本文關(guān)鍵詞： 鼻咽癌 pH值 酸激活性氯電流 氯通道 胞外酸化　出處：《暨南大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：酸激活性氯電流存在于多種正常的細(xì)胞中，并參與細(xì)胞生理功能的調(diào)控。腫瘤細(xì)胞外pH值低于正常細(xì)胞，但其胞外酸化對(duì)其自身氯通道有何影響？腫瘤細(xì)胞酸激活性氯電流有何特性？介導(dǎo)此電流的通道蛋白分子本質(zhì)是什么？目前仍未明了。本研究主要觀察細(xì)胞外酸化對(duì)低分化鼻咽癌CNE-2Z細(xì)胞氯電流的影響，分析酸激活性氯電流的多種特性、探討介導(dǎo)腫瘤細(xì)胞酸激活性氯電流的通道蛋白分子本質(zhì)。 方法：用全細(xì)胞膜片鉗技術(shù)記錄鼻咽癌CNE-2Z細(xì)胞的氯電流；改變細(xì)胞外灌流液的pH值，觀察其對(duì)CNE-2Z細(xì)胞氯通道活動(dòng)的影響；利用離子置換技術(shù)和改變細(xì)胞外灌流液的滲透壓來(lái)分析酸激活性氯電流的生理學(xué)特性；應(yīng)用多種氯通道阻斷劑來(lái)分析該電流的藥理學(xué)特性；用RT-PCR技術(shù)和Western blot技術(shù)檢測(cè)氯通道（ClC家族的不同亞型）在CNE-2Z細(xì)胞的表達(dá)；用siRNA技術(shù)干擾不同亞型的氯通道的表達(dá)，分析和探討介導(dǎo)CNE-2Z細(xì)胞酸激活性氯電流的通道蛋白分子本質(zhì)。 結(jié)果： 1、在正常的pH值（胞外7.4）和滲透壓條件下，可在CNE-2Z細(xì)胞記錄到一個(gè)微弱而穩(wěn)定的背景氯電流，當(dāng)將灌流液的pH值由7.4降到6.6時(shí)，可迅速激活氯通道，產(chǎn)生一個(gè)具有較弱外向整流特性的氯電流，該電流可被高滲灌流液誘導(dǎo)的細(xì)胞縮小和NPPB(5-nitro-2-3-phenylpropylamino benzoic acid)、 tamoxifen、 DIDS (4,4'-diisothiocyanatostilbe-ne-2,2'-disulfonic acid disodium salt hydrate)等多種氯通道阻斷劑抑制，其對(duì)多種陰離子的選擇性為I- Br- Cl- gluconate-； 2、將灌流液的pH值由7.4直接降到5.8，不能誘導(dǎo)出酸激活性氯電流；將灌流液的pH值由6.6進(jìn)一步降低至5.8可抑制酸（pH6.6）激活的氯電流； 3、ClC氯通道家族中，在CNE-2Z細(xì)胞上表達(dá)的亞型有ClC-3、ClC-5和ClC-7，但ClC-5的表達(dá)量很少； 4、采用siRNA技術(shù)干擾ClC-3的表達(dá)后，pH6.6誘導(dǎo)的酸激活性氯電流被明顯抑制，，但干擾ClC-7的表達(dá)，則不影響該電流的激活。 結(jié)論：CNE-2Z細(xì)胞存在酸激活性氯通道的表達(dá)，該通道介導(dǎo)的氯電流有容積敏感性，并與容積敏感性氯電流有許多相似的特征。ClC-3氯通道介導(dǎo)或調(diào)節(jié)CNE-2Z細(xì)胞酸激活性氯電流。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R739.63

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