Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection

A nucleic acid aptamer and screening method technology, applied in biochemical equipment and methods, measuring devices, individual particle analysis and other directions, can solve the problems that restrict the development of targeted diagnosis and treatment of prostate cancer, and it is difficult to determine the site and location of tumor recurrence, It is difficult for clinicians to choose a treatment plan to achieve the effect of short cycle, small molecular weight and strong binding ability

Active Publication Date: 2014-03-19
GUANGZHOU SHIWEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, as far as patients with advanced prostate cancer are concerned, the treatment methods are limited and the effect is poor. The reason is that the drug dosage is high and the side effects are large. In addition to the development of new drugs, targeted therapy has become the most important method.
In addition, for patients with biochemical recurrence after radical prostatectomy, there is currently a lack of effective targeted diagnostic measures, and it is difficult to determine the site and location of tumor recurrence, making it very difficult for clinicians to choose a reasonable treatment plan
At present, the lack of highly specific targeting mediators is a bottleneck restricting the development of targeted diagnosis and treatment of prostate cancer

Method used

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  • Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection
  • Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection
  • Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Screening of prostate cancer cell-specific nucleic acid aptamers

[0081] 1. Design and synthesis of random nucleic acid library

[0082] Design and synthesize a nucleic acid sequence library containing 20 nucleotides (primers) at both ends and a random sequence of 45 nucleotides in the middle as follows:

[0083] 5'-ACGCTCGGATGCCACTACAG-(45N)-CTCATGGACGTGCTGGTGAC-3'.

[0084] 2. Cell-SELEX screening to obtain nucleic acid aptamers

[0085] 2.1 Incubation: use binding buffer solution (PBS, containing 0.45% glucose, 5mM MgCl 2 , 0.1mg / mL herring sperm DNA) to dissolve the above random nucleic acid library, shake at 95°C for 5 minutes, and quickly put it on ice; then incubate with the cultured and pretreated prostate cancer cell line PC-3 on ice for 1 hour .

[0086] 2.2 Dissociation: After the incubation, pour out the liquid in the incubation flask, wash with washing buffer (PBS, containing 0.45% glucose, 5mM MgCl 2 ) to wash the cells in the incubation f...

Embodiment 2

[0107] Example 2: Detection of cell specificity

[0108] A total of 12 cell lines were selected for specificity analysis: three prostate cancer cell sublines were PC-3, LNCap, DU-145, and 22RV-1; immortalized normal prostate epithelial cells RWPE-1; liver cancer cells SMMC -7721; cervical cancer cells HeLa, lung cancer cells A549, MCF-7; colon cancer sub-cell line LoVo; leukemia cells Jurkat, K562.

[0109] Each cell line was subjected to the following steps (1) and (2), repeated three times.

[0110] 1. By cell counting, take 5×10 5 The cells were dispersed in the binding buffer, and then the labeled nucleic acid aptamer was added with a final concentration of 0.25 μM.

[0111] 2. Incubate the mixture of cells and nucleic acid aptamers on ice for 20 minutes, centrifuge and wash twice, and use flow cytometry to detect the combination of nucleic acid aptamers and the above-mentioned different cell lines. The cells were incubated with the FITC-labeled random library and then ...

Embodiment 3

[0116] Example 3: Targeted inhibitory effect of doxorubicin-loaded aptamers on cells

[0117] Inoculate 2000 cells per well in a 96-well plate, and after culturing for 24 hours, add 2 μM doxorubicin and 0.5 μM doxorubicin-loaded aptamer every 4 hours (1 molecular sequence and 2 aptamers can carry 4 molecules of doxorubicin After incubation for 15 minutes, the drug-containing medium was withdrawn, the cells were rinsed once, and 100 μL of fresh medium was added, a total of 4 times. After continuing to cultivate for 48 hours, add 10 μL cck-8 to each well, incubate at 37°C for 40 minutes, and measure the absorbance on a microplate reader at a wavelength of 450 nm ( Figure 4 ).

[0118] Depend on Figure 4 It can be seen that the drug-loaded aptamer significantly enhanced the inhibition of the proliferation of the target cell PC-3 (1), but the inhibitory effect on HeLa (2) and SMMC-7721 (3) cells was not obvious.

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Abstract

The present invention discloses a nucleic acid aptamer, wherein the sequence of the nucleic acid aptamer comprises a DNA segment represented by any one sequence selected from a sequence 1, a sequence 2 and a sequence 3. The nucleic acid aptamer can further be various similar sequences with high homology or a derivative obtained from the sequence of the present invention. The invention further discloses a nucleic acid aptamer screening method, which comprises: synthesizing a random single-stranded DNA library and primers, carrying out SELEX screening, carrying out PCR amplification of library, preparing a DNA single strand library, and finally carrying out repeated screening, negative screening and multi-round screening to obtain the nucleic acid aptamer. The nucleic acid aptamer and the derivative thereof can be used in recognition of the prostate cancer cell strain PC-3 or preparation of kits, molecular probes and targeted mediums for prostate cancer detection, and can further be used in design and preparation of prostate cancer treatment drugs. Compared with the protein antibody, the nucleic acid aptamer of the present invention has advantages of high affinity, high specificity, no immunogenicity, capability of being chemically synthesized, small molecular weight, stability, easy storage, easy labeling and the like.

Description

technical field [0001] The invention relates to a nucleic acid aptamer and its screening and application, in particular to a nucleic acid aptamer which can be used to detect prostate cancer cells and its screening method and application. Background technique [0002] Prostate cancer (PCa) is one of the most common malignant tumors in men in European and American countries, and its mortality rate is second only to lung cancer. In recent years, with the increase of the aging population and the change of diet structure, the incidence of PCa in my country has shown an obvious upward trend. Domestic research reports show that the incidence of PCa in Shanghai has jumped to the first place in male urological tumors. Due to the insidious onset of prostate cancer, most prostate cancers are already in the advanced or advanced stage when they are diagnosed. At present, endocrine therapy is used clinically for prostate cancer that has developed to this stage. Although endocrine therapy ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68G01N15/14
Inventor 高新上官棣华罗云邴涛王元元
Owner GUANGZHOU SHIWEN BIOTECHNOLOGY CO LTD
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