A miRNA-182 inhibitor and its application in the preparation of drugs for preventing and treating immune rejection in heart transplantation

A technology of mirna-182 and 1. mirna-182 is applied in the field of medical bioengineering to achieve the effect of protecting the function of the heart graft and preventing the failure of the heart graft

Active Publication Date: 2017-07-28
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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  • A miRNA-182 inhibitor and its application in the preparation of drugs for preventing and treating immune rejection in heart transplantation
  • A miRNA-182 inhibitor and its application in the preparation of drugs for preventing and treating immune rejection in heart transplantation
  • A miRNA-182 inhibitor and its application in the preparation of drugs for preventing and treating immune rejection in 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 Using a Mouse Model of Heterotopic Heart Transplantation

[0021] 1. Synthesis of the 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 (abbreviated as Skm-182 in the schematic diagram), the RNA sequence of the miRNA-182 inhibitor is 5'-CGGUGUGAGUUCUACCAUUGCCAAA-3'. The connection modification of RNA and cholesterol groups was carried out in a controlled-pore glass solid support. During the synthesis of miRNA-182 inhibitors, the modification of the phosphorosulfuryl bond at the designated position is accomplished by the oxidation reaction of phosphite and phenylacet...

Embodiment 2

[0032] Embodiment 2: Observing the pathological changes of the heart graft with the method of HE staining

[0033] 1. Experimental method: 1) on the 7th day after transplantation, each 3 recipient mice in the control group and the experimental group were sacrificed to obtain transplanted heart tissue pieces, which were fixed with 10% neutral formalin for 12- After 24 hours, they were routinely embedded in paraffin and sectioned at 4 μm with a Leica microtome. 2) Sections were dewaxed with xylene and washed with various levels of ethanol to water. The dewaxing steps are: xylene (I) 5 min→xylene (II) 5 min→100% ethanol for 2 min→95% ethanol for 1 min→80% ethanol for 1 min→75% ethanol for 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) Differentiate in hydrochloric acid ethanol solution for 30s. 5) Soak in tap water for 15 minutes or warm water (about 50°C) for 5 minutes. 6) Place the e...

Embodiment 3

[0035] Example 3: Detection of expression changes of miRNA-182 in mouse in vivo tissues by fluorescent quantitative PCR

[0036] 1. Extraction of Total miRNAs from Heart Grafts, Spleen, Liver, and PBMCs

[0037] On the 7th day after transplantation, 3 recipient mice in each of the control group and the experimental group were sacrificed, and part of the heart graft, spleen and liver tissues were removed respectively. At the same time, mouse whole blood was collected in EP tubes containing EDTA anticoagulant for the extraction of peripheral blood mononuclear cells (PBMC). Total miRNAs were then extracted from heart grafts, spleen, liver and PBMCs, respectively.

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

[0039] PBMCs were separated by Ficoll density gradient centrifugation. First add 2ml of lymphocyte separation medium to a 15ml centrifuge tube, mix 1ml of blood and 1ml of 1640 medium evenly, slowly add to the upper layer of lymphocyte separation medium, centrifuge at 20...

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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 the preparation of drugs for preventing and treating immune rejection in heart transplantation. Background technique [0002] Heart transplantation is an effective means of treating various end-stage heart diseases. The incidence of acute rejection has decreased with the use of newer immunosuppressants, but it remains a major cause of death in recipients within 1 year of heart transplantation. Acute cellular rejection (acute cellular rejection, ACR) often occurs 3-6 months after surgery or occasionally at any time after surgery, and is mediated by T cells to infiltrate the graft with lymphocytes and macrophages, which not only causes short-term graft damage , and involved in cardiac allograft vascular disease (cardiac allograft vasculopathy, CAV). CAV causes concentric thickening of arterial intima and diffuse narrowing of vascu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C12N15/113A61K31/7105A61P37/06
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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