本文選題：ClC-3氯通道 + IK1鉀通道��； 參考：《暨南大學(xué)》2012年博士論文

【摘要】：目的：1）研究ClC-3氯通道和IK1鉀通道在人低分化鼻咽癌（CNE-2Z）細(xì)胞和高轉(zhuǎn)移人肝癌（MHCC97-H）細(xì)胞基礎(chǔ)容積穩(wěn)定和調(diào)節(jié)性容積減�。╮egulatory volume decrease，RVD）中的作用；2）探討RVD過程中Cl~-和K~+跨膜轉(zhuǎn)運(yùn)動(dòng)力學(xué)特征，以及H+跨膜轉(zhuǎn)運(yùn)及pH值改變對(duì)鉀、氯通道活動(dòng)和RVD的調(diào)節(jié)；3）研究IK1鉀通道在細(xì)胞遷移過程中的作用，探討IK1鉀通道在細(xì)胞容積調(diào)節(jié)和遷移過程中的空間和極性分布及活動(dòng)的改變。 方法：全細(xì)胞膜片鉗技術(shù)記錄CNE-2Z細(xì)胞和MHCC97-H細(xì)胞氯電流和鉀電流；分別將ClC-3siRNA和IK1siRNA轉(zhuǎn)染細(xì)胞，Western blotting檢測(cè)ClC-3氯通道和IK1鉀通道蛋白表達(dá)；Scion image圖像記錄分析軟件檢測(cè)細(xì)胞容積；非損傷微測(cè)技術(shù)記錄細(xì)胞K~+、Cl~-、H~+和Ca~2+跨膜流動(dòng)；H~+選擇性微電極檢測(cè)細(xì)胞外表面pH值，pH敏感性熒光探針BCECF檢測(cè)細(xì)胞內(nèi)pH值；構(gòu)建綠色熒光蛋白（enhanced green fluorescent protein，EGFP)與IK1鉀通道的融合蛋白表達(dá)質(zhì)粒，紅色熒光探針DiI標(biāo)記細(xì)胞膜，激光共聚焦顯微鏡觀察IK1鉀通道空間分布及動(dòng)態(tài)變化過程；趨化因子誘導(dǎo)細(xì)胞遷移；細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力。 結(jié)果：1）等滲條件下，，用全細(xì)胞膜片鉗技術(shù)可以在CNE-2Z細(xì)胞中分別記錄到穩(wěn)定的背景氯電流和背景鉀電流，細(xì)胞外47%高滲液可使細(xì)胞縮小，抑制背景氯電流和背景鉀電流；用siRNA技術(shù)下調(diào)ClC-3氯通道或IK1鉀通道表達(dá)，分別抑制背景氯電流和背景鉀電流，并使等滲液中的細(xì)胞容積增大；嘌呤受體阻斷劑RB2在正�；蚣�(xì)胞外無Ca~2+條件下均可使細(xì)胞容積增大；細(xì)胞外加入嘌呤受體阻斷劑RB2阻斷P2Y受體、用ATP酶分解細(xì)胞外ATP或用GdCl3抑制ATP經(jīng)通道的釋放均可顯著抑制背景氯電流，細(xì)胞外微摩爾濃度的ATP可以激活氯電流并且該電流可被47%高滲誘導(dǎo)的細(xì)胞縮小抑制，ATP可逆轉(zhuǎn)GdCl3對(duì)背景氯電流的抑制作用。 2）非損傷微測(cè)結(jié)果表明，在不接觸細(xì)胞、不干擾細(xì)胞活動(dòng)的條件下，低滲刺激下CNE-2Z細(xì)胞中K~+、Cl~-和H~+均為外流，但它們的跨膜轉(zhuǎn)運(yùn)動(dòng)力學(xué)特征不同，K~+外流激活較早且持續(xù)時(shí)間較短，Cl~-和H~+外流激活較晚且持續(xù)時(shí)間較長；低滲刺激引起細(xì)胞內(nèi)pH降低，激活H~+外流，使細(xì)胞外pH降低，質(zhì)子泵抑制劑奧美拉唑可抑制低滲引起的H+外流和細(xì)胞外pH降低；改變細(xì)胞外pH對(duì)低滲激活性鉀、氯電流的影響不同，適度降低細(xì)胞外pH值抑制低滲引起的K~+外流和RVD過程，而對(duì)低滲引起的Cl~-外流無抑制作用，抑制質(zhì)子泵或者增強(qiáng)細(xì)胞外pH緩沖能力可以促進(jìn)低滲引起的K~+外流和RVD過程；膜片鉗技術(shù)觀察到的低滲激活性鉀、氯電流時(shí)程相似，低滲激活的K~+、Cl~-外流可能分別由鉀通道和氯通道介導(dǎo)，低滲激活的K~+、Cl~-外流和電流可分別被鉀通道和氯通道阻斷劑抑制；IK1鉀通道和ClC-3氯通道可能分別是低滲激活的鉀通道和氯通道的主要成份，IK1siRNA和ClC-3siRNA分別抑制低滲引起的K~+外流和Cl~-外流。 