Cl~-通道阻断剂DIDS抑制T淋巴细胞活化增殖机制的研究

目的 :探讨Ｃｌ- 通道开放在Ｃａ2 依赖性Ｔ淋巴细胞活化增殖信号转导中的作用。方法 :采用ＣｏｎＡ诱导的成人外周血Ｔ细胞活化增殖模型 ,应用Ｆｕｒａ - 2 /ＡＭ荧光探针 ,观察Ｃｌ- 通道阻断剂ＤＩＤＳ以及Ｃａ2 通道阻断剂ＳＫ＆Ｆ 96365对活化Ｔ淋巴细胞Ｃａ2 调控的影响 ,并加以比较 ;应用核酸酶保护分析法 (ＲＰＡ)测定以上药物对人白细胞介素 2 (ｈＩＬ - 2 )ｍＲＮＡ表达的影响。结果 :ＤＩＤＳ( 0 5,1 ,2 μｍｏｌ·Ｌ- 1 )浓度依赖性地抑制活化Ｔ细胞引起的持续性Ｃａ2 内流 ,抑制率分别为 1 8 4%± 6 0 % ,2 5 1 %± 1 0 …     

K+通道在正常与慢性缺氧大鼠缺氧性肺血管收缩反应中的作用

目的和方法 :探讨Ｋ 通道在慢性缺氧致缺氧性肺血管收缩反应降低中的作用 ,采用离体肺灌流方法 ,探讨了 4-ＡＰ( 4 -ａｍｉｎｏｐｙｒｉｄｉｎｅ ,电压依赖性Ｋ 通道 -Ｋｖ阻滞剂 )、ＴＥＡ(ｔｅｔｒａｅｔｈｙｌａｍｏｎｉｕｍ ,Ｃａ2 激活性Ｋ 通道 -Ｋｃａ阻滞剂 )、ＧＬＩＢ(ｇｌｉｂｅｎｃｌａｍｉｎｄｅ ,ＡＴＰ敏感性Ｋ 通道 -ＫＡＴＰ阻滞剂 )对正常与慢性缺氧大鼠肺血管缺氧反应的影响。结果 :对于正常大鼠 4-ＡＰ、ＴＥＡ均可使肺动及基础压上升 ,且使血管缺氧反应明显增强 ;对于慢性缺氧大鼠 ,其肺血管对缺氧反应明显低下 ,4-Ａ…     

Ox-LDL与血液粘度的关系及其对脑梗塞影响的研究

目的 :探讨脑梗塞病人 (ＣＩ)氧化型低密度脂蛋白 (Ｏｘ ＬＤＬ)和其它血脂成分与血液粘度的相关性。方法 :测定 49例非急性期的脑梗塞患者和 2 9例健康人Ｏｘ ＬＤＬ、ＬＤＬ Ｃ、ＨＤＬ Ｃ、ａｐｏＡ、ａｐｏＢ、Ｃｈｏ ｃ、ＴＧ以及血液粘度等指标 ,进行统计学分析。结果 :ηｂ、ηｐ、ηｒｅ、ＨＣＴ、ＥＡＩ、ＴＫ与Ｏｘ ＬＤＬ、ＬＤＬ Ｃ、Ｃｈｏ ｃ呈明显正相关 ;与 ηｂ(ｒ =0 .6 3)、ＴＫ(ｒ=0 .49)的关系最为密切 (Ｐ <0 .0 1)。结论 :Ｏｘ ＬＤＬ、ＬＤＬ Ｃ、ＨＤＬ Ｃ、Ｃｈｏ ｃ等脂质的变化 ,能引起ＣＩ患者血液粘度的改变 …     

