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Establishment and application method of immortalized Hu sheep rumen epithelial cell line

A technology of epithelial cells and establishment methods, which is applied in the field of immortalized Hu sheep rumen epithelial cell lines and their construction, can solve the problems of long culture time and cumbersome and complicated process of primary cells, and achieve long culture time, fast growth speed, The effect that the upbringing method is simple

Inactive Publication Date: 2019-05-17
ZHEJIANG 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 live Hu sheep can be sampled. This process is cumbersome and complicated, and the primary cell culture takes a long time

Method used

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  • Establishment and application method of immortalized Hu sheep rumen epithelial cell line
  • Establishment and application method of immortalized Hu sheep rumen epithelial cell line
  • Establishment and application method of immortalized Hu sheep rumen epithelial cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Establishment of Immortalized Hu sheep rumen epithelial cell line

[0044] (1) Primary culture

[0045] The collected Hu sheep rumen epithelial tissue was repeatedly washed with PBS containing 2 times of penicillin and streptomycin, then cut into pieces, and then washed with PBS containing 2% P / S, 1% amphotericin B and 1% gentamicin Wash the epithelial tissue at least 10 times with washing solution, and discard the supernatant for later use. The shredded and cleaned Hu sheep rumen epithelial tissue was digested with 0.25% trypsin-0.02% EDTA trypsin digestion solution in a 37°C water bath for 10 minutes, and the digestion solution containing cell clusters was absorbed, and fetal bovine serum was added to terminate the digestion. Continue to add trypsin digestion solution to digest for 4-6 times, collect the digestion solution containing cell mass obtained each time, and terminate the digestion with fetal calf serum. Pass the collected digestion solution conta...

Embodiment 2

[0048] Example 2 Identification of Immortalized Hu sheep rumen epithelial cell line

[0049] (1) Cell slide

[0050] Take 3 pieces of glass slides in a 24-well plate, add 1 mL of culture medium to each well, and add cells 2×10 4 pcs / hole. Place in the incubator for 2 hours or overnight.

[0051] (2) Fixed

[0052] After the cells climbed the slices, the medium was aspirated, washed once with PBS, and fixed with 4% PFA at 4°C for 30 min. Wash with PBS 3×5min / time. It is also possible not to suck out the PBS for the last time, and put it at 4°C overnight.

[0053] (3) Membrane closure

[0054] Remove water from the glass slide, place it on the petri dish support, glass slide sealing solution configuration: 0.5% Trition X-100 mixed with PBS 1:1, add 10% serum, take 50uL membrane rupture sealing solution and drop it on the waterproof membrane , cover the cell side of the slide for 2h.

[0055] (4) Primary antibody incubation

[0056] Preparation of primary antibody CK19: ...

Embodiment 3

[0063] Example 3 Determination of Growth Velocity of Immortalized Hu Sheep Rumen Epithelial Cell Line

[0064] The 23rd passage cells were divided into 5×10 3 The density of each well was planted in 96-well culture plates, divided into 7 groups, and each group had 8 replicates. Add 10 μl of CCK8 solution to each group of cells every day, and measure the absorbance at 450 nm with a microplate reader after incubating at 37° C. for 1 hour. Absorbance is proportional to the number of cells, with the culture time as the abscissa and the absorbance as the ordinate, draw the cell growth curve (see Figure 6 ).

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Abstract

The invention relates to the technical field of biology and aims to provide an establishment and application method of an immortalized Hu sheep rumen epithelial cell line. The method comprises the following steps: taking rumen epithelial tissues of newborn Hu sheep, cutting and cleaning the tissues, and digesting them with trypsin digestion liquid; purifying cells by a differential adherence method to obtain primary Hu sheep rumen epithelial cells; transfecting virus liquid carrying SV40T antigen gene into primary Hu sheep rumen epithelial cells to obtain infected Hu sheep rumen epithelial cells; and further subculturing the infected Hu sheep rumen epithelial cells to obtain an immortalized Hu sheep rumen epithelial cell line. A test cell model can be provided for rumen physiological regulation and nutrient absorption mechanism research of Hu sheep. The culture method is simple, the growth speed is high, and the Hu sheep rumen epithelial cell line with physiological functions and capable of continuous passage can be obtained. By utilizing the cell line, sampling of living Hu sheep can be reduced, and the problems of long primary cell culture time, high cost and complicated processat present are solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an immortalized Hu sheep rumen epithelial cell line and a construction method thereof. Background technique [0002] Hu sheep is a unique breed of white lambskin sheep in my country. It belongs to the national first-class local protected livestock and poultry breeds. Geographical protection sign. Hu sheep have the characteristics of being resistant to rough feeding, suitable for captivity, fast growth, and high reproductive performance. Lamb is a meat product with high protein content and low cholesterol content. Its feed quality is natural, and there is no antibiotics in the feeding process. Products are favored by more and more consumers. Hu sheep are ecological and grain-saving livestock. They can use the waste left over from agricultural planting as feed sources, such as farmland straw, stems and leaves after harvesting vegetables, and leftovers from agricultural pro...

Claims

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Application Information

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IPC IPC(8): C12N5/10
Inventor 杨春蕾兰伟
Owner ZHEJIANG UNIV
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