中 文 摘 要
缺血性心脏病（Ischemic Heart Disease IHD）是由于心脏冠状循环改变，引起冠状血流和心肌需求之间不平衡而导致的心肌损害。其最常见的病因是冠状动脉粥样硬化引起的冠状动脉狭窄和闭塞。无论在发达国家还是在发展中国家，IHD都是重要的致死性疾病，严重地威胁着人类的健康。目前临床所应用的各种药物均有一定的局限性，还不能满足需要，寻找新的、安全有效的药物用于防治 IHD 成为医学研究的重要课题之一。
Rbl属于人参二醇组皂苷的一个单体，现代医学研究证明人参皂苷Rb1可以通过改善缺血心肌代谢，减少左室做功， 降低心肌耗氧量， 增加心肌供血，对抗自由基氧化损伤，抑制心肌细胞凋亡等环节发挥抗心肌缺血作用。为了探讨人参皂苷单体Rb1对缺血心肌细胞的直接作用，本项研究采用酶解加机械分离的方法，取得了具有正常生理活性的耐钙心室肌细胞，通过模拟缺血环境，应用全细胞膜片钳技术观察Rb1对缺血心室肌细胞动作电位(AP)，L-型钙通道电流（ICa-L），ATP敏感钾通道电流（IKATP）的影响，从而进一步阐明其对缺血心肌细胞保护作用的分子机制，为临床应用提供理论依据。
采用Langendorff 灌流法，先用台氏液灌流30s ，再用无钙台氏液灌流5-7min， 其后换含胶原酶0. 16-0.20 g/ L 的酶液消化约8-10min，剪下心肌组织，KB液中37 ℃剪碎吹打，温育5min后，低速离心1min，换液后进行膜片钳实验，液体均用O2饱和，温度控制在37℃。结果：注意控制水、酶、温度等各种因素后，可得到杆状、横纹清晰、膜良好的耐钙心肌细胞。
采用全细胞电流钳（Current Clamp）模式记录单个豚鼠心室肌动作电位，给予细胞0.5-1.0nA内向电流，刺激持续5mS，诱发细胞产生动作电位，并记录动作电位幅度（APA）、静息电位（RP）、动作电位复极50%、90%时程（APD50、 APD90）。稳定以后给Rb1200μmol/L，给药5分钟后记录上述指标，Rb1分别将正常心肌细胞APD50从381.48±28.23mS缩短至291.72±22.6mS，APD90从398.64±26.77mS缩短至310.21±20.38mS。与加药前相比，分别缩短了23.52%和22.19%。Rb1将缺血心肌细胞APD50从 212.42±15.32mS缩短至148.35±12.56mS，APD90从228.6±11.34mS缩短至164.47±10.25mS。对正常组及缺血组细胞动作电位的动作电位幅度（APA）、静息电位（RP）均无明显影响。提示Rb1可能对动作电位复极化相的Ca2+通道电流和K+通道电流有影响，对Na+通道电流可能无作用。对于缺血心肌细胞而言，动作电位时程的缩短，导致Ca2+内流减少，使心肌收缩力减弱，降低心肌氧耗，产生心脏保护作用。
采用全细胞电压钳方法记录Rb1对缺血心肌细胞L型钙通道电流的影响，将保持电压设置在-50mV，命令电压从-50mV ~ +60mV，阶跃10mV，持续时间300mS，可成功记录到ICa-L，将细胞外液更换为模拟缺血浴液记录到缺血细胞的ICa-L，Rb1可显著降低ICa-L，使缺血细胞ICa-L的峰值从-0.394±0.093nA降至－0.282±0.113nA，I-V曲线上移，但不发生偏移，对激活电位，峰值电位，反转电位均无明显影响，此结果说明人参皂苷Rb1对缺血心肌细胞的钙通道电流有阻滞作用。
Ischemic heart disease is caused by coronal blood circulation changes and induces the imbalance of myocardial supply and demand. its most common pathogenesis is the straightening and obstruction of the coronary arteries from atherosclerosis. Whether in developed countries or in developing countries, IHD is a deadly and deforming disease .so widely epidemic that it imperils the whole of humankind’s health. The therapeutic arsenal that is currently available to physicians in combating IHD targets is deficient in direct cardio protective agents. A safe and effective new therapy that shows the progression of myocardial ischemic injury and protects the myocardial cell would therefore be expected to synergies with existing therapies and provide considerable benefit.
