多重耐药基因在睫状体上皮细胞容积调节中的作用

目的：研究多重耐药（ＭＤＲ１）基因与细胞容积调节的关系。方法：用反义寡核苷酸阻抑牛眼睫状体非色素上皮细胞ＭＤＲ１基因表达,在激光共聚焦显微镜下检测ＭＤＲ１基因反义寡核苷酸（ＭＤＲ－ａｎｔｉ）的导入,用反射／散射技术测量由低渗液引起的细胞容积变化。结果：阳离子脂质体促进ＭＤＲ－ａｎｔｉ导入细胞,ＭＤＲ－ａｎｔｉ导入量与剂量呈依赖性增强关系（ｒ＝０９７,Ｐ＜００１）。人ＭＤＲ－ａｎｔｉ使低渗液引起的细胞调节性容积减小反应延迟、缓慢,容积恢复不完全（６０％）,而鼠ＤＭＲ－ａｎｔｉ没有此作用。结论：人ＭＤＲ－ａｎｔｉ抑制细胞调节性容积减小,提示ＭＤＲ１基因参与细胞容积调节。     

电压门控氯通道生物学功能与疾病的关系

电压门控氯通道是表达于细胞膜或细胞器质膜上的一类阴离子跨膜通道蛋白,具有调节细胞容积、参与细胞迁移、增殖与凋亡以及氧化应激等生物学功能,本综述总结近年来该通道生物学功能及其与疾病的相关研究进展。     

门脉高压形成,调节的细胞机制研究进展

肝星状细胞是肝非实质细胞中的一种,具有贮存脂肪、维生素A和参与维生素A代谢的功能。近年发现肝星状细胞具有收缩性,能调节肝窦血流,体外培养及体内研究发现,肝星状细胞的收缩性受内皮素及一氧化氮等多种血管活性物质调节,活化的肝星状细胞与血管活性物质的相互作用,可能是门脉高压形成的重要病理机制之一,对肝星状细胞收缩性进行调节有可能成为降低门静脉高压的新途径。     

ClC-3氯通道蛋白调控细胞增殖机制的研究进展

Cl C-3是电压门控性氯离子通道家族的成员,主要作为容积激活性氯通道通过调节容积激活性氯电流[Cl,（vol）],调节细胞体积,细胞膜电位等影响细胞增殖。近年的研究发现Cl C-3也可通过调节有丝分裂前凝集（premitotic condensation,PMC）过程,或维持细胞囊泡酸化和细胞内氧压,或作为调控因子通过Akt-GSK-3β信号通路和Ca MKII调节的信号通路等途径参与细胞增殖的调控。     

ClC氯离子通道与细胞容积调节及细胞迁移的关系

Cl 是体内最为丰富和常见的阴离子 ,氯离子通道 (ClC)广泛分布于哺乳动物的组织器官和各种细胞中 ,对维持细胞的容积平衡、调节细胞电活动、细胞迁移等多种生理、病理过程发挥重要的作用。综述ClC氯离子通道的特点、生理作用 ,及其与细胞容积调节和细胞迁移之间的关系。     

皮肤中的P物质及其作用

P物质是一种高活性的神经肽类物质,广泛地分布于神经系统和其他外周组织器官内,具有多种生理功能。在皮肤,P物质主要由感觉神经末梢释放,它通过趋化免疫细胞、调节细胞因子的产生以及舒张微血管等参与皮肤局部免疫炎症反应;还可促进皮肤内多种细胞的分裂、增殖以及细胞DNA的合成,参与皮肤创伤的修复;此外,P物质作为一种重要的瘙痒介质,与多种瘙痒性皮肤疾病的发生和发展有关。     

电压门控氯通道3（ClC-3）的生物学作用及其与疾病的关系

ClC-3属于电压门控氯通道家族,存在于细胞膜和细胞质,以及某些肿瘤细胞的细胞核内,高表达于中枢神经系统、肾脏和肠道。ClC-3参与离子转运、容积调节、免疫应答、细胞迁移、增殖、分化、凋亡等生理生物学活动,近年发现ClC-3与肿瘤、糖尿病等疾病的发生密切相关。     

胃肠胰内分泌系统

胃肠胰内分泌系统(gastro-encro-pancreatic endocrine system,GEP)由分布于胃肠道与胰的内分泌细胞组成。系由 Fujita 1973年命各的。这些细胞具有 APUD 细胞的特征,属于 APUD 系。它们参与整合消化系统各部之间的机能活动,调节分泌、消化、吸收与物质代谢等     

