Upregulated voltage-gated potassium channel Kvl.3 on CD4＋CD28null T lymphocytes from patients with acute coronary syndrome

Objective The purpose of our study is to observe the voltage-gated potassium channel Kvl.3 expressed on CD4＋CD28~ T cells from the peripheral blood of acute coronary syndrome （ACS） patients by the patch clamp technique. Methods Kvl.3 potassium channels expression from 17 patients with ACS and 11 healthy age-match controls was detected in single cell（CD4＋CD28null T cells and CD4＋CD28＋T cells） by fluorescence mieroscopy and patch clamp. Results The percentage of CD4＋CD28mullT cells was higher in the ACS patients [（6.97±2.05）%] than that in the controls [（1.38±0.84）%, P〈0.05]. The concentration of hsCRP was directly correlated with the number of the CD4~CD28nul~ T cells in the ACS patients （r=0.52, P〈0,05）. The conductance （6.89±1.17ns vs 3.36±0.66ns）, dens （1.95±0.80 μm2 vs 1.13±0.57 μm2） and numbers （574.5±97.6 n/cell vs. 280.3±55.3 n/cell） of the Kv1.3 channels on the CIM＋CD28null T cells were significantly higher than those on the CD4＋CD28＋ T cells （all P〈0.01） in ACS patients, but were similar on CD4＋CD28＋T betweenACS patients and controls. Conclusion The CD4＋CD28nullT cells and the numbers of Kvl.3 channels on the CD4＋CD28nullT cells from patients with ACS are significantly upregulated and might contribute to the pathogenesis of ACS （d Geriatr Cardio12010; 7：40-46）.     

急性冠状动脉综合征患者的CD4+CD28null T淋巴细胞电压门控性Kv1.3钾通道数量增加

目的 运用膜片钳技术检测急性冠状动脉综合征(ACS)患者的CD4+CD28nullT淋巴细胞Kv1.3钾通道的数量是否增加,探讨CD4+CD28nullT淋巴细胞上Kv1.3钾通道的表达.方法 选择17例ACS患者,11例年龄、性别匹配的正常体检者作为对照.运用荧光染色标记及膜片钳技术检测单个T淋巴细胞(CD4+CD28nullT淋巴细胞和CD4+CD28+T淋巴细胞)上Kv1.3钾通道的数量,比较Kv1.3通道在两种细胞上的差别.结果 ACS患者的CD4+CD28nullT淋巴细胞比例为(6.97±2.05)%,明显高于对照组的(1.38±0.84)%(P<0.05).ACS患者的CD4+CD28nullT淋巴细胞数量与hsCRP浓度呈正相关(r=0.52,P<0.05).ACS患者CD4+CD28nullT淋巴细胞Kv1.3通道的电导、密度、数量明显高于对照组CD4+CD28+T淋巴细胞(P<0.01).而两组CD4+CD28+T淋巴细胞间Kv1.3通道的电导、密度、数量差异无统计学意义(P>0.05).结论 ACS患者CD4+CD28nullT淋巴细胞明显增多.ACS患者CD4+CD28nullT淋巴细胞上Kv1.3钾通道数量比自身和对照的CD4+CD28+T淋巴细胞明显增多,CD4+CD28nullT淋巴细胞的功能可能与Kv1.3钾通道增多相关.     

