自上世纪以来的流行病学研究表明，心血管疾病的发病率和死亡率存在着性别差异。绝经前女性心血管疾病的发病率明显低于同龄男性。而且，不管是由于自然的因素还是外科手术抑或是卵巢功能受损从而导致雌激素分泌的停滞，都会使女性患心血管疾病的危险性增加。女性心脏疾病的发病比男性平均晚10年，绝经后妇女心血管疾病的发病率是同龄的绝经前妇女的4倍以上。雌激素水平下降是绝经后妇女最主要的生理变化。实验研究证实，天然雌激素如17β-雌二醇可直接作用于心脏或影响血管系统从而对心血管疾病的发生产生影响。近年来研究认为雌激素可通过防止动脉硬化形成和血管重塑而起保护作用，包括雌激素的抗凋亡、抗氧化，改变脂蛋白代谢、血小板的粘附和聚集，血管内皮激活一氧化氮合成从而导致血管舒张，抑制心肌肥大和减少心肌缺血等。因此，男性与绝经前同龄女性之间心血管疾病的发病率和死亡率之间的差异主要是由于雌激素的保护作用。然而，绝经后女性大样本随机临床试验的研究表明雌激素和孕激素的联合使用结果对激素替代的心血管保护作用提出了质疑，认为激素替代不仅不对心血管有保护作用，反而增加心血管疾病的发病率和死亡率，引起心脏疾病、乳腺癌、脑血栓和痴呆等发病的危险性。这些有关雌激素对心血管作用负面研究结果引发了人们对激素替代治疗的质疑。为更好地解决激素替代治疗引发的公共卫生危机，关键是弄清楚雌激素对心血管系统的快效应和慢效应的复杂机制，因此需要对雌激素的正效应和负作用进行全面评估。雌激素对血管系统的作用已有较全面的认识，但对心脏的直接作用还知之甚少。近年来，研究的焦点己开始从血管系统转向心肌细胞。一般认为，细胞内离子稳态对维持心肌细胞的正常功能起着重要的作用，心脏正常的收缩功能有赖于心肌细胞离子稳态和激活-收缩耦联。雌激素对心肌细胞离子通道的瞬时效应已有广泛的研究，然而对其长期效应却知之甚少，因此阐明雌激素影响心肌细胞内离子稳态作用机制是十分必需的。 在我们以往的研究中，利用cDNA微阵列技术观察到：去势3个月后，左室心肌细胞的上百个基因的表达发生改变，其中包括Na⁺，K⁺-ATP酶、Ca²⁺-ATP酶、碳酸酐酶、L型钙通道蛋白、延迟整流钾通道蛋白和电压门控钠通道蛋白以及Na⁺-H⁺交换蛋白和Na⁺-Ca²⁺交换蛋白等的表达。心肌细胞电解质稳定对其功能发挥起至关重要的作用。对心肌细胞H₉c₂的研究发现生理浓度的雌激素能上调Na⁺，K⁺-ATP酶、肌浆网Ca²⁺-ATP酶和碳酸酐酶的表达和活性，这些酶均可能参与细胞内外电解质平衡的调节。上述的实验结果提示，生理浓度的雌激素水平可能维护心肌细胞内的离子浓度、pH值和膜电生理等特性而保护心肌细胞；否则，会导致电解质紊乱、电生理异常、心肌细胞损伤乃至死亡。因此，本研究的目的是寻找雌激素对心肌细胞内游离离子稳态影响的直接证据。我们用鼠H₉c₂心肌细胞为研究对象，检测不同浓度雌激素对细胞内Ca²⁺、Na⁺、K⁺、H⁺和Cl⁻等离子浓度和I〓、I〓 〓、I〓等离子电流的影响。 第一章 17β-雌二醇对心肌细胞H₉c₂内游离离子浓度的影响 目的 研究不同浓度的17β-雌二醇对心肌细胞H₉c₂内Ca²⁺、Na⁺、K³、 H⁺、Cl⁻等游离离子浓度的影响。 方法 H₉c₂心肌细胞分别在0，0.01 nM，1 nM，100 nM，1000 nM 17β-雌二醇(E₂)条件下培养24小时，接着分别用荧光探针Fluo-3，SBFI， PBFI， SNARF-1， MQAE孵育细胞，用荧光法测定细胞内游离离子(Ca²⁺、Na⁺、K⁺、H⁺、Cl⁻)浓度。 结果 心肌细胞H₉c₂分别在 0、0.01 nM、1 nM、100 nM、1000 nM17β-雌二醇中培养24小时后，心肌细胞内的游离钙离子浓度分别是(157.5±5.7) nM，(153.8±6.2) nM，(148.5±8.7) nM， (144.5±7.8) nM，(167.5±9.0) nM。实验结果显示：与无17β-雌二醇组相比，0.01 nM、1 nM 和 100 nM17β-雌二醇组的细胞内[Ca²⁺]下降(p<0.05)。无E2组和0.01 nM E2组细胞内[Ca²⁺]显著高于1 nM E2组(p<0.05)。 1 nm和100 nM17β-雌二醇组之间细胞内[Ca²⁺]无显著性差别(p> 0.05)，而1000nM17β-雌二醇组细胞内[Ca²⁺]却比无E2组和0.01 nM E₂组，1nM E₂组和100nM E₂组有显著性升高(p<0.05)。 心肌细胞 H₉c₂ 分别在0、0.01 nM、1 nM、100 nM、1000nM17β-雌二醇中培养24小时后，心肌细胞内的游离钠离子浓度分别是(8.9±0.5) mM，(8.5±0.8)mM，(7.8±0.8)mM，(7.7±0.9)mM，(9.9±0.7) mM。0.01 nM17β-雌二醇组细胞内[Na⁺]与无17β-雌二醇组无显著性差异(p>0.05)。当17β-雌二醇浓度为1 nM、100nM时，H₉c₂细胞内[Na⁺]比无17β-雌二醇时存在显著降低(p<0.05)。无17β-雌二醇组和0.01 nM17β-雌二醇组细胞内[Na⁺]比1 nM 17β-雌二醇组有显著性升高(p<0.05)。但是1 nM 17β-雌二醇组与100 nM17β-雌二醇组之间[Na⁺]无显著性差异(p>0.05)。1000 nM17β-雌二醇组细胞内[Na⁺]显著性的高于无E₂组，0.01 nM E₂组，1 nM E₂组和100nM E₂组(p<0.05)。 心肌细胞 H₉c₂ 分别在 0、0.01 nM、1nM、100 nM、1000nM17β-雌二醇中培养24小时后，心肌细胞内的游离钾离子浓度分别是(134.4±8.3) mM，(137.2±7.5) mM，(140.0±7.5) mM， (142.0±6.8) mM， (133.7±9.6)mM。对H₉c₂细胞内的[K⁺]，0.01 nM E₂组和无E2组之间无显著性差异(p>0.05)。1nM和100nM₂组细胞内[K⁺]显著性高于无E₂组(p<0.05)。1nM E2和100nM E₂组之间亦无显著性差异(p >0.05)。但是当 H₉c₂细胞分别在 0、0.01 nM、1000 nM E₂培养 24h时，细胞内[K⁺]比1nM E₂组有显著性的下降(p<0.05)。 