本文關(guān)鍵詞：細(xì)胞內(nèi)鈣濃度變化對(duì)心房肌小電導(dǎo)鈣激活鉀通道電流的影響　出處：《瀘州醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小電導(dǎo)鈣激活鉀電流 胞內(nèi)鈣 L-型鈣通道 心房顫動(dòng) 人心房肌細(xì)胞 穿孔膜片鉗技術(shù)

【摘要】：目的：心房纖顫(Atrial Fibrillation，AF)是最常見(jiàn)的快速性心律失常，隨著年齡的增加其發(fā)病率增加,小于60歲的人群發(fā)病率為0.5％，大于80歲的人群發(fā)病率增加到10％。房顫可使死亡率增加1.5～1.8倍。AF患者可以產(chǎn)生明顯的癥狀，例如喪失活動(dòng)能力或使活動(dòng)能力下降，且發(fā)生腦卒中的發(fā)病率增加到15％，心力衰竭的危險(xiǎn)性明顯增加。然而，目前房顫發(fā)生的機(jī)制仍未完全闡明。AF的發(fā)生與動(dòng)作電位（Action potential，AP）的有效不應(yīng)期（effective refractory period，ERP）縮短密切相關(guān)，而動(dòng)作電位時(shí)程(action potential duration,APD)則由心肌細(xì)胞不同離子通道活動(dòng)決定。因此，離子通道功能的變化可能與AF的發(fā)生及維持有關(guān)。小電導(dǎo)鈣激活鉀通道（small conductance Ca~(2+)-activated K~+channels, SK通道）是鈣激活鉀通道（Ca~(2+)-activated K~+channels, KCa）中的一種，具有電壓不敏感、鈣離子敏感的特性。近幾年研究發(fā)現(xiàn)SK通道存在于心肌細(xì)胞上，且分子生物學(xué)證據(jù)表明在SK通道的4個(gè)亞型(SK1-4)中，SK_2通道在人和鼠的心房和心室表達(dá)存在差異，主要表達(dá)在心房。SK2通道為KCa通道，對(duì)胞內(nèi)游離鈣離子（[Ca~(2+)]i）高度敏感，發(fā)生反應(yīng)迅速，可快速將細(xì)胞內(nèi)鈣離子濃度的變化轉(zhuǎn)換成細(xì)胞膜電位變化。因此我們推測(cè)AF時(shí)胞內(nèi)鈣超載可能影響SK2通道功能，深入討論這些問(wèn)題對(duì)揭示AF發(fā)生的病理生理機(jī)制具有重要意義。本實(shí)驗(yàn)應(yīng)用穿孔膜片鉗技術(shù)（perforated Patch clamp，PPR）記錄全細(xì)胞電流，觀察竇性心律患者（Sinus Rhythm，SR）與AF心律患者心房肌細(xì)胞SK2通道電流變化及其對(duì)Ca~(2+)的敏感性的差異，以及觀察L-鈣通道的激動(dòng)劑Bay-K8644、抑制劑維拉帕米（verapamil）對(duì)SK2通道電流的影響，探討AF發(fā)生發(fā)展過(guò)程中可能出現(xiàn)的離子通道之間的相互作用，為AF機(jī)制的闡明提供更多的實(shí)驗(yàn)依據(jù)。方法：42例接受體外循環(huán)手術(shù)的患者分為兩組：心房顫動(dòng)組(AF)11例，竇性心律組（SR）31例。心肌細(xì)胞的獲得：取體外循環(huán)術(shù)中切除的右心耳，在氧飽和Cardiplegic液中將組織剪成約1mm3的小塊，EGTA脫鈣，再進(jìn)行兩步酶消化：酶I(XXIV蛋白酶2-3U/ml，V膠原酶150U/ml，BSA1mg/ml)，酶II (V膠原酶150U/ml，BSA l mg/ml)。選取貼壁良好，折光性強(qiáng)，具有較強(qiáng)立體感，表面光滑的心肌細(xì)胞進(jìn)行實(shí)驗(yàn)。SK2通道電流的記錄：取細(xì)胞置于浴液中，用穿孔膜片鉗技術(shù)記錄全細(xì)胞電流，所得電流通過(guò)膜片鉗放大器（CEZ-2300, Nihonkonden, Japan)放大，濾波（1KHz）后輸入計(jì)算機(jī)，經(jīng)Clampex10.0軟件采集電流，Clampfit10.1、OriginPro8.0軟件進(jìn)行數(shù)據(jù)分析作圖。實(shí)驗(yàn)分組：（1）以人心房肌細(xì)胞為研究對(duì)象，用二性霉素B和/或β-escin作為穿孔電極液，進(jìn)行穿孔膜片鉗實(shí)驗(yàn)，并用胞內(nèi)鈣成像系統(tǒng)驗(yàn)證穿孔前后胞內(nèi)鈣變化，以建立鈣離子可以進(jìn)入細(xì)胞內(nèi)的穿孔膜片鉗技術(shù)。（2）在全細(xì)胞穿孔膜片鉗模式下觀察SR組與AF組SK2通道電流的差異。（3）不同的胞內(nèi)鈣離子濃度對(duì)AF組與SR組SK2通道電流的影響。（4）全細(xì)胞穿孔膜片鉗模式下觀察L-型鈣通道激動(dòng)劑Bay-K8644、抑制劑（verapamil）對(duì)SK通道電流的影響。結(jié)果：（1）混合使用穿孔電極液6.88μg/ml β-escin和150μg/ml二性霉素B能形成穩(wěn)定的穿孔膜片鉗記錄模式，且胞內(nèi)鈣測(cè)試系統(tǒng)檢測(cè)可觀察到穿孔后電極液至細(xì)胞內(nèi)的游離鈣離子濃度增加，F(xiàn)340/F380增強(qiáng)。（2）人心房肌細(xì)胞SK_2通道特性：①在全細(xì)胞穿孔膜片鉗模式下，施以SK_2通道電流的刺激方案，可記錄到一個(gè)內(nèi)向整流混合電流，在浴液中加入SK_2通道的特異性阻斷劑apamin，加藥前后的內(nèi)向整流綜合電流相減即可獲得apamin敏感的電流（SK2通道電流）。本實(shí)驗(yàn)記錄到的人心房肌細(xì)胞SK2通道電流具有電壓不敏感、內(nèi)向整流的特性。