七氟醚处理后的低温缺氧-复氧心脏成纤维细胞培养液对心肌细胞的影响

Effects of hypothermic hypoxia-reoxygenation fibroblast culture medium treated with sevoflurane on cardiomyocytes

目的观察七氟醚对低温缺氧-复氧心脏成纤维细胞(CFs)培养液中心肌细胞搏动及缝隙连接蛋白43(Cx43)表达的影响。方法培养4 d的正常原代CFs随机分为三组:对照组(C组)、缺氧-复氧损伤组(IR组)和七氟醚组(Sev组),每组约5×10^5个细胞。C组CFs持续培养5 h;IR组和Sev组CFs低温缺氧1 h(4℃,95%N 2+5%CO2),复氧(37℃,5%CO2+95%空气)4 h,Sev组复氧过程中加入含2.5%七氟醚的培养液培养。培养结束时分别收集各组培养液,加入正常心肌细胞培养16 h。免疫细胞化学SP染色法鉴定CFs与心肌细胞;HE染色观察CFs与心肌细胞形态;磁带式录像机观察并记录心肌细胞搏动频率;台盼蓝染色法测心肌细胞死亡率;Western blot法测定Cx43蛋白及p-Cx43蛋白相对含量。结果与C组比较,IR组心肌细胞搏动频率明显减慢,IR组和Sev组心肌细胞死亡率明显升高,Cx43蛋白和p-Cx43蛋白相对含量明显降低(P<0.05)。与IR组比较,Sev组心肌细胞搏动频率明显加快,心肌细胞死亡率明显降低,Cx43蛋白和p-Cx43蛋白相对含量明显升高(P<0.05)。结论七氟醚可加快低温缺氧-复氧CFs培养液中心肌细胞搏动频率,上调Cx43和p-Cx43蛋白表达,降低心肌细胞死亡率,增强心肌细胞活性。

Objective To observe the effects of sevoflurane on cardiomyocytes pulsation and Connexin 43(Cx43)expression in hypothermic hypoxia-reoxygenation fibroblasts culture medium.Methods Normal primary fibroblasts(CFs)cultured for 4 days were randomly divided into three groups:control group(group C),hypoxia-reoxygenation group(group IR)and sevoflurane group(group Sev),each group has 5×10^5 cells.CFs in group C continued to be cultured for 5 hours.CFs under hypothermia hypoxia(4℃,95%N 2+5%CO2)for 1 hour and reoxygenation(37℃,5%CO2+95%air)for 4 hours in group IR and group Sev,and 2.5%sevoflurane was added to the culture medium during reoxygenation in group Sev.At the end of culture,the culture media of each group were collected and cultured with normal cardiomyocytes for 16 hours.Immunocytochemical SP staining was used to identify fibroblasts and cardiomyocytes.HE staining was used to observe the morphology of fibroblasts and cardiomyocytes.Tape recorder was used to observe and record the beating frequency of cardiomyocytes.Trypan blue staining was used to determinate the mortality of cardiomyocyte.Western blot was used to determine the relative expression of Cx43 and p-Cx43.Results Compared with group C,the beating frequency of cardiomyocytes in group IR was significantly decreased,and the mortality of cardiomyocyte was significantly higher in group IR and group Sev,the relative expression of Cx43 protein and p-Cx43 protein in group IR and group Sev was significantly lower(P<0.05).Compared with group IR,the beating frequency of cardiomyocytes in group Sev was significantly increased,the mortality of cardiomyocyte in group Sev was significantly decreased,and the relative expression of Cx43 and p-Cx43 in group Sev was significantly increased(P<0.05).Conclusion Sevoflurane can increase the beating frequency of myocytes in hypothermic hypoxia-reoxygenation fibroblast culture medium,up-regulate the expression of Cx43 and p-Cx43,reduce the mortality of cardiomyocytes and enhance the activity of cardiomyocytes.