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Establishment and application method of ovary cell line of cynoglossus semilaevis

A technology of semi-smooth tongue sole and construction method, which can be applied to artificial cell constructs, animal cells, vertebrate cells, etc., and can solve problems such as unestablished

Active Publication Date: 2014-04-30
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment of ovarian tissue cell lines of seawater fish has always been a difficult point in fish cell culture. It was only reported in Atlantic salmon and sockeye salmon in the early days. So far, the ovarian tissue cell lines of seawater fish in my country have not yet been established.

Method used

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  • Establishment and application method of ovary cell line of cynoglossus semilaevis
  • Establishment and application method of ovary cell line of cynoglossus semilaevis
  • Establishment and application method of ovary cell line of cynoglossus semilaevis

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Experimental program
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Effect test

Embodiment 1

[0029] Healthy half-smooth tongue sole was sterilized in 70% ethanol for 1 minute, the ovarian tissue was removed in a sterile ultra-clean bench, the outermost membrane of the ovarian tissue was peeled off, and the tissue was cut into 1mm in 5% FBS-DMEM / F12 complete culture medium 3 Rinse 3 times with PBS, digest with 0.25% trypsin for 15 minutes, centrifuge at 1000rpm / min for 10 minutes to remove trypsin, suspend the cells with 1ml of 20% FBS-DMEM / F12 special culture medium to make a cell suspension, and inoculate Put the six-well cell culture plates treated with 0.1% type Ⅰ collagen and culture them in a biochemical incubator. Prepare 100ml of special culture medium, take 80ml of DMEM / F12l liquid medium, add 20ml of fetal bovine serum, 1-4μg of human recombinant epidermal growth factor (EGF), 0.5-1.5μg of human recombinant basic fibroblast growth factor (bFGF) ), 0.5-1.5 μg insulin-like cell growth factor Ⅰ (IGF-1), 500-2000 IU human chorionic gonadotropin (HCG). After 24 h...

Embodiment 2

[0031] Healthy half-smooth tongue sole was sterilized in 70% ethanol for 1 minute, the ovarian tissue was removed in a sterile ultra-clean bench, the outermost membrane of the ovarian tissue was peeled off, and the tissue was cut into 1mm in 5% FBS-DMEM / F12 complete culture medium 3 Rinse 3 times with PBS, digest with 0.4% trypsin for 10 minutes, centrifuge at 1000rpm / min for 10 minutes to remove trypsin, suspend cells with 1.25ml of 20% FBS-DMEM / F12 special culture medium to make cell suspension, Inoculate into six-well cell culture plates treated with 0.1% type Ⅰ collagen, and culture in a biochemical incubator; the preparation method for 20% FBS-DMEM / F12 special culture is the same as above; after 24 hours, add 1ml of special culture medium, and after 72 hours Replace half of the special culture medium; after the ovarian cells grow into a monolayer, suck out the culture medium in the culture wells, add 0.5 ml of trypsin solution with a concentration of 0.25% to each culture we...

Embodiment 3

[0033] Healthy half-smooth tongue sole was sterilized in 70% ethanol for 1 minute, the ovarian tissue was removed in a sterile ultra-clean bench, the outermost membrane of the ovarian tissue was peeled off, and the tissue was cut into 1mm in 5% FBS-DMEM / F12 complete culture medium 3 Rinse 3 times with PBS, digest with 0.25% trypsin for 20 minutes, centrifuge at 1000rpm / min for 10 minutes to remove trypsin, suspend cells with 1.5ml of 20% FBS-DMEM / F12 special culture medium to make cell suspension, Inoculate into the treated six-well cell culture plate and culture in a biochemical incubator; the 20% FBS-DMEM / F12 special culture preparation method is the same as above, add 1ml of special culture medium after 24 hours, and replace half of the special culture medium after 72 hours. After the ovarian cells grow into a single layer, suck out the culture medium in the culture wells, add 0.5 ml of trypsin solution with a concentration of 0.5% to each culture well, and let it stand for ...

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Abstract

The invention aims at providing an establishment method of an ovary cell line of cynoglossus semilaevis, which comprises the following steps: 1) removing the outermost membrane of the ovary tissue of cynoglossus semilaevis under a sterile condition, cutting the tissue into small blocks in a culture solution, flushing the tissue blocks with a PBS solution and digesting with a trypsin solution, wherein the solution after trypsin digestion contains cells, cell clusters and tissue blocks not completely digested; and 2) centrifuging the solution to remove the supernatant trypsin, and suspending the cells, cell clusters and small tissue block precipitates with a culture solution; after a suspension is obtained, inoculating into a culture plate treated by the type-I collagen; adding a culture solution, and culturing in a biochemical incubator at 24 DEG C; and performing subculture to finish establishment of the ovary cell line. In the establishment method provided by the invention, an ovary cell line of cynoglossus semilaevis is successfully established by use of the ovary tissue of cynoglossus semilaevis, the established cell line can be continuously passed, the passage cells obtained by the method can be passed for over 60 generations, and a great quantity of ovary cells can be provided.

Description

technical field [0001] The invention belongs to the technical field of seawater fish cell culture, and in particular relates to a construction and application method of a seawater fish Cynoglossus semilaevis ovarian tissue cell line. Background technique [0002] The cell types in fish ovaries are mainly germ cells and somatic cells surrounding them. Interactions between germ cells and somatic cells play an important role in early sex determination and ovarian development in fish. The main role of somatic cells in the ovary is to provide nutrition for germ cells and synthesize a large number of steroid hormones to regulate the self-renewal of egg cells and the development and differentiation of mature eggs. [0003] Therefore, the establishment of ovarian somatic cell lines has important theoretical significance for the study of the regulation mechanism of egg cell self-renewal and differentiation into mature eggs, and the cell lines can normally express ovarian-specific fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 孙爱陈松林王天姿王娜刘肖锋沙珍霞
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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