②在全細(xì)胞穿孔膜片鉗模式下，100nM apamin可以阻斷部分內(nèi)向整流混合電流，使內(nèi)向整流混合電流減小，用浴液灌流將apamin洗脫后，被阻斷的電流可以恢復(fù)。⑵SR組與AF組SK2通道電流的差異：在全細(xì)胞穿孔膜片鉗模式下，電極液中鈣離子濃度為5×10－7mol/L時(shí)，記錄到的AF組的SK2通道電流明顯大于SR組，尤其是在超極化方向。膜電位在-130mV時(shí)，SR組與AF組的SK2通道電流密度分別為：-2.92±0.35pA/pF、-6.83±0.19pA/pF（nSR=6，nAF=3，p＜0.05）。⑶不同濃度的胞內(nèi)游離鈣離子對(duì)SK2通道電流的影響：在電極液游離鈣離子濃度為0mol/L、5×10~(－7)mol/L、10~(－6)mol/L膜電位為-130mV時(shí)， SR組SK2通道電流密度分別為-1.43±0.33pA/pF、-2.92±0.35pA/pF、-10.11±2.15pA/pF (n0=7，n_(5×10－7)=6，，n10－6=8，p＜0.05）；AF組SK_2通道電流密度分別為-2.17±0.40pA/pF、-6.83±0.19pA/pF、-14.47±2.89pA/pF (n0=4，n_(5×10－7)=3，n_(10－6)=4,p＜0.05）。（4）L-型鈣通道與SK2通道電流的相關(guān)性研究：①L-型鈣通道激動(dòng)劑Bay-K8644對(duì)SK2通道電流的影響：在全細(xì)胞穿孔膜片鉗模式下，在電極液游離鈣離子濃度為5×10－7mol/L，膜電位為-130mV時(shí)，Bay-K8644能增大內(nèi)向整流混合電流。②L-型鈣通道抑制劑verapamil對(duì)SK2通道電流的影響：在全細(xì)胞穿孔膜片鉗模式下，在電極液游離鈣離子濃度為5×10~(－7)mol/L，膜電位為-130mV時(shí)，在浴液中加入verapamil的基礎(chǔ)上，apamin對(duì)內(nèi)向整流混合電流無(wú)抑制作用。結(jié)論：⑴適當(dāng)濃度的二性霉素B與β-escin混合使用進(jìn)行穿孔膜片鉗實(shí)驗(yàn)可形成穩(wěn)定的全細(xì)胞記錄模式，本技術(shù)方法即可保證胞內(nèi)環(huán)境相對(duì)穩(wěn)定，又可使電極液內(nèi)的鈣離子進(jìn)入細(xì)胞內(nèi)，從而改變細(xì)胞內(nèi)的鈣離子濃度，進(jìn)而研究不同[Ca2+]i濃度對(duì)SK_2通道電流的調(diào)控。⑵電極液內(nèi)游離鈣離子在各濃度下(0mol/L、5×10~(－7)mol/L、10~(－6)mol/L)，AF組的SK2通道電流密度均大于SR組。⑶在AF狀態(tài)下，SK2通道電流對(duì)Ca~(2+)的敏感性增強(qiáng)，與SR組比較，差異顯著，提示SK_2通道電流可能參與了AF的發(fā)生發(fā)展。⑷L-型鈣通道的激動(dòng)劑Bay-K8644使SR組內(nèi)向整流混合電流增大的趨勢(shì)，在浴液中加入L-型鈣通道的抑制劑verapamil時(shí)，apamin對(duì)內(nèi)向整流混合電流無(wú)抑制作用，提示L-型鈣通道與SK2通道之間存在著相關(guān)性。
[Abstract]:Objective: atrial fibrillation (Atrial Fibrillation AF) is the most common arrhythmia, with the increase of age, the incidence rate of incidence increased, less than 60 years old to 0.5% years old, more than 80 people to the increased incidence of atrial fibrillation in 10%. mortality could increase 1.5 ~ 1.8 times of.AF were significant the symptoms, such as loss of activity or the activity decreased, and the stroke incidence rate increased to 15%, the risk of heart failure increased significantly. However, the mechanism has not yet been completely elucidated AF occurrence and action potential of.AF (Action potential AP) the effective refractory period (effective refractory period. ERP) are closely related, and the action potential duration (action potential, duration, APD) is determined by the activity of myocardial cells of different ion channels. Therefore, changes in the function of ion channels may be related to the occurrence and maintenance of a AF . the small conductance calcium activated potassium channel (small conductance Ca~ -activated (2+) K~+channels, SK channel) is a calcium activated potassium channel (Ca~ (2+) -activated K~+channels, KCa) in one, with a voltage insensitive, calcium sensitive characteristics. In recent years, studies have found that SK channels are present in the myocardial cells, and the molecular biology evidence in 4 subtypes of SK channels (SK1-4), SK_2 channels are differentially expressed in human and rat atria and ventricles, mainly expressed in atrial.SK2 channels as KCa channels on intracellular free calcium ([Ca~ (2+)]i) highly sensitive reaction can rapidly. The rapid changes of the intracellular calcium concentration changes into cell membrane potential. So we speculate that AF intracellular calcium overload may affect the function of SK2 channels, in-depth discussion of these issues is of great significance to reveal the pathophysiological mechanisms of AF. The experimental application of perforated patch Clamp technique (perforated Patch clamp, PPR) to record the whole cell current, observation of patients with sinus rhythm (Sinus Rhythm, SR) and the changes of the SK2 channel AF in patients with atrial cardiac muscle cells and the current of Ca~ (2+) the difference of sensitivity, and the observation of L- calcium channel agonist Bay-K8644 inhibitor (verapamil) influence on Vera Pammy the SK2 channel current, the interaction between ion channels may appear on the occurrence and development of AF process, and provide more experimental basis for clarifying the mechanism of AF. Methods: 42 patients undergoing cardiopulmonary bypass surgery were divided into two groups: atrial fibrillation group (AF) in 11 cases, sinus rhythm group (SR) 31 cases. Cardiomyocytes were obtained: take the right atrial appendage resection during cardiopulmonary bypass, and cut into 1mm3 in Cardiplegic liquid oxygen saturation will be small, EGTA decalcification, and then a two step enzyme digestion enzyme I (XXIV protease 2-3U/ml, V collagenase 150U/ml, BSA1mg/m L II (V) enzyme, collagenase 150U/ml, BSA L mg/ml). Selected adherent, high refractive index, with a strong sense of three-dimensional, smooth surface of the myocardial cell experiments of.SK2 channel current records: cells were placed in a bath with the perforated patch clamp technique to record whole cell current, the current through the patch clamp amplifier (CEZ-2300 Nihonkonden, Japan), amplification, filtering (1KHz) after input to the computer by Clampex10.0 software to collect current, Clampfit10.1, OriginPro8.0 software to analyze the data mapping. Experimental