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Targeted aptamer for human poorly differentiated gastric carcinoma cells and application of targeted aptamer

A nucleic acid aptamer and gastric cancer cell technology, applied in the field of biomedicine, to achieve the effect of low differentiation tissue specificity, high specificity and high affinity

Active Publication Date: 2019-04-02
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there is no report on the screening of human poorly differentiated gastric cancer cell-specific nucleic acid aptamers using poorly differentiated gastric cancer cells as target cells and moderately and highly differentiated gastric cancer cells as non-target cells

Method used

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  • Targeted aptamer for human poorly differentiated gastric carcinoma cells and application of targeted aptamer
  • Targeted aptamer for human poorly differentiated gastric carcinoma cells and application of targeted aptamer
  • Targeted aptamer for human poorly differentiated gastric carcinoma cells and application of targeted aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Screening of nucleic acid aptamers.

[0024] (1) Preparation of random screening library.

[0025] Entrust Sangon Bioengineering Co., Ltd. to synthesize a random single-stranded DNA library (5'-AAGGAGCAG CGTGGAGGATANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTAGGGTGTGTCGTCGTGGT -3'); take 1 tube of 10OD random single-stranded DNA library, add binding buffer, vortex to dissolve, and cover the centrifuge tube Cover, bathe in water at 95°C for 5 minutes, quickly add to ice for 8 minutes, and set aside.

[0026] (2) Human gastric cancer cell BGC-823 positive screening: BGC-823 cells (purchased from Shanghai Chinese Academy of Sciences Cell Bank) were cultured to logarithmic growth phase, washed twice with washing buffer, and the random library prepared in step (1) was added to BGC- In 823 cells, shake at 4°C for 1 hour; discard the supernatant; wash the BGC-823 cells three times with washing buffer; Transfer to a centrifuge tube, put in a water bath at 95°C for 5 minut...

Embodiment 2

[0034] Example 2 The specific binding of the nucleic acid aptamer to the target cell BGC-823 was detected by flow cytometry.

[0035] Human gastric cancer cells BGC-823 and SGC-7901 in the logarithmic growth phase were digested with enzyme-free digestion solution and blown into a single cell suspension, centrifuged at 1000rpm for 5min to remove the supernatant, and washed with 4°C pre-cooled washing buffer Wash cells twice. Then add 200 μl of binding buffer containing 250 nM nucleic acid aptamer, incubate gently on a shaker at 4 °C for 30 min, centrifuge at 1000 rpm for 5 min at room temperature, remove the supernatant, and wash the cells twice with 4 °C pre-cooled washing buffer. Finally, 300 µl PBS was added for flow cytometry detection, and the random library sequence was used as a control. The flow cytometry detection results of the nucleic acid aptamer and the target cell BGC-823 and the counter-screened cell SGC-7901 are as follows: figure 1 As shown, the nucleic acid ...

Embodiment 3

[0036] Example 3 Fluorescence microscopy was used to analyze the specific binding of the nucleic acid aptamer to the target cell BGC-823.

[0037] Human gastric cancer cells BGC-823 and SGC-7901 were cultured in confocal culture dishes respectively. After culturing for 24 hours, all the liquid in the culture dish was aspirated, washed twice with 4°C pre-cooled washing buffer, and then the nucleic acid containing 250nM The binding buffer of the aptamer was incubated with the above two kinds of cells at 4° C. for 30 min with gentle shaking. After incubation, aspirate all the liquid in the culture dish, wash twice with 4°C pre-cooled washing buffer, and finally add 300 µl PBS for flow cytometry detection, random library sequence as a control, the results are as follows figure 2 shown. Observation under a confocal fluorescence microscope showed that the surface of BGC-823 cells showed obvious fluorescence, but no fluorescence was seen on the surface of SGC-7901 cells, suggesting...

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Abstract

The invention belongs to the technical field of biomedical science, and particularly relates to targeted aptamer for human poorly differentiated gastric carcinoma cells and an application of the targeted aptamer. The sequence of the aptamer is 5'-ACACCAAAATCGTCCGTTTCGTTTTAGTCCGTCTCTTTAGGGTGT-3'. The aptamer disclosed by the invention has high specificity and high affinity, and can maintain favorable activity at 4 DEG C, 25 DEG C and 37 DEG C. The aptamer disclosed by the invention can be used as a molecular probe, and can perform specificity imaging and detection on the human poorly differentiated gastric carcinoma cells. The aptamer has favorable application prospects in the respects of prediction and diagnosis and treatment of poorly differentiated gastric carcinoma.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a nucleic acid aptamer targeting human poorly differentiated gastric cancer cells and its application. Background technique [0002] Gastric cancer is a common malignant tumor with high incidence and the third leading cause of cancer death worldwide. According to the degree of differentiation of the glands, gastric cancer can be divided into differentiated and undifferentiated types. Clinical studies have shown that moderately and well-differentiated gastric cancer has a lower degree of malignancy and a better prognosis; while poorly differentiated gastric cancer has a higher degree of malignancy and a poorer prognosis. In addition, the differentiation status of gastric cancer is also correlated with the effect of chemotherapy. Therefore, it is of great significance to accurately distinguish the differentiation state of gastric cancer tissue for judging the prog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/574A61K49/00
CPCA61K49/0067G01N33/57446C12N15/115C12N2310/16
Inventor 方瑾李婉明
Owner 中国医科大学
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