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研究生(外文):Hsiao Yun Kuo
論文名稱(外文):Chitosan Coated Superparamagnetic Iron Oxide as Potential Magnetic Resonance Contrast Agents
指導教授(外文):C. R. Shen
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超順磁性氧化鐵奈米粒子 (superparamagnetic iron oxide nanoparticles) 因具有超順磁性,而被應用在磁振造影,增強磁共振對比。本研究專題所使用的CSPIO (chitosan coated superparamagnetic iron oxide),為表面包覆幾丁聚醣的超順磁性氧化鐵,由於幾丁聚醣具有良好的生物相容性及生物可分解性,與市售常用的SPIO以葡萄聚醣包覆的粒子做比較,具有胺基的化學基團可與其他常見的生物分子做結合,應有更多的應用潛力。實驗結果則證明了CSPIO在磁振造影中具有隨濃度增加而T2影像訊號下降的性質,其R2/R1值也顯示出CSPIO具有較好的鬆弛對比能力。緊接著在吞噬CSPIO小鼠巨噬細胞及其所造成細胞型態及功能影響的分析上,發現單純以光學顯微鏡或進一步以電子顯微鏡皆可觀察到巨噬細胞進行吞噬現象的行為。所以進一步檢測CSPIO對吞噬細胞的細胞毒性,結果證明低濃度或高濃度的CSPIO,並未明顯造成細胞死亡,而這些細胞正常分泌IL-6、IL-10及MCP-1的分泌功能雖出現抑制的現象,但其分泌IL-12與TNF-α的功能則無顯著的影響。同時CSPIO標幟的活體試驗評估,也得到成功的MRI影像結果,顯示這些低細胞毒性、以幾丁聚醣包覆的超順磁性氧化鐵是極具潛力的磁振造影對比顯影劑。
Superparamagnetic iron oxide (SPIO) nanoparticles have been developed as MRI contrast agents. Upon systemic application, SPIO particles are preferentially internalized by macrophages. Chitosan coated superparamagnetic iron oxide (CSPIO) was developed and utilized in the study due to the unique characteristics including nature material, good biocompatibility and biodegradability. In contrast to the FDA approved SPIO, Resovist ®, CSPIO have the amine function group (-NH2) which enables lots of the chemical modification leading to a variety of future applications of MRI. First, CSPIO were tested for the imaging modality of MRI with a 3T scanner. It showed that CSPIO apparently harbor the property of descending with the density increment and T2 image signal. As a result, its R2/R1 value displays the better ground to relax contrast ability. When the macrophage cells, RAW 264.7, were cultured in the presence of CSPIO, internalization of CSPIO was visualized and recorded under microscope analysis. The effects of CSPIO internalization on cell toxicity and function were also investigated. No or little cytotoxicity of CSPIO was noted while high doses of Resovist ® presented relatively high cytotoxic effect. The phagocytic function was significantly reduced as a result of SPIO internalization. Moreover, the secretion of inflammatory cytokines such as IL-6, IL-10 and MCP-1 was partially inhibited although there seemed to be less inhibitory effect on IL-12 and TNF-α production. Although the CSPIO internalization mechanisms and efficiency by different cell types remain unclear, several cells were found capable of uptaking CSPIO. In vivo study of CSPIO labeled islet grafting further indicated the potent contrast agent of CSPIO on cell tracking by MRI. In conclusion, CSPIO with the high-relaxivity of MRI and low cytotoxicity is with the great potential as a MR contrast agent.
授權書 iii
誌謝 iv
摘要 v
縮寫表 vii
目錄 viii
第一章 研究動機與背景 1
第一節 磁振造影 2
第二節 磁振造影之對比顯影劑 4
第三節 超順磁性氧化鐵 5
第四節 幾丁聚醣 9
第五節 巨噬細胞 10
第二章 研究目的 14
第三章 實驗設計 15
第一節 CSPIO材料性質 15
第二節 In vitro實驗 15
第三節 In vivo實驗 16
第四章 材料與方法 17
第一節 CSPIO材料性質 17
第二節 In vitro實驗 18
第三節 In vivo實驗 22
第四節 統計方法 22
第五章 實驗結果 23
第一節 CSPIO材料性質 23
第二節 In vitro實驗 24
第三節 In vivo實驗 28
第六章 討論 29
第七章 參考文獻 36
表1. 超順磁性氧化鐵奈米粒子之基本特性 40
圖1. Resovist與SPIO不同劑量與T2訊號關係之結果 41
圖2. 評估CSPIO對組織訊號強度的影響 ( R2/R1比值 ) 42
圖3. 普魯士藍染色觀察小鼠巨噬細胞株對SPIO的吞噬情況 43
圖4. 標幟Resovist和CSPIO的TEM觀察結果 44
圖5. 標幟Resovist和CSPIO的SEM觀察結果 45
圖6. 標幟Resovist和CSPIO的細胞毒性分析結果 46
圖7. 標幟Resovist和CSPIO的巨噬細胞吞噬能力分析 47
圖8. 標幟不同濃度SPIO的RAW 264.7 分泌IL-10之分析 48
圖9. 標幟不同濃度CSPIO的primary cell分泌IL-10之分析 49
圖10. 標幟不同濃度SPIO的RAW 264.7分泌TNF-α之分析 50
圖11. 標幟不同濃度SPIO的RAW 264.7之CBA分析 51
圖12. In vivo小鼠胰島移植MRI影像 52
圖13. In vivo 小鼠胰島移植之TEM與普魯士藍染色 53
圖14. 幾丁聚醣之SEM觀察 54
圖15. 小鼠纖維母細胞 (3T3) 之普魯士藍染色 55
圖16. 小鼠大腸癌細胞株 (CT26) 之普魯士藍染色 56
附錄一. 幾丁質與幾丁聚醣之化學結構式 57
附錄二. 單核巨噬細胞於不同器官之名稱 58
附錄三. 巨噬細胞的吞噬作用 59
附錄四. 巨噬細胞的毒殺機制 60
附錄五. TEM常溫超薄樣品固定包埋之藥品配製 61
Textbook:Ray H. Hashemi, William G. Bradley, Christopher J. Lisanti, (2006). MRI the basics
Textbook :王政光 等著, 免疫學, (2004). 新文京開發出版社
Textbook : 許清祥 校閱, KUBY 免疫學, (2002). 合記圖書出版社
Data sheet : Schering AG, Germany, Resovist (Information for radiologists)
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