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  • 1. Induction of apoptosis by Meretrix lusoria through reactive oxygen species production, glutathione depletion, and caspase activation in human leukemia cells Origin : Life Sciences 79 (2006) 1140–1152 Authors : P, M-H , H, Y-T , H, C-J , C, C-I , H, P-C , P, B-S Impect Impact factor : 2.512 Speaker : Ching-Sheng Hung MT Date : 2007/11/09
  • 2. Introduction
  • 3. 學名 Meretrix lusoria 中文名 文蛤 中文俗名 普通文蛤、麗文蛤、粉蟯  英文俗名 Hard clam (HC)   型態特徵 貝殼呈亞三角形,黃褐色而有深褐色之斑紋,殼質堅 厚,殼面光滑,大者可長至 12 公分 棲所生態 生活在低潮區及稍深的淺海細砂的海底,棲息深度可達 20 公尺 地理分布 東亞一帶皆產,台灣盛產於西海岸泥沙底,且大量被人養殖 漁業利用 肉質鮮味美,殼可供藥用或做燒石灰之原料 Meretrix lusoria
  • 4. Morphology of apoptosis Single cell Shrinking Chromatin condensation Nuclear fragment protuberances Apoptotic bodies formation Phagocytosis
  • 5. ROS 氧分子能夠連續接受電子形成一系列中間產物,這些反應性較強的含氧分子稱為活化氧 (activated oxygen) ,有些活化氧亦是自由基
  • 6. Extrinsic ( Death Receptor-Initiated ) Pathway DNA fragmentation DNA repair ↓ Apoptosis FADD Pro-cas-8 cas-8 Apoptosis PARP DFF-45 CAD CAD Fas Fas L cas-3 Nucleus
  • 7. Anti-apoptotic proteins Bcl-2, Mcl-1 , Bcl-X L Bad, Bid , Bim, Bax Pro-apoptotic proteins 抗凋亡蛋白 存在於粒線體膜、內質網膜以及外核膜上, 但主要定位於粒線體外膜,它抵抗促凋亡蛋白的功能 促凋亡蛋白 則定位於細胞質,一旦受到凋亡因子的誘導 ,它們可以向粒線體靠近,在粒線體外膜形成跨膜通道 ,或者開啟粒線體的 PT 孔,而導致粒線體中的凋亡因子 釋放,活化 Caspase ,導致細胞凋亡 Bcl-2 family
  • 8. Intrinsic ( Mitochondrial ) Pathway Apoptosis Mitochondria Cytochrome c Mcl-1 ↓ Bax cleavage Bcl-x L ↓ cas-9 ROS↑ GSH↓ DFF-45 CAD cas-3 Nucleus Apoptosis Δ Ψm↓
  • 9. Mcl-1 ↓ Bax cleavage Bcl-x L ↓ caspase-3 Fas Fas L cas-8 tBid Bid Mitochondria cas-9 Apoptosis
  • 10. Purpose
  • 11. ? Apoptosis Hard Clam
  • 12. Meterials and Methods
  • 13. MTT assay terazolium formazan 570 nm 黃色 紫色 dehydrogenase hello
  • 14. Analysis of apoptosis by cell dying Acridine orange stain 利用 Acridine orange 染劑能與核酸親和的特性,其會嵌入 DNA 與 RNA 中,使用螢光顯微技術下可清楚觀測細胞經處理後整體型態與核內染色質的改變 Morphology hello
  • 15. look me Analysis of apoptosis by cell dying PI Stain 可以和細胞內的 DNA 結合,且結合量與 DNA 的量成正比,使用 Flow cytometer 來計算各細胞的 PI 含量,可以了解處於不同細胞週期的細胞數目多寡。 Flow cytometry
  • 16. Analysis of apoptosis by cell dying Annexin V-FITC stain Fluorescence ↑ Apoptosis cells ↑ ; Fluorescence ↓ Apoptosis cells ↓ Flow cytometry Annexin V-FITC PS
  • 17. Determination of ROS generation happy Flow cytometry ROS DCF Flow cytometry DCFH DCFH-DA Fluorescence ↑ ROS ↑ ; Fluorescence ↓ ROS ↓
  • 18. GSH CMF Flow cytometry CMF CMF-DA Determination of GSH depletion Cell esterase Flow cytometry Fluorescence ↑ GSH ↑ ; Fluorescence ↓ GSH ↓
  • 19. Analysis of mitochondrial transmembrane potential DiOC 6 stain 粒線體膜電位的存在,會使一些親脂性陽離子螢光染料:《 DiOC 6 》結合到粒線體基質,然後再利用流式細胞計檢測細胞的螢光強度 其螢光的增強或減弱說明粒線體膜電位的增高或降低 Flow cytometry
  • 20. specific substrate release (AMC) Fluorescence spectrophotometer 產生黃綠色螢光 Caspase caspase specific substrate-(AMC) caspase specific substrate-(AMC) Fluorescence ↑ active caspase ↑ ; Fluorescence ↓ active caspase ↓ 激發光 360 nm Caspase 發射光 460 nm Fluorogenic assay
  • 21. Results
  • 22. 抑制 50% 酵素活性所需要的藥物濃度 COLO 205 : 結腸癌細胞 SK-Hep 1 : 肝癌細胞 HL-60 : 人類骨髓白血病細胞株 AGS : 胃癌細胞 THP-1 : 人類白血病單核球細胞株 PMNs : 健康人類的多形核中性球 6 hrs MTT assay Table 1
  • 23. agarose gel electrophoresis Fig. 1 dose dependent time dependent
  • 24. /6 hrs 染色質皺縮 Acridine orange 觀測細胞經處理後整體型態與核內染色質的改變 Fig. 1
  • 25. annexin V-FITC staining and flow cytometry Right shift Apoptosis ↑ Fig. 1
  • 26. dose time Fig. 2
  • 27. left shift Δ Ψm ↓ Δ Ψm↓ Cytochrome c cas-9 cas-3 Fig. 3 Mitochondria
  • 28. Δ Ψm↓ Cytochrome c cas-9 cas-3 Fig. 3 Mitochondria
  • 29. Right shift ROS ↑ left shift GSH ↓ Fig. 4
  • 30. caspase 9 在 1 hr 可看到明顯的上升 caspase 3,8 在 2,3 hr 看到上升 9 Fig. 5 3 8 1,6 2
  • 31. -> caspase inhibitor Fig. 5
  • 32. a -> allopurinol b -> catalase c -> DPI d -> NAC e -> PDTC f -> SOD , GSH c a f b d e Fig. 5
  • 33. dose time DNA repair ↓ cas-3 PARP Apoptosis Fig. 6
  • 34. cas-3 ( inhibitor of CAD ) DNA fragmentation Fig. 6 DFF-45 CAD DFF-45 CAD
  • 35. Fig. 7
  • 36. tBid Bid cas-8 Fig. 8 Fas Fas L FADD Pro-cas-8
  • 37. Fig. 8 Fas Fas Ligand
  • 38. Discussion
  • 39. Intrinsic Pathway Extrinsic Pathway
  • 40. 市售蜆精一覽表 80 元 綠川黃金蜆精 60 元 蒜味重 ★ ( 多 3%) 白蘭氏蜆精 ★ 50 元 ★ 台糖蜆精 零售價格 調味 ( 口味 ) 蜆精濃度
  • 41. Thank you for your attention