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Application of DNA (Deoxyribonucleic Acid) tetrahedron to preparation of medicament for promoting neural restoration

A technology of nerve repair and tetrahedron, which is applied in the field of nerve repair, can solve the problems that the application has yet to be developed, and achieve the effect of promoting nerve repair, good biocompatibility, and increasing proliferation

Active Publication Date: 2019-05-28
CHENGDU GENREZE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, TDNs have appeared in some studies as drug carriers, but their application in nerve repair has yet to be developed

Method used

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  • Application of DNA (Deoxyribonucleic Acid) tetrahedron to preparation of medicament for promoting neural restoration
  • Application of DNA (Deoxyribonucleic Acid) tetrahedron to preparation of medicament for promoting neural restoration
  • Application of DNA (Deoxyribonucleic Acid) tetrahedron to preparation of medicament for promoting neural restoration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0067] Synthesis of Example TDNs

[0068] 1. Synthesis

[0069] Four kinds of DNA single strands (S1, S2, S3, S4) are dissolved in the TM buffer solution with the same final concentration, wherein, the solute of the TMbuffer solution is Tris-HCl and MgCl , and their concentrations are respectively 10mM and 50mM, and adjusted The pH of the solution was 8.0. Then the mixture was vortexed, mixed, and centrifuged, then placed in a PCR instrument, and the temperature was rapidly raised to 95°C and stabilized for 10-15 minutes, then cooled to 4°C and stabilized for 20 minutes. That is synthesized as figure 1 The tetrahedral structure shown.

[0070] The sequence of the DNA single strand is shown in Table 1.

[0071] Table 1 Sequence of TDNs single strand

[0072]

[0073]

[0074] 2. Identification

[0075] 2.1 Gel electrophoresis

[0076] a. Preparation of polyacrylamide gel: 40wt% acrylamide solution, 10×TAE, 10wt% ammonium sulfate solution, distilled water, tetrameth...

experiment example 1

[0087] Experimental Example 1 Identification of Neural Stem Cells

[0088] Immunofluorescence technique was used to identify mouse neural stem cells, once including the following steps:

[0089] A. Inoculate the cell suspension in a confocal small dish and place it in an incubator for 24 hours. Aspirate off the culture medium whose component is DMEM+10% serum+1% double antibody, wash with PBS 3 times, 5 minutes each time;

[0090] B. After fixing with 4% paraformaldehyde for 25 minutes, absorb the paraformaldehyde and wash with PBS 3 times, 5 minutes each time;

[0091] C. Treat with 0.5% Triton-100 for 20-25 minutes, absorb Triton-100, wash with PBS 3 times, 5 minutes each time;

[0092] D. Treat sheep serum for 1 hour, absorb sheep serum, wash with PBS 3 times, 5 minutes each time;

[0093] E. Primary antibody (anti-nestin antibody) treatment, overnight at 4°C. On the second day, rewarm at 37°C for 0.5 hours, recover the primary antibody, and wash with PBS 3 times, 5 min...

experiment example 2

[0098] Experimental Example 2 Neural Stem Cell Uptake Experiment

[0099] Being able to be taken up by cells in large quantities is the prerequisite for most drugs to play a therapeutic role. This experiment example detects the uptake ability of neural stem cells to TDNs.

[0100] (1) Flow cytometry

[0101] a. Inoculate the cell suspension in a 6-well plate, and pre-cultivate it in an incubator for 24 hours (37°C, 5% (v / v) CO2); then reduce the serum concentration in the medium from 10% to 6%, and then Continue culturing in the incubator for 6 hours (37°C, 5% (v / v) CO2); then reduce the serum concentration in the medium from 6% to 0, and continue culturing in the incubator for 1 hour (37°C, 5% (v / v) / v)CO2).

[0102] B, the negative control group does not do any treatment, the positive control group adds the single-stranded DNAS1 that the concentration is the Cy5 modification of 250nM, the TDNs that the experimental group adds the Cy5 modification that the concentration is...

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Abstract

The invention provides application of a DNA (Deoxyribonucleic Acid) tetrahedron to preparation of a medicament for promoting neural restoration. The DNA tetrahedron is assembled into a tetrahedron structure from four single-chain DNAs in a complementary base pairing manner; each edge of the tetrahedron is of a dual-chain DNA structure. The DNA tetrahedron provided by the invention can remarkably promote the proliferation, differentiation and migration of mouse neural stem cell, and has high biocompatibility and bioavailability.

Description

technical field [0001] The invention relates to the field of nerve repair, in particular to the use of DNA tetrahedrons in the preparation of drugs for promoting nerve repair. Background technique [0002] At present, nervous system diseases, such as neurodegenerative diseases and nerve injuries, are difficult problems in the medical field. Because the self-repair and renewal of nerve cells is difficult, neural stem cell (neural stem cells, NSCs) therapy has attracted more and more attention. Although there are still some technical difficulties to be overcome in the clinical application of neural stem cell therapy, the most critical thing at present is whether the implanted cells themselves can proliferate, differentiate and migrate. [0003] Some researchers have found that some drug molecules (such as prostaglandin E2, tenuigenin, etc.) can improve the proliferation and differentiation ability of NSC, but their biocompatibility and utilization are not high, and there are ...

Claims

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Application Information

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IPC IPC(8): A61K31/711A61P25/02A61P25/00
CPCA61K31/7088A61K31/711A61P25/00A61P25/02C07H21/04
Inventor 林云锋马文娟蔡潇潇邵晓茹谢雪萍
Owner CHENGDU GENREZE GENE TECH CO LTD
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