【作者】 孙燕；

【导师】 刘莹；

【作者基本信息】 大连医科大学 ， 内科学， 2015， 硕士

【摘要】 研究背景:目前心脑血管疾病己成为威胁我国人民健康和小康建设的最主要的公共卫生问题。我国每年因心血管疾病包括冠心病和脑卒中的死亡人数约为150万。在心肌梗塞患者发病时,其局部的缺血缺氧会导致局部较严重的酸中毒,由于血液循环障碍时,不利于代谢产物的排出,而且酸中毒时对局部组织的影响也非常大,特别是对冠脉平滑肌和心肌细胞,因而,在临床上及时的纠正酸中毒将极大的减少了对心梗心肌细胞的损害。揭示酸中毒对心肌细胞损害的机制,将有助于对某些心脏疾病的预后提供理论基础。在某些疾病导致机体酸中毒时,会改变心肌的窦性心率,心输出量减少等症状。在正常情况下窦房结是生理起搏点,控制着心脏的跳动,窦房结细胞自动除极频率决定着心率的快慢。参与窦房结自动除极的离子流目前认为有以下几种:超极化激活的内向阳离子电流If、持续性内向离子流IST、内向钙电流ICa-T、钠钙交换电流INa/Ca、延迟整流性钾电流Ik、背景电流(包括钠流和钾流)、D型ICa-L。超极化激活的环核苷门控离子通道(hyperpolarization-activated cyclic nucleotide-gated channel,HCN),目前认为主要有钠离子和钾离子流通过,近期有研究发现还有钙离子流的成分。HCN通道介导的起搏电流引起窦房结细胞舒张期去极化,从而在心脏自主搏动及心律的调节等方面起重要的生理作用。体内HCN通道在心脏起博细胞中呈高表达,参与心脏起博的产生及心率调控。有实验表明,在机体酸中毒时,心率是减慢的,但是对HCN通道的功能变化尚不明确。HCN通道包括四个亚基,其中心脏中包括3个亚基(HCN1,HCN2,HCN4),这三个亚基在心脏中有可能是同聚体或者是异聚体,为了揭示细胞外不同PH值对HCN通道不同亚基的影响,本实验利用爪蟾卵母细胞建立表达HCN通道不同亚基的同聚体及异聚体表达模型,来观察细胞外酸性液对HCN通道不同亚基同聚体或者是异聚体通道电流的影响。目的:建立爪蟾卵母细胞表达的HCN通道同聚体或者异聚体的细胞模型,观察不同PH酸性液对HCN通道电流的影响。方法:将HCN1、HCN2的c DNA体外转录为c RNA后,分别注射至去除滤泡膜的非洲爪蟾卵母细胞中,表达1-3天后使用双电极电压钳技术记录HCN通道电流,非选择性阻断剂Cs Cl可将其完全阻断,成功建立了爪蟾卵母细胞表达的HCN通道细胞模型,使用不同PH酸性液观察对HCN通道同聚体及异聚体电流的影响。结果:在爪蟾卵母细胞表达的HCN1及HCN2通道的同聚体细胞模型上,记录到了超极化激活的内向阳离子电流,可被非选择性阻断剂Cs Cl所阻断。在1 mmol/L的Cs Cl作用下,HCN通道产生的电流显著减小,在-140 m V水平,HCN1电流值减少率为83.4%?9.5%(n=5,P<0.001);HCN2产生的电流减少率为99.7%?0.6%(n=4,P<0.001)。TRIP8b是HCN1的辅助亚基,可使HCN1的电流幅度显著增大。与HCN1组相比,HCN1的m RNAs稀释4倍后等比例与辅助亚基TRIP8b注入细胞后,其电流幅度仍明显增大,且HCN1+TRIP8b组电流幅度的增大有统计学意义。在不同p H酸性液作用下,HCN1及HCN2同聚体和异聚体通道电流被抑制,且其电流幅度随p H值的降低而减小。在-140 m V水平,从p H 6.5至p H 5.0,HCN1电流值减少率为24.6%?8.1%(n=6,P<0.001);HCN2产生的电流减少率为25.6%?6.8%(n=4,P<0.01);HCN1+HCN2的电流减少率为21.3%?6.5%(n=4,P<0.001);在不同p H碱性液作用下,HCN1及HCN2同聚体和异聚体通道电流幅度改变并无统计学意义。在-140 m V水平,从p H7.5至p H8.0时,HCN1电流减少率为1.9%?0.1%(n=3,P>0.05);HCN1+HCN2异聚体电流减少率为5.6%?2.0%(n=3,P>0.05)。结论:1.HCN1及HCN2的同聚体和异聚体通道能够在爪蟾卵母细胞中成功表达;2.在不同p H值的酸性液中HCN1及HCN2的同聚体和异聚体通道电流幅度随p H值的降低而减小;3.在不同p H值的碱性液中,HCN1及HCN2的同聚体和异聚体通道电流幅度并无明显变化;4.机体在酸中毒情况下心率的改变,可能与HCN通道电流在酸性液中被抑制有关。更多还原

