Construction method of bastard halibut brain cell system

A technology for the establishment of flounder brain and method, which is applied in the field of construction of flounder brain cell lines, can solve problems such as the establishment of cell lines with no brain tissue, and achieve the effects of easy grasp and operation, easy operation, and strong repeatability

Inactive Publication Date: 2015-07-22
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there are only flounder fin ray cell lines at home and abroad (Ocean University of China, Tong Shangliang, Aquaculture, 1997, 156:327-333.), embryonic cell lines (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Chen Songlin, Diseases of Aquatic Organisms, 2004, 60: 241-246.), kidney cell line (Huanghai Fisheries Research Institute, China Fisheries Research Institute, Wang Na, Journal of Fish Diseases, 2011, 34: 81-85.), etc. Cell lines, there is no establishment of brain tissue cell lines of this fish species

Method used

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  • Construction method of bastard halibut brain cell system
  • Construction method of bastard halibut brain cell system
  • Construction method of bastard halibut brain cell system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The method for establishing a cell line derived from Paralichthys olivaceus brain is as follows:

[0027] 1) Prepare cell culture medium: Take Hyclone MEM culture medium, add 20% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, and 10ng / mL human basic fibroblasts to the culture medium. Growth factor, pH value is 7.0-7.4, stored at 4°C for later use.

[0028] 2) Primary culture: Take the brain tissue of 250g Japanese flounder aseptically and place it in a sterile glass plate containing 400U / mL penicillin and 400μg / mL streptomycin in 4mL Hyclone's MEM culture medium for 3-5 minutes. Aspirate the culture medium, wash the brain tissue twice with 1-1.2mL PBS (pH7.2), aspirate the PBS, and cut the brain tissue into about 1mm 3 Add 1 mL of the above step 1) to prepare the cell culture solution, suspend the small pieces of brain tissue, and then transfer to 25cm 2 Place the culture flask upright in the incubator at 25±0.2℃. On the next day, add 1 mL of the cell cultur...

Embodiment 2

[0031] Identification and application of Paralichthys olivaceus brain cell line

[0032] 1) Cryopreservation and recovery of cells

[0033] Cryopreservation of cells:

[0034] Select the above-mentioned subcultured brain cells that are in the exponential growth phase and have a cell density of more than 90%, and digest them according to conventional methods: aspirate the culture solution, rinse with PBS (pH 7.2), aspirate PBS, and add 1 mL of 0.25% trypsin solution for digestion. Observe the cell shrinkage under the microscope and aspirate the trypsin solution. Continue to observe under the microscope. When the cells become round, add 2 mL of freshly prepared cell culture solution to the original bottle in step 1) of the above example to prepare a cell suspension. Transfer the cell suspension to a 15mL centrifuge tube, and use 2200r p Centrifuge for 2 min at m and remove the supernatant. Suspend the cells in 2 mL of pre-chilled cryopreservation medium (ie Hyclone MEM cell culture m...

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PUM

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Abstract

The invention discloses a method for establishing a bastard halibut brain tissue-derived cell system. The method comprises brain tissue primary culture, subculture, cryopreservation, recovery and identification, wherein cell culture fluid adopted is obtained by adding fetal calf serum and human basic fibroblast growth factor to basal cell culture fluid. The established bastard halibut brain cell system is represented as an epithelioid-like astrocytic cell, which can support continuous passage to provide a great amount of bastard halibut brain cells and can be directly used for research of bastard halibut functional gene. The construction method disclosed by the invention not only is expected to serve as a platform for theoretical research of bastard halibut molecule cellular level but also can be used as an ideal material for research on fish hemadenology, reproductive biology and environmental internal-secretion interfering-substance toxicology.

Description

Technical field [0001] The invention relates to a marine fish cell culture technology, in particular to a method for constructing a Japanese flounder brain cell line. Background technique [0002] The construction and cultivation of animal cell lines has long become a powerful tool for the study of developmental biology, endocrinology, toxicology, genetics, virology, and immunology. Up to now, more than 280 different fish cell lines have been established one after another, playing important roles in many fields such as physiology, virology, toxicology, tumor and genetic engineering. But relatively speaking, there are still fewer cell lines in marine fish, including only about 100 in saltwater fish. [0003] Fish brain tissue is an important tissue and organ of fish. The regulation and control of all its life activities are subject to the nervous system and endocrine system. Reproductive activities such as gonadal differentiation, development, maturation, gamete discharge, etc. rel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079C12R1/91
Inventor 尤锋郑媛彭丽敏邹玉霞吴志昊谭训刚焦爽
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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