Establishment and application of epinephelus lanceolatus frontal bone tissue osteoblast line

Abstract

The invention discloses establishment and application of an epinephelus lanceolatus frontal bone tissue osteoblast line, and relates to the technical field of biology. The cell line is an epinephelus lanceolatus frontal bone tissue osteoblast line ELFBOB, the preservation number is CCTCC NO: C2025132, the cell line is derived from epinephelus lanceolatus fry frontal bone tissue, and the cell line is established after tissue separation, primary culture with a dry pasting method, subculture and cryopreservation resuscitation. The biological characteristics are stable, 1-40 generations are mainly long fusiform, the chromosome karyotype is 2n = 48, strong alkaline phosphatase activity is achieved, the transfection efficiency is about 75%, and the activity is good after cryopreservation and resuscitation. The establishment method is high in repeatability, the obtained cell line can serve as an in-vitro research model and is applied to epinephelus lanceolatus skull malformation cause analysis, osteogenesis-related gene function verification, improved variety breeding and healthy culture strategy optimization, the blank of the epinephelus lanceolatus bone tissue cell line is filled, and the method has important scientific research value and industrial application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a gill arch osteoblast cell line of Epinephelus lanceolatus and application thereof. BACKGROUND

[0002] Epinephelus lanceolatus, commonly known as dragon grouper, belongs to Perciformes, Epinephelidae and Epinephelus, is a warm water coral reef fish, mainly distributed in the Indian-Pacific Ocean, from the east coast of Africa and the Red Sea in the west, to southern Japan in the north, and to northwestern Australia in the south. It is distributed in the waters near the Nansha Islands in Taiwan, China, and is relatively rare in quantity. This species is the largest fish in the Epinephelus group, with a maximum recorded body length of 2.7 meters and a maximum recorded body weight of 400 kilograms. Due to its fast growth rate, strong disease resistance and delicious meat, it is widely favored by the consumer market and is an important marine economic fish in China, with an annual output of about 5000-8000 tons. In addition, due to its excellent characteristics, it is often used as a parent for hybrid grouper. Tiger dragon, mouse dragon, and green dragon, which are hybrid gouramis with it as a parent, have been mass-produced in artificial breeding. Market research shows that whether it is the gourami itself or the hybrid gourami with it as a parent, it is prone to deformity during fry breeding, especially skull deformity, with a deformity rate as high as 82% (Lü X. Growth characteristics and bone development and deformity of Scophthalmus maximus and Epinephelus lanceolatus[D]. Chinese Academy of Sciences (Institute of Oceanology, Chinese Academy of Sciences), 2018), which significantly reduces the survival rate of fry and causes serious economic losses. Even if some individuals survive, the deformity phenotype will affect the commodity value, further exacerbating the loss of breeding income.

[0003] Normal development of fish skull highly depends on the proliferation, differentiation and mineralization activity of osteoblasts. As the core functional cells of bone formation and remodeling, osteoblasts directly participate in the morphological construction of craniofacial bones by constructing the framework of collagen matrix and hardening through calcium and phosphorus deposition. Studies have shown that abnormal differentiation or functional defects of osteoblasts may lead to reduced bone density, incomplete mineralization and imbalance of absorption-formation coupling of skull, ultimately resulting in skull morphological deformity. Fish cell lines, as a simple and reproducible in vitro culture system, have been widely used in basic research, developmental biology, biotechnology and other fields. So far, only heart cell line (Guo C, Huang Y, et al. Establishment of Epinephelus lanceolatus heart cell line and its application in cytotoxicity and virology [C] / / Chinese Academy of Sciences. Chinese Academy of Sciences, 2013), skin tissue cell line (Ouyang Z, Chen R, Ji S, et al. Epinephelus lanceolatus skin tissue cell line and its construction method: CN201510217925.6 [P]) and head kidney cell line (Huang Y, Huang X, Liu Z, et al. A kind of Epinephelus lanceolatus head kidney cell line and its construction method and application: CN202110088381.3 [P]) have been reported from E. lanceolatus tissue since 2013, 2018 and 2023, respectively. However, there is no report on cell lines derived from bone tissue. It is worth noting that the skull deformity of E. lanceolatus and its hybrid offspring is mostly manifested as abnormal bulging of the dorsal head. As the key craniofacial bone above the fish eye orbit, abnormal development of frontal bone is likely to be closely related to this phenotype. Similarly, in the same 'humpback' characteristic of Cromileptes altivelis, the allometric growth of frontal bone has been confirmed as one of the main reasons for the dorsal head bulging. Therefore, the establishment of frontal bone-derived osteoblast cell line of E. lanceolatus not only provides an ideal research model for analyzing the molecular mechanism of its craniofacial bone deformity, but also further explores the growth characteristics in the process of its skeletal development, and thus provides a theoretical basis for the optimization of good strain selection and healthy breeding strategies. SUMMARY

[0004] The purpose of the present application is to provide a frontal bone tissue-derived osteoblast cell line of E. lanceolatus, which can not only provide an ideal research model for analyzing the molecular mechanism of its craniofacial bone deformity, but also further explore the growth characteristics in the process of its skeletal development, and thus provide a theoretical basis for the optimization of good strain selection and healthy breeding strategies.