3）MHCC97-H細(xì)胞中存在穩(wěn)定的背景鉀電流和較明顯的低滲激活性鉀電流，且均可被鉀通道阻斷劑克霉唑（CLT）抑制；用siRNA技術(shù)下調(diào)IK1鉀通道蛋白表達(dá)，可以抑制背景鉀電流和低滲激活性鉀電流，并且使等滲條件下細(xì)胞容積增大；EGFP-IK1鉀通道融合蛋白表達(dá)質(zhì)粒轉(zhuǎn)染后細(xì)胞可表達(dá)綠色熒光，背景鉀電流增大，且該電流可被鉀通道阻斷劑CLT抑制；等滲條件下EGFP-IK1鉀通道在細(xì)胞中可呈不均勻分布，且IK1鉀通道數(shù)量多的區(qū)域細(xì)胞局部容積減小較明顯；低滲刺激誘導(dǎo)胞漿中EGFP-IK1鉀通道呈囊泡樣聚集，并以胞吐方式轉(zhuǎn)移到細(xì)胞膜上，使細(xì)胞膜上EGFP-IK1鉀通道增多；鉀通道阻斷劑CLT和IK1siRNA均可抑制細(xì)胞遷移；K~+、H~+和Ca~2+跨膜轉(zhuǎn)運(yùn)在遷移細(xì)胞前、后極間不同，前極K~+內(nèi)流，后極K~+外流，前極H+外流大于后極，前極無明顯Ca~2+跨膜流動(dòng)，后極有明顯Ca~2+內(nèi)流；遷移細(xì)胞后極的K~+外流主要由通道活動(dòng)介導(dǎo)，前極的K~+內(nèi)流可能與通道無關(guān)，鉀通道阻斷劑CLT抑制后極的K~+外流，不影響前極的K~+內(nèi)流；IK1鉀通道在遷移細(xì)胞后極多于前極，且在誘導(dǎo)活細(xì)胞遷移過程中連續(xù)動(dòng)態(tài)觀察到IK1鉀通道在細(xì)胞后極分布逐漸增多，并在前極皺褶處聚集，IK1鉀通道在胞漿與胞膜之間的轉(zhuǎn)移現(xiàn)象在遷移活細(xì)胞前、后極也不同，在后極從胞漿向胞膜轉(zhuǎn)移，在前極從胞膜向胞漿轉(zhuǎn)移。 結(jié)論：ClC-3氯通道和IK1鉀通道在細(xì)胞基礎(chǔ)容積穩(wěn)定和RVD過程中發(fā)揮重要作用。等滲條件下細(xì)胞自分泌釋放的ATP通過嘌呤受體信號(hào)途徑激活ClC-3氯通道，參與細(xì)胞生理?xiàng)l件下的基礎(chǔ)容積調(diào)節(jié)。低滲刺激下，細(xì)胞RVD過程中H~+外流致細(xì)胞外pH降低，引起K~+、Cl~-外流的非同步。IK1鉀通道參與MHCC97-H細(xì)胞遷移過程，遷移細(xì)胞前、后極K~+跨膜轉(zhuǎn)運(yùn)不同，呈前極內(nèi)流、后極外流的極化狀態(tài)。IK1鉀通道通過在胞漿與細(xì)胞膜間的轉(zhuǎn)位以及在細(xì)胞膜上的運(yùn)動(dòng)，使整體或局部細(xì)胞膜上通道蛋白的數(shù)量發(fā)生改變，從而對(duì)細(xì)胞容積和遷移過程進(jìn)行調(diào)節(jié)。遷移細(xì)胞前后兩極H~+和Ca~2+跨膜流動(dòng)呈極化分布，可能使IK1鉀通道活動(dòng)在細(xì)胞前后極分布不同，從而影響細(xì)胞遷移過程。
[Abstract]:Objective: 1) to study the role of ClC-3 chloride channel and IK1 potassium channel in the volume stability and regulatory volume reduction (regulatory volume decrease, RVD) of human low differentiated nasopharyngeal carcinoma (CNE-2Z) cells and high metastatic human hepatoma (MHCC97-H) cells (regulatory volume decrease, RVD). (2) to explore the mechanical characteristics of Cl~- and K~+ trans membrane transmembrane movement in RVD process, and the transfer of H+ transmembrane and alteration value Change to potassium, chloride channel activity and regulation of RVD; 3) study the role of IK1 potassium channel during cell migration, and explore the spatial and polar distribution and activity changes of IK1 potassium channel during cell volume regulation and migration.