心肌细胞 H₉c₂ 分别在 0、O.01nM、1nM、100nM、1000nM17β-雌二醇中培养24小时后，心肌细胞内pH分别是(7.0±0.2)， (7.3±0.3)， (7.4±0.2)，(7.4±0.2)，(6.4±0.3)。0.01nM、1nM、100 nM E₂ 组的 H₉c₂细胞内的pH显著性的高于无E₂组(p<0.05)。但是在0.01 nM、1 nM、 100nM E₂组之间细胞内pH值无显著性差异(p>0.05)。另外，1000 nM E₂组的细胞内pH显著性低于无E₂组，0.01 nM E₂组，1 nM E₂组和 100nM E₂组(p<0.05)。 心肌细胞 H₉c₂ 分别在 0、0.01nM、1nM、100nM、1000nM17β-雌二醇中培养24小时后，心肌细胞内的游离氯离子浓度分别是(32.3±3.2) mM， (30.6±4.8) mM， (27.6±4.5) mM， (28.5±4.4) mM， (35.7±4.0)mM。H₉c₂细胞内[Cl⁻]在0.01nME₂组和无E₂组之间无显著性差异(p>0.05)。1 nM E₂组和100 nM E₂组的[Cl⁻]i显著性的低于无 E₂ 组(p<0.05)而 1 nM E₂ 组和 100 nM E₂之间 H₉c₂ 细胞内[Cl⁻]无显著性差异(p>0.05)。但是，1000nM E₂组[Cl⁻]分别比无E₂组， 0.01 nM E₂组，1 nM E₂ 组和 100nM E₂ 组显著上升(p<0.05)。 结论 研究表明1 nM和100nM的17β-雌二醇能维持H₉c₂细胞内Ca²⁺、 Na⁺、K⁺、H⁺、Cl⁻等离子的浓度在生理浓度范围，1 nM和100nM17β-雌二醇组间细胞内离子浓度无显著性差异。H₉c₂细胞在无E2、0.01nM E2或1000 nM E2条件下，细胞内游离离子平衡出现紊乱。因此，17β-雌二醇对H₉c₂细胞内Ca²⁺、Na⁺、K⁺、H⁺、Cl⁻等游离离子浓度的稳定起着至关重要的调节作用。 关键词 H₉c₂心肌细胞；17β-雌二醇；细胞内离子浓度 第二章 17β-雌二醇对H₉c₂心肌细胞的电压门控钠电流、 L型钙电流、延迟整流钾电流等离子通道电流的影响 目的 研究不同浓度的17β-雌二醇对H₉c₂心肌细胞的电压门控钠电流(I〓)、L型钙电流(I〓)、延迟整流钾电流(I〓)等离子通道电流的影响。 方法 H₉c₂ 心肌细胞分别在 0，0.01 nM，1 nM，100 nM，1000 nM 17β-雌二醇(E₂)条件下培养24小时，用全细胞膜片钳技术测定H₉c₂细胞的电压门控钠电流(I〓)、L型钙电流(I〓)、延迟整流钾电流(I〓)等离子通道电流。 结果 电压依赖的I〓的测定：维持电位(holding potential)为-80mV，每隔10mV，连续除极至20mV。内向I〓大约在-70mV出现，在-30mV达到最大，而在+20mV 逆转。用 0、0.01 nM、1nM、100nM、1000 nM E₂处理H₉c₂细胞24h后，其平均最大I〓分别是(3.61±0.42) (pA/pF)，(2.80±0.29) (pA/pF)，(2.27±0.30) (pA/pF)，(2.08±0.31) (pA/pF)，(1.51±0.20) (pA/pF)。与无E₂ 组相比，0.01 nM、1nM、 100nM、1000nM E₂处理组的平均最大I〓有显著性下降(p<0.05)， 17β-雌二醇能以浓度依赖的方式抑制I〓。另外，17β-雌二醇不改变I-V曲线(n=12-16)。 17β-雌二醇对L型钙电流(I〓)的影响维持电位-40mV，每隔10mV连续除极至+50mV，时间180ms。I〓在0mV达最大。用0、 0.01 nM、1nM、100 nM 和 1000 nM E₂ 处理 H₉c₂ 细胞 24h 后，其平均最大 I〓分别是(9.54±0.75) (pA/pF)，(7.70±0.81) (pA/pF)， (6.28±0.62) (pA/pF)，(5.01±0.38) (pA/pF)，(4.45±0.57) (pA/pF)。与无E₂组相比，0.01 nM、1nM、100nM、1000 nM E₂处理组的平均最大I〓有显著性下降(p<0.05)。同样的，17β-雌二醇能以浓度信赖的方式抑制I〓。与I〓相类似，17β-雌二醇亦不影响I-V关系(n=12-16)。 17β-雌二醇对延迟整流钾电流的影响延迟整流钾电流I〓测定：维持电位-40mV，每隔10mV，连续除极至+80mV，时间为260ms。用 0、0.01nM、1nM、100nM、1000 nM E₂ 处理 H₉c₂ 细胞 24h 后，其平均最大 I〓 分别是(8.19±0.65) (pA/pF)、(4.8±0.52) (pA/pF)、 (3.8±0.33) (pA/pF)、(2.87±0.17) (pA/pF)、(2.14±0.13) (pA/pF)。与无E₂组相比，0.01nM、1nM、100nM、1000 nM E₂处理组的平均最大I〓有显著性下降(p<0.05)。17β-雌二醇能以浓度依赖方式抑制I〓。同样的，17β-雌二醇不改变I-V曲线(n=12-16)。 结论 用 0、0.01 nM、1 nM、100 nM 和 1000 nM E₂ 处理 H₉c₂ 细胞 24h后，I〓、I〓、I〓随17β-雌二醇浓度升高而降低。研究表明电压门控钠电流(I〓)、L型钙电流(I〓)、延迟整流钾电流(I〓)等离子通道电流以浓度依赖的方式随17β-雌二醇浓度升高而降低。 关键词 H₉c₂心肌细胞；17β-雌二醇；电压门控钠电流；L型钙电流；延迟整流钾电流 第三章 不同浓度的17β-雌二醇对心肌细胞H₉c₂细胞形态、损伤及死亡程度的影响 目的 研究不同浓度的17β-雌二醇对心肌细胞H₉c₂细胞形态及损伤程度的影响。 方法 H₉c₂心肌细胞分别在 0，0.01 nM，1 nM，100nM，1000 nM 17β-雌二醇(E₂)条件下培养24小时，接着分别用荧光探针Fluo-3，SBFI， PBFI，SNARF-1，MQAE孵育细胞，用激光扫描共聚焦显微镜进行细胞形态观察。通过释放到培养基的LDH活性测定和PI染色细胞观察17β- 雌二醇对细胞损伤和细胞死亡的影响。 结果 将H₉c₂与不同的荧光探针孵育后，用激光共聚焦显微镜进行形态学观察，发现在无E₂或在1000nM E₂时有细胞形态学的改变，细胞变得平而圆。