Recombinant protein for treating myocardial hypertrophy and application thereof to preparation of medicine for resisting myocardial hypertrophy
A technology of cardiac hypertrophy and recombinant protein, applied in the field of medicine, can solve the problems such as the anti-cardiac hypertrophy effect of galectin 1 that has not been reported, and achieve the improvement of intracellular calcium and calcium-related signal protein disorders, improve signal pathways, and inhibit expression Effect
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Embodiment 1
[0031] Production of hypertrophic cardiomyocyte model
[0032] (1) Isolation of neonatal rat ventricular myocytes (NRVMs): SD neonatal rats within 48 hours of birth were collected regardless of sex. The heart tissue was excised and placed in ice water bath containing Ca 2+ , Mg 2+ Hanks' Balanced Salt Solution (HBSS, Gibco, Thermo Fisher). The blood in the heart chamber was washed away with HBSS, and the non-myocardial tissue was removed under the microscope using a stereo microscope. Carefully cut the myocardial tissue into a uniform size, about 1mm, with curved scissors in a sterile centrifuge tube 3 tissue fragments. Myocardial tissue was digested with an enzyme complex containing 0.06% trypsin (Sigma-Aldrich, Merck) and 0.04% type II collagenase (Sigma-Aldrich, Merck), formulated in calcium- or magnesium-free HBSS solution. After several times of digestion at 37°C, cardiomyocytes were obtained. Cardiac fibroblasts were removed by differential attachment for 1 hour, a...
Embodiment 2
[0035] Determination of surface area and membrane capacitance of cardiomyocytes
[0036] (1) Using anti-α-actinin antibody (1:100 dilution) to perform immunofluorescence staining on cardiomyocytes treated with galectin 1, and scan cell fluorescence pictures under a confocal laser microscope (LSM710, Zeiss, Germany), the The obtained images were measured by Image J software (NIH) for cell surface area.
[0037] (2) Since the cell membrane capacitance represents the size of the cell, the present invention uses the patch clamp technique to measure the myocardial cell membrane capacitance after galectin-1 treatment. The formulation of the whole cell voltage clamp electrode external solution is as follows: (unit: mM) 140TEA-Cl, 10HEPES, 1MgCl 2 , 5CaCl 2 , 10glucose, pH 7.4, osmotic pressure 300-310mOsm / L. The inner solution of the electrode contains (unit: mM) 130CsCl, 5EGTA, 1MgCl 2 , 10HEPES, 2Na 2 ATP, 0.5GTP, 10glucose, pH 7.2, osmotic pressure 290-300mOsm / L. Use Axopatc...
Embodiment 3
[0039] Detection of intracellular calcium level in cardiomyocytes
[0040]The detection of intracellular calcium in cardiomyocytes uses the Fluo-4AM probe (Molecular Probe, ThermoFisher). The specific method is: the cardiomyocytes are planted in a cell culture dish pre-coated with laminin, and cultured in DMEM medium for 24 hours . After the cells adhered to the wall, the medium was replaced, and the cells were washed 3 times with bench-top solution. Tyrode solution contains: 140NaCl, 5.4KCl, 1.8CaCl 2 , 1MgCl 2 , 10HEPES, and 10glucose, the pH was adjusted to 7.4 with NaOH. Afterwards, Fluo-4AM fluorescent probe was added to make the final concentration 4 μM. After incubating at 37°C for 30 minutes, the cells were washed with Tyrode's solution to wash away excess dye. A laser confocal microscope (LSM710, Zeiss, Germany) was used for shooting, using the Time series continuous recording mode, the quality was 7, the sampling size was 512×512, and the scanning speed of each ...
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