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Small nucleic acid molecule capable of inhibiting vascular restenosis and applications of small nucleic acid molecule

A kind of restenosis, molecular technology, applied in cardiovascular system diseases, organic active ingredients, genetic material components, etc., can solve the problem of lumen affecting the long-term efficacy of surgery and so on

Inactive Publication Date: 2017-02-01
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the hyperplasia of the grafted vein intima, restenosis and even occlusion of the lumen have seriously affected the long-term efficacy of the operation.

Method used

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  • Small nucleic acid molecule capable of inhibiting vascular restenosis and applications of small nucleic acid molecule
  • Small nucleic acid molecule capable of inhibiting vascular restenosis and applications of small nucleic acid molecule
  • Small nucleic acid molecule capable of inhibiting vascular restenosis and applications of small nucleic acid molecule

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Experimental program
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Effect test

preparation example Construction

[0070] Various methods can be used to prepare RNA, such as: chemical synthesis, in vitro transcription, enzyme-cut long-chain dsRNA, vector expression of RNA, PCR synthesis of RNA expression elements, etc. The emergence of these methods provides researchers with options. Better access to gene silencing efficiency.

[0071] The RNA molecule of the present invention can be used as an active ingredient of drugs for inhibiting restenosis of blood vessels, especially as an active ingredient of drugs for anti-restenosis of transplanted blood vessels.

[0072] For application purposes, RNA molecules can be directly administered as drugs to specific parts of the recipient's body, such as grafted blood vessels.

[0073] The dosage form of the drug of the present invention can be in various forms, as long as it is suitable for the administration of the corresponding disease and properly maintains the activity of the RNA molecule. For example, for an injectable drug delivery system, the...

Embodiment 1

[0080] 1. Cell culture

[0081] Human aortic smooth muscle cells HA-VSMC (preserved at the Affiliated Hospital of Nantong University) were stored in DMEM / F12 medium containing 10% FBS (Thermo Fisher Scientific, USA) at 37°C, 5% CO 2 cultured in an incubator (ThermoFisher Scientific, USA).

[0082] 2. siRNA design and synthesis

[0083] Design and synthesize the siRNA sequences targeting the Nur77 gene, respectively: siRNA1, which has a sense strand of SEQ ID NO:5 and an antisense strand of SEQ ID NO:6; siRNA2, which has a sense strand of SEQ ID NO:11 and an antisense strand of SEQ ID NO:12; siRNA3 with sense strand SEQ ID NO:17 and antisense strand SEQ ID NO:18. The sense strand and the corresponding antisense strand were annealed into siRNA double strands, and the concentration was 20 μM before transfection.

[0084] In addition, NC siRNA (Negetivecontrol siRNA), which is not homologous to human genes, was designed and synthesized as a negative control, and its sequence is...

Embodiment 2

[0102] Detection of gene protein expression level by western blot

[0103] Cell culture and siRNA transfection were carried out according to the method of Example 1.

[0104] Divide the cells into 1×10 6 Cells / well were placed in a 6-well plate, grown to 70-80% confluence after 24 hours, and the cells were divided into 5 groups, namely siRNA1 treatment group, siRNA2 treatment group, siRNA3 treatment group, negative control group, and normal group.

[0105] After 48 hours of treatment, pre-cooled 1×SDS buffer (50mM Tris-HCl, pH7.6; 1% SDS; 150mM NaCl; 0.5% Triton-X 100; 5mM EDTA; 5% β-mercaptoethanol (BME); 1mM PMSF) Cells were lysed with protein lysate, placed at 4°C, centrifuged at 10,000rpm for 20min, the supernatant was diluted and subjected to polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride filter (PVDF) membrane (Millipore, USA) ), and blocked with 5% skimmed milk at room temperature for 2h, and then used rabbit anti-human Nur77 monocl...

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Abstract

The invention discloses a small nucleic acid siRNA molecule capable of inhibiting the expression of orphan nuclear receptor Nur77. The small nucleic acid siRNA molecule is composed of a positive-sense strand and an antisense strand with the following sequences: the positive-sense strand: 5'-GCUCAGGCCUGGUGCUACANn-3' (SEQ ID NO:13), and the antisense strand: 5'-UGUAGCACCAGGCCUGAGCNn-3' (SEQ ID NO:14), N in the positive-sense strand and N in the antisense strand are the same or different, and cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT is independent; n is the number of N, and is 0, 1 or 2. The siRNA molecule provided by the invention can be used for inhibiting the grafted vascular restenosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a small nucleic acid siRNA molecule capable of inhibiting orphan nuclear receptor Nur77 and its application in anti-restenosis of transplanted blood vessels. Background technique [0002] Coronary artery bypass grafting is currently an important method for the treatment of severe cardiovascular diseases such as coronary arteriosclerosis. Among them, autologous veins such as the great saphenous vein are often the preferred grafts due to factors such as superficial anatomy and multiple sources. However, postoperative intimal hyperplasia, restenosis and even occlusion of the grafted vein have seriously affected the long-term efficacy of the operation. A large number of clinical and experimental results show that the patency rate of vein graft grafts is 85% and 78% in 1 year and 5 years respectively, while the patency rate in 10 years is only 50%. Therefore, how to inhibit the ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P9/10
CPCC12N15/1138C12N2310/14
Inventor 刘锟尤庆生施炜陈宏林王德丰
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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