IL-7与IL-2、PHA或抗CD3联用诱导人外周血LAK细胞的研究

ＰＨＡ ＬＡＫ细胞和ＣＤ3ＡＫ细胞的活性均优于ＩＬ 2培养的常规ＬＡＫ细胞[1,2 ] 。本文分析比较ＩＬ 7对ＰＨＡ ＬＡＫ、ＣＤ3ＡＫ细胞诱导和活性的影响。1　材料与方法1 1　主要试剂　ＩＬ 7(Ｐｅｐｒｏ Ｔｅｃｈ Ｉｎｃ,美国 ) ;余参见文献 [3]。1 2　效应细胞的诱生与培养　常规方法从Ｏ型血分离单个核细胞 ,分别用ＰＨＡ (12 5 μｇ/ｍｌ)预刺激 48ｈ或抗ＣＤ3(1μｇ/ｍｌ)包被 4ｈ ,然后与含ｒＩＬ 2 (2 5 0ｕ/ｍｌ)的ＲＰＭＩ 16 40培基 (含ＡＢ型血清 )共培养 ,诱生ＰＨＡ ＬＡＫ和ＣＤ3ＡＫ细胞 ,与ＩＬ 7(10ｎｇ/ｍｌ)诱生的Ｉ…     

心肌钾通道的研究进展

心肌细胞膜上的钾通道亚型多，性质复杂。各个钾通道的电导值、门控动力学特征、离子动力学特征、通道激动剂、阻断剂均不同，在动作电位的形成中有着各自的作用。心脏几种重要的钾通道包括ＩＫ１、ＩＫ、Ｉｔｏ、ＩＫ（ＡＴＰ）。心脏疾病时ＩＫ１、Ｉｔｏ、ＩＫ（ＡＴＰ）的活性和功能发生改变。各个钾通道的全面认识有赖于膜片钳技术和分子生物学的深入研究。     

黄芪总皂甙对CVB_3感染心肌细胞cTnI、SERCA活性及mRNA表达的影响

目的 :探讨黄芪提取物 -黄芪总皂甙对柯萨奇Ｂ3(ＣＶＢ3)感染的培养ＳＤ乳鼠心肌细胞损伤模型的保护作用 ,对病毒引起的肌浆网钙泵 (Ｓａｒｃｏ/ＥｎｄｏｐｌａｓｍｉｃｒｅｔｉｃｕｌｕｍＣａ2 ＡＴＰａｓｅ,ＳＥＲＣＡ)活力及其ｍＲＮＡ表达变化的影响。方法 :将培养ＳＤ乳鼠心肌细胞分为正常组、模型组及黄芪组 ,模型组及黄芪组以柯萨奇Ｂ3亲心肌病毒株 (ＣＶＢ3ｍ,ＴＣＩＤ50 为 1 0 5 .83)感染心肌细胞。 72～ 96ｈ后 ,观察各组细胞病变作用 (ｃｙｔｏｐａｔｈｉｃｅｆ ｆｅｃｔ,ＣＰＥ) ,检测其培养上清心肌肌钙蛋白Ｉ(ｃＴｎＩ)含…     

CD27介导小鼠NK细胞活化

细胞活化不仅要有抗原特异性ＴＣＲ参与还要有共刺激信号 ,包括ＣＤ2 8或ＴＮＦ受体超家族分子 (ＣＤ2 7、ＣＤ3 0、ＣＤ1 3 4、ＣＤ1 3 7等 ) ;近来也有报道ＣＤ2 8、ＫＩＲ、ＮＫＰ Ｐ、ＣＤ94 ＮＫＧ2、ＣＤ2、ＣＤ1 6等ＮＫ细胞表面分子参与ＮＫ细胞活化。但迄今为止有关ＴＮＦ受体超家族分子与ＮＫ细胞活化关系仍少有报道。文章作者对ＮＫ细胞表面ＣＤ2 7分子的表达及功能进行了深入研究。雄性 6周龄Ｃ57ＢＬ/ 6(Ｂ6)小鼠 ,纯化的抗 ＣＤ1 6/ 3 2ＭｃＡｂ、ＦＩＴＣ标记的抗小鼠ＮＫＭｃＡｂ、生物素标记的抗小鼠ＣＤ2 7ＭｃＡｂ、Ｎ…     

