MDR1基因在睫状体色素上皮细胞容积激活性氯电流中的作用

用膜片钳,反义寡核苷酸,免疫荧光及激光共聚焦显微镜等技术,研究MDR1基因在牛睫状体色素上皮（pigmented ciliary epithelial,PCE)细胞容积激活性氯电流中的作用,PCE细胞表达MDR1基因产物-P糖蛋白（P-gp),反义MDR1寡核苷酸抑制MDR1基因的表达（P-gp免疫荧光减少93%）,延缓容积激活性氯电流的出现（潜伏期延长109%）,并导致激活率降低62%及电流峰值减小56%,而核酸转染剂阳离子脂质体和非配对性的寡核苷酸对电流没有显著性影响,上述观察结果表明,睫状体色素上皮细胞容积激活性氯电流与内源性MDR1表达有关。     

鼻咽上皮细胞的调节性容积回缩能力及机制

用图像分析系统和通道阻断法研究了原代人胎儿鼻咽上皮细胞的调节性容积回缩(regulatoryvolumedecrease,RVD)能力及其机制。结果发现,低渗刺激可诱发鼻咽上皮细胞产生RVD,在160-240mOsmol/L范围内,RVD强弱与渗透压呈“S”形负相关(r=-0.99,P<0.05),与细胞肿胀程度呈“S”形正相关(=0.99,P<0.05)。Cl~-通道阻断剂tamoxifen(20μmol/L),ATP(10mmol/L)或NPPB(100μmol/L)对RVD阻抑率分别为100%(P<0.01),76.3%(P<0.01)和62.7%(P<0.01)。本研究表明,鼻咽上皮细胞受到低渗刺激时可产生RVD,Cl~-通道开放是其RVD的关键机制。     

迁移的鼻咽癌细胞容积激活性氯电流

用膜片钳技术研究了Transwell小室趋化迁移后的鼻咽癌CNE-2Z细胞容积激活性CT电流。47％低渗刺激迁移后的CNE-2Z细胞诱发容积激活性氯电流,与未迁移细胞相比,其特性以及其对氯通道阻断剂的敏感性发生明显的变化,此电流的密度明显高于未迁移细胞,而且该电流几乎完全被氯通道阻断剂adenosine-5'-triphosphate(ATP,10 mmol/L)、5-nitro-2-3-phenylpropylamino benzoic acid(NPPB,100μmol/L)和他莫昔芬(30μmol/L)抑制,其中NPPB和他莫昔芬对迁移细胞的抑制作用明显强于未迁移细胞。迁移后的CNE-2Z细胞容积激活性氯通道对阴离子的通透性为:Br>Cl>I>葡萄糖酸,与未迁移细胞(I>Br>Cl>葡萄糖酸)不同。结果提示,容积激活性氯通道可能参与CNE-2Z细胞的迁移过程。     

鼻咽上皮细胞的调节性容积回缩能力及机制

用图像分析系统和通道阻断法研究了原代人胎儿鼻咽上皮细胞的调节性容积回缩（regulatory volume decrease,RVD）能力及其机制。结果发现,低渗刺激可诱发鼻咽上皮细胞产生RVD,在160-240 mOsmol／L范围内,RVD强弱与渗透压呈“S”形负相关（r=-0．99,P〈0．05）,与细胞肿胀程度呈“S”形正相关（r=0．99,P〈0．05）。Cl-通道阻断剂tamoxifen（20μmol／L）,ATP（10mmol／L）或NPPB（100μmol／L）对RVD阻抑率分别为100％（P〈0．01）,76．3％（P〈0．01）和62．7％（P〈0．01）。本研究表明,鼻咽上皮细胞受到低渗刺激时可产生RVD,Cl-通道开放是其RVD的关键机制。     

人胎儿鼻咽上皮细胞的背景氯电流

采用膜片钳和图像分析技术,研究人胎儿鼻咽上皮细胞背景电流的特性及其与容积激活性氯电流的关系。在等张溶液中,可记录到一背景电流,该电流呈微弱的外向整流性,无明显时间依赖性失活,其翻转电位为(?0.73±1.7)mV(n=21),接近氯离子平衡电位(?0.9mV)。细胞外高张刺激(440mOsmol/L)明显抑制此电流(59.6±7.1)%,而低张刺激(160mOsmol/L)则诱发细胞产生容积激活性氯电流。氯通道阻断剂tamoxifen和5-硝基-2-(3-苯丙胺基)苯甲酸[5-nitro-2-(3-phenylpropylamino)benzoicacid,NPPB]显著地抑制背景电流并使细胞基础容积增大。上述结果表明,人胎儿鼻咽上皮细胞的背景Cl?电流是背景电流的重要成分,此Cl?电流与容积激活性氯电流及细胞基础容积调节有关。     

