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Aptamer for typing different subtypes of non-small cell lung cancer and method for screening the same

A non-small cell lung cancer and nucleic acid aptamer technology, which is applied in library screening, biochemical equipment and methods, and microbial measurement/testing, can solve the problems of insufficient specificity and sensitivity, and achieve accurate diagnosis

Active Publication Date: 2009-09-23
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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  • Aptamer for typing different subtypes of non-small cell lung cancer and method for screening the same
  • Aptamer for typing different subtypes of non-small cell lung cancer and method for screening the same
  • Aptamer for typing different subtypes of non-small cell lung cancer and method for screening the same

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, 150mM NaCl, 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 templ...

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

[0076] Example 3. Detection of the binding of the nucleic acid aptamers listed in Table 1 to lung cancer tissue sections

[0077] 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 aptamer for typing different subtypes of non-small cell lung cancer and a method for screening the same. The aptamer provided by the invention is any DNA segment of nucleotide expressed by sequence 1 to sequence 9 in a sequence table. The aptamer is applied for typing different subtypes of non-small cell lung cancer. The aptamer of the invention can differentiate the different subtypes of non-small cell lung cancer on the molecule response signal under the condition of not knowing tumor markers of the non-small cell lung cancer. Using the aptamer of the invention for identifying the combined target is good for discovering the tumor markers of the different subtypes of non-small cell lung cancer, and earlier and exactly diagnosing and typing non-small cell lung cancer and discovering the tumor for treating new drug action targets.

Description

technical field [0001] 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 [0002] 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 level, the 5-year survival rate of patients drops from 60% in stage I to 1% in stage IV. Although great progress has been made in the detection methods of lung cancer, about 70% of the patients are still in the advanced stage of lung cancer when they are diagnosed....

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12N15/10C40B30/04C12P19/34C12N15/115
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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