[0005] To achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The application provides a goggle grouper (Epinephelus lanceolatus) frontal bone osteoblast, which is named as a goggle grouper frontal bone tissue osteoblast cell line ELFBOB, and has been preserved in the China Center for Type Culture Collection on May 17, 2025, with a preservation number of CCTCC NO: C2025132.

[0007] The application adopts the frontal bone tissue from goggle grouper fry as a starting material, removes adherent blood cells by treating with a red blood cell lysate, inoculates by using a dry sticking method and adds complete culture solution for primary culture, and establishes the goggle grouper frontal bone osteoblast cell line by purifying the cell culture technology for multiple passages, which is named as the goggle grouper frontal bone tissue osteoblast cell line ELFBOB. The cell line has been preserved in the China Center for Type Culture Collection (CCTCC) on May 17, 2025, with a preservation address of China. Wuhan. Wuhan University, and a preservation number of CCTCC NO: C2025132.

[0008] The cell strain has the following biological characteristics:

[0009] 1) Morphological characteristics: during the passage culture for 1-40 generations, the cells are mainly long spindle-shaped, and the morphology is stable;

[0010] 2) Growth characteristics: the cells grow well in the L-15 complete culture solution containing specific additives at 27 DEG C, and can be subcultured once every 2-3 days, and the proliferation capacity is stable;

[0011] 3) Karyotype characteristics: the karyotype analysis shows that 72% of the metaphase cells have the chromosome number of 2n = 48, and the karyotype is stable;

[0012] 4) Osteogenic characteristics: the alkaline phosphatase staining is positive, and has strong alkaline phosphatase activity, which meets the core characteristics of osteoblasts;

[0013] 5) Transfection characteristics: the cells can efficiently receive exogenous genes, and the transfection efficiency is about 75% after electrically transfecting the pEGFP-N1 plasmid, and are suitable for experiments related to exogenous gene expression;

[0014] 6) Freezing and recovery characteristics: after the programmed gradient cooling and freezing, the adhesion rate of the cells is 60%-80% after 24h of recovery, and the cell morphology and proliferation capacity after recovery have no obvious difference with those before freezing, and the cells can be normally subcultured.

[0015] The application obtains the goggle grouper frontal bone osteoblast cell line by separating the frontal bone tissue of the goggle grouper, culturing by using a dry sticking method, starting primary culture, starting passage culture, freezing and recovery.

[0016] The application further provides a method for establishing the bone tissue cell line ELFBOB of the forehead bone of Epinephelus lanceolatus, which comprises the following steps:

[0017] 1) Obtaining the forehead bone tissue of Epinephelus lanceolatus

[0018] The Epinephelus lanceolatus fry is placed on ice until it is no longer responsive to stimulation stress; the fish body surface is wiped with a sterilized gauze block, and then the fish body surface is wiped with a gauze soaked with alcohol, and then the forehead bone tissue of the Epinephelus lanceolatus is taken out and placed in a PBS solution containing penicillin, streptomycin and amphotericin B (100 mL of PBS solution is added with 1 mL of 100x concentrated solution of commercial three-antibodies).

[0019] 2) Primary culture of the forehead bone tissue of Epinephelus lanceolatus

[0020] The forehead bone tissue of Epinephelus lanceolatus is cut into small tissue blocks of about 2.0 mm, rinsed with PBS solution for three times. After rinsing, the small tissue blocks are transferred to a 15 mL centrifuge tube, and a commercial red blood cell lysis solution is used to lyse the blood cells attached to the tissue; the tissue blocks are then washed with PBS solution for three times, and are evenly attached to a cell culture bottle, and the bottom of the culture bottle is resuspended and moistened with 1 mL of complete culture solution, and the primary culture is started by incubation at 27°C under 5% CO2; 1 mL of culture solution is added on the second day; 1 mL of culture solution is added on the third day; and the complete culture solution is replaced once on the fourth day, and 5 mL of complete culture solution is supplemented for primary culture.

