PART Ι. 藉由離氨酸及半胱氨酸標定的二維差異電泳分析紫外光照射所引起之蛋白質體的表現量變化及巰基變化

紫外線波長位於280-320 nm是到達地球表面上最具能量且最具傷害性的紫外線種類。很多文獻指出暴露在紫外光 (UVB) 下會導致一些皮膚病徵，例如光老化作用和皮膚癌。這些原因可能是由於紫外光 (UVB) 直接破壞細胞內的去氧核糖核苷酸或是促使細胞產生活性氧物質 (Reactive oxygen species, ROS) 而導致在目標蛋白質的半胱氨酸殘基上產生氧化修飾作用，使蛋白質失去功能，進而干擾下游訊息傳遞路徑。然而，紫外線如何產生皮膚癌的分子作用機制尚未明瞭。因此，在此研究中，我們將正常的人類皮膚纖維母細胞 (CCD-966SK) 照射不同劑量的紫外光，並利用離氨酸及半胱氨酸標定的二維差異電泳結合基質輔助雷射脫附游離飛行式質譜儀 (MALDI-TOF-MS) 鑑定出具有表現量差異且具有巰基變化的