臺灣博碩士論文加值系統

中 文 摘 要
粒線體腦肌病（encephalomyopathy）的發生與神經細胞或肌肉細胞的損失有關。因此，我假設粒線體呼吸鏈的功能缺陷是粒線體疾病患者肌肉細胞或神經細胞損失的原因。呼吸鏈的功能缺陷原因可能緣於粒線體DNA（mitochondrial DNA, mtDNA）與細胞核內DNA的基因突變。粒線體腦肌病相關的mtDNA突變中，4977-bp大片段斷損突變最為常見。在本研究中，我首先探討mtDNA減除（depletion）對細胞凋亡行為的影響，藉此模擬呼吸鏈功能嚴重缺陷的情況，細胞在staurosporine（STS）處理下所表現出之凋亡行為。STS為一種廣效性的PKC抑制劑，常被用來誘使細胞發生細胞凋亡。為了排除細胞核內基因所造成的影響，我選擇經EtBr處理而減除mtDNA的r0細胞作為實驗材料，探討其細胞凋亡行為與原始143B母細胞的差異。結果顯示，在STS的處理下，r0細胞的死亡率較143B細胞高，且梯狀DNA斷裂的情形也較為嚴重。分析造成r0細胞容易死亡的原因，我發現在STS處理下，r0細胞有較高的caspase 3活性，並伴隨著細胞色素c釋放至細胞質中。所以，我推測mtDNA減除的影響層次應在caspases活化的上游。另外，藉著抗氧化酵素抑制劑的處理，我發現r0細胞並未較143B細胞敏感；且r0細胞的能量來源為以培養液中所含的丙酮酸進行無氧呼吸產生ATP，所以產生較少的活性氧分子（reactive oxygen species, ROS），因此我推測ROS並非造成r0細胞較容易走向細胞凋亡的主要原因。
再者，本實驗室先前已建立細胞質融合的技術，可將CPEO病人的細胞中所帶有之4977-bp斷損突變送入r0細胞中。我乃利用此技術所製造的細胞質融合細胞株，探討高百分比4977-bp斷損突變對細胞凋亡的影響，希望藉此可以更進一步瞭解粒線體腦肌病之致病機轉。在STS處理下，我發現不論野生型的1-3-16細胞株，或帶有高百分比4977-bp mtDNA斷損突變的51-10細胞株，都有caspase 3活性大量增加的現象，但細胞死亡率卻有明顯的差異。進一步利用自由基清除酵素抑制劑處理細胞，我發現51-10細胞株對ROS的刺激較1-3-16細胞株來得敏感。因此，我推測高百分比4977-bp mtDNA斷損突變會使細胞對ROS的耐受能力降低，並因此而較容易導致細胞凋亡或細胞壞死。
根據以上的研究結果，我推測帶有4977-bp mtDNA斷損突變的病人對氧化壓力較為敏感，給予抗氧化傷害之藥物可能會有保護性的治療效果。另一方面，mtDNA的減除對活體細胞的影響與在細胞培養所觀察到之凋亡行為應有所不同，因為在活體組織內細胞並無法獲得大量的丙酮酸。所以，需要對肌肉細胞與神經細胞的能量代謝有更清楚的瞭解，才能釐清mtDNA減除是否會在粒線體疾病患者病變組織內造成細胞凋亡。

Abstract
The pathology of mitochondrial encephalomyopathies is associated with abnormal cell loss of neurons and muscle fibers. Is it possible that mitochondrial respiratory chain dysfunction results in cell loss, which is commonly found in patients with encephalomyopathies? The respiratory chain impairment may be caused by DNA mutations in mitochondrial or nuclear genome. Among the mitochondrial DNA (mtDNA) mutations associated with mitochondrial diseases, 4977-bp deletion is the most common one. In this study, the effects of mtDNA depletion on the apoptotic behaviors of human cells were first investigated. In order to exclude the possible contributions of nuclear background on the possible effect of mtDNA itself, r0 cells derived from 143B osteosarcoma cells were used. r0 cells that are devoid of mtDNA were obtained by long-term treatment of osteosarcoma cells with ethidium bromide (EtBr). Under the treatment of staurosporine (STS), the viability, DNA fragmentation, caspase 3 activities and ROS production of r0 and parental 143B cells were compared. The results showed that r0 cells exhibited lower viability, higher caspase 3 activity and increased cytochrome c release to the cytosol. These findings suggest that mtDNA depletion exerts damaging effects on an upstream effector of caspase activation. In addition, it was also found that r0 cells were not as sensitive to the inhibitors of scavenger enzymes as 143B parental cells. Moreover, r0 cells utilize pyruvate in culture media to provide energy needed and thus produce less ROS. Taken together, these findings suggest that it is not ROS increase per se that leads r0 cells to be more susceptible to apoptosis.
By using cytoplasmic fusion techniques, 4977-bp mtDNA deletion from skin fibroblasts of a CPEO patient was introduced into r0 cells and created the so-called cybrids. The cybrids containing 0% and 85% of mtDNA with 4977-bp deletion as an experimental system were used as an experimental system to investigate the effects of high proportion of 4977-bp mtDNA deletion on apoptotic behaviors of human cells. Under the treatment of STS, both the wild-type cell line 1-3-16 and the high mutant cell line 51-10 exhibited high caspase 3 activities. However, they can be distinguished by cell viability. Moreover, by the treatment of antioxidant enzyme inhibitors, such as mercaptosuccinate and 3-amino-1, 2, 4-trizole, 51-10 cells were more susceptible to apoptosis than 1-3-16 cells. Taken together, these findings suggest that high proportion of 4977-bp mtDNA deletion may reduce the ability of cells to deal with ROS and result in apoptosis or necrosis.
Based on the results of this study, it is proposed that 4977-bp mtDNA deletion renders human cells more vulnerable to oxidative stress and that antioxidant therapy can be useful for patients with CPEO syndrome. On the other hand, the apoptotic effects of mtDNA depletion in vivo are different from those observed in cultured cells because of the supplementation of pyruvate and uridine in the medium. It is warranted to better understand the pathological changes of muscles and neurons in relation to the role of mtDNA depletion in patients with MERRF syndrome.

中文摘要..................1
英文摘要..................3
縮寫表....................5
緒論......................7
材料與方法...............18
結果.....................27
討論.....................37
參考文獻.................48
圖與表...................57

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