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研究生:彭羿菱
論文名稱:建立窗型觀測腔結合活體顯微鏡術系統以比較原發及再復發腫瘤上早期血管形成的過程
論文名稱(外文):Establishment of intravital microscopy combined with window chamber system for the study of the process of tumor vascular formation in primary and recurrent tumors
指導教授:江啟勳洪志宏洪志宏引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生醫工程與環境科學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:65
中文關鍵詞:窗型觀測腔活體顯微鏡術血管新生預先性放射線
外文關鍵詞:Window chamberIntravital microscopyAngiogenesisPre-irradiation
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血管新生是腫瘤生長、發展、及轉移時所必須的,本研究主要著重在腫瘤早期生長及治療後復發時的血管變化,以預先性放射線治療可用來模擬臨床上腫瘤復發生長的環境。結合窗型觀測腔、活體顯微鏡術的使用,可以在相同動物上進行持續12天的觀察。實驗中植入帶有綠色螢光蛋白的TRAMP-C1-GFP攝護腺癌細胞,以螢光顯微鏡、共軛焦雷射掃瞄式顯微鏡監測腫瘤細胞與周圍正常組織血管間的交互作用,分析無放射線治療及預先性放射線治療時,比較明視野影像、螢光影像、共軛焦螢光影像中腫瘤內新生血管分布及型態差異。在無放射線治療的Control group中,第6~7天腫瘤開始進行血管新生,從螢光顯微鏡中可以看到紅色出血點,共軛焦螢光影像血管出芽扭曲變形,血管密度為25.43%;第10~11天,影像可一致性的觀察到細絲狀血管佈滿整個腫瘤,血管密度為49.82%。在預先性放射線治療的Pre-IR group中,第6~7天與Control group情況相似,腫瘤內有出芽的新血管;第10~11天腫瘤內充斥著膨漲的血管聯結成完整的血管網路,血管密度為35.55%,其血管型態上及分布與Control group有明顯的差異。結果顯示本實驗建立一套良好的觀測系統,可連續觀察腫瘤生長與血管的變化,做定性及定量上的分析,未來運用在放射線治療合併抗血管新生治療,對腫瘤早期血管新生有更進一步的瞭解。
Angiogenesis is essential for tumor growth, progression and metastasis. To gain a more complete understanding of angiogenic mechanisms, it is important to have an animal model for examining time-lapse imaging series and real-time functional analysis of tumor vasculature. In this study, we used the window chamber and intravital microscopy systems to serially monitor the initial process of tumor vascular formation in primary and recurrence tumors on the same observational area for 12-days. Pre-irradiation is frequently used to simulate the clinical environment for the tumor relapse after the radiotherapy. To explore the relationship of tumor cells and surrounding host vasculature, murine prostate TRAMP-C1-GFP tumors expressing green fluorescence protein were implanted into non-irradiated (non-IR) or pre-irradiated (pre-IR) tissue. The vascular distribution and morphological variation of tumor neovasculature in these two groups were compared under bright-field image, fluorescence image and confocal image using fluorescence microscopy and confocal laser scanning microscopy. Tumor cells grown in non-IR tissue (control group) begin the initial tumor angiogenesis obviously at 6-7 days after implantation with the evidence of the appearance of red hemorrhage spot in the bright-field images and irregular sprouting neovessles in the confocal images. The microvessel density (MVD) is around 25.43%. By day 10~11, the tread-like blood vessels distribute among whole tumor densely, and the MVD is increased to 49.82%. In pre-IR group, the onset of tumor angiogenesis also occurs 6-7 days, which is similar to what have been seen in control tumors. However, by day 10~11, the vessels are larger and more dilated, but the MVD (35.55%) is less than that in control tumor. In conclusion, we have established a suitable monitor system to not only qualitatively, but also quantitatively analyze the tumor growth and vascular change in vivo serially. It can certainly be an useful system for understanding the working mechanism when radiation therapy is combined with other forms of anti-angionesis or anti-vascular therapy.
