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目的研究乳鼠心室肌细胞起搏电流(If)的特点,并运用实时定量聚合酶链式反应(PCR)分析乳鼠心室肌If基因的表达。方法通过酶消化法分离乳鼠心室肌细胞,用光镜、透射电镜、免疫组化鉴定心肌细胞;用全细胞膜片钳技术记录If并研究其特性;采用SYBR-GreenI染料进行实时定量PCR,测定乳鼠心室肌If基因即超极化激活的环核苷酸门控通道(mHCN)亚型2和mHCN4的表达。结果光电显微镜下见搏动频率50~100次/分的细胞团;透射电镜看到丰富的线粒体、肌丝明显;用抗肌动蛋白α-actin的单克隆抗体鉴定心肌细胞,95%以上呈现阳性。全细胞膜片钳记录到心室肌细胞的If,并得到电流密度-电压曲线,其激活电压约为-75mV;实时定量PCR检测mHCN2∶mHCN4为(5.15±0.19)∶1。结论乳鼠心室肌细胞有超极化激活、可被Cs+阻断的If,其基因表达以mHCN2为主。
Objective To study the characteristics of pacemaker current (If) in neonatal rat ventricular myocytes and analyze the expression of If gene in neonatal rat ventricular myocardium using real-time quantitative polymerase chain reaction (PCR). Methods Cardiomyocytes were isolated by enzymatic digestion, and cardiomyocytes were identified by light microscopy, transmission electron microscopy and immunohistochemistry. Whole cell patch clamp technique was used to record If and study its characteristics. Real-time quantitative PCR was performed with SYBR-GreenI dye Expression of the If gene in neonatal ventricular myocardium, hyperpolarization-activated cyclic nucleotide gateways (mHCNs) subtype 2 and mHCN4. Results The cells with pulsating frequency of 50 ~ 100 beats per minute were observed under light microscope. The abundant mitochondria and myofilament were observed by transmission electron microscopy. Cardiomyocytes were identified by anti-actin α-actin monoclonal antibody and more than 95% positive . Whole cell patch clamp recorded to If of ventricular myocytes, and the current density - voltage curve, the activation voltage of about -75mV; real-time quantitative PCR detection of mHCN2: mHCN4 (5.15 ± 0.19): 1. Conclusion The neonatal rat ventricular myocytes are hyperpolarized and can be blocked by Cs + if the gene is mHCN2.