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Rabbit intestinal epithelial cell line and preparation method thereof

A technology for small intestinal epithelial cells and cells, which is applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of less research on the isolation and culture of rabbit intestinal epithelial cells.

Inactive Publication Date: 2011-10-12
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, although the culture of human and poultry small intestinal epithelial cells has been successful, there are no detailed reports on the cultivation of rabbit intestinal epithelial cells in China, and there are few studies on the isolation and culture of rabbit intestinal epithelial cells abroad, mainly about colonic epithelial cells. Culture, the transport characteristics of the cells, the expression of enzymes and transmembrane resistance and other indicators are relatively more reflective of colonocytes than small intestinal cells

Method used

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  • Rabbit intestinal epithelial cell line and preparation method thereof
  • Rabbit intestinal epithelial cell line and preparation method thereof
  • Rabbit intestinal epithelial cell line and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Experimental animals The Japanese white rabbits were purchased from the Experimental Animal Center of Jilin University.

[0020] Instruments and Reagents

[0021] Carbon dioxide incubator (SELECTA, Spain), inverted microscope (Olympus, Japan), ultra-clean bench (SANYO, Japan), autoclave (SANYO, Japan), blast drying oven (Shanghai Shenxian Constant Temperature Equipment Factory), 96-well cell culture plate (COSTAR) DMEM / F12 medium (GIBCO), fetal bovine serum (PAA), cytokeratin 8 monoclonal antibody, DAB color development kit purchased from Wuhan Boster Company, glutamine, trypsin All are AMRESCO products, collagenase IV, penicillin, and streptomycin are all Sigma products.

[0022] 1) Rabbit IEC isolation and culture

[0023] Newborn unbreasted rabbits were sacrificed by neck dislocation, immersed in 75% alcohol for 5 minutes, the abdominal cavity was aseptically opened, the small intestine and ileum were removed and the surrounding connective tissue was removed, washe...

Embodiment 2

[0030] Example 2 The growth curve was measured by the MTT method.

[0031] The IEC suspensions with good growth conditions were inoculated in 96-well cell culture plates at a density of 1 × 10 5 1 / mL, 200 μL per well, 6 replicates per day, continuous measurement for 7 days. Add 20 μL MTT solution to each well of the cells to be tested every day, continue to culture for 4 h, stop the culture, remove the culture medium, add 150 μL DMSO to each well and shake for 10 min, and measure OD with an enzyme-linked immunosorbent assay (ELISA) 570 value. Only the culture medium was added to the blank zero-adjusted wells, and no cells were added. See IEC growth curve Figure 7 .

Embodiment 3

[0032] Example 3 Identification of cytokeratin 8 monoclonal antibody

[0033] The cell slides were fixed in 4% paraformaldehyde for 1 h, and then rinsed three times with PBST for 5 min each. Repair with citric acid repair solution for 10min, 95°C, 3% hydrogen peroxide solution for 10min to eliminate endogenous peroxidase, block with BSA for 30min, rinse with distilled water for 3 times, 5min each time, add anti-keratin antibody Incubate for 30min. Add goat anti-mouse IgG antibody and incubate at 37°C for 30min. Then add the color developer and develop color for 10min. After each step, rinse with PBST for 3 times, 5min each time. The purified IEC was identified with cytokeratin 8 monoclonal antibody and the result was positive, see Figure 5 , the cells are arranged in brown, which shows that the obtained cells are small intestinal epithelial cells. Negative control IEC was not stained, see Image 6 .

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Abstract

The invention provides a rabbit intestinal epithelial cell line. The preparation method comprises the following steps: the ileum part of a newborn baby rabbit is taken to be mechanically cut to pieces, collagenase IV is used to digest the cut ileum part to obtain primary cultured rabbit intestinal epithelial cells, the phase contrast digestion method is combined with the phase contrast adherent method to perform primary purification on the rabbit intestinal epithelial cells; then the monoclonal method is adopted to further purify the cells, and the obtained cell line grows well and stably; the shape of the cells is regular, the cells show a flagstone appearance, and the keratin 8 identification result of the cell line is positive; and a cell line is screened out through subculture and thesubculture is kept stable. The rabbit intestinal epithelial cell line comes from the ileum part of the newborn Japanese big-ear rabbit, the preservation number is CGMCC ((China General Microbiological Culture Collection Center) No.4784; and the cell line can be used in the absorption pathway of nutrient substances in the intestinal tract, have the function of resisting nutrient substances and be used in the researches of the microecological nutrition, the signal transduction between cells, the proliferation and differentiation mechanisms of various cells in a crypt-villus system, etc.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a rabbit small intestine epithelial cell line and a preparation method thereof. Background technique [0002] The small intestine is an important part of the selective digestion and absorption of nutrients in the body, and 90% of the absorption of the entire gastrointestinal tract occurs in the small intestine. The intestinal mucosal epithelium is a highly dynamic system that reflects important biological phenomena such as cell proliferation, migration, differentiation, and apoptosis. Intestinal epithelial cells (IEC) are specialized cells for intestinal digestion and absorption, maintaining the metabolic renewal of the entire villous epithelium. Due to the dense and large number of microvilli on the surface of small intestinal mucosal epithelial cells, it is conducive to the activity of enzymes and the absorption of nutrients; in addition, IEC is also mainly involved in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12R1/91
Inventor 车东升刘飞飞穆成龙孙泽威秦贵信杨连玉张海全赵元鲍男赵晗程传锋卢加成张春
Owner JILIN AGRICULTURAL UNIV
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