退变椎间盘细胞外基质的改变

细胞外基质(extracellular matrix，ECM)是指位于上皮或内皮细胞下层、结缔组织细胞周围，为组织、器官甚至整个机体的完整性提供力学支持和物理强度的物质。ECM是一种动态物质，不仅仅是细胞机械支持组织，而且是供给营养和免疫应答的场所，参与调节胚胎发育进程，决定细胞的粘附与迁移，在创伤修复和纤维化、细胞的生长、分化、代谢和肿瘤发生及转移中起重要作用。     

空间生命科学中细胞骨架的研究进展

真核细胞骨架由三种基本纤维和上百种纤维结合蛋白组成。除了保持细胞形状、分隔固定细胞内部结构和参与构成某些细胞器外,真核细胞骨架还具有物质运输、信号传递、参与细胞运动、分化、增殖以及调节基因表达等作用。既然细胞骨架功能如此多样,那么它的变化必然会从多方面影响细胞行为。细胞骨架对重力变化相当敏感,微重力刺激对微管/微丝有破坏作用,进而影响了细胞生长、分裂、信号传导、新陈代谢等过程。     

Phosphorylation and functional regulation of CIC-2 chloride channels expressed in Xenopus oocytes by M cyclin-dependent protein kinase

Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl⁻ channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume-sensitive Cl⁻ channel, ClC-2 is regulated by the M-phase-specific cyclin-dependent kinase, p34^cdc2/cyclin B. ClC-2 channels were phosphorylated by p34^cdc2/cyclin B in both in vitro and cell-free phosphorylation assays. ClC-2 phosphorylation was inhibited by olomoucine and abolished by a ⁶³²Ser-to-Ala (S632A) mutation in the C-terminus, indicating that ⁶³²Ser is a target of phosphorylation by p34^cdc2/cyclin B. Injection of activated p34^cdc2/cyclin B attenuated the ClC-2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC-2 currents attenuated by p34^cdc2/cyclin B were increased by application of the cyclin-dependent kinase inhibitor, olomoucine (100 μ m ), an effect that was inhibited by calyculin A (5 n m ) but not by okadaic acid (5 n m ). A yeast two-hybrid system revealed a direct interaction between the ClC-2 C-terminus and protein phosphatase 1. These data suggest that the ClC-2 channel is also counter-regulated by protein phosphatase 1. In addition, p34^cdc2/cyclin B decreased the magnitude of ClC-2 channel activation caused by cell swelling. As the activities of both p34^cdc2/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC-2 channel could be regulated physiologically by these factors.     

沉默ClC-3氯通道基因对HeLa细胞周期分布的影响

目的:探讨沉默HeLa细胞的ClC-3氯通道基因后细胞周期分布的变化及其作用机制。方法:依照siRNA设计原则构建沉默ClC-3基因的ClC-3 siRNA并转染HeLa细胞;实验分为空白对照组(control组)、转染试剂对照组(Lipo组)、阴性对照组(negative siRNA组)和ClC-3 siRNA组。采用real-time PCR检测ClC-3 siRNA的沉默效率;流式细胞术检测细胞周期分布情况;Western blot检测ClC-3蛋白及相关细胞周期蛋白(cyclin)D1、细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)4、CDK6、P21和P27等表达。结果:CIC-3 siRNA成功沉默HeLa细胞的ClC-3基因。和其它组相比,ClC-3 siRNA组的细胞周期被阻抑在G_0/G_1期。CIC-3 siRNA组的cyclin D1、CDK4和CDK6蛋白表达水平明显下降,P21和P27蛋白表达水平明显上升。结论:沉默HeLa细胞ClC-3氯通道基因可影响cyclin D1、CDK4、CDK6、P21和27蛋白的表达水平胆抑HeLa细胞周期停滞在G_0/G_1期。     

