鼻咽癌细胞酸激活性氯电流的研究

发布时间：2018-03-02 10:55

本文关键词： 鼻咽癌 pH值酸激活性氯电流氯通道胞外酸化　出处：《暨南大学》2012年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective: acid-activated chloride currents exist in many normal cells and participate in the regulation of cell physiological function. The extracellular pH value of tumor cells is lower than that of normal cells, but what effect does extracellular acidification have on its own chloride channels? What are the characteristics of acid-activated chloride currents in tumor cells? What is the nature of the channel protein that mediates this current? The effects of extracellular acidification on chloride currents in poorly differentiated nasopharyngeal carcinoma (NPC) CNE-2Z cells were observed, the characteristics of acid-activated chloride currents were analyzed, and the molecular nature of channel proteins mediating acid-activated chloride currents in tumor cells was investigated. Methods: the chloride currents of nasopharyngeal carcinoma (NPC) CNE-2Z cells were recorded by whole-cell patch clamp technique, and the pH value of extracellular perfusate was changed to observe its effect on the chloride channel activity of CNE-2Z cells. The physiological characteristics of acid-activated chloride current were analyzed by using ion replacement technique and changing the osmotic pressure of extracellular perfusion fluid, and the pharmacological characteristics of the current were analyzed by using a variety of chloride channel blockers. RT-PCR and Western blot techniques were used to detect the expression of chloride channels in different subtypes of ClC family in CNE-2Z cells, and siRNA technique was used to interfere with the expression of chloride channels of different subtypes, and to analyze and explore the molecular nature of channel proteins mediating the acid-activated chloride currents in CNE-2Z cells. Results:. 1. Under normal pH (extracellular 7.4) and osmotic pressure, a weak and stable background chlorine current could be recorded in CNE-2Z cells. When the pH value of perfusion fluid was reduced from 7.4 to 6.6, chloride channels could be activated rapidly. A chloride current with a weak outward rectifier is produced, which can be inhibited by various chloride channel blockers, such as NPPB(5-nitro-2-3-phenylpropylamino benzoic acidine, tamoxifen, DIDS 4 isothiocyanocyanatostilbe-ne-2n2C 2- disulfonic acid disodium salt hydrate, and its selectivity for various anions is IBr-Cl-gluconate-. (2) when the pH value of perfusion fluid was reduced directly from 7.4 to 5.8, the acid-activated chloride current could not be induced, and the chloride current activated by acid-activated chloride could be inhibited by further lowering the pH value of perfusion fluid from 6.6 to 5.8. The subtypes of ClC-3ClC-5 and ClC-7 were expressed in CNE-2Z cells, but the expression of ClC-5 was very small. (4) the acid-activated chloride current induced by pH 6.6 was significantly inhibited by interfering with the expression of ClC-3 by siRNA technique, but interfering with the expression of ClC-7 did not affect the activation of the current. Conclusion there is an acid-activated chloride channel in CNE-2Z cells. The chloride current mediated by this channel is volume-sensitive and has many similar characteristics to that of volume-sensitive chloride current. The chloride channel of ClC-3 mediates or regulates the acid-activated chloride current in CNE-2Z cells.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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