臺灣博碩士論文加值系統

太陽光的紫外線是引起皮膚傷害的主要環境來源。UVA 紫外線 ( 320~400 nm ) 可透過活性氧生成傷害微小分子，例如：核酸、脂質與蛋白質。膠原蛋白是脊椎動物組織提供張力的成分。也可維持皮膚的正常結構與功能。 而 vimentin屬於中間絲蛋白質的其中之ㄧ，vimentin修飾的生物影響跟纖維母細胞收縮能力有關，由中間絲系統的結構破壞所引起的變化與老化的過程也有相關。 此研究目的主要使用雷射掃描式共軛焦顯微鏡研究正常人類纖維母細胞與葡萄糖六磷酸氫酶缺乏之纖維母細胞在接收不同劑量UVA紫外線後，第一型膠原蛋白及vimentin在細胞中的分布情形，並且用影像軟體做半定量，提供兩種標的蛋白影像資訊，取代傳統的西方墨點法。在照UVA紫外線前，第一型膠原蛋白與vimentin在正常人類纖維母細胞的表現比葡萄糖六磷酸氫酶缺乏之纖維母細胞多。處理UVA紫外線後，第一型膠原蛋白或vimentin的螢光強度表現反映了第一型膠原蛋白或vimentin在兩株細胞內有依UVA劑量增加而下降的趨勢。有趣的是，在影像圖片中，葡萄糖六磷酸氫酶缺乏之纖維母細胞由UVA引起螢光強度的減弱比正常人類纖維母細胞還嚴重。整體而言，新發展的共軛焦顯微影像技術應用於皮膚纖維母細胞的形態生物標記也可以延伸到其它的生物化學研究。

The ultraviolet ( UV ) radiation in sunlight is the major environmental cause of skin damage. The UVA ( 320-400 nm ) irradiation may damage the micromolecules, such as nucleic acids, proteins and lipids, chiefly through the generation of reactive oxygen species ( ROS ). Collagen has been known as the principal tensile element of vertebrate tissues, such as tendon, cartilage, bone and skin. Collagen also maintains the normal structure and function of skin. Vimentin is one of intermediate filament proteins, and its biological impact of the identified vimentin modification is related to the loss of contractile capacity of fibroblasts caused by the structural breakdown of the intermediate filament system, leading to acceleration of aging process. This study aims to use laser-scanning confocal microscope to investigate the distribution of type I collagen and vimentin and semi-quantitate their content by image processing software to provide imaging information of the two target proteins in place of traditional western blot method in normal human foreskin fibroblast ( HFF3 ) and G6PD-deficient human foreskin fibroblast ( HFF1 ) receiving different fluence UVA irradiation. Prior to UVA irradiation, the type I collagen and vimentin expression in HFF3 cells are distinctly higher than those in HFF1. After UVA treatment, both types of the cells exhibited a fluence-dependent drop in fluorescence intensities of type I collagen -FITC or vimentin-FITC, which reflected a decrease of content of type I collagen or vimentin in a dose-dependent manner. Interestingly, the extent of lost fluorescence intensity induced by UVA from acquired images in HHF1 cells is more severe than in HHF3. Collectively, our newly developed confocal imaging technique for phenotypic biomarkers of skin fibroblasts can also be extended to other biochemical researches.

指導教授推薦書 …………………………………………………… i
口試委員會審定書 ………………………………………………… ii
授權書 ……………………………………………………………… iii
誌謝 ………………………………………………………………… iv
中文摘要 …………………………………………………………… v
英文摘要 …………………………………………………………… vi
目錄 ……………………………………………………………… viii
第一章 簡介 ……………………………………………………………1
1.1 皮膚的組成及構造 …………………………………………… 1
1.2皮膚的老化 …………………………………………………… 1
1.3紫外線 ………………………………………………………… 2
1.4紫外線產生活性氧物質 …………………………………………3
1.5膠原蛋白的結構及組成 ……………………………………… 7
1.6脊椎動物膠原蛋白分類 ……………………………………… 7
1.7膠原蛋白家族 ………………………………………………… 10
1.8第一型膠原蛋白 ……………………………………………… 13
1.9 Vimentin ……………………………………………………… 15
1.10葡萄糖六磷酸去氫酶 ……………………………………… 16
1.11研究目的 ………………………………………………… 19
第二章 實驗材料與方法 …………………………………………… 20
2.1 材料 ………………………………………………………… 20
2.1.1 細胞株 ………………………………………………… 20
2.1.2 細胞培養基材料 ……………………………………… 21
2.1.3 UV box ………………………………………………… 21
2.1.4 抗體 …………………………………………………… 22
2.2 實驗方法 …………………………………………………… 22
2.2.1 細胞培養 ……………………………………………… 22
2.2.2 流式細胞儀 …………………………………………… 22
2.2.3 雷射掃描式共軛焦顯微鏡 …………………………… 23
第三章 結果 ………………………………………………………… 24
3.1鑑定細胞來源為人類纖維母細胞 …………………………… 24
3.2觀察正常人類纖維母細胞 ( HFF3 ) 及葡萄糖六磷酸氫酶缺乏
之纖維母細胞 ( HFF1 ) 膠原蛋白的表現及含量 ……………24
3.3不同UVA劑量對HFF1與HFF3細胞表現膠原蛋白的差異 … 24
3.4觀察HFF1與 HFF3表現vimentin的分佈及含量 …………… 25
3.5不同UVA劑量對HFF1與HFF3細胞表現vimentin的差異 … 26
第四章 結論 ………………………………………………………… 27
第五章 討論 ………………………………………………………… 28
第六章 參考資料 …………………………………………………… 31
第七章 圖表 ………………………………………………………… 37

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