Thimerosal (硫柳汞)是一種含汞藥劑常見於疫苗製劑中作為防腐劑。它會藉由不同的受體及離子通道於不同的細胞內造成鈣離子經細胞膜移動。
細胞內游離鈣離子濃度的上升於許多病生理過程中扮演重要訊息如凋亡及壞死。 Thimerosal會造成不同種類細胞內游離鈣離子濃度的上升且藉由不同的基轉。
本實驗在於探討thimerosal對人類攝護腺癌細胞株(PC3)的細胞內游離鈣離子濃度的效應。WST-1螢光法測定不同藥劑濃度條件下的細胞存活率及以 propidium iodide染色法測定其凋亡的情況。本研究之結果有助於了解thimerosal對身體重要器官細胞的藥理毒理作用。
研究表明thimerosalu以濃度依賴方式來增加細胞內游離鈣離子濃度。若將細胞外鈣離子移除會部分降低鈣離子訊息。Thimerosal造成鈣離子內流會被下列藥劑抑制含econazole、SKF963656及protein kinase C modulators (含PMA及GF109203X)。於無鈣離子培養液中，若以BHQ(一種endoplasmic reticulum Ca2+ pump inhibitor)前處理則thimerosal誘發細胞內游離鈣離子濃度的上升會部分受抑制。Thimerosal以濃度依賴方式造成細胞死亡且不受鈣離子螯合劑BAPTA影響。Propidium iodide染色顯示凋亡是細胞死亡的原因。
總結，.thimerosal造成PC3細胞細胞內游離鈣離子濃度上升是由於鈣離子自內質網釋出及鈣離子內流以protein kinase C及phospholipase A2來調控store-operated Ca2+ channels。Thimerosal 造成細胞死亡是以與鈣離子無關的凋亡方式。

Thimerosal is a mercury-containing component found in many vaccine preparations and used as a preservative. It causes Ca2+ movement across cell membrane in different cells that may be mediated via effects on different receptors and ion channels.
A rise in intracellular free Ca2+ concentrations ([Ca2+]i) is a key signal for many pathophysiological processes in cells, including apoptosis and necrosis. Thimerosal increases [Ca2+]i in different cell types. The mechanisms underlying thimerosal-induced Ca2+ signal vary with cell types.
The present study evaluated the effects of thimerosal on [Ca2+]i in human prostate cancer cells (PC3). WST-1 reduction assays and propidium iodide-staining assays were used to determine cell viability and apoptosis in the presence of thimerosal. The experimental results may be helpful to understand the pahrmocological and toxicological effects of thimerosal on cells from important organs.
Results showed that thimerosal (10–200μM) increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal-induced Ca2+ influx was inhibited by econazole, SKF963656, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, a 200-mM thimerosal-induced [Ca2+]i rise was partly inhibited after pretreatment with 2,5-di-tert-butylhydroquinone (BHQ) (an endoplasmic reticulum Ca2+ pump inhibitor). Thimerosal at 1–7μM induced cell death in a concentration-dependent manner that was not reversed when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Propidium iodide staining suggests that apoptosis played a role in the death.
Collectively, in PC3 cells, thimerosal induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Thimerosal also induced cell death in a Ca2+-independent apoptotic manner.

摘要 3
ABSTRACT 5
LIST OF ABBREVIATIONS 7
CHAPTER 1 INTRODUCTION 11
1.INTRODUCTION 11
1.1. The characteristics of thimerosal 11
1.2. The calcium signaling with dynamics, homeostasis and cell death 14
1.3. Forms of cell death: apoptosis, necrosis and autophage 16
1.4. The role of Ca2+ signaling in apoptosis 19
1.5. Thimerosal increases [Ca2+]i in different channels, and cell types and induces cytotoxicity 21
1.6. Ca2+ homeostasis of advanced prostate cancer cells 22
1.7. Various ligands increase [Ca2+]i in PC3 cell line 23
1.8. Pharmacological analysis of intracellular Ca2+ signaling 23
1.9. Cell viability assays 24
CHAPTER 2 AIM AND STUDY STRATEGY 36
CHAPTER 3 MATERIALS AND METHODS 38
3.1. Materials 38
3.2. Cell culture 38
3.3. Solutions 38
3.4. [Ca2+]i measurements 38
3.5. Cell viability assays 39
3.6. Flow cytometry 40
3.7. Statistics 40
CHAPTER 4 EFFECT OF THIMEROSAL ON Ca2+ MOVEMENT AND APOPPTOSIS IN PC3 PROSTATE CANCER CELLS. 42
4.1. INTRODUCTION 42
4.2. RESULTS 43
4.2.1. Effect of thimerosal on [Ca2+]i in fura-2-loaded PC3 cells 43
4.2.2. Effect of thimerosal on Ca2+ influx by measuring Mn2+ quench of fura-2 fluorescence 44
4.2.3. Effect of Ca2+ channel blockers and protein kinase C modulators on thimerosal-induced [Ca2+]i rise 44
4.2.4. Intracellular Ca2+ stores of thimerosal-induced Ca2+ release 45
4.2.5. Cytotoxic effect of thimerosal on PC3 cells 45
4.2.6. Effect of thimerosal on apoptosis of PC3 cells 45
4.3. DISCUSSION 54
CHAPTER 5 GENERAL CONCLUSION 58
LIST OF FIGURES
Figure 1.1. The structural formulas of thimerosal and of thiosalicylic acid. 26
Figure 1.2. The effect of thimerosal and thiosalicylic acid on the level of intracellular calcium in the neutrophil. 27
Figure 1.3. Calcium-signaling dynamics and homeostasis. 28
Figure 1.4. Calcium-mobilizing messengers and modulators. 30
Figure 1.5. Signalling pathway that lead to apoptosis in mammalian cells. 32
Figure 1.6. Crosstalk between calpains and caspases during apoptosis. 33
Figure 1.7. Scheme summarizing the changes in Ca2+ homeostasis in epithelial cells of late prostate cancer stages. 34
Figure 1.8. The structure formula of fura-2. 35
Figure 1.9. Cleavage of the tetrazolium salt WST-1 to formazan. 35
Figure 4.1. Effect of thimerosal on [Ca2+]i in fura-2-laoded PC3 cells. 47
Figure 4.2. Effect of thimerosal on Ca2+ influx by measuring Mn2+ quench of fura-2 fluorescence. 49
Figure 4.3. Effect of Ca2+ channel blockers and protein kinase C modulators on thimerosal-induce [Ca2+]i rise. 50
Figure 4.4. Intracellular Ca2+ stores of thimerosal-induced Ca2+ release. 51
Figure 4.5. Cytotoxic effect of thimerosal on PC3 cells. 52
Figure 4.6. Effect of thimerosal on apoptosis of PC3 cells. 53
REFERENCES 59
APPENDIX 1
Effect of thimerosal on Ca2+ movement and apoptosis in PC3 prostate cancer cells 76
APPENDIX 2
Effect of diallyl disulfide on Ca2+ movement and viability in PC3 human prostate cancer cells 83
APPENDIX 3
Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells. 91
APPENDIX 4
Maptotiline-induced Ca2+ fluxes and apoptosis in human osteosarcoma cells. 99
PUBLICATIONS 106

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