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Application of miRNA to treat myocardial fibrosis diseases

A technology for myocardial fibrosis, disease, applied in the field of biomedical specialty

Active Publication Date: 2014-09-10
GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More than 400 kinds of miRNAs have been identified in recent years, but there are still many kinds that have not yet been discovered [Berezikov E, Thuemmler F, van Laake LW, et al.Diversity of microRNAs in human and chimpanzee brain[J]. Nat Genet, 2006, 38( 12): 1375-1377.]

Method used

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  • Application of miRNA to treat myocardial fibrosis diseases
  • Application of miRNA to treat myocardial fibrosis diseases
  • Application of miRNA to treat myocardial fibrosis diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Neonatal rat cardiomyocyte culture and cardiomyocyte total RNA and total protein extraction

[0054] l. Primary culture of neonatal rat cardiomyocytes

[0055] 1.1 Take the heart of a newborn rat 1-3 days old and place it in pre-cooled Hanks balanced salt solution (HBSS, Invitrogen).

[0056] 1.2 Separate the myocardial tissue at about 1 / 3 of the lower end of the ventricle, and cut it into small pieces by surgery.

[0057] 1.3 Put a small piece of myocardial tissue into 5 mL of digestion solution containing 75 U / mL collagenase II (Worthington), incubate at 37°C for 20 minutes, collect the digestion solution and add fresh digestion solution to continue digestion. This digestion process was repeated 6 times.

[0058] 1.4 After the cells were centrifuged at 283 g for 5 minutes, they were resuspended in DMEM-F12 cell culture medium (1:1, Invitrogen) containing 10% fetal bovine serum and 1% antibiotics.

[0059] 1.5 Cultivate the cells on the cell culture dish for 1 hour,...

Embodiment 2

[0078] Cardiomyocytes transfected with miR-19b precursor (pre-mir-19b) and stimulated with angiotensin Ang II to induce cardiomyocyte fibrosis

[0079] 1. Extraction of total RNA and total protein after pre-mir-19b transfected cardiomyocytes

[0080] 1.1 Cells were seeded in a 24-well culture plate, the number of cells per well was 4×10 4 .

[0081] 1.2 Mix 1 ul of transfection reagent siPORT NeoFX (AM4510, Ambion) and 25 ul of OPTI-MEM I culture solution (Invitrogen) and incubate at room temperature for 10 minutes.

[0082] 1.3 Dilute pre-mir-19b (AM17100, Ambion) with OPTI-MEM I culture medium (Invitrogen), mix the two and incubate at room temperature for 10 minutes. Using a non-functional miRNA sequence pre-miR TM -Negative Control (AM17110, Ambion) treated cells as a control group (Control).

[0083] 1.4 Mix the diluted pre-mir-19b and transfection reagent, and incubate at room temperature for 10 minutes to form a transfection complex.

[0084] 1.5 Add the culture sol...

Embodiment 3

[0093] Analysis of the expression levels of miRNA-19b and CTGF in cardiomyocytes

[0094] 1. qRT-PCR detection of miRNA-19b and CTGF mRNA expression in cardiomyocytes.

[0095] 1.1 Using the collected cardiomyocyte total RNA as a template, the miR-19b gene was amplified with miR-19b-specific mirVana qRT-PCR primers (Ambion). The real-time TaqMan miRNA analysis and detection kit (ABI) was used to perform quantitative PCR to detect the expression of miR-19b in cells, and the U6snRNA gene was used as the internal reference for detection. See the kit instructions for detailed operating principles and methods.

[0096] 1.2 With the total RNA of cardiomyocytes collected as a template, CTGF mRNA was amplified with CTGF gene-specific PCR primers, real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect intracellular CTGF mRNA expression, and GAPDH gene was used as Check the internal reference. PCR products were visualized and quantified by 1% agarose gel electr...

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Abstract

The invention aims at providing application of miRNA to treat or diagnose myocardial fibrosis diseases. The sequence of miRNA is shown as SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 or SEQ ID 6. By applying miRNA to treat myocardial fibrosis diseases, substantial effect is obtained especially when different human derived miRNA-19b sequences and non-human derived miRNA-19b sequences are mixed for application, namely a human derived miRNA-19b sequence of SEQ ID 1 or SEQ ID 2 and one or more of the non-human derived miRNA-19b sequences of SEQ ID 3, SEQ ID 4, SEQ ID 5 and SEQ ID 6 are mixed.

Description

technical field [0001] The invention belongs to the professional field of biomedicine, and relates to miRNA, miRNA precursor RNA, miRNA antisense oligonucleotide sequence and its expression carrier, and its application in diagnosis and treatment of myocardial fibrosis. Background technique [0002] Cardiovascular disease is a serious disease that endangers human health and is one of the main causes of death. At present, in the death spectrum of Chinese residents, cardiovascular disease has become the second killer after malignant tumors, and tens of thousands of people are disabled every year due to this disease. The disease is of a great variety and its etiology is complex. Among them, the continuous and / or repeated aggravation of ischemia and hypoxia of myocardial fibers caused by moderate and severe coronary atherosclerotic stenosis will lead to myocardial fibrosis. Early in the disease, left ventricular diastolic function is impaired. If left unchecked, the normal str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11A61K48/00A61P9/00
CPCA61K48/005C12Q1/6883C12Q2600/158C12Q2600/178
Inventor 聂瑛洁高松池洪杰陈辉安宇
Owner GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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