- 學位論文
以量子點感測及成像探討薑黃素誘導人類造骨細胞株-hFOB1.19之細胞凋亡與細胞壞死
Using Quantum Dots to Detect and Image in hFOB1.19 Cells by Curcumin nducing Apoptosis and Necrosis
摘要
摘 要 薑黃素是一種取自薑黃科植物被廣泛使用的黃色色素，已被證實對許多癌細胞具有抗發炎及抗癌的效果，但對於造骨細胞的影響仍不清楚，所以本研究以不同濃度之薑黃素刺激人類造骨細胞株-hFOB1.19。結果發現，薑黃素在低濃度時(12.5和25 μg/mL)會使造骨細胞內的活性氧(reactive oxygen species, ROS)上升，導致粒線體失去膜電位，並且活化凋亡的相關蛋白質JNK(c-Jun N-terminal kinases)、caspase 3、PARP(Poly(ADP-ribose) polymerase)和PAK2(p21Cdc42/Rac-activated kinase 2)；但在更高濃度薑黃素刺激下(50、100和200μg/mL)上述凋亡相關蛋白質未活化，卻有明顯的細胞死亡，同時發現隨著薑黃素濃度的升高，細胞內的ATP明顯下降，因此我們認為，薑黃素在低濃度時誘導造骨細胞進行細胞凋亡，但隨者劑量的增加由細胞凋亡轉為細胞壞死。 接著，利用新興的奈米材料-量子點可標定生物分子並且發出螢光的特性，將量子點接枝上可標定凋亡細胞的蛋白質Annexin V，使接枝後的量子點探針得以標定細胞凋亡時外翻的磷脂質，使用螢光顯微鏡觀察凋亡的細胞。實驗結果發現，接枝後的量子點可以清楚的分辨出細胞凋亡，並且配合上無法穿透細胞膜的有機染劑PI後，可區分出早期的細胞凋亡及壞死。將兩種表面改質的量子點與市售的螢光探針FITC-Annexin V相比，可發現量子點探針專一性較佳。並且由細胞存活率得知量子點在包附蛋白質後，即使在高能量的UV光照射下也不會造成細胞毒性，實為標的細胞凋亡時的有效工具。
並列摘要
Abstract Human osteoblast cell line (hFOB1.19) was treated with different concentration of curcumin, the yellow pigment of Curcuma longa that is known to have anti-oxidant and anti-inflammatory properties. Curcumin was found to cause cell death in a dose-dependent manner. At low concentrations, curcumin induced osteoblast apoptosis is through reactive oxygen species (ROS)formation, loss mitochondrial membrane potential and JNK(c-Jun N-terminal kinases), caspase 3, PARP(Poly(ADP-ribose) polymerase) and PAK2(p21Cdc42/Rac-activated kinase 2) activation. The cell death is increased at high dose, but the apoptotic protein markers is inactivation and decrease in cellular adenosine triphosphate (ATP).Results indicate that curcumin induces apoptosis at low concentrations (12.5 and 25 μg/mL) and switching apoptosis to necrosis at high concentrations (50, 100 and 200μg/mL) in osteoblast. Fluorescent quantum dots (QDs) which could track subcellular structures in living cells represents a powerful tool in cell biology. We have demonstrated that Annexin V-conjugated quantum dots could be a probe to detect apoptotic cells. Annexin V, which binds to phosphatidylserine (PS), is a lipid normally facing the cytoplasm which flips and faces the extracellular fluid early in apoptosis. We tested different dose of curcumin to induce osteoblast apoptosis and necrosis, and than detected and imaged apoptotic cells by quantum dots-Annexin V (QD-AV). The quantum dots-Annexin V and Propidium Iodide (QD-AV/PI) can clear to detect apoptosis and necrosis, and the sensitive of QD-AV is better than the FITC - AV. We also investigate the cytotoxicity of protein modified quantum dot would not have significant differents, even expose to UV light.