臺灣博碩士論文加值系統

中文摘要
五胺基戊酮酸(5-ALA)是一個光感物質前驅物，可用於治療或診斷癌症。5-ALA在粒線體中代謝為真正的光感物質protoporphyrin IX (PpIX)並且選擇性的累積在癌細胞中。當PpIX 受到630 nm紅光激發後可產生單態氧產生細胞毒殺作用。在本篇實驗證實，HT1080 human fibrosarcoma與1 mM 5-ALA於不含血清培養液中培養3小時後，以共軛焦顯微鏡觀察PpIX大多產生在粒線體中。照光後以電子顯微結果看到粒線體的破壞，但其他的胞器仍保持完整，除了粒線體外觀上的變化，也偵測到粒線體的膜電位喪失、cytochrome c釋放以及粒線體去氫酶活性的下降。
HT1080細胞在五胺基戊酮酸-光動力處理後，約4小時後有少數細胞核濃縮，但卻沒有偵測到caspase 3的活化，給予廣效性caspase 抑制劑，zVAD也不能增加存活率，但是再8小時後，細胞失去表面的完整性，LDH (lactate dehydrogenase)釋放增加，推測像這樣選擇性破壞粒線體處理，會造成HT1080細胞壞死(Necrosis)。在光動力處理後，MAPK成員ERK、JNK、P38均被活化，給予MEK1、P38抑制劑PD98059和SB202190均不能抑制其死亡，所以ERK和p38或許沒有參與在5-ALA光動力處理引起的死亡機轉中。
光動力處理後，細胞對substratum附著力增加，具光劑量相依性，且會隨著時間增加而恢復。這個效應可以被抗氧化劑glutathione、N-acetyl-cystein及單態氧清除劑NaN3所抑制，另外，有趣的是，給予PD98059也可以抑制此現象。在F-actin的染色中，直接在顯微鏡下可以觀察到actin的分布改變，並且大部分細胞在表面有一個巨大突起，以流式細胞儀偵測，5ALA-PDT後F-actin的總量增加，螢光免疫染色也看到在光動力處理後細胞周圍的Vinculin也有增加變密的現象，推測可能是因為actin和integrin之間的協同作用，使得細胞在ALA-PDT之後附著力增加。綜合以上所述，在ALA-PDT 選擇性破壞粒線體的狀況下，會造成HT1080細胞壞死。另外，光動力處理造成細胞附著性增加，可能是透過增加actin和integrin之間的作用而造成。

英文摘要
(Abstract)
5-aminolevulinic acid (5-ALA), a new prodrug of photosensitizer, can be effectively used in photodynamic diagnosis and therapy of cancer. 5-ALA is metabolized in mitochondria to protoporphyrin IX, which selectively accumulates to a greater extent in cancer cells and displayed its cytotoxicity via singlet oxygen after irritation with 630nm light. Using HT1080 human fibrosarcoma cells, we demonstrated that the production of protoporphyrin IX mostly occurs in mitochondria under confocal microscopy. Electromicroscopy analysis further revealed the destruction of mitochondria and the intactness of other organelles. In addition to morphological changes, we also detect the breakdown of mitochondrial membrane potential, release of cytochrome c and decreased activity of mitochondrial dehydrogenase.
Hoechst staining reveals apoptotic character 4h post-ALA-PDT. After PDT (photodynamic therapy) there is no activation of caspase, and the caspase inhibitor c (zVAD) did not protect cell from death. Furthermore, lactate dehydrogenase (LDH) release from cytosol by losing surface intact about 8h post-ALA-PDT. This result suggests that the damage of mitochondria may cause cell necrosis in HT1080 cells. The three main members of MAPK -ERK, JNK and p38 are activated after PDT. However, ERK and p38 specific inhibitors (PD98059 and SB202190) have no significant effect on ALA-PDT induced cell death. These observations indicate that the p38 and ERK MAPK pathway may not play an important role in resistance against PDT-induced cell death.
After ALA-PDT, there is significant number of cells which could not be removed by trypsin compared to control cell without ALA-PDT. The increased adhesion is in a dose-dependent manner and gradually reverses post photodynamic treatment. The antioxidants glutathione, N-acetyl-cysteine and singlet oxygen scavenger NaN3 inhibit the cellular adherence after ALA-PDT. Furthermore, PD98059 can also inhibit PDT-induced cell attachment to substratum. Increasing F-actin and vinculin demonstrated that PDT-induced cell resistance to trypsinization may work through integrin and cytoskeleton network.

縮寫表
英文摘要
中文摘要
緒論
附圖
實驗材料與方法
結果
第一部分
第二部分
第三部分
討論
圖表
參考文獻

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