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Takifugu bimaculatus ovarian tissue cell line and application thereof

A technique for ovarian cells and ovarian tissue, which is applied to the ovarian tissue cell line of the double-spotted pufferfish and its application field, can solve problems such as the establishment of the double-spotted pufferfish cell line that has not been reported, and achieves improved mitotic activity, short passage time, and stability. good effect

Pending Publication Date: 2021-11-09
FUJIAN AGRI & FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the establishment of two-spot puffer cell lines; only the establishment of some cell lines of other puffer species and the isolation and culture of a small part

Method used

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  • Takifugu bimaculatus ovarian tissue cell line and application thereof
  • Takifugu bimaculatus ovarian tissue cell line and application thereof
  • Takifugu bimaculatus ovarian tissue cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A method for establishing a puffer puffer ovary cell line comprises the following steps:

[0044] (1) The 7-month-old puffer juveniles with a length of 8-10 cm were temporarily raised in sterilized seawater containing three antibodies for 1 to 2 hours, and the surface of the fish was treated with sterilized seawater added with 1wt‰ sodium hypochlorite. Disinfect for 15 min. At the end, drip 0.005 - 0.015% of eugenol in the volume of sterilized seawater until the fish turns over, the abdomen is upward, and there is no stress response to the stimulation. Wipe off the mucus on the surface of the fish with a sterilized gauze piece. Wipe the surface of the fish twice with gauze soaked in 75vol% alcohol. Move the fish body into the ultra-clean bench, take out the ovarian tissue with a dissecting instrument, and rinse it in PBS solution containing the third antibody for 3-4 times.

[0045] The above-mentioned third antibody is the 100 × concentrate of the third antibody, wh...

Embodiment 2

[0057] Cryopreservation and recovery of the puffer puffer ovary cell line prepared in Example 1.

[0058] (1) Frozen storage: Take a bottle of 75cm 2 The cells of the puffer puffer ovary cell line that grew vigorously and covered the bottom of the culture bottle were digested with 0.25wt% trypsin, and the cell pellet was collected after centrifugation, and 3 mL of the prepared cell freezing solution was slowly added, and gently pipetted Cells, so that they are evenly dispersed in the cell freezing solution, use a pipette gun to transfer the liquid into the cryovial. Place the cryopreservation tube at 4°C for 1 hour, then place it in a programmed gradient cooling freezer box (temperature drop rate is 1°C / min), place the freezer box at -80°C for 1 day, and finally take out the cryopreservation tube and dry it. Immersed in liquid nitrogen, can be frozen for a long time.

[0059] Freezing solution formula, by volume percentage: 10% dimethyl sulfoxide (DMSO) + 90% fetal bovine se...

Embodiment 3

[0063] The impact of the different passage ratio proliferation curves of the puffer puffer ovary cell line of embodiment 1:

[0064] From the puffer puffer ovary cell line established in Example 1, cells with good morphology and vigorous proliferation were selected, 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, add 2.5 mL, 1.6 mL, 1 mL, and 0.5 mL of cell suspension to the four culture flasks, and then use complete culture solution to make up the total amount of the solution in each bottle to 5 mL). Photographs were taken every 24 hours from the 24th hour of subculture, and the cultured cells were taken by inverted microscope using the classic five-point cross sampling method to count the number of cells observed in the visual field, and the sum of the number of cells observed at five points in each group was plotted for analysis.

[0065] Such as Figure 4 It was shown that for the ovarian cell line of p...

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Abstract

The invention provides a Takifugu bimaculatus ovarian tissue cell line and application thereof, and belongs to the technical field of biology. According to the method, ovaries of the takifugu bimaculatus are subjected to isolated culture to obtain primary cells of the ovaries, and continuous subculture is performed to finally establish the stable ovarian cell line of the takifugu bimaculatus. 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 ovary 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 an ovarian tissue cell line of puffer puffer and an application thereof. Background technique [0002] Two-spot pufferfish ( Takifugu bimaculatus ), belonging to the order Fuguformes, the family Fuguidae, and the genus Fugu, are offshore warm-temperate bottom fishes, distributed in the South my country Sea, the East China Sea and the southern part of the Yellow Sea. It is a rare aquatic product with high economic value and great development potential. Two-spot puffer is the main species of puffer fish cultured in Fujian Province. It is the main breeding species for the ecological polyculture model of fish, shrimp and shellfish. It plays an important role in blocking the horizontal spread of shrimp diseases and improving the survival rate of culture. has reached a certain scale. In 2015, the breeding area was more than 80,000 mu, the output was about 11,000 tons, and th...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12R1/91
CPCC12N5/0682C12N2509/10Y02A40/81
Inventor 张子平钟照威王艺磊赵丽萍姜永华
Owner FUJIAN AGRI & FORESTRY UNIV
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