Humanized intestinal cancer precancerous lesion immortalized epithelial cell line and construction method and application thereof

A technology of epithelial cells and construction method, applied in the field of cell engineering, to achieve the effect of good application prospect and improvement of survival rate

Active Publication Date: 2020-05-19
YUEYANG INTEGRATED TRADITIONAL CHINESE & WESTERN MEDICINE HOSPITAL SHANGHAI UNIV OF CHINESE TRADITIONAL MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report about a humanized intestinal precancerous lesion immortalized

Method used

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  • Humanized intestinal cancer precancerous lesion immortalized epithelial cell line and construction method and application thereof
  • Humanized intestinal cancer precancerous lesion immortalized epithelial cell line and construction method and application thereof
  • Humanized intestinal cancer precancerous lesion immortalized epithelial cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 lentiviral overexpression vector construction

[0064] The purpose of the experiment: to construct the SV40LT gene into the pLVX-EF1α-IRES-Puro vector.

[0065] 1. Materials and Instruments

[0066] Main Reagents and Instruments

[0067] Table 1

[0068]

[0069] 2 Experimental methods

[0070] 2.1 Primer design

[0071] Design a pair of specific primers for SV40LT gene. The 5' and 3' ends of the target gene were amplified with target fragments with homologous sequences to both ends of the cloning vector pLVX-EF1α-IRES-Puro, and the primer sequences are shown in Table 2:

[0072] Table 2

[0073]

[0074] 2.2 Target gene PCR amplification

[0075] 2.2.1 Centrifuge the primers to the bottom of the Ep tube, and resuspend the primers in water to a concentration of 100 μM stock solution.

[0076] 2.2.2 Use pBABE-Puro-SV40 LT as a template to carry out PCR amplification according to the system shown in Table 3:

[0077] table 3

[0078] ...

Embodiment 2

[0097] Example 2 lentiviral packaging

[0098] 1. Purpose of the experiment

[0099] Using the calcium phosphate transfection method, the target plasmid and the lentiviral packaging plasmid were co-transfected into HEK293T cells to package the lentivirus and concentrate to the finished lentivirus.

[0100] 2. Materials and Instruments

[0101] 2.1 Sample situation

[0102] The pLVX-EF1α-IRES-Puro and pLVX-EF1α-SV40LT-IRES-Puro high-concentration endotoxin-free plasmids were constructed previously;

[0103] Lenti-Mix (pMDLg / pRRE, pVSV-G and pRSV-Rev);

[0104] HEK293T cell line, Escherichia coli DH5α competent.

[0105] 2.2 Reagents and instruments

[0106] table 5

[0107]

[0108]

[0109] 3. Experimental method

[0110] 3.1 Lentivirus packaging and concentration

[0111] 3.1.1 Dividing HEK293T cells: When HEK293T cells grow to about 80%-90% density in a 10cm cell culture dish, digest with 10% trypsin (trypsin stock solution 0.25% trypsin EDTA), collect HEK293T ...

Embodiment 3

[0163] Example 3 Establishment and testing of immortalized epithelial cell line for humanized intestinal precancerous lesions

[0164] 1. Equipment and reagents required for the test

[0165] (1) See Table 9 for the instruments.

[0166] Table 9

[0167]

[0168] (2) Table 10 for reagent consumables.

[0169] Table 10

[0170]

[0171]

[0172] 2. Construction method

[0173] 2.1 Isolation and culture of human polyp cells

[0174] 1) The polyp tissue removed during the operation is transported in sterile PBS buffer at low temperature;

[0175] 2) Take out the tissue in the ultra-clean workbench, soak it in 75% alcohol for about 2 minutes, and place it in PBS containing P / S;

[0176] 3) Cut the tissue block into a square with a side length of about 0.1 cm, discard the supernatant after repeated washing, place it in a petri dish containing complete medium, and incubate at 37°C for 30-60 min;

[0177] 4) Clip it into the culture bottle with tweezers, and place it ...

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Abstract

The invention relates to a humanized intestinal cancer precancerous lesion immortalized epithelial cell line and a construction method and application thereof. The construction method comprises the following steps: firstly, transfecting primarily separated human colorectal adenoma polyp epithelial cells by using an SV40 overexpression lentiviral vector, then performing screening by using puromycin, and finally amplifying the screened cells to obtain the humanized intestinal cancer precancerous lesion immortalized epithelial cell line. The humanized intestinal cancer precancerous lesion immortalized epithelial cell line constructed by the invention overcomes the problems that conventional adenoma polyp cannot be subjected to passage in vitro, is low in cell proliferation and poor in cell activity, and cannot meet the cell passage requirement. According to the invention, the humanized intestinal cancer precancerous lesion immortalized epithelial cell line is established for the first time, an important cell experiment tool is provided for developing an in-vitro experiment of intestinal cancer precancerous lesions, and preclinical researches such as new drug screening and drug effectcomponents are also convenient to develop.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a humanized intestinal precancerous lesion immortalized epithelial cell line, a construction method and application thereof. Background technique [0002] Immortalized cells have the ability to proliferate indefinitely. Immortalized cells can provide a stable and uniform source of cells, which is convenient for widespread use, and can reduce the cost of cell materials and facilitate standardization. This technology is currently used in retinal cells, liver cells, alveolar epithelial cells, etc. This technology requires that the cells are free of pollution. The liver, lung tissue, and retinal tissue are solid organs, and there is no possibility of contact with bacterial contamination. It is relatively easy to establish immortalization cell line. [0003] The primary human intestinal adenomatous polyposis cells are benign lesions, which are precancerous lesions of the in...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0679C12N15/86C12N2510/04C12N2531/00C12N2740/15043C12N2800/107
Inventor 付晓伶张华月熊光苏郭翠徐祎敏龚亚斌李园
Owner YUEYANG INTEGRATED TRADITIONAL CHINESE & WESTERN MEDICINE HOSPITAL SHANGHAI UNIV OF CHINESE TRADITIONAL MEDICINE
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