Effects Of T-cadherin Against APN Attenuates Hypoxia/reoxygenation (H/R) Induced Myocardial Apoptosis

Part1:Culture of neonatal rat cardiomyocytes and transfection of Adenovirusobjective:The culture of neonatal rat cardiomyocytes and investigation of the optimum MOI about the transfection of adenovirusMethods:(1). The culture of neonatal rat cardiomyocytesNeonatal SD rat of24-48hours were disinfected,opened-heart, and clamped the ventricular portion, then chopped in D-Hanks washing liquid in ice bath.Digested with collagenase Ⅱ3-4times, the myocardial cells were collected trough centrifugation, and purified by treatment with the differential adhesion teehnique and the chemical inhibition method. Then cultured in the Dulbeeeo’s modified eagle culture medium consisting of20%fetal bovine serum, changed medium after48hours.The condition of cell growth and spontaneous beating were observed under the inverted microscope.(2) To the α-actin and cTnT in cytoplasm, the myocardial cells was identificated by immunofluorescence and immunocytochemistry.(3) the experiment of gene transfer targeting with cardiomyocyte in vitro.The neonatal rat cardiomyocyte were isolated and cultured, transfection ability and efficiency was examined at various MOI by fluorescence microscope and flow cytometer.Results:(1). High purity neonatal rat cardiomyocytes were obtained by primary culture, the microscope showed:after24hours, the primary cultured cells attached to the wall; after48hours, the cells began spontaneous fluctuation; after72hours, with the formation of cell junction, and the cells grew into clusters and beat regularly.(2). Cardiomyocytes were confirmed by immunofluorescence and immunocytochemistry, and the purity was more than95%.(3). Inverted fluorescence microscope and flow cytometer demonstrated:After72hours, over90%of cardiomyocytes were infected at MOI100,the expression of red report gene and transfection ability and efficiency reached to the highest level after72hours.Conclusion:The neonatal rat cardiomyocytes was cultured, and the optimum MOI value of adenovirus transfection were confirmed.Part2:Effects of T-cadherin against APN attenuates hypoxia/reoxygenation (H/R) induced myocardial apoptosisobjective:The aim of the prenent study was to establish myocardial cell hypoxia/reoxygenation (H/R) model, and silence the expression T-cadherin through the adenovirus mediated interference RNA technology. Then observe the influence of myocardial cell injury.Methods:We isolated primary cardiomyocytes from neonatal rats and established an model of hypoxia/reoxygenation(H/R) in vitro. The cardiomyocytes were randomly divided into5groups:(1)the control group:(2) H/R group;(3) H/R+APN group,(4) H/R+APN group+Ad-T-cadherin-siRNA group;(5) H/R group+APN+Ad-siRNA group (Adenovirus negative control). The indexes for observation were as follows: First of all, the cardiocyte morphology and spontaneous beating rate were observed by inverted microscope. Secondly, the expression of T-cadherin was detected by RT-PCR and Western blot. The apoptosis rate was analyzed through the flow cytometry and TUNEL(Terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling). Results:(1).The morphology of cardiocyte in every groupr:In the H/R group, the cells grew poorly, the beating rate was significantly slow. However,xompared with H/R tgroup,most cells in the H/R+APN group grew well.But the beating rate of cells in the H/R+APN+Ad-T-cadherin-siRNA group was slow when compared with H/R+APN group,and was not obviously when compared with H/R group.(2). RT-PCR and Western blotting analysis showed:the expression of T-cadherin mRNA and protein could detected in all of groups.Compared with control group,the expresstion of T-cad mRNA and the protein was decreased in H/R group.Pretreatment with APN,the above changes were significantly reversed.compared with H/R group,the indexes improved obviously(P<0.05),and the expression of T-cadherin could significantly reduced in H/R+APN+Ad-T-cadherin-siRNA group, compared with H/R+APN group, the difference was statistically significant(P<0.05).(3). The flow cytometry and TUNEL analysis showed:Compared with control group,the rate of apoptosis in H/R group was significantly increased, the TUNEL-positive cells(green)increased significantly.Pretreatment with APN, compared with H/R group,the above changes were significantly reversed (P<0.05). In silencing T-cadherin gene group, Cell apoptosis rate significantly increased, compared with H/R+APN group,the difference was statistically significant(P<0.05). compared with H/R group, the difference was not statistically significant(P>0.05).Conclusion:(1)The myocardial cells could was efficientively infected by Ad-T-cadherin-siRNA in vitro and the expression of this receptor was successfully reduced.(2)Adiponectin can reduce the apoptosis of myocardial cell which induced by hypoxia/reoxygenation.(3).T-cadherin may play a strong role for adiponectin-mediated cardioprotection in vitro model of hypoxia/reoxygenation...