Immortalized dairy cow rumen endothelial cell line and construction method thereof

A technology of epithelial cells and construction methods, applied in the field of immortalized dairy cow rumen epithelial cell lines and their construction, can solve the problems of cumbersome and complicated processes, BRECs are not commercialized, and the time for culturing primary cells with trypsin is long, etc., to achieve fast proliferation , cost-saving effect

Inactive Publication Date: 2018-04-06
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If there is no cell material, only living cows can be sampled. This process is cumbersome and complicated, and it takes a long time to culture primary cells with trypsin
At the same time, BRECs are not commercialized at present, so it is imminent to establish functional immortalized cow rumen epithelial cells

Method used

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  • Immortalized dairy cow rumen endothelial cell line and construction method thereof
  • Immortalized dairy cow rumen endothelial cell line and construction method thereof
  • Immortalized dairy cow rumen endothelial cell line and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Establishment of Immortalized Dairy Cow Rumen Epithelial Cell Line

[0039] (1) Primary culture

[0040] The rumen epithelial tissue of dairy cows came from the experimental farm of Yangzhou University. Use scissors to select the appropriate rumen epithelial tissue, then repeatedly wash the rumen with PBS containing 5 times of penicillin, streptomycin, gentamicin and amphotericin B, and wash away the incompletely digested pellet feed on the rumen until it is clean. Next, put it into DMEM complete medium containing 5 times penicillin, streptomycin, gentamicin and amphotericin B, and bring it back to the laboratory immediately within 30 minutes. Use blunt scissors and tweezers to separate the rumen epithelial tissue, and wash it repeatedly with the above-mentioned cleaning solution containing 5 times of antibiotics. Subsequently, these rumen epithelial tissues were minced with small scissors to about ~1mm 3 organization block. Next, transfer the shredded rum...

Embodiment 2

[0043] Example 2 Western blot detection of SV40T expression in cow rumen epithelial cells

[0044] (1) Use trypsin to separate and digest the cells, and collect the cell pellet by centrifugation. Add 100 μL RIPA lysis buffer (V protease inhibitor: V RIPA=1:49) to suspend the pelleted cells; place on ice for 30 min, and vortex every 10 min for 30 s. Centrifuge at 12000g at 4°C for 10min, transfer the supernatant to a new 1.5mL finger tube, and obtain the total cell protein product.

[0045] (2) Take 200 μL protein standard preparation solution and add it to 5 mg BSA to fully dissolve to prepare 25 mg / mL protein standard. Take 20μL of 25mg / mL protein standard and add 980μL of diluent to prepare 0.5mg / mL protein standard.

[0046] (3) Add 0.5 mg / mL protein standard substance to standard wells of 96-well culture plate at 0, 1, 2, 4, 8, 12, 16 and 20 μL, and add PBS to make up to 20 μL.

[0047] (4) The sample to be tested was diluted 15 times with PBS, and 20 μL was added to ea...

Embodiment 3

[0062] Example 3 Identification of Cow Rumen Epithelial Cytokeratin 18

[0063] BRECs according to 1×10 4 cells / cm 2 Seed in 8-well Chamber Slide, and wash the cells 3 times with pre-cooled PBS when the cell density reaches 60%. Fix with pre-cooled acetone:methanol for 15 min, and wash the cells 3 times with PBS. 3% horse serum was blocked at room temperature for 60 min, and the serum was discarded. Add mouse cytokeratin 18 primary antibody and place in 4°C refrigerator overnight. Wash with PBS three times, add FITC-linked goat anti-mouse secondary antibody, and incubate at room temperature for 45 minutes in the dark. Washed 3 times with PBS, stained with DAPI for 5 min at room temperature. Wash 3 times with PBS, 1 time with distilled water, mount the slides overnight, and store in the refrigerator at 4°C in the dark for at least 6 months. Next, cytokeratin 18 green fluorescence was observed using a confocal microscope (see image 3 ), further proving that BRECs are der...

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Abstract

The invention provides an immortalized dairy cow rumen endothelial cell line and a construction method thereof. The construction method comprises the following steps: (a) cutting, washing, and digesting collected dairy cow rumen endothelial tissues in a culture medium, and carrying out primary culture to obtain rumen endothelial cells; step (b) incubating rumen endothelial cells obtained in the step (a) with a viral liquid containing an SV40T antigen gene to obtain infected rumen endothelial cells; and step (c) culturing the infected rumen endothelial cells obtained in the step (b) to obtain the immortalized dairy cow rumen endothelial cell line. The provided immortalized dairy cow rumen endothelial cell line can provide a test cell model for the research on physiological regulation and nutrient absorption mechanism of dairy cow rumen. The culture method is simple, the growth speed is quick, and the construction method can obtain BRECs, which have physiological functions and can carryout continuous passage.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to the technical field of animal cell engineering, and relates to an immortalized dairy cow rumen epithelial cell line and a construction method thereof. Background technique [0002] The rumen is an important place for ruminants to digest, absorb and metabolize nutrients. The rumen epithelium has important physiological functions, such as the transport and absorption of nutrients, the absorption and metabolism of short chain fatty acids (SCFAs), and the adhesion of rumen microorganisms. The absorption of SCFAs in the rumen epithelium is the most important physiological function, because SCFAs can provide 70% of the main energy source for ruminants and provide precursors for the synthesis of milk fat. The research on the mechanism of nutrient transport and absorption by the rumen epithelium and the research on the function of the SCFAs transporter and receptor protein genes inv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/37C12N5/10C12R1/91
CPCC12N15/86C07K14/005C12N5/0679C12N2510/04C12N2710/22022
Inventor 赵国琦贡笑笑詹康姜茂成
Owner YANGZHOU UNIV
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