中文摘要
本研究乃探討心血管用藥carvedilol對兔角膜上皮細胞內游離鈣離子濃度[Ca2+]i及其對細胞存活率的影響作用。鈣離子濃度和細胞存活率的測定分別是採用螢光染料fura-2和WST-1。研究顯示carvedilol濃度在1~30μM的範圍內時，能增加細胞內鈣離子濃度，且會隨carvedilol濃度增加而上升，故具有濃度依存關係。去除細胞外的鈣離子時，會使carvedilol 產生鈣離子增加的訊號下降。carvedilol可以誘發鈣離子湧入到細胞內，當錳離子與鈣離子經由同一離子通到湧入到細胞內時，錳離子則會降解 fura-2螢光，使fura-2螢光下降。當蛋白激酶C的活性受到抑制時，鈣離子在細胞內的運輸也會受到抑制。在去除細胞外鈣離子的實驗中，先以1μM thapsigargin（一種內質網鈣離子幫浦抑制劑） 進行前處理，會使carvedilol誘發的鈣離子濃度上升現象減少；而且相反地，carvedilol抑制了 thapsigargin誘導鈣離子濃度上升的作用。實驗中以2 μM U73122來抑制磷脂酶C，發現並未改變carvedilol誘導鈣離子濃度的上升。而在5至70 μM 這個濃度範圍內，carvedilol會依濃度增加而具有毒殺細胞的作用。濃度20 μM carvedilol的細胞毒性，並不會因為細胞內鈣離子與BAPTA /AM所產生的螯合作用而改變。實驗顯示5至70 μM carvedilol 可導致SIRC角膜上皮細胞的凋亡。正確來說， carvedilol誘導Ca2+離子濃度升高，主要是由內質網鈣離子釋放，而非單獨由磷脂酶C，或鈣離子的湧入，透過蛋白激酶C的調節等方式，使鈣離子進入細胞內。Carvedilol所引起之細胞毒性作用，與其誘導鈣離子濃度的增加，可能是因為carvedilol本身毒性所造成。

英文摘要
The effect of the cardiovascular drug carvedilol on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was examined in SIRC corneal epithelial cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations between 1-30 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Carvedilol induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was inhibited by suppression of protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), carvedilol-induced [Ca2+]i rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca2+]i rise. At concentrations between 5 and 70 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 μM carvedilol was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-70 μM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via protein kinase C-regulated Ca2+ channels. Carvedilol-caused cytotoxicity was mediated by Ca2+-independent apoptosis in a concentration-dependent manner.

中文摘要 I
英文摘要 II
目 錄 IV
圖目錄 VII
第一章 緒論 8
第一節 細胞週期 8
第二節 細胞週期調控相關因子 11
(一)、Cyclins與Cdks 11
(二)、Cyclin-dependent kinases inhibitors(CdkIs) 12
第三節 細胞凋亡 13
(一)、細胞凋亡的型態與特徵 13
(二)、細胞凋亡的變化 14
第四節 細胞凋亡訊息傳導路徑 15
(一)、細胞凋亡外在路徑(Extrinsic Pathway) 15
(二)、細胞凋亡之內在路徑space (Intrinsic pathway) 16
(三)、鈣離子二級傳遞路徑 17
(四)、細胞凋亡的基因調節 20
第五節 β-阻斷劑 (β-blocker) 23
第六節 鬱血性心衰竭 30
第七節 Carvedilol 33
(一)、藥效學以及藥物動力學特性 33
(二)、Carvedilol的副作用 34
第二章 研究目的 35
第三章 材料與實驗方法 36
第一節 實驗材料 36
(一)、試藥 36
(二)、實驗用溶液 36
(三)、實驗儀器 37
(四)、細胞培養 37
第二節 實驗方法 38
(一)、鈣離子測定 38
(二)、細胞活性測試 39
(三)、流式細胞儀測量細胞凋亡 40
(四)、統計與分析 40
第四章 結果 41
(一)、Carvedilol對鈣離子的影響 41
(二)、Carvedilol對Mn2+ 細胞內轉移的作用 44
(三)、蛋白激酶C抑制carvedilol誘導鈣離子的上升 45
(四)、鈣離儲存庫和Carvedilol誘導的鈣離子上升 47
(五)、磷脂酶C不參與在Carvedilol誘導鈣離子的上升 49
(六)、Carvedilol對細胞存活率的影響 51
(七)、Carvedilol誘導鈣離子的升高和細胞死亡缺乏必然關係 52
(八)、Carvedilol誘導的細胞死亡的機制 54
第五章 討論 55
第六章 參考文獻 57

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