腺病毒载体介导的人β_2-AR在心肌细胞特异性表达的研究

目的 :观察心肌细胞特异性表达腺病毒载体介导的人β2 肾上腺素能受体 (简称 β2 -ＡＲ)基因在心肌细胞中的表达。方法 :构建含β2 -ＡＲ基因的心肌特异性表达腺病毒载体 (简称Ａｄｍｌｃβ2 ＡＲ) ,转染大鼠心肌细胞、3Ｔ3细胞、ＨＦＣＬ细胞及ＨｅＬａ细胞 ,检测心肌细胞对人β2 -ＡＲ的表达。结果 :ＲＴ -ＰＣＲ显示只有转染的心肌细胞表达人 β2 ＡＲｍＲＮＡ ;Ｗｅｓｔｅｒｎ免疫印迹杂交分析表明转染的心肌细胞有人β2 -ＡＲ表达 ;放射性配基检测显示转基因心肌细胞的 β-ＡＲ密度 ( 1 53 0± 5.2ｆｍｏｌ/ｍｇｐｒｏｔｅｉｎ)明显高…     

肾癌的PHA-LAK/IL-2治疗——50例肾癌患者的临床试验分析

目的 :我们发现 ,经过ＰＨＡ预刺激的ＬＡＫ(ＰＨＡ -ＬＡＫ)细胞较常规ＬＡＫ(Ｃ -ＬＡＫ)具有更高、维持更久的增埴能力和细胞毒活性。在实验研究的基础上 ,将取自健康“Ｏ”型供血者的ＰＢＭＣ ,经ＰＨＡ预刺激 48ｈ后用ｒＩＬ - 2 (ＴＧＰ - 3,日本武田 )激活扩增 ,制成ＰＨＡ -ＬＡＫ ,用于临床治疗试验。本文主要报道以ＰＨＡ -ＬＡＫ及ＩＬ - 2全身疗法对 50例肾癌患者的临床疗效分析。方法和结果 :接受本ＰＨＡ -ＬＡＫ/ＩＬ - 2疗法的50例肾癌患者 ,男 39例、女 1 1例 ,平均年龄 54 0岁 ( 1 1～ 73岁 ) ,病理诊断为肾透明细胞癌 42…     

用无血清培养基体外扩增CIK细胞的研究

ＣＩＫ (ｃｙｔｏｋｉｎｅ ｉｎｄｕｃｅｄｋｉｌｌｅｒ)细胞是一类细胞因子诱导的杀伤细胞。ＣＩＫ细胞能大量扩增 ,而且具有比ＬＡＫ细胞更强的肿瘤杀伤活性。为了获得临床应用的ＣＩＫ细胞 ,必须用自身血浆或无血清培养取代传统的牛血清培养方式 ,以减少病人意外感染的机会。本实验对有血清和无血清培养条件下ＣＩＫ细胞的生物活性进行了研究。将人脐血用Ｆｉｃｏｌｌ密度梯度分离后得单个核细胞 (ＭＮＣ) ,ＭＮＣ分别用含胎牛血清或自身血浆的ＲＰＭＩ 16 40培养基、或ＡＩＭ Ⅴ无血清培养基进行培养。培养ＣＩＫ细胞时 ,先加ＩＦＮ γ …     

Swimming training can affect intrinsic calcium current characteristics in rat myocardium

