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Nucleic acid aptamer for detecting bladder cancer and its application

A nucleic acid aptamer and bladder cancer technology, applied in the field of tumor molecular biology, can solve the problems of high antibody immunogenicity, damage, systemic infection, etc.

Active Publication Date: 2020-10-30
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional surgery, radiotherapy and chemotherapy can easily lead to a series of complications and other side effects, and cause irreparable damage to other healthy tissues and organs of the human body at the same time
Immunotherapy, although very effective, uses antibodies that are highly immunogenic and can lead to medical emergencies such as systemic infections

Method used

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  • Nucleic acid aptamer for detecting bladder cancer and its application
  • Nucleic acid aptamer for detecting bladder cancer and its application
  • Nucleic acid aptamer for detecting bladder cancer and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 The nucleic acid aptamer was screened by cell-SELEX technology.

[0055] (1) Design of the nucleic acid library and primers used:

[0056] Random ssDNA library:

[0057] 5'-CGTACGGTCGACGCTAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCACGTGGAGCTCGGATCC-3' (N represents four arbitrary bases of A, T, G and C) (SEQ NO.8)

[0058] Upstream primer: 5'-fluorescein isothiocyanate-CGTACGGTCGACGCTAGC-3'(SEQ NO.9)

[0059] Downstream primer: 5'-biotin-GGATCCGAGCTCCACGTG-3' (SEQ NO.10)

[0060] (2) Screening process:

[0061] In the present invention, human bladder transitional cell carcinoma cell 5637 is used as a positive screening target, and human bladder urothelial cell SV-HUC-1 is used as a reverse screening target.

[0062] 2.1 Positive screening:

[0063] a. Incubation: Dissolve the above random DNA library with binding buffer, denature at 95°C for 5 minutes, refold on ice for 10 minutes, and then combine with human bladder transitional cell carcinoma cell 5637, whic...

Embodiment 2

[0070] Example 2 Flow cytometric detection of the binding of the nucleic acid aptamer spl3 to human transitional bladder cancer cell 5637 and normal bladder urothelial cells SV-HUC-1

[0071] First, 5637 cells and SV-HUC-1 cells were cultured for 24 hours to make the cell density reach 90%, and then the adherent cells were digested from the culture dish with 0.2% EDTA respectively. Prepare 250nM synthetic FAM-labeled spl3 with 200μl of binding buffer, denature at 95°C for 5min, and refold on ice for 10min. Incubate with 300,000 5637 cells or SV-HUC-1 cells at 4°C for 1 hour. Wash the incubated cells 2-3 times with wash buffer, then resuspend the cells in 300 μl wash buffer. Fluorescent detection was performed by flow cytometry, and an initial random library of DNA was used as a control. The nucleic acid aptamer only binds to the target cell 5637, but not to the normal bladder urothelial cell SV-HUC-1. The results are as follows figure 2 shown.

Embodiment 3

[0072] Example 3 The binding of the obtained nucleic acid aptamer spl3 to other solid tumor cancer cells was detected by flow cytometry.

[0073] Firstly, different types of cells were cultured for 24 hours to make the cell density reach 90%, and then the adherent cells were digested from the culture dish with 0.2% EDTA respectively. Prepare 250nM synthetic FAM-labeled spl3 with 200μl of binding buffer, denature at 95°C for 5min, and refold on ice for 10min. Incubate with 300,000 cells for 1 h at 4°C. Wash the incubated cells 2-3 times with wash buffer, then resuspend the cells in 300 μl wash buffer. Fluorescent detection was performed by flow cytometry, and an initial random library of DNA was used as a control. Nucleic acid aptamer spl3 does not bind to other solid tumor cells, the results are as follows image 3 shown.

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Abstract

The invention discloses a nucleic acid aptamer for detecting bladder cancer and an application thereof. The nucleic acid aptamer for detecting bladder cancer has a unique stem ring structure under theconditions of 25 DEG C, 1.0mM Na+ and 0.5mM Mg2+, and has high recognition specificity and strong binding ability. Compared with the prior art, the present invention has the advantages that the aptamer screening does not need to define the conformation of the target molecule in advance, and molecular probes capable of specifically recognizing bladder cancer are directly screened from living cells. Nucleic acid aptamer has no immunogenicity. It can be chemically synthesized in vitro and can be modified and substituted in different parts. The sequence is stable and easy to be preserved.

Description

technical field [0001] The invention belongs to the technical field of tumor molecular biology, and relates to a nucleic acid aptamer that can be used for the detection of bladder cancer cells and tumor living bodies and its application. Background technique [0002] Bladder cancer (transitional cell carcinoma of the bladder) refers to malignant tumors that occur on the bladder mucosa. It is the most common malignant tumor of the urinary system and one of the ten most common cancers in the world. It is the sixth most common cancer and the ninth leading cause of cancer death in men worldwide, accounting for the first place in the incidence of urogenital tumors in my country. There are now approximately 549,000 new cases and 200,000 deaths worldwide. Bladder cancer is more common in men than in women, and the incidence and mortality rates of men are 9.6 and 3.2 / 100000, respectively, which are about 4 times that of women worldwide. Southern Europe (highest in men in Greece; ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/574
CPCC12N15/115C12N2310/16G01N33/57407
Inventor 叶茂谭蔚泓谢琳邓堂刚
Owner HUNAN UNIV
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