肝細胞癌為人類常見的肝癌，同時是致死率前十大的癌症。目前對於肝癌的發生路徑研究還並未非常明確，但已知肝纖維化為肝細胞癌最嚴重的致癌因子，約80~90%肝細胞癌患者同時患有肝纖維化。近期研究發現了一種新型肝癌原致癌基因ISX，此種基因會被肝纖維化相關細胞因子IL-6激發，並對癌細胞生長,轉移與免疫抑制等功能有調控的效果。對ISX進一步的研究可能可以對肝癌的致癌機制獲得突破性的了解，或對早期肝癌診斷提供協助。本團隊藉由結合氧化鐵奈米粒子、螢光顯影劑CyTE777與對ISX專一性抗體，合成出具有標靶化顯影功能的多功能顯影奈米粒子。合成出來的粒子大小約為20奈米，其在細胞實驗測試中顯示出良好的生物相容性與物理穩定度，並在之後的細胞影像測試中展現出良好的標靶化螢光顯影與磁振造影解析度強化功能。在異體腫瘤移植的裸鼠上同樣表現出良好的活體核磁共振影像解析度增強的效果。

Hepatocellular carcinoma or HCC is the most common primary liver cancer. Although detailed oncogenesis is unknown, cirrhosis is considered to be the highest risk factor for the development of HCC. About 80 to 90% HCC cases was reported to occurred in the setting of cirrhosis. Recent research suggests that intestine specific homeobox (ISX) is a proto-oncogene for hepatocellular carcinoma which is triggered by cirrhosis related cytokines IL-6. Also, ISX is reported to have regulator effect on multiple cancer related cell functions including proliferation, transformation and immunosuppression. Further research on ISX could be important for advancing knowledge about HCC oncogenic pathway and possible early diagnostic of hepatocellular carcinoma. Nanoparticles (MnMEIO-(ISX)-CyTE777-mPEG) with fluorescence dye and iron oxide nanoparticals that specifically targeting ISX homeodomain were synthesized. Diameter of this particle is identified as approximately 20 nm which is suitable to enter the cell. And was studied for in vivo and in vitro imagine property. The result demonstrated that this nanoparticle is able to target and mark ISX homeodomain in vitro and in vivo, and have confirmed functional on both optical and MR imiging contrast enhancement.

VII

Contents
摘 要 ..................................................................................................... I
Abstract ................................................................................................... III
致 謝 .................................................................................................... V
Contents ................................................................................................. VII
Table of Figures ...................................................................................... IX
List of abbreviations .............................................................................. XI
1. Introduction ........................................................................................ 1
1.1. Hepatocellular carcinoma ....................................................... 1
1.2. Diagnosis method of hepatocellular carcinoma .................... 1
1.3. Intestine specific homeobox (ISX) .......................................... 2
1.4. MR imaging .............................................................................. 3
1.5. MnMEIO nanoparticle............................................................ 4
2. Project Aim ......................................................................................... 6
3. Material and Method ......................................................................... 7
3.1. Instrumentation ....................................................................... 7
3.2. Reagents .................................................................................... 7
3.3. Antibodies ................................................................................. 8
3.4. Animals ..................................................................................... 8
3.5. Methods .................................................................................... 8
3.5.1. Cell culture ............................................................................ 8
3.5.2. Whole cell lysates .................................................................. 9
3.5.3. Protein assay ......................................................................... 9
3.5.4. SDS-polyacrylamide gel electrophoresis ............................ 9
3.5.5. Western blot analysis ......................................................... 10
3.5.6. Confocal Microscopy.......................................................... 11
3.5.7. Confocal microscopy using living cell .............................. 11
3.5.8. Synthesis of MnMEIO-(ISX)-CyTE777-mPEG
nanoparticle ...................................................................................... 11
3.5.9. Dynamic light scattering analysis ..................................... 12
3.5.10. UV absorbance analysis ..................................................... 12
3.5.11. Cell viability ........................................................................ 12
3.5.12. Prussian blue stain ............................................................. 13
3.5.13. Cell preparation for in vitro optical and MR imaging ... 13
3.5.14. In vivo MR imaging study ................................................. 14

4. Results and Discussion ..................................................................... 14
4.1. The property of tetracycline inducible ISX system of
Sk-Hep1 cell ...................................................................................... 15
4.2. Characterization of MnMEIO-(ISX)-CyTE777-mPEG
nanoparticles..................................................................................... 18
4.3. MnMEIO-(ISX)-CyTE777-mPEG nanoparticle has no cell
toxicity ............................................................................................... 21
4.4. MnMEIO-(ISX)-CyTE777-mPEG nanoparticle is able to
specifically target ISX ...................................................................... 22
4.5. MnMEIO-(ISX)-CyTE777-mPEG nanoparticle can target
and mark ISX in cytoplasm of living cells ..................................... 24
4.6. In vivo MRI imaging analysis ............................................... 30
5. Conclusion......................................................................................... 32
6. References ......................................................................................... 33

33

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