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Single-stranded deoxyribonucleic acid (ssDNA) aptamer targeting human highly metastatic hepatoma carcinoma cell and application thereof

A technology of liver cancer cells and aptamers, which can be used in recombinant DNA technology, DNA/RNA fragments, medical preparations with non-active ingredients, etc.

Active Publication Date: 2012-11-07
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, so far, there has been no report on the successful application of SELEX screening technology to the development of reagents for the diagnosis and treatment of liver cancer metastasis and recurrence.

Method used

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  • Single-stranded deoxyribonucleic acid (ssDNA) aptamer targeting human highly metastatic hepatoma carcinoma cell and application thereof
  • Single-stranded deoxyribonucleic acid (ssDNA) aptamer targeting human highly metastatic hepatoma carcinoma cell and application thereof
  • Single-stranded deoxyribonucleic acid (ssDNA) aptamer targeting human highly metastatic hepatoma carcinoma cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Subtractive cell-SELEX screening of nucleic acid aptamers that specifically bind to highly metastatic liver cancer cells

[0083] 1.1 Pre-screening treatment of high metastatic potential liver cancer cells HCCLM9 (target cells) and low metastatic potential liver cancer cells MHCC97-L (subtractive cells)

[0084] Cells in good growth condition were treated with enzyme-free cell digestion solution, and 5×10 6 Cells were inoculated in a culture dish and cultured overnight. When the fusion rate of the cells reached 95%, they could be used for library screening.

[0085]1.2 The first round of cell selection

[0086] 1.2.1 Select HCCLM9, a monolayer adherent overnight growth cell with a cell fusion rate of 95%, as a screening target, use trypan blue to identify its activity, and use 1×PBS-1mmol / LMgCl 2 -0.1%BSA rinse cells twice;

[0087] 1.2.2 Incubate the random ssDNA library with the target cells on ice for 60 minutes, so that the ssDNA library can fully inte...

Embodiment 2

[0147] Example 2. Recognition of liver cancer cells based on quantum dot-aptamer functionalized biomolecular probes

[0148] 2.1 Animal model making:

[0149] According to the method of this group, the animal model of lung metastasis of human liver cancer was made, and the brief description is as follows:

[0150] 2.1.1 Observe that the MHCC97-L and HCCLM9 cells grow well, and the cell fusion rate reaches 80%. Trypsinize them respectively, put them in 15ml centrifuge tubes, centrifuge at 1000rpm×10min at 4°C, discard the supernatant, and press 5×10 6 / 0.2mL dose was inoculated subcutaneously on the back of nude mice, and subcutaneously formed tumors after 2 weeks, with a diameter of about 0.5cm.

[0151] 2.1.2 Orthotopic implantation: Simultaneously excise the subcutaneous tumors on the back of MHCC97-L and HCCLM9 nude mice, and place them in culture dishes added with 1×PBS respectively. Intrahepatic orthotopic tumor inoculation, the method of MHCC97-L and HCCLM9 is the same...

Embodiment 3

[0202] Example 3. Peripheral blood simulated environment circulating liver cancer cell enrichment test

[0203] 3.1 Red blood cell lysis

[0204] 10 respectively 4 a, 10 2 Put a highly metastatic liver cancer cell HCCLM9 into 1mL peripheral whole blood of a healthy person, add 2 times the volume of PBS, mix well, 1500r / min, 5min, remove the supernatant, add red blood cell lysate NH 4 Cl, incubate at room temperature for 10min, 1500r / min, 5min, remove the supernatant; repeat lysis of residual red blood cells once, 1500r / min, 5min, remove the supernatant, add 10 times the volume of PBS to wash once, remove the supernatant, add the adapter Sub-binding solution 1mL, mix the cell pellet.

[0205] 3.2 Incubation of aptamers and mixed cells

[0206] Add a certain concentration of aptamers to make the final concentration 500pM, incubate on ice for 30min with shaking, wash with ice-cold PBS 3 times / 5min, 1500r / min, 5min, remove the supernatant for the last time, and cell pellet for...

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Abstract

The invention discloses a single-stranded deoxyribonucleic acid (ssDNA) aptamer targeting human highly metastatic hepatoma carcinoma cell and an application of the ssDNA aptamer, and relates to the field of diagnosis and treatment of the recurrence and metastasis of the hepatocellular carcinoma, so that the high-efficient novel specific molecules are provided for the diagnosis and treatment of the recurrence and metastasis of the hepatocellular carcinoma. According to the invention, a subtractive cell-SELEX technology is applied, two hepatoma carcinoma cell systems with same genetic background and obviously different metastatic potentials are a subtractive target and a screening target, respectively, and the hepatoma carcinoma cell systems are MHCC97-L (low metastasis) and HCCLM9 (high metastasis), so that the ssDNA aptamer is capable of specifically recognizing the highly metastatic potential hepatoma carcinoma cell HCCLM9. The aptamer is a single-stranded DNA molecular probe capableof specially combining with a surface of a human highly metastatic hepatoma carcinoma cell membrane, and a nucleotide sequence of aptamer is represented by SEQ ID No.1. The aptamer is expected to play an important role in preparation of a reagent for diagnosing or treating the hepatocellular carcinoma.

Description

technical field [0001] The invention relates to the technical field of diagnosis and treatment of liver cancer metastasis and recurrence, in particular to a single-stranded DNA aptamer targeting human high-metastatic liver cancer cells and its application, which can be used for targeted diagnosis and treatment of liver cancer metastasis and recurrence. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, accounting for 6% of all malignant tumors. China is the country with the highest mortality rate of liver cancer in the world, accounting for 53% of the total number of deaths from liver cancer in the world. Become the second cause of death of malignant tumors. Although great progress has been made in the diagnosis, treatment and basic research of liver cancer in recent decades, overall, the prognosis of liver cancer has not been significantly improved, and the 5-year survival rate is only about 5%. At present,...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/68A61K31/713A61K47/48A61P35/00A61K47/54
Inventor 汪付兵李雁刘少平
Owner WUHAN UNIV
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