Method for constructing Kareius bicoloratus liver cell line

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...

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