急性冠状动脉综合征患者外周血CD4+T细胞及CD28null/CD28+亚型活化后Kv1.3钾通道的表达和意义

目的 研究急性冠状动脉综合征(ACS)患者外周血中CD4+T细胞及CD28null/CD28+亚型活化前后Kv1.3钾通道数目的 变化以及Kv1.3钾通道阻滞剂对CD4+T细胞活化表达的影响,探讨Kv1.3钾通道在不稳定斑块中的意义.方法 用免疫磁珠法分离出27例ACS患者外周血中的CD4+T细胞,其中12例进一步分出亚型CD4+CD28null和CD4+CD28+T细胞,采用全细胞膜片钳技术记录细胞活化前及经CD3抗体活化72 h后的Kv1.3钾电流.CD4+T细胞活化时分别加入终浓度为0.1、1、10 nmol/L特异性Kv1.3钾通道阻滞剂rMargatoxin(rMgTX),共同培养72 h后用反转录-PER法检测干扰素-γ、肿瘤坏死因子(TNF)-α及颗粒酶B mRNA的表达.结果 活化后CD4+、CD4+CD28null、CD4+CD28+T细胞的Kv1.3钾通道的峰电流均明显增加,细胞平均通道数分别增加约90%、60%、80%[活化前后每个细胞的通道数分别为(402±88)个比(752±275)个、(553±328)个比(874±400)个、(392±133)个比(716±251)个,均P<0.05].活化前CD4+CD28nullT细胞Kv1.3钾通道的平均数目比CD4+CD28+T细胞多约40%(P<0.05),活化后两者差异无统计学意义(P=0.102).不同浓度的rMgTX均下调CD4+T细胞活化后干扰素-γ、TNF-α、颗粒酶B mRNA的表达,各浓度组间干扰素-γ、TNF-α、颗粒酶B mRNA的表达差异均有统计学意义(均P<0.01),浓度越高,各mRNA表达越低.结论 ACS患者外周血CD4+T细胞及CD28null/CD28+亚型活化后Kv1.3钾通道表达增加,特异性Kv1.3通道阻滞剂rMgTX呈浓度依赖性地抑制CD4+T细胞活化时干扰素-γ、TNF-α及颗粒酶B mRNA的表达,提示CD4+T细胞特别是CD4+CD28nullT细胞的Kv1.3钾通道可作为预防动脉粥样斑块不稳定的潜在治疗靶点.     

急性冠状动脉综合征患者外周血CD4~+及CD4~+ CD28~(null) T细胞活化后IKCa1通道的表达及意义

目的:研究急性冠状动脉综合征(ACS)患者外周血中CD4+T细胞及CD4+CD28null亚型活化前后IKCa1钾通道数目的变化以及IKCa1钾通道阻滞剂对活化CD4+T细胞效应分子表达的影响,探讨IKCa1钾通道对不稳定斑块的意义.方法:用免疫磁珠法分离出24例ACS患者外周血中的CD4+T细胞,其中12例进一步分出亚型CD4+CD28nullT细胞,采用全细胞膜片钳技术记录细胞活化前及活化3 d后的IKCa1钾电流.CD4+T细胞活化3 d后分别加入终浓度为0 2、1、5 μmol/L特异性IKCa1钾通道阻滞剂TRAM-34,再继续活化3 d,用反转录-PCR法检测干扰素-γ及颗粒酶B mRNA的表达.结果:活化后CD4+、CD4+CD28nullT细胞的IKCa1通道数目分别增加了9倍和8倍[活化前后2种细胞的通道数分别为(45±3)∶(439±33), (56±4)∶(497±45),均P<0 01].2种细胞的通道密度也分别增加了约3倍(P<0 01).活化前及活化后2种细胞的通道数目及通道密度均无差别.不同浓度的TRAM-34均下调CD4+T细胞活化后干扰素-γ、颗粒酶B mRNA的表达,各浓度组间干扰素-γ、颗粒酶B mRNA的表达差异均有统计学意义(P<0 01),浓度越高,各mRNA表达越低.结论:ACS患者外周血CD4+及CD4+CD28nullT细胞活化后IKCa1的通道数目及通道密度均明显增加,特异性IKCa1通道阻滞剂TRAM-34呈浓度依赖性地抑制CD4+T细胞活化时干扰素-γ及颗粒酶B mRNA的表达,提示CD4+T细胞的IKCa1钾通道可作为预防动脉粥样斑块不稳定的潜在治疗靶点.     

