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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

Active Publication Date: 2022-02-18
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the immortalized dairy cow rumen epithelial cell line of the present invention provides a test cell model for the study of dairy cow rumen physiological regulation and nutrient absorption mechanism, the culture method is simple and the growth rate is fast, the construction method of the present invention can obtain BRECs with physiological functions and continuous passage , but due to the significant differences in the species of cows and yaks, this method cannot grow immortalized yak rumen epithelial cell lines
After searching, there is no report on the establishment of immortalized yak rumen epithelial cell line

Method used

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  • Immortalized yak rumen epithelial cell line and construction method thereof
  • Immortalized yak rumen epithelial cell line and construction method thereof
  • Immortalized yak rumen epithelial cell line and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses an immortalized yak rumen epithelial cell line and a construction method thereof. The preservation number of the cell line is CCTCC No. C2021245, and the cell line is classified and named as the immortalized yak rumen epithelial cell line SV40T-YREC-hTERT. The method comprises the following steps: performing adherent culture on tissue digested by primary yak rumen epithelial papilla type I collagenase to obtain primary rumen epithelial cells; with the cells as host cells, infecting the cells with lentiviruses carrying SV40T and hTERT genes; and performing screening and enlarged culture to obtain the immortalized yak rumen epithelial cell line. Through the establishment of the cell line, the defects that the primary rumen epithelial cells are difficult to culture and the number of passage times is limited are overcome, and yak source cell line resources are enriched; and the cell line can be stably passaged, can keep the morphological characteristics and normal functions of the primary rumen epithelial cells, and provides a useful in-vitro cell model for researching rumen epithelial nutrition digestion, absorption and metabolism, cell signal path mechanisms and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an immortalized yak rumen epithelial cell line and a construction method thereof. Background technique [0002] Yak is a unique breed of cattle in the world that can adapt to the special and harsh natural environment of the Qinghai-Tibet Plateau region. It is an important means of life and production for local herdsmen to survive, and it is also the main pillar industry for pastoral areas to get rid of poverty and become rich. my country is a big yak breeding country in the world, accounting for more than 92% of the world's total yak breeding, mainly distributed in my country's Tibet, Qinghai, Sichuan, Gansu, Yunnan and Xinjiang and other places. my country is at the leading level in the world in basic research and applied basic research on yak genetics and breeding, nutrition and feed, and disease prevention and control. So far, there are more and more studies on the ru...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/54C12N15/38C12R1/91
CPCC12N5/0679C12N15/86C12N9/1276C07K14/005C12Y207/07049C12N2510/04C12N2509/00C12N2501/11C12N2501/115C12N2501/105C12N2501/39C12N2740/15043C12N2800/107C12N2710/16622C12N2740/16043C12N2710/22022C12N2510/00C12Y304/24003
Inventor 王之盛王俊梅胡瑞王森
Owner SICHUAN AGRI UNIV
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