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Screening method for lncRNA relevant to myocardial ischemia reperfusion and application

A technology of myocardial ischemia and screening method, which is applied in the application of lncRNA and the field of lncRNA screening, can solve the problems such as the inability to understand the function of lncRNA, and achieve the effect of protecting myocardial cells from hypoxia and reoxygenation injury

Active Publication Date: 2015-03-11
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Screening method for lncRNA relevant to myocardial ischemia reperfusion and application
  • Screening method for lncRNA relevant to myocardial ischemia reperfusion and application
  • Screening method for lncRNA relevant to myocardial ischemia reperfusion and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] An in vitro cardiomyocyte hypoxia-reoxygenation model was constructed, and the Rat LncRNAArray v2.0 (4x44K, Arraystar) chip was used to screen out lncRNAs differentially expressed during cardiomyocyte hypoxia-reoxygenation. The steps are as follows:

[0032] a. Cardiomyocyte hypoxia and reoxygenation treatment and normal cell treatment: the cardiomyocyte cell line (H9c2) was placed in the cell culture medium of sugar-free DMEM without FBS, and put into the hypoxic incubator (2 , 5%CO 2 , about 95% N 2 ) for 1 hour, and then put the two groups of cells into the normoxic incubator (21% O 2 , 5%CO 2 , 74%N 2 ) for reoxygenation for 2 hours to establish a cardiomyocyte model of cardiomyocyte hypoxia and reoxygenation injury. 2 , 5%CO 2 , 74%N 2 ) for 3 hours.

[0033] b. Monolayer adherent cell homogenate: add 1ml TRIZOL reagent directly to the culture dish to lyse the cells, and pipette several times with a gun during lysis.

[0034] c. Add 0.2ml chloroform to the T...

Embodiment 2

[0045] An in vitro cardiomyocyte hypoxia-reoxygenation model and a Langendorff isolated heart ischemia-reperfusion model were constructed, and real-time quantitative PCR was used to verify the results of the chip.

[0046] a. Construct an in vitro cardiomyocyte hypoxia-reoxygenation model, which is the same as that described in a in Example 1.

[0047] b. Constructing the Langendorff isolated heart ischemia-reperfusion model: 6 male SD rats (SD rats with a body weight between 280-330g) were randomly divided into 2 groups using the random number table method, and the rats were weighed and weighed. Anesthetized by intraperitoneal injection of 10% chloral hydrate. After the animals were fully anesthetized, they were placed on the animal table and fixed. Open the thoracotomy to expose the aortic arch, gently lift the confluence of the aorta and the innominate artery, cut the aorta from below the aortic arch, lift the broken end of the aorta, cut off the complete heart, quickly put...

Embodiment 3

[0060] (1) Use siRNA to interfere with lncRNA (NR_002154), observe the changes in cardiomyocytes after hypoxia and reoxygenation in vitro, use real-time quantitative PCR to observe the effect of interference, CCK-8 to detect cell viability, and WB to detect changes in autophagy-related indicators LC3B Condition

[0061]a. siRNA interferes with lncRNA (NR_002154) in cardiomyocytes, and selects the most suitable siRNA for subsequent experiments: Synthesis of small interference fragments (3 pieces) targeting lncRNA (NR_002154), sequence 1: sense strand: 5'GCAUCCUGGUGCUAAGUUA dTdT 3 ', antisense strand: 3'dTdT CGUAGGACCACGAUUCAAU 5'; Sequence 2: sense strand: 5'GCGCCAUUGCUGCAAAUUA dTdT 3', antisense strand: 3'dTdT CGCGGUAACGACGUUUAAU 5'; Sequence 3: sense strand: 5'GCAGGACACAGUUAAGAAU dTdT 3'anti Sense strand: 3'dTdT CGUCCUGUGUCAAUUCUUA 5'. Spread H9c2 cells in a 6-well plate, and start transfection with siRNA when the cell density reaches 60%. For each transfection sample, prep...

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Abstract

The invention discloses a screening method for lncRNA relevant to myocardial ischemia reperfusion. The method comprises the following steps: constructing an extracorporeal myocardial cell anoxia reaeration model, and screening lncRNA with differential expression in a myocardial cell anoxia reaeration process by using a Rat LncRNA Array v2.0 chip; constructing an extracorporeal myocardial cell anoxia reaeration model and a langendorff isolated heart ischemia reperfusion model, and verifying the result of the chip by using a real-time quantitative PCR. Interference lncRNA is used, so that expression is reduced, an autophagia level in cardiac muscle cells are adjusted and controlled, and the purpose of protecting myocardial anoxia reaeration damage is realized; therefore, the invention provides the method of protecting the cardiac muscle cell anoxia reaeration damage of the cardiac muscle cells by adjusting and controlling the lncRNA, and has an important significance for further researching lncRNA relevant to the myocardial ischemia reperfusion in the aspect of predicting, and preventing and treating miocardial infarction.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a screening method for lncRNA related to myocardial ischemia-reperfusion, and also relates to an application of lncRNA 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. Reperfusion therapy can rescue ischemic myocardium, but it can also transform reversible ischemic injury into irreversible reperfusion injury. How to reduce the degree of myocardial cell damage and improve the prognosis has become an important topic in the field of cardiovascular diseases. The mechanism of myocardial cell injury caused by ischemia-reperfusion has not been fully elucidated. The currently recognized mechanism may be: mitochondrial dysfunction, intracellular calcium ion overload, and a large number of...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 黄周青叶浡之黄伟剑蔡雪黎单培仁孔繁奇吴圣杰
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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