Method for establishing blunt-snout bream caudal fin-induced pluripotent stem cell line and application thereof

Abstract

The present invention discloses a method for establishing and applying a caudal fin-induced pluripotent stem cell line of Erythroculter ilishaeformis. By the tissue adherence method, a cell line with high proliferation performance and stemness is isolated from the caudal fin of Erythroculter ilishaeformis. In the early stage, the cells of this cell line are significantly fibroblast-like. Later, the cells become smaller but still maintain a spindle shape, without obvious contact inhibition phenomenon, and can spontaneously form cell clusters similar to induced pluripotent stem cells. Alkaline phosphatase staining is positive and it expresses stemness marker genes such as Oct4, Sox2, Klf4, Nanog, and c-Myc. The present invention uses a single cAMP agonist small molecule compound to reverse the phenomenon of weakened stemness after continuous subculture of this cell line, enabling it to maintain the characteristics of high proliferation performance and high stemness, which is suitable for future research on the induction and differentiation of fish stem cells and provides more choices for the research and application of fish stem cells.

Description

Technical Field

[0001] The invention belongs to the technical field of fish cell biology, and in particular relates to a method for establishing an induced pluripotent stem cell line of the caudal fin of Erythroculter ilishaeformis and an application thereof. Background Art

[0002] Topmouth culter, formerly known as red croaker, belongs to the class of bony fish, order of Cypriniformes, family of Cyprinidae, subfamily of Cteninae, genus of Cteninae. It is also known as striped fish, big white fish, topmouth, and topshell. It is an important economic fish widely distributed in the main water bodies of the middle and lower reaches of the Yangtze River in China. It is large in size, grows fast, has tender and delicious meat, and is rich in nutrition. It is one of the "three treasures" of Taihu Lake. It is known as one of the four famous fishes in China together with Songjiang perch, Yellow River carp, and Songhua River salmon. It is one of the main economic fishes of Taihu Lake and is deeply favored by consumers. On December 12, 2007, it was included in the "List of Key Economic Aquatic Animal and Plant Resources Protected by the State of China (First Batch)". Its artificial breeding technology has been widely promoted, forming an industry worth tens of billions of yuan.

[0003] Induced pluripotent stem cells (iPSCs) are a type of cell that regains stem cell characteristics from mature cells through genetic reprogramming technology. They have abilities similar to embryonic stem cells, can self-renew and have the potential to differentiate into various different types of cells. The history of induced pluripotent stem cells (iPSCs) can be traced back to 2006, when Japanese scientist Shinya Yamanaka and his team successfully transformed mature skin cells into omnipotent stem cells for the first time through genetic reprogramming technology by transfecting a set of specific transcription factors (Oct4, Sox2, Klf4, c-Myc). Yamanaka's research results have created a new field of stem cell research, providing important tools and resources for stem cell therapy, disease modeling, drug screening and other fields. Since then, research on iPSCs has received widespread attention and continued development, constantly promoting progress in the field of stem cells and regenerative medicine.

[0004] Chemical reprogramming is a method of cell reprogramming using small molecule compounds to replace traditional transcription factors. In 2009, a Japanese research team first reported a chemical that can replace transcription factors to induce cell reprogramming to generate pluripotent stem cells. This study showed that a chemical called BIX-01294 can promote cell demethylation, thereby achieving cell reprogramming. Subsequently, researchers have discovered more chemicals that can replace traditional transcription factors to induce the generation of pluripotent stem cells. These chemicals involve mechanisms such as regulating cell signaling pathways and changing epigenetic modifications. By optimizing the combination and use conditions of these chemicals, researchers have gradually achieved efficient conversion of mature cells into totipotent stem cells. The advantage of chemical reprogramming is that it can avoid the problems of gene mutations and insertion mutations that may be caused by the introduction of transcription factors using genetic engineering technology, and it can also improve the efficiency and safety of cell reprogramming. By screening and optimizing the combination of chemicals, it is possible to efficiently convert mature cells into iPSCs. The development of chemically reprogrammed induced pluripotent stem cells provides new possibilities for stem cell research and is expected to further promote the application of stem cells in fields such as regenerative medicine and disease treatment. With the continuous advancement and optimization of technology, the method of chemically reprogramming induced pluripotent stem cells is expected to become a safer and more efficient cell reprogramming strategy.

