Takifugu bimaculatus testis tissue cell line and application thereof

Abstract

The invention provides a takifugu bimaculatus testis tissue cell line and application thereof, and belongs to the technical field of biology. According to the method, the testis of the takifugu bimaculata is subjected to isolated culture, primary cells of the testis are obtained, continuous subculture is carried out, and finally the stable testis cell TBTc of the takifugu bimaculata is established. The testis cell TBTc of the takifugu bimaculatus is preserved in China Center for Type Culture Collection on October 11, 2021, and the preservation number is CCTCC NO: C2021230. The cell line is excellent in cell character, mainly comprises long fibrous cells, and is vigorous in division, short in passage time, easy to digest, good in adherence rate and resistant to hunger. The takifugu bimaculatus testis cell line provides a new in-vitro model for research on a gonad development mechanism of takifugu bimaculatus.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a testicular tissue cell line of the double-spotted pufferfish and its application. Background technique

[0002] Double-spotted pufferfish ( Takifugu bimaculatus ), belonging to the order pufferfish, puffer family, genus Oriental puffer, is an offshore warm temperate bottom fish, distributed in the South my country Sea, the East China Sea and the southern part of the Yellow Sea. It is a treasure of aquatic products with high economic value and great development potential. The establishment of fish cell lines can provide great convenience for the research of fish virus isolation, anti-virus mechanism, vaccine development, transgenic technology, chimeric tissue engineering, etc., and the requirements of facilities, human resources and other resources are lower than in vivo experiments, and the repeatability is good. The research period is short. Cell lines provide impor...

Examples

Embodiment 1

[0042] A method for establishing a testicular tissue cell line (TBTc) of double-spotted pufferfish, comprising the following steps:

[0043] (1) Obtainment of testis tissue of the double-spotted pufferfish: 7 months old and 8-10 cm long juveniles of the double-spotted pufferfish were temporarily cultured in sterilized seawater containing three antibodies for 1 to 2 hours, using the addition of 1wt‰ The sterilized seawater of sodium hypochlorite was used to disinfect the fish surface for 15 minutes. At the end, drop 0.005 - 0.015% of eugenol in the volume of sterilized seawater until the fish is turned over, the abdomen is up, and there is no stress response to the stimulation, wipe the mucus on the surface of the fish with a sterilized gauze piece, and then soak it with alcohol After wiping the surface of the fish body with gauze, the fish body was moved into the ultra-clean bench, and the gonad tissue was taken out with a dissecting instrument, and a small part was placed in ...

Embodiment 2

[0073] Example 1 Cryopreservation and recovery of the testis cell line of the double-spotted pufferfish.

[0074] (1) Frozen storage: take a bottle of 75cm 2 The cells of the testicular cell line of the double-spotted pufferfish that grew vigorously and covered the bottom of the culture flask were digested with 0.25wt% trypsin. After centrifugation, the cell pellet was collected, and 3 mL of the prepared cell freezing solution was slowly added, and gently pipetted The cells are dispersed evenly in the cell cryopreservation solution, and the liquid is transferred into the cryovial using a pipette. Using a cell characterization analyzer, analyze and count the cell suspension ( Figure 4 A), adjust the cell concentration so that the number of cells per mL of cryopreservation solution is 1 × 10 6 , use a pipette to transfer the liquid into the cryovial. The cryovials were placed at 4 °C for 1 h, then placed in a programmed gradient cooling freezing box (the temperature drop ra...

Embodiment 3

[0079] The influence of the proliferation curve of different passage ratios of the testicular cell line of the double-spotted pufferfish in Example 1:

[0080] Cells with good shape and vigorous proliferation were selected from the testicular cell line established in Example 1, digested and passaged, and passaged to 25 cm at 1:2, 1:3, 1:5 and 1:10 respectively. 2 Cell culture flasks (that is, inoculate 5 × 10 5 , 3.3×10 5 , 2×10 5 , 1 × 10 5 6 cells in each group in parallel, and then make up the total volume of the solution in each flask to 5 mL with complete culture medium). From 24 h of passage, one flask of cells in each group was digested every 24 h, and the cells in each group were counted by a cell characteristic analyzer and analyzed by graph.

