摘要
前言
房颤(atrial fibrillation，AF)是临床上最常见的持续性心律失常，也是危害人类健康的重要疾病。目前，AF的形成机制尚无定论，大量临床实践和流行病学研究表明，心房电重构对房颤的发生和维持起重要作用，因而抑制通道重构是治疗AF的热点之一。
心房电重构会对复极电流产生重要影响，钾通道电流是心肌动作电位复极的主要电流，所以钾通道电流是AF的重点研究对象。超快速激活型(ultrarapid delayed rectifier potassium current，I_(Kur))是一种延迟整流钾电流(delayed rectifier potassium current，I_k)，它在心房肌中表达较多，而在心室肌中表达很少或不表达，选择性抑制I_(Kur)将可能把对AF的治疗作用与作用于心室的致心律失常作用完全分离开来，从而达到有效而安全的治疗目标。因此，高效、特异性的I_(Kur)阻断药在抗AF等房性心律失常方面有着重大意义。
甲壳质广泛存在于甲壳类动物、昆虫和其它无脊椎动物的外壳中。壳聚糖是甲壳质脱去乙酰基的产物。实验表明壳聚糖具有抗感染、抗肿瘤、防治高血压和抗心律失常等方面的作用。羧甲基壳聚糖(carboxymethyl chitosan，CMCS)是壳聚糖经羧甲基化而得的一种水溶性多糖，本实验以前的实验曾观察到CMCS具有抑制豚鼠单一心室肌细胞延迟外向钾电流的作用，本实验应用膜片钳技术首次研究CMCS对大鼠单一心房肌细胞I_(Kur)的作用，进一步揭示其在心血管尤其抗房性心律失常方面的作用机制。
实验方法
1．大鼠单一心房肌细胞的制备
Wistar大鼠，200～250 g，雌雄不限，中国医科大学实验动物部提供。戊巴比妥钠20 mg·kg~(-1)腹腔麻醉，取出心脏，主动脉插管，在37℃恒温和供氧条件下用Langendorff装置进行灌流。用二步酶解法分离出单个心房肌细胞，当数量最多、质量最好时收集细胞。将分离的心房肌细胞放在KB液
中，置于4℃冰箱稳定lh后即可使用。
2.膜片钳制技术全细胞模式记录IKur
将分离的心肌细胞置于容积1. smL平放于倒置显微镜上的浴槽中，挑
选表面光滑，横纹清晰，无收缩的心房肌细胞行千兆封接，全细胞状态形感
后，用pCLAMPS.5·‘软件程序对细胞发放刺激并记录电流信号轨迹。_根铎
文献方法，采用经典的双脉冲刺激方案获得‘Ku一即首先给一个时程为乡叩
ms，保持电位一60 mV，去极化到+30mV的条件脉冲，间隔20Ins后，再给
予波宽1200
ms的方波刺激，保持电位一60mV，刺激电压从一40mV一+50
mv，步阶电压
10 mV。当记录的电流稳定后(1一2而n)开始累积给药观察
电流的变化并实时记录。
3.统计分析一
记录的电流在pCLAMPS .5 .1中进行分析和电流大小的测量.。IKur的幅
度是刺激结束时相对于零电流水平的幅值，实验结果用又士s表示，实验数
菇死理是将细胞给药前后的电流值进行配对资料t检验，P<。.05为有统
计学意义。
实验结果
CMCS抑制IKur，当其浓度为1和29·L一，时，指令电位+50 mV的电流
值分别由给药前的(1.8士O
.7)和(2.0土0.6)nA下降(14土6)%(n二
12，P<0 .01)和(23土3)%(n二7，P<0.01)。
其他指令电位下的IKur的改
变也符合此趋势。增加刺激频率及指令电压IKur
的变化均不显著，提示
cMCs对IKur的抑制作用木具有电压依赖性和频率依赖性。
讨论
1.膜片钳技术及其在心血管研究中的应用
膜片钳技术是一种以记录通过离子通道的离子电流来反映细胞膜单一
的(或多个的)离子通道分子活动的技术。
该技术是对细胞和分子水平的
生理学研究方法的一次革命，使昔日“肉汤生理学(broth
physiofo留)”与“闪
电生理学(lightuing physiology)”在分子水平上结合起来，
使人们对膜通道
的认识耳目一新。全细胞记录(whole一eeu recording)是膜片钳技术的一种
记录方式，近年来在研究离子通道的变化对细胞功能的影响，以及在药物作
用机制的研究中被广泛采用。
随着膜片钳技术在心血管方面的广泛应用，为了解不同疾病机理，为研
制新的更为特效的药物开辟了道路。本实验应用膜片钳技术研究了CMCS
对大鼠单一心房肌细胞IKur的作用，阐明了其在抗房性心律失常方面的作用
机制。
2.大鼠单一心房肌细胞的制备
心房肌细胞直径明显小于心室肌细胞，高阻封接的难度要大于心室肌，
因而对单个心房肌细胞质量的要求更高。目前关于心肌(心房肌和心室
肌)细胞分离的技术已经成熟，但取得数量多、活性好的细胞还是不容易
的，本实验按文献方法，经两次消化孵育能获得成活率较高(>70%)活性
较好的心房肌细胞。经多次实验观察发现温度、酶浓度和消化时间、PH值
对分离细胞的成活率影响较大:(l)温度在36.5一37.5℃;酶浓度在0.3%
左右;PH为7 .2一7.3;消化时间第一次为20一25而n，第二次在5一8而n，同
时满足以上条件时才能分离出活性好、数量多、耐钙性好的心房肌细胞。
3 .AF电重构与心肌细胞钾电流
目前，AF的形成机制还不是十分清楚，临床实验表明，心房电重构对房
颤的发生和维持起重要作用，其主要表现为心房有效不应期(atrial effective
refractory Period，AERP)缩短
Introduction
Atrial fibrillation ( AF) is the most prevalent sustained tachyarrhythmia and is a threatening disease to human health. Nowadays, the mechanism of AF is still undefined. A great deal of clinical studies and epidemiology research showed that atrial electrid remodeling is very important to occurrence and maintain of AF. Therefore, the inhibiting channel remodeling is studing focus.
Atrial electrid remodeling can effect the repolarization currents. Potassium currents are the major outward currents of action potential repolarization, so potassium currents are important investigate target of AF. IKur is a subtype of Ik which expresses in atrial myocytes, but very little or not in ventricle myocytes. The treatment to AF may be compeletely separated from leading arrhythmia by slective inhibition of IKur. Therefore, high - powered specific inhibitor of IKur plays a significant role in antiarrhythmia.