急性冠状动脉综合征患者外周血T淋巴细胞钾通道表达的差异

目的:探讨急性冠状动脉综合征(ACS)患者外周血CD_4~+CD28~(null)T细胞和CD_4~+CD28~+T细胞上电压依赖性钾离子通道(Kv1.3)和钙依赖激活的钾离子通道(IKCa1)的表达.方法:入选对象共75例,分为3组:其中ACS组27例,稳定型心绞痛组20例,对照组28例.分离外周血单个核细胞(PBMCs),用ELISA法测定PBMCs中NF-κB的活性,流式细胞仪计数CD_4~+CD28~(null)T细胞占CD4+T细胞的比例.免疫荧光染色标记结合膜片钳技术记录ACS组CD_4~+CD28~(null)T细胞和CD_4~+CD28~+T细胞上Kv1.3和IKCa1的电流,计算单细胞上通道的数目.结果:ACS组NF-κB的活性和CD_4~+CD28~(null)T细胞的比例显著高于稳定型心绞痛组和对照组(P<0.05).NF-κB的活性和CD4+CD28null T细胞的比例具有正相关关系(r=0.369,P<0.05).CD_4~+CD28~(null)T细胞上Kv1.3的数目和电流密度显著高于CD_4~+CD28~+T细胞(P<0.05).CD_4~+CD28~(null)T细胞、CD_4~+CD28~+T细胞上IKCa1的数目差异无统计学意义(P>0.05).结论:Kv1.3通道对CD_4~+CD28~(null)T细胞的功能具有重要作用,Kv1.3通道有可能成为AS治疗的新靶点.     

大鼠动脉粥样硬化斑块中T淋巴细胞亚群及Kv1.3通道蛋白的表达

目的探究Kv1.3通道蛋白与动脉粥样硬化(AS)模型中活化的T淋巴细胞间的关系。方法 Wistar雄性大鼠24只,随机分为对照组(n=10)和AS组(n=14),采用高脂饲料喂养方法建立AS模型。分别于实验开始前,实验第8周,实验第12周观察各组大鼠体重变化。于第12周处死大鼠前取血,检测血清中总胆固醇(TC)、低密度脂蛋白(LDL-L)、高密度脂蛋白(HDL-L)和甘油三酯(TG)的水平。通过病理HE染色及免疫组织化学方法观察AS斑块内T淋巴细胞亚群分布和Kv1.3通道蛋白表达的改变。结果 AS组体重、TC、LDL-C较对照组明显升高(P均<0.05);HDL-C和TG两组无差异。AS组主动脉管壁可见明显斑块形成,对照组血管壁各层的组织结构正常。AS组动脉斑块部位内膜下及中膜层可见CD4+与CD8+T淋巴细胞聚集,以CD4+T淋巴细胞聚集为主,在病变部位Kv1.3通道蛋白表达增加。对照组血管内膜、中膜中未见T淋巴细胞聚集及KV1.3通道蛋白的表达。结论 Kv1.3通道可能在调节AS斑块部T淋巴细胞亚群的激活中起着重要作用。     

替米沙坦对电压依赖性的Kv1.3和Kv1.5通道电流阻断作用的实验研究

目的 通过观察替米沙坦对电压依赖性的Kv1.3和Kv1.5的阻断作用,探讨替米沙坦对此类通道的阻断可能具有的临床作用.方法 使用双电极电压钳技术记录表达于非洲爪蟾卵母细胞的Kv1.3和Kv1.5钾通道电流,不同浓度灌流观察其对电流影响.结果 (1)替米沙坦浓度依赖性的阻断Kv1.3通道,其阻断的IC50是2.05 μmol/L.替米沙坦对Kv1.3电流的阻断具有电压依赖性.(2)替米沙坦浓度依赖件的阻断Kv1.5通道,其阻断的IC50是2.37 μmol/L.替米沙坦对Kv1.5电流的阻断具有更显著的电压依赖性.结论 替米沙坦阻断开放状态的Kv1.3可能是其发挥免疫调节和抗动脉粥样硬化作用的机制之一.替米沙坦对开放状态的Kv1.5钾通道的阻断可能是其减少心房颤动发生率的作用机制之一.     