[0005] In terms of efficiency, chemical reprogramming has the following defects: (1) Low efficiency: Chemical reprogramming usually requires high concentrations of chemicals or a combination of multiple chemicals to achieve cell reprogramming, resulting in low efficiency of the entire process. This means that more time and resources are required to complete cell reprogramming, and it also increases cell instability. (2) Cell heterogeneity: Due to the low efficiency of chemical reprogramming, different cells may have different responses to chemicals, resulting in heterogeneity in the cell population. This heterogeneity may affect the subsequent application of cells and the reliability of research results. (3) Multi-step process: Chemical reprogramming usually requires multiple steps and complex operation procedures, including optimization of chemicals and adjustment of cell culture conditions. This increases the complexity and uncertainty of the experiment and also increases the difficulty of experimental operation. (4) Difficult to standardize: Since the effect of chemical reprogramming is affected by many factors, including cell type, choice and concentration of chemicals, it is difficult to establish a universal standardized scheme to achieve efficient cell reprogramming. In general, chemical reprogramming has some challenges and defects in terms of efficiency, and further research and technical improvements are needed to improve its efficiency and achieve wider application. Summary of the invention

[0006] Aiming at the deficiencies of the prior art, the technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art, and provide a topmouth culter caudal fin cell line that can be simply induced back to a highly stem cell state by a single small molecule compound and its construction method.

[0007] To solve the above technical problems, the technical solution proposed by the present invention is as follows:

[0008] A method for establishing an induced pluripotent stem cell line from topmouth culter caudal fin, comprising the following steps:

[0009] (1) After cutting a small piece of fin ray from the topmouth culter caudal fin, disinfect and rinse it, and then cut it into small pieces of caudal fin tissue.

[0010] (2) Digest the small pieces of caudal fin tissue obtained in step (1) with trypsin for 10 minutes. After attaching them to a culture dish, perform primary culture in a primary culture medium.

[0011] (3) When the cells obtained in step (2) grow to a monolayer, add trypsin for digestion for 3 - 5 minutes. After terminating the digestion, perform subculture using a subculture growth medium at a ratio of one well to three wells.

[0012] (4) In the subcultured caudal fin cell line, add a cAMP agonist to induce it into induced pluripotent stem cells and maintain its stem cell state.

[0013] Further, the disinfection in step (1) is specifically to soak in a 1:10000 potassium permanganate solution for half an hour, disinfect with 75% alcohol for 5 - 15 seconds, and repeatedly rinse with D-hanks buffer containing 2% (v / v) penicillin-streptomycin, and then cut it into caudal fin small tissue pieces of 0.5 - 1 mm2.

[0014] Specifically, attaching to the culture dish in step (1) is specifically to attach the tissue blocks to a culture dish coated with L-type polylysine solution, and place it in an incubator for inverted culture for 30 minutes.

[0015] Specifically, the trypsin in step (2) is 0.25% (w / v) trypsin-EDTA digestive solution.

[0016] Specifically, the primary culture medium in step (2) is DMEM / F12 medium containing 15% - 20% (v / v) fetal bovine serum, 5 - 40 ng / ml bFGF, 200 U / ml penicillin, and 200 μg / ml streptomycin.

[0017] Specifically, the subculture medium in step (3) is DMEM / F12 medium containing 10% - 20% (v / v) fetal bovine serum, 5 - 40 ng / ml bFGF, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0018] Specifically, the concentration range of the cAMP agonist in step (4) is 0 - 20 μM.

[0019] Specifically, the temperature range of the incubator for primary culture and subculture is 25°C - 28°C, and the volume concentration range of carbon dioxide is 3% - 7%.

[0020] The second aspect of the present invention: The application of an E. ilishaeformis caudal fin-induced pluripotent stem cell line in fish stem cell lines.