Methods: whole cell patch clamp technique was used to record the chlorine current and potassium current of CNE-2Z and MHCC97-H cells, ClC-3siRNA and IK1siRNA were transfected into cells, Western blotting was used to detect the expression of ClC-3 chloride channel and IK1 potassium channel protein, Scion image image recording analysis software was used to detect cell volume, and cell K~+ and Cl~-, H~+ and Ca~2+ were transmembrane flow; H~+ selective microelectrode was used to detect the pH value on the outer surface of the cell, and the pH sensitivity fluorescence probe BCECF was used to detect the intracellular pH value; the fusion protein expression plasmid of the green fluorescent protein (enhanced green fluorescent protein, EGFP) and the potassium channel was constructed, the red fluorescent probe labeled cell membrane, and the laser confocal microscope view The spatial distribution and dynamic process of IK1 potassium channel were observed; chemokines induced cell migration; cell scratch test was used to detect cell migration ability.
Results: 1) under the isosmotic condition, the stable background chlorine current and background potassium current can be recorded in CNE-2Z cells by whole cell patch clamp technique, and the 47% hypertonic liquid can reduce the cell, inhibit the background chlorine current and background potassium current, and reduce the expression of ClC-3 chloride channel or IK1 potassium channel with the technique of siRNA, and inhibit the background chlorine electricity respectively. The flow and background potassium current can increase the cell volume in the isosotic fluid, and the purinergic receptor blocker RB2 can increase the cell volume in normal or extracellular Ca~2+, and the addition of the purinergic receptor blocker RB2 to the P2Y receptor, the ATP enzyme decomposition of the extracellular ATP or the release of the ATP via the channel can be significantly inhibited by the ATP enzyme. ATP can activate the chlorine current and the current can be reduced by 47% hyperosmotic cells, and ATP can reverse the inhibitory effect of GdCl3 on the background chlorine current.
