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Method for separating epithelial cells of intestinal tract of fish

A technology for intestinal epithelial cells and fish, applied in the biological field, can solve the problems of cumbersome operation of low vertebrate cell culture, unstable state of primary intestinal epithelial cells, low success rate, etc., and achieve the stability of fish intestinal epithelial cells. , The effect of stable intestinal epithelial cells and high success rate

Active Publication Date: 2016-03-09
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the cumbersome operation of lower vertebrate cell culture, easy pollution and high cost, no published patents have been seen yet.
Existing literature only refers to mammalian intestinal primary cell culture for fish cell culture, which is not fully applicable, resulting in unstable state of primary intestinal epithelial cells, low success rate, and easy contamination.

Method used

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  • Method for separating epithelial cells of intestinal tract of fish
  • Method for separating epithelial cells of intestinal tract of fish
  • Method for separating epithelial cells of intestinal tract of fish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Isolation of fish intestinal epithelial cells

[0049] (1) Implementing animals: about 80g tilapia

[0050] (2) Experimental reagents: Hanks balanced salt (sigma company), DMEM (gibco company), fetal bovine serum (gibco company), collagenase XI (sigma company), trypsin (sigma company), insulin (sigma company), transfection Ferritin (sigma company), HEPES (sigma company)

[0051] (3) Implementation steps: pass the reagents required for the experiment through a 0.22 μm sterile sieve; spread the six-well cell plate required for the experiment with 0.01 g / ml gelatin and incubate at 37° C. for 1 hr, discard the gelatin. Take 80g tilapia and starve it in aerated still water overnight at 28°C for about 24 hours, then anesthetize. Take out the whole intestine of the fish under a sterile environment, soak it briefly in 75% alcohol, remove the end 2cm, soak the whole intestine in Hanks balanced salt solution, cut it into small pieces of about 1cm, wash the contents, a...

Embodiment 2

[0054] Example 2 Separation of fish intestinal epithelial cells (different cleaning methods and no cleaning medium)

[0055] (1) Implementing animals: about 80g tilapia

[0056] (2) Experimental reagents: Hanks balanced salt (sigma company), DMEM (gibco company), fetal bovine serum (gibco company), collagenase XI (sigma company), trypsin (sigma company), insulin (sigma company), transfection Ferritin (sigma company), HEPES (sigma company)

[0057] (3) Implementation steps: pass the reagents required for the experiment through a 0.22 μm sterile sieve; spread the six-well cell plate required for the experiment with 0.01 g / ml gelatin and incubate at 37° C. for 1 hr, discard the gelatin. Take 80g tilapia and starve it in aerated still water overnight at 28°C for about 24 hours, then anesthetize. Take out the whole intestine of the fish under a sterile environment, soak the whole intestine in Hanks balanced salt solution, cut into small pieces of about 1mm, and wash eight times i...

Embodiment 3

[0059] Example 3 Fish intestinal epithelial cell separation (cell digestion solution is a single collagenase XI)

[0060] (1) Implementing animals: about 80g tilapia

[0061] (2) Experimental reagents: Hanks balanced salt (sigma company), DMEM (gibco company), fetal bovine serum (gibco company), collagenase XI (sigma company), trypsin (sigma company), insulin (sigma company), transfection Ferritin (sigma company), HEPES (sigma company)

[0062] (3) Implementation steps: pass the reagents required for the experiment through a 0.22 μm sterile sieve; spread the six-well cell plate required for the experiment with 0.01 g / ml gelatin and incubate at 37° C. for 1 hr, discard the gelatin. Take 80g tilapia and starve it in aerated still water overnight at 28°C for about 24 hours, then anesthetize. Take out the whole intestine of the fish under a sterile environment, soak it briefly in 75% alcohol, remove the end 2cm, soak the whole intestine in Hanks balanced salt solution, cut it in...

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Abstract

The invention discloses a method for separating and culturing the epithelial cells of intestinal tract of fish. The method comprises the following steps: separating and washing intestinal tracts of fishes, digesting the tissue blocks by protease; collecting cells, separating enzyme liquid and cells; and culturing cells. For the first time, a feasible method for culturing epithelial cells of intestinal tracts of fish is provided; and the method has the characteristics of simple operation, low cost, high success rate, high efficiency, and stable separated epithelial cells of intestinal tracts of fish, and solves the problem that the intestinal epithelial cells can be easily polluted.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating and culturing fish intestinal epithelial cells. Background technique [0002] Fishes are widely distributed and inhabit almost all aquatic environments, from freshwater lakes to saltwater oceans. It is also a rare delicacy on the Chinese table. It can provide rich protein and is the only natural source of polyunsaturated fatty acids (n3). In recent years, people's increasing demand for fish has promoted the development of aquaculture industry, and also attracted the attention of a large number of outstanding researchers. The gut is an important immune organ in fish, containing many lymphocytes, macrophages, eosinophils and neutrophils. At the same time, the digestive ability and absorption function of the fish body depend on the health of the intestinal tract. Using the fish intestinal epithelium at the cellular level, it is possible to conduct research on n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N9/50
Inventor 张美玲刘钰锟薛敬东
Owner EAST CHINA NORMAL UNIV