Culture method and application of duck intestine epithelial cells

A technology of small intestinal epithelial cells and culture methods, applied in the establishment of duck small intestinal epithelial cell oxidative stress model, the cultivation of duck primary small intestinal epithelial cells, and can solve the problem of inability to obtain highly active and treatable duck primary small intestinal epithelial cells, etc. problems, to achieve scientific and reasonable training methods

Active Publication Date: 2019-11-01
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] A technical problem to be solved in the present invention is the problem that the existing method for isolating small intestinal epithelial cells cannot obtain highly active and treatable duck primary small intestinal epithelial cells. A culture method for duck small intestinal epithelial cells is established, which specifically includes the following steps:

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  • Culture method and application of duck intestine epithelial cells
  • Culture method and application of duck intestine epithelial cells
  • Culture method and application of duck intestine epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Isolation, Purification and Culture of Duck Small Intestinal Epithelial Cells

[0036]Take duck eggs hatched for 26 days, take out the small intestine tissue of duck embryos by aseptic operation, and place them in DPBS (38.8mL PBS + 1.2mL double antibody, where PBS (phosphate buffer) is HyClone PBS (Thermo Fisher, USA) ); after removing the mesentery and pancreas, dissect the small intestine, rinse with DPBS until the supernatant is clear; use ophthalmic scissors to cut the small intestine into pieces of 1-3mm 3 Tissue block; use 20ml, 1mg / ml type I collagenase digestion solution to digest 20mL tissue block, 37°C, 80 r / min shaking digestion for 70min; use DPBS to gently wash the tissue block twice, discard the DPBS, and keep the tissue block ; Gently pipet the tissue mass with DPBS at 37°C, collect the upper cell suspension, keep the tissue block and continue to wash with DPBS, repeat this step 7-8 times until the supernatant is clear; collect the obtained upp...

Embodiment 2

[0037] Example 2 Identification of duck intestinal epithelial cells.

[0038] 1) Identification of duck intestinal epithelial cells screened by CK18 protein

[0039] Small intestinal epithelial cells cultured in 6-well plates for 18 hours were washed twice with PBS; fixed with 400 μL of 4% paraformaldehyde at room temperature for 30 minutes; washed twice with PBS; permeabilized with 0.5% Triton-X100 for 15 minutes; washed twice with PBS; Block with BSA for 20min; add 1:50 dilution of CK18 rabbit polyclonal antibody (primary antibody), incubate at 37°C for 1h; after washing with PBS, add 1:40 dilution of FITC-treated goat anti-rabbit Ig secondary antibody, and incubate at 37°C for 1h ; Observation by fluorescence microscope after incubation. Cell immunofluorescence results see figure 2 .

[0040] 2) Verification of intestinal-specific expression genes

[0041] The cultured cells are identified by detecting the mRNA expression level specifically expressed in the duck small ...

Embodiment 3

[0043] Embodiment 3 Different hydrogen peroxide treatments affect the cell viability

[0044] Using the cultured duck small intestinal epithelial cells, a 6×5 factorial design was adopted, divided into 30 treatments, and 6 hydrogen peroxide concentration gradients (0, 50, 100, 200, 500, 1000 μmol / ml) and 5 time points were designed (1, 4, 12, 24, 48h), the cell viability was measured by the CCK8 method.

[0045] 1) Cell viability was measured by CCK8 method, and the test results were as follows: Figure 4 As shown, at 4h, at 200μM hydrogen peroxide concentration, the cell viability was around 70%, indicating that it had a certain impact on the cells, and the hydrogen peroxide concentration at other time points caused too much or less damage to the cells, so it was determined that the peroxidation The hydrogen action time is 4h, and the action concentration is 200μM.

[0046] 2) Observation of the cell state showed that after the cells were treated for 4 hours at various conc...

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Abstract

The invention discloses a culture method and application of duck intestine epithelial cells, and belongs to the technical field of cell separating culture and the technical field of cell physiology. According to the cell culture method, an enzymic digestion method and a tissue block blowing and beating method are combined to obtain duck intestine epithelial primary cells, the problems that when the duck primary intestine epithelial cells are cultured through an existing method, activity is low, and cell transfection cannot be conducted are solved, the primary cells are cultured successfully, and the cell purity reaches 95% or above, and the culture method of the duck intestine epithelial cells is established successfully; and by utilizing the cultured primary duck intestine epithelial cells and through aging and dose-effect tests, the influences of the concentration and action time of hydrogen peroxide on the cell activity and the cell apoptosis situation are tested, a model making scheme suitable for the intestine epithelial cells is determined, an oxidative stress model for the duck primary intestine epithelial cell is established, and the basis is provided for a molecular mechanism for later researching duck intestinal injury.

Description

technical field [0001] The invention belongs to the technical field of cell separation and culture and the technical field of cytotoxicity, and in particular relates to a method for culturing duck primary small intestinal epithelial cells, and also relates to a method for establishing an oxidative stress model of duck small intestinal epithelial cells. Background technique [0002] With the development of large-scale breeding of livestock and poultry, group transfer, immunization, transportation, and restricted feeding have become important links in standardized breeding. These production links can induce oxidative stress in animals. In addition, the small environment of the breeding house is affected by external environmental factors, such as high temperature in summer and low temperature in winter, which can also induce oxidative stress in the body. Oxidative stress in the body is due to the excessive production of reactive oxygen free radicals or reactive nitrogen free ra...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N5/0679C12N2509/00C12N2501/91C12N2501/11C12N2501/33C12N2500/84C12N2500/05Y02A50/30
Inventor 张昊陈芳吴艳袁杰梁振华皮劲松申杰潘爱銮杜金平孙静蒲跃进
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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