A kind of immortalized tree shrew intestinal epithelial cell line and its construction method and application

A technology of small intestinal epithelial cells and construction methods, applied in the field of immortalized tree shrew small intestinal epithelial cell lines and their construction, can solve the problem of introducing new antigens and failing to successfully establish immortalized cell lines and primary tree shrew intestinal epithelial cell lines Set up problems such as unsatisfactory results to achieve good cell morphology, avoid low transfection efficiency, and avoid transfection failures

Active Publication Date: 2021-10-01
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to solve the problem that the traditional method has an unsatisfactory effect on the establishment of the tree shrew primary intestinal epithelial cell line, the established cell line is a transformed cell, new antigens will be introduced, and the problem of being unable to successfully establish an immortalized cell line is provided by the present invention. Immortalized tree shrew small intestinal epithelial cell line and its construction method and application. Through optimization of a large number of experiments, the applicant finally innovatively used lentivirus-mediated transfection of hTERT gene on the premise of continuously exploring and screening conditions, and determined the virus transfection After 15 days, add the screening agent puromycin 5 μg / mL. After all the cells in the control group are killed, the concentration of the screening agent is halved, and the culture is continued for 3 generations.

Method used

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  • A kind of immortalized tree shrew intestinal epithelial cell line and its construction method and application
  • A kind of immortalized tree shrew intestinal epithelial cell line and its construction method and application
  • A kind of immortalized tree shrew intestinal epithelial cell line and its construction method and application

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Embodiment 1

[0046] A method for constructing an immortalized tree shrew intestinal epithelial cell line, comprising the steps of:

[0047] In step (1), 18 hours before lentiviral transfection, the tree shrew primary intestinal epithelial cells were mixed with 1×10 4 / well spread to cell culture plate, and make the number of cells at the time of lentiviral transfection be 2×10 4 / hole;

[0048] Wherein, the medium used for culturing is a complete medium;

[0049] The complete medium is DMEM high glucose medium supplemented with 2% (v / v) fetal bovine serum, 1% double antibody, 1% ITS-G, 1% HEPES and 10ng / ml recombinant human epidermal growth factor;

[0050] Step (2): Aspirate the original culture medium of the cells, add 250 μL of maintenance solution to each well, convert the volume of the virus stock solution according to MOI=2, and add the virus stock solution to the cells according to the volume, shake well, and place in a 37°C incubator to infect for 4 After that, add 250 μL maint...

Embodiment 2

[0058] A method for constructing an immortalized tree shrew intestinal epithelial cell line, comprising the steps of:

[0059] In step (1), 24 hours before lentiviral transfection, the tree shrew primary intestinal epithelial cells were mixed with 1×10 4 / well spread to cell culture plate, and make the number of cells at the time of lentiviral transfection be 2×10 4 / hole;

[0060] Wherein, the medium used for culturing is a complete medium;

[0061] The complete medium is DMEM high glucose medium supplemented with 5% (v / v) fetal bovine serum, 1% double antibody, 1% ITS-G, 1% HEPES and 10ng / ml recombinant human epidermal growth factor;

[0062] Step (2): Aspirate the original culture medium of the cells, add 250 μL of maintenance solution to each well, convert the volume of the virus stock solution according to MOI=100, and add the virus stock solution to the cells according to the volume, shake well, and place in a 37°C incubator to infect for 4 After that, add 250 μL of ...

Embodiment 3

[0071] A method for constructing an immortalized tree shrew intestinal epithelial cell line, comprising the steps of:

[0072] In step (1), 20 hours before lentiviral transfection, the tree shrew primary intestinal epithelial cells were mixed with 1×10 4 / well spread to cell culture plate, and make the number of cells at the time of lentiviral transfection be 2×10 4 / hole;

[0073] Wherein, the medium used for culturing is a complete medium;

[0074] The complete medium is DMEM high glucose medium supplemented with 3% (v / v) fetal bovine serum, 1% double antibody, 1% ITS-G, 1% HEPES and 10ng / ml recombinant human epidermal growth factor;

[0075] Step (2), absorb the original medium of the cells, add 250 μL of maintenance solution to each well, convert the volume of the virus stock solution according to MOI=10, and add the virus stock solution to the cells according to the volume, shake well, and place in a 37°C incubator to infect for 4 After that, add 250 μL maintenance so...

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Abstract

The invention relates to an immortalized tree shrew small intestinal epithelial cell line and its construction method and application, belonging to the field of cell technology. The present invention activates the telomerase activity of tree shrew intestinal epithelial cells by using lentivirus-mediated hTERT gene transfection to obtain the cell line, and can rapidly and effectively establish immortalized tree shrew intestinal epithelial cells, and the established immortalized Tree shrew intestinal epithelial cells have successfully overcome replicative senescence and acquired immortality, and the immortalized tree shrew intestinal epithelial cells have maintained a phenotype very similar to that of primary tree shrew intestinal epithelial cells, maintaining cell contact inhibition and cell proliferation. Density is limited, and CPE can appear after infection with reovirus, so it can be used for research on virus infection and the like.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to an immortalized tree shrew intestinal epithelial cell line and its construction method and application. The cell line maintains the biological characteristics of primary cells. Background technique [0002] The intestine is one of the largest immune systems in the body, with about 80% immune cells. The intestinal epithelium is a natural physical barrier in the intestinal tract, and is closely related to many factors, such as the occurrence of diseases, the interaction with intestinal immune cells, the immune tolerance in the intestinal tract, and the occurrence of inflammation. However, the in vitro culture of primary intestinal epithelial cells generally does not exceed 15 days, which affects the conduct of many experiments and the repeatability of experiments. The establishment of immortalized tree shrew small intestinal epithelial cell lines can be applied in many a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10C12Q1/02
CPCC12N5/0625C12N5/0679C12N15/86C12N2510/00C12N2740/15043G01N33/5044G01N2800/06
Inventor 孙晓梅代解杰尹博文仝品芬王文广罕园园陈玲霞陆彩霞匡德宣李娜宋庆凯
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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