大鼠壁细胞基底膜容积致敏感氯通道电流

为研究胃粘膜壁细胞容积致敏感氯通道电流的定位、电生理特征及药物效应并推断其在壁细胞病理生理过程中所起作用,对急性分离的大鼠胃粘膜壁细胞进行全细胞膜片钳记录,将电极液设置为高渗（Δ≈７０ｍＯｓｍ）,使细胞出现稳定的体积增大后,在其基底膜上记录到容积致敏感氯通道电流（ｖｏｌｕｍｅ－ｓｅｎｓｉｔｉｖｅｃｈｌｏｒｉｄｅｃｈａｎｎｅｌｃｕｒｅｎｔ,ＶＳＣｌＣＣ）,该电流具有外向整流性及电压和时间依赖性,可被１０μｍｏｌ／Ｌ花生四烯酸（ａｒａｃｈｉｄｏｎｉｃａｃｉｄ,ＡＡ）可逆性抑制（７１．３±１０．９％）。细胞外液ｐＨ值由７．４降低至４．０,ＶＳＣｌＣＣ的幅度显著下降（６３．１±１４．０％）,而其激活及失活动力学无改变;ｐＨ升高至９．０对ＶＳＣｌＣＣ无影响。壁细胞ＶＳＣｌＣＣ对细胞内外钙离子浓度均呈现明显的依赖性。结果表明,壁细胞基底膜上存在本底氯通道和容积致敏感氯通道两种不同的氯离子通道。ＶＳＣｌＣＣ可能参与某些胃粘膜疾病的病理生理过程。     

色素上皮细胞衍生因子

色素上皮细胞衍生因子(pigment epithelium-derived factor,PEDF)最初从视网膜色素上皮细胞分离,是维持角膜、玻璃体无血管状态的主要血管增生抑制因子。体内外试验证明：PEDF具有高效的抗血管生成活性,成为治疗血管增生性疾病的候选药物。     

ATP激活鼻咽癌细胞氯电流并减小细胞容积

采用全细胞膜片钳技术和细胞容积测量技术,在低分化鼻咽癌细胞株CNE-2Z上观察ATP 诱导的Cl- 电流的特性及其对细胞容积的影响。细胞外微摩尔水平的ATP 以剂量依赖性的方式激活一个具有弱外向整流特性,没有时间依赖性失活的电流,此电流的反转电位 [(-0.05 ± 0.03) mV]接近Cl- 的平衡电位(-0.9 mV)。用葡萄糖酸置换细胞外液Cl- 后, ATP 激活的电流明显减小并且反转电位发生改变。氯通道抑制剂NPPB (200 μmol/L)可以抑制这一电流 [(81.03 ± 9.3)%] 。此电流亦可被嘌呤受体(P2Y) 拮抗剂反应蓝 2 抑制 [(67.39 ± 5.06)%]。50 μmol/L 的 ATP 使在等渗状态下的细胞容积缩小, 替代和耗竭细胞外、内的Cl- 后, ATP 的这一作用消失。这些结果提示细胞外微摩尔水平的 ATP 可通过兴奋 P2Y 受体激活氯通道而产生与细胞容积调节相关的Cl- 电流。     

重组人色素上皮细胞衍生因子融合蛋白的制备及其抑制新生血管

目的：人色素上皮细胞衍生因子 (pigment epithelium-derived factor, PEDF)是一种有效的新生血管形成抑制因子和神经营养因子。本文通过原核细胞表达人PEDF蛋白,鉴定其抑制新生血管的生物学活性。方法：采用PCR法扩增人PEDFcDNA,将其克隆到pET32a载体中,在大肠杆菌BL21中表达人PEDF蛋白。经SDS-PAGE和Western-blot鉴定后,镍柱亲和层析法变性条件下纯化重组融合蛋白。Bradford法测蛋白浓度,采用鸡胚脲囊膜法测其对新生血管形成的影响。结果：成功构建了pET32a-PEDF表达载体。重组人PEDF蛋白在BL21宿主菌中获得了稳定高效表达,鸡胚脲囊膜实验结果显示在重组蛋白浓度为0.4、0.04 ng/ml时均有对新生血管的显著抑制作用（P<0.01）,而在4 ng/ml时无抑制作用。结论： 成功高效表达及纯化了重组人PEDF蛋白,鉴定其抑制新生血管的生物学活性,并且证实该活性在一定范围内有效,为进一步研究其功能及推广应用奠定了基础。     