[0021] The technical solutions provided by the embodiments of the present application may include the following beneficial effects:

[0022] The induction method of the present invention uses an induction medium containing only a single small molecule compound for induction, without the need to introduce foreign genes, improving the safety of future applications. Induction with a single small molecule compound eliminates the need for a combination of small molecule compounds and multi-step long-term induction and differentiation. It can significantly increase the expression level of stemness marker genes in as little as 1 - 3 days. The induction process is flexible and controllable, reducing the induction period and improving the induction efficiency. Moreover, the induction method is highly operable and repeatable, and can stably induce pluripotent stem cells, thus being suitable for the induction application of future fish pluripotent stem cells. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 Cell morphology diagrams of the 6th day of primary culture, the 3rd passage, and the 20th passage of the present invention; Scale bar: 80 μM

[0025] Figure 2 Cell growth curve diagram of the 20th passage cells drawn by the CCK8 method of the present invention;

[0026] Figure 3 Karyotype analysis diagram of the 20th passage cells of the present invention;

[0027] Figure 4 Diagram of embryoid body cell clusters spontaneously formed by the 10th passage cells of the present invention;

[0028] Figure 5 Alkaline phosphatase staining diagrams of the 1st, 3rd, 5th, and 7th days after the 10th passage cells of the present invention are cultured to a monolayer; Scale bar: 80 μM;

[0029] Figure 6 RT-PCR diagram of the stem cell marker genes of the 10th passage cells of the present invention;

[0030] Figure 7 Alkaline phosphatase staining diagram of the 20th passage cells of the present invention after being treated with 5 μM F (forskolin) for 3 days;

[0031] Figure 8 Diagram of the change in the relative expression levels of the stem cell marker genes detected by qRT-PCR after the 30th passage cells of the present invention were treated with 5 μM F (forskolin), 5 μM cAMP (8-Bromo-cAMP sodium salt), and 5 μM Lysipressin for 3 days. Detailed implementation manners

[0032] Here, the exemplary embodiments will be described in detail, and the examples are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the present application.

[0033] The terms used in the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "the", and "said" used in the present application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0034] It should be understood that although the terms first, second, third, etc. may be used in the present application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "when" or "while" or "in response to determining".

[0035] In order to more clearly and clearly elaborate the technical solutions and advantages of the present invention, the present invention will be further described in detail with examples in combination with the drawings. Note that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or can be prepared by existing methods.

[0036] Example:

[0037] A method for establishing and applying a topmouth culter caudal fin-induced pluripotent stem cell line, including the construction of a topmouth culter caudal fin cell line and the induction of pluripotent stem cells, comprising the following steps:

[0038] Primary culture of caudal fin cells: Soak the cut caudal fin tissue in a potassium permanganate solution with a concentration of 1:10,000 for 30 minutes, and then rinse it 5 times with D-Hanks buffer containing 2×PS (penicillin-streptomycin double antibody) to remove excess potassium permanganate. Next, soak the caudal fin tissue in 75% alcohol for 15 seconds for disinfection, and then rinse it 5 times with D-Hanks buffer again to remove mucus and epithelium. Finally, cut the caudal fin into 1-mm tissue blocks and attach them to a six-well plate coated with L-type polylysine solution using the tissue adherence method. To promote the adherent growth of the tissue, first invert and dry it for 30 minutes, then digest it with 0.5 mL of 0.25% trypsin-EDTA digestive solution for 10 minutes; then terminate the digestion with 0.5 mL of DMEM / F12 containing 1×PS and 10% fetal bovine serum; finally, add 2 mL of DMEM / F12 culture medium containing 20% fetal bovine serum, 10 ng / ml bFGF, and 1×PS, and place it in a 5% CO2 incubator for culture at 27°C. The results of the primary culture are shown in Figure 1 .

[0039] Subculture of caudal fin cells: When the primary cells migrate out and reach 90% confluence, aspirate the culture medium, add 1 mL of D-Hanks and rinse 2 times, then digest with 0.5 mL of digestive solution containing 0.25% trypsin-EDTA, add 0.5 mL of DMEM / F12 medium (subculture medium) containing 10% fetal bovine serum, 10 ng / ml bfgf, and 1×PS to terminate the digestion. After pipetting down the cells, transfer them to a 1.5-mL centrifuge tube, centrifuge at 300 g for 3 minutes, remove the supernatant, and subculture them into the subculture medium at a ratio of one to three, and place it in a 5% CO2 incubator for culture at 27°C. The results of the subculture are shown in Figure 1 , and the spontaneous aggregation phenomenon of the 10th-generation cells is shown in Figure 4 .

[0040] Cryopreservation of caudal fin cells: Suspend the cells to be cryopreserved with 1 mL of DMEM / F12 medium containing 20% FBS and 5% DMSO, place them in an -80°C refrigerator for programmed cooling, and then transfer them to liquid nitrogen for long-term storage.

