臺灣博碩士論文加值系統

本論文以研究台灣產番荔枝科植物所分離出來的乙醯生合成物 (Annonaceous acetogenin) Squamocin之藥理活性。除了以小白鼠之毒性實驗探討其半致死劑量 (LD50)，更進一步以大白鼠神經膠質瘤細胞 (rat glioma C6 cell)研究其抗癌活性及其誘發細胞凋亡(apoptosis)之相關機轉。
以背部皮下方式將Squamocin 之不同劑量(0.1-2.0 mg/Kg)注入小白鼠(20-25 g)之體內，觀察小白鼠死亡情形，發現其死亡率與劑量成正相關之反應，且Squamocin 之半致死劑量(LD50)約為0.375 mg/kg。
在細胞毒殺方面，將C6細胞以不同濃度(10-4 —1.0μM)之Squamocin 作用72小時後，以Methylene blue assay的方法測定。發現其與劑量成正相關之反應，其50 % 之抑制生長濃度(IC50)約為51.2 nM，且細胞以藥物處理後其型態均有萎縮、產生空泡之現象。
C6 細胞經加入Squamocin 不同濃度(1或100 nM)作用24、48及72小時後，以流式細胞儀分析其細胞週期的變化，發現其呈現出停滯於G2/M期的現象，而且隨時間之增加，細胞變化之百分比更為明顯。
C6 細胞在加入不同濃度(1或100 nM)之Squamocin 24、48及72小時後，經 由原位DNA片段測定法(TUNEL)分析其DNA片段後發現隨著Squamocin 之劑量與反應時間的增加，它所造成之C6細胞凋亡數目也隨之增加。
而目前對有關引起細胞凋亡之途徑,被認為可經由腫瘤壞死因子(TNF-α)與腫瘤壞死因子之接受體(TNF-R1)結合後，而進行機轉中腫瘤壞死因子之接受體相關死亡區段(TRADD)之活化，引起一連串瀑布式反應進而導致細胞凋亡作用。
以流式細胞儀分析發現Squamocin (1或100 nM)作用24、48及72小時後可增加C6細胞之細胞膜上TNF-R1的表現及細胞所釋放之TNF-α。經由西方點漬法分析，亦發現以Squamocin (1或100 nM)作用24小時後，細胞內Bcl-2蛋白質的表現有降低之現象。
本研究之結果發現，Squamocin 的細胞毒性主要是經由細胞凋亡之作用。Squamocin 會使C6細胞之細胞週期停滯在G2/M期，並使細胞內之DNA斷裂百分比、TNF-α及TNF-R1 之表現增加，且降低細胞內Bcl-2蛋白質之表現。

Squamocin is an active acetogenin purified from Formosa Annonaceous plants. The aim of this study was to investigate cytotoxic effects and apoptotic mechanisms of squamocin in rat glioma C6 cells.
Mice (20-25 g) were administrated back subcutaneous with different dosage (0.1—2 mg/Kg) of squamocin. Our results have indicated that the lethal dose 50 % (LD50) of squamocin is approximated 0.375 mg/Kg.
Squamocin possess potent antitumor effect in rat glioma C6 cells. The cytotoxic IC50 of squamocin in C6 cells is 51.2 nM, which is more potent than other commercial anti-neoplastic agents. In addition, squamocin also induced the morphological changes of C6 cell including cells shrunken and membrane blebbing phenomenon.
We hypothesized that the cytotoxic effect of squamocin may be due to the apoptotic signal modulation. It was confirmed by a flow cytometric analysis, treatment squamocin with (1 & 100 nM) for different time (24, 48 or 72 hours) caused an accumulation in the G2/M phase of the cell cycle.
In situ detection of fragmented DNA (TUNEL assay) of C6 cells, squamocin (1 & 100 nM) dose- and time-dependents increased the percentage of DNA fragmentation.
Most apoptotic induction of TNF-α is mediated by TNF-R1 through interaction of its death domain protein (TRADD). Therefore, in TNF-α level and TNF-R1 expression determination by flow cytometry. Our results have demonstrated squamocin increase the TNF-α level and TNF-R1 expression in the C6 cell apoptotic induction.
The determination of signal protein on apoptosis pathway (Bcl-2) was analysis by western blot assay in C6 cells. When treated with squamocin (1 & 100 nM) for 24 hours, C6 cells contained lower concentration of Bcl-2 protein as compared to control.
In conclusion, our data have indicated the effect on cell cycle modulation (G2/M phase arrest) and apoptotic induction of squamocin. Meanwhile, squamocin increase the TNF-α level, TNF-R1 expression and decrease Bcl-2 protein in the C6 cell apoptotic induction.

目 錄
頁次
中文摘要 ………………………………………………1
英文摘要 ………………………………………………3
縮寫表 …………………………………………………5
一.緒論 ………………………………………………...6
二.實驗材料與方法……………………….……………11
三.結果… ………………………………………………23
四.討論… ………………………………………………50
五.參考文獻….…………………………………………59
六.附表… ………………………………………………70
圖表目錄
頁次
表 1 Squamocin及抗癌藥物對C6細胞之IC50……...28
表 2 Squamocin對C6細胞之細胞週期影響………...29
表 3 Squamocin對C6細胞之DNA斷裂片段影響…30
表 4 Squamocin對C6細胞之TNF-α表現影響.. …..31
表 5 Squamocin對C6細胞之TNF-R1表現影響...…32
附表1 目前可分離到Squamocin之番荔枝科植物...…..70
附表2 Squamocin對不同癌細胞之IC50及選擇性..……71
頁次
圖1 Annona squamosa 之果實....………………………..33
圖2 Squamocin 之化學結構式...………………………..34
圖3 Squamocin 引起小白鼠之死亡率...………………..35
圖4 Methylene blue 細胞數目檢量線圖.………………36
圖5 以Methylene blue方法偵測不同濃度的.
Squamocin 對C6細胞的毒殺作用.………………37
圖6 以光學顯微鏡觀察Squamocin處理72
小時引起C6細胞型態改變……………………….38
圖7 以流式細胞儀觀察Squamocin對C6
細胞型態改變………………………………………39
圖8 以流式細胞儀觀察Squamocin對C6
細胞之細胞週期改變………………………………40
圖9 以流式細胞儀觀察Squamocin對C6
細胞作用24小時之細胞週期改變………………..41
圖10 以流式細胞儀觀察Squamocin對C6
細胞作用48小時之細胞週期改變………………..42
圖11 以流式細胞儀觀察Squamocin對C6
細胞作用72小時之細胞週期改變………………..43
圖12 以流式細胞儀觀察Squamocin對C6
細胞之DNA斷裂片段改變………………………44
圖13 以流式細胞儀觀察Squamocin對C6
細胞之DNA斷裂片段與細胞大小改變…………45
圖14 以流式細胞儀觀察Squamocin對C6
細胞之TNF-α改變……………………………….46
圖15 以流式細胞儀觀察Squamocin對C6
細胞之TNF-R1改變………………………………47
圖16 以SDS-PAGE觀察Squamocin對C6
細胞作用24小時之蛋白質改變………………….48
圖17 以Western-blotting觀察Squamocin對C6
細胞作用24小時之Bcl-2蛋白改變……………..49
圖18 Squamocin引起細胞凋亡之分子相關機轉圖……58

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