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Aptamer for recognizing adriamycin-resistant breast cancer cells as well as screening method and application of aptamer

A breast cancer cell and nucleic acid aptamer technology, which is applied in the field of identifying adriamycin-resistant breast cancer cell nucleic acid aptamers and its screening, can solve the problems of cumbersome steps, high cost, and long time consumption, and achieve high affinity and high specificity sex, increase the success rate of chemotherapy

Active Publication Date: 2015-04-08
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the mainstream method for detecting tumor drug resistance is RT-PCR technology, which uses real-time quantitative PCR to relatively quantitatively detect the expression level of related drug resistance gene mRNA. Although this method has high sensitivity, the steps are cumbersome, time-consuming, and expensive. , and it is impossible to observe the position of cancer drug resistance directly and in situ

Method used

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  • Aptamer for recognizing adriamycin-resistant breast cancer cells as well as screening method and application of aptamer
  • Aptamer for recognizing adriamycin-resistant breast cancer cells as well as screening method and application of aptamer
  • Aptamer for recognizing adriamycin-resistant breast cancer cells as well as screening method and application of aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1, Screening and Preparation of Nucleic Aptamers

[0055] 1. Culture of doxorubicin-resistant breast cancer cells and sensitive breast cancer cells

[0056] Doxorubicin-resistant breast cancer cells (MCF-7 / Adr) (purchased from Shanghai Aiyan Biotechnology Co., Ltd.) were treated with RPMI 1640 (containing 10% fetal bovine serum, 1% penicillin / streptomycin) supplemented with 1 μg / mL doxorubicin prime) culture, inoculated into doxorubicin-free medium before use and cultivated for one generation;

[0057] Sensitive breast cancer cells (MCF-7) (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in DMEM (containing 10% fetal bovine serum, 1% penicillin / streptomycin). All cells were routinely cultured in an incubator (37°C, 5% CO 2 ), passaged every two days.

[0058] 2. Design of Random Nucleic Acid Library

[0059] Design a random nucleic acid library with 20 fixed nucleotides at both ends and 45 nucleotides in the middle: 5'-AAGGAGCAG...

Embodiment 2

[0080] Example 2, flow cytometric analysis to detect the binding ability of nucleic acid aptamers to doxorubicin-resistant breast cancer cells MCF-7 / Adr

[0081] The obtained nucleic acid aptamer 2 to nucleic acid aptamer 6 and the control sequence (SEQ ID No. 7) were labeled with a fluorescein (FAM) molecule at the 5' end. Each single-stranded DNA was dissolved with binding buffer, and the concentration was calibrated according to ultraviolet absorption, heated at 95°C for 5 minutes, placed on ice for 10 minutes, and left at room temperature for 30 minutes. The denatured-refolded DNA is diluted with binding buffer to a DNA solution with a concentration gradient of 1nmol / L, 2nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 50nmol / L, 100nmol / L and 200nmol / L . Take a plate of doxorubicin-resistant breast cancer cells in the logarithmic growth phase, digest them with 0.2% EDTA into a monodisperse cell suspension, divide them into several parts, incubate with the above DNA solution on ice fo...

Embodiment 3

[0083] Embodiment 3, the specificity of nucleic acid aptamer detection by flow cytometry

[0084] 1. Nucleic acid aptamer probe pretreatment

[0085] Dissolve the synthesized fluorescein (6-FAM)-labeled aptamer 2, aptamer 3, aptamer 4, aptamer 5 and aptamer 6 and the control sequence (SEQ ID No.7) in A 20 μM DNA solution was obtained in the binding buffer, denatured by heating at 95° C. for 5 minutes, placed on ice for 10 minutes, and left at room temperature for 30 minutes.

[0086]2. Pretreatment of cell lines

[0087] Human doxorubicin-resistant breast cancer cells (MCF-7 / Adr), human breast cancer cells (MCF-7), human breast cancer cells (SK-BR-3), human breast cancer cells (MDA-MB-231), Human vincristine-resistant oral cancer cells (KB / VCR), human paclitaxel-resistant lung cancer cells (A549T), human paclitaxel-resistant colon cancer cells (HCT-8T), human embryonic kidney cells (HEK293), human oral cancer cells (KB) , human lung cancer cells (A549), human colon cancer c...

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Abstract

The invention discloses an aptamer for recognizing adriamycin-resistant breast cancer cells. The aptamer can be a single-stranded DNA molecule. Particularly, the aptamer can be an aptamer (1) formed by nucleotide molecules as shown in SEQ ID No. 1, an aptamer (2) formed by nucleotide molecules as shown in SEQ ID No. 2, an aptamer (3) formed by nucleotide molecules as shown in SEQ ID No. 3, an aptamer (4) formed by nucleotide molecules as shown in SEQ ID No. 4, an aptamer (5) formed by nucleotide molecules as shown in SEQ ID No. 5 and an aptamer (6) formed by nucleotide molecules as shown in SEQ ID No. 6. By adopting the aptamer disclosed by the invention, the resistance to medicines of tumors can be monitored in real time according to molecular response signals under the condition that the medicine resistance mechanism is unknown so that the treatment scheme can be changed timely and the success rate of chemotherapy can be increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer for recognizing doxorubicin-resistant breast cancer cells and its screening method and application. Background technique [0002] Breast cancer is the most common malignant tumor in women, mainly including ductal carcinoma and lobular carcinoma. In 2012, the 2009 breast cancer incidence data released by the National Cancer Center and the Ministry of Health's Bureau of Disease Control and Prevention showed that the incidence of breast cancer in the national tumor registration area ranked first among female malignancies, and it showed a trend of increasing year by year. Doxorubicin is one of the common first-line chemotherapy drugs for breast cancer; however, during the course of treatment, tumor cells gradually become resistant to doxorubicin and at the same time develop resistance to other chemotherapy drugs, that is, multidrug resistance . The emergence of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68
Inventor 上官棣华张楠邴涛刘祥军
Owner INST OF CHEM CHINESE ACAD OF SCI
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