[0041] Recovery of cryopreserved cells: Place the cryopreserved cells in a 37°C water bath to thaw until only rice grain-sized ice cubes remain, add 5 volumes of subculture medium, centrifuge at 300 g for 3 minutes, remove the supernatant, and subculture them into the subculture medium at a ratio of one to one.

[0042] Drawing the growth curve by CCK8 method: Inoculate 4000 topmouth culter caudal fin cells per well into a 96-well plate, culture at 27°C with 5% CO2. After 1 hour of inoculation and when the cells adhere to the wall, remove the culture medium, add 100 μL of DMEM / F12 medium containing 10% hypersensitive cell proliferation detection reagent (CCK-8) and treat for 4 hours. Detect the absorbance at 450 nm, and then use CCK8 to detect once every 24 hours. The actual absorbance = absorbance of the experimental group - absorbance of the blank control group. The experimental results are shown in Figure 2 。

[0043] Karyotype analysis: Inoculate 10^6 cells into a T25 cell culture dish. When the cell confluence reaches 80%, add 5 mL of passage medium containing 1 μg / ml colchicine. After incubating at 27°C for 5 hours, remove the culture medium, add 1 mL of 0.25% trypsin-EDTA digestive solution and digest for 5 minutes. After digestion is completed, add 1 mL of passage medium to terminate digestion. Pipette to blow down the cells, transfer them to a 15 mL centrifuge tube, centrifuge at 300 g for 3 minutes, remove the supernatant, add 1 mL of D-PBS and wash once. After centrifuging again to remove the supernatant, add 4 mL of 0.075 M KCl and treat for 20 minutes. Add 1 mL of Carnoy's fixative (ethanol: acetic acid = 3:1) and pre-fix for 5 minutes. Centrifuge at 500 g for 5 minutes to remove the supernatant, add 1 mL of Carnoy's fixative, fix for 30 minutes, then gently pipette to resuspend. Centrifuge at 500 g for 5 minutes to remove the supernatant, repeat the fixation once. After centrifugation, remove 800 μL of the fixative, resuspend the cells with the remaining 200 μL of the fixative, use the cold dropping method to drop the cells onto the slide, place it at 37°C to dry for 30 minutes, stain with 5% Giemsa stain for 30 minutes, wash away the excess stain with running water and then seal the slide with neutral resin in water. Observe under a 100× oil immersion objective. The experimental results are shown in Figure 3 。

[0044] Alkaline phosphatase staining: Inoculate 2×10^5 cells into a 12-well plate, add 1 mL of passage medium and culture in an incubator at 27°C with 5% CO2. After the cell confluence reaches 90%, continue to culture for 1, 3, 5, and 7 days respectively. Remove the culture medium, add 1 mL of PBS and rinse twice, then add 0.5 mL of 4% PFA and fix for 30 minutes. Remove the 4% PFA, add 1 mL of PBS and rinse three times, then use an alkaline phosphatase staining kit (red) to stain for 1 hour. After removing the stain, add 1 mL of PBS and rinse three times, and take pictures using an inverted phase contrast microscope. The experimental results are shown in Figure 5 。

[0045] Induction of pluripotent stem cells from caudal fin cells 1: Inoculate 5×10^5 cells into a 6-well plate. The control group adds 2 mL of passage medium, and the experimental group adds 5 μM forskolin on the basis of the passage medium. Replace half of the medium every 2 days. The images of alkaline phosphatase staining after induction are shown inFigure 7 , where CK is the control group, F is the experimental group (treated with 5 μM forskolin for 3 days), scale bar: 80 μM; fluorescence quantitative PCR was performed after 3 days of induction, and the qRT-PCR results are shown in Figure 8 .

[0046] Induction of pluripotent stem cells in caudal fin cells 2: 5×10^5 cells were seeded in a 6-well plate. The control group was added with 2 mL of passage medium, and the experimental group was added with 5 μM cAMP (8-Bromo-cAMP sodium salt) on the basis of the passage medium. Half of the medium was changed every 2 days, and fluorescence quantitative PCR was performed after 3 days of induction. The qRT-PCR results are shown in Figure 8 .

