Fluorescent cell model for screening of antitumor drugs, labeling method and application thereof

An anti-tumor drug and cell model technology is applied to a fluorescent cell model for anti-tumor drug screening and its labeling and application fields, and can solve the problems of inability to detect the absolute number of cells, influence on the accuracy of experimental results, and increased workload. To achieve the effect of clear drug action mechanism, small error and easy operation

Inactive Publication Date: 2011-10-19
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

However, since the formazan product produced by the reduction of MTT is insoluble in water, it needs to be dissolved before detection, which not only increases the workload, but also affects the accuracy of the experimental results.
Moreover, MTT is carcinogenic, and the organic solvent that dissolves formazan is also harmful to the experimenter.
Most importantly, MTT can only be used to detect the relative number and relative viability of cells, but not the absolute number of cells

Method used

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  • Fluorescent cell model for screening of antitumor drugs, labeling method and application thereof
  • Fluorescent cell model for screening of antitumor drugs, labeling method and application thereof
  • Fluorescent cell model for screening of antitumor drugs, labeling method and application thereof

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Experimental program
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Embodiment 1

[0030] Establishment of U2OS cell line (U2OS-EGFP cell line) efficiently and stably expressing enhanced green fluorescent protein (EGFP)

[0031] 1. Transfection of U2OS cells with neo-resistant EGFP plasmid

[0032] 1) U2OS cells were subcultured for 2-3 times in order to maintain the cells in a good growth state and prepare for plating;

[0033] 2) Spread 24-well plates, a 90% full 10cm dish cells can be plated in 24 wells, and spread in 24-well cell culture plates according to the amount of 500 μL medium per well. Observe the cell adhesion every 20 minutes, and shake the plate to spread the cells in the well as evenly as possible;

[0034] 3) Incubate at 37°C for 1-2 hours in 5% CO2, and prepare one well of a 24-well plate for transfection after the cells adhere to the wall;

[0035] 4) Transfection step: according to DNA concentration of 0.4 μg / mL and PEI concentration of 0.8 μg / mL, dissolve in 200 μD DEM basal medium to prepare transfection solution; after incubating at...

Embodiment 2

[0048] Establishment of a method for screening antitumor drugs using U2OS-EGFP cell model

[0049] 1. Screening of anti-tumor drug test scheme by fluorescence analysis method based on U2OS-EGFP cells

[0050] The first stage: use high-concentration samples to analyze the cell growth inhibition rate of the sample, and initially determine whether the sample has the activity of inhibiting cell growth;

[0051] The second stage: In this stage, the samples are divided into high, medium and low concentration groups, and the statistics of the cell growth inhibition rate and the significant difference of the samples are carried out to accurately determine whether the sample has cell growth inhibition activity, and to screen the cells with a cell growth inhibition rate greater than 50%. Samples are hits;

[0052] The third stage: In this stage, the gradient dilution method is used to divide the samples into 9 concentration groups, analyze the relationship between the sample concentrat...

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Abstract

A fluorescent cell model for the screening of antitumor drugs in the bioengineering technology field, and a labeling method and application thereof. The fluorescent cell model is a fluorescent substance-containing cell model; the fluorescent substance is a green fluorescent protein; and the cells are U2OS human osteosarcoma cells. According to the labeling method provided by the invention, stable cells for expressing an enhanced green fluorescent protein are established by the transfection of enhanced green fluorescent protein into cells. The application: the fluorescent cell model for the screening of antitumor drugs can be used to detect the fluorescence value change of a cell lysate. According to the invention, the change of cell survival and growth is directly reflected by the determination of fluorescence values. The established screening method of antitumor drugs has a clear pharmacological mechanism, is simple, rapid, direct and economical with little error and high sensitivity, and is suitable for accurate high throughput screening of antitumor drugs.

Description

technical field [0001] The invention relates to a cell model in the technical field of bioengineering and a labeling method thereof, in particular to a fluorescent cell model for screening antitumor drugs, a labeling method and application thereof. Background technique [0002] Cancer, a general term for various malignant tumors, whose cells grow and divide faster than normal cells, and can often metastasize to other tissues. Tumor is one of the diseases that seriously threaten human health. However, anticancer drugs used in clinical practice generally have problems such as poor selectivity, high toxicity and side effects, and easy drug resistance. Therefore, we continue to search for anticancer drugs with high selectivity and low toxicity. It is the focus of anti-tumor drug research. [0003] Current screening methods for antineoplastic drugs include in vitro and in vivo screening methods. The in vivo screening method, that is, animal experiments, is a method in which dru...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/63C12N15/11C12Q1/02G01N21/64C12R1/91
Inventor 李大伟D·柯伊拉腊徐维果武正华吴凤娟
Owner SHANGHAI JIAO TONG UNIV
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