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Construction of lung cancer drug screening cell line

A technology for human lung cancer cell lines and lung cancer, which is applied in the field of cell biology and can solve problems such as sensitive lung cancer cell lines

Active Publication Date: 2012-01-18
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, looking at the drug screening models of human lung cancer cells at home and abroad, there is still a lack of a lung cancer cell line that is sensitive to lung cancer drugs, can be stably expressed in vivo and in vitro for a long time, has high experimental repeatability, and is fluorescently labeled to meet the requirements of rapid screening of new lung cancer drugs. need

Method used

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  • Construction of lung cancer drug screening cell line
  • Construction of lung cancer drug screening cell line
  • Construction of lung cancer drug screening cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Packaging pseudo-lentiviral particles

[0056] 1. Lentiviral vector: Lentiviral vector system (Shanghai Jikai Gene Chemical Technology Co., Ltd.) is used. The viral packaging system consists of GV225 ( figure 1 ), pHelper1.0 ( figure 2 ), pHelper 2.0 ( image 3 ). Among them, GV225 (vector component) contains GFP gene and Puromycin (Puromycin) resistance gene, which is driven by the mU6 promoter; pHelper 1.0 contains the gag gene of HIV virus, which encodes the main structural protein of the virus; pol gene, which encodes the virus-specific enzyme; the rev gene, which encodes a regulator that regulates the expression of the gag and pol genes. pHelper2.0 contains the VSV-G gene derived from herpes simplex, which provides the envelope protein required for virus packaging.

[0057] 2. Amplification of plasmid DNA: Plasmids GV225, pHelper 1.0 and pHelper 2.0 were transformed into Escherichia coli DH5a by conventional methods, positive clones were screened wi...

Embodiment 2

[0062] The mensuration of embodiment 2 virus titer

[0063] Use 4×10 4 / 100uL / well of 293T cells was plated in a 96-well plate. Virus particles were serially diluted 10-fold with serum-free medium. Select the required cell wells, suck out 90uL of the medium, and add 90uL of the diluted virus solution. After being cultured in the incubator for 24 hours, 100uL of complete medium was added, and the resistant drug Puromycin (Sigma-Aldrich, Cat.P9620) was added 2 days after virus infection to maintain the drug concentration at 25ug / mL. After continuing to culture for 2 days, the fluorescence expression was observed under a fluorescent microscope (Olympus), and the titer value was judged by the number of living cells after infection.

Embodiment 3

[0064] Example 3 Obtaining double-labeled human lung cancer cell lines

[0065] 1. Virus infection of H1299 cells: human lung cancer H1299 cells (purchased from ATCC) in logarithmic growth phase were trypsinized to make cell suspension (cell density 5×10 4 / mL) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI=10), add an appropriate amount of virus, set 3 replicate wells in each group, replace the medium after 24 hours of culture, expand the cells after 72 hours of infection, and add 25ug / mL of virus to the expanded cells. Puromycin (Puromycin) was used for screening, and the cells after screening were observed in time for the positive rate of fluorescent expression cells to determine whether to continue to add drugs.

[0066] 2. Amplification of fluorescence expressing cells and identification of expression stability: Fluorescence expressing H1299 cells were continuously passaged in vitro f...

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Abstract

The invention belongs to the field of cell biology, and discloses a human lung cancer cell line. The present invention also discloses a construction method for the cell line and a use of the cell line. The human lung cancer cell line provided by the present invention has characteristics of high chromosome integration, high fluorescence intensity, stable genetic character and sensitivity to the lung cancer drug, and is an ideal study model for screening the lung cancer drugs.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to the establishment of a cell strain which can be used for lung cancer drug screening. Background technique [0002] Lung cancer is a disease that seriously threatens human health, and it is also a malignant tumor with the highest morbidity and mortality. The incidence of lung cancer in my country ranks first in the world and is on the rise. Although dozens of lung cancer drugs (including nucleic acid drugs, small molecule drugs, protein drugs, etc.) have been used in clinical treatment so far, most lung cancer drugs can only alleviate the disease, but cannot cure it. Therefore, there is an urgent need to screen out new and effective lung cancer drugs. [0003] High-throughput drug screening is currently the most popular method of drug development in the world, and the establishment of a high-throughput drug screening platform depends on the realization of a high-throughpu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/01C12Q1/06G01N21/64C12R1/91
Inventor 曹跃琼朱向莹杨松翁仕强谢胜华
Owner SHANGHAI GENECHEM
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