Thimerosal對人類口腔癌細胞株OC2細胞內鈣離子濃度的影響目前未知。本研究主要探討在含Fura-2螢光物的細胞培養液中，thimerosal是否能改變OC2鈣離子濃度。人類口腔癌細胞株OC2加入濃度1 –50 μM 之thimerosal後，其鈣離子的濃度的變化隨thimerosal濃度增加而上升。當移去細胞外培養液中的鈣離子後，鈣離子的訊號則部份下降。由thimerosal誘發細胞外之鈣離子內流，不受L-型鈣離子通道抑制劑及蛋白質激酶調控劑(PMA)及GF109203的影響。在不含鈣離子的細胞培養液中，對於先在細胞中加入thapsigargin(一種內質網上鈣離子幫浦抑制劑)，再加入50μM 的thimerosal則細胞內的鈣離子濃度並不會上升。用U73122來抑制磷脂酶C，再加入thimerosal後鈣離子濃度依然上升。Thimerosal在濃度5-10μM造成OC2細胞死亡的數量隨濃度上升而增加。濃度 8μM之thimerosal的細胞毒性則由於在細胞液中加入BAPTA/AM將鈣離子抓走而加強。細胞流式儀實驗發現1-7μM thimerosal引起細胞凋亡。總之，在人類口腔癌細胞株OC2中加入thimerosal後鈣離子濃度上升是藉由非依賴磷脂酶C之途徑由內質網釋放；同時鈣離子由細胞外內流則經由非L-型鈣離子通道。Thimerosal會引起藉由凋亡路徑之細胞死亡。

The effect of thimerosal on cytosolic free Ca2+ concentrations ([Ca2+]i) in human oral cancer cells (OC2) is unclear. This study explored whether thimerosal changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2. Thimerosal at concentrations between 1-50μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 50μM thimerosal failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with U73122 did not change thimerosal-induced [Ca2+]i rises. At concentrations between 5 and 10μM thimerosal killed cells in a concentration-dependent manner. The cytotoxic effect of 8μM thimerosal was potentiated by prechelating cytosolic Ca2+ with the Ca2+ chelator BAPTA/AM. Flow cytometry data suggested that 1-7μM thimerosal induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, thimerosal induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Thimerosal killed cells in a concentration-dependent manner via apoptosis.

誌謝 …………………………………………………………………4
中文摘要 ……………………………………………………………6
Abstract ……………………………………………………………...7
1. Introduction ………………………………………………………9
1.1. Thimerosal………………………………………………………9
1.2. Ca2+ as a pivotal intracellular signal in cells…………………..11
1.3. Apoptosis………………………………………………………..14
1.4. The OC2 human squamous cell line…………………………..15
2. Aims……………………………………………………………….17
3. Materials and methods…………………………………………...18
3.1. Cell culture ……………………………………………………..18
3.2. Solutions used in [Ca2+]i measurements…………………….18
3.3. [Ca2+]i measurements…………………………………………..18
3.4. Mn2+ quench of fura-2 fluorescence……………………………21
3.5. Cell viability assays …………………………………………….21
3.6. Apoptosis assays…………………………………………………22
3.7. Chemicals………………………………………………………..23
3.8. Statistics………………………………………………………….23
4. Results …………………………………………………………….25
4.1. Effect of thimerosal on [Ca2+]i …………………………………25
4.2. Effect of thimerosal on Mn2+ influx…………………………….26
4.3. Exploration of thimerosal-induced Ca2+ influx pathways…….26
4.4. Intracellular stores of thimerosal-induced [Ca2+]i rise………..27
4.5. Lack of a role of phospholipase C in thimerosal-induced [Ca2+]i rise…………………………………………………………………….27
4.6. Effect of thimerosal on cell viability……………………………28
4.7. Role of Ca2+ in thimerosal-induced cell death…………………28
4.8. The role of apoptosis in thimerosal-induced cell death……….29
5. Figure legends……………………………………………………..30
Figure 1: A. Effect of thimerosal on [Ca2+]i in fura-2-loaded OC2 cells…………………………………………………………………….31
Figure 2: Effect of thimerosal on Ca2+ influx by measuring Mn2+
quench of fura-2 fluorescence……………………………………….32
Figure 3: Lack of effect of L-type Ca2+ channel blockers and protein kinase C modulators on thimerosal-induced[Ca2+]i rise…………………………………………………………………….33

Figure 4: Intracellular Ca2+ stores of thimerosal-induced Ca2+ release…………………………………………………………………35
Figure 5: Intracellular Ca2+ stores of thimerosal-induced Ca2+ release…………………………………………………………………36
Figure 6: A tetrazolium assay of effect of thimerosal on viability of OC2cells………………………………………………………………38
Figure 7: Thimerosal-induced apoptosis……………………………40
6. Discussion ………………………………………………………….41
7. Conclusion………………………………………………………….44
8. References …………………………………………………………45

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