急性冠状动脉综合征患者B7∶CD28/CTLA4共刺激分子的表达

目的:探讨急性冠状动脉综合征(ACS)患者外周血中单核细胞共刺激分子CD28、细胞毒T淋巴细胞抗原(CTLA)4、CD80(B7-1)的变化,探讨这些分子在发病中的意义。方法:采用直接荧光标记流式细胞仪测定23例ACS患者(ACS组)和31例稳定型心绞痛(SA)患者(SA组)入院时外周血CD4 ,CD8 T淋巴细胞CD28、CTLA4、B7-1分子的表达,同时选健康人群15例作为对照(对照组)。结果:与对照组相比,SA组及ACS组发病时CD4 ,CD8 T淋巴细胞表面共刺激分子CD28均显著升高(均P<0.01);SA组与ACS组比较差异无统计学意义。与对照组相比,SA组CD4 ,CD8 T淋巴细胞表面CTLA4表达均显著升高(P<0.01);而ACS组CD4 ,CD8 T淋巴细胞表面CTLA4表达均显著下降(P<0.01)。各组B7-1的表达差异无统计学意义。结论:①共刺激分子B7-1:CD28/CTLA4途径参与了冠心病的发病过程;②ACS的强烈的炎症反应与CT-LA4的低表达有关。     

Unusual CD4+CD28null T lymphocytes and recurrence of acute coronary events.

OBJECTIVES: We hypothesized that the expansion of unusual T lymphocytes, CD4+CD28null T cells, might represent a key pathogenetic mechanism of recurrent instability. BACKGROUND: Clinical presentation of acute coronary syndromes (ACS) is variable. Some patients have recurrent episodes of instability, despite optimal treatment, whereas others have a single acute event in their life. The CD4+CD28null T cells, with a functional profile that favors vascular injury, have recently been found both in peripheral blood and in unstable coronary plaques of patients with ACS. METHODS: Peripheral blood T cells from 120 consecutive unstable angina (UA) patients were analyzed for the distribution of T-cell subsets by flow cytometry. Patients were subgrouped according to the occurrence of prior (during the 24 months before the study enrollment) and subsequent (during the 24 months of follow-up) acute coronary events. For 51 patients, the index event was the first ever (G1); 30 patients had prior events (G2); and 39 patients had further events at follow-up (death, myocardial infarction, or UA) or both before and after the index event (G3). RESULTS: The CD4+CD28null T-cell frequency was higher in G3 than in G2 and G1 (median 9.5% [range 2.4% to 48.0%] vs. 5.1% [range 0.4% to 27.8%] and 2.3% [range 0.2% to 22.8%], respectively; p < 0.001). The expansion of these unusual T lymphocytes was higher in patients with elevated C-reactive protein levels, and it was reduced by statin therapy. On multivariate logistic regression analysis, CD4+CD28null T-cell frequency was an independent predictor of future acute coronary events (odds ratio 3.01, 95% confidence interval 1.1 to 8.25; p = 0.023). CONCLUSIONS: A perturbation of T-cell repertoire is strongly associated with the recurrence of acute coronary events, conceivably playing a key pathogenetic role.     

普罗帕酮对Kv1.4ΔN钾通道的作用及细胞外钾离子和pH浓度变化时的影响

目的:探讨抗心律失常药物普罗帕酮对Kv1.4△N钾通道的作用,以及细胞外钾离子和pH浓度变化时对该作用的影响,并探讨该作用可能的机制.方法:将Kv1.4ΔN的mRNA注射入非洲爪蟾卵母细胞并使用双电极钳制法观察普罗帕酮对Kv1.4ΔN电生理特性的影响,以及细胞外钾离子和pH变化时的电生理特性改变.结果:pH7 4状态下,普罗帕酮对Kv1.4ΔN通道的峰电流有抑制作用,这种阻滞作用具有电压依赖性、浓度依赖性以及频率依赖性,并且随电位的升高而作用加强,符合单指数和线性关系.普罗帕酮加速电流的失活过程.在不同的钾离子浓度下,这种阻滞作用具有pH依赖性,细胞外高钾pH7 4时,不同浓度普罗帕酮灌流显示IC50为121 μmol/L;细胞外酸性环境下(pH6 0)IC50提高到463 μmol/L,碱性化的环境(pH8 0)降至58 μmol/L.结论:普罗帕酮是Kv1.4ΔN的阻滞剂,可能与作用于细胞内的某些位点有关.     

Inhibitory effects of candesartan on KCa3.1 potassium channel expression and cell culture and proliferation in peripheral blood CD4+T lymphocytes in Kazakh patients with hypertension from the Xinjiang region

Background and aim: Increasing evidence confirms that potassium channels are essential for lymphocyte activation, suggesting an involvement in the development of hypertension. Moreover, chronic inflammation is regarded as a direct or indirect manifestation of hypertension, highlighting the theoretical mechanisms. In this study, we investigated changes in KCa3.1 potassium channel expression in the blood of hypertensive and healthy Kazakh people in north-west China.

