臺灣博碩士論文加值系統

粒線體的型態與粒線體的功能具有高度的相關性。在細胞週期中，粒線體的型態與功能隨著細胞週期的不同而改變，這些改變通常也反映特殊的生理條件。在細胞分裂的過程，粒線體會隨著時間變成高度融合的型態以提高粒線體的活性，產生足夠的能量提供細胞分裂使用。

為了分析粒線體在三度空間下的型態變化，我們發展了一套高含量分析系統分析共軛焦顯微鏡所擷取的 normal rat kidney細胞 (NRK cells) 粒線體，利用影像的螢光亮度切割出粒線體的位置，並計算粒線體的體積、緊密度等等各種不同的形態特徵值。接著利用計算得到的粒線體型態特徵值將粒線體分成五種不同的子型態。並利用擷取的各種細胞層級的特徵值，以Kolmogorov-Smirnov Test 比較細胞週期之間的差異。

從計算的結果顯示，細胞中粒線體的總體積從G0開始不斷的增加，到 S 時期變成兩倍，但緊接著的 G2 時期突然驟降成原本的體積，並在M時期稍微提高。但粒線體的截面積在M時期並沒有上升，表示粒線體的體積上升，並不是因為粒線體即將啟動自我吞噬的膨脹造成。另外螢光亮度在這段時期中沒有明顯變化，也表示粒線體在這個過程中沒有自我吞噬導致粒線體的螢光蛋白轉移。因此我們推論，G0 時期到S時期，與 G2 到 M 時期的粒線體體積上升，主要是因為粒線體的生成造成，而不是因為粒線體的自噬行為產生。而在G2時期的粒線體體積下降，可能是由於粒線體為了在進入細胞分裂之前，維持並更新粒線體的功能，因此在G2時期產生大量的分裂與粒線體自我吞噬，使粒線體保持完整的功能。

我們的系統可以自動化的計算NRK細胞週期中粒線體型態變化與特徵值，並將粒線體分類成五種子型態。另外，透過我們的系統可以找到粒線體在細胞週期中有生質合成與品質控管的行為，從這份研究顯示，粒線體的型態可以做為潛在的細胞週期生物標記。

The function of mitochondria is highly correlated with mitochondrial morphology. Mitochondrial morphology and functions change during cell cycle, and these changes usually imply specific physiological conditions. In the mitotic phase, mitochondria become hyperfused to reach maximal activity.

To analyze mitochondrial 3D morphological changes, we established a high content analysis system to apply to normal rat kidney cell image stacks. By using our system, we calculated mitochondrial morphological features to classify mitochondria into 5 different subtypes. And we extracted various cell-level mitochondrial morphological features to find the difference between cell cycle steps.

In this study, we found the total mitochondrial volume increased to two-fold from G0 phase to S phase, abruptly drops to original size at G2 phase, and then increased again at M phase. Changes in mitochondrial cross section area and fluorescence intensity of fluorescence-tagged mitochondrial proteins show that the volume increase is not due to swelling of dysfunctional mitochondria. This indicated that mitochondrial biogenesis occurred at the transition from G0 phase to S phase and from G2 phase to M phase. Decrease of mitochondrial volume at G2 phase implies that mitochondrial quality control occurs at G2 phase.

By classifying mitochondria into 5 representative morphological subtypes our system reveals specific mitochondrial morphological composition in different cell cycle steps of natural rat kidney cells. Moreover, using our system, we discovered that there are two phases of mitochondrial biogenesis and one phase of quality control during cell cycle implied by cell-level feature analysis. In this study, we show that mitochondrial morphology can be a potential biomarker for cell cycle.

內容
中文摘要 iv
Abstract v
1. 緒論 1
1.1. 粒線體 1
1.2. 粒線體功能與型態 2
1.3. 粒線體型態與功能在細胞週期的改變 5
1.4. 粒線體定量分析 6
2. 研究目標 7
3. 材料與方法 8
3.1. 影像來源 8
3.2. 影像前處理 11
3.3. 影像分割 13
3.4. 特徵值擷取 16
3.5. 高斯混合模型 22
4. 結果 23
4.1. 使用高斯混合模型之分群結果 23
4.2. 各個細胞週期之粒線體子型態 25
4.3. 盒狀圖與統計分析 26
4.4. 多元尺度分析 28
4.5. 支持向量機 29
5. 討論 32
6. 參考文獻 33
7. 附錄 i
 
圖 1: 粒線體藉由融合與分裂來互補受損的粒線體DNA(Youle and van der Bliek, 2012) 2
圖 2: 粒線體動力學失能導致神經元病變(Chen and Chan, 2009) 4
圖 3: Mitra Kasturi拍攝之螢光影像 8
圖 4: 細胞週期控制流程(Mitra et al., 2009) 10
圖 5: 以Median Filter模糊化處理後的圖 11
圖 6: 利用內插法計算每張螢光影像之間的變化量。 12
圖 7: Otsu法與ALT切割方法使用於2D螢光影像之比較(Peng et al., 2010) 14
圖 8: 切割方法之比較。 15
圖 9: 3D 12-Subiteration Thinning Algorithm所使用的樣板(Palágyi and Kuba, 1999) 17
圖 10: 骨架化之結果比較。 18
圖 11: 以貝氏資訊準則比較高斯混合模型之收斂結果 23
圖 12: 粒線體子型態 25
圖 13: 細胞週期與其粒線體子型態。 26
圖 14: 粒線體特徵值之盒狀圖與統計分析結果 27
圖 15: 粒線體特徵值之盒狀圖與統計分析結果 28
圖 16: 多元尺度分析之分類結果 29
 
表 1: 細胞影像數量 10
表 2: 細胞層級粒線體形態特徵值 20
表 3: 粒線體形態特徵值 21
表 4: 使用高斯混合模型之分群結果 24
表 5: 3D粒線體子型態 25
表 6: SVM結果之混淆矩陣（Confusion Matrix） 30
表 7 個別細胞週期之SVM準確率 31

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