小檗碱激活人结肠癌细胞容积敏感的氯通道

本文旨在研究小檗碱对人结肠癌细胞(SW480)氯通道的作用.采用膜片钳技术记录小檗碱激活的SW480全细胞氯电流,并用高渗和低渗灌流液、以及氯通道阻断剂研究该电流的生理学和药理学特性.结果显示,当细胞处在等渗液中,可在SW480细胞膜上记录到微弱且稳定的背景电流;小檗碱(10 nmol/L)可诱发SW480细胞迅速产生...     

Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells

Summary The apical membrane of epithelial cells from the A6 cell line grown on impermeable substrata was studied using the patch-clamp technique. We defined the apical membrane as that membrane in contact with the growth medium. In about 50% of the patches, channels with single-unit conductances of 360±45 pS in symmetrical 105mm NaCl solutions, and characteristic voltage-dependent inactivation were observed. Using excised membrane patches and varying the ionic composition of the bathing medium, we determined that the channels were anion selective, with a permeability ratio for Cl^– over Na⁺ of about 91, calculated from the reversal potential using the constantfield equation. The channel was most active at membrane potentials between ±20 mV and inactivated, usually within a few seconds, at higher potentials of either polarity. Reactivation from this inactivation was slow, sometimes requiring minutes. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to one or more submaximal conductance levels. The channel was inhibited by the disulfonic stilbene SITS in a manner characteristic of reversible open-channel blockers.     

血管内皮细胞容量激活的氯通道

氯通道是血管内皮细胞上主要的离子通道,容量激活的氯通道是其中一种主要类型并广为研究。已经主宰容量激活的氯通道在维持静息膜电位,调节细胞内钙、ｐＨ值,影响细胞增殖和分化中起着重要的作用。本文综述了血管内皮细胞容量激活氯通道的基本电生理及分子生物学特性,并初步探讨该通道的调节机制。     

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene

Summary The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl^– channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl^– currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotomic-induced chloride conductance in neuroblastoma cells.     

Volume-activated chloride currents in pancreatic duct cells

We have used the patch clamp technique to study volume-activated Cl^– currents in the bicarbonatesecreting pancreatic duct cell. These currents could be elicited by a hypertonic pipette solution (osmotic gradient 20 mOsm/l), developed over about 8 min to a peak value of 91 ± 5.8 pA/pF at 60 mV (n = 123), and were inhibited by a hypertonic bath solution. The proportion of cells which developed currents increased from 15% in freshly isolated ducts to 93% if the ducts were cultured for 2 days. The currents were ATP-dependent, had an outwardly rectifying current/voltage (I-V) plot, and displayed time-dependent inactivation at depolarizing potentials. The anion selectivity sequence was: ClO₄ = I = SCN > Br = NO₃ > Cl > F > HCO₃ > gluconate, and the currents were inhibited to a variable extent by DIDS, NPPB, dideoxyforskolin, tamoxifen, verapamil and quinine. Increasing the intracellular Ca²⁺ buffering capacity, or lowering the extracellular Ca²⁺ concentration, reduced the proportion of duct cells which developed currents. However, removal of extracellular Ca²⁺ once the currents had developed was without effect. Inhibiting protein kinase C (PKC) with either the pseudosubstrate PKC (19–36), calphostin C or staurosporine completely blocked development of the currents. We speculate that cell swelling causes Ca²⁺ influx which activates PKC which in turn either phosphorylates the Cl^– channel or a regulatory protein leading to channel activation.We thank David Stephenson for skilled technical assistance, and Dr. Malcolm Hunter for useful discussions. This work was funded by the National Institutes of Health (grant No. DK 43956), and the Cystic Fibrosis Trust.