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Cell line used for susceptibility detection of lung cancer chemotherapy drugs and establishment thereof

A chemotherapeutic drug and sensitivity technology, which is applied to the measurement/testing of cells modified by the introduction of foreign genetic material, microorganisms, and the use of vectors to introduce foreign genetic material, etc. It can solve the problems of no lung cancer chemotherapy sensitivity prediction method, etc.

Inactive Publication Date: 2010-03-10
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no mature method for predicting chemotherapy sensitivity of lung cancer in the world at present.

Method used

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  • Cell line used for susceptibility detection of lung cancer chemotherapy drugs and establishment thereof
  • Cell line used for susceptibility detection of lung cancer chemotherapy drugs and establishment thereof
  • Cell line used for susceptibility detection of lung cancer chemotherapy drugs and establishment thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Establishment of the cell line of the present invention

[0016] The NIH3T3 cells were cultured with 10% calf serum plus DMEM (GIBCO, product of invitrogen), and placed at 37°C, 5% CO 2 Cultivate in an incubator for 3 days, plant a 12-well plate, and replace with antibiotic-free medium to continue culturing for 24 hours. Use Lipofectamine 2000 TM Transfect 2μg of eIF3a eukaryotic expression vector pCβA-eIF3a at a ratio of 1:2.5. The specific construction method is to amplify the cDNA of eIF3a, with Not I and Kpn I restriction sites on both ends, and then connect to pCβA On an empty carrier. The specific sequence is shown in the following sequence table) into the cell. 48 hours after transfection, it was diluted and passaged into a 100mm cell culture dish at a volume ratio of 1:10, and the inoculum was 1ml. 600μg / ml G418 was added for screening for 2 weeks, and single clones were selected and expanded. After verification by Real-time PCR and Western Blot, it w...

Embodiment 2

[0017] Example 2: Specific detection method

[0018] (1) Take the logarithmic growth phase NIH3T3 cells, adjust the concentration of the cell suspension, plant them in a 96-well plate, 100ul per well, 5000 cells / well;

[0019] (2) 37℃, 5% CO 2 Cultivate for 24 hours;

[0020] (3) Add different concentrations of test drugs (different types of lung cancer chemotherapy drugs), and continue to culture for 3 days. The concentration used is 0, 0.01, 0.1, 1, 10, 100uM;

[0021] (4) Select the cells on day 1, 2, and 3, add 20ul MTT / well, and continue to culture for 4-6 hours;

[0022] (5) Use a microplate reader to detect the absorbance at 490nm;

[0023] (6) Draw a dose-effect curve based on the absorbance of different concentrations of drugs, and calculate the IC50 of different groups, and use analysis of variance to compare the IC50 of cells with and without eIF3a expression to determine whether there is a difference. If there is a difference, it means that patients with different expressio...

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Abstract

The invention provides a cell line used for susceptibility detection of lung cancer chemotherapy drugs and establishment thereof. The establishment thereof comprises the following processes: culturingNIH3T3 cell by using 10 percent of calf serum added with DMEM, placing the NIH3T3 cell into an incubator with the temperature of 37 DEG C and 5 percent of CO2 for culturing for 3 days, growing 12-hole plate, and changing with a culture medium without antibiotics to continue culturing for 24h; according to the ratio of 1:2.5, carrying out transfection of 2 he of eIF3a eukaryotic expression vectorpCbA-eIF3a to the NIH3T3 cell, wherein the sequence of the vector pCbA-eIF3a is shown in a sequence table; and after transfection for 48h, according to the volume ration of 1:10, diluting transfer ofculture to a cell culture dish of 100mm, with 1ml of inoculation quantity; adding G418 of 600he / ml, screening for two weeks, selecting monoclone and expanding culture, and establishing the cell line.The establishing method of the cell line is simple and convenient in operation, steady in conditions and low in cost; and the established cell line has stable expression and good prediction effect.

Description

Technical field [0001] The method relates to a stable expression cell line and its establishment. The cell line can detect the drug sensitivity of the existing lung cancer chemotherapy drugs and predict the chemotherapy efficacy of the patient. Background technique [0002] Lung cancer is a common tumor that endangers human life and health in my country and worldwide. In the world, the incidence and mortality of lung cancer ranks first among cancers. The 5-year survival rate is only about 10%, making the prevention and treatment of lung cancer the most important and difficult task. The incidence and mortality of lung cancer in my country have been showing a significant upward trend. Compared with the 1970s in the 1990s, the mortality rate of lung cancer in my country increased by 111.85%. At present, the incidence of lung cancer in my country has exceeded 3 per 10,000, reaching 35 per 100,000, making it the malignant tumor with the highest incidence. At the same time, the mor...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12Q1/02
Inventor 刘昭前尹继业黄琼欧阳冬生张建亭周宏灏
Owner CENT SOUTH UNIV
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