臺灣博碩士論文加值系統

對藥物毒理學而言，若能有效研發抵禦癌症侵襲之臨床藥物具有顯著研究價值，而以往含毒藥物的研發多採用二維樣本，以檢測癌細胞株對應相異藥物導致之細胞凋亡現象。然其卻有著相較三維樣本的缺憾: 不自然細胞型貌、偏離複雜生物體內條件、無法精準預測真實生物之細胞反應。
本研究主旨為探討藥物毒性對A2058細胞株三維形貌的影響，並由實驗室自行架設之超高解析Mirau-based全域式光學同調斷層掃描術(FF-OCT)佐三維細胞培養技術，以獲細胞因受含毒藥物侵襲，其隨時間流逝(24hrs; 48hrs; 72hrs; 96hrs)的凋亡影像，從中擷取單顆細胞三維投影以利特徵參數量化分析與凋亡階段判別。
全域式OCT影像方面，除定時觀測三維視域影像做形貌判別外，更藉單顆細胞三維投影與降噪演算法進一步擷取細部資訊與特徵，並以七項特徵參數(峰值訊號強度、均值訊號強度、訊號強度均方差、訊號強度變異數、胞內動態範圍、細胞像素加總、訊號強度加總)對99顆細胞進行量化分析而佐證推論: 24hrs-48hrs區段產生細胞凋亡初期的固縮、皺縮現象；48hrs-72hrs區段細胞膜碎裂情形加劇；最終於72hrs-96hrs區段至末期成凋亡小體。
而對照共焦螢光顯微鏡之染色影像，其中歧異甚微之48hrs-72hrs區段，全域式OCT影像不僅可展示大幅度分歧(六項特徵參數p值遠小於0.05)；更能以所選七項特徵參數完整劃分24hrs-48hrs區段(p值皆小於0.05)；而儘管72hrs-96hrs區段散射訊號微弱導致歧異甚微，仍有兩項p值小於0.05的特徵參數，而冀望最終可藉此項光學技術協助現有染色技術對細胞凋亡現象做出相對精細之階段判別。

For drug toxicology, significant research values have shown in developing anti-cancer drugs to resist the invasion of cancers. In the past, the researches and developments mostly utilised two-dimensional samples to detect the apoptosis of cancer cell lines from toxic drugs. However, comparing with three-dimensional samples, two-dimensional samples have relative disadvantages, e.g., unnatural cell morphology, deviation from complex in vivo conditions and inability to predict the cell response of real organisms accurately.
The main purpose of this research is to explore effects of drug toxicity on three-dimensional morphology of A2058 cell line. By utilising three-dimensional cell culture technology and ultra-high resolution of homemade Mirau-based full-field optical coherence tomography (FF-OCT), we acquire OCT images of apoptosis from the cells with toxic drug in regularly-observed samples, 24hrs, 48hrs, 72hrs and 96hrs. For facilitating quantitative analysis of characteristic parameters and distinguishing apoptosis stages, three-dimensional projection of single cell is extracted from the images.
Besides regular observation from OCT images, three-dimensional projection of single cells and noise reduction algorithms have been used to capture detailed information and characteristics. Applying seven characteristic parameters, peak signal intensity, mean signal intensity, standard deviation signal intensity, variance signal intensity, intra-cell dynamic range, total cell pixels and total signal intensities, for quantitative analysis of 99 cells to support the inference: 24hrs-48hrs period has feature of cell shrinkage and pyknosis, early-stage phenomenon of apoptosis; cell membrane fragments drastically during the 48hrs-72hrs period; and apoptotic bodies are formed during the 72hrs-96hrs period, final-stage phenomenon of apoptosis.
Comparing 48hrs-72hrs period OCT images with the images, stained by confocal fluorescence microscope (CFM), OCT images demonstrate not only enormous divergences (p-values of the six characteristic parameters are much less than 0.05); it can also be completely divided in 24hrs-48hrs period (p-values are all less than 0.05); although the back-scattering light during 72hrs-96hrs period is faint and causes hardly differences in images, there are still two characteristic parameters with p-values less than 0.05. Therefore, one may be expected to assist recently-utilised optical technology, CFM, for having a more accurate result about distinguishing different stages of apoptosis.

誌謝 I
摘要 II
Abstract III
目錄 V
圖目錄 VII
表目錄 XI
第一章 緒論 1
1.1 研究動機 1
1.2 內文概述 2
第二章 Mirau-based全域式光學同調斷層掃描術 3
2.1 光學低同調干涉術 3
2.2 摻鈰釔鋁石榴石寬頻光源 11
2.3 Mirau-based全域式OCT系統架構 15
2.3.1 全域式OCT系統架構概述 15
2.3.2 全域式OCT系統之橫/縱向解析度 22
2.3.3 全域式OCT影像處理法 26
第三章 黑色素瘤與細胞凋亡 30
3.1 皮膚癌細胞株 30
3.1.1 皮膚各層架構 30
3.1.2 皮膚癌 35
3.1.3 黑色素瘤細胞株 40
3.2 細胞凋亡 44
3.2.1 凋亡之型態特徵 44
3.2.2 凋亡現象檢測 47
3.3 三維細胞培養技術 49
第四章 三維細胞培養與全域式OCT影像 52
4.1 定時樣本製備 52
4.2 空腔體實驗 55
4.3 三維視域影像 60
第五章 全域式OCT影像量化分析 64
5.1 單顆細胞三維投影 64
5.2 降噪演算法 68
5.3 各凋亡階段之判別分析 74
5.3.1 三維定時投影對照 74
5.3.2 特徵參數與量化分析 79
第六章 研究結論與未來展望 91
6.1 研究結論 91
6.2 未來展望 92
參考文獻 93
附錄1 單顆細胞三維投影 102
附錄2 共焦螢光顯微鏡影像 106
附錄3 特徵參數之分析檢定 108

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