心肌细胞H₉c₂分别在0、0.01 nM、1nM、100 nM、1000 nM17β-雌二醇中培养24小时后，细胞培养基中LDH释放率分别是(11.9±1.1)%，(11.4±1.5)%，(10.2±0.9)%，(9.9±1.6)%，(13.4±1.7)%。无E₂组和0.01nM E₂组之间，培养基中LDH释放率无显著性差异(p> 0.05)，而1nME₂组和100 nM E₂组中LDH释放率显著低于无E₂组和0.01 nM E₂组(p<0.05)。但1 nM E₂和100nM E₂组之间无显著性差异(p >0.05)。1000 nM E₂组的H₉c₂细胞LDH释放率都显著性的高于无E₂组，0.01 nM E₂组，1 nM E₂组和 100nM E₂组(p<0.05)。 心肌细胞 H₉c₂ 分别在 0、0.01 nM、1 nM，100 nM，1000nM17β-雌二醇中培养24小时后，细胞死亡率分别是(3.1±0.3)%，(2.9±0.4)%， (2.8±0.5)%，(2.9±0.5)%，(3.8±0.4)%。0.01 nM E₂ 组、1 nM E₂ 组和100nM E₂组细胞细胞死亡率与无E₂组相比显著性的下降(p<0.05)。而1 nM E₂组和100 nM E₂组之间细胞细胞死亡率无显著性差异(p> 0.05)。当H₉c₂细胞用1000 nM E₂处理时，其细胞死亡率显著性的高于无 E₂ 组，0.01 nM E₂ 组，1 nM E₂组和 100 nM E₂ 组(p<0.05 )。 结论 研究表明H₂细胞在无E2、0.01 nM E2或1000 nM E2条件下，有细胞形态学的改变，并引起细胞损伤和细胞死亡的增加。 关键词 H₉c₂心肌细胞；17β-雌二醇；细胞形态；细胞损伤；细胞死亡

 During the last century, gender-based differences in the morbidity and mortality of cardiovascular disease have been revealed by epidemiological studies. These studies showed that cardiovascular mortality of pre-menopausal women is significantly smaller than that of men of the same age. Indeed, the risk of cardiovascular disease is increased when the estrogen production stops, either naturally or after surgery or in woman with impaired ovarian function. Cardiovascular disease develops in women on average 10 years later in life compared with men. The decrease of the estrogen level is the main physiological change of the postmenopausal women. On the other hand, experimental studies have shown that natural estrogens, such as 17β-estradiol(E2), can influence cardiovascular disease by acting directly on the heart or by affecting the vascular system. The beneficial effects of estrogen on the development of arteriosclerosis and vascular remodeling are currently under investigation, including its antiapoptotic effects, antioxidant effects, beneficial changes in lipoprotein metabolism, platelet adhesion and aggregation, endothelium-dependent vasodilatation through activation of endothelial nitric oxide synthase (eNOS) with increased expression of eNOS, limiting cardiac hypertrophy and reducing myocardial ischemia. Thus, the differences of morbidity and mortality of cardiovascular disease between men and pre-menopausal women may be due to a protective effect of estrogen. In contrast to these observations, large randomized clinical trials in postmenopausal women with cardiovascular risk factors or coronary artery disease(CAD), both using conjugated equine estrogens and medroxyprogesterone acetate as hormone "replacement"have recently questioned these atheroprotective effects, because the results showed no effects or even an increase in cardiovascular morbidity and mortality . Also, estrogen was reported to increase the risk of heart attack, breast cancer, blood clots, and dementia. These divergent findings resulted in confusion about whether a substitution therapy with natural or novel synthetic sex steroids could represent a therapeutic option for the treatment of atherosclerosis and its complications. One key to solving such an important public health issue would be to better understand the complex mechanisms of acute and chronically administered estrogen action in the cardiovascular system. Therefore, it is important that further studies be carried out to evaluate the beneficial effects of estrogen and its adverse side effects. The effects of estrogen on vascular function have been studied extensively, however, the direct influences of estrogen on heart have received much less consideration. Nowadays, the focus on the beneficial influence of estrogen is gradually shifting from the vascular system to myocardium . It is well known that intracellular ions homeostasis plays an important role in maintaining the functions of the cardiomyocytes. Proper contractile function of the heart depends on ions homeostasis of the cardiac myocytes and intact excitation-contraction. The transient effects of estrogen on single ion channel in the cardiomyocytes have been extensively studied, however, much less is known regarding long term effects of estrogen on several main cardiac ions together. Thus, further studies are required to evaluate the ionic mechanism of estrogen on the cardiomyocytes. In our previous research, the effect of ovarian sex hormones was investigated using 3-month ovariectomized rat heart. The results of DNA microarray indicated a series of changes in gene expression, including enzymes(Na⁺,K⁺-ATPase, Ca²⁺-ATPase, carbonic anhydrase), ion channel proteins (L-type calcium channel, delayed rectifier potassium channel, voltage-gated sodium channel), exchanger proteins (Na⁺-H⁺-exchanger, Na⁺-Ca²⁺ exchanger), which were up(down)-regulated more than 2 folds compared with the control. Also, physiological concentration of E2 can enhance gene expression and the activity of Na⁺,K⁺-ATPase, sarcoplsmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) and carbonic anhydrase(CA) in H9c2 cells. Interestingly, all these proteins or enzymes may associate with communication of electrolytes between intracellular and extracellular apartment. It was therefore hypothesized that physiological concentration of estrogen may maintain normal intracellular ion concentration, acid-base balance and electrophysiological property and play a protective role of estrogen to cardiomyocytes. Otherwise, estrogen may