臺灣博碩士論文加值系統

實驗室先前的研究發現，腫瘤在經過放射治療後會改變微環境中巨噬細胞像缺氧區聚集的現象，利用高度聚集於腫瘤環境的巨噬細胞作為藥物載體，可能會有提升治療效果的潛力。為了探討這個可行性，本研究利用實驗室先前建立的臨床前放射治療後攝護癌(TRAMP-C1)再復發模型，在放射治療前24小時尾靜脈注射帶有綠色螢光蛋白(EGFP)的骨髓細胞與其由體外分化的單核球，分別在治療後一天、一周、兩周和三周，利用流式細胞儀與免疫螢光染色分析骨髓細胞和單核球在周邊血與腫瘤微環境中聚集趨勢。實驗結果發現骨髓細胞和單核球皆在注射後一週有最大數量滲透聚集於腫瘤微環境中，且經過放射治療比正常的微環境更為顯著，但在缺氧區域並沒有明顯聚集。為了釐清缺氧環境對單核球移動能力的影響，在單核球體外分化過程中分別給予24、48和72小時的缺氧條件，結果顯示缺氧化單核球在移動能力上的確有所抑制，不同程度的缺氧化在細胞表現特徵也有所差異。由於放療後的腫瘤微環境確實有大量單核球滲透聚集，我們認為單核球具有做為細胞載體以提升放射治療效果的潛力。

Previous studies have known that radiotherapy could course the aggregation of macrophages in hypoxia areas. We hypothesize that the tumor-homing ability of tumor-associated macrophages (TAM) rendered it as a potential cellular carrier. To investigate this hypothesis, we used an established pre-clinical recurring prostate tumor (TRAMP-C1) model to study the infiltration pattern of bone marrow derived cells (BMDCs). 24 hours before the irradiation, 2×106 BMDCs or differentiated monocytes- expressing EGFP were injected i.v. The peripheral bloods and tumor microenvironment were analyzed by the flow cytometry and immunofluorescence staining. The results show that the largest number of BMDCs and monocytes infiltrated into tumor microenvironment occurred 1 week after cell injection, which was more notable in irradiated tumor, but not found at hypoxia regions. To investigate the hypoxic effect for migrated ability of monocytes, monocytes were differentiated in hypoxia conditions (2% O2) for the duration of 24, 48 or 72 hours. The results show that the migration rate of monocyts was restrained under hypoxia conditions, and the degrees of hypoxia condition have different effects on monocytes differentiation. The significant recruitment of monocytes into irradiated tumor microenvironment suggests that monocytes could be a potential cellular carrier for target therapy to enhance the efficiency of radiotherapy.

第一章、緒論
1.1 攝護腺瘤
1.1.1 攝護腺瘤簡介
1.1.2 攝護腺癌的臨床診斷
1.1.3 攝護腺癌的臨床治療
1.2 腫瘤微環境與放射治療
1.2.1 腫瘤微環境簡介
1.2.2腫瘤缺氧性區域與再復發
1.2.3血管新生
1.2.2 放療後的腫瘤微環境
1.3 骨髓細胞與單核球
1.3.1 骨髓細胞簡介
1.3.2 骨髓細胞與腫瘤微環境
1.3.3 單核球簡介
1.3.4 單核球與腫瘤微環境
1.3.5 單核球和缺氧性區域
1.4 研究目的與內容
第二章、材料與方法
2.1 細胞培養
2.1.1 配製DMEM細胞培養液
2.1.2 配製RPMI-1640細胞培養液
2.1.3 細胞培養液的配方
2.1.4 細胞繼代
2.1.5 細胞上清液(Condition Medium)收集
2.2 小鼠骨髓細胞萃取與單核球分化
2.2.1 動物來源
2.2.2 小鼠骨髓細胞萃取
2.2.3 單核球分化
2.3 動物實驗
2.3.1 動物來源
2.3.2 腫瘤植入
2.3.3 放射治療
2.3.4 動物分組
2.3.5 動物犧牲
2.4.6 周邊血萃取
2.4.7 螢光固定與腫瘤組織包埋
2.4.8 腫瘤細胞均質化
2.4.9 流式細胞儀分析
2.4.10 腫瘤組織冷凍切片
2.4.11 組織免疫螢光染色
2.4 體外缺氧實驗
2.4.1 缺氧條件
2.4.2 細胞分組
2.4.3 細胞存活分析 (Survival)
2.5 細胞移動實驗 (此實驗由胡桓軒同學協助完成)
2.5.1 條件培養液製備
2.5.2 實驗分組
2.5.3 實驗流程
第三章、實驗結果
3.1 骨髓細胞滲透分析
3.1.1 骨髓細胞定性分析
3.1.2 周邊血分析
3.1.3 腫瘤微環境分析
3.1.4 放射照射後注射分析
3.1.5 器官分析
3.2 單核球滲透分析
3.2.1 單核球定性分析
3.2.2 周邊血分析
3.2.3 腫瘤微環境分析
3.2.4 加強注射分析
3.3 缺氧化實驗
3.3.1 腫瘤缺氧微環境分析
3.3.2 缺氧化單核球生存曲線與定性分析
3.3.3 缺氧化單核球移動能力分析
第四章、結論與討論
第五章、圖表與說明
附錄
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