Endurance exercise is widely assumed to improve cardiac function in humans, but the mechanisms involved in such changes are not clearly understood. The purpose of this study is to determine whether training elicits adaptations at the level of the L-type Ca(2+) channel. Sprague-Dawley rats performed swimming training at either moderate intensity (MOD) or high intensity (HIGH) during 8 weeks. The trained rats were studied by echocardiography and the whole-cell L-type Ca(2+) currents (I (Ca,L)) characteristics in a single cell were measured by standard whole-cell patch-clamp recording technique. Echocardiography showed that septal and posterior wall thickness in MOD and HIGH increased with the increased LV mass by 43 and 41%, respectively (P < 0.05). Training (P < 0.05) increased mean myocyte capacitance (approximately 38% in MOD and HIGH) and myocyte length (approximately 20% longer in MOD and 26% longer in HIGH), thus providing electrophysiological and morphological evidence that the training elicited LV cardiocyte hypertrophy. Mean peak I (Ca,L) was not different in three groups. However, whole-cell I (Ca,L) density was decreased in MOD and HIGH versus sedentary (P < 0.05), but there was no significant difference between MOD and HIGH. The present study provides the evidence of a training adaptation in intrinsic I (Ca,L) characteristics in ventricular myocardium, which demonstrates a remarkable adaptive plasticity of L-type channel characteristics in training rat heart.     

慢性心力衰竭的细胞信号转导机制

慢性心力衰竭（chronicheartfailure，CHF）是指各种原因引起心脏泵功能受损，致使心输出量减少，不能满足机体组织代谢需要，并出现心室充盈压升高、体（肺）循环淤血的临床综合征。近年来随着人口老龄化的出现，以及冠心病急性心肌梗塞死亡率的下降，CHF的发病率呈上升趋势，在美国每年有50万人发生心力衰竭’‘’。CHF严重影响老年人生活质量，是发达国家患病率和死亡率最高的疾病之一。CHF发生与发展过程中，多种基因、生理和环境因素共同作用，造成与心肌收缩蛋白功能和兴奋．收缩耦联相关的分子发生变化，如肌球蛋白头部CaZ”－…     

Sustained beta-adrenergic stimulation increased L-type Ca2+ channel expression in cultured quiescent ventricular myocytes

The abundance of voltage-gated L-type Ca2+ channels is altered by beta-adrenergic receptor (beta-AR) stimulation and by an elevation of the intracellular Ca2+ concentration in cardiac myocytes. In whole animal, chronic beta-AR stimulation or pacing heart results in various changes in the abundance of the channel, but it reduces the beta-AR responsiveness of the L-type channel. Because beta-AR stimulation facilitates the L-type calcium channels, it is difficult in the whole animal to study the effects of beta-AR and Ca2+ influx on the upregulation of the L-type channel independently of each other, which makes the culture of nonbeating adult myocytes an attractive model. We found that culturing quiescent adult rabbit ventricular myocytes with isoproterenol (ISO, 2 microM) for 72 h or more caused a significant increase in the expression of mRNA coding for the L-type channel alpha(1C) subunit by approximately twofold as compared to time-matched controls, and it was followed by a 1.8-fold increase in the Ca2+ current density at 96 h. Somewhat surprisingly, an acute application of 1 microM ISO increased the current amplitude even in ISO-treated cells. The increase in the current density, induced by sustained beta-AR stimulation, was blocked by a beta-AR antagonist, propranolol (10 microM), but not by a Ca2+ antagonist, nitrendipine (10 microM). In addition, the effects were reproduced by forskolin (10 microM), but not by a Ca2+ agonist, Bay-K 8644 (2 microM). Taken together, these results suggest that sustained beta-AR stimulation upregulates L-type channel expression, but does not alter the beta-AR responsiveness of the channel in quiescent myocytes.     

舒张性心力衰竭兔Ca2+调控蛋白mRNA和蛋白质的表达

目的:探讨心肌细胞内Ca²⁺超负荷以及Ca²⁺调控蛋白在舒张性心力衰竭(DHF)发生中的作用。方法:采用RT-PCR和Westernblot技术测定实验兔Ca²⁺调控蛋白mRNA和蛋白质表达的变化。结果:⑴DHF兔心肌细胞内Ca²⁺含量显著高于假手术组(P＜0.01);⑵DHF兔SRCa²⁺-ATPase活性明显低于假手术组(P＜0.01);⑶DHF兔SRCa²⁺-ATPase和细胞膜L型Ca²⁺通道的mRNA水平明显低于假手术组(P＜0.05),而磷酸受纳蛋白、兰尼碱受体和肌集钙蛋白的mRNA转录无明显差异(P＞0.05);⑷DHF兔SRCa²⁺-ATPase的蛋白质表达明显低于假手术组(P＜0.05);磷酸受纳蛋白的相对含量与假手术组无明显差异(P＞0.05)。结论:SRCa²⁺-ATPase的mRNA和蛋白质表达减低以及细胞膜L型Ca²⁺通道mRNA转录减低是导致心肌细胞内Ca²⁺超负荷及DHF发生的重要因素。     

Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes

Increasing evidence suggests that oxidative and nitrosative stress play an important role in regulation of cardiac myocyte growth and survival. The cardiovascular system is continuously exposed to both reactive oxygen species (ROS) and nitrogen species (RNS), collectively termed reactive inflammatory species (RIS), and imbalances between the enzymes that regulate their bioavailability are associated with cardiac hypertrophy and the pathogenesis of cardiomyopathies, myocardial infarction and heart failure. It is now clear that RIS act as critical regulators of cardiac myocyte hypertrophy and apoptosis through control of redox-sensitive signaling cascades, such as tyrosine kinases and phosphatases, protein kinase C, and mitogen-activated protein kinases. This review will focus on the mechanisms by which ROS/RNS modulate cardiac myocyte growth and apoptosis induced by neurohormones and cytokines, and will discuss evidence for a role in the pathophysiology of heart failure.     

Changing pattern of gene expression is associated with ventricular myocyte dysfunction and altered mechanisms of Ca2+ signalling in young type 2 Zucker diabetic fatty rat heart

The association between type 2 diabetes and obesity is very strong, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The aim of this study was to investigate early changes in the pattern of genes encoding cardiac muscle regulatory proteins and associated changes in ventricular myocyte contraction and Ca(2+) transport in young (9- to 13-week-old) type 2 Zucker diabetic fatty (ZDF) rats. The amplitude of myocyte shortening was unaltered; however, time-to-peak shortening and time to half-relaxation of shortening were prolonged in ZDF myocytes (163 ± 5 and 127 ± 7 ms, respectively) compared with age-matched control rats (136 ± 5 and 103 ± 4 ms, respectively). The amplitude of the Ca(2+) transient was unaltered; however, time-to-peak Ca(2+) transient was prolonged in ZDF myocytes (66.9 ± 2.6 ms) compared with control myocytes (57.6 ± 2.3 ms). The L-type Ca(2+) current was reduced, and inactivation was prolonged over a range of test potentials in ZDF myocytes. At 0 mV, the density of L-type Ca(2+) current was 1.19 ± 0.28 pA pF(-1) in ZDF myocytes compared with 2.42 ± 0.40 pA pF(-1) in control myocytes. Sarcoplasmic reticulum Ca(2+) content, release and uptake and myofilament sensitivity to Ca(2+) were unaltered in ZDF myocytes compared with control myocytes. Expression of genes encoding various L-type Ca(2+) channel proteins (Cacna1c, Cacna1g, Cacna1h and Cacna2d1) and cardiac muscle proteins (Myh7) were upregulated, and genes encoding intracellular Ca(2+) transport regulatory proteins (Atp2a2 and Calm1) and some cardiac muscle proteins (Myh6, Myl2, Actc1, Tnni3, Tnn2, and Tnnc1) were downregulated in ZDF heart compared with control heart. A change in the expression of genes encoding myosin heavy chain and L-type Ca(2+) channel proteins might partly underlie alterations in the time course of contraction and Ca(2+) transients in ventricular myocytes from ZDF rats.     