play a completely inverse role on cardiomyocytrs and result in intracellular electrolytes disorder, electrophysiological abnormalities, cardiac injury and even death. In the present, we are attempting to find direct evidence how the estrogen exerts influence on intracellular free ions homeostasis. Using rat myogenic H9c2 cells as an in vitro model, we recently examined intracellular free ion concentrations of Ca²⁺, Na⁺, K⁺, H⁺ and Cl⁻ and ionic currents of voltage-gated sodium current (I〓), L-type calcium current (I〓) and delayed rectifier potassium current (I〓). Chapter One: Long-Term Effect of Estrogen on Concentration of Intracellular Free Ions in H9c2 Cardiac Cells Objective: To study the long term effect of 17β-estradiol on intracellular free ions concentration of Ca²⁺, Na⁺, K⁺, H⁺, Cl⁻ in H9c2 cardiac cells. Methods: H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM, 1000 nM E2 for 24h. Then H9c2 cells were incubated with fluorescent probes, intracellular free ion concentrations ([Ca²⁺]i, [Na⁺]i, [K⁺]i, [H⁺]i, [Cl⁻]i) were measured by fluorescence assay Results: When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [Ca²⁺] was (157.5±5.7) nM, (153.8±6.2) nM, (148.5±8.7) nM, (144.5±7.8) nM, (167.5±9.0) nM, respectively. It was demonstrated that the intracellular free calcium ion concentration of H9c2 cells cultured with 0.01 nM, 1 nM, or 100 nM 17β-estradiol decreased compared with that in the no E2-treated group(P＜0.05). The [Ca²⁺]i in H9c2 cells cultured with no E2 or 0.01 nM E2 was significantly higher than that in the group cultured with 1 nM E2 treated group(P＜0.05). There was no significant difference between groups cultured with 1 and 100nM E2(P＞0.05) . However, [Ca²⁺]i of H9c2 treated with 1000 nM 17β-estradiol was significantly higher than that treated with no E2, 0.01 nM E2, 1 nM E2 and 100 nM E2 groups, respectively (P＜0.05). When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the [Na⁺]i was (8.9±0.5) mM, (8.5±0.8) mM, (7.8±0.8) mM, (7.7±0.9) mM, (9.9±0.7) mM, respectively. The [Na⁺]i of H9c2 cells treated with 0.01 nM 17β-estradiol was similar to that of no E2 treated group (P＞0.05). When H9c2 cells were cultured with 1 nM, 100 nM 17β-estradiol, the [Na⁺]i decreased compared with that of cells treated with no E2 group (P＜0.05). The [Na⁺]i in H9c2 cells cultured with no E2 or 0.01 nM E2 was significantly higher than that in the group cultured with 1 nM E2 treated group(P＜0.05). But no significant difference was observed between groups treated with 1 nM and 100 nM E2(P＞0.05). Interestingly, [Na⁺]i of H9c2 incubated with 1000 nM 17β-estradiol was significantly enhanced compared with the no E2 group, 