[0021] 3) Subculture of the forehead bone tissue cells of Epinephelus lanceolatus

[0022] When the adherent cells proliferate to 80-90% coverage in the primary culture, the old culture solution is removed, 3 mL of PBS is added for cleaning, and the residual serum and divalent metal ions are removed, and the waste liquid is discarded; (1 mL of gun head is used for suction). Then, the cells are subcultured by 0.25% trypsin digestion method to make the cells in the bottle bottom all immersed in the solution; the bottle mouth is covered, shaken back and forth for 2-3 min, and then placed under an inverted microscope to observe the cells. With the passage of time, the originally adherent cells gradually tend to be round, and the trypsin is discarded before the cells float, and 10 mL of culture solution is added to terminate the digestion. The observation of digestion can also be done by naked eye, and the digestion is terminated when the bottle bottom appears white and there are fine needle hole gaps. Generally, the digestion time at room temperature is about 1-3 min. The adherent cells are blown into a suspension with a pipette, and are divided into two to three other bottles, the bottle mouth and bottle cap are roasted above the flame of an alcohol lamp for a while, the bottle cap is covered, and the culture is continued at 27°C. The adherent growth is observed the next day. The subculture is carried out at a ratio of 1:2; the subculture is carried out once every 2-3 days thereafter, until 210 days and 40 generations, and the bone tissue cell line of the forehead bone of Epinephelus lanceolatus is successfully established.

[0023] 4) Freezing of Epinephelus lanceolatus frontal bone tissue cells

[0024] The application also provides a freezing method of the Epinephelus lanceolatus frontal bone osteoblast cell line ELFBOB, a bottle of Epinephelus lanceolatus frontal bone osteoblast cell line growing vigorously and covering the bottom of the culture bottle is taken, a trypsin digestion method is used, and the cell precipitate is collected after centrifugation; the serum-free cell freezing solution is slowly added, and the cells are gently blown and beaten to uniformly disperse in the cell freezing solution, and the liquid is moved into the freezing tube using a pipette gun; the freezing tube is placed at 4℃ for 1 h, and then placed in a program gradient cooling freezing box, the temperature of the program gradient cooling is reduced at a speed of 1℃ / min, the freezing box is placed at -80℃ for 1 day, and finally the freezing tube is taken out and immersed in liquid nitrogen, and can be long-term frozen.

[0025] 5) Resuscitation of Epinephelus lanceolatus frontal bone tissue cells

[0026] The application also provides a resuscitation method of the Epinephelus lanceolatus frontal bone tissue osteoblast cell line ELFBOB, the freezing tube is taken out from the liquid nitrogen and quickly placed in a water bath pot at 40℃, the freezing tube should be constantly shaken during the process of thawing the cells to quickly and uniformly thaw, until the cells are completely thawed, the thawed cell suspension is transferred to a culture bottle centrifuge tube, the complete culture solution is added to the culture bottle, and the culture is carried out at 27℃ and 5% CO2; the complete culture solution is replaced after 24 h, and the culture is continued.

[0027] 6) Application of Epinephelus lanceolatus frontal bone tissue cells in gene function

[0028] The Epinephelus lanceolatus frontal bone osteoblast cells of the application are subjected to alkaline phosphatase activity identification, transfection efficiency analysis and double luciferase experiment analysis, and the function verification analysis of related genes is preliminarily obtained, which lays a foundation for analyzing the function of genes related to bone development and formation of Epinephelus lanceolatus, and provides an ideal model for analyzing the mechanism of bone development and the cause of cranial bone malformation, and has important value for good strain selection and healthy breeding.

[0029] The above complete culture solution: L-15 culture solution is used as a basic culture solution, and the addition amounts of fetal bovine serum, β-mercaptoethanol, human basic fibroblast growth factor (Human FGF-basic), human epithelial cell growth factor (Human EGF), human hepatocyte growth factor (Human HGF), human leukemia inhibitory factor (Human LIF), penicillin, streptomycin and fish serum are 15vol%-20vol%, 0.5vol‰, 10 μg / L, 5 μg / L, 1 μg / L, 1 μg / L, 100 IU / mL, 100 μg / mL and 1vol% respectively.

[0030] The preparation method of the fish serum is as follows: 10 mL syringes are soaked with 15% (w / v) EDTA, blood is taken from the caudal vein of Epinephelus lanceolatus into 15 mL centrifuge tubes placed on ice, centrifuged at 3,500 g for 15 min, the supernatant is transferred into a 50 mL centrifuge tube, incubated at 4 DEG C overnight, centrifuged at 3,500 g for 30 min again, the supernatant is filtered with a 0.2 mu m filter to remove bacteria, and one tube of 10 mL is stored in a 15 mL centrifuge tube and stored at -20 DEG C.

[0031] The frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can be applied to the establishment of a cell model for studying cranial bone deformity (abnormal development of frontal bone) of the Epinephelus lanceolatus.

[0032] The frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can be applied to the establishment of a cell model for studying the differentiation mechanism of osteoblasts of the Epinephelus lanceolatus, the cytological basis of cranial bone development and the regulation mechanism of deformity.