Methods: Flow cytometry technology was used for T-lymphocyte subtype analysis. Changes in the messenger RNA and protein expression of the KCa3.1 potassium channel in CD4⁺ T lymphocytes were detected using real-time quantitative polymerase chain reaction and western blots, using CD4⁺ T-cell samples from hypertensive Kazakh patients divided into candesartan and TRAM-34 treatment groups, and healthy case controls. Peripheral blood CD4⁺ T lymphocytes were activated and proliferated in vitro and then incubated for 0, 24, and 48 h under various treatment conditions. Changes in CD4⁺ T-lymphocytic proliferation were determined using Cell Counting Kit-8 and electron microscope photography.

Results: Expression of KCa3.1 was significantly higher in the hypertensive patients than in the controls (p < 0.05). Compared with the healthy group, Kazakh hypertensive patients had a reduced proportion of CD4⁺ T lymphocytes (p < 0.05).Candesartan and TRAM-34 intervention for 24 h and 48 h inhibited the expression of Kv1.3 and KCa3.1 at mRNA and protein level (p < 0.05).

Conclusions: Increase in functional KCa3.1 channels expressed in CD4⁺ T lymphocytes of Kazakh patients with hypertension was blocked by candesartan, providing theoretical support for hypertension treatment at the cellular ion channel level. Candesartan may potentially regulate hypertensive inflammatory responses by inhibiting T-lymphocytic proliferation and KCa3.1 potassium channel expression in CD4 ⁺ T lymphocytes.     

Glutamate levels and activity of the T cell voltage-gated potassium Kv1.3 channel in patients with systemic lupus erythematosus

OBJECTIVE: Alterations in glutamate homeostasis and Kv1.3 voltage-gated potassium channel function have been independently associated with T cell dysfunction, whereas selective blockade of Kv1.3 channels inhibits T cell activation and improves T cell-mediated manifestations in animal models of autoimmunity. Because low extracellular glutamate concentrations enhance the activity of this channel in normal T cells ex vivo, we undertook this study to examine serum glutamate concentrations and Kv1.3 channel activity in patients with systemic lupus erythematosus (SLE). METHODS: We used high-performance liquid chromatography for glutamate measurements, and we used the whole-cell patch-clamp technique for electrophysiologic studies performed in freshly isolated, noncultured peripheral T cells. RESULTS: Mean +/- SD serum concentrations of glutamate were lower in patients with either clinically quiescent SLE (77 +/- 27 microM [n = 18]) or active SLE (61 +/- 36 microM [n = 16]) than in healthy controls (166 +/- 64 microM [n = 24]) (both P < 0.0001). The intrinsic gating properties of the Kv1.3 channels in lupus T cells were found to be comparable with those in healthy control-derived T cells. Notably, electrophysiologic data from SLE patient-derived T cells exposed to extracellular glutamate concentrations similar to their respective serum levels (50 microM) demonstrated Kv1.3 current responses enhanced by almost 20% (P < 0.01) compared with those subsequently obtained from the same cell in the presence of glutamate concentrations within control serum levels (200 microM). CONCLUSION: Based on the key role of Kv1.3 channel activity in lymphocyte physiology, an enhancing in vivo effect of low serum glutamate concentrations on the functional activity of this channel may contribute to lupus T cell hyperactivity. Studies to further elucidate Kv1.3 responses in SLE, as well as the possible pathogenetic role of this unsuspected metabolic abnormality, may have therapeutic implications for SLE patients.     

