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Nonalcoholic fatty liver cell aptamer and application thereof

A non-alcoholic, cellular nucleic acid technology, applied in the field of nucleic acids, can solve problems such as inability to effectively reach the target, achieve easy synthesis and labeling, ensure natural properties, and achieve the effect of small molecular weight

Active Publication Date: 2016-09-07
XIANGYA HOSPITAL CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no specific drugs and methods, and the target cannot be effectively reached

Method used

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  • Nonalcoholic fatty liver cell aptamer and application thereof
  • Nonalcoholic fatty liver cell aptamer and application thereof
  • Nonalcoholic fatty liver cell aptamer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: In vitro screening of nucleic acid aptamers specifically binding to NAFLD cells

[0052] (1) The first round of screening:

[0053] 1.1. Induction and differentiation of NAFLD cells:

[0054] HepG 2 Cells were inoculated on 10 cm culture dishes, using complete culture medium (high glucose DMEM containing 10% FBS, 100 μg / mL penicillin, and 100 μg / mL streptomycin in complete culture medium), at 37 °C, 5% CO 2 Culture under conditions, change the medium once every 2 days, and start to induce differentiation when the cell density reaches 60%-70%. Pour off the original culture solution, and use a final concentration of 100mM oleic acid, 50mM palmitic acid, 1×10 ﹣ 6 M dexamethasone and 1 x 10 ﹣ 12 The induction medium of M insulin continued to culture the cells, changing the medium every 2 days until 1 week, and it could be seen under the microscope that more than 90% of the cells had been induced to differentiate into NAFLD cells.

[0055] 1.2, using 20nmol...

Embodiment 2

[0145] Investigate the effect of temperature on the nucleic acid aptamer NFD-1.

[0146] Because the screening conditions were carried out at 4°C, and the binding test was also carried out at 4°C, but the internal environment temperature of the human body is 37°C, so we studied whether the nucleic acid is suitable for use at 4°C and 37°C. Whether the affinity between the body and the target cell is affected.

[0147] Specific steps are as follows:

[0148] (1) The synthesized FITC-labeled nucleic acid aptamer and random control library were prepared into a solution with a concentration of 250 nM using a binding buffer and stored at 4°C.

[0149] (2) Select NAFLD cells in good growth state, in the logarithmic growth phase, and whose cell abundance reaches more than 90% to induce differentiation, remove the medium, and wash twice with washing buffer at 4°C.

[0150] (3) Add 3 mL of non-enzymatic digestion solution and digest at 37°C for 5-15 minutes. When the cells become roun...

Embodiment 3

[0158] The effect of protease treatment on the nucleic acid aptamer NFD-1 was investigated.

[0159] (1) Collect differentiated NAFLD cells in good growth state, in the logarithmic growth phase, and with a cell abundance of more than 90%, digest with proteinase K or trypsin at 37°C for 30 min, and wash the cells 3 times with washing buffer.

[0160] (2) Count 1×10 6 Collect the cells in flow cytometry tubes, add 100 μL of 250 nM nucleic acid escape to each tube of cells, and incubate at -4°C for 30 min.

[0161] (3) After the incubation, the cells were washed again 3 times, and the cells were resuspended with 200 μL of washing buffer, and tested on the machine.

[0162] Nucleic acid aptamers screened by Cell-SELEX technology usually bind specifically to proteins on the cell membrane. In order to verify the binding of NFD-1 to cell membrane proteins, we used trypsin and Proteinase K as protease experiments. We used trypsin and Proteinase K. The protease was incubated with the...

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Abstract

The invention provides a nonalcoholic fatty liver cell aptamer and an application thereof. The nucleotide sequence of the nonalcoholic fatty liver cell aptamer has a DNA segment as shown in SEQ ID NO.1. The nonalcoholic fatty liver cell aptamer disclosed by the invention is high in specificity and affinity, and the nonalcoholic fatty liver disease cell aptamer is applicable to preparation of nonalcoholic fatty liver disease therapeutic drugs, preparation of a detection kit for detecting the nonalcoholic fatty liver and to tissue section imaging of the nonalcoholic fatty liver.

Description

technical field [0001] The invention relates to the field of nucleic acid technology, in particular to a nonalcoholic fatty liver cell nucleic acid aptamer and its application. Background technique [0002] Nonalcoholic fatty liver disease (NAFLD) refers to the clinicopathological syndrome characterized by excessive fat deposition in liver cells caused by alcohol and other definite liver damage factors. Characteristic manifestations in the liver. With the economic development of the society and the change of people's lifestyle and dietary structure, the prevalence of NAFLD has increased rapidly, and the age of onset has gradually become younger. It has become one of the most important public health problems in the world in the 21st century. NAFLD has become the first cause of chronic liver disease in developed countries such as Europe and the United States and my country. It can not only lead to liver cirrhosis, liver cancer and liver failure, but also the occurrence, devel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68C12Q1/02A61K48/00A61K31/7088A61P1/16
CPCA61K31/7088C12N15/115C12N2310/16C12Q1/02G01N33/68G01N2333/47
Inventor 刘波刘俊廖洁蒲颖刘慧霞谭蔚泓
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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