SD大鼠乳鼠心房肌细胞间歇性低氧模型制备

Establishment of intermittent hypoxia model of atrial cardiomyocyte of SD neonate rat

目的采用间歇性低氧(IH)干预SD大鼠乳鼠原代心房肌细胞,为探索阻塞性睡眠呼吸暂停与心房颤动之间的关系提供体外实验模型和依据。方法选择1~3 d龄SD大鼠乳鼠30只,雌雄不限。分离、纯化、培养、鉴定SD大鼠乳鼠心房肌细胞。按常氧(N)、6 h IH、12 h IH、24 h IH和48 h IH随机分组,以体积分数5%O230 min+体积分数21%O230 min为1个循环,对心房肌细胞实施6、12、24、48 h的IH干预。四唑氮化合物(MTS)法检测细胞活力;收集细胞总蛋白,使用Western blot检测各组缺氧诱导因子-1α(HIF-1ɑ)、含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved caspase-3)、缝隙连接蛋白43(Cx43)的表达量。结果随IH刺激时间延长,细胞活力在6 h IH时明显下降,随IH时间延长,细胞活力逐渐升高(1.66±0.12、1.38±0.14、1.69±0.08、1.70±0.18、2.19±0.17。P<0.001)。HIF-1ɑ蛋白表达的上调呈时间依赖性(0.19±0.06、0.50±0.26、0.84±0.31、1.10±0.28、1.62±0.36。P<0.001)。cleaved caspase-3表达量在6 h IH组最高,以后逐渐下降并趋于稳定(0.65±0.05、0.73±0.11、0.46±0.08、0.43±0.13、0.49±0.13。P<0.001)。IH下调Cx43的表达,但随着IH时间延长,Cx43表达趋于稳定(1.05±0.24、0.64±0.12、0.55±0.09、0.55±0.14、0.55±0.09。P<0.001)。结论成功建立了SD大鼠乳鼠心房肌细胞IH模型,为进一步研究提供方法学基础。

Objective To established the intermittent hypoxia(IH)model of atrial cardiomyocyte in SD neonate rat,and provide experimental model and basis for exploring the relationship between obstructive sleep apnea and atrial fibrillation in vitro.Methods Thirty male or female SD rats aged 1-3 day were sacrificed,and SD rat atrial myocytes were isolated,purified,cultured and identified.All of the cells were randomly divided into 5 groups,named normoxia(N)group,6-hour intermittent hypoxia(6 h IH)group,12-hour intermittent hypoxia(12 h IH)group,24-hour intermittent hypoxia(24 h IH)group and 48-hour intermittent hypoxia(48 h IH),respectively.The model(5%O230 minutes,21%O230 minutes as a cycle)was provided to carry out IH intervention on atrial myocytes for 6,12,24 and 48 hours.The[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt,MTS]was used to evaluate cell viability.The protein expression of hypoxia inducible factor-1α(HIF-1α),cleaved cysteinyl aspartate specific proteinase-3(cleaved caspase-3)and connexin 43(Cx43)were detected by Western blot.Results With the prolongation of IH stimulation time,the cell viability decreased significantly at 6h IH,and increased gradually with prolongation of IH stimulation time(1.66±0.12,1.38±0.14,1.69±0.08,1.70±0.18,2.19±0.17.P<0.001).The protein level of HIF-1αwas time-dependent(0.19±0.06,0.50±0.26,0.84±0.31,1.10±0.28,1.62±0.36.P<0.001).The expression of cleaved caspase-3 reached peak in 6 h IH group,then gradually declined and stabilized(0.65±0.05,0.73±0.11,0.46±0.08,0.43±0.13,0.49±0.13.P<0.001).The IH down-regulated the expression of Cx43,but with prolongation of IH time,the expression of Cx43 tended to be stable(1.05±0.24,0.64±0.12,0.55±0.09,0.55±0.14,0.55±0.09.P<0.001).Conclusion The intermittent hypoxia model of SD rat atrial myocytes is successfully established,which provides the methodological basis for further study.