臺灣博碩士論文加值系統

硼中子捕獲治療（BNCT）為目前正在進行臨床實驗的新式放射性腫瘤治療方法。當硼-10被送入腫瘤細胞後，在中子照射時會捕捉中子，產生核反應裂解成具有高能的分子，進而殺死腫瘤細胞，對於腫瘤周圍的一般正常細胞卻只有甚少或者無負面影響，目前此治療已被應用於多種癌症的治療上。
　　2009年國立清華大學周鳳英教授實驗室發現除了現今已被運用於BNCT治療上的borophenylalanine (BPA)跟borocaptate (BSH)兩種藥物外，硼酸也可以做為硼的攜帶藥物，並且可能做為BNCT治療肝腫瘤的良好硼藥物。不過目前對於硼酸送入癌細胞的機制，以及調節肝腫瘤及周邊正常細胞吸收硼酸具差異性的原因，尚未有完整的研究。
　　文獻指出，腫瘤微環境，例如癌細胞表面醣蛋白的特異性分佈與表現、醣類代謝活性、及微環境中的酸鹼度會與一般正常肝組織的細胞不同，而硼酸也在一些研究中被證實可以與醣類結合形成化合物。基於上述原因，我們推測在硼酸為藥物的BNCT治療下，造成腫瘤與一般細胞吸收硼-10的差異性可能是因為腫瘤與一般細胞的微環境不同，導致腫瘤可以吸收較多的硼-10。實驗結果顯示，當環境中含有比較高的葡萄糖，或微環境酸鹼值偏酸時，可能促進肝癌細胞吸收硼-10。
　　另外，為了加速臨床前研究的相關藥理分析，我們也致力建立新式斑馬魚BNCT研究平台。對於使斑馬魚體內發生腫瘤，我們嘗試將老鼠黑色素瘤細胞B16-F10進行異種轉殖入斑馬魚體，另外也嘗試使用高度表現黑色素瘤致癌基因的轉殖基因癌症魚，來取得可產生膠質黑色素瘤的癌症魚。我們也確認了斑馬魚可以透過眼窩注射傳遞及吸收硼酸藥物，並設計了固定裝置盤，使斑馬魚可以在BNCT的照射過程中維持麻醉及存活。這個新式的癌症斑馬魚平台在未來可以幫助我們加速以硼酸為藥物的BNCT轉譯研究。

Boron neutron capture therapy (BNCT) is a targeted radiation therapy under clinical trial in Taiwan. Boron-10 delivered into the tumor cells disintegrates after capturing a neutron, and the high energy heavy charged particles produced from the nuclear event destroy cancer cells, with only marginal damage to the surrounding normal tissues.
BNCT has been used in clinical trials to treat various types of cancers. In 2009, Prof. Fong-In Chou of National Tsing Hua University found, in addition to borophenylalanine (BPA) and borocaptate (BSH), boric acid (BA) could also be the boron carrier for BNCT. Specifically, boric acid could be a promising agent for BNCT treatment of hepatocellular carcinoma (HCC). However, the underlying mechanism that influence tumor to normal tissue (T/N) ratio of the BA uptake and the transport process is still unclear.
There are numerous reports showing that tumor microenvironment is different from the surrounding normal cells, including the glycoprotein profile, glucose metabolism and microenvironment pH value. Other reports also show that BA could form complexes with carboxylates, which involve covalent interactions that are reversible in aqueous solution. For these reasons, we hypothesized the differences between tumor and normal cells microenvironment may cause differential uptake of boron/BA into the liver cancer cells prior neutron irradiation. Consequently, our experimental data implied glucose metabolic condition and acidic environment might regulate the uptake of boron/BA by the liver cancer cells.
Meanwhile, in order to accelerate the preclinical study of BA-mediated BNCT in established a zebrafish xenograft model with mouse melanoma cell line B16-F10 and adapted a transgenic zebrafish line that could develop spontaneous melanoma. We demonstrated that zebrafish could absorb boron in the delivery form of BA by orbital injection. A holding device was developed to immobilize the zebrafish during neutron irradiation. This novel zebrafish BNCT platform shall help us to understand the BA-mediated BNCT mechanism and speed up its translational application in the future.

中文摘要 I
Abstract II
致謝 IV
List of Figures VIII
List of Supplemental VIII
List of Table VIII
List of Abbreviations IX
1. Introduction 1
1.1 Type of cancer treatment 1
1.2 Boron neutron capture therapy 2
1.3 Hepatocellular carcinoma 3
1.4 Melanoma 5
1.5 Boric acid-mediated BNCT on mouse and rabbit liver cancer 5
1.6 Boron-carbohydrate interaction and cancer microenvironment 6
1.7 The benefit of zebrafish model for drug screening 7
1.8 The objectives of this study 8
2. Materials and methods 9
2.1 Cell and cell culture 9
2.2 Zebrafish line 9
2.3 Preparation of BA solution 9
2.4 Drug treatment 9
2.5 Evaluation of boric acid uptake in vitro 10
2.6 Evaluation of boric uptake in zebrafish 11
2.7 Xenotransplantation assay 11
2.8 Zebrafish melanoma tumor cell isolation assay 12
2.9 Zebrafish melanoma tumor cell transplant assay 12
2.10 Zebrafish melanoma tumor preservation 12
3. Results 13
3.1 Cell membrane N-glycans could not influence the uptake of boron under boric acid treatment. 13
3.2 Effect of glucose on boron uptake under boric acid treatment. 15
3.3 Acidic environment might influence HepG2 boron uptake of boron under boric acid treatment. 16
3.4 Novel zebrafish platform for the preclinical study of BNCT 16
3.4.1 Zebrafish tumor xenograft assay for BNCT 16
3.4.2 Transgenic Zebrafish could develop spontaneous melanoma 17
3.4.3 Zebrafish could uptake boron after boric acid treatment 18
3.4.4 Zebrafish holding device for BNCT 18
4. Discussion 20
4.1 Selection of assay standard for inductively coupled plasma-mass spectrometer influenced the analysis 22
4.2 N-glycans might not influence the boron uptake in liver cancer cell culture under boric acid treatment 22
4.3 Tumor glycolytic metabolism might affect boron uptake and the tumor-to normal tissue ratio 23
4.4 The acidic tumor microenvironment might enhance boron uptake 24
4.5 The aquaporin might act as a transporter of boric acid into tumor, normal tissue and other organs 25
4.6 Effect of sampling time point and transient boron retention in the cells 25
4.7 Zebrafish model for boron drug research and development 26
4.8 Summary and perspective of this study 27
Reference 28

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