2) the results of noninvasive micromeasurement showed that K~+, Cl~- and H~+ were Exodus in CNE-2Z cells under hypotonic stimulation without contact cell and cell activity, but their transmembrane movement mechanics characteristics were different, K~+ Exodus activated early and duration was shorter, Cl~- and H~+ Exodus activated late and lasted longer; low permeability stimulation was caused by low permeability. PH decreased in cells, activated H~+ Exodus and reduced extracellular pH. Omeprazole, a proton pump inhibitor, inhibited H+ Exodus induced by hypotonic and extracellular pH; the effects of pH on hypotonic activation potassium, chlorine current were different, and the extracellular pH values were moderated to inhibit the K~+ Exodus and RVD process induced by low permeability, and Cl~- for hypotonic Cl~-. The external flow has no inhibitory effect. The inhibition of the proton pump or the enhancement of the pH buffer capacity can promote the K~+ Exodus and RVD processes caused by low permeability; the patch clamp technique has observed the hypotonic activation potassium, the chlorine current time history similar, the low permeability activated K~+, the Cl~- Exodus may be mediated by the potassium channel and the chloride channel, the low permeability activated K~+, the Cl~- Exodus and the electricity. The flow can be inhibited by potassium channel and chloride channel blocker respectively; IK1 potassium channel and ClC-3 chloride channel may be the main components of low permeability activated potassium channel and chlorine channel respectively. IK1siRNA and ClC-3siRNA inhibit the K~+ Exodus and Cl~- Exodus caused by hypotonic respectively.
3) there are stable background potassium current and obvious hypotonic activation potassium current in MHCC97-H cells, and can be inhibited by potassium channel blocker clotrimazole (CLT). Down regulation of IK1 potassium channel protein expression by siRNA technology can inhibit the background potassium current and hypotonic activated potassium current, and increase the cell volume under isosmotic conditions; EGFP-IK1 potassium. After transfection of the channel fusion protein expression plasmid, the cells could express green fluorescence, the background potassium current increased, and the current could be inhibited by potassium channel blocker CLT. Under isosmonic conditions, the EGFP-IK1 potassium channel could be distributed inhomogeneous in the cells, and the local volume of the cells in the region with more IK1 potassium channels decreased obviously, and the hypotonic stimulation induced the cytoplasm. The EGFP-IK1 potassium channel is vesicular aggregation and is transferred to the cell membrane by exocytosis, which increases the EGFP-IK1 potassium channel on the cell membrane, the potassium channel blocker CLT and IK1siRNA can inhibit cell migration, and K~+, H~+ and Ca~2+ transtransport before the migration of cells, the posterior polar K ~ + influx, the backward K~+ Exodus, and the exodus of the front pole H+ are larger than the posterior pole. There is no obvious Ca~2+ transmembrane flow in the front, and there is an obvious Ca~2+ inflow in the posterior pole; the K~+ exodus of the migratory cells is mainly mediated by channel activity, and the K~+ influx of the front pole may not be related to the channel. The potassium channel blocker CLT inhibits the K~+ exodus of the pole, and does not affect the K~+ inflow in the front pole; IK1 potassium channel is more than the anterior pole after the migration of the cells and is induced to live. In the process of cell migration, it was observed continuously that the IK1 potassium channel increased gradually in the post pole distribution, and gathered at the front polar fold. The transfer of IK1 potassium channel between the cytoplasm and the membrane was very different before the migration of the living cells, and transferred from the cytoplasm to the cytoplasm at the back pole and transferred from the membrane to the cytoplasm at the front pole.
Conclusion: ClC-3 chloride channel and IK1 potassium channel play an important role in cell base volume stability and RVD process. Under isosmotic conditions, the autocrine and released ATP activates the ClC-3 chloride channel through the purinergic receptor signaling pathway and participates in the basal volume regulation under the cell physiological conditions. Under the hypotonic stimulation, the H~+ Exodus induces the extracellular pH in the cell RVD process. The non synchronous.IK1 potassium channel of K~+, Cl~- exodus is involved in the migration process of MHCC97-H cells. Before the migration of the cells, the trans membrane transport of the K~+ transmembrane is different, and the transmembrane of the posterior polar exodus is in the forward flow. The polarization state of the posterior polar Exodus,.IK1 potassium channel, through the transposition between the cytoplasm and the cell membrane and the movement on the cell membrane, makes the whole or local cell membrane protein on the membrane. The number of the cell volume and the migration process are regulated. The polarization distribution of the H~+ and Ca~2+ transmembrane flow in the two poles of the migrated cells may lead to the different distribution of the IK1 potassium channel activity before and after the cell, thus affecting the cell migration process.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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