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Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof

A technology of oral mucosal epithelium and establishment method, which is applied in the field of immortalized piglet oral mucosal epithelial cell line and its establishment, can solve the problems of cell stability, poor uniformity, limited number of culture passages, etc., and achieve the effect of clear boundary and uniform shape

Inactive Publication Date: 2016-04-13
张彦明
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide an immortalized piglet oral mucosal epithelial cell line and its establishment method, aiming to solve the problems of limited number of in vitro culture and passage of primary porcine oral mucosal epithelial cells and poor cell stability and uniformity

Method used

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  • Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof
  • Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof
  • Immortalized piglet oral mucosa epithelial cell line and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Establishment method of immortalized piglet oral mucosal epithelial cell line

[0062] 1. Primary culture of oral mucosal epithelial cells of piglets: After anesthetizing non-lactating piglets, blood was collected from the heart to death, disinfected with alcohol, and the oral epithelial tissue corresponding to the cheek was cut, and PBS (containing 600IU / ml double antibody, 5 μg / mL amphotericin B) Rinse several times, soak at 4°C for 30 minutes, and cut the oral epithelial tissue into 3 Rinse in PBS three times, centrifuge at 1000r / min for 5min, inoculate in 96-well culture plate, let stand for 10min, add growth medium for primary culture, place at 37°C, 5% CO 2 Cultivate in an incubator, observe for 24 hours whether there is bacterial contamination, change the liquid every 3 days, observe the cell morphology and growth every day, and cultivate for 7-12 days.

[0063] 2. Transfect the primary piglet oral mucosal epithelial cells: extract and purify the eukar...

Embodiment 2

[0079] Example 2.RT-PCR detection of exogenous hTERT expression in transfected cells

[0080] 1. Primer design and synthesis

[0081] According to the published mRNA reference sequence of hTERT in GenBank. One pair of specific detection primers was designed using PrimerPremere5.0. The upstream and downstream primers are P1: 5′-GTATGCCGTGGTCCAGAAGG-3′; P2: 5′-CGTGGGTGAGGTGAGGTGTC-3′, the size of the amplified fragment is 228bp, and the primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0082] 2. Extraction of total cellular RNA

[0083] (1) The transfected cells and primary cells were digested with 0.25% trypsin and inoculated into a 6-well plate. After the cells covered the monolayer, they were taken out, washed twice with sterilized PBS, and the liquid was discarded.

[0084] (2) Add 1mL Trizol reagent, let it stand for 1min, blow back and forth with a gun, suck the cell lysate into a 1.5mL sterilized EP tube treated with DEPC water, and let it sta...

Embodiment 3

[0102]Example 3. Identification of Piglet Oral Mucosa Epithelial Cells by Indirect Immunofluorescence

[0103] 1. Plating: Place a sterile coverslip of 0.5 cm × 0.6 cm in the well of a 24-well cell culture plate, digest ZYM-OMEC into single cells with trypsin, and adjust the cell concentration to 2 × 10 after counting. 5 cells / mL, inoculate 0.5 mL of cell suspension in each well.

[0104] 2. Fixation: When the cells grow to a near-confluent state (low-density monolayer), wash 3 times with PBS (0.01mol / L, pH7.4), add 0.4g / L paraformaldehyde phosphate buffer (pH7.4 ), with the cells facing up, fixed at room temperature for 10 min, and washed 3 times with PBS, 3 min each time.

[0105] 3. Breakthrough: add 1% Triton breakthrough solution (prepared in PBS), act at 37°C for 15 minutes, wash with PBS 3 times, 3 minutes each time.

[0106] 4. Blocking: 5% skimmed milk powder in PBS solution, 37° C., blocking for 1 hour.

[0107] 5. Incubate the primary antibody: shake off the bloc...

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Abstract

The invention discloses an immortalized piglet oral mucosa epithelial cell line and an establishment method and application thereof. The establishment method comprises the following steps: carrying out primary culture of piglet oral mucosa epithelial cells; extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes; replacing the culture medium of the cells 24 hours after the cells are transfected with the eukaryotic expression plasmids pCI-neo-hTERT; after 48 hours, adding a DMEM / F12 culture medium containing 500 mu g / ml of G418 for screening; marking positive clones under a high power lens, and transferring into a culture plate for culture; carrying out enlarged culture; screening for 1 month and not adding G418 into the culture medium any more, so that the cells obtained after culturing for over 50 generations are the immortalized cell line. The immortalized piglet oral mucosa epithelial primary cells are consistent and uniform in morphology.

Description

technical field [0001] The invention belongs to the technical field of animal cell engineering, and in particular relates to an immortalized piglet oral mucosa epithelial cell line and its establishment method and application. Background technique [0002] Oral mucosal epithelial cells, as the first line of defense of the mammalian immune system, are the earliest type of cells encountered by bacterial virus-infected hosts. Oral mucosal epithelial cells can generate immune responses like myeloid cells and play an important role in recognizing microbial antigens. There are great difficulties in the in vitro culture of oral mucosal epithelial cells. It is not easy to obtain materials, time-consuming and laborious, and the number of passages is limited. The stability and uniformity of cells are poor. research and application. Therefore, it is of great significance to establish a stable oral mucosal epithelial cell line to provide a good cell model for the research on the patho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12R1/91
CPCC12N5/0632C12N2510/04
Inventor 张彦明崔红杰郭抗抗
Owner 张彦明
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