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Nucleic acid aptamer for classification of different-subtype non-small cell lung cancers and screening method thereof

A nucleic acid aptamer and cell technology, applied in biochemical equipment and methods, individual particle analysis, material excitation analysis, etc., can solve the problems of insufficient specificity and sensitivity

Inactive Publication Date: 2012-04-18
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although tumor markers such as neuron-specific enolase, cytokeratin fragment 21-1, carcinoembryonic antigen, and carbohydrate antigen are currently used in the detection and postoperative evaluation of lung cancer, these tumor markers have different effects on lung cancer, especially lung cancer. Insufficient specificity and sensitivity for lung cancer subtypes

Method used

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  • Nucleic acid aptamer for classification of different-subtype non-small cell lung cancers and screening method thereof
  • Nucleic acid aptamer for classification of different-subtype non-small cell lung cancers and screening method thereof
  • Nucleic acid aptamer for classification of different-subtype non-small cell lung cancers and screening method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Screening of nucleic acid aptamers for typing different subtypes of non-small cell lung cancer

[0045] 1. Design and synthesis of random nucleic acid library

[0046] Design and synthesize a random nucleic acid sequence library comprising 20 nucleotides at both ends and 45 nucleotides in the middle as follows: 5'-ACGCTCGGATGCCACTACAG(N 45 ) CTCATGGACGTGCTGGTGAC-3'.

[0047] 2. Screening of nucleic acid aptamers

[0048] Dissolve the random nucleic acid library in binding buffer (PBS, 150mM NaCl, 5mM MgCl 2 , 1mg / ml yeast transfer RNA), incubated with lung adenocarcinoma cell line A549 cells on ice for 1 hour; washed with buffer solution (PBS, 150mMNaCl, 5mM MgCl 2 ) after washing, the A549 cells were scraped off, and then the DNA bound to the cell surface was dissociated by heating; the dissociated DNA was incubated with the large cell lung cancer cell line HLAMP cells on ice for 1 hour, and the supernatant was collected and used DNA was used as a templa...

Embodiment 2

[0061] Example 2. Applying nucleic acid aptamers to type different subtypes of small cell lung cancer

[0062] The four aptamers obtained in Example 1 were labeled with fluorescein isothiocyanate (FITC). The random nucleic acid library of Example 1 was labeled with fluorescein isothiocyanate (FITC).

[0063] Different cultured tumor cells were washed twice with PBS, then treated with 0.02% EDTA for 3 minutes, and washed twice with PBS.

[0064] Each tumor cell was subjected to the following four groups of treatments, and each treatment was repeated three times, and the results were averaged:

[0065] Treatment 1: by cell counting, about 300,000 cells were respectively dispersed in the binding buffer solution, and then fluorescein isothiocyanate (FITC) was added to label the nucleic acid aptamer 1 (the final concentration was 100 nM).

[0066] Treatment 2: by cell counting, about 300,000 cells were collected and dispersed in the binding buffer solution, and then fluorescein i...

Embodiment 3

[0075] Example 3. Detection of the binding of the nucleic acid aptamers listed in Table 1 to lung cancer tissue sections

[0076] Formaldehyde-fixed paraffin-embedded tissue sections from different volunteers with lung cancer were dewaxed twice in xylene at room temperature for 20 minutes each time, and then immersed in a series of ethanol for hydration (100%, twice, 1 minute each time) ; 95% ethanol, 1 time, 1 minute; 70% ethanol, 1 time, 1 minute). The hydrated tissue sections were then immersed in buffer solution and heated at 95°C for 15 minutes. After the tissue sections returned to room temperature, they were incubated with binding buffer containing 20% ​​fetal bovine serum and calf thymus DNA for 1 hour at room temperature. After washing, the tissue sections were respectively combined with tetramethylrhodin-labeled nucleic acid aptamers (200nM) Incubate in buffer for 1 hour at room temperature. After staining, the tissue sections were washed 3 times with washing buffe...

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Abstract

The invention discloses a nucleic acid aptamer for classification of different-subtype non-small cell lung cancers and a screening method thereof. The nucleic acid aptamer provided by the invention is a DNA segment which comprises nucleotide shown as any one of sequences 1 to 9 in a sequence table. The nucleic acid aptamer can be applied to the classification of the different-subtype non-small cell lung cancers. By using the nucleic acid aptamer of the invention, the differentiation of the different-subtype non-small cell lung cancers can be realized on a molecular response signal under the condition that tumor markers of the non-small cell lung cancers are unknown. When used for identifying a target combined with the nucleic acid aptamer, the nucleic acid aptamer is favorable for findingout the tumor markers of the different-subtype non-small cell lung cancers, performing earlier diagnosis and more accurate classification of the non-small cell lung cancers and finding out new medicament acting target points for tumor therapy.

Description

[0001] This application is a divisional application with an application number of 200910083075.X, an application date of April 28, 2009, and an invention titled "nucleic acid aptamers and screening methods for different subtypes of non-small cell lung cancer". technical field [0002] The invention relates to a nucleic acid aptamer for typing different subtypes of non-small cell lung cancer and a screening method thereof. Background technique [0003] Lung cancer is one of the deadliest cancers, histologically divided into small cell lung cancer and non-small cell lung cancer, with the latter accounting for 80% of lung cancer cases. Non-small cell lung cancer is divided into three subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Adenocarcinoma, the most dominant subtype, accounts for 40% of NSCLC. The prognosis of non-small cell lung cancer depends heavily on the stage of the disease at the time of diagnosis. According to the current treatment lev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68C12Q1/02G01N15/10G01N21/64
Inventor 方晓红赵子龙徐丽上官棣华谭蔚泓
Owner INST OF CHEM CHINESE ACAD OF SCI
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