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Application of TCRP1 gene in the preparation of tumor cell platinum resistance reversal agent

A tumor cell, drug resistance reversal technology, applied in anti-tumor drugs, gene therapy, drug combination and other directions, can solve the problems of weakened cell damage ability, unclear biological function, abnormal DNA methylation signal transduction pathway, etc. Tolerance, the effect of improving efficacy

Active Publication Date: 2013-04-24
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

4. The ability of platinum drugs to damage cells is weakened

Method used

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  • Application of TCRP1 gene in the preparation of tumor cell platinum resistance reversal agent
  • Application of TCRP1 gene in the preparation of tumor cell platinum resistance reversal agent
  • Application of TCRP1 gene in the preparation of tumor cell platinum resistance reversal agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Cell Culture

[0047] A549 (human lung cancer cell line), A549 / DDP (drug-resistant substrain of A549 cell), Coc1 (human ovarian cancer cell line), Coc1 / DDP (drug-resistant substrain of Coc1 cell), Tca8113 (human tongue squamous cell carcinoma cell line) , purchased from China Center for Type Culture Collection. Tca8113 / PYM was induced by our laboratory. Tongue cancer cell line Tca8113 cells were treated with 4 doses of pingyangmycin (PYM) (1, 2, 5 and 10 μg / mL) repeatedly intermittently for 24 h or 48 h, until the cells could be treated with 100 ng / mL of PYM. It grows exponentially in the medium, and the drug-resistant cell line is named Tca8113 / PYM.

[0048] Human lung cancer tumor cell lines, SK-MES-1 (lung squamous cell carcinoma cells), LTEP-a-2 (lung adenocarcinoma cells), SPC-A-1 (lung adenocarcinoma cells), 95D (highly metastatic lung cancer cells), NCI-H1299 (non-small cell lung cancer cells), NCI-H1975 (lung adenocarcinoma cells), NCI-H446...

Embodiment 2

[0049] Example 2: Cell line drug resistance analysis

[0050] Select the cultured cells grown to the logarithmic phase, discard the cell culture medium, rinse twice with 1*PBS solution, digest and make a cell suspension, count with a counter and inoculate 1000-5000 per well into a 96-well culture plate (180 μl / Wells) were incubated at 37°C in a 5% CO2 incubator until the cells adhered to the wall. Add different concentrations of drugs (such as cisplatin, oxaliplatin) to 96-well culture plate, 5 wells of each concentration, and incubate in a 37°C incubator for 72 hours; add 20 μl of MTS to each well, mix well and incubate for 3 hours. The OD value of each group at 490 wavelength was detected on a microplate reader. Calculate the average cell survival rate (Survival Rate), draw a regression curve with the drug concentration as the abscissa, and the cell survival rate (Survival Rate) as the ordinate, and calculate the IC50 value through the curve. The above experiments we...

Embodiment 3

[0053] Example 3: Real-time quantitative PCR analysis of the expression of TCRP1 in various cell lines

[0054] Various cultured cells grown to logarithmic phase were taken, and total RNA was extracted by TRIZOL method. After DNaseI digestion, measure RNA. Use oligdT as a primer to take an equal amount of RNA and reverse transcribe it into cDNA according to the procedure of Fermentas Reverse Transcription System. Using cDNA as a template, according to the SYBGreen operating instructions, use the following primers for real-time quantitative PCR amplification, primers for the coding region of the TCRP1 gene:

[0055] SEQ ID NO: 6 Forward: 5'-CCAATAGTCCCAGTTATGCTCCA-3'

[0056] SEQ ID NO: 7 Reverse: 5'-TGCTTGGTAAGTTCGGTTCTCG-3'

[0057] The amplified fragment is 134bp

[0058] GAPDH gene coding region primers:

[0059] SEQ ID NO: 8 Forward: 5'-ATTCCATGGCACCGTCAAGGCTGA-3'

[0060] SEQ ID NO: 9 Reverse: 5'-TTCTCCATGGTGGTGAAGACGCCA-3'

[0061] The amplified fragmen...

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Abstract

The invention discloses a self-cloning new human tumor drug-resistant related gene, namely TCRP1, wherein the gene and drug resistance generation of tumor chemotherapy platinum drugs has confidential relation. The expression of TCRP1 in platinum secondary drug-resistant tumor cell strains such as Tca8113 / PYM, A549 / DDP, Coc1 / DDP is raised, and the expression of TCRP1 in primary platinum drug-resistant cell strain such as lung cancer 95D is obviously superior to that of other sensitive cell strains of lung cancer. The gene is located in human chromosome 11q13.4, wherein the total length of sequence is 1834bp, the open reading frame is 708bp, and 235 amino acids are encoded. The application not only provides new information for tumor drug-resistant mechanism, but also provides new target for the design of anti-tumor drug-resistant drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new application of TCRP1 gene in preparing a tumor cell platinum resistance reversal agent. Background technique [0002] Since cisplatin (cisplatin) was discovered to have antitumor activity in 1967, the synthesis, application and research of platinum-based metal antitumor drugs have developed rapidly. At present, its development has experienced three generations: cisplatin, carboplatin and oxali platinum. With its good curative effect, platinum drugs have been widely used in the first-line treatment of various malignant solid tumors, such as ovarian cancer, prostate cancer, testicular cancer, lung cancer, colon cancer, nasopharyngeal cancer and other head and neck solid tumors. Indispensable drug for cancer chemotherapy. According to statistics, platinum-based chemotherapy regimens or chemotherapy regimens with platinum drugs account for 70%-80% of all chemotherapy ...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17C12N15/113A61P35/00
Inventor 贺智敏谷依学郑国沛张志杰
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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