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研究生:林志壕
研究生(外文):Chih-Hao Lin
論文名稱:探討紫杉醇誘導人類口腔癌症細胞株細胞凋亡之機制
論文名稱(外文):The Mechanism of Paclitaxel-induced apoptosis in human oral cancer cell lines
指導教授:蕭振仁蕭振仁引用關係黃步敏
指導教授(外文):Jenn-Ren HsiaoBu-Miin Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:細胞生物及解剖學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:67
中文關鍵詞:紫杉醇口腔癌症細胞株細胞凋亡
外文關鍵詞:Oral cancer cell linesApoptosisPaclitaxel
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  細胞凋亡在多細胞生物的體內平衡扮演了重要的角色,而且硫胱氨酸蛋白脢(caspase)、Bcl-2家族蛋白酶和絲裂原活化蛋白激酶 (mitogen-activated protein kinase, MAPK)都有參與調控細胞的機制。目前有很多種抗癌藥物可以引起癌症細胞進行細胞凋亡中,如:紫杉醇 (paclitaxel)。紫杉醇是一種抗微小管的藥劑,可以抑制正常細胞分裂週期的進行而達到治療癌症的效果。口腔癌在台灣的發生率有逐年提高的現象,並且紫杉醇已經被用來治療頭頸部癌症。然而紫杉醇誘導頭頸部癌症細胞凋亡的機制仍然不清楚,了解抗癌藥物誘導細胞凋亡的機制對於臨床上的用藥是很重要的。因此,在我們要研究紫杉醇是如何誘導口腔癌症細胞株 (HSC-3, OEC-M1和OC3) 進行細胞凋亡。值得特別關注的是OEC-M1和OC3為台灣本土性的口腔癌細胞株。我們發現紫杉醇 (50和500 nM)處理這三株口腔癌症細胞株48小時後,可以引起口腔癌細胞株型態上的改變,並可以降低細胞的存活率。除此之外,紫杉醇可引起三株口腔癌細胞株去氧核醣核酸的斷裂。OEC-M1及OC3的細胞分裂週期有明顯的停留在G2/M週期,而且三株口腔癌症細胞株的subG1週期都是呈現增加的現象,指出紫杉醇引起口腔癌症細胞死亡是透過細胞凋亡機制。在OEC-M1以及OC3口腔癌症細胞株中,我們發現有硫胱氨酸蛋白脢 (caspase-3, -6, -7, -8和-9) 的活化,而且有看到poly (ADP-ribose) polymerases (PARP)失去活性。除此之外,絲裂原活化蛋白激酶家族中的JNK和Bcl-2都有被磷酸化的現象。另外我們使用硫胱氨酸蛋白脢的抑制劑 (Z-VAD) 去抑制口腔癌症細胞株 (OEC-M1和OC3) 細胞凋亡,結果發現口腔癌症細胞株的存活率可以增加,但是卻無法使細胞死亡的情形回復到百分之百。總結之,紫杉醇可以誘導口腔癌症細胞株進行細胞凋亡,並且硫胱氨酸蛋白脢、Bcl-2蛋白酶以及JNK有參與在其中。
  Cell suicide or apoptosis plays an important role in the maintenance of cellular homeostasis in multicellular organisms, and the caspase cascades, Bcl-2 family proteins and mitogen-activated protein kinases (MAPKs) are involved in apoptosis. In cancer cell lines, apoptosis can be induced by many kinds of therapy-related inducer, such as paclitaxel (Taxol), a microtubule-stabilizing agent, suppressing several kinds of tumors by arresting cell-cycle progression. In Taiwan, the incidence of oral cancer increases in recent years, and paclitaxel has been successfully used for treatment of head and neck cancers. However the detail mechanism is still not sufficient. Understanding the molecular mechanisms of a chemotherapeutic agent on cancer cells is indispensable for clinical applications. Hence, we investigated the effect and mechanism of paclitaxel on oral cancer cell lines (HSC-3, OEC-M1, and OC3). It should be noted that, OEC-M1 and OC3 cells are indigenous oral cancer cell lines in Taiwan. We observed that 50 nM and 500 nM paclitaxel could cause morphological changes and significantly decreased cell viability after 48 hours treatment in all three cell lines. Additionally, paclitaxel induced the DNA fragmentation of these cell lines, in which OEC-M1 and OC3 cells significantly arrested at G2/M phase. In addition, the amount of subG1 increased among three cell lines, which indicated that paclitaxel triggered cell death through apoptosis. In OEC-M1 and OC3 cells, the activation of the caspases-3, -6, -7, -8, -9 and the cleavage of poly ADP-ribose polymerase (PARP) were detected. Moreover, the JNK and Bcl-2 were phosphorylated in OEC-M1 and OC3 cell lines. In caspase general inhibitor experiment, the general inhibitor (Z-VAD-FRM) could increase the cell viability, but could not prevent all cell death. In summary, paclitaxel could induce oral cancer cell lines apoptosis through caspase and MAPKs cascades in OEC-M1 and OC3 cells.
TABLE OF CONTENTS
ABSTRACT
Chinese……………………………………………………………..i
English……………………………………………………………..ii
INTRODUCTION……………………………………………………..1
MATERIALS AND METHODS……………………………………8
Chemicals………………………………………………………….8
Cells and cell culture………………………………………………9
Morphology study…………………………………………………9
MTT assay………………………………………………………..10
Flow cytometry analysis………………………………………….10
Immunoblot analysis……………………………………………...11
Caspase inhibitors treatment……………………………………...12
Statistics…………………………………………………………..12
RESULTS
Effects of paclitaxel on morphological changes in human
oral cancer cell lines……………………….……………………..13
Effects of paclitaxel on cell viability in human oral cancer
cell lines………………………………….……………………….13
Effects of paclitaxel on cell cycle progression in human oral
Cancer cell lines………………………………………………......14
The percentage of paclitaxel-induced subG1 and G2/M phases
of cell cycle progression in human oral cancer cell lines……...…15
Effects of paclitaxel on caspases-8 and -9 protein expression
in human oral cancer cell lines…………………………………...16
Effects of paclitaxel on effector caspase proteins expression
in human oral cancer cell lines………………………………...…17
Effects of paclitaxel on PARP protein cleavage in human
oral cancer cell lines……………………………………………...18
The effects of paclitaxel on the activation of MAPK pathway
in human oral cancer cell lines…………………………………...19
The effects of paclitaxel on the expression of p-Bcl-2 paclitaxel in
human oral cancer cell lines……………………………………...20
The effects of general caspase inhibitor (z-VAD-fmk)
in paclitaxel-induced cell death in human oral cancer cell lines....21
DISCUSSION………………………………………………………...23
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