ClC-3氯通道在二甲双胍抑制鼻咽癌细胞周期进程中的作用

目的:探讨ClC-3氯通道在二甲双胍抑制鼻咽癌细胞周期进程中的作用。方法:采用不同浓度二甲双胍处理低分化鼻咽癌细胞CNE-2Z,CCK-8法检测细胞活力,流式细胞术检测细胞周期分布,Western blot法检测ClC-3氯通道蛋白表达,全细胞膜片钳技术检测细胞氯电流。构建高表达ClC-3氯通道蛋白的质粒pEZ-M03-ClC-3转染CNE-2Z细胞,流式细胞术检测ClC-3氯通道对细胞周期分布的影响。结果:5、10和20 mmol/L浓度的二甲双胍均可有效抑制CNE-2Z细胞的活力。10 mmol/L二甲双胍可阻抑CNE-2Z细胞周期于G0/G1期,并抑制CNE-2Z细胞氯电流及ClC-3氯离子通道蛋白的表达。ClC-3氯通道蛋白高表达可逆转二甲双胍对CNE-2Z细胞周期分布的影响。结论:二甲双胍抑制鼻咽癌CNE-2Z细胞周期进程可能与抑制ClC-3氯通道功能和蛋白表达有关。     

氯通道ClC-2融合蛋白的原核表达及体外磷酸化

目的：检测ClC-2是否能被促分裂原活化蛋白激酶（MAPK）磷酸化，为进一步研究其在细胞增殖、分化过程中的调控机制提供基础。方法：从含有家兔ClC-2cDNA的质粒pSPORT1中PCR扩增ClC-2胞浆内的羧基端DNA编码序列，构建重组载体pGEX-4T-1／ClC-2CT；重组质粒经酶切、测序鉴定正确后转化大肠杆菌BL21菌株；经异丙基β-D-硫代半乳糖（IPTG）诱导后，通过Gluthathion Sepharose4B亲和层析柱纯化融合蛋白；并行融合蛋白的体外磷酸化实验。结果：酶切、测序鉴定重组载体pGEX-4T-1／ClC-2CT构建正确，并纯化得到GST／ClC-2CT融合蛋白。而且该融合蛋白能被MAPK磷酸化，而GST不能被MAPK磷酸化。结论：氯通道ClC-2能被MAPK磷酸化。     

敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 目的: 探讨ClC-3氯通道是否为IK1钾通道的调节靶点,重点研究鼻咽癌细胞IK1钾通道对ClC-3氯通道功能及蛋白表达的影响。方法: 采用siRNA转染技术抑制低分化鼻咽癌上皮细胞(CNE-2Z) IK1 基因的表达;real-time PCR技术检测ClC-3 mRNA的表达;Western blot检测ClC-3的蛋白表达;细胞免疫荧光结合激光共聚焦显微镜技术检测ClC-3和IK1蛋白在细胞内分布;全细胞膜片钳记录细胞氯电流。结果: IK1 siRNA可以成功转染CNE-2Z细胞,有效抑制鼻咽癌细胞IK1钾离子通道的表达;用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后, ClC-3的mRNA表达上调而ClC-3蛋白却表达减少:在低分化鼻咽癌上皮细胞,低渗刺激可激活氯通道,产生一个较大的氯电流,在成功转染IK1 siRNA的细胞,此氯电流明显减弱。结论: 敲低IK1钾离子通道可抑制ClC-3氯离子通道的表达和功能。     

Single-channel properties of volume-sensitive Cl- channel in ClC-3-deficient cardiomyocytes

It is controversial whether the ClC-3 protein, which is one of the voltage-dependent chloride channel ClC family members, is a candidate for the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel per se or its regulator. Here, for the first time, we examined the single-channel properties of the VSOR Cl(-) channel in ventricular myocytes isolated from ClC-3-deficient mice. The single-channel current induced by cell swelling exhibited Cl(-) selectivity, mild outward rectification, and an intermediate unitary conductance (around 38 pS). A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibited the outward current. These single-channel properties were identical with those in ClC-3 expressing wild-type ventricular myocytes. These results indicate that the single-channel activity of the VSOR Cl(-) channel is independent of the expression of ClC-3 proteins in mouse ventricular myocytes.     

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl− channel gene

Salivary gland acinar cells shrink when Cl⁻ currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl⁻ channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl⁻ channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl⁻ currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca²⁺-calmodulin-dependent protein kinase II; however, the magnitude of the Ca²⁺-dependent Cl⁻ current was unchanged in the Clcn3 ^−/- animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3 ^−/- mice. Our results demonstrate that the ClC-3 Cl⁻ channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.     

Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.     

Anion transport in heart

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.