Chitin is extensively consisted in shell of shellfish, insect and other nonver-tebrate. Chitosan is the product of chitin shucking off acetyl . The studying showed that chitosan could cure infection, tumor and prevent hypertension, as well as antiarrhythmia. The previous studies had observed that CMCS could inhibit Ik in single isolated ventricle myocyte of guinea pig. The studying observed firstly the effect of CMCS on IKur in rat single atrial myocyte using patch - clamp technique in order to further reveal ionic mechanism to atrial arrhythmia.
Methods
1. Isolation of single atrial cell
Male of female Wistar rats ( 200 - 250g) , supplied by Laboratory Animal
Center of CMU, were anesthetized by ip injection of a dose (20 mg kg -1 ) of Pentobarbital Sodium. Heart were mounted on a Langendorff apparatus and were retrogradely perfused via the aorta with oxygenate at 37 C. Singal myocyte was i-solated with enzymatic dissociation techniques and the cells with best quality and quantity and was collected and stored in KB at 4 C for Ih before use.
2. Whole cell reording IKur with patch - clamp method
Isolated atrial cells were placed in a 1. 5ml chamber on invert microscope. Only relaxed, spindle - shaped cells were used for experiments. Single cell was voltage - clamped, and membrane currents were measured using the whole - cell configuration of the patch - clamp technique. IKur was recorded as described previously : The stimulating protocol was holding potential - 60 mV, stimulating potential -40 mV to +50 mV, step pulse +10 mV, duration 1200 ms, stimulating interval 3 s and stimulating frequency 2 Hz, preceded by a 200 ms pre-pulse to + 30 mV; the distance between the prepulse and test pulse was 20 ms. We observe the changes of currents and record at all times after I Kur stabilize 1 -2min. '
3. Statistics analysis
All data analysis was performed with pclamp 5.5.1 software. IKur was measured as the amplitude of the current remaining at the end of the test pulse relative to the zero current level. Data were presented as x s. Statistical significance was assessed using a paired t - test and p <0. 05 was considered significant.
Results
When holding potential was 60 mV and stimulating potential was + 50 mV, IKur reduced from (1.8 0.7) and (2.0 0.6) nA by (14 6)% (n = 12, P <0.01) and (23 3)% (n=7, P<0. 01), respectively, after 1 and 2 g L -1 CMCS were added to extracellular solution. The changes of IKur were consistent with this under other commanding potentials. With the increases of stimulating frequency and commanding potential, no significant change of IKur was seen, which suggested CMCS inhibited IKur in a voltage - independency and frequency
- independency manner.
Discussion
1. Patch -clamp technique and its application in cardiovascular studies Patch - clamp technique is a technique that reports molecular activity of
singal of several ion channel by recording currents. To physiological research method of cell and molecular level, this technique is a revolutionary
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