臺灣博碩士論文加值系統

粒線體是一種具有高度動態變化的胞器，粒線體會不斷進行融合、分裂以及粒線體的品質管控（粒線體自噬），這些行為簡稱為「粒線體動態」。粒線體動態變化是維持粒線體功能的重要的角色，適當的粒線體動態變化能減少粒線體受損也能維持細胞的完整性。當細胞分裂時，不只有遺傳物質DNA會被完整無誤的送到子細胞當中，細胞中的粒線體也需要被分配到子細胞當中，目前在哺乳類細胞中發現粒線體在細胞分裂的過程中會先執行過度分裂後再被分配至子細胞中，但感到矛盾的是，過度分裂後的小顆粒狀粒線體往往被認為產能總量極低，這樣極低產能狀態的粒線體要如何提供足夠的能量給細胞進行有絲分裂，也或許是在有絲分裂過程中的某個時間點，粒線體會呈現產能較高的長條狀型態來暫時提供能量讓細胞進行有絲分裂。然而，到目前為止尚未建立一個良好的定量方法去分析粒線體的型態，所以現在對於整個細胞週期的過程中，粒線體的生合成與品質管控是如何去影響粒線體型態變化還不是很清楚。在我的研究中，利用共軛焦顯微鏡拍攝倉鼠卵巢上皮細胞（CHO-K1）的z軸堆疊影像，進一步擷取粒線體型態並利用統計方法計算出粒線體型態的種類以建立一套用來定量粒線體3D型態的自動化分析系統。從高斯混合模型與貝葉斯信息準則以及先前的文獻資料中做粒線體型態上的歸納與分類，結果可以將3D粒線體型態分為六大類；利用我們建立好的分析系統，我們可以了解在細胞分裂時粒線體的生合成以及品質管控是如何影響粒線體型態。為了縮短細胞拍攝時間，利用細胞週期同步化的方法，使細胞處於飢餓狀態(serum free starvation)，細胞週期同步在G0時期，並擷取特定細胞週期的時間點，以免過度曝露在雷射下造成粒線體損傷。從細胞同步實驗中觀察到G0-G1時間的粒線體會快速進行融合作用，由小顆粒狀變為條網狀的型態。
從長時間的3D影像當中我們可以看到在不同細胞週期中特定的粒線體型態。特別是細胞分裂的細胞我們發現粒線體型態呈現較小條狀的構造而不是小顆粒狀的型態，而且粒線體分裂會特別發生在細胞分開的位置；此外，也定量了綠螢光標定的粒線體基質蛋白隨著細胞分裂時的變化，發現粒線體基質蛋白質以及粒線體總體積會在有絲分裂早期降低，又會在有絲分裂後期及末期慢慢增加，這樣的結果顯示在細胞有絲分裂時粒線體有生合成以及品質管控的變化。

Mitochondria are dynamic organelles that undergo fusion, fission and mitochondria quality control (mitophagy). Proper mitochondrial dynamics regulates mitochondrial damages to ensure cellular integrity. During cell division, not only genetic materials but also organelles are evenly distributed to daughter cells. Current model for mitochondrial inheritance in mammalian cells suggested that mitochondria undergo complete fission for even partition to daughter cells. Paradoxically, extensive mitochondrial fission may result in too low production of energy to support enough energy for mitosis; mitotic cells may still keep tubular at a particular time point of mitosis. Nowadays, no quantitative method for mitochondrial morphology, exact cell cycle-dependent mitochondrial morphological changes are not well-defined and the mechanism how bioenergetics status and mitochondrial quality control affects mitochondrial morphology is still unclear. In this study, we used 3D confocal image stacks of CHO-K1 cells to establish the automated system to extract morphological features and calculate distribution of morphological subtypes for numerical 3D mitochondrial morphological analysis. Combining literature information, we employed Gaussian Mixture Model (GMM) and Bayesian Information Criterions (BIC) to identify 6 major mitochondrial morphological subtypes. Using this system, we can know that how the dynamics work and biogenesis status and quality control during cell division. To shorten the time of cell images acquirement, using serum starvation to synchronize cell in G0 phase, and get each cell cycle step time point to reduce mitochondrial damage in excessive laser exposure. From the result of cell synchronization, we observed the mitochondrial fusion occur rapidly, and mitochondrial fragments change into mitochondrial tubular networks in G0-G1 phase. From our 3D time-lapsed videos, we find mitochondrial morphology at specific to different cell cycle stages. Especially, the mitochondrial morphology of mitotic cell is in smaller tubular network instead of fragmentation. Mitochondrial fission usually occurs at the division furrow. Besides, intensity of GFP-tagged mitochondrial matrix protein and total mitochondrial volume are reduced at early mitotic phase and gradually increased in anaphase and telophase, and imply biogenesis status and quality control are changed in mitosis.

目錄
摘要 1
Abstract 3
目錄 5
中英文對照表 7
縮寫表 8
緒論 10
一、粒線體動態變化 10
二、粒線體品質管控 13
三、細胞分裂與粒線體分配 15
四、目的與假說 18
材料與方法 20
一、實驗材料與藥品 20
二、研究方法 21
（一）細胞培養 21
（二）質體DNA純化（Transformation） 22
（三）細胞轉染（Transfection） 22
（四）病毒感染 （Infection） 23
（五）螢光顯微影像擷取 24
（六）細胞週期同步之粒線體型態拍攝 25
（七）2D粒線體與細胞核型態分析 26
（八）3D粒線體分割 26
（九）3D粒線體型態分類 26
（十）3D粒線體與細胞核型態之數值分析 27
實驗結果 28
一、篩選各種穩定表達的細胞株 28
二、3D粒線體型態分析系統之建立 28
三、在G0-G1時期粒線體會由小顆粒狀變為條網狀型態 29
四、在G0-G1時期，粒線體會快速進行融合作用 31
五、有絲分裂時的粒線體大多呈現條狀型態 32
六、在有絲分裂期發生粒線體蛋白降解和生合成 33
研究討論 35
參考文獻 39
圖 44
圖1 ：利用螢光顯微鏡觀察成功轉染與感染的細胞 45
圖2：建立出3D粒線體型態分析系統將粒線體分類成六種型態 46
圖3：不同型態的線粒體在單一細胞內的呈現 47
圖4：細胞在G0-G1時期的粒線體的型態 49
圖5：在G0-G1時期各型態的粒線體在細胞中的分佈情況 50
圖6：六種不同粒線體在細胞G0-G1時期不同時間點的型態變化 52
圖7：在G0-G1時期會進行粒線體融合 56
圖8：粒線體型態在細胞有絲分裂時的變化 57
圖9：利用3D粒線體分析系統呈現出有絲分裂時期粒線體型態的變化 58
圖10：3D粒線體的六大類型態在有絲分裂時期體積上的變化 60
圖11：有絲分裂時期粒線體的特徵值變化 62
附錄 63
附錄1：單一細胞影像的切割步驟 64
附錄2：利用micro-P分析軟體進行粒線體型態分析 67
附錄3：觀察軟體分析結果的正確性 67
附錄4：把Micro-P分析所得到的數據，利用excel做出統計圖。 68
附錄5：2D粒線體分析軟體可把粒線體分類成六種型態（Subtype） 69
附錄6：從共軛焦顯微鏡拍攝的之影像切割出單一細胞 70
附錄7：3D粒線體型態分析之步驟 73

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