Rb1,a monomer of panaxadiol saponins，has been proved that it can decrease the heart rate, myocardial oxygen consumption, coronary arterial resistance. Rb1 could ameliorate the index of hemodynamics, decrease the raised myocardial enzyme, the positive percent of cardiocytes apoptosis and reduce the infarction area. All of this we stated above showed that Rb1 have the effect of protect the cardiocytes. In order to investigate the direct function of Rb1 on ischemic cardiocytes and to provide theoretical reason for the clinical, we obtained single active ca2+ tolerance guinea pig ventricular myocytes by acute enzymatic dissociation. L-type calcium channel current (IL-ca), adenosine triphosphate-sensitive potassium channel current (IKATP) and action potential were recorded using the whole cell patch-clamp technique in ischemic cardiocytes.
1. Isolation of ca2+ tolerant cardiocytes
Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode’s solution, then with Ca2+ free Tyrode’s solution and subsequently with enzyme solution containing collagenase 0.16-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37℃. Finally hearts were washed by Ca2+ free Tyrode’s solution, after which the ventricle were minced into small pieces in KB solution, dispersed and filtered. The isolated cardiocytes were stored in KB solution at room temperature for 1h before patch clamp experiments. Results: When all the factors such as water, enzyme, Ca2+, pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30 %-50 % came out.
2. Effect of Rb1 on the action potential in guinea pig ventricular myocytes
Action potential (AP) curve was recorded by current clamp technique. AP was elicited by a series of depolarizing pulse (5mS range, 0.5-1.0nA). We recorded action potential amplitude (APA), resting potential (RP), action potential duration 50% (APD50), action potential duration 90% (APD90). Rb1 can significantly abbreviate APD50 from 381.48±28.23mS to 291.72±22.6mS，APD90 from 398.64±26.77mS to 310.21±20.38mS in normal cardiocytes, Rb1 can significantly abbreviate APD50 from 212.42±15.32mS to 148.35±12.56mS, APD90 from 228.6±11.34mS to 164.47±10.25mS in ischemic cardiocytes. Rb1 have no effect on APA, RP.
3. Effect of Rb1 on the L-type calcium channel current in ischemic guinea pig ventricular myocytes.
Standard whole cell IL-ca was recorded by patch clamp technique. IL-Ca was elicited by a series of depolarizing pulse (300mS range from –50mV to +60mV, each steps to from a holding potential of –50mV). After recording Ca2+ current, Ca2+ blocker verapamil was added in the bath solution to determine that the current is IL-Ca. recording normal IL-Ca to be control and replacing the bath solution to simulated ischemic solution; we viewed the effect of Rb1 on IL-Ca in ischemic cardiocytes. the amplitude of peak potential decreased from -0.394±0.093nA to 0.282±0.113nA.
4. Effect of Rb1 on the IKATP in ischemic guinea pig ventricular myocytes
KATP channel current were recorded in the whole-cell configuration of the patch clamp technique by the isolated cell under conditions of hypoxia. In our experiment, sodium and calcium current could be inhibited when holding potential was -40mV. KATP channel’s outward current can be inhibited by glibenclamide, a peculiarly blocker of KATP channel. Compared with sustained ischemia, IKATP was not changed significantly at the concentration of Rb1 (200μmol/L).
1. All the factors should be well controlled, which ensured the isolated cells Ca2+ tolerant and appropriate for patch clamp experiments.
2.Rb1 remarkably shortened the action potential duration (APD) both normal and ischemic cardiocytes; Rb1 has no effect on resting potential (RP), action potential amplitude (APA).
3.Rb1 decreased L-type calcium channel current of ischemic cardiocytes, which may play a role in the mechanism of anti-myocardial ischemia.
4. The results suggested that Rb1 has no effect on sarcolemmal KATP. Whether or not has effect on mitochondrial KATP need more investingation.