- 學位論文
五胺基酮戊酸光動力效應導致粒線體傷害造成細胞骨架重組之分子機制探討
Mitochondrial photodamages by ALA-PDT result in cytoskeleton reorganization
摘要
光動力治療，結合光感物質與特殊波長光源以達到治療的效果。五胺基酮戊酸 （5-ALA） 為光感物質前驅物質，經由血色素代謝過程，細胞將外加五胺基酮戊酸 (ALA) 代謝成光感物質 Protoporphyrin IX （PpIX），並且在癌症細胞內部產生較高的累積量。當 PpIX 受630 nm紅光激發後，促使細胞內部產生單態氧與活性氧分子，進一步對細胞產生毒殺效應。本論文中發現在老鼠胚胎纖維母細胞 （NIH3T3）給予不同濃度與不同時間的培養條件下，可以觀察到細胞將外加的 ALA 代謝成 PpIX，累積在細胞內部。並且在 1 mM ALA 培養 3 小時的實驗條件下，PpIX 在細胞內部的累積量即達到飽和。更進一步觀察 PpIX 在細胞內的位置，發現 PpIX 主要累積在粒線體內部。ALA-PDT 光動力處理後，產生大量的單態氧與活性氧造成細胞的光照傷害。同時，觀察到細胞粒線體呈現漲大的型態，進一步檢測細胞粒線體弁遄A細胞粒線體去氫酶活性下降，膜電位喪失並且粒線體生成 ATP 能力受損。這些證據進一步顯示 ALA-PDT 光動力處理，致使細胞粒線體受到光動力傷害而造成細胞死亡。 ALA-PDT 光動力處理後，細胞型態產生劇烈變化，細胞呈現圓縮 （Round up），同時細胞對於附著基質 （Substratum） 的附著力增加，產生Resistance to trypsinization 的現象，此現象隨光照劑量增加，增加細胞 Resistance to trypsinization 的比例，並且隨時間而逐漸減緩。除此之外，進一步觀察細胞骨架變化情形，觀察細胞骨架分子Actin 與細胞骨架支持分子 Paxillin， Vinculin 及
並列摘要
Photodynamic Therapy (PDT) is the new modality for cancer treatment, which involves photosensitiser excited by specific wavelength of light. 5-Aminolevulinic acid (5-ALA), a precursor in the heme biosynthetic pathway and of the subsequently activated photosensitiser, shows selective damage to tumor tissue after light irradiation. Studies have shown that 5-ALA is metabolized to protoporphrin IX, which selectively accumulates to greater extent in tumor cells and exhibits its cytotoxicity via singlet oxygen and ROS after irradiation of 630 nm light source. Using mouse embryonic fibroblast cells (NIH3T3), we found ALA was metabolized to PpIX in mitochondria by confocal microscope and the accumulation of PpIX reached to its plateau within 3 hr. After ALA-PDT, quantity of ROS and singlet oxygen were generated within the cells and consequently caused mitochondrial swelling which led to mitochondrial damage. Further studies have shown photo-induced mitochondrial damages with supporting evidences as following: (1) reduced mitochondria dehydrogenase activity (2) breakdown of mitochondrial membrane potential, and (3) decreased intracellular ATP content. These evidences indicate that ALA-based PDT induces photocytoxicity via selective mitochondrial damage. Meanwhile, following ALA-PDT, cells exhibit dramatically morphological change and decrease deattachment to substratum by trypsinization. The pattern of resistance to trypsinization following ALA-PDT was in a dose-dependent manner and gradually reversed post ALA-PDT. Moreover, the cytoskeleton, actin, and cytoskeleton adaptor protein, paxillin, vinculin, and