Scar and tissue fibration resistant oligomeric double-stranded nucleotide medicine and its application
A technology of double-stranded nucleotides and nucleic acid drugs, which can be used in drug combinations, antipyretics, anti-inflammatory agents, etc., and can solve problems such as difficult to achieve
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Embodiment 1
[0039] Embodiment 1, preparation and analysis of decoy nucleic acid drug
[0040] Synthesize four of the following thio double-stranded oligomeric decoy nucleic acid drugs: AFODN1: Mix equal amounts of SEQ ID NO.1 and SEQ ID NO.2 in PBS buffer, denature at 94°C for 5 minutes, and anneal to form a double-stranded oligo Polymeric DNA; AFODN2: SEQ ID NO.3 and SEQ ID NO.4 are mixed in equal amounts in PBS buffer, denatured at 94°C for 5 minutes, and then annealed into double-stranded oligomeric DNA; AFODN3: SEQ ID NO.5 and SEQ ID NO.5 Mix equal amounts of ID NO.6 in PBS buffer, and anneal to double-stranded oligomeric DNA after denaturation at 94°C for 5 minutes; AFODN4: mix equal amounts of SEQ ID NO.7 and SEQ ID NO.8 in PBS buffer , after denaturation at 94°C for 5 minutes, annealed into double-stranded oligomeric DNA.
[0041] Scramble negative control sequence sense strand: 5′-GACGCAAGCAGTAGCTATTGCTCAGTCTACCATC-3′
[0042] Antisense strand: 5′-GATGGTAGACTGAGCAATAGCTACTGCTT...
Embodiment 2
[0048] Example 2. Growth inhibition of L929 fibroblasts by four kinds of AFODN series decoy nucleic acids
[0049] Resuscitate and inoculate L929 fibroblasts in cell culture flasks at 37°C, 5% CO 2 Under certain conditions, culture cells with RPMI 1640 complete culture medium containing 10% FBS; when the cells reach 70%-80% confluence, trypsinize the cells, count the viable cells and adjust the cell concentration to 2×10 5 Cells / ml; Inoculate cells with 100 μL cell suspension per well in a 96-well plate and incubate for 12 hours; then replace with serum-free medium, and randomly divide cells into blank control group, positive control group, negative control group and four AFODN drugs group, and each group was provided with 3 multiple wells, wherein only 0.6 μl / well liposome was added to the blank control group, and 0.6 μl / well liposome and different decoy nucleic acids with a final concentration of 100 nM were added to the rest; after 5 hours of transfection, Replace it wit...
Embodiment 3
[0051] Example 3, ELISA method to detect the competition of decoy nucleic acid for the binding of AP-1 and Smad to its cis-acting elements Inhibition experiment
[0052] Dilute 10 pmol of AFODN series drugs in 30 μl of complete binding buffer, and add 30 μl per hole to a 96-well enzyme-linked plate coated with Ap-1 or Smad cis-acting elements (purchased from TransAM from Active Motif, USA). TM Transcription factor assay kits); then dilute 5 μg TPA-induced K-562 nuclear extract (for Smad, use TGF-β1-induced L929 fibroblast nuclear extract) in 20 μl complete binding buffer, and then add Mix well in a 96-well ELISA plate and incubate at room temperature for 1 h. Note that the detection of various samples was carried out in 3 duplicate wells, and the experimental groups were as follows: blank negative control only added 50 μl complete binding buffer without AFODN drug and cell nucleus extract; blank positive control was added without AFODN drug, only containing 50 μl of comp...
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