Regulation Mechanism Of GFA On Cardiac Sodium Channel And Auxiliary Subunits

Part Ⅰ Primary culture of rats myocardial cellObjective: To provide qualified cardiac myocytes for drug interventionexperiment. Methods: Use rats as experimental animals within24hours of birth,sterile conditions using enzyme separation method to separate singlecardiomyocytes, differential adherent method of purification of myocardialcells.Use containing10%fetal bovine serum DMEM/F12culture based on37℃constant temperature,5%CO2develop myocardial cells.Once every24hours inhalf volume broth, when abnormal cells rate after100times/min, the second partcan be carried out the experiment. Results: Get myocardial cells whose pulsefrequency beyond100times/min successfully for more than25times, and thenext part of drug experiment could be done. Conclusions: The shorter time ofborn, the stronger cell is. Strict aseptic operation is the necessary premise of cellculture success, reducing blocking injury in training process, to ensure thestability of cell environment, can effectively improve the probability of success. Part Ⅱ GFA and Amio on neonatal rat cardiac myocytes in vitrointerventionObjective: To explore two different concentrations of GFA and Amio, whichspontaneously beating cardiac myocytes were incubated frequency differences.Methods: In the first part of the experiment of the proceeds of the cardiacmyocytes, for this part is experimental material. Using different concentrationsof GFA and Amio incubation cells, respectively in the incubation for3hours,6 hours,12hours,24hours. Observe pulsation frequency count of cardiacmyocytes. Observation contrast different times and different concentrations afterdrug intervention, cardiac autonomic pulsation frequency difference. After eachtime point to observe, collect cells to-80℃, as the third part of theexperimental materials. Results: GFA and Amio on cardiac myocytesautonomous pulsation have inhibition. GFA inhibition and the time and theconcentration were positively correlated. When the incubation time reaches acertain length, the two kinds of drugs on cardiac myocytes autonomousinhibition pulsation frequency are no longer statistically significant.Conclusions: GFA and Amio can inhibit cardiac myocytes in vitro independentpulsation, and the inhibition effect is not statistically difference. Part Ⅲ Impact of sodium channel and its auxiliary protein incardiac myocytes intervene by GFA and AmioObjective: Detection of the two drugs on cardiac myocytes sodium channelsand the influence of auxiliary subunits expression. Methods: Extract mRNAspecimens of each group, using reverse transcription (RT) and polymerase chainreaction (PCR), select GAPDH as internal, detection of sodium channel SCN5A,SCN1B, SCN3B, SCN4B gene expression. Results: GFA and Amio inhibitsSCN1B and SCN3B expression (P <0.01), and drug concentration is related tothe intensity of inhibition. GFA and Amio has no work on SCN4B (P>0.05).GFA shows time dependence on SCN1B SCN3B and SCN5A.(P <0.01).Conclusions: GFA and Amio inhibits SCN5A, SCN1B SCN3B expression, anda concentration and time dependence.