Study On An Optimized Culture Method Of Neonatal Rat Cardiomyocytes And The Effect Of CTRP3 Anti-MIRI

Objective:(1)To optimize and improve the isolation and culture method of neonatal rat cardiomyocytes(NRCMs),and to find an efficient method for isolation and culture of neonatal rat cardiomyocytes;(2)to study the role of CTRP3 in myocardial ischemia/reperfusion injury.Methods:(1)Isolation and culture of neonatal rat cardiomyocytes.The heart of suckling rat was quickly obtained by aseptic operation,and then the heart was cut into myocardial tissue blocks by mechanical treatment.Two digestion methods were used to digest myocardial tissue respectively:(1)double enzyme digestion method: 0.08% trypsin and 0.125% collagenase mixture(1:1)digested myocardial tissue.(2)0.08% trypsin+mechanical digestion: first digested myocardial tissue with 0.08% trypsin and then digested the remaining myocardial tissue by mechanical digestion;The cell suspension filtered by 100?m cell sieve was used for differential adherent culture,the number and survival rates of isolated cells were detected by trypan blue staining,the morphology and characteristics of cells were observed under optical inverted phase contrast microscope,and the purity of neonatal rat cardiomyocytes was identified by confocal immunofluorescence staining.(2)Establishment of hypoxia/reoxygenation injury model: the hypoxia/reoxygenation injury model of NRCMs was established by nitrogen oxygen avoidance method(three-gas incubator).(3)MTT method was used to detect the response of NRCMs to drugs(CTRP3).(4)The activity of LDH,SOD and the content of MDA were analyzed by colorimetry,the percentage of apoptosis were detected by annexin V-FITC/PI double staining.Results:(1)There was no significant difference in cell morphology and the time when spontaneous pulsation began to occur in the primary cardiomyocytes obtained by the two digestion methods.The non-adherent cardiomyocytes were round and refractive.After adhering to the wall,the cardiomyocytes protruded pseudopodia,showing a diamond-shaped,fusiform,or irregular shape.At 24 hours after laying,some cardiomyocytes adhered to the wall and spontaneous pulsation could be observed.98% of the cells adhered to the wall at 48 h and the same amplitude pulsation between the cell clusters could be observed.(2)The number,survival rate,purity and vitality of NRCMs cultured by 0.08% trypsin + mechanical digestion method was better than that of double enzyme digestion method(p < 0.05).(3)Compared with the double enzyme digestion method,0.08% trypsin + mechanical digestion cultured NRCMs had better drug response.(4)Compared with H/R group,pretreatment with CTRP3 could significantly increase cell survival and the activity of SOD,and decrease the activity of LDH,the content of MDA,and the cell apoptosis rate.Conclusion:(1)The neonatal rat cardiomyocytes cultured by 0.08% trypsin + mechanical digestion method have a large number,high survival rate,high purity,good viability and good activity to drug,which is an optimized method for isolation and culture of neonatal rat cardiomyocytes;(2)CTRP3 may protect myocardial ischemia-reperfusion injury by reducing oxidative stress injury and inhibiting apoptosis.