[0033] The frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can be applied to the analysis of functions of genes related to bone development and formation of the Epinephelus lanceolatus and the exploration of causes of cranial bone deformity.

[0034] The frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can be applied to the establishment of a cell model for the function verification of osteoblast-related genes (such as En1b The frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can be applied to the establishment of a cell model for the function verification of osteoblast-related genes (such as

[0035] The frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can be applied to the establishment of a cell model for the function verification of osteoblast-related genes (such as

[0036] In addition, the frontal bone tissue osteoblast cell line ELFBOB of the Epinephelus lanceolatus can also be applied to the large-scale preparation, long-term preservation and recovery of the frontal bone osteoblast cell line of the Epinephelus lanceolatus.

[0037] The technical problem solved by the present application is:

[0038] The construction method of the frontal bone tissue osteoblast cell line of the Epinephelus lanceolatus has high repeatability, and the cultured frontal bone tissue cell line has good stability.

[0039] The frontal bone tissue osteoblast cell line of the Epinephelus lanceolatus is transfected by using the electroporation method, and the transfection efficiency is high.

[0040] The frontal bone tissue of the Epinephelus lanceolatus has excellent cell characteristics; the chromosome karyotype analysis is stable.

[0041] The frontal bone osteoblasts of the Epinephelus lanceolatus of the application are subjected to alkaline phosphatase activity identification, transfection efficiency analysis and double luciferase experiment analysis, and the function verification analysis of related genes is preliminarily obtained, which lays a foundation for analyzing the functions of genes related to the development and formation of the skeleton of the Epinephelus lanceolatus, and provides an ideal model for analyzing the development mechanism of the skeleton and the causes of cranial deformity, and has important value for good strain selection and healthy breeding.

[0042] Compared with the prior art, the application has the following advantages:

[0043] The complete culture medium used in the construction method of the application is based on L-15 culture solution, fetal bovine serum, beta-mercaptoethanol, human basic fibroblast growth factor (Human FGF-basic), human epithelial cell growth factor (Human EGF), human hepatocyte growth factor (Human HGF), human leukemia inhibitory factor (Human LIF), penicillin, streptomycin and grouper serum. The L-15 culture solution and fetal bovine serum FBS provide sufficient nutrients for cell growth; the addition of beta-mercaptoethanol, human FGF-basic, human EGF and human HGF can stimulate cell activity, accelerate cell division and proliferation, and at the same time provide a good buffer environment for in vitro cell culture, so that the cells can maintain stable pH during long-term culture; fish serum helps to improve the mitotic activity of cells in fish cell culture, and may be one of the key additives in the current culture medium; the addition of LIF can effectively inhibit the differentiation of frontal bone osteoblasts and promote cell proliferation.

[0044] The frontal bone tissue osteoblast cell line of the Epinephelus lanceolatus is established for the first time, which fills the research gap of bone tissue cell lines of the species and provides a special cell model for the research on the mechanism of cranial deformity; the construction method has good repeatability, and the cell line maintains stable morphology, karyotype and growth characteristics after being subcultured for 40 generations, and the activity is stable after being frozen and recovered, so that the cell line can be used for a long time for experiments; the cell line has excellent transfection characteristics (transfection efficiency of 75%) and osteoblast specificity, and can meet the experimental needs of gene function verification, osteogenesis mechanism analysis and the like, and is especially suitable for the research on genes related to cranial deformity; the formula of the complete culture medium is optimized, and L-15 is added with various growth factors and grouper serum, in which the fetal bovine serum and L-15 provide basic nutrients, various growth factors can stimulate cell activity, accelerate proliferation and maintain stable pH, fish serum improves cell mitotic activity, and LIF can inhibit osteoblast differentiation and promote proliferation, thereby ensuring stable in vitro cell culture; the application provides a key tool for analyzing the causes of cranial deformity of the Epinephelus lanceolatus and exploring the characteristics of skeletal development, and can directly guide the optimization of good strain selection and healthy breeding strategies, significantly reduce economic losses in breeding, and has important industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1Image of primary cultured frontal bone tissue cells of saddle-banded grouper after 5 days;

[0046] Figure 2 Image of primary cultured frontal bone tissue cells of saddle-banded grouper after 7 days;

[0047] Figure 3 Image of first-generation passaged cells from the frontal bone tissue of the saddle-banded grouper;

[0048] Figure 4 Image showing second-generation passaged culture of frontal bone tissue cells from the saddle-banded grouper;

[0049] Figure 5 Figure 5 shows the passaged cells of the frontal bone tissue of the saddle-banded grouper.

[0050] Figure 6 Image showing the 10th generation passage of frontal bone tissue cells from the saddle-banded grouper.

