Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells

An intestinal epithelium, isolation and culture technology, applied in the biological field, can solve the problem of not being able to clearly reflect the direct effect of target stimuli, and achieve the effects of reducing the risk of pollution, great innovation and uniqueness, and shortening the time.

Active Publication Date: 2015-02-18
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Mixed culture does not clearly reflect the direct effect of target stim

Method used

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  • Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells
  • Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells
  • Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells

Examples

Experimental program
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Effect test

Embodiment 1

[0040] In this example, intestinal epithelial γδT cells were isolated and cultured from 30-day-old 1.5kg healthy white-feathered broiler chickens. Fast for 24 hours first, remove dirt and dander, remove abdomen and leg feathers, and soak for 15 minutes; then soak in 0.1% bromogeramine solution for 10 minutes; take out the broiler chicken, bind it on the operating table, and spray 75% alcohol on the chicken For the abdomen and legs, the abdominal cavity was opened; 1% double-antibody solution was sprayed on the surface of each abdominal organ, and the middle section of the jejunum was found with surgical forceps, and a 3 cm intestinal segment was taken in 1% double-antibody PBS solution.

[0041] Move the solution into the ultra-clean bench, cut the intestinal wall longitudinally, wash it in 1% double-antibody PBS solution three times, transfer it into double-antibody-free PBS solution and wash it twice, peel off the intestinal wall fat and connective tissue with surgical scisso...

Embodiment 2

[0047] ——Method of the present invention and traditional method comparative test

[0048] For comparison experiments, traditional 0.05% collagenase IV digestion was used at 37°C, 5% CO 2 , placed on a shaker and shaken for 45 minutes to promote the complete separation of cells. After density gradient centrifugation and magnetic bead secondary antibody sorting, chicken intestinal epithelial lymphocytes were obtained, and cell viability was detected.

[0049] The contriver uses this method and the method described in comparative example, has all done ten tests and compares, and has carried out the data statistics of cell separation situation, compares as table 1:

[0050] Table 1 Method of the present invention and traditional method separation cell comparative test

[0051]

[0052] After using this method to isolate chicken intestinal epithelial γδT cells, after 48 hours of culture, the cell viability and purity were identified by flow cytometry and data statistics, as sh...

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Abstract

The invention relates to an isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells. Physical grinding is matched with a chemical enzyme process to dissociate chicken intestinal tract epithelial cells, thereby enhancing the intestinal tract cell dissociation degree, shortening the cell treatment time and ensuring the high activity of the cells; the use of the cell separation solution with the optimum density ensures the purification of the intestinal tract epithelial lymphocytes; the magnetic bead secondary antibody separation process is utilized to ensure the higher purity of the target cells; and the cell culture detects that the purity of the target cells obtained by isolated culture is up to 90%. The method overcomes the defects in the isolated culture process of the chicken intestinal tract epithelial GammaDeltaT cells, thereby laying the foundation for chicken intestinal tract epithelial GammaDeltaT cell culture and biological and immunological researches thereof.

Description

technical field [0001] The invention relates to a method for separating and culturing chicken intestinal epithelial γδT cells, belonging to the field of biotechnology. Background technique [0002] γδT cells are a newly recognized T cell subset in the past 20 years, and they respond more rapidly to abnormal changes in the body than αβT cells. Different from the γδT cells in the peripheral blood of humans and other mammals, which only have a small proportion (3%-5%), the γδT cells in the peripheral blood of poultry account for about 50%. γδT cells in poultry mucosal epithelium and peripheral blood play an important role in the initial defense of the body when invaded by pathogens. However, the isolation and culture of chicken intestinal epithelial γδT cells has been difficult for a long time. Traditional studies mostly focus on expanding lymphocytes in vitro and selectively counting γδT cells (Dong Siyuan. In vitro expansion of γδT cells, i-IELs separation and establishment...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 张海军杨金玉武书庚齐广海岳洪源王晶
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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