cell line, characterized in that: Under sterile conditions, the rostral tissue of Lateolabrax japonicus was obtained, cut into pieces and digested with enzymes to obtain primary cells; the cells were resuspended in complete culture medium after enzymatic digestion of the primary cells, inoculated into a cell culture bottle, and cultured and passaged at a constant temperature of 28°C to obtain the rostral cell line of Lateolabrax japonicus.

2. The method for constructing the Lateolabrax japonicus cell line according to claim 1, characterized in that: The sea bass snout tissue is digested with collagenase and trypsin successively; the primary cells are digested with trypsin.

3. The method for constructing the Lateolabrax japonicus cell line according to claim 1, characterized in that: The primary cells were passaged when they adhered to and grew to 90-100% confluence.

4. A Lateolabrax japonicus cell line, characterized in that: The method is constructed by the following method: under sterile conditions, the body snout tissue of the striped sea bass is obtained, cut into pieces and then digested with enzymes to obtain primary cells; the cells are resuspended in complete culture medium after the enzymatic digestion of the primary cells, inoculated into a cell culture bottle, and cultured and passaged at a constant temperature of 28°C to obtain the snout cell line of the striped sea bass.

5. The Lateolabrax japonicus cell line according to claim 4, characterized in that: The sea bass snout tissue is digested with collagenase and trypsin successively; the primary cells are digested with trypsin.

6. The Lateolabrax japonicus cell line according to claim 4, characterized in that: The primary cells were passaged when they adhered to and grew to 90-100% confluence.

7. Application of the Lateolabrax jacquard cell line described in any one of claims 4 to 6 in the toxicity assessment of environmental pollutants, microplastics.

8. A method for assessing the toxicity of environmental pollutants microplastics, characterized by: The cells of the Lateolabrax japonicus cell line described in any one of claims 4 to 6 are placed in a culture medium containing environmental pollutant microplastics for culture, and the cell conditions are observed to evaluate the toxicity of environmental pollutant microplastics.

9. The method for evaluating the toxicity of environmental pollutants microplastics according to claim 8, characterized in that: The observed cell conditions include the state of the cell nucleus, survival rate, and cell proliferation time to evaluate the toxicity of environmental pollutants such as microplastics.

10. The method for evaluating the toxicity of environmental pollutants microplastics according to claim 9, characterized in that: The cells of the Lateolabrax japonicus cell line have undergone at least 20 passages.

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