[0051] Figure 7 Image of frontal bone tissue cells from saddle-banded grouper passaged for 15 generations;

[0052] Figure 8 Image showing the 20th generation passage of frontal bone tissue cells from the saddle-banded grouper.

[0053] Figure 9 Image of frontal bone tissue cells from saddle-banded grouper cultured for 25 generations;

[0054] Figure 10 Image of frontal bone tissue cells from saddle-banded grouper after 30 passages;

[0055] Figure 11 Image of frontal bone tissue cells from saddle-banded grouper passaged for 35 generations;

[0056] Figure 12 Image of frontal bone tissue cell line from saddle-banded grouper 24 h after cryopreservation and revival;

[0057] Figure 13 Image showing the species origin identification results of the frontal bone tissue cell line of the saddle-banded grouper;

[0058] Figure 14 Image showing the karyotype analysis results of the frontal bone tissue cell line of the saddle-banded grouper;

[0059] Figure 15 Image showing alkaline phosphatase staining of frontal bone tissue cells from the saddle-banded grouper;

[0060] Figure 16 Image showing the results of transfecting EGFP into frontal bone tissue cells of the saddle-banded grouper.

[0061] Figure 17 The image shows the results of a dual-luciferase assay using frontal bone tissue cells from the saddle-banded grouper. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments will be used in conjunction with the accompanying drawings to further illustrate the invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0063] Unless otherwise specified, the experimental methods and reagents described in the following examples are all conventional methods.

[0064] Example 1: Establishment of the frontal bone tissue cell line of the grouper of the present invention

[0065] (1) Place the juvenile grouper on ice until the fish show no stress response to stimulation. Wipe off the mucus on the fish's body surface with sterile gauze. Wipe the fish's body surface twice with gauze soaked in 75% alcohol. Transfer the fish to a clean bench, remove the frontal bone tissue with dissecting instruments, and rinse 3-4 times in PBS solution containing 1X commercial triple antibodies (penicillin, streptomycin, and amphotericin B) (i.e., 100 mL of PBS solution with 1 mL of 100X concentrated commercial triple antibodies).

[0066] (2) The frontal bone tissue block was cross-cut into small pieces of approximately 2 mm using two No. 11 scalpels. The cut tissue pieces were rinsed three times with PBS solution containing triple antibodies. After rinsing, the tissue pieces were transferred to 15 mL centrifuge tubes. The attached blood cells were lysed using commercially available erythrocyte lysis buffer. The tissue pieces were then washed three times with PBS solution. The tissue pieces were evenly placed into a cell culture flask, and the bottom of the flask was resuspended and moistened with 1 mL of complete culture medium. Primary culture was initiated at 27°C. 1 mL of culture medium was added on the second day, and another 1 mL was added on the third day. On the fourth day, the complete culture medium was replaced once, and 5 mL of complete culture medium was added to continue primary culture. Figures 1 to 2 As shown, adherent cells migrated from the frontal bone tissue of the saddle-banded grouper as early as day 5 after the initiation of primary culture. Figure 1 By day 9, the cell count had increased to 70-80%. Figure 2 ).

[0067] (3) When the coverage of the primary culture adherent cells of the Epinephelus lanceolatus frontal bone cell line reaches 80-90% or higher, the primary cells may exhibit obvious contact inhibition, at which time the passaging culture is initiated by trypsin digestion. The classic cell digestion procedure is as follows: remove the old culture solution in the original culture bottle; add 5 mL of PBS solution and wash once to remove the serum and divalent metal ions in the original culture solution (such substances can affect the digestion of trypsin); pour out the PBS solution, add 0.5 mL of 0.25% commercial trypsin digestion solution containing EDTA, shake the bottle to allow the trypsin solution to fully contact the bottom cells, and pour out the trypsin digestion solution (this step should be performed quickly, otherwise it is easy to cause excessive digestion; when removing the trypsin digestion solution, enough solution should be left to keep the flat bottom wet, otherwise the bottom may be partially dried, affecting digestion); remove the culture bottle and observe under an inverted microscope until most of the cells are rounded and detached from the bottom, then immediately transfer to a clean bench and add 5 mL of complete culture solution containing serum to terminate digestion; blow and check the blowing effect under a microscope; if there are too many residual cells, repeat the digestion once; inoculate the cell suspension into a new culture bottle at a ratio of 1:2, add complete culture solution to 5 mL, and then place the culture bottle in a constant temperature incubator at 27°C; passaging is performed every 2-3 days thereafter. As shown in FIG. 1, the Epinephelus lanceolatus frontal bone cell line stably presents a long spindle shape from the 1st generation to the 40th generation. Figures 3 to 11

[0068] The above experiments prove that the cell line culture method established by the present application is stable and reliable, and can be applied in the large-scale preparation of the Epinephelus lanceolatus frontal bone osteoblast cell line.

