低温缺氧复氧时大鼠心肌成纤维细胞对H9c2细胞Cx43表达的影响

Effect of rat cardiac fibroblasts on expression of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation

目的评价低温缺氧复氧时大鼠心肌成纤维细胞(RCF)对H9c2细胞缝隙连接蛋白43(Cx43)表达的影响。方法体外培养的H9c2细胞,采用随机数字表法分为4组(n=12):对照组(C组)、低温缺氧复氧组(HHR组)、RCF共培养组(Co组)和RCF共培养+低温缺氧复氧组(Co+HHR组)。C组于37℃、5%CO_(2)+95%空气条件下培养5 h。HHR组于4℃、5%CO_(2)+95%N_(2)条件下培养1 h,然后于37℃、5%CO_(2)+95%空气条件下培养4 h。Co组和Co+HHR组将0.3×10^(5)个/孔H9c2细胞接种于Transwell©小室下层培养皿、0.6×10^(5)个/孔RCF接种于上层小室。Co组于37℃、5%CO_(2)+95%空气条件下培养5 h;Co+HHR组于4℃、5%CO_(2)+95%N_(2)条件下培养1 h后,于37℃、5%CO_(2)+95%空气条件下培养4 h。采用台盼蓝染色法测定H9c2细胞死亡率,免疫荧光法测定Cx43和细胞外调节蛋白激酶1/2(ERK1/2)的表达水平,Western blot法测定Cx43、磷酸化Cx43、ERK1/2及磷酸化ERK1/2的表达水平。结果与C组相比,HHR组H9c2细胞死亡率升高,Cx43表达及磷酸化水平降低,ERK1/2表达及磷酸化水平升高(P<0.05),Co组H9c2细胞死亡率、Cx43及ERK1/2的表达与磷酸化水平差异无统计学意义(P>0.05);与Co组相比,Co+HHR组H9c2细胞死亡率升高,Cx43及ERK1/2的表达和磷酸化水平降低(P<0.05);与HHR组相比,Co+HHR组H9c2细胞死亡率升高,Cx43及ERK1/2的表达和磷酸化水平降低(P<0.05)。结论低温缺氧复氧时RCF可降低H9c2细胞Cx43的表达水平和活性,其机制可能与下调ERK1/2的表达、抑制ERK1/2的活性有关。

Objective To evaluate the effect of rat cardiac fibroblasts(RCF)on the expression of connexin43(Cx43)in H9c2 cells during hypothermic hypoxia/reoxygenation.Methods H9c2 cells cultured in vitro were divided into 4 groups(n=12 each)using the random number table method:control group(group C),hypothermic hypoxia/reoxygenation group(group HHR),RCF co-culture group(group Co)and RCF co-culture plus hypothermic hypoxia/reoxygenation group(group Co+HHR).Group C was incubated at 37℃in 5%CO_(2)+95%air for 5 h.Group HHR was incubated at 4℃in 5%CO_(2)+95%N_(2) for 1 h and then at 37℃in 5%CO_(2)+95%air for 4 h.In group Co and group Co+HHR,H9c2 cells 0.3×10^(5) cells/well were inoculated in the lower chamber and RCF 0.6×10^(5) cells/well in the the upper chamber of a transwell©culture dish.Group Co was incubated at 37℃in 5%CO_(2)+95%air for 5 h.Group Co+HHR was incubated at 4℃in 95%N_(2)+5%CO_(2) for 1 h,and then incubated at 37℃in 5%CO_(2)+95%air for 4 h.The mortality rate of H9c2 cells was measured by trypan blue staining,the expression of Cx43 and extracellular signal-regulated protein kinases 1/2(ERK1/2)by immunofluorescence,and the expression of Cx43,phosphorylated Cx43,ERK1/2 and phosphorylated ERK1/2 by Western blot.Results Compared with group C,the mortality rate of H9c2 cells was significantly increased,the expression and phosphorylation of Cx43 were decreased,and the expression and phosphorylation of ERK1/2 were increased in group HHR(P<0.05),and no significant change was found in the mortality rate of H9c2 cells or expression and phosphorylation of Cx43 and ERK1/2 in group Co(P>0.05).Compared with group Co,the mortality rate of H9c2 cells was significantly increased,and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+HHR(P<0.05).Compared with group HHR,the mortality rate of H9c2 cells was significantly increased,and the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group Co+HHR(P<0.05).Conclusions RCFs can decrease the expression and activity of Cx43 in H9c2 cells during hypothermic hypoxia/reoxygenation,and the mechanism may be related to the down-regulation of ERK1/2 expression and inhibition of ERK1/2 activity.