[0047] Induction of pluripotent stem cells in caudal fin cells 3: 5×10^5 cells were seeded in a 6-well plate. The control group was added with 2 mL of passage medium, and the experimental group was added with 5 μM Lysipressin on the basis of the passage medium. Half of the medium was changed every 2 days, and fluorescence quantitative PCR was performed after 3 days of induction. The qRT-PCR results are shown in Figure 8 .

[0048] Maintenance of stemness of caudal fin cells: The cells were cultured and passaged using DMEM / F12 medium (stemness maintenance medium) containing 10% fetal bovine serum, 10 ng / ml bfgf, 0.5 μM forskolin, and 1×PS, which can maintain the stemness and high proliferation performance of the cell line.

[0049] RNA extraction: After 3 days of culture of the experimental group and the control group, the medium was removed, 1 mL of PBS was added for rinsing once, 1 mL of RNAiso plus was added to lyse the cells. After standing for 10 minutes, the RNAiso plus was collected into a 1 mL centrifuge tube, 200 μl of chloroform was added, and it was shaken vigorously for 15 seconds and then left standing at room temperature for 10 minutes. Centrifugation was performed at 12000 g at 4°C for 10 minutes. The supernatant was taken, an equal volume of isopropanol was added, and it was left standing at room temperature for 10 minutes. Centrifugation was performed at 12000 g at 4°C for 10 minutes. The supernatant was removed, rinsed once with 75% alcohol, centrifuged at 12000 g at 4°C for 5 minutes, the alcohol was removed and dried for 5 minutes, and the RNA was dissolved with DEPC water;

[0050] RT-PCR: Using PrimeScript TM II 1st Strand cDNA Synthesis Kit to reverse transcribe RNA into the first strand of cDNA, using PrimeSTAR The target fragment was amplified by HSDNA Polymerase, and stemness marker genes such as Sox2, Nanog, Oct4, c-Myc, Klf4, and Nestin were successfully amplified, proving that this cell line itself has a certain degree of stemness. The electrophoresis results of the amplification products are shown in Figure 6 (1: marker, 2: Sox2, 3: Nanog, 4: Oct4, 5: c-Myc, 6: Klf4, 7: Nestin), and the RT-PCR primers are shown in Table 9;

[0051] Table 9

[0052]

[0053]

[0054] qRT-PCR: The first-strand cDNA was synthesized according to the method in RT-PCR. qRT-PCR was performed using CFX ConnectTM Optics Module (BIO-RAD) and TB green, with β-actin as the internal reference, and the relative expression levels of each gene were calculated by the 2 -△△CT -method. The relative expression levels of Sox2, Nanog, Oct4, and Klf4 were successfully calculated, as shown in Figure 8 , and the vertical coordinate (Foldchange) represents the fold change in differential expression; Figure 8 In it, CK is the control group, and the experimental groups are 5 μM F (forskolin), 5 μM cAMP (8-bromo-cAMP sodium salt), and 5 μM Lysipressin. Standard deviation, ns P >= 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, and the qRT-PCR primers are shown in Table 9.

[0055] In summary, the results show that the blunt snout bream caudal fin cell construction method and induction method described in the present invention can successfully induce the acquisition of blunt snout bream induced pluripotent stem cells. A stable induced pluripotent stem cell line has been obtained through the identification of pluripotency, which has important scientific value and provides good materials for the further research of fish induced pluripotent stem cells.

[0056] Each component involved in the culture medium used in the present invention was purchased from:

[0057] 100× penicillin-streptomycin double antibody was purchased from Macklin, product number P917928;

[0058] D-Hanks buffer was purchased from Meilunbio, product number MA0039;

[0059] The L-type polylysine solution was purchased from Sangon, product number E607015;

[0060] The 0.25% trypsin-EDTA digestion solution was purchased from Cienry, product number CR25200;

[0061] Fetal bovine serum was purchased from Gibco, product number 10099141C;

[0062] DMEM / F12 was purchased from Gibco, product number C11330500BT;

[0063] bFGF was purchased from Beyotime, product number P5453;

[0064] The hypersensitive cell proliferation detection reagent (CCK-8) was purchased from Abbkine, product number BMU106;

[0065] Colchicine was purchased from MACKLIN, product number C11856468;

[0066] The alkaline phosphatase staining kit (red) was purchased from Applygen, product number E1041-50;

[0067] Forskolin was purchased from Targetmol, product number T2939;

[0068] Lysipressin was purchased from MCE, product number HY-P0004;

[0069] 8-Bromo-cAMP sodium salt was purchased from Targetmol, product number T6747;

[0070] RNAiso plus was purchased from Takara, product number D9108A;

[0071] PrimeScript TM The PrimeScript II 1st Strand cDNA Synthesis Kit was purchased from TaKaRa, product number 6210A;

[0072] The HS DNA Polymerase was purchased from TaKaRa, product number DR044A;

[0073] TB green was purchased from TaKaRa, product number RR420A.