【Abstract】 Background: The cardiovascular disease has become a major threat to people’s health in our country. About 1.5 million patients are died of cardiovascular disease including coronary heart disease and stroke every year. On the pathogenesis of myocardial infarction patients, the severe acidosis due to ischemia hypoxia, which can lead to circulation disorder and decrease the excretion of metabolic product, mainly influence on local tissue, especially for coronary artery smooth muscle and cardiac muscle cells [1,2]. Therefore, correct acidosis timely can greatly reduce the myocardial infarction myocardial cell damage in clinic.Therefore, revealed the mechanism of acidosis of myocardial cell injury, will help to offer a theoretical basis for some heart disease prognosis. In some diseases, acidosis will change myocardial sinus heart rate and decrease the cardiac output. The sinoatrial node is physiology pacemaker and dominate the heart beat by automatically depolarization of sinoatrial node cells.The ion currents participated in the sinoatrial node automatic depolarization has the following kinds:hyperpolarization activated introverted cationic current If, delayed rectifier potassium current Ik, inward calcium current ICa-T and sodium calcium exchange current INa/Ca,continuous ion current introversion IST, background current(including sodium and potassium flows) and type D ICa-L etc. If hyperpolarization-activated cyclic nucleotide-gated channel(HCN), which is mainly by sodium and potassium ion flow on and recent studies have found that it also has calcium ion flow components.Hyperpolarization-activated cyclic nucleotide-gated channel(HCN channel)mediated pacemaker current cause diastolic depolarization of sinoatrial node cells, thus play an important physiological role in the regulation of cardiac autonomic beat and rhythm of the heart. HCN channel in the body is high expression in cells of cardiac pacing. With the experimental results show that when the heart acidosis, the heart rate is slow, but the mechanism of the change in function of the HCN channel is unclear.HCN channel includes four subunits, in the heart including three subunits(HCN1,HCN2,HCN4), which may be the same polymers or the different polymers in the heart, therefore, this experiment using clawed frog oocyte establish express HCN channel of polymers with different expression model, to observe the effect of channel current of the extracellular acid liquid on HCN channel subunits with same polymers or different polymers.Aim:To observe the effect of different p H value on hyperpolarization—activated cyclic nucleotide—gated cation channel(HCN channel), we build HCN channel the different subunit expression in Xenopus laevis oocytes.Method: The two-electrode voltage clamp recording technique was applied to observe voltage-gated HCN ion channel currents expressed in Xenopus laevis oocytes without follicles. c DNAs, including HCN1, HCN2 and HCN3 transcribed in vitro. And then different c RNA was microinjected into the oocyte cytoplasm to express HCN channel currents. After 2-3 days, currents were recorded by the two-electrode voltage clamp and the effects of p H on HCN channel currents were observed.Result: The currents of HCN homopolymer and heteropolymer expressed in Xenopus laevis oocytes were recorded, which was inhibited by use of Cs Cl, a non-selective blocker of HCN channel. In the level of-140 m V, the amplitude of HCN1 channel currents was inhibited compared with control(83.4 % ± 9.5 %, n = 5,P < 0.001);and the inhibition rate of HCN2 channel current amplitude reached 99.7%±0.6%(n=4,P < 0.001).TRIP8 b is a auxiliary subunit that regulate the current amplitude of HCN1 in xenopus oocytes.The current amplitude of HCN1+TRIP8b( 1.4ng+1.4ng) group issignificant increase compare with HCN1(5.5ng) group.With the p H value decreased, the amplitude of HCN channel currents was inhibited in the level of-140 m V from p H 6.5to p H 5.0. The amplitude of HCN1 channel currents was inhibited compared with control(24.6% ± 8.1%, n=6,P<0.001);the amplitude of HCN2 channel current was inhibited compared with control(25.6% ± 6.8%,n=4,P<0.01);the amplitude of HCN1 and HCN2 heteropolymer channel current was inhibited compared with control(21.3%±6.5%, n=4,P<0.001);In a different alkaline solution,there is not significant difference in amplitude of different HCN subunit channel current. In the level of-140 m V, from p H 7.5 to p H 8.0, the amplitude of HCN1 channel current wasn’t inhibited compared with control(1.9% ± 0.1%, n=3,P>0.05);the amplitude of HCN1 and HCN2 heteropolymer wasn’t inhibited compared with control(5.6%±2.0%,n=3,P>0.05).Conclusion: HCN channel currents were blocked by application of HCN channel non–selective blocker in oocytes and it was demonstrated that HCN channel expressed in oocytes successfully. With decrease of PH value, the amplitude of HCN channel currents was inhibited. The The amplitude of HCN channel currents was not altered by the application of alkalinity PH. HCN channel currents were inhibited by decreased p H value was likely to involved in the change of heart rate in acidosis.更多还原