- 學位論文
細胞內pH值與大鼠心肌細胞凋亡機制關係之探討
The Relationship between Intracellular pH and Mechanism of Apoptosis in Rat Cardiomyocytes
摘要
細胞要維持正常的生理現象，需要仰賴大量的生化反應，而生化反應的進行，細胞內環境的pH值（pHi）扮演很重要的角色。而pHi會受到釵h內在及外在因素的影響，例如細胞本身對氫離子的緩衝能力、細胞外pH值（pHo）和在遭遇一些生理或病理刺激等。近年的研究發現，當細胞發生不可逆的病理過程，例如心肌細胞的缺血傷害後再灌流導致的細胞凋亡時，pHi在此過程扮演著重要的角色：心肌缺血時的酸化現象被認為對於心肌細胞具有保護作用，而此ㄧ保護作用將隨著再灌流細胞pHi回到正常值而失效導致細胞死亡，而其機制尚有爭議；除了上述之病理現象，受到其他藥物刺激所產生的酸化現象卻被認為也能造成細胞的死亡，而非保護細胞。細胞內的鹼化對於細胞的作用同樣尚未有一明確的定論，一般而言鹼化被認為是對細胞體有害的刺激，但是其促使細胞死亡的機制也尚不明朗。在本研究中，我們以較廣泛的角度，觀察初代培養的心肌細胞內的酸化或是鹼化對於細胞產生死亡機制的影響。我們發現若將pHi固定在酸化的狀態pHi=6.5，細胞凋亡（非細胞壞死）現象會有小幅度的提高，但是與pHi固定在鹼化的狀態（pHi=8.0, 8.5）相比，鹼化造成的細胞凋亡的現象則明顯得多。此外，我們也模擬心肌缺血再灌流的酸鹼變化，發現單純的細胞酸化而後回復至正常pHi即會使細胞發生凋亡反應，且引發凋亡的路徑與鈣離子內流無關─此結果也印證了心肌缺血後再灌流造成的細胞凋亡是來自pHi改變的說法。最後，，我們也利用數位式CCD攝影機，來觀察心肌細胞粒線體內pHm（藉由螢光蛋白質mito-EYFP的螢光變化）在上述處理中的變化。我們發現隨著我們調控pHi，pHm也會隨著pHi酸化或鹼化，這意味著pHi改變所引發的凋亡可能有部份是透過影響pHm而達成。
並列摘要
Intracellular pH ( pHi ) plays an important role in biochemincal reactions in order to maintain normal cellular functions . Intracellular pH can be varied by many factors, including intracellular buffering power, extracellular pH ( pHo ) and various physiology or pathological stimulation. Recently, it has been proven that pHi is a significant factor during pathogenesis of irreversible cell injury, such as apoptosis induced by reperfusion after ischemic treatment. Acidification during ischemia is thought to have a protective effect and this effect on ischemic cardiomyocytes is eliminated after the restoration of normal pHi by reperfusion. However, intracellular acidification stimulated by some chemicals is thought to be able to induce cell death, not. Similarly, there is no definite conclusion about the effect of cellular alkalinization. It is well known that alkaline conditions are cytotoxic, but the mechanisms leading to alkaline-induced cytotoxicity are still unclear. In the present study, we investigate the effect of acidification and alkalinization on primary culture of rat cardiomyocytes. When we clamped pHi at acidic level, there was a slight increase in ratio of apoptosis ( not necrosis ) . However, clamping pHi at alkaline level ( pHi=8.0, 8.5 ) caused much more severe apoptotic cell death. Furthermore, by simulating the pHi variation during ischemia-reperfusion conditions, we found that pHi recovery from acidification causes apoptosis and is calcium-independent. Our findings supports the hypothesis that apoptosis is induced by pHi recovery during ischemia-reperfusion injury. We also used digital CCD（charge-coupled device）camera imaging techniques to explore the variation of mitochondria matrix pH ( pHm ). We found that pHm varies in parallel to the change of pHi. Therefore, pHm could partly cause the apoptosis induced by pHi variation.