0.01 nM E2 group, 1 nM E2 group and 100 nM E2 group, respectively(P＜0.05). When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [K⁺] was (134.4±8.3) mM, (137.2±7.5) mM, (140.0±7.5) mM, (142.0±6.8) mM, (133.7±9.6) mM, respectively. The [K⁺]i in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 treated group(P＞0.05). The [K⁺]i in H9c2 cells cultured with 1 nM and 100 nM E2 was significantly higher than that in the group cultured with no E2 treated group(P＜0.05). There was no significant difference between groups cultured with 1 nM and lOOnM E2 (P＞0.05). When H9c2 cells were cultured with 0, 0.01 nM, 1000 nM E2 for 24h, the [K⁺]i decreased compared with that of cells treated with 1 nM E2 (P＜0.05). When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular pH was (7.0±0.2), (7.3±0.3), (7.4±0.2), (7.4±0.2), (6.4±0.3), respectively. The [pH]i of H9c2 cells treated with 0.01 nM, 1 nM, 100 nM 17β-estradiol was significantly enhanced compared with no 17β-estradiol group (P＜0.05). But there was no significant difference among groups cultured with 0.01 nM, 1 nM and 100 nM 17β-estradiol (P＞0.05). Also, the [pH]i in H9c2 cells of the group cultured with 1000 nM 17β-estradiol decreased compared that cultured with 0, 0.01 nM E2, InM E2, 100 nM E2 groups, respectively (P＜0.05). When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [Cl⁻] was (32.3±3.2) mM, (30.6±4.8) mM, (27.6±4.5) mM, (28.5±4.4) mM, (35.7±4.0) mM, respectively. The [Cl⁻]i in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 treated (P＞0.05). The [Cl⁻]i of H9c2 cells cultured with 1 nM and 100 nM 17β-estradiol decreased compared with that in the no E2 treated group(P＜0.05) . There was no significant difference between groups cultured with 1 nM and 100 nM E2(P＞0.05). However, [Cl⁻]i in H9c2 cells cultured with 1000 nM increased compared with that in the no E2, 0.01 nM E2, 1 nM E2 and 100 nM E2 groups, respectively (P＜0.05). Conclusion: The results indicated that 1 nM and 100 nM E2 can maintain the intracellular free ion concentrations of Ca²⁺, Na⁺, K⁺, H⁺, Cl⁻ in physiological range. No significant difference was observed between groups treated with 1 nM and 100 nM E2 for intracellular free ion concentrations. The treatment of H9c2 cells with no E2, 0.01 nM E2 or 1000 nM E2 can disturb the intracellular free ions balance and result in cell injury. It was found that E2 may have important regular role on intracellular free ions homeostasis which is essential to normal function of cardiac cells. Key Words: H9c2 cardiac cells; 17β-estradiol; intracellular free ion concentration Chapter Two: Effect of 17β-estradiol on ionic currents of voltage-gated sodium