臺灣博碩士論文加值系統

以酵母菌為模式生物探討舒瓦克曼綜合症中活性氧的生成
舒瓦克曼綜合症是一種體染色體隱性遺傳疾病，主要是由於第七對體染色體上的SBDS基因突變所造成，而酵母菌中的SDO1基因與人類SBDS基因為同源基因。前人的研究指出SDO1基因與核糖體生合成相關。本文藉由酵母菌為模式生物，探討SDO1基因突變株，對於氧化壓力的反應與野生株酵母菌的不同之處，並探討可能的原因。研究發現，sdo1突變株比野生株酵母菌具有較差的細胞壁完整性，但在不同的環境壓力誘導下，都產生了較少的活性氧（Reactive oxygen species，ROS）。以過氧化氫處理時，sdo1突變株更具有較高存活率。測定了抗氧化酵素的活性發現，sdo1突變株的超氧化歧化酶（Superoxide dismutase，SOD）活性與野生株酵母菌並無明顯差異，但過氧化氫酶活性比野生株高，經過氧化氫誘導氧化壓力時，sdo1突變株的過氧化氫酶活性更被大量誘導；以過氧化氫酶抑制劑處理後，活性氧的生成會增加。因此，推測過氧化氫酶於sdo1突變株過量表現，為sdo1突變株在環境壓力刺激下產生較少的活性氧(ROS)、並具有較高的存活率的原因。

以微流道為系統探討肺癌細胞中活性氧的生成
微流道是一種在毫米甚至微米級別的流道，它可以在時間和空間上操縱細胞的生長，並進行一些刺激。本研究設計了能創造濃度及剪切力(shear stress)梯度的微流道，將肺癌細胞培養在微流道中，來分析肺癌細胞在不同濃度的過氧化氫、剪切力、或抗氧化劑環境中活性氧的生成情形。實驗結果發現本研究設計的微流
道可以產生一個穩定的濃度梯度，並且可以產生梯度性的剪切力。首先利用過氧化氫來製造一個過氧化氫濃度梯度(0-200 μM），隨著過氧化氫濃度的升高，肺癌細胞產生的ROS越來越多。並且在剪切力越大的地方，產生的ROS也比剪切力小的地方多。接著本研究利用抗氧化劑維他命E (α-tocopherol)製造一個濃度梯度
(0-25 μg/ml)，維他命E在22 μg/mL的濃度下，可清除肺癌細胞因剪切壓力產生的ROS，但超過22 μg/mL以後，反而會增加細胞中ROS的量。但是在靜置培養時，相同濃度梯度的維他命E並沒有看到這一現象。這些結果顯示細胞於靜置或流體中培養條件下對環境刺激有不同的反應，而微流道能模擬細胞於體內中有體液或血液流動的環境，因此更能反應體內中細胞的真實狀況。

Studies on the production of reactive oxygen species (ROS) in SBDS mutant using a yeast model
Shwachman–Bodian–Diamond syndrome (SBDS) is an autosomal recessive genetic disease which is caused by the mutation of SBDS gene on the 7th chromosome. SDO1 is the yeast ortholog. Several studies indicate that Sdo1 has function in 60S ribosome biogenesis. In this study,the ROS production of sdo1 mutant in response to environmental stresses was studied. According to the data, interestingly, the sdo1 mutant produced much less ROS than wild type when exposed to different stimulations even its cell wall integrity is weaker than that of wild type., And under treatment of hydrogen peroxides, sdo1 mutant has higher viability. The activity of the antioxidant enzymes was measured and demonstrate that the sdo1 mutant has similar superoxide dismutase (SOD) activity, but much higher catalase activity than that of wild type. After treatment of catalase inhibitor, 3-AT, the production of ROS could be restored in sdo1 mutant. In conclusion, the higher catalase activity is one of the reasons that sdo1 mutant produces lower ROS.

Studies on the production of ROS in lung cancer cells using microfluidic chips
The microfluidics is a millimeter or micrometer level device that can be used to culture cells and treated with different stimulations simultaneously. In this study, a microfluidic chip was designed to generate gradient of concentration and shear stress.
It was applied to investigate how human lung cancer cells response to stimulus of oxidative stress, antioxidants, and fluidic shear stress. With the increasing concentrations of H2O2 (0-200 μM) or shear stress, the human lung cancer cells produced more ROS Interestingly, addition of a gradient of α-tocopherol (0-25 μg/mL) with shear stress, lung cancer cells showed decreasing ROS response under increasing concentration of α-tocopherol until the concentration of 22 μg/mL, however, the ROS increased when the concentration exceeded above 22 μg/mL. These situations can’t be observed under the stationary culture condition with the same α-tocopherol condition.

