| 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/β-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,β-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 collagenaseⅡ.(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 ofβ-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,β-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 ofβ-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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