When calcium turns arrhythmogenic: intracellular calcium handling during the development of hypertrophy and heart failure

Alterations of intracellular Ca2+ handling in hypertrophied myocardium have been proposed as a mechanism of ventricular tachyarrhythmias, which are a major cause of sudden death in patients with heart failure. In this review, alterations in intracellular Ca2+ handling and Ca2+ handling proteins in the development of myocardial hypertrophy and the transition to heart failure are discussed. The leading question is at what stage of hypertrophy or heart failure Ca2+ handling can turn arrhythmogenic. During the development of myocardial hypertrophy and the transition to failure, Ca2+ handling is progressively altered. Recordings of free myocyte Ca2+ concentrations during a cardiac cycle (Ca2+ transients) are prolonged early in the development of hypertrophy. However, resting (or diastolic) Ca2+ does not increase before end-stage heart failure has developed. These alterations are due to progressively defective Ca2+ uptake into the sarcoplasmic reticulum that seems to be caused by quantitative changes of gene expression of the Ca2+ ATPase of the sarcoplasmic reticulum. Increased expression and activity of the Na+/Ca2+ exchanger might compensate for this defective Ca2+ uptake, probably at the expense of increased arrhythmogenicity. When the Ca2+ handling proteins no longer efficiently counterbalance increasing intracellular Ca2+ - during stress conditions, resulting Ca2+ overload can lead to spontaneous intracellular Ca2+ oscillations, after depolarizations. Thus, after the transition to heart failure, Ca2+ overloaded sarcoplasmic reticulum, increasing resting intracellular Ca2+, and increased Na+/Ca2+ activity may all provoke afterdepolarizations, triggered activity, and finally, life-threatening ventricular arrhythmias. This increased susceptibility to ventricular arrhythmias in heart failure should not be treated with calcium antagonists.     

卡托普利对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制

目的：观察卡托普利晚期预处理对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制。方法:建立培养乳鼠心肌细胞缺氧/复氧损伤模型。设正常对照组、缺氧/复氧组、缺氧预适应组和卡托普利组。经Flou-3/AM负载染色后，采用流式细胞分析技术，测定细胞内钙离子浓度(［Ca²⁺］i)；利用膜片钳技术，观察L-型钙通道和钠钙交换电流的变化。结果:（1）缺氧/复氧时，［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于正常对照组（P<0.01），L-型钙电流（I_Ca-L）峰值下降，I-V曲线上移，半数失活电压(V_0.5)减小，ICa-L失活曲线左移。（2）晚期预处理和卡托普利使缺氧/复氧时［Ca²⁺］i低于缺氧/复氧组（P<0.01）；I_Ca-L增加，I-V曲线下移，V_0.5增大及稳态失活曲线右移；Na⁺/Ca²⁺ 交换电流减少；但［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于对照组（P<0.05）。（3）卡托普利组与缺氧预适应组比较上述指标均无显著差异 。结论：心肌细胞缺氧/复氧，通过Na⁺/Ca²⁺交换电流的异常增加可引起［Ca²⁺］i的异常升高及其钙超载；卡托普利通过轻度增加Na⁺/Ca²⁺交换电流及其［Ca²⁺］i而触发晚期预处理，抑制后续缺氧/复氧引起的Na⁺/Ca²⁺交换电流及其［Ca²⁺］i的异常增加。     

自发性高血压大鼠肥大心室肌细胞膜离子流的研究

目的 研究自发性高血压大鼠肥大左心室肌细胞的膜离子流并探讨其意义。方法 选用自发性高血压大鼠，以正常血压Wistar大鼠左心室肌细胞作为对照，采用膜片钳全细胞记录技术记录膜离子流，观察除极电流（钠流和L型钙流）及复极电流（内向整流性钾流，延迟整流性钾流和瞬间外向性钾流），并比较其差异。结果 自发性高血压大鼠的心脏重量，心脏重量与体重比及平均细胞膜电容均显增大，钠流，L型钙流及延迟整流性钾流密度无改变，然而L型钙流慢失活时间常数显延长。内向整流性钾流的内向电流密度显降低，瞬间外向性钾流密度显降低但通道的激活和失活动力学无改变。结论 心脏肥大使左心室肌细胞的重要钾流和钙流发生重构引起复极延迟，此在室性心律失常的发生中具有重要意义。