急性冠脉综合征患者血钾情况分析

目的研究急性冠脉综合征患者血钾（血K^＋）浓度的变化情况。方法记录294例急性冠脉综合征患者胸痛发作距急检采血的间隔时间以及急检化验血U的结果。依据应用B受体阻滞剂情况以及是否患糖尿病对患者进行分组研究。结果患者血钾浓度于发病≤2h时最低[（4．0±0．5）mmol／L]，2-4h逐渐上升[（4．3±0．6）mmol／L]，4-6h最高[（4．4±0．3）mmol／L]，〉6h后略有下降[（4．3±0．5）mmol／L]，构成胸痛时间-血钾浓度曲线。应用13受体阻滞剂者该曲线变得平坦，糖尿病患者该曲线变得异常，不稳定型心绞痛患者该改变表现得更明显。结论急性冠脉综合征患者血钾浓度于发病早期较低，6h内迅速上升，6h后略有下降，构成胸痛时间-血钾浓度曲线。该曲线在糖尿病及应用岱受体阻滞剂者中受到抑制，心肌损害程度较轻者抑制改变表现得更明显。     

新疆哈萨克族高血压患者外周血淋巴细胞Kv1.3通道的表达

目的研究新疆哈萨克族高血压患者外周血淋巴细胞Kv1.3电压门控钾通道表达的变化。方法于2010-09-2011-06在新疆医科大学第一附属医院高血压门诊随机入选未经降压药物治疗的新疆哈萨克族高血压患者20例为高血压组,年龄(50.2±3.8)岁,选择同期健康体检者20名为对照组,年龄(49.1±3.6)岁,两组男女各10名。采集这两组外周血淋巴细胞,运用实时荧光定量PCR(RT-PCR)技术和Western blotting技术检测外周血淋巴细胞Kv1.3通道基因和蛋白表达水平。结果高血压组Kv1.3通道mRNA相对表达量及蛋白相对表达量均高于对照组[(0.063±0.008)比(0.008±0.007),(0.835±0.067)比(0.250±0.068),均P<0.01]。结论新疆哈萨克族高血压患者淋巴细胞上Kv1.3通道表达增多。     

系统性红斑狼疮患者外周血CD4+ CD8+和CD22+淋巴细胞活化分子CD69的表达

目的探讨系统性红斑狼疮(SLE)患者外周血淋巴细胞(PBL) T细胞(CD4+、CD8+)和B细胞(CD22+)活化分子CD69的表达.方法应用双染色流式细胞术检测CD4、CD8、和CD22细胞亚群CD69分子;在植物凝集素(PHA)刺激后20 h淋巴细胞亚群CD69分子的表达.结果①SLE患者PBMC的CD69分子活动期高于静止期(P＜0.001)和正常对照组(P＜0.01)的表达,SLE静止期患者与正常对照组CD69表达差异无显著性(P＞0.05).②进一步分析CD4+、CD8+和CD22+淋巴细胞亚群的CD69的表达,其中,SLE活动期患者CD4+细胞的CD69表达显著高于静止期(P＜0.001)和正常对照组(P＜0.01)的表达,SLE静止期患者与正常对照组CD69表达差异无显著性(P＞0.05);CD8+细胞活动期高于静止期患者(P＜0.05),其余组间差异无显著性(P＞0.05);CD22+B细胞各组间差异无显著性.③PHA刺激20 h后,CD4+、CD22+B细胞的CD69表达,活动期显著高于静止期患者和正常对照组(P＜0.01).结论 SLE患者外周血CD4+T细胞和CD22+B细胞存在着异常的活化,这种淋巴细胞的异常活化是SLE重要的发病机制之一.     

急性冠状动脉综合征患者急诊介入治疗临床研究

目的分析本中心急性冠状动脉综合征患者的急诊介入治疗详细特征及趋势,以便更及时、有效地救治患者。方法对在我中心实施急诊介入治疗的230例急性冠状动脉综合征患者进行分析,总结病例的临床特征、介入治疗时间窗、治疗情况及近期预后。结果平均年龄增大、高龄及高危患者增多,治疗时间窗中各时间段(发病→急诊科→导管室→首次球囊扩张)均有缩短,其中导管室→首次球囊扩张时间缩短显著;药物支架、远端保护装置、血栓抽吸器、预防性冠状动脉联合注射防止无复流药物在近两年增长迅速,无血流发生率、主要心血管事件发生率及平均住院日显著下降,同时溶栓后介入治疗较直接介入治疗各种严重出血并发症无明显增加。结论急诊介入治疗是急性冠状动脉综合征的最有效治疗方法之一,各种新型辅助技术的使用可有效预防严重并发症的发生。