- 學位論文
五胺基酮戊酸光動力效應誘發PC12細胞粒線體傷害的相關生物效應探討
Studies of the Biological Effects of 5-Aminolevulinic Acid Photodynamic Therapy-Induced Mitochondrial Damage in PC12 Cells
摘要
光動力治療 (Photodynamic therapy, PDT) 為一新興的癌症治療技術，主要是藉由適當波長的光激發光感物質產生光化學作用，對細胞產生毒殺的效果。光化學作用會產生單態氧 (Singlet oxygen) 與其他活化態的氧 (Reactive oxygen species, ROS)。光動力治療主要是經由單態氧的作用，攻擊細胞的脂質、蛋白質或核酸而殺死細胞。五胺基酮戊酸 (5-aminolevulinic acid, ALA) 本身非光感物質，可經由原血紅素合成路徑 (Heme biosynthetic pathway) 代謝為內生性的光感物質 ─ Protoporphyrin IX (PpIX)。五胺基酮戊酸目前已被廣泛應用於癌症的治療與診斷。有研究結果顯示，腫瘤細胞因為原血紅素路徑的酵素活性表現不同，例如：Ferrochelatase (FC) 於腫瘤細胞的活性降低，造成 PpIX 會累積於腫瘤細胞，使得五胺基酮戊酸光動力效應 (ALA-PDT)，可選擇性殺死腫瘤細胞。本研究採用的老鼠嗜鉻細胞瘤 (Rat pheochromocytoma cells, PC12 cells)，常被用來探討神經生物 (Neurobiology) 及神經化學 (Neurochemistry) 特性。因此，本研究預探討五胺基酮戊酸光動力效應引發PC12細胞粒線體傷害後，會導致類似神經的PC12細胞有哪些生物效應產生？研究結果顯示： (1) 五胺基酮戊酸光動力效應造成PC12細胞粒線體弁鈳鄍╮A包括粒線體去氫酶活性 (Mitochondrial dehydrogenase) 降低，膜電位喪失以及ATP生成能力減弱。(2) 五胺基酮戊酸光動力效應造成PC12細胞凋亡。 (3) 反覆五次ALA-PDT處理的PC12細胞，粒線體膜電位與ATP生成能力減弱。此外，反覆五次ALA-PDT處理的PC12細胞，分化的能力與程度皆受到抑制。 (4) 五胺基酮戊酸光動力效應造成分化的PC12細胞膜電位喪失。 (5) 分化的PC12細胞經五胺基酮戊酸光作用後，神經突 (Neurite) 受到傷害而退化。
並列摘要
Photodynamic therapy (PDT) has been developed as a modality for cancer treatment, which is based on the administration of photosensitizers to induce cytotoxicity after light irradiation. It has been shown that singlet oxygen and other reactive oxygen species (ROS) are responsible for the PDT induced cell killing that leads to tumor ablation. As the primary oxidant associated with the PDT reaction is singlet oxygen, photosensitization has been found to oxidize cellular macromolecules including lipids, proteins, and nucleic acids that result in cell death. 5-aminolevulinic acid (ALA) has been successfully used to diagnose and treat neoplastic tissue. ALA itself is not a photosensitizer and serves as the biological precursor of the endogenous photosensitizers, protoporphyrin IX (PpIX), in the heme biosynthetic pathway. It has been shown that ALA–PDT induced selective destruction of neoplastic lesions attribute to the aberration of heme biosynthesis, such as the reduction of ferrochelatase (FC) activities, in tumor cells. Rat pheochromocytoma cells (PC12 cells), a kind of tumor cell, is a useful model of studying in neurobiology and neurochemistry. The purpose of this study is to investigate the effects of ALA-PDT induced mitochondrial damage in PC12 cells. We found: (1) ALA-PDT results in mitochondrial dysfunctions as indicated by the decreased activity of mitochondrial dehydrogenase, reduced ability of ATP generation and disruption of membrane potential. (2)ALA-PDT induced apoptotic death of PC12 cells judged by Hoechst 33258 and TUNEL staining. (3) After 5 times of ALA-PDT (5PDT) treatment, mitochondrial function has changed as indicated by the decreased ability of ATP generation and membrane potential. Moreover, the differentiation ability of PC12 cells has reduced and inhibited. (4) ALA-PDT results in the disruption of membrane potential in differentiated PC12 cells. (5)ALA-PDT caused neurite damage by morphology and microtubules immunostaining observation in PC12 cells.