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Application of DNA tetrahedron to induction of autophagy

A technology for inducing cells and tetrahedrons, applied to medical preparations containing active ingredients, pharmaceutical formulas, organic active ingredients, etc., can solve the problem of less research on cell physiological activities, and achieve the effect of improving proliferation and migration

Pending Publication Date: 2018-04-27
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many studies on DNA tetrahedron, there are few studies on various physiological activities of cells, especially on autophagy.

Method used

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  • Application of DNA tetrahedron to induction of autophagy
  • Application of DNA tetrahedron to induction of autophagy
  • Application of DNA tetrahedron to induction of autophagy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Synthesis and characterization of embodiment 1 DNA tetrahedron

[0049] 1. Synthesis of DNA tetrahedron

[0050] DNA tetrahedron is composed of uniquely designed four DNA single strands (S1, S2, S3, S4) through a fast, simple and specific PCR program (95°C for 10min, rapid cooling to 4°C for 20min, 4°C for long-term storage) synthesized by self-assembly. The four single strands were added into a 200μl EP tube containing 100ul of TM buffer (10mM Tris-HCl, 50mM MgCl2, pH 8.0) in an equimolar ratio, and the reaction solution was heated to 95°C for 10min, and then rapidly cooled to 4°C to synthesize DNA tetrahedrons body.

[0051] The specific sequences of the four DNA single strands are as follows:

[0052] S1:

[0053] 5'-ATTTATTCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3' (SEQ ID NO: 1);

[0054] S2:

[0055] 5'-ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG-3' (SEQ ID NO: 2);

[0056] S3:

[0057] 5'-ACTACTATGGCGGGTGATAAAACGTGTAG...

Embodiment 2

[0064] Example 2 Transmission Electron Microscopy Detects the Induction Effect of DNA Tetrahedron on Chondrocyte Autophagy

[0065] 1. In vitro culture of chondrocytes

[0066] The cells used in this experiment were rat chondrocytes, which were obtained from the knee joints of 3-5-day-old SD suckling mice, and the culture medium was high-glucose DMEM containing 10% fetal bovine serum (fetal bovine serum, FBS, Gibco) And 1% double antibody (Penicillin-Streptomycin liquid penicillin and streptomycin mixture). at 37°C, 5% CO 2 Carry out cell culture in an incubator, and when the cells grow to occupy 80-90% of the bottom of the culture dish, they are digested with 0.25% trypsin-EDTA and passaged, and generally transferred to P1-P2 for use.

[0067] 2. Detection of DNA tetrahedron-induced autophagy in chondrocytes

[0068] The ability of DNA tetrahedron to induce autophagy in chondrocytes was detected by transmission electron microscopy. The chondrocytes of the P1 generation we...

Embodiment 3

[0070] Example 3 Confocal microscope detection of LC3 protein expression to observe the induction of DNA tetrahedron on chondrocyte autophagy

[0071] LC3 is a marker protein of autophagy and an important indicator for detecting autophagy. LC3 has two subtypes, namely LC3I and LC3II. The two subtypes switch between each other. When there is no autophagy, the LC3I subtype mainly exists. On the contrary, when autophagy occurs, the LC3II subtype mainly exists. Type exists. Since the LC3Ⅰ subtype protein is water-soluble protein, it will exist in a diffuse state in cells; while the LC3Ⅱ subtype protein is water-insoluble and mainly exists in an aggregated state in water. Therefore, the strength of autophagy in cells can be judged by observing the changes in LC3II content in cells (that is, the distribution of green fluorescence).

[0072] experiment method:

[0073] (1) Digest the chondrocytes of the P1 generation and plant them in a 6-well plate, at 37°C, 5% CO 2 After cultur...

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Abstract

The invention provides application of a DNA tetrahedron to induction of autophagy. The DNA tetrahedron is synthesized from four single DNA strands through self-assembling, wherein the sequences of thefour single DNA strands are shown in SEQ ID No. 1-4. The DNA tetrahedron can activate a PI3K / AKT / mTOR signaling pathway so as to conduct induction effect on autophagy. Specifically, the DNA tetrahedron can act on the upstream protein AKT of mTOR and conducts inhibition effect on the expression of the protein mTOR by activating the expression of AKT; meanwhile, DNA tetrahedron can negatively act on mTOR by up-regulating the expression of the upstream protein PI3K of mTOR so as to allow the expression of mTOR to be inhibited, so autophagy is further induced and biomolecules in cells are highlyrecycled for the cells, and thus, the proliferation and migration of cells are improved.

Description

technical field [0001] The invention belongs to the technical field of cell autophagy, and in particular relates to the application of a DNA tetrahedron in inducing cell autophagy. Background technique [0002] Autophagy is an important regulatory process in cells that degrades damaged or unnecessary organelles and proteins to provide intracellular nutrients and maintain intracellular balance. During this process, a double-layer membrane wraps part of the cytoplasm, organelles, and proteins fuse to form double-membrane vesicles called autophagosomes. Autophagosomes are the most important hallmark of autophagy. Eventually, autophagosomes and lysosomes combine and fuse together to form autolysosomes and degrade the organelles and macromolecules inside for recycling. Autophagy is recognized as an efficient circulatory system, and more and more studies have proved that autophagy plays a very important role in maintaining cell metabolism and homeostasis (including cell survival...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/711A61P43/00
CPCA61K31/711
Inventor 林云锋石思容蔡潇潇
Owner SICHUAN UNIV
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