Porcine trachea epithelial cell line and building method thereof

An epithelial cell and a method for establishing a technology, applied in the field of biological animal cell lines, can solve the problems of high requirements on nutritional conditions, few monoclonal positive cells, and small amount of cell growth.

Inactive Publication Date: 2011-09-07
张彦明 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no research on the establishment of porcine tracheal epithelial cell lines, and the immortalized porcine vascular endothelial cells obtained by the existing cell line establishment methods, due to the few monoclonal positive cells obtained during the screening process, the cell growth The amount is also very small, and the proliferation is slow, the nutritional requirements are high, and the production cost is high, which is not conducive to popularization

Method used

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  • Porcine trachea epithelial cell line and building method thereof
  • Porcine trachea epithelial cell line and building method thereof
  • Porcine trachea epithelial cell line and building method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Aseptically obtain the trachea of ​​newborn piglets, remove the adherent tissue, and use the pronase protease perfusion cold digestion method to digest and separate high-purity tracheal epithelial cells, which are spherical in shape, mostly single, and cilia can be seen under the microscope.

[0038] 2. Suspend the isolated primary tracheal epithelial cells in DF12 medium, place at 37°C, 5% CO 2 cultivated under conditions. In 6-8 hours, the cells adhered to the wall; in about 24 hours, the cells grew in a single layer of adherence; in 2-4 days, the cells covered the bottom of the plate. The indirect immunofluorescence identification of type 8 keratin showed that the nuclei had no specific fluorescence, but the cell membrane had fluorescence. Combined with the observation of cell morphology, it was confirmed that the isolated and cultured cells were porcine tracheal epithelial cells.

[0039] 3. Extract and purify the eukaryotic expression plasmid pCI-neo-hTERT cont...

Embodiment 3

[0046] The screening process of the above-mentioned positive cells and the purification process of the transfected cells, the specific steps are as follows:

[0047] 1. G418 screening and acquisition of positive cells

[0048] (1) After 48 hours of transfection, when the cells reached 80% confluence, subculture and digest the cells into another 12-well plate, and then add 800 μg / mL G418 for screening. On the 7th day, trypsinize the cells in the original dish, discard Remove the digestion solution and add the screening solution (complete DF12 medium plus G418). The culture was continued for two weeks, and the same concentration of G418 screening solution was changed every two days, and the cell growth and death were observed every day.

[0049] (2) After all the cells in the control wells died, the concentration of G418 was halved to 400 μg / mL.

[0050] (3) Continue to screen cells for about a month, changing the solution every two days until positive clones are visible.

[...

Embodiment 4

[0060] Cell identification test process:

[0061] 1. Morphological observation of transfected cells

[0062] Primary cultured STECs ( figure 1 ) and immortalized STECs ( figure 2 and image 3 ) are similar in shape and are typical epithelioid. The central nucleus of the cells is obvious, and there are often more than 2 nucleoli. The cells can be covered with a monolayer. The monolayer cells are arranged in a typical cobblestone paving stone shape, and there is contact inhibition between cells.

[0063] 2. RT-PCR detection of exogenous hTERT expression in immortalized cells

[0064] (1) The extraction method of total cellular RNA is as follows:

[0065] ①When the 3rd generation of primary generation, the 15th generation and 35th generation of immortalized cells reached 70%-80%, the cells were digested and transferred to a 1.5mL Eppendorf tube.

[0066] ② Centrifuge to remove the cell culture medium, add 750 μL Trizol, shake and mix, and let stand at room temperature for ...

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Abstract

The invention discloses a building method of a porcine trachea epithelial cell line. The method comprises the following steps of: (1) primary culture: sterilely acquiring newborn piglet tracheas, clearing adhesive tissues, performing digestion and separation by adopting a pronase perfusion cold digestion method to obtain high-purity trachea epithelial cells, suspending the separated primary trachea epithelial cells in a DF12 culture medium, and culturing the cells at the temperature of 37 DEG C under the condition of 5 percent CO2; (2) transfection and screening: extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes, and guiding the eukaryotic expression plasmids pCI-neo-hTERT for coding hTERT and neo genes to the porcine trachea epithelial cell primarily cultured in the step (1) by adopting a liposome transfection method to perform transfection; and (3) screening and amplified culture. The method is easy in culture, the growth speed is high, and the operating method is simple and convenient.

Description

technical field [0001] The invention relates to the field of biological animal cell lines, in particular to a porcine tracheal epithelial cell line and a method for establishing the same. Background technique [0002] The tracheal epithelium is located on the inner surface of the tracheal lumen and is a pseudostratified ciliated columnar epithelium, which is the first barrier for the body to defend against harmful substances. Its constituent cells - tracheal epithelial cells, in addition to having a mechanical defense function, can also synthesize and secrete a variety of mucus components and cytokines, which are used to regulate the growth of airway epithelial cells, maintain the integrity of the tracheal epithelium, moisten and purify the respiratory tract, resist In addition, tracheal epithelial cells also play an important role in participating in the immune response of airway mucosa and helping to eliminate tracheal inflammation. [0003] At present, there is no resear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12R1/91
Inventor 张彦明
Owner 张彦明
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