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Deoxyribozyme for effective degradation of esoderma native-1mRNA and uses in reducing acute ischemic arrhythmia

A deoxyribozyme and nucleotide technology, applied in medical preparations containing active ingredients, hydrolytic enzymes, cardiovascular system diseases, etc., can solve problems related to non-specific activity toxicity, design limitations, etc., to reduce arrhythmia, Effects of reducing damage and inhibiting hypertrophy of cardiomyocytes

Inactive Publication Date: 2009-08-19
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering the chemistry and ability of antisense molecules to form stable heteroduplexes with their target mRNAs, this dependence on RNAse H limits the design of antisense molecules
Antisense DNA molecules also have problems associated with non-specific activity and even toxicity at higher concentrations

Method used

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  • Deoxyribozyme for effective degradation of esoderma native-1mRNA and uses in reducing acute ischemic arrhythmia
  • Deoxyribozyme for effective degradation of esoderma native-1mRNA and uses in reducing acute ischemic arrhythmia
  • Deoxyribozyme for effective degradation of esoderma native-1mRNA and uses in reducing acute ischemic arrhythmia

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1 Design and in vitro screening of ET-1 10-23 deoxyribozyme

[0061] The ET-1 10-23 deoxyribozyme designed by the present invention contains a catalytic core of 15 nucleotides and a substrate binding region of 9 nucleotides, and two nucleotides are thiolated at its 5' and 3' ends grooming. The cleavage site is all AU and GU connections from the start site AUG of rat ET-1 mRNA to +219 nucleotides downstream, and 5 deoxyribozymes with a substrate-binding entropy value <-24kcal / mol were selected, They were named DZ1-DZ5 according to the cutting sites.

[0062] The full length of rat ET-1 mRNA is 1385 nucleotides, and its initiation codon AUG begins at the 184th nucleotide of rat ET-1 mRNA, and thereafter a total of 609 nucleotides encode preproendothelin-1 ( Prepro ET-1, ppET-1). The region of ETT-1 mRNA encoding mature ETT-1 is located between +156 and +219 after the start codon. Find all AU and GU junctions between the start codon and +219 as cutting sites...

Embodiment 2

[0067] Example 2 Effect of DZ4 on the expression of ET-1 gene in cardiomyocytes

[0068] The uptake of DZ4 labeled with fluorescein FAM at the 5′ end of the primary cultured neonatal rat cardiomyocytes was transfected with liposome-labeled DZ4 (0.2umol / L) for 12 hours, and there was no fluorescence in the cardiomyocytes, and fluorescence in the nuclei after 24 hours Stronger; the cardiomyocytes transfected with calcium phosphate precipitation marked DZ4 had fluorescence after 12 hours, and increased after 24 hours, and the fluorescence was stronger than liposome transfection. The results of confocal laser microscopy showed that 24 hours after transfection of cardiomyocytes by calcium phosphate precipitation, there was fluorescence distribution in the cytoplasm and nucleus of the cells.

[0069]After 24 hours of liposome transfection into cardiomyocytes, semi-quantitative RT-PCR results showed that the integrated optical density value of the ET-1 / β-actin band in the serum co...

Embodiment 3

[0070] Example 3 Protective effect of DZ4 on hypoxic injury of cardiomyocytes

[0071] To observe the effects of DZ4 on ET-1mRNA in hypoxic cardiomyocytes simulated by cobalt chloride, the content of malondialdehyde (MDA) in culture medium supernatant, and the activity of superoxide dismutase (SOD). The content of ET-1mRNA in the hypoxia group increased to 288±37% of that in the normoxia group (n=3, p<0.05), the MDA content in the supernatant of the culture medium increased to 141±7% (n=10, p<0.05), and the SOD Activity decreased to 52±7% (n=10, p<0.05). 24 hours after transfection with 0.1umol / L DZ4, compared with the hypoxic group, the ET-1 mRNA content decreased by 27% (n=3, p<0.05), and the MDA content in the culture supernatant decreased by 6% (n=10, p<0.05 ), the SOD activity increased by 19% (n=10, p<0.05); after 24 hours of 0.2umol / L DZ4 transfection, the ET-1 mRNA content decreased by 35% (n=3, p<0.05), and the supernatant MDA content of the culture medium Decrea...

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Abstract

The 10-23 deoxyribozyme constructed in the invention utilizes in-vitro-transcription ET-1 full-length RNA as s substrate to filter the 10-23 deoxyribozyme with cut ET-1 mRNA, and can reduce the expression of an ET-1 gene of myocardial cells through transfection, inhibit hypertrophy of serum induced myocardial cells, and reduce the damage of hypoxic myocardial cells; the ET-1 10-23 deoxyribozyme can obviously reduce the occurrence of arrhythmia of isolated rat hearts after acute ischemia and improve heart functions. The invention provides tools for further clearing the functions of endogenous ET-1 in acute ischemic arrhythmia, and also provides theoretical basis and new ways of thinking for explaining the mechanism and prevention of and development of new medicaments for the acute ischemic arrhythmia.

Description

technical field [0001] The invention relates to the fields of basic medicine and physiology, in particular to a class of deoxyribozyme, which has the functions of degrading ET-1 mRNA and alleviating acute ischemic arrhythmia. Background technique [0002] Endothelin (endothelin, ET) is a strong vasoconstriction peptide, its family includes ET-1, ET-2, ET-3 and ET-4 four isomeric peptides. Except for the difference in chromosomal location and sequence, the expression process of each isomeric peptide gene of ET is similar. The ET gene transcribes mRNA, which enters the cytoplasm, and under the action of ribosomes, translates to produce prepro-endothelin (prepro ET, ppET), and ppET generates pre-endothelin (preET) or preET under the action of double amino acid endonuclease Big endothelin (big ET), then forms a mature ET consisting of 21 amino acids or 31 amino acids under the action of different enzymes. The ET receptors currently cloned in mammals are divided into ET A and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22A61K38/46A61P9/06
Inventor 任安经潘秀颉林丽李楠袁文俊
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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