【作者】 杨涛；

【导师】 葛郁芝；

【作者基本信息】 南昌大学 ， 内科学， 2009， 硕士

【摘要】 目的:观察不同浓度甘松挥发油对大鼠心室肌细胞膜钠通道电流(I_Na)的影响,以及甘松挥发油对I_Na电流-电压（I-V）曲线、激活曲线和失活曲线的影响,探讨甘松挥发油在离子通道水平抗心律失常的作用机制。方法:1.用急性酶解法分离大鼠心室肌细胞。以肝素抗凝,戊巴比妥钠麻醉大鼠,取大鼠心脏悬挂与Langendorff灌流仪行逆行灌流,先以无钙台氏液灌流心脏约5分钟,再以50微摩尔/升（μmol/L）低钙酶液灌流心脏,待心脏质地明显松软,则停止酶液消化,冲洗灌流系统及心脏中残留酶液,将心室肌组织块置于盛有KB液的烧杯中充分剪碎、轻轻吹打、过滤后置于KB液中室温下孵育1²小时。2.应用全细胞膜片钳记录技术,保持细胞钳制电位（holding potential,HP）在-90mV,选取纹理清晰、杆状和大小适中的细胞在室温下进行实验,以电阻为1.0³.0MΩ的玻璃微电极封接大鼠心室肌细胞,待封接电阻至吉欧（GΩ）水平破膜,补偿串联电阻和细胞膜电容（Cm）,记录不同浓度的甘松挥发油对I_Na的影响。结果:1.分别以浓度为1、3、5、10、100μg/g甘松挥发油作用大鼠心室肌细胞钠通道电流,其抑制率分别为8.0±2.9%、20.9±3.5%、53.9±7.8%、74.9±3.4%、94.7±3.6%（每个浓度n=5,P<0.05）,采用I=ImaxCn/(Cn+EC₅₀n)方程进行拟合,半数有效浓度EC₅₀值为4.74±0.59,斜率因子为2.08±0.50;2.以浓度为5μg/g甘松挥发油灌流液作用大鼠心室肌细胞进行对照研究,保持细胞HP为-90mV,给药前I_Na的激活电位为-70mV,电流密度在膜电位为-30mV时达最大值,其值为（-44.7±10.3）pA/pF,翻转电位为20mV;在浓度为5μg/g甘松挥发油灌流法给药后,I_Na的激活电位仍为-70mV,电流密度在膜电位在-30mV时达最大值,值为（-20.3±4.6）pA/pF,翻转电位为20mV。给药前后I_Na激活电位、峰电位和翻转电位无改变,峰值电流密度变化有显著的统计学意义（n=5,P<0.05）;3.以浓度为5μg/g甘松挥发油作用钠通道,使激活曲线半数激活电压（V1/2）从（-48.66±0.76）mV右移至（-43.94±1.02）mV（n=5,P<0.05）;4.以浓度为5μg/g甘松挥发油作用I_Na,使失活曲线半数失活电压(V_1/2)从（-100.48±0.71）mV左移至（-109.82±0.86）mV （n=5,P<0.05）。结论:1.甘松挥发油可通过浓度依赖性地抑制大鼠心肌细胞膜I_Na;在不同膜电位水平对钠通道电流均具有抑制作用;2.甘松挥发油使I_Na的I-V曲线明显上移,不改变I_Na的激活电位、峰值电位和反转电位;3.使钠通道激活曲线右移;4.使钠通道失活曲线左移。更多还原

【Abstract】 OBJECTIVE: To investigate the effects of the volatile oil of Nardostachys chinensis with different dosages on sodium channel of isolated ventricular myocytes of rat.And the effects of the volatile oil of Nardostachys chinensis on I-V curve; activation cureve and inactivation curve were also investigated.METHODS: Ventricular myocytes were isolated enzymatically from rat which was sacrificed after anesthesia with pentobarbital, The heart was quickly removed and hung up a Langendorff apparatus and perfused initially with Ca²⁺ free Tyrode’s solution for 5 minutes, then perfused digest enzyme solution for about 20 minutes at 37℃. As soon as the heart became softened, the ventricle was cut into nubs and blown with sucker in KB solution to obtain single cells. Only rod-shaped cells with smooth surfaces and cross-striations were selected for patch clamp. The tip resistance of the micro electrodes is ranging from 1.0³.0 MΩ.RESULTS:1. The volatile oil of Nardostachys chinensis reduced sodium current in a dose-dependent block. The concentration of volatile oil of chinensis at 1、3、5、10、100μg/g suppressed I_Na current which inhibition rate respectively was 8.0±2.9%、20.9±3.5%、53.9±7.8%、74.9±3.4%、94.7±3.6% （n=5,P<0.05）2. The concentration of volatile oil of Nardostachys chinensis at 5μg/g suppressed I_Na current remarkably. The average maximal peak current density is （-20.3±4.6）（pA/pF） after the exposure to drug vs control is （-44.7±10.3） （pA/pF）. On the other hand. The volatile oil of Nardostachys chinensis did not shift the I-V curve.3. The activity curve have significant change by using the volatile oil of Nardostachys chinensis at 5μg/g. thier V1/2 is （-48.66±0.76）mV vs （-43.94±1.02） mV（n=5,P<0.05）.4. The inactivation curve have significant change by using volatile oil of Nardostachys chinensis at 5μg/g concentration.their V1/2 is （-100.48±0.71）mV vs （-109.82±0.86）mV （n=5,P<0.05）. CONCLUSION:1、The volatile oil of Nardostachys chinensis reduced the sodium channel current in a dose-dependent block.2、The volatile oil of Nardostachys chinensis lifted the I-V curve of I_Na without changing the threshold , peak and reversal potentials.3、The activation curve is shifted towards the depolarizing direction after exposuring to the drug.4、The inactivation curve is shifted toward to the hyperpolarizing direction after exposuring to the drug.更多还原