Method for constructing Kareius bicoloratus liver cell line

The technology of a liver cell and a construction method is applied in the field of construction of a flounder liver cell line, and can solve the problems of a small number of cells migrating out of a liver tissue block, a small number of cells, and difficulty in adhering to the wall.

Inactive Publication Date: 2012-01-18
SHANDONG ANALYSIS & TEST CENT
View PDF3 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Fish liver tissue contains a lot of collagen and fibrous tissue. In the fish cell line construction method in the existing patent, the mechanical shear force generated when the tissue is quickly shredded will have a great impact on the liver cells and cannot make the liver The primary cells adhere to the wall and proliferate normally. After the various enzyme digestion methods provided, the number of cells in the liver tissue block migrated out is small, the number of digested cells is small, and it is not easy to adhere to the wall, and the liver tissue of fish is different from that of mammals. Liver, unable to perform cell line establishment using the perfusion digestion method currently used to generate mammalian liver cell lines

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Soak fresh live stone flounder in a container filled with 75% alcohol for 5-10 minutes, wipe off the mucus on the body surface with gauze, move it into an ultra-clean workbench, wipe the fish body surface with a cotton ball, dissect out the liver tissue, and place it in the Rinse twice in a beaker filled with phosphate buffer solution; transfer to a penicillin vial containing 5% fetal bovine serum DMEM / F12 culture medium and roughly cut into tissue pieces of about 50-100 cubic millimeters; centrifuge at 800rpm to collect liver tissue pieces, Add digestion solution I (phosphate buffer containing 0.1% trypsin and 0.1% EDTA) to digest the liver tissue block for 5 minutes; add 2 drops of fetal bovine serum to neutralize the reaction, and collect the tissue block by centrifugation at 1000rpm; In II (DMEM / F12 culture medium with 0.1% type Ⅳ collagenase), blow the tissue block slowly with a dropper to mix the enzyme solution with the tissue block evenly, after standing for 0.5 ...

Embodiment 2

[0020] Soak fresh live stone flounder in a container filled with 75% alcohol for 5-10 minutes, wipe off the mucus on the body surface with gauze, move it into an ultra-clean workbench, wipe the fish body surface with a cotton ball, dissect out the liver tissue, and place it in the Rinse twice in a beaker filled with phosphate buffer solution; transfer to a penicillin vial containing 5% fetal bovine serum DMEM / F12 culture medium and roughly cut into tissue pieces of about 50-100 cubic millimeters; centrifuge at 800rpm to collect liver tissue pieces, Add digestion solution I (phosphate buffered saline containing 0.1% trypsin and 0.2% EDTA) to digest the liver tissue block for 10 minutes; add 2 drops of fetal bovine serum to neutralize the reaction, and collect the tissue block by centrifugation at 1000rpm; In II (DMEM / F12 culture medium with 0.2% type Ⅳ collagenase), blow the tissue block slowly with a dropper to mix the enzyme solution with the tissue block evenly. After standin...

Embodiment 3

[0024] Soak fresh live stone flounder in a container filled with 75% alcohol for 5-10 minutes, wipe off the mucus on the body surface with gauze, move it into an ultra-clean workbench, wipe the fish body surface with a cotton ball, dissect out the liver tissue, and place it in the Rinse twice in a beaker filled with phosphate buffer solution; transfer to a penicillin vial containing 5% fetal bovine serum DMEM / F12 culture medium and roughly cut into tissue pieces of about 50-100 cubic millimeters; centrifuge at 800rpm to collect liver tissue pieces, Add digestion solution I (phosphate buffer solution containing 0.2% trypsin and 0.2% EDTA) to digest the liver tissue block for 5 minutes; add 2 drops of fetal bovine serum to neutralize the reaction, and collect the tissue block by centrifugation at 1000rpm; In II (DMEM / F12 culture medium with 0.2% type Ⅳ collagenase), blow the tissue block slowly with a dropper to mix the enzyme solution with the tissue block evenly. After standing...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a method for constructing a Kareius bicoloratus liver cell line. The method comprises the following steps of: starting primary culture by a two-step enzymatic digestion method by taking liver tissue cells of Kareius bicoloratus as a material, and culturing in a dulbecco's modified eagle medium (DMEM)/F12 culture solution which contains fetal calf serum, liver cell growth factor, chondroitin sulfate, N-acetyl glucose hydrochloride and Kareius bicoloratus liver extracting solution and has a pH value of between 7.0 and 7.4; and subculturing by a trypsin digestion method.The process is scientific and reasonable, the damage of an enzymatic digestion method to liver cells of the Kareius bicoloratus in the construction of the conventional cell line is avoided, the cellscan normally adhere to walls to grow, various culture solution additives effectively promote the proliferation of the liver cells, the Kareius bicoloratus liver cell line constructed by the method ispassed to 110th generation at present, the proliferation state of the cells is good, and the cell line is expected to be applied to the research on the antiviral action mechanism of active ingredients of a plurality of kinds of Chinese medicinal herbs on fish viruses at molecular and cellular levels, so that an effective therapeutic medicine or an immunopotentiator in disease control is developed, and various aquaculture diseases which become increasingly serious are solved.

Description

technical field [0001] The invention relates to a method for establishing a liver cell line by using stone flounder liver tissue cells—a method for constructing a stone flounder liver cell line. Background technique [0002] Stone plaice ( Kareius bicoloratus ) is a bottom-dwelling fish in warm and tropical oceans, rich in the Yellow Sea and Bohai Sea in my country, and is an important economic fish for marine aquaculture in my country. Frequent outbreaks of various viral diseases have had a serious impact on the stone flounder aquaculture industry, resulting in large-scale death of the diseased stone flounder, serious economic losses, and may threaten human health through the food chain. At present, there is no appropriate medicine or vaccine for effective prevention and treatment, and it is urgent to develop and produce safe and efficient pollution-free fishing medicine. Among them, the application of active ingredients of various Chinese herbal medicines with broad ant...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 魏云波刘书花张瑞凌李福伟王兴民徐建荣
Owner SHANDONG ANALYSIS & TEST CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap