【作者】 马文波；

【导师】 王立伟； 陈丽新；

【作者基本信息】 暨南大学 ， 生理学， 2012， 硕士

【摘要】 目的：酸激活性氯电流存在于多种正常的细胞中，并参与细胞生理功能的调控。肿瘤细胞外pH值低于正常细胞，但其胞外酸化对其自身氯通道有何影响？肿瘤细胞酸激活性氯电流有何特性？介导此电流的通道蛋白分子本质是什么？目前仍未明了。本研究主要观察细胞外酸化对低分化鼻咽癌CNE-2Z细胞氯电流的影响，分析酸激活性氯电流的多种特性、探讨介导肿瘤细胞酸激活性氯电流的通道蛋白分子本质。方法：用全细胞膜片钳技术记录鼻咽癌CNE-2Z细胞的氯电流；改变细胞外灌流液的pH值，观察其对CNE-2Z细胞氯通道活动的影响；利用离子置换技术和改变细胞外灌流液的渗透压来分析酸激活性氯电流的生理学特性；应用多种氯通道阻断剂来分析该电流的药理学特性；用RT-PCR技术和Western blot技术检测氯通道（ClC家族的不同亚型）在CNE-2Z细胞的表达；用siRNA技术干扰不同亚型的氯通道的表达，分析和探讨介导CNE-2Z细胞酸激活性氯电流的通道蛋白分子本质。结果：1、在正常的pH值（胞外7.4）和渗透压条件下，可在CNE-2Z细胞记录到一个微弱而稳定的背景氯电流，当将灌流液的pH值由7.4降到6.6时，可迅速激活氯通道，产生一个具有较弱外向整流特性的氯电流，该电流可被高渗灌流液诱导的细胞缩小和NPPB(5-nitro-2-3-phenylpropylamino benzoic acid)、 tamoxifen、 DIDS (4,4’-diisothiocyanatostilbe-ne-2,2’-disulfonic acid disodium salt hydrate)等多种氯通道阻断剂抑制，其对多种阴离子的选择性为I-> Br-> Cl-> gluconate-；2、将灌流液的pH值由7.4直接降到5.8，不能诱导出酸激活性氯电流；将灌流液的pH值由6.6进一步降低至5.8可抑制酸（pH6.6）激活的氯电流；3、ClC氯通道家族中，在CNE-2Z细胞上表达的亚型有ClC-3、ClC-5和ClC-7，但ClC-5的表达量很少；4、采用siRNA技术干扰ClC-3的表达后，pH6.6诱导的酸激活性氯电流被明显抑制，但干扰ClC-7的表达，则不影响该电流的激活。结论：CNE-2Z细胞存在酸激活性氯通道的表达，该通道介导的氯电流有容积敏感性，并与容积敏感性氯电流有许多相似的特征。ClC-3氯通道介导或调节CNE-2Z细胞酸激活性氯电流。更多还原

【Abstract】 Objective: Acid-activated chloride currents have been reported in several normal cells and mayplay important roles in regulation of cell function. The extracellular pH of tumor cells is lowerthan normal cells, but the effects of extracellular acidification on chloride channels, thecharacteristics of the acid-sensitive current and the molecular identity of the channel that mediatethe current are still unclear in tumor cells. In this study, the activation and properties of theacid-induced chloride current and the possible candidates of the acid-activated chloride channelwere investigated in human nasopharyngeal carcinoma cells (CNE-2Z).Methods: Whole-cell patch clamp technique was used to record the chloride current innasopharyngeal carcinoma cells (CNE-2Z). The effects of extracellular acidification wereobserved by changing the pH in bath solutions. Hypertonic bath solutions, anionic substitutionand Cl-channel blockers were used to observe the physiological and pharmacologicalcharacteristics of the acid induced chloride current. RT-PCR and Western blot techniques wereused to analyze the expression of the ClC chloride channel families in nasopharyngeal carcinomacells. Specific siRNAs were employed to knock down the expression of various chloridechannels, and were used to identify the candidate protein of the acid-induced chloride channeltogether with the patch clamp technique.Results:1. A chloride current was activated when extracellular pH was reduced to6.6from7.4.The current was weakly outward–rectified and was suppressed by hypertonicity-induced cellshrinkages and by the chloride channel blockers5-nitro-2-3-phenylpropylamino benzoic acid(NPPB), tamoxifen and4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid disodium salt hydrate(DIDS). The permeability sequence of the channel to anions was I-> Br-> Cl-> gluconate.2. The isotonic bath solution with pH5.8could not activate a chloride current. A furtherdecrease of extracellular pH to5.8from6.6inhibited the acid (pH6.6)-induced chloride current.3. Among the ClC families, ClC-3ClC-5and ClC-7mRNA were expressed in CNE-2Zcells, but ClC-5presented a weak expression.4. Knock-down of ClC-3expression with ClC-3siRNA prevented the activation of theacid-induced current. While knock-down of ClC-7expresion with ClC-7siRNA showed noobvious effect on this current. Conclusion: The acid-induced chloride current is expressed in CNE-2Z cells. The chloridechannel mediating the current is volume-sensitive and shares various properties with thevolume-sensitive chloride channel. ClC-3is a candidate of the channel protein that mediates orregulates the acid-activated chloride current in nasopharyngeal carcinoma cells.更多还原