Oligonucleotides and usage thereof in preparation of medicine for preventing and curing myocardial hypertrophy and heart failure
An oligonucleotide, myocardial hypertrophy technology, applied in the directions of drug combination, gene therapy, pharmaceutical formulation, etc., can solve the problems of ventricular remodeling and myocardial hypertrophy that have not been seen with oligonucleotide, and achieve the effect of inhibiting the occurrence and development
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Embodiment 1
[0046] Inhibitory effect of oligonucleotide (ODN) on the mRNA expression of ANF and β-MHC during neonatal rat cardiomyocyte hypertrophy
[0047] 1 Isolation of neonatal rat cardiomyocytes, induction of cardiac hypertrophy model and treatment of oligonucleotides
[0048] (1) Instruments, equipment, reagents and materials
[0049] Low-temperature refrigerators, carbon dioxide incubators, ultra-clean benches, inverted microscopes, cell culture bottles, horizontal centrifuges, sample injectors, droppers of various specifications, etc. ODN: All were synthesized by Takara Company, OPC grade, and their phosphate backbones were all thio-modified unless otherwise specified. Dissolve it in PBS (for the recipe, refer to "Molecular Cloning Experiment Guide (Third Edition)"), and quantify with a UV spectrophotometer. Isoproterenol (isoproterenol), trypsin, collagenase type II, ITS and Laminin were purchased from Sigma. HBSS, DMEM, double antibody (PS) and FBS were purchased from Gibco. ...
Embodiment 2
[0088] Inhibitory effect of oligonucleotide (ODN) on ANF protein expression during cardiac hypertrophy
[0089] 1. Separation of neonatal rat cardiomyocytes and induction of cardiac hypertrophy: the method is the same as in Example 1, and the cells are inoculated into a culture dish covered with a 20mm cover glass.
[0090] 2. Indirect immunofluorescence detection of ANF protein expression
[0091] (1) Instruments, equipment, reagents and materials
[0092] PI (sigma), ANF monoclonal antibody (mouse) was purchased from Santa Cruz, FITC-fluorescent secondary antibody was purchased from Invitrogen, and confocal microscope (Leica, Germany).
[0093] (2) Method:
[0094] Aspirate the culture medium, add appropriate amount of PBS to wash 3 times; add 4% formaldehyde solution, fix at room temperature for 20 min; add appropriate amount of PBS to fully wash, add 0.5% Triton100, and permeabilize at -20°C for 10 min; 2% BSA for blocking for 1 h; add ANF primary antibody (1:200 diluti...
Embodiment 3
[0099] Oligonucleotide (ODN) Dose Curves for Inhibiting ANF and β-MHC Expression in Cardiac Hypertrophy Model
[0100] 1. Separation of neonatal rat cardiomyocytes and induction of cardiac hypertrophy: the method is the same as in Example 1.
[0101] In the dose curve experiment, neonatal rat cardiomyocytes were stimulated with RJL01 at a final concentration of 20, 10, 5, 2, 1, and 0.5 μg / ml respectively. PBS was added to the control group and model group, and after 12 hours of action, isoproterenol was added. to a final concentration of 10 μmol / ml, and continued to incubate for 48 h before performing subsequent experiments.
[0102] 2. Extraction, reverse transcription and real-time quantitative PCR of total RNA: the method is the same as in Example 1.
[0103] 3. Experimental results
[0104] The results showed that ODN RJL01 at a concentration of 1 μg / ml could effectively inhibit the up-regulation of the expression of ANF and β-MHC in the process of cardiac hypertrophy, a...
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