| Objective:This experiment aims to build hypoxia/reoxygenation models of SD rat primary cardiomyocytes to find out whether the wnt/beta-catenin signaling pathway effects on them and the different expressions among hypoxia/reoxygenation cardiomyocyte models.And we also want to know that what may lay the foundation for the relationship of cardiomyocyte apoptosis,and try to explain the mechanism of myocardial ischemia by Wnt/?-catenin signaling pathway.Methods:(1)Cardiomyocytes of Neonatal rat culture:40 Neonatal Spague-Dawley rats were operated to expose their hearts on aseptic operation table.After fascia was cleaned away,cut the heart into pieces,transfer them to a new Dish filled with 0.25%trypsin 3 ml.Cardiomyocytes were digested by trypsin overnight(14 hours)under4?,and then lightly clean away trypsin.Cardiomyocytes were digested by collagenase II for 15 min at 37?to get cardiomyocytes.Myocardial cells were plated into petri dish at a density about 1×10~5/cm~2 for about 80minutes.Then culture cardiomyocytes with DMEM which contained 10%FBS and Brdu at 37?,5%CO2 humidifier incubator for 48 hours.(2)Hypoxia/reoxygen model of cardiomyocytes:cells were divided into 7groups randomly including control group,6 groups put into anoxic environment for 2h,4h,6h,8h,10h,12h and then 2 hours'reoxygenation.Lactate Dehydrogenase Kit(colorimetric method)tested LDH(lactate dehydrogenase)of each group.Total superoxide dismutase(hydroxylamine)kit tested SOD(Superoxide Dismutase)activity.(3)Real-time PCR:Divided cardiomyocytes into 4 groups randomly,including control group,group H/R,group H/R+A,group H/R+D.All cardiomyocytes besides that in control group were put into anoxic environment for 8 hours and then 2 hours'reoxygenation,add with DKK-1 and Agonist 1 to intervene group H/R+A and group H/R+D before reoxygenation.Total RNA was extracted from different groups and transcribed into cRNA by RT-PCR kit,then use SYBR Green finish quantitative PCR to test Wnt3a mRNA,?-catenin mRNA,c-myc mRNA.(4)Western blotting:Total protein was extracted from different cardiomyocytes groups.BCA method was used to measure the concentration of the protein.After total protein was separated by SDS-PAGE electrophoresis,transferred to the PVDF membrane,primary antibodies were incubated,the spicific bands were imaged by ECL method and were analyzed by Quantity One software and Bio-Rad.(5)Flow cytometry:Cardiomyocytes of each group apoptosis was evaluated by flow cytometry.Cardiomyocyte samples were cleaned with PBS for 3times,and then digested by 0.25%trypsin for 3 minutes,were centrifugated by centrifugal machine.Then binding buffer was used to suspended cells for 3 times.Dye samples with Annexin V-FITC and PI.Flow cytometry was used to detect cell apoptosis rat.Results:(1)Neonatal rats were isolated by trypsin and collagenase?.(2)Real-time PCR:Effect of hypoxia/reoxygenation on wnt3a,beta-catenin and c-myc:Compared with,Wnt3a mRNA expression of hypoxia/reoxygenation group was higher than that of the control group(P<0.05),lower than that of group H/R+A(P<0.05),higher than that of H/R+D group.The expression of?-catenin mRNA of the hypoxia/reoxygenation group was higher than that in the control group(P<0.05),was lower than H/R+A group(P<0.05),and was higher than H/R+D group,c-myc mRNA expression of hypoxia/reoxygenation group was higher than that of the control group(P<0.05),lower than that of H/R+A group(P<0.05),higher than that of H/R+D group.(3)Western blotting:Effect of hypoxia/reoxygenation on wnt3a,?-catenin and c-myc:Compared with,Wnt3a protein expression of hypoxia reoxygenation group was higher than that of the control group(P<0.05),lower than that of H/R+A group(P<0.05),higher than that of H/R+D group.The expression of?-catenin protein of the hypoxia/reoxygenation group was higher than that in the control group(P<0.05),was lower than H/R+A group(P<0.05),and was higher than H/R+D group.C-myc protein expression of hypoxia/reoxygenation group was higher than that of the control group(P<0.05),lower than that of H/R+A group(P<0.05),higher than that of H/R+D group.(4)Apoptosis rate detection:Early apoptosis rate:Rate of group H/R was higher than that of the control group(P<0.05),lower than that of group H/R+A(P<0.05),lower than H/R+D(P<0.05).Late apoptosis rate:H/R group was higher than control group(P<0.05),lower than that of group H/R+A(P<0.05),lower than H/R+D(P<0.05).Conclusion:(1)Wnt3a,beta-catenin and c-myc are involved in the process of hypoxia/reoxygenation in SD rat cardiomyocytes.Hypoxia/reoxygenation activates the Wnt signaling pathway in SD rats.(3)Wnt3a,beta-catenin and c-myc aparticipate in the regulation of cell apoptosis during hypoxia/reoxygenation. |
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