current, L-type calcium current and delayed rectifier potassium current of H9c2 cardiac cells Objective: To study the effect of 17β-estradiol on ionic currents of voltage-gated sodium current (I〓), L-type calcium current (I〓) and delayed rectifier potassium current (I〓) of H9c2 cardiac cells. Methods: H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM, 1000 nM E2 for 24h. Whole cell patch-clamp technique was used to study ion channel currents (I〓, I〓,I〓) in H9c2 cells. Results: The voltage dependent activation of I〓 was measured by 200ms test potential (Vt) that 10mV increased stepwise to 20mV from a holding potential (Vh) of -80mV. This inward current appeared at about -70mV, reached a maximal value around -30mV and reversed at about +20mV. The average maximal peak current density of H9c2 cells in the groups cultured with 0, 0.01 nM, 1 nM, 100 nM, 1000 nM E2 were (3.61 ±0.42), (2.80±0.29), (2.27±0.30), (2.08±0.35), (1.51±0.20) (pA/pF) . The average maximal peak current density in cells cultured with 0.01 nM, 1 nM, 100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p＜0.05). 17β-estradiol suppressed I〓 in a concentration dependent manner. On the other hand, the 17β-estradiol did not shift the I-V curve (n= 12-16/group). The voltage dependent activation of I〓 was tested by a 180ms test potential (Vt) that 10mV increased stepwise to 50mV from a holding potential (Vh) of -40mV, reaching a maximal value about 0mV. The average maximal peak current density of H9c2 cells in the groups cultured with 0, 0.01 nM, 1 nM, 100 nM, 1000 nM E2 were (9.54±0.75), (7.70±0.81), (6.28±0.62), (5.01±0.38), (4.45±0.57) (pA/pF). Similarly, the average maximal peak current density in cells cultured with 0.01 nM, 1 nM, 100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p＜0.05). 17β-estradiol suppressed I〓 in a concentration dependent manner. Similar to the effect on I〓, the 17β-estradiol decreased the amplitude of I〓 without shifting the I-V relationship (n=12- 16/group). I〓 was induced by 260ms depolarizing pulses from -40mV to +80mV in 10mV steps. The average maximal peak current density of H9c2 cells in the groups cultured with 0, 0.01 nM, 1 nM, 100 nM, 1000 nM E2 were (8.19±0.65), (4.80±0.52), (3.81±0.33), (2.87±0.17), (2.14±0.13) (pA/pF). The average maximal peak current density in cells cultured with 0.01 nM, 1 nM, 100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p＜0.05). 