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任課老師 : 詹于誼 學 生 : 唐若廷 & 陳玉貞 日 期 : 2008 06 10

The apoptosis effect of hispolon from Phellinus linteus (Berkeley & Curtis)Teng on human epidermoid KB cells. Journal of Ethnopharmacology 105 (2006) 280–285. 任課老師 : 詹于誼 學 生 : 唐若廷 & 陳玉貞 日 期 : 2008 06 10. Results. Fig. 2. Effect of hispolon on the growth of KB cells.

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任課老師 : 詹于誼 學 生 : 唐若廷 & 陳玉貞 日 期 : 2008 06 10

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  1. The apoptosis effect of hispolon from Phellinus linteus (Berkeley & Curtis)Teng on human epidermoid KB cells Journal of Ethnopharmacology 105 (2006) 280–285 任課老師:詹于誼 學 生:唐若廷 &陳玉貞 日 期: 20080610

  2. Results

  3. Fig. 2. Effect of hispolon on the growth of KB cells. (A)Dose-dependent effects of hispolon on the cell growth inhibition of KB cells (exposure: 24h). (B)Time-Dependent effects of hispolon on the inhibition of KB cell growth (hispolon:5mg/ml). Cell viability was determined by a MTT assay as described in the text. Results are expressed as means ± S.E.M. of data obtained in three independent experiments.

  4. con 2.5mg/ml 5mg/ml 10mg/ml Fig.3. Dose-dependence of DNA fragmentation. KB cells were exposed to the Indicated concentrations of hispolon for 24h. Cells were harvested by centrifugation and DNA was extracted. The DNA fragments was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light after staining with ethidium bromide. Lane1: control; lane2: 2.5mg/ml; lane3: 5mg/ml; lane4: 10mg/ml.

  5. 11.29% 24hr 1.04% Fig.4. Flow cytometric analysis of DNA fragmentation for KB cells after hispolon treatment. KB cells were exposed to the indicated concentrations of hispolon for 24h or not. The harvested cells were fixed in 70% ethanol, andstained with PI, followed by flow cytometric analysis. The percentages of cells with hypodiploid DNA contents(sub-G1) represent the fractions undergoing apoptotic DNA degradation. 67.90% 24.52%

  6. rhodamine 123 (Rh123) 粒線體在呼吸氧化過程中，將所產生的能量以電化學位能儲存於粒線體內膜 (約 250 mV/5~10 nm)，稱之為 mitochondrial membrane potential (mmp)，並以此位能來進行電子傳遞鏈，最終產生 ATP 以供細胞使用。而此一電位差形成於粒線體內膜，為外正內負的形式。 許多親脂性的陽離子化合物會結合到粒線體內膜，在雷射激發光下放出螢光，可以非常方便地使用螢光顯微鏡或共軛焦顯微鏡來觀察粒線體的形態和移動的變化情形。最早被發現的粒線體染劑為 rhodamine 123 (Rh123)，Dr. Lan-Bo Chen 的實驗室發現 Rh123 可特異性的染在活細胞的粒線體上，而一些抑制電子傳遞鏈的藥物 都會降低 mmp，並會減少粒線體攝入 Rh123 的量。

  7. Cyt c 粒線體膜電位 Fig. 5. Measurements of mitochondrial membrane potential and Cyt C release in KB cells during the hispolon treatment. (A). KB cells were treated with 10mM Rh123 for 10 min, and then incubated with 2.5, 5 and 10 mg/ml hispolon for a different period of time. At the end of incubation, the mitochondrial membrane potential was measured as described in the text. (B). KB cells were incubated with 2.5, 5 and 10 mg/ml hispolon for the indicated time. Cyt C release was determined by immunoassay as described in the text. C: control; H2.5: 2.5 mg/ml; H5: 5 mg/ml; H10: 10 mg/ml.

  8. 桑黃中的活性物質hispolon可以誘導KB cell • 細胞凋亡，凋亡路徑是粒線體 • 2. 作著先確定細胞是否凋亡就是DNA ladder • 跟 sub-G1期的增加並發現粒線體膜電位的改變與 cyt C 的釋放有time與dose dependent

  9. Thank you for your attention

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