Application of dna tetrahedron in preparation of medicine for treating corneal injury
A technology of corneal injury and tetrahedron, which is applied in the field of ophthalmic medicine, can solve the problems of insufficient treatment effect and side effects, and achieve the effect of simple synthesis method, promoting tissue regeneration, promoting proliferation and migration
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[0030] Preparation and Identification of Example DNA Tetrahedron
[0031] 1. Method
[0032] 1.1 Preparation method of DNA tetrahedron (TDN)
[0033] TDN is self-assembled by four uniquely designed 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, long-term storage at 4°C) Synthetic. The four single strands were added to 96 μl TM buffer (10 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0) in a 200μl EP tube, the reaction solution was heated to 95°C for 10min, and then rapidly cooled to 4°C to synthesize TDN.
[0034] 1.2 The specific sequences of the four DNA single strands are as follows:
[0035]
[0036] 1.3 Characterization of TDN by polyacrylamide gel electrophoresis, dynamic light scattering (DLS), atomic force microscopy (AFM), transmission electron microscopy (TEM) and charge determination:
[0037] ①DLS: The synthesized TDN was diluted to 250nM with twice distilled water and then observed on...
experiment example 1
[0043] Experimental Example 1 Cell Migration Experiment
[0044] Wound healing is a dynamic, strictly ordered biological process, in which re-epithelialization plays a very important role. Wound re-epithelialization mainly depends on the migration of epithelial cells from the wound edge to the wound center. In order to investigate whether TDN contributes to the migration of human corneal epithelial cells, the inventors conducted the following experiments.
[0045] 1. Method
[0046] The effect of TDN on the migration of human corneal epithelial cells was detected by scratch test, transwell test and RTCA method.
[0047] Scratch test: Cells were divided into 1.5×10 5 Cells / well were inoculated into 12-well plates for culture. When the cells were 80-90% full, use a sterile gun tip to draw two vertically intersecting scratches. After washing away the cell debris, add TDN at a concentration of 125nM. , 250nM, 375nM cell culture medium was the test group, and the cell culture m...
experiment example 2
[0054] Experimental Example 2 Cell Proliferation Experiment
[0055] 1. Method
[0056] To examine the effect of TDN on the proliferation behavior of corneal epithelial cells, we conducted three experiments.
[0057] CCK-8 assay: Cells were divided into 5×10 3 The density of each well was inoculated in a 96-well plate, and after using the growth medium for 24h, it was replaced with a serum-free medium containing different concentrations (0nM, 125nM, 250nM, 375nM) of TDN, at 6h, 12h, 24h, 36h, According to the cck-8 product use method, the cck-8 absorbance photometry analysis test was carried out respectively, and the statistical analysis was carried out by using SPSS.
[0058] BrdU cell proliferation test: corneal epithelial cells were treated with 1×10 4 The density of cells / ml was inoculated in a confocal small dish, and after culturing with the growth medium for 24 hours, the medium was replaced with a medium containing 10 μm BrdU and different concentrations (0 nM, 250 ...
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