目錄
致謝 i
中文摘要 ii
ABSTRACT iv
目錄 vi
表目錄 x
圖目錄 xi
縮寫表 xii
Part I、以酵母菌為模式生物探討舒瓦克曼綜合症中活性氧的生成
壹、 前言 1
一、 釀酒酵母（Saccharomyces cerevisiae）簡介 1
二、 舒瓦克曼綜合症簡介 2
三、 活性氧（Reactive oxygen species，ROS）簡介 3
（一） 內生性ROS的產生 3
（二） 外源性ROS的產生 4
（三） ROS對細胞的損傷 4
（四） 粒線體ROS與酵母菌細胞凋亡 5
（五） 在酵母菌中表達哺乳動物細胞抗凋亡基因導致細胞凋亡 5
四、 抗氧化系統簡介 6
（一） 非酵素型系統 6
（二） 酵素型系統 6
五、 研究動機 8
參、 實驗材料與方法 9
一、 實驗材料 9
二、 實驗方法 9
（一） 細胞壁完整性測試 9
（二） 利用流式細胞儀測定細胞中的ROS 9
（三） 酵母菌存活率測試 10
（四） 超氧歧化酶的活性染色分析 10
（五） 超氧歧化酶活性定量分析 11
（六） 過氧化氫酶的活性染色分析 11
（七） 過氧化氫酶活性定量分析 12
（八） 抑制過氧化氫酶活性對ROS生成影響分析 12
（九） 利用螢光共軛焦顯微鏡觀察酵母菌粒線體的形態分佈 13
（十） 利用西方墨點法觀察在氧化壓力下Sdo1蛋白表現量 13
參、 實驗結果 15
（一） sdo1突變株酵母菌於不同環境壓力下活性氧生成的反應 15
（二） sdo1突變株酵母菌在過氧化氫誘導的氧化壓力下產生的活性氧比野生株少 16
（三） sdo1突變株酵母菌的細胞壁完整性比野生株差 17
（四） sdo1突變株酵母菌在過氧化氫誘導的氧化壓力下存活率比野生株高 18
（五） sdo1突變株的過氧化氫酶活性比野生株高 19
（六） 經過氧化氫酶抑制劑處理的sdo1突變株酵母菌能產生較高的ROS 20
（七） 野生株與sdo1突變株酵母菌在氧化壓力下粒線體分佈情況 22
（八） 野生株與sdo1突變株酵母菌在氧化壓力下Sdo1蛋白的表現量 23
肆、 討論 24
Part II、以微流道為系統探討肺癌細胞中活性氧的生成
壹、 簡介 27
一、 活性氧與癌症之間的關係 27
二、 肺癌簡介 27
三、 肺癌細胞簡介 27
四、 維他命E簡介 28
五、 微流道簡介 28
六、 研究目的 29
貳、 實驗材料與方法 30
一、 實驗材料 30
二、 實驗方法 30
（一） 肺癌細胞在微流道中對於過氧化氫濃度梯度的反應 30
（二） 肺癌細胞在微流道中對於α-tocopherol濃度梯度的反應 31
（三） 肺癌細胞在靜置培養時對於α-tocopherol濃度梯度的反應 32
（四） 肺癌細胞在微流道中對於ferulic acid濃度梯度的反應 32
參、 實驗結果 33
（一） 微流道濃度梯度的穩定性 33
（二） 微流道中肺癌細胞ROS的產生量隨著過氧化氫濃度及剪切力的上升而升高 34
（三） 微流道中肺癌細胞ROS的產生量隨著維他命E濃度的上升而減少但高於22 μg/ml時反而大量增加 35
（四） 肺癌細胞在靜止狀態時ROS的產生情況不會隨著維他命E濃度的上升而改變 36
（五） 微流道中肺癌細胞ROS的產生量不隨阿魏酸濃度的上升而減少 37
肆、 討論 38
參考文獻 39

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