腦部腫瘤缺氧區(Tumor Hypoxia Area)的形成在臨床上被視為造成腫瘤治療抗性的主要原因之一。根據過往對於腫瘤缺氧區的研究，缺氧區的產生容易造成免疫抑制、放射治療抗性以及化療阻礙等現象，使得腦部腫瘤在治療中得不到預期中的成效，甚至加劇腫瘤浸潤的能力。本論文利用小鼠星狀膠質瘤(ALTS1C1)以及神經膠質瘤(GL261)兩種目前常用於研究小鼠腦腫瘤的模型去深入探討非均質外圍缺氧區的存在和其對於放射治療以及藥物治療成效的影響。當腫瘤細胞經由顱內注射進入腦部環境時，腫瘤細胞藉由對周圍微環境調整來適應不利的環境，進而使得腫瘤內部產生大量的缺氧區域。缺氧環境誘使腫瘤產生相對應的代謝提供腫瘤所需要的能量。此外當星狀膠質細胞偵測到腦部異常現象產生即被活化並開始產生移動、黏附甚至浸潤至腦腫瘤周圍並形成血管板模誘使上皮細胞的附著進而產生血管新生以達到缺氧區修復的效果。然而隨著腫瘤不斷地成長擴大，正常腦組織也不斷的受到擠壓甚至產生外圍出血情形。在腦腫瘤生長的後期中，腦腫瘤產生了周圍缺氧區以及內部缺氧區。由於兩種缺氧區內部的血管功能與結構並不盡相同，故本論文推論為兩種不同型態的缺氧區。而經由腫瘤細胞凋亡的數量量化後，數據顯示出在腦腫瘤周圍的缺氧區域對放射線或藥物治療所造成的腫瘤細胞凋亡數目明顯的少於其他區域，換句話說這些地方已形成所謂的治療抗性區。總結來說，本論文研究結果發現當腦腫瘤生長至可偵測的大小後，腫瘤內有兩種不同型態的缺氧區。因為血管功能與結構的不同，這兩種缺氧區的成因可能不同。另外更證明外圍缺氧區的產生為一種腦部腫瘤在成長過程中的特有現象，而且此現象也確實會造成腦部腫瘤在治療過程的阻礙。

Hypoxic tumor region is deemed as a critical source for therapy resistant in clinic. Previous studies indicate that tumor hypoxia frequently results in immunosuppression, radio- and chemo-resistance. In addition, tumor hypoxia can also enhance tumor invasion and tumor mobility. Astrocytes are the prime glial cells in brain tissue. They play important roles in angiogenesis and immune-regulation during the process of brain restoration. In this research, we found the appearance of a non-homogeneous peripheral hypoxia in brain tumor and studied their consequent effects on the response of brain tumor to therapies. Using GL261 glioma and ALTS1C1 astrocytoma tumor models, two frequently used brain tumor research models, we found that tumor cells could adjust themselves to adapt the constant change of brain microenvironments during tumor progression. Meanwhile astrocytes detect the tumor-induced microenvironmental changes and are subsequently activated. Activated astrocytes start to adhere to tumor edge and further invade into tumor core to become vascular template that consequently induces endothelial cell adhesion for angiogenesis to re-oxygenate tumors. On the other hand, normal brain tissue is continuously oppressed and become hemorrhage during brain tumor progression. When tumors grow to detectable size, tumor hypoxia could be separated into two parts, peripheral hypoxia and inner hypoxia. Their vasculatures are different, and so do the vessel function. It is therefore concluded that they are different types of hypoxia and formed by different mechanism. By quantifying therapy-induced apoptotic cells at tumor edge versus tumor core, this study also shows that hypoxia indeed reduces cytotoxic effect of irradiation or chemotherapy. In summary, the study found that two types of tumor hypoxia could be generated in brain tumor. The formation of peripheral hypoxic tumor region is a unique phenomenon during brain tumor progression and it is indeed a barrier for brain tumor therapy.

Table of Contents
中文摘要 I
Abstract II
致謝 III
Table of Contents IV
Chapter I. Introduction 1
1.1 Glioma 1
1.2 Tumor Hypoxia 2
1.3 Astrocytes 3
1.4 Tumor metabolism 3
Chapter II. Materials and Methods 5
2.1 Mice 5
2.2 RNA isolation and Reverse transcription PCR (RT-PCR) 5
2.3 Cell lines cultures 6
2.4 Immunocytochemistry analysis 7
2.5 In vitro cell growth curve (cell doubling time) 7
2.6 Tumor Implantation in orthrotopic tumor model 8
2.6.1 Intra-cranial (I.C.) Injection 8
2.6.2 Intra-muscle (I.M.) Injection 9
2.6.3 Subcutaneous (S.C.) injection 9
2.7 Irradiation and chemotherapy in orthrotopic tumor model 9
2.7.1 Radiotherapy 9
2.7.2 Chemotherapy 10
2.8 Vascular function analysis 10
2.8.1 Biotinylated 70-kDa dextran fluorescence profiling 10
2.8.2 Lectin injection and Fixation by vascular perfusion 11
2.9 Process of embedding brain tumor samples 11
2.10 Immunhistochemical analysis 12
2.11 Statistics
 15
Chapter III. Result 16
3.1 Proliferating cells at tumor edge versus tumor core 16
3.2 Hypoxia pattern in ALTS1C1 tumor progression 16
3.3 The cause of non-homogenous hypoxia at tumor peripheral 18
3.4 The patterns of tumor hypoxia area in alternative injection systems 19
3.5 Pattern of astrocytes activation and adhesion 19
3.6 Metabolism could be altered by tumor peripheral hypoxia 20
3.7 Vascular structure and function at tumor edge versus tumor core 21
3.8 Hemorrhage in brain tumor progression 23
3.9 Response to irradiation of cells at tumor edge versus tumor core 24
3.10 Response to HSV-tk/GCV treatment of cells at tumor edge versus tumor core 24
3.11 Hypoxia pattern in different brain tumor model 25
Chapter IV. Discussion 27
4.1 Tumor peripheral hypoxia area leads to radio-resistance at tumor invasion front 27
4.2 Distribution of hypoxia in ALTS1C1 brain tumor progression 28
4.3 Non-homogenous tumor peripheral hypoxia is caused by re-oxygenating tumor 30
4.4 Tumor peripheral hypoxia area is unique in brain stroma 30
4.5 Evidences of the existence of tumor peripheral hypoxia in brain tumor model 31
4.6 Vascular characteristic in tumor peripheral hypoxia versus tumor inner hypoxia 31
4.7 Tumor peripheral hypoxia is an obstruction of brain tumor therapy at tumor edge 33
4.8 Conclusion 34
Figure 35
Reference 75

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