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Screening method of tsRNA related to myocardial ischemia reperfusion

A technology for myocardial ischemia and screening methods, applied in the field of biomedicine, can solve problems such as the inability to understand the function of tsRNA

Inactive Publication Date: 2020-11-06
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not clear which tsRNAs are involved in the process of myocardial ischemia-reperfusion injury, and the functions of these tsRNAs are still unclear

Method used

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  • Screening method of tsRNA related to myocardial ischemia reperfusion
  • Screening method of tsRNA related to myocardial ischemia reperfusion
  • Screening method of tsRNA related to myocardial ischemia reperfusion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Construct an in vitro myocardial ischemia-reperfusion model, and use sequencing to screen out differentially expressed tsRNAs during myocardial ischemia-reperfusion. The steps are as follows:

[0099] a. Grouping and treatment of myocardial ischemia-reperfusion in vitro model animals: 6-week-old Sprague-Dawley (SD) male rats (body weight 260-280g), kept at temperature (22-25°C) and humidity (relative humidity 50%) ) in an animal room with a 12h / 12h light-dark cycle and allowed free access to food and water. The SD rats were randomly divided into two groups: a sham operation group (sham operation, n=4), and a myocardial ischemia-reperfusion group (I / R, n=4). Rats in the myocardial ischemia-reperfusion group were anesthetized with 2% sodium pentobarbital (50 mg / kg intraperitoneally), and artificially ventilated (80 times / min) using a rodent mask. After the anesthesia was completed, at the fourth intercostal space on the left side of the sternum, the skin and muscle tissu...

Embodiment 2

[0110] The rat myocardial ischemia-reperfusion model and myocardial cell hypoxia-reoxygenation model were constructed, and the sequencing results were verified by real-time quantitative PCR.

[0111] a. Construct a myocardial ischemia-reperfusion model, as described in a in Example 1.

[0112] b. Construct the cardiomyocyte hypoxia-reoxygenation model: place the cardiomyocyte cell line H9c2 in the cell culture medium of sugar-free DMEM without FBS, put them into the hypoxic incubator and culture them for 2 hours, and then put the two groups of cells into the normal The oxygen incubator was reoxygenated for 3 hours to establish a cardiomyocyte model of cardiomyocyte hypoxia and reoxygenation injury. The cells in the normal group were cultured in the normoxic incubator for 5 hours; among them, the hypoxic incubator was 2 , 5%CO 2 , about 95% N 2 ; Normoxic incubator is 21% O 2 , 5%CO 2 , 74%N 2 ;

[0113] c. Tissue sample homogenization: For every 50-100 mg tissue sample, a...

Embodiment 3

[0124] To construct cardiomyocyte hypoxia-reoxygenation model, tiRNA-Met-CAT-002-mimics were used to overexpress tiRNA-Met-CAT-002.

[0125] Cell viability was detected by CCK-8.

[0126] Western blot was used to detect autophagy-related indicators; the ratio of LC3-II / LC3-I and the expression of p62 protein.

[0127] Electron microscopy was used to observe the formation of autophagosomes.

[0128] a. Construct an in vitro cardiomyocyte hypoxia-reoxygenation model, as described in b in Example 2. The cells were randomly divided into four groups: Control group; H / R group; H / R+tiRNA-Met-CAT-002-mimics-NC group; H / R+tiRNA-Met-CAT-002-mimics group.

[0129] b. Overexpress tiRNA-Met-CAT-002 using tiRNA-Met-CAT-002-mimics: add 150ul OPTI-MEM and 1.5ul lipo2000 to reagent tube a; add 150ul OPTI-MEM and 5ul mimics-NC to reagent tube b and mimics; after standing for 5 minutes, mix the two tubes a and b; after mixing and standing for 15 minutes, add the above mixed solution to 1ml of...

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Abstract

The invention discloses a screening method of tsRNA related to myocardial ischemia reperfusion. The method is characterized by comprising the following steps of building a rat myocardial ischemia reperfusion model, and screening out differentially expressed tsRNA in a myocardial ischemia reperfusion process by adopting a sequencing method; and building an in-vivo rat myocardial ischemia reperfusion model and an in-vitro myocardial cell hypoxia reoxygenation model, and adopting real-time quantitative PCR for sequencing verification. According to the scheme, the tsRNA participates in the myocardial ischemia reperfusion injury process, and an application of the tsRNA in the aspect of myocardial ischemia reperfusion injury treatment is provided, so that a new idea is provided for people to research a pathophysiology process of myocardial ischemia reperfusion injury, and a new molecular basis or a treatment target is provided for prevention and treatment of myocardial ischemia reperfusion injury.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a screening method for tsRNA related to myocardial ischemia-reperfusion. Background technique [0002] Coronary atherosclerotic heart disease (CHD) is the most common cardiovascular disease, and acute myocardial infarction is one of the main causes of human death. In recent years, early reperfusion therapy using thrombolytic agents and percutaneous coronary intervention (PCI) has achieved some effect in reducing myocardial infarction. To a certain extent, reperfusion therapy can save ischemic myocardium, but it can also transform reversible ischemic injury into irreversible reperfusion injury, thereby increasing the incidence of heart failure. The mechanism of myocardial cell damage caused by ischemia-reperfusion injury is multifactorial and complex, and has not yet been fully elucidated. Various processes such as intracellular calcium ion overload, oxidative st...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6869C12Q1/6883
CPCC12Q1/6851C12Q1/6869C12Q1/6883C12Q2600/158C12Q2600/178C12Q2563/107C12Q2531/113C12Q2545/101
Inventor 叶浡之肖方毅黄伟剑翁雅雯林爽钟凌峰
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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