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Construction method of Epinephelus fuscoguttatus heart cell line

A technology of heart cells and construction methods, applied in the direction of animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of untargeted detection effects, research obstacles, lack, etc.

Inactive Publication Date: 2012-11-28
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, due to the lack of marine fish cell lines, especially the various tissue cell lines of the brown grouper, the research on the virology and environmental toxicology of the brown grouper is hindered, and other marine fish cell lines cannot be used effectively. Isolation, identification, and propagation of the brown-spotted grouper virus, the development of virus vaccines, and the study of the interaction between the virus and host cells cannot specifically detect the impact of various environmental pollutants in the ocean on the brown-spotted grouper. And to study the mechanism of action of pollutants on the cells of brown spotted grouper

Method used

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Examples

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preparation example Construction

[0008] 1. Preparation of heart tissue block of brown spotted grouper: fresh and live brown spotted grouper was kept in high double-antibody seawater with penicillin and streptomycin at a concentration of 1000 units / ml, and then sterilized in 75% alcohol for 24 hours. ~2 minutes. The heart tissue was aseptically dissected in an ultra-clean workbench, rinsed twice with phosphate buffered saline, and cut into tissue pieces of about 1 cubic millimeter in DMEM / F12 culture medium containing 5% fetal bovine serum; 800 rpm centrifugation to collect heart tissue blocks, the present invention takes into account the tissue characteristics of the heart of the brown grouper, and specially selects trypsin with a concentration of 0.0625% to 0.125% to digest the heart tissue blocks for 5-10 minutes; 1000 rpm The tissue block was collected by centrifugation; the heart tissue block was treated with DMEM / F12 culture solution (Gibco) containing 5% fetal bovine serum (Hyclone) and chondroitin sulf...

Embodiment 1

[0014] Put the fresh and live brown grouper in high double-antibody seawater with penicillin and streptomycin at a concentration of 1000 units / ml for 24 hours, then disinfect it in 75% alcohol for 2 minutes, wipe the surface of the fish with a cotton ball, and then dissect it Take out the heart tissue, put it in a beaker filled with phosphate buffered saline, grab the heart tissue with tweezers and rinse twice to wash away the blood stains on the surface of the heart tissue; transfer it to a penicillin vial containing 5% fetal bovine serum DMEM / F12 culture solution Cut into tissue pieces of 1 cubic millimeter; centrifuge at 800 rpm to collect heart tissue pieces, add trypsin with a concentration of 0.0625% to digest the heart tissue pieces for 5 minutes; centrifuge at 1000 rpm to collect tissue pieces; use the heart tissue pieces Sufficiently suspend the DMEM / F12 culture solution containing 5% fetal bovine serum and 0.25‰ chondroitin sulfate with a pH value of 7.2, and inoculat...

Embodiment 2

[0017] Put the fresh and live brown grouper in high double-antibody seawater with penicillin and streptomycin at a concentration of 1000 units / ml for 24 hours, then disinfect it in 75% alcohol for 2 minutes, wipe the surface of the fish with a cotton ball, and then dissect it Take out the heart tissue, put it in a beaker filled with phosphate buffered saline, grab the heart tissue with tweezers and rinse twice to wash away the blood stains on the surface of the heart tissue; transfer it to a penicillin vial containing 5% fetal bovine serum DMEM / F12 culture solution Cut into tissue pieces of 1 cubic millimeter; centrifuge at 800 rpm to collect heart tissue pieces, add trypsin with a concentration of 0.125% to digest the heart tissue pieces for 5 minutes; centrifuge at 1000 rpm to collect tissue pieces; use the heart tissue pieces Sufficiently suspend the DMEM / F12 culture solution containing 5% fetal bovine serum and 0.5‰ chondroitin sulfate with a pH value of 7.4, and inoculate ...

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Abstract

The invention relates to a method for constructing a heart cell system of blotchy rockcod, which comprises: taking a heart tissue of the blotchy rockcod as a material, adopting a trypsin digestion method to start primary culture, and culturing the heart tissue in a DMEM / F12 liquid medium which contains fetal calf serum, basic fibroblast growth factors, I-type insulin-like growth factors, chondroitin sulfate and culture supernatant of fin cells of the blotchy rockcod in the log phase and has a pH value of between 7.0 and 7.4; and adopting the trypsin digestion method for subculturing. The heart cell system of the blotchy rockcod constructed by the method is subcultured for 70 generations currently. The technology is scientific and reasonable, is hopeful to be applied to separation and breeding of fish viroids, preparation of virus vaccines and research in the aspects of interaction of viruses and host cells, and so on, can also be taken as a model for researching environmental pollutants in environmental toxicology, and performs pollution monitoring and safety evaluation on various environmental pollutants in the sea such as genic toxins, mutagen, carcinogens, environmental hormones and endocrine disruptors.

Description

technical field [0001] The invention relates to a method for establishing a heart cell line by using heart tissue cells of the brown-spotted grouper—a method for constructing the heart cell line of the brown-spotted grouper. Background technique [0002] Grouper is an important marine fish cultured in my country, widely distributed in the tropical waters of the Indian Ocean and the Pacific Ocean, and distributed in the South my country Sea and the south of the East China Sea. Brown spot grouper (Epinephelus fuscoguttatus), commonly known as "tiger spot", is a rare grouper with high economic value. In recent years, large-scale outbreaks of various fish viral diseases have caused huge economic losses in the grouper aquaculture industry, and have become one of the important factors restricting the sustainable development of the fish aquaculture industry. The fish cell line is an important research system for studying the virus infection route, infection mechanism and the devel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/077
Inventor 樊廷俊魏云波姜国建
Owner OCEAN UNIV OF CHINA
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