[0069] Example 2 Freezing and recovery of the Epinephelus lanceolatus frontal bone osteoblast cell line

[0070] (1) Freezing: take a 75 cm 2 culture bottle of the Epinephelus lanceolatus frontal bone osteoblast cell line with vigorous growth and full coverage of the bottom of the culture bottle, use trypsin digestion method, centrifuge to collect the cell precipitate, slowly add 3 mL of prepared cell freezing solution, and gently blow the cells to uniformly disperse in the cell freezing solution, and use a pipette to transfer the liquid into a freezing tube. The freezing tube is placed at 4°C for 1 h, then placed in a program gradient cooling freezing box (temperature reduction speed is 1°C / min), the freezing box is placed at -80°C for 1 day, and finally the freezing tube is taken out and immersed in liquid nitrogen for long-term freezing.

[0071] (2) Take the freezing tube out of the liquid nitrogen and quickly place it in a water bath pot with adjusted temperature (40°C), constantly shake the freezing tube during the process of thawing the cells to make them thaw quickly and uniformly until completely thawed. Transfer the thawed cell suspension to a 25 cm 2 ​In the culture bottle centrifuge tube, 5 mL of complete culture solution is added to the culture bottle, and the culture is carried out at 27 DEG C, 5% CO2. After 24 h, new complete culture solution is replaced, and the culture is continued. As shown in Table 1, the adhesion rate of the Epinephelus lanceolatus frontal bone osteoblast cell line after cryopreservation and recovery for 24 h reaches 60%-80%, and there is no obvious difference in cell morphology before and after cryopreservation. The recovered Epinephelus lanceolatus frontal bone osteoblast cells can be normally subcultured. Figure 12

[0072] The above experiments prove that the cell cryopreservation and recovery method of the application has excellent effect, and can be applied in the long-term preservation and subsequent recovery use of the Epinephelus lanceolatus frontal bone osteoblast cell line.

[0073] Example 3: Species identification verification of the Epinephelus lanceolatus frontal bone cell line

[0074] The Epinephelus lanceolatus frontal bone cell line is identified by using an RT-PCR technique, En1b as shown in Table 2, the expression of the gene in the frontal bone tissue and the frontal bone cells is high, after gel purification, the sequencing sequence is compared with the sequence of the Epinephelus lanceolatus Figure 13 The comparison result of the sequence of the gene is more than 99%. En1b En1b

[0075] Example 4: Chromosome analysis of the Epinephelus lanceolatus frontal bone cell line

[0076] The Epinephelus lanceolatus frontal bone cells with vigorous division (30 generations, the cell state is good when the cell growth is 48 h, and the cell basically adheres to the bottle bottom) are taken, inoculated into 75 cm 2 cell culture bottles, when the cell growth is in the logarithmic phase, 20 μg / mL of colchicine is added, the cells are collected after being cultured for 6-8 h to obtain a cell suspension. The cell suspension is moved to a 15 mL centrifuge tube, centrifuged at 1,000 g for 10 min, the supernatant is gently sucked out, 4 mL of 0.075 M KCl is added for low penetration for 30 min; 0.5 mL of freshly prepared pre-cooled Carnoy's fixing solution is added for pre-fixing for 10 min, and centrifuged at 2,000 g for 10 min; the cell precipitate is taken, 0.5 mL of Carnoy's fixing solution is added to resuspend the precipitate by using a pipette; 1 mL of fixing solution is added; the glass slide (pre-treated at -20 DEG C) is dropped at a height of 30 cm, and placed horizontally to make it completely spread, and dried at 65 DEG C; then, the glass slide is immersed in a staining tank containing Giemsa staining solution working solution for staining for 10 min, washed with double-distilled water to remove the slag on the surface of the glass slide, dried, and sealed with neutral resin, and observed and counted under a 1000x oil immersion lens.​​​

[0077] As shown in Figure 14 chromosome analysis of the cell line of the frontal bone of Epinephelus lanceolatus, 72% of the observed mitotic phase cells had 48 chromosomes.

[0078] The above experiments prove that the cell line established by the application has stable karyotype and can be applied in the study of cell genetics of Epinephelus lanceolatus and the analysis model of the mechanism of cranial deformity related karyotype.

