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
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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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