[0074] Other embodiments of the present application will be readily contemplated by those skilled in the art upon consideration of the specification and practice of the disclosure herein. The present application is intended to cover any variations, uses, or adaptations of the present application, which follow the general principles of the present application and include known common general knowledge or conventional technical means in the technical field not disclosed in the present application.

[0075] It should be understood that the present application is not limited to the exact structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for establishing an induced pluripotent stem cell line from the caudal fin of Erythroculter ilishaeformis, characterized in that: The following steps are involved: (1) Cut off small pieces of fin rays from the caudal fin of the red bream, disinfect and rinse them, and then cut them into small pieces of caudal fin tissue; (2) digesting the caudal fin tissue piece obtained in step (1) with trypsin for 10 minutes, attaching it to a culture dish, and then performing primary culture in primary culture medium; (3) After the cells obtained in step (2) have grown to a monolayer, trypsin is added for digestion for 3-5 minutes. After digestion is terminated, subculture is performed using subculture growth medium at a ratio of one well to three wells; (4) Adding cAMP agonist to the caudal fin cell line after subculture to induce it into induced pluripotent stem cells and maintain its stem state; the induction of induced pluripotent stem cells of the caudal fin cells is specifically as follows: 5×10 5 The cells were seeded in a 6-well plate, 5 μM forskolin was added to the culture medium, half of the culture medium was replaced every 2 days, and the induced pluripotent stem cells of the tail fin cells were obtained after 3 days of induction; the maintenance of the stemness state was specifically carried out by culturing and passage using a DMEM / F12 culture medium containing 10% fetal bovine serum, 10 ng / ml bFGF, 0.5 μM forskolin, and 1×PS to maintain the stemness and high proliferation performance of the cells.

2. The method for establishing the caudal fin induced pluripotent stem cell line of Erythroculter ilishaeformis according to claim 1, characterized in that: The disinfection in step (1) is specifically to soak in a 1:10000 potassium permanganate solution for half an hour, disinfect with 75% alcohol for 5-15 seconds, rinse repeatedly with D-hanks buffer containing 2% by volume of blue chain bispecific antibody, and cut into 0.5-1 mm 3 A small piece of tissue from the tail fin.

3. The method for establishing the caudal fin induced pluripotent stem cell line of Erythroculter ilishaeformis according to claim 1, characterized in that: The step (2) of attaching the tissue block to a culture dish specifically involves attaching the tissue block to a culture dish coated with an L-polylysine solution and placing the dish in an incubator and inverting the dish for 30 minutes.

4. The method for establishing the caudal fin induced pluripotent stem cell line of Erythroculter ilishaeformis according to claim 1, characterized in that: The trypsin in steps (2) and (3) is 0.25% W / V trypsin-EDTA digestion solution.

5. The method for establishing the caudal fin induced pluripotent stem cell line of Erythroculter ilishaeformis according to claim 1, characterized in that: The primary culture medium in step (2) is a DMEM / F12 culture medium containing 15%-20% V / V fetal bovine serum, 5-40 ng / ml bFGF, 200 U / ml penicillin, and 200 μg / ml streptomycin.

6. The method for establishing the caudal fin induced pluripotent stem cell line of Erythroculter ilishaeformis according to claim 1, characterized in that: The subculture growth medium in step (3) is a DMEM / F12 medium containing 10%-20% V / V fetal bovine serum, 5-40 ng / ml bFGF, 100 U / ml penicillin, and 100 μg / ml streptomycin.

7. The method for establishing the caudal fin induced pluripotent stem cell line of Erythroculter ilishaeformis according to claim 1, characterized in that: The temperature range of the incubator for primary culture and subculture is 25° C.-28° C., and the volume concentration range of carbon dioxide is 3%-7%.

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