[0079] Example 5 Alkaline phosphatase staining analysis of the cell line of the frontal bone of Epinephelus lanceolatus

[0080] Take the mitotically active frontal bone cells of Epinephelus lanceolatus (30 generations, the cells are in good condition when the cell growth is 48 h, and the cells are basically attached to the bottom of the bottle), and culture the frontal bone cell line of Epinephelus lanceolatus to an appropriate density (when the cells are expanded to 80%, the subsequent experiments are carried out), discard the culture medium, and gently wash twice with pre-cooled PBS. Add 4% PFA and fix at room temperature for 15-20 min. Wash with PBS for 3 times, 5 min each time. Add 0.1% Triton X-100 (prepared with PBS), and incubate at room temperature for 10 min. Wash with PBS for 2 times, 5 min each time. Prepare BCIP / NBT staining solution according to the instructions (operate in the dark). Uniformly cover the cells with the staining solution, and place them in a light-proof box, and incubate at 37°C or room temperature for 30-60 min (observe the color development under a microscope). After color development (blue-purple precipitate appears), terminate the reaction with PBS or deionized water, and wash for 3 times. Figure 15 As shown in

[0081] Example 6 Transfection efficiency of egfp transfection analysis of the cell line of the frontal bone of Epinephelus lanceolatus

[0082] Vigorously dividing frontal bone cells from *Siniperca saddleback* (passage 30, cells in good condition and largely adhering to the bottom of the flask at 48 h) were used for transfection experiments using a BEX™ CUY21 EDIT II electroporator. The pEGFP-N1 plasmid was electroporated into the *Siniperca saddleback* frontal bone cell line. The culture medium was changed after 24 h, and cell status was observed after 48 h. Cells were gently washed twice with pre-cooled PBS, and fixed with 4% PFA at room temperature for 10 min. They were then washed three times with PBS for 3 min each time. 0.1% Triton X-100 (prepared with PBS) was added, and the cells were incubated at room temperature for 10 min. They were then washed twice with PBS for 2 min each time. DAPI staining solution was prepared according to the manufacturer's instructions (protected from light), and the staining solution was evenly applied to the cells. The cells were incubated in a light-protected container at room temperature for 15 min for nuclear staining. Cells were washed three times with PBS for 1 min each time. The expression of green fluorescence in the cells was observed and photographed under an inverted fluorescence microscope. Figure 16 As shown, 48 h after transfection, the cell nuclei stained blue, and the pEGFP-N1 plasmid expressed green fluorescence in the cell line, with a transfection efficiency of approximately 75%. This indicates that the established frontal bone cell line of *S. saddleback* can adapt to transfection experiments of exogenous genes. The above experiments demonstrate that the cell line established in this invention has high transfection efficiency and can be applied in the functional verification of exogenous genes in *S. saddleback* and in the research model of gene regulation mechanisms related to skull malformations.

[0083] Example 7: Dual-luciferase assay of frontal bone cell line from grouper with saddle band.

[0084] The plasmids used for transfection in this experiment were the target plasmid (PGL3-El.en1b-basic, pCDNA3.1-El.En1b, pCDNA3.1-Ca.En1b), the internal control plasmid (TK), the negative control (PGL3-basic), and the positive control (PGL3-El.en1b-basic). Transfection was performed when cells were in good growth condition after passage and reached 70%–80% confluency. After cell digestion, the mixture was not discarded; an appropriate amount of fresh cell culture medium was added directly. After repeated pipetting, the cells were seeded into 48-well culture plates and placed back in an incubator. After 24 hours, microscopic observation showed that if cells adhered to the well and the density reached 70%–80%, transfection was performed. Lipofectamine 3000 was used as the transfection reagent, and the ratio of internal control plasmid to target plasmid was 1:20. The specific procedures are as follows:

[0085] Before transfection, the concentration of the transfected plasmid must be determined, and... En1bThe promoter plasmid and en1b overexpression plasmid were diluted to approximately 200 ng / μL, and the internal control plasmid was diluted to 50 ng / μL. 1 μg (5 μL) of plasmid was added to 25 μL of Opti-MEM medium, mixed well, and incubated at room temperature for 5 min. Simultaneously, 1 μL of Lipo3000 transfection reagent was added to 25 μL of Opti-MEM medium in a new 1.5 mL EP tube, mixed by pipetting, and incubated at room temperature for 5 min. After 5 min, the plasmid and Opti-MEM medium mixture was added dropwise to the transfection reagent and Opti-MEM medium mixture, mixed by pipetting, and incubated at room temperature for 20 min. During this time, the old cell culture medium was aspirated from the culture plate, and 200 μL of Opti-MEM medium was slowly added. The mixture was carefully suspended and added dropwise to the culture plate, 50 μL per well, and the bottom of the plate was gently tapped to mix the solution thoroughly. After transfection, the 48-well plate was returned to the incubator for 24 hours of incubation. h, then cells can be collected for activity assays. Dual-luciferase activity was measured using the Promega Dual-Luciferase® Reporter Assay System kit and the Berthold LB9508 tube chemiluminescence analyzer. The activities of firefly luciferase and Renilla luciferase were recorded separately; the ratio of their activities represents the relative activity of the plasmid dual-luciferase reporter gene. For example... Figure 17 As shown, it can be seen that overexpression of pCDNA3.1-El.En1b... En1b The promoter activity was not significantly different from that of the positive control group (p>0.05), indicating that the established frontal bone cell line of the saddle-banded grouper can adapt to overexpression experiments and experiments such as promoter interactions.