目錄
摘要 ……………………………………………………………………Ⅰ
Abstract ………………………………………………………………Ⅱ
致謝 ……………………………………………………………………Ⅳ
目錄 ……………………………………………………………………Ⅴ
第一章 序論 ……………………………………………………………1
1.1 血管新生與腫瘤生長………………………………………………1
1.1.1 血管新生作用……………………………………………………1
1.1.2 腫瘤生成…………………………………………………………3
1.2 活體顯微鏡術………………………………………………………6
1.2.1 活體影像…………………………………………………………6
1.2.2 窗型觀測腔………………………………………………………6
1.2.3 活體顯微鏡術……………………………………………………8
1.2.4 發展與應用 ……………………………………………………10
1.3 放射線治療 ………………………………………………………13
1.3.1 放射線治療 ……………………………………………………13
1.3.2 預先性放射線治療 ……………………………………………14
1.3.3 攝護線癌 ………………………………………………………15
1.4 研究目的 …………………………………………………………17
第二章 材料與方法……………………………………………………18
2.1 架設窗型觀測腔 …………………………………………………18
2.1.1 動物來源 ………………………………………………………18
2.1.2 實驗分組 ………………………………………………………18
2.1.3 架設手術 ………………………………………………………18
2.2 放射線照射 ………………………………………………………21
2.3 腫瘤細胞植入 ……………………………………………………22
2.3.1 細胞培養 ………………………………………………………22
2.3.2 細胞繼代 ………………………………………………………22
2.3.3 植入腫瘤細胞株 ………………………………………………23
2.4 螢光顯微鏡 ………………………………………………………24
2.5 共軛焦雷射掃描式顯微鏡 ………………………………………25
第三章 實驗結果 ……………………………………………………28
3.1 對照組:無放射線照射治療 ……………………………………28
3.1.1 明視野影像 ……………………………………………………28
3.1.2 螢光影像 ………………………………………………………29
3.1.3 共軛焦螢光影像 ………………………………………………30
3.2 實驗組:預先放射線照射治療 …………………………………33
3.2.1 明視野影像 ……………………………………………………33
3.2.2 螢光影像 ………………………………………………………33
3.2.3 共軛焦螢光影像 ………………………………………………34
3.3 綜合比較 …………………………………………………………36
第四章 討論……………………………………………………………37
圖表及說明 ……………………………………………………………44
圖2.1 架設窗型觀測腔手術過程 ……………………………………26
圖2.2 背部皮膚預先放射線照射 ……………………………………26
圖2.3 螢光顯微鏡觀察用自製載臺 …………………………………27
圖2.4 腫瘤在窗型觀測腔內的三度空間及upper layer、middle layer、lower layer的相對位置示意圖 ……………………………27
圖3.1 螢光顯微鏡觀察影像 …………………………………………44
圖3.2 Control group連續螢光顯微鏡觀察影像……………………46
圖3.3 Control group第6天之共軛焦螢光影像連續切面圖 ………48
圖3.4 Control group第6天之紅色螢光血管影像連續切面圖 ……49
圖3.5 Control group第6天之各層共軛焦螢光影像 ………………50
圖3.6 Control group第11天之共軛焦螢光影像連續切面圖………51
圖3.7 Control group第11天之紅色螢光血管影像連續切面圖……52
圖3.8 Control group第11天之各層共軛焦螢光影像………………53
圖3.9 Pre-IR group連續螢光顯微鏡觀察影像 ……………………54
圖3.10 Pre-IR group第6天之各層共軛焦螢光影像 ………………57
圖3.11 Pre-IR group第10天之共軛焦螢光影像連續切面圖………58
圖3.12 Pre-IR group第10天之紅色螢光血管影像連續切面圖……59
圖3.13 Pre-IR group第10天之各層共軛焦螢光影像………………60
表3.1 Control group第6~7天、第10~11天與Pre-IR group第6~7天、第10~11天之共軛焦螢光影像綜合比較………………………………61
參考文獻 ………………………………………………………………62

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