摘要
胰臟無論是在內、外分泌系統上都扮演重要的角色。其中，位於胰臟組織中的蘭氏小島含有β細胞，負責分泌胰島素以代謝血糖。β細胞因免疫系統攻擊死亡，為第一型糖尿病型成的重要原因。然而造成蘭氏小島功能異常與β細胞死亡的因素很多，其中因蘭氏小島發炎而產生的血管與神經組織結構變化，一直是糖尿病型原因的研究重點。然而，以傳統的二維組織切片方式，進行蘭氏小島的顯微影像擷取，會造成組織變形與切割破壞，無法保持完整的樣本結構。並且，以切片方式所獲得的平面組織影像，無法觀察到血管與神經網路在空間中的完整結構。本實驗以小鼠胰臟組織為樣本，結合螢光標記、染色技術，共軛焦顯微鏡技術，與組織澄清技術，提供了一個不需要使用切片機，即能進行胰臟內蘭氏小島組織觀察的方法，建立觀察蘭氏小島組織的三維顯微影像技術。本研究所發展的立體組織影像獲得方式，可清楚地觀察到蘭氏小島血管與神經網路在空間中的形態。此一影像技術將可提供胰臟與糖尿病領域一項新的研究觀察工具。

Abstract
The pancreas is a unique organ where the endocrine microorgans are embedded in the exocrine tissue. To visualize the spatial distribution of pancreatic cells in situ also with three dimensions observation could provide a better understanding of the overall organization of endocrine cells and the relationship between different structures in the pancreas. In this research, we apply an optical-clearing solution, FocusClear, to minimize the random light scattering in the mouse pancreas and show the overall spatial organization of β-cells and islets in the pancreas. This technique, with the advantage than conventional fluorescence microscopy and alleviates technical difficulties such as collection and operating for serial histological sections and avoids structural defects during the sectioning process. Our results thus establish the basis for a new platform which is a noninvasive three-dimensional microscopic imaging method for in situ visualization and assessment of pancreatic islet microstructure.
The three-dimensional images of the fluorescence-labeled structure. Specifically, the islet vasculature and sympathetic nerve fibers were labeled by vessel painting (lipophilic dye labeling of blood vessels) and immunofluorescence staining of neural markers, respectively, for confocal microscopy. Optical sectioning and reconstruction of bulk tissue are simple and rapid. Volume and number of β-cells within a block of tissue can be automatically quantified by freely available software.

論文目錄
第一部分 文獻回顧.......................................................................1
論文架構……………………………………………………….……....1
1.1 小鼠胰臟蘭氏小島之三維影像………………………….….…2
1.2 生物醫學影像技術..................................................................3
1.2.1 共軛焦顯微鏡...................................................................3
1.2.2 生物組織澄清試劑..........................................................4
1.3 小鼠胰臟蘭氏小島組織..........................................................6
1.3.1 小鼠胰臟蘭氏小島組織之血管系統.................................6
1.3.2 小鼠胰臟蘭氏小島組織之神經網路系統..........................7
1.4 糖尿病簡介............................................................................9
1.4.1 糖尿病之動物模型..........................................................10
1.4.2一streptozotocin (STZ).....................................................11
1.5 研究動機…………………………………………..……………….11
第二部分 實驗材料和儀器.............................................................13
2.1 實驗材料...............................................................................13
2.2 實驗儀器...............................................................................13
第三部分 實驗方法.......................................................................14
3.1 動物實驗...............................................................................14
3.1.1 實驗動物.........................................................................14
3.1.2 灌流之動物模型..............................................................14
3.2 FocusClearTM應用於小鼠胰臟組織之分析……………...….15
3.2.1 光學穿透度定性分析.......................................................15
3.2.2 光學穿透度定量分析......................................................15
3.3 組織染色……………………………………………….………..16
3.3.1 組織螢光免疫染色..........................................................16
3.3.2 組織螢光染劑染色..........................................................16
3.4 封片......................................................................................16
第四部分 結果討論....................................................................18
4.1 FocusClearTM應用於小鼠胰臟組織之特性........................18
4.2 小鼠胰臟蘭氏小島之二維影像...........................................19
4.3 小鼠胰臟蘭氏小島之三維影像...........................................20
4.4 小鼠胰臟蘭氏小島之血管網路..........................................21
4.5 小鼠胰臟蘭氏小島之神經系統...........................................23
4.6 Streptozotocin誘導型糖尿病之三維影像…………………..…24
第五部分 總結..........................................................................26
第六部分 未來工作...................................................................27
第七部分 實驗數據...................................................................28
第八部分 參考文獻...................................................................40

Figure 目錄
Figure 1 光學穿透度定性分析....................................................28
Figure 2 光學穿透度定量分析....................................................29
Figure 3 光學穿透光強度量化....................................................30
Figure 4 Anti-insulin小鼠胰臟蘭氏小島免疫螢光染色..............31
Figure 5 小鼠胰臟蘭氏小島組織二維和三維影像.......................32
Figure 6 小鼠胰臟蘭氏小島組織三維影像後製...........................33
Figure 7 小鼠胰臟蘭氏小島組織三維血管影像...........................34
Figure 8 小鼠胰臟蘭氏小島組織血管長度計算...........................35
Figure 9 小鼠胰臟蘭氏小島組織Tyrosine hydroxylase之三維影像..36
Figure 10 小鼠胰臟蘭氏小島組織S100β之三維影像......................37
Figure 11 Streptozotocin誘導型糖尿病之三維影像…………...……..38
Figure 12 Streptozotocin誘導型糖尿病之血管三維影像………..…..39

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