[0086] The above experiments demonstrate that the cell line established in this invention can be used to detect osteogenic genes (such as those in grouper) in saddle-banded grouper. En1b It is applied in promoter activity analysis, gene interaction and functional verification models of genes, providing key technical tools for elucidating the causes of skull deformities.

[0087] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A cell line of osteoblasts derived from the frontal bone of Epinephelus lanceolatus, characterized in that, It is named as the forehead bone tissue osteoblast cell line ELFBOB of Epinephelus lanceolatus, which has been preserved in China Center for Type Culture Collection on May 17, 2025, and the preservation number is CCTCC NO: C2025132.

2. The method for establishing an osteoblast cell line from the frontal bone tissue of a grouper with a saddle band according to claim 1, characterized in that, The method comprises the following steps: (1) Tissue acquisition: the fry of Epinephelus lanceolatus is placed on ice until no stress reaction, and then the forehead bone tissue is taken out after disinfection and placed in PBS liquid containing penicillin, streptomycin and amphotericin B; 1 mL of 100x commercial three-antibody concentrate is added to 100 mL of the PBS liquid; (2) Primary culture: the forehead bone tissue is cut into about 2.0 mm, washed with PBS, treated with red blood cell lysis solution, and then dried and pasted into a culture bottle, complete culture solution is added, and primary culture is started at 27 DEG C and 5% CO2, and the culture solution is gradually supplemented and replaced; (3) Subculture: when the primary cells grow to 80%-90% coverage, PBS is used for cleaning, 0.25% trypsin is used for digestion, and the culture solution is used for stopping digestion, then subculture is carried out at a ratio of 1:2, 27 DEG C culture is carried out, subculture is carried out every 2-3 days, and culture is carried out to 210 days and 40 generations; (4) Freezing and recovery: logarithmic phase cells are taken, digested and centrifuged, serum-free freezing solution is added, placed at 4 DEG C for 1 h, programmed gradient cooling (1 DEG C / min) to-80 DEG C for 1 day, and finally stored in liquid nitrogen; when recovered, rapid thawing is carried out in a 40 DEG C water bath, and then cultured at 27 DEG C and 5% CO2 after inoculation, and the culture solution is replaced after 24 h.

3. The method for establishing an osteoblast cell line from the frontal bone tissue of a grouper with a saddle band according to claim 2, characterized in that, In step (2), the complete culture solution is based on L-15, and 15vol%-20vol% fetal bovine serum, 0.5vol‰ beta-mercaptoethanol, 10 ug / L human basic fibroblast growth factor, 5 ug / L human epithelial cell growth factor, 1 ug / L human hepatocyte growth factor, 1 ug / L human leukemia inhibitory factor, 100 IU / mL penicillin, 100 ug / mL streptomycin and 1 vol% grouper serum are added.

4. The method for establishing an osteoblast cell line from the frontal bone tissue of a grouper with a saddle band according to claim 3, characterized in that, The preparation method of the grouper serum is as follows: a syringe is soaked in 15% (w / v) EDTA, blood is taken from the tail vein of the grouper, the supernatant is obtained by centrifugation at 3500 g for 15 min, the supernatant is incubated at 4 DEG C overnight, then centrifuged at 3500 g for 30 min, the supernatant is filtered through a 0.2 um filter to remove bacteria, and then stored at-20 DEG C.

5. The forehead bone tissue osteoblast cell line of Epinephelus lanceolatus according to claim 1 is applied in the analysis model of cell genetics and the mechanism of the related karyotype of cranial deformity of Epinephelus lanceolatus.

6. The forehead bone tissue osteoblast cell line of Epinephelus lanceolatus according to claim 1 is applied in the analysis model of the osteogenic mechanism and the related osteogenic function of cranial deformity of Epinephelus lanceolatus.

7. The forehead bone tissue osteoblast cell line of Epinephelus lanceolatus according to claim 1 is applied in the cell model for studying the differentiation mechanism of osteoblasts, the cytological basis of cranial development and the regulation mechanism of deformity of Epinephelus lanceolatus.

8. The forehead bone tissue osteoblast cell line of Epinephelus lanceolatus according to claim 1 is applied in the analysis model of the promoter activity of the related gene of osteogenesis, gene interaction and function verification of Epinephelus lanceolatus.

9. Use of the cell line of osteoblasts of the frontal bone of Epinephelus lanceolatus according to claim 1 in a cellular model for the species traceability verification of cell lines of Epinephelus lanceolatus origin.

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