Immortalized yak rumen epithelial cell line and construction method thereof
A technology of epithelial cells and construction methods, which is applied in the field of immortalized yak rumen epithelial cell lines and their construction, can solve the problems that yak rumen epithelial cell lines cannot be cultured, achieve good cell proliferation morphology, reduce the culture period, and have strong cell activity Effect
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Embodiment 1
[0038] Embodiment 1: a kind of construction method of immortalized yak rumen epithelial cell line, it comprises the following steps:
[0039] S1. Sample pretreatment: a slaughterhouse in Sichuan Province collected rumen epithelial tissues of healthy adult yaks, and put them into antibiotics (200 U / ml penicillin, 0.2 mg / ml streptomycin, 100 μg / ml gentamicin and 1 μg / ml amphoteric Place the ziplock bag of the Hanks solution of mycin B) on ice and bring it back to the laboratory, cut the rumen papilla in a sterile ultra-clean bench, add antibiotic-containing (200U / ml penicillin, 0.2mg / ml streptomycin, 100 μg / ml ml gentamicin and 1 μg / ml amphotericin B) in Hanks solution for 4 to 5 times, each time for 3 to 5 minutes;
[0040] S2. Enzyme digestion and cultivation: add 0.1% type Ⅰ collagenase at a volume ratio of 5:1 to a 250ml glass bottle equipped with a rumen nipple, put it in a constant temperature air bath shaking box at 37°C for 30 minutes, and discard the supernatant , use ...
Embodiment 2
[0047] Example 2: Immunofluorescence identification of keratin Cytokeratin 18 in the first generation of primary yak rumen epithelial cells and the fifth generation of immortalized yak rumen epithelial cell lines
[0048] 1 × 10 rumen epithelial cells 6 Cells / mL were inoculated in a 6-well plate, and the cell growth was observed after the cells were stably adhered to the wall. When the cells grew uniformly and there were gaps between the cells, and the cells were not connected into sheets, take out the culture dish, add pre-cooled PBS buffer, and put Wash 3 times on a shaker for 10 min each time. Add pre-cooled 4% paraformaldehyde fixative solution for 15 minutes, add pre-cooled PBS after removing the fixative solution, wash 3 times on a shaker for 10 minutes each time. Add pre-cooled 0.1% Triton×100 to treat the cells for 10 min, remove the dialysate, add pre-cooled PBS, wash 3 times on a shaker for 10 min each time. Add 5% BSA, block at room temperature for 30min. Remove ...
Embodiment 3
[0050] Example 3: Cell Proliferation Growth Curve
[0051] The 10th generation yak rumen epithelial cells in good growth state were taken, the cells were digested to make a cell suspension, and the cells were divided into 5×10 3 Inoculate / mL density in 96-well plate for culture, set up 6 replicates per plate, inoculate 10 plates and place at 37°C, 5% CO 2 cultured in a humidified cell culture incubator. From the second day onwards, only one plate was taken out for measurement every day, and 10% CCK8 solution was added to each well, and after standing for 2 hours, the OD value at 450 nm was measured with a microplate reader. Take the culture time as the abscissa and the average OD value as the ordinate to draw the growth curve of the cells, such as Figure 6 shown. The results showed that the growth curve of cell proliferation presented an "S" shape, which was in line with the growth law of epithelial cells.
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