Novel bifunctional cicatrix and tissue fibrosis resistant oligonucleotide medicament

A functional equivalent, nucleic acid technology, applied in the field of medicine, can solve difficult-to-reach problems

Inactive Publication Date: 2011-08-10
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to achieve this goal by interfering with the upstream and downstream pathways of Smads and AP-1 activation

Method used

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  • Novel bifunctional cicatrix and tissue fibrosis resistant oligonucleotide medicament
  • Novel bifunctional cicatrix and tissue fibrosis resistant oligonucleotide medicament
  • Novel bifunctional cicatrix and tissue fibrosis resistant oligonucleotide medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, preparation and analysis of decoy nucleic acid drug

[0040] Eight of the following thio-decoy nucleic acid drugs were synthesized: ASCES101A: Sequence 2; ASCES101B: Sequence 3; ASCES101C: Sequence 4; ASCES101D: Sequence 5; ASCES102A: Sequence 7 and Sequence 8; ASCES102B: Sequence 9; 12; ASCES103B: Sequence 13.

[0041] Negative control sequence: Sense: 5′-TGTGGTCATGTGGTCATGTGGTCA-3′

[0042] Antisense: 5′-TGACCACATGACCACATGACCACA-3′

[0043] AP-1 positive control sequence: 5′-TGACGTCATGACGTCATGACGTCA-3′

[0044] Smad Positive Control Sequence: Sense: 5′-ATGCAGACAATGCAGACAATGCAGACA-3′

[0045] Antisense: 5′-TGTCTGCATTGTCTGCATTGTCTGCAT-3′

[0046] The designed thio-decoy nucleic acid was synthesized by the phosphoramidite solid-phase synthesis method using the 391 DNA synthesizer of the American PE company. The main raw materials are: four diisopropyl β-cyanoethyl phosphoramidite monomers: adenosine (A), guanosine (G), cytidine (C) and thymidine (T);...

Embodiment 2

[0047] Embodiment 2, the growth inhibition of eight kinds of ASCES series decoy nucleic acids to L929 fibroblasts

[0048] 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 eight ASCES 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, Repla...

Embodiment 3

[0050] 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

[0051] 10 pmol of the ASCES series drugs were diluted in 30 μl of complete binding buffer, and added to 96-well enzyme-linked plates coated with Ap-1 or Smad cis-acting elements (purchased from TransAM from Active Motif, USA) in an amount of 30 μl per well. 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 ASCES drug and cell nucleus extract; blank positive control was added without ASCES drug, onl...

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Abstract

The invention relates to the field of medicaments, in particular to a novel bifunctional cicatrix and tissue fibrosis resistant oligonucleotide medicament. The nucleic acid contains an AP-1 cis-acting component in high affinity binding with AP-1 and a Smad cis-acting component in high affinity binding with Smad.

Description

technical field [0001] The present invention relates to the field of medicine, in particular to a decoy oligonucleotide for anti-scar and tissue fibrosis treatment, and more specifically, to a decoy oligonucleotide capable of synchronizing with transcription factor activator protein-1 (Activator protein-1, AP-1) and Smad3 specifically bind to decoy oligonucleotides that inhibit the activity of these two transcription factors. The invention also relates to the preparation of the oligonucleotide and its application in the treatment of scar and tissue fibrosis. Background technique [0002] Pathological scar is the result of excessive wound repair and tissue fibrosis, which not only affects the appearance, but also can lead to varying degrees of tissue and organ dysfunction, and is even the main reason for the failure of certain diseases, such as anti-glaucoma filtration surgery, filtration The formation of over-bubble scar is the main reason for the failure of anti-glaucoma s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10A61K48/00A61P17/02A61P19/08A61P29/00A61P37/02A61P43/00
Inventor 徐祥袁洪峰黄宏郭韡邢伟梁华平
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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