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研究生:陳佳欣
研究生(外文):Chia-hsin Chen
論文名稱:放射線合併化學藥劑誘發細胞凋亡及細胞週期調控影響之機制探討
論文名稱(外文):The molecular mechanisms of apoptosis and cell cycle regulation induced by ionizing radiation combined Staurosporin
指導教授:王應然王應然引用關係
指導教授(外文):Ying-Jan Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:43
中文關鍵詞:細胞凋亡staurosporine放射線治療放射線敏感度細胞週期
外文關鍵詞:apoptosisradiotherapyradiosensitivitystaurosporinecell cycle
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  過去的研究指出,癌細胞於放射線照射後,會誘發細胞週期G2/M停滯現象和細胞凋亡。但是,如果癌細胞對放射線產生抵抗性(radioresistance),使得細胞凋亡能力減低,導致癌細胞對放射治療效果不好。研究也指出,阻斷蛋白質磷酸激酉每 C(protein kinase c)路徑能夠誘發細胞凋亡。因此本篇研究欲合併處理低量蛋白質磷酸激酉每C抑制劑(protein kinase c inhibitor -PKC inhibitor)staurosporine,誘發較多細胞凋亡數量,以增加對放射線敏感度,來抵抗radioresistance。此外,更進一步探討,合併低劑量staurosporine及低劑量放射線照射處理之後,誘發細胞凋亡及細胞週期調控影響之機制探討。結果顯示,在流氏細胞儀分析下,細胞合併處理後,會減少G2/M停滯現象,增加細胞週期G1現象,並且增加細胞凋亡。細胞減少G2/M停滯現象是經由減少G2/M相關的蛋白質表現量,例如,增加Cyclin A和Cyclin B蛋白質表現量;增加細胞週期G1現象是經由增加G1/S負調節因子p21蛋白質表現量;細胞凋亡是經由caspase-8和caspase9相關的細胞死亡機制所誘發出來的。再者,細胞合併處理後,是經由活化JNK蛋白質的訊息傳遞路徑,進而誘發下游的細胞死亡機制。
  Irradiation induces apoptosis and G2/M arrest in cell cycle. However, cellular radiation resistance(radioresistance)may diminish the ability to undergo apoptosis in vivo and in vitro and decrease the efficiency of radiotherapy in clinical trials. It has been suggested that protein kinase C inhibitor, staurosporine, is an inducer of apoptosis in tumor cells. To overcome radioresistance, we use low-dose staurosporine in combination with irradiation to induce more apoptosis and increase radiosensitivity. In addition, we investigated the molecular mechanisms of apoptosis and cell cycle regulation induced by combined treatment. Our results indicated that decreased G2/M arrest, increased G1 phase, and apoptosis could be found in tumor cells treated with combination of staurosporine and irradiation analyzed by Flow Cytometry. Combined treatment overrides G2/M arrest induced by irradiation through decreasing the expression of G2/M -related proteins such as Cyclin A, Cyclin B, and CDK1. Combined treatment increases G1 phase through increasing the expression of G1/S phase regulator, p21. Furthermore, combined treatment induces apoptosis by both of caspase-8 and caspase-9 related death pathway, and the activation of JNK pathway contributes to upstream signaling of apoptosis after combined treatment.
摘 要..................................................................I
Abstract...................................................................II
Introduction...............................................................1
Materials and methods......................................................5
Cell culture, drug treatments, and irradiation conditions..................5
DNA fragmentation assay....................................................5
Flow cytometry.............................................................5
Western blotting analysis..................................................6
Caspase activity...........................................................6
Analysis of mitochondrial transmembrane potentional........................7
Statistical analysis.......................................................7
Results....................................................................8
Cell death mechanisms of combined treatment in U937 cells..................8
Cell cycle progression and apoptosis after combined treatment..............8
Effect of combined treatment with STP and IR on expression of regulators of cell cycle......................................................................9
Apoptotic pathway of combined treatment with STP and IR in U937 Cells......9
Analysis of mitochondrial transmembrane potential and cytochrome c release in U937 cells with combined treatment.........................................10
JNK pathway activation was observed after combined treatment in U937 Cells ...........................................................................11
Discussion.................................................................13
Combined treatment with STP and IR induced more apoptosis and overrode G2/M arrest in cells treated with irradiation...................................13
p21, a critical cell cycle regulator in U937 cells treated with STP and IR ...........................................................................15
The mechanism of apoptotic pathway in cells with combined treatment........17
The prolonged phosphorylation of JNK and apoptotic pathway in cells with combined treatment..................................................................17
The prolonged phosphorylation of JNK and G2/M arrest.......................19
The relevance of STP and the induction of p21 in the comprehensive signaling network in cells with combined treatment...................................20
Conclusion.................................................................21
Reference..................................................................22
Figure.....................................................................35
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