MiRNA-182 inhibitor and application thereof in preparation of drug for preventing and treating immunological rejection of heart transplantation

A technology of miRNA-182 and inhibitors, applied in the field of medical bioengineering, to achieve the effect of protecting the function of heart grafts and preventing the failure of heart grafts

Active Publication Date: 2016-07-13
成都仕康美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective treatment for CAV clinically, so early diagnosis and prevention of CAV are important links to reduce the risk of chronic rejection and prolong the survival time of heart grafts

Method used

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  • MiRNA-182 inhibitor and application thereof in preparation of drug for preventing and treating immunological rejection of heart transplantation
  • MiRNA-182 inhibitor and application thereof in preparation of drug for preventing and treating immunological rejection of heart transplantation
  • MiRNA-182 inhibitor and application thereof in preparation of drug for preventing and treating immunological rejection of heart transplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Detecting the effect of miRNA-182 inhibitors on the survival time of transplanted hearts through a mouse heterotopic heart transplantation model

[0021] 1. Synthesis of miRNA-182 inhibitor of the present invention

[0022] 5'-O-(4,4-Dimethoxytrityl)-2'-O-methylcytidine-3'-(2-cyanoethyl-N,N-diisopropyl )(5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-(2-cyanoethyl-N,N-diisopropyl)) modified RNA phosphoramidite monomer A, G, C, U, using a DNA synthesizer to chemically synthesize the miRNA-182 inhibitor of the present invention (referred to as Skm-182 in the schematic diagram), and the RNA sequence of the miRNA-182 inhibitor is 5'-CGGUGUGAGUUCUACCAUUGCCAAA-3'. The linking modification of RNA and cholesterol group is carried out in a controlled-pore glass solid support. In the synthesis process of miRNA-182 inhibitors, the modification of the phosphorosulfuryl bond at the specified position is accomplished by the oxidation reaction of phosphite and phenylacetyl disul...

Embodiment 2

[0032] Example 2: Using HE staining method to observe the pathological changes of heart grafts

[0033] 1. Experimental method: 1) On the 7th day after transplantation, 3 recipient mice in the control group and the experimental group were sacrificed to obtain the transplanted heart tissue block and fix it with 10% neutral formalin for 12- After 24 hours, conventional paraffin embedding, 4μm section with Leica microtome. 2) The sections are deparaffinized with xylene, and then washed with various levels of ethanol. The dewaxing steps are: xylene (I) 5 min → xylene (II) 5 min → 100% ethanol 2 min → 95% ethanol 1 min → 80% ethanol 1 min → 75% ethanol 1 min → distilled water washing for 2 min. 3) Harris hematoxylin semen was stained for 5 minutes and washed with tap water for 1 minute. 4) Differentiation of hydrochloric acid ethanol solution for 30s. 5) Soak in tap water for 15min or warm water (about 50℃) for 5min. 6) Set the eosin aqueous solution for 2 minutes. 7) Routine deh...

Embodiment 3

[0035] Example 3: Detecting the expression changes of miRNA-182 in the tissues of mice by fluorescence quantitative PCR

[0036] 1. Extract total miRNA from heart graft, spleen, liver and PBMC

[0037] On the 7th day after transplantation, 3 recipient mice in the control group and the experimental group were sacrificed, and part of the heart graft, spleen, and liver tissue were taken out. At the same time, EP tubes containing EDTA anticoagulant were used to collect mouse whole blood for the extraction of peripheral blood mononuclear cells (PBMC). Then, the total miRNA in heart graft, spleen, liver and PBMC were extracted.

[0038] (1) Extraction of PBMC and its miRNA

[0039] PBMC was separated by Ficoll density gradient centrifugation. First add 2ml of lymphocyte separation solution to a 15ml centrifuge tube, mix 1ml of blood and 1ml of 1640 medium uniformly, slowly add to the upper layer of lymphocyte separation solution, centrifuge at 2000rpm for 20 minutes, naturally slow down. ...

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Abstract

The invention discloses a miRNA-182 inhibitor.The sequence of the inhibitor is as follows: 5'-CGGUGUGAGUUCUACCAUUGCCAAA-3', wherein each nucleotide is modified by 2'-OMe, first two basic groups of the 5' end and first 4 basic groups of the 3' end are modified by phosphorus sulfuryl bonds, and the 3' end is modified with a cholesterol group.The miRNA-182 inhibitor can be used for preparing a drug for preventing and treating immunological rejection of heart transplantation.The miRNA-182 specific targeting inhibitor can be beneficial for reducing an allogeneic immune response and prolonging the graft survival time by inhibiting expression of miRNA-182 in a targeted mode after heart transplant surgery and can supply a basis to preparation of the drug for preventing heart graft dysfunction and protecting the heart graft functions.

Description

Technical field [0001] The invention relates to the field of medical bioengineering, in particular to a miRNA-182 inhibitor and its application in preparing medicines for preventing and treating heart transplantation immune rejection. Background technique [0002] Heart transplantation is an effective means to treat various end-stage heart diseases. With the use of new immunosuppressive agents, the incidence of acute rejection has decreased, but it is still the main cause of death within one year after heart transplantation. Acute cellular rejection (ACR) usually occurs 3-6 months after surgery or occasionally at any period after surgery. T-cell-mediated lymphocytes and macrophages infiltrate the graft, which not only causes short-term graft damage, but also Participate in cardiac graft vascular disease (cardiacallograftvasculopathy, CAV). CAV causes the concentric thickening of the arterial intima layer and the diffuse narrowing of the vascular lumen, which is an important man...

Claims

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Application Information

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IPC IPC(8): A61K31/7105A61K48/00A61P37/06
CPCA61K31/7105
Inventor 魏亮卢俊龚雪
Owner 成都仕康美生物科技有限公司
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