17β-estradiol suppressed I〓 in a concentration dependent manner . Similarly, the 17β-estradiol did not shift the I-V curve (n=12-16/group). Conclusion Ionic currents of voltage-gated sodium current (I〓), L-type calcium current (I〓) and delayed rectifier potassium current (I〓) of H9c2 cardiac cells were suppressed via 17β-estradiol in a concentration-dependent manner. Key Words H9c2 cardiac cells; 17β-estradiol; voltage-gated sodium current; L-type calcium current; delayed rectifier potassium current Chapter Three: Effect of 17β-estradiol on morphological change, cell injury and cell death of H9c2 cardiac cells Objective: To study the effect of 17β-estradiol on morphological change, cell injury and cell death of H9c2 cardiac cells. Methods: H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM, 1000 nM E2 for 24h. Then H9c2 cells were incubated with fluorescent probes, morphological change of the cells was observed by laser scanning confocal microscope. The activity assay of LDH released into the medium and propidium iodide (PI) staining were used to assess the E2 induced cell injury and cell death. Results: Laser scanning confocal microscope images in H9c2 cells incubated with different fluorescent probes showed that no E2 or 1000 nM E2 can resulted in H9c2 cardiac cells morphological change. The cells became flat and round When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the percent LDH release(% of Max) in the medium was (11.9±1.1), (11.4±1.5), (10.2±0.9), (9.9±1.6), (13.4±1.7), respectively. The percent LDH release in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 group(P＞0.05). The percent LDH release in H9c2 cells cultured with 1 nM and 100 nM E2 was significantly decreased compared with the no E2 treated group(P＜0.05). There was no significant different between groups cultured with 1 nM and 100 nM E2(P＞0.05). Interestingly, the percent LDH release in H9c2 cells cultured with 1000 nM E2 was higher than in the groups cultured with 0, 0.01 nM E2, InM E2 and 100 nM E2 groups, respectively(P＜0.05). When H9c2 cardiac cells were cultured with 0, 0.01 nM, 1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h, the percent cell death(%) was (3.1±0.3), (2.9±0.4), (2.8±0.5), (2.9±0.5), (3.8±0.4), respectively. The percent cell death in H9c2 cultured with 0.01 nM, 1 nM, 100 nM E2 decreased compared with that in the no E2 group (P＜0.05). No significant difference was observed between groups treated with 1 nM and 100 nM E2(P＞0.05). When H9c2 cells were incubated with 1000 nM E2, the percent cell death increased compared with that of cells treated with no E2 group, 0.01 nM E2 group, 1nM E2 group or 100 nM group(P＜0.05). Conclusion: The treatment of H9c2 cells with no E2, 0.01 nM E2 or 1000 nM E2 can resulted in H9c2 cardiac cells morphological change and increase in cell injury, death. Key Words: H9c2 cardiac cells; 17β-estradiol; cell morphology; cell injury; cell death