groups: (1) in human atrial muscle cells as the research object, using amphotercin B and / or -escin beta as perforating electrode solution, perforated patch clamp experiment and intracellular calcium imaging system to verify the changes of intracellular calcium before and after perforation, in order to establish the calcium ions can enter the perforated patch clamp technique in cells. (2) in the whole cell perforated patch clamp mode was observed in the SR group and AF group SK2 The differences in channel current. (3) effects of different intracellular calcium ion concentration of AF group and SR group of SK2 channel current. (4) observed by patch clamp mode of L- type calcium channel agonist Bay-K8644 perforated whole cell inhibitor (verapamil) effect on SK channel current. Results: (1) mixed with perforation the electrode solution 6.88 g/ml beta -escin and 150 g/ml of amphotericin B can form a perforated patch recording mode is stable, and the intracellular calcium detection test system can be observed, the concentration of free calcium ion electrode solution to intracellular perforation after enhanced F340/F380. (2) human atrial muscle cell SK_2 channel characteristics: 1. In the whole cell perforated patch clamp mode, with the stimulus of SK_2 currents, recorded an inwardly rectifying mixed current SK_2 channel in the bath in a specific inhibitor of apamin, the inward rectifier current subtraction before and after comprehensive dosing can be obtained apamin sensitivity The current (SK2 current). Human atrial myocytes SK2 channel currents recorded in this experiment is insensitive to voltage characteristics of inward rectifier. The whole cell perforated patch clamp mode, 100nM apamin could block the inward rectifier part of mixed current, the inward rectifier hybrid current decreases, the apamin eluted by bath perfusion after current interruption can be restored. The difference between SR group and AF group: SK2 currents in whole cell perforated patch clamp mode, the electrode liquid calcium ion concentration of 5 * 10 7mol/L, SK2 channel current AF Group recorded significantly more than group SR, especially in the hyperpolarizing direction. The membrane potential at -130mV, SK2 channel current density of SR group and AF group respectively: -2.92 + 0.35pA/pF, -6.83 + 0.19pA/pF (nSR=6, nAF=3, P < 0.05). The different concentration of intracellular free calcium ion effect on SK2 channel current in the electrode liquid free calcium 紱誨瓙嫻撳害涓

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