Anti-arrhythmic medicine screening method and platform thereof
An anti-arrhythmic drug screening method technology, which is applied in the field of anti-arrhythmic drug screening method and platform, can solve the problems of long drug screening cycle, high instability, and high randomness, and achieve clear drug action mechanism, simple operation, reliable results
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Embodiment 1
[0057] Example 1 Construction of Cardiac Arrhythmia Model
[0058] 1. Take the heart, collect the ventricle, and cut into small pieces of male rats (Wistar) born 1 to 2 days old.
[0059] 2. Place the small pieces in the Tyrode digestion solution in a shaker at 37°C for 30 minutes. The digestion solution components are as follows:
[0060] Calcium-free Tyrode solution (40ml) + 50μM CaCl 2 (10 ml) + 1 mg / ml BSA (40 mg) + Collagenase (30 mg) + Protease II (1.5 mg).
[0061] 3. The cell suspension was centrifuged with Percoll gradient, the cells were collected, and the cells were resuspended in the culture medium (DMEM: M199 (4:1), 10% horse serum, 5% calf serum), and then according to the inoculation density 1.8×10 4 ~3.4×10 4 Cells / cm 2 , Inoculate into a petri dish with fibronectin on the bottom.
[0062] 4. Cells are grown in culture medium at 37℃ with 5% CO 2 When the cells are connected into a small network and begin to beat, the calcium fluorescent dye is loaded:
[0063] Weigh 10 mg...
Embodiment 2
[0066] Example 2 Determination of intracellular calcium ion concentration and cell membrane displacement in a cardiac arrhythmia model
[0067] 1. Drawing of microelectrodes for scanning conductance microscope
[0068] Application of boronized glass electrode, outer diameter 1.5mm inner diameter 0.86mm, containing guide wire
[0069] Apply Sutter P1000 drawing instrument
[0070] The drawing procedure is:
[0071]
[0072] 2. Put the cells of the arrhythmia model induced by different concentrations of taurocholate (taurocholate) prepared in Example 1 into a culture dish and place them on a microscope.
[0073] 3. Use SICM probes to record the beating of myocardial cells in real time. Here, the piezoelectric ceramic of the SICM displacement in the z direction is on the support of the petri dish. Apply a constant voltage (6V) to the electrode. Under the control of the micromanipulator, the glass probe filled with electrode solution steps closer to the cell membrane of the beating cell. W...
Embodiment 3
[0078] Example 3 Determination of intracellular calcium ion concentration and cell membrane displacement of cardiomyocyte arrhythmia model under the action of drugs
[0079] 1. The stained cells prepared in Example 1 were incubated with taurocholate (taurocholic acid) at a concentration of 1 mM and dexamethasone (a commonly used drug for treating arrhythmia) and placed in a microscope. on.
[0080] 2. Use SICM probes to record the beating of cardiomyocytes in real time. Here, the piezoelectric ceramic of the SICM displacement in the z-direction is on the support of the petri dish. Apply a constant voltage (6V) to the electrode. Under the control of the micromanipulator, the glass probe filled with electrode solution steps closer to the cell membrane of the beating cell. When the electrode approaches the cell membrane, the current will suddenly decrease, indicating that the electrode is working Area. Continue to move the electrode close to the cell membrane until the current is r...
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