[0081] like Figure 5 As shown in A, for the testis cell line of the double-spotted pufferfish, different passage ratios have a great influence on the cells. Except for the 1:2 and 1:3 passage groups, the proliferation of...

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a testicular tissue cell line of the double-spotted pufferfish and its application. Background technique

[0002] Double-spotted pufferfish ( Takifugu bimaculatus ), belonging to the order pufferfish, puffer family, genus Oriental puffer, is an offshore warm temperate bottom fish, distributed in the South my country Sea, the East China Sea and the southern part of the Yellow Sea. It is a treasure of aquatic products with high economic value and great development potential. The establishment of fish cell lines can provide great convenience for the research of fish virus isolation, anti-virus mechanism, vaccine development, transgenic technology, chimeric tissue engineering, etc., and the requirements of facilities, human resources and other resources are lower than in vivo experiments, and the repeatability is good. The research period is short. Cell lines provide impor...

Examples

Embodiment 1

[0042] A method for establishing a testicular tissue cell line (TBTc) of double-spotted pufferfish, comprising the following steps:

[0043] (1) Obtainment of testis tissue of the double-spotted pufferfish: 7 months old and 8-10 cm long juveniles of the double-spotted pufferfish were temporarily cultured in sterilized seawater containing three antibodies for 1 to 2 hours, using the addition of 1wt‰ The sterilized seawater of sodium hypochlorite was used to disinfect the fish surface for 15 minutes. At the end, drop 0.005 - 0.015% of eugenol in the volume of sterilized seawater until the fish is turned over, the abdomen is up, and there is no stress response to the stimulation, wipe the mucus on the surface of the fish with a sterilized gauze piece, and then soak it with alcohol After wiping the surface of the fish body with gauze, the fish body was moved into the ultra-clean bench, and the gonad tissue was taken out with a dissecting instrument, and a small part was placed in ...

Embodiment 2

[0073] Example 1 Cryopreservation and recovery of the testis cell line of the double-spotted pufferfish.

[0074] (1) Frozen storage: take a bottle of 75cm 2 The cells of the testicular cell line of the double-spotted pufferfish that grew vigorously and covered the bottom of the culture flask were digested with 0.25wt% trypsin. After centrifugation, the cell pellet was collected, and 3 mL of the prepared cell freezing solution was slowly added, and gently pipetted The cells are dispersed evenly in the cell cryopreservation solution, and the liquid is transferred into the cryovial using a pipette. Using a cell characterization analyzer, analyze and count the cell suspension ( Figure 4 A), adjust the cell concentration so that the number of cells per mL of cryopreservation solution is 1 × 10 6 , use a pipette to transfer the liquid into the cryovial. The cryovials were placed at 4 °C for 1 h, then placed in a programmed gradient cooling freezing box (the temperature drop ra...

Embodiment 3

[0079] The influence of the proliferation curve of different passage ratios of the testicular cell line of the double-spotted pufferfish in Example 1:

[0080] Cells with good shape and vigorous proliferation were selected from the testicular cell line established in Example 1, digested and passaged, and passaged to 25 cm at 1:2, 1:3, 1:5 and 1:10 respectively. 2 Cell culture flasks (that is, inoculate 5 × 10 5 , 3.3×10 5 , 2×10 5 , 1 × 10 5 6 cells in each group in parallel, and then make up the total volume of the solution in each flask to 5 mL with complete culture medium). From 24 h of passage, one flask of cells in each group was digested every 24 h, and the cells in each group were counted by a cell characteristic analyzer and analyzed by graph.

[0081] like Figure 5 As shown in A, for the testis cell line of the double-spotted pufferfish, different passage ratios have a great influence on the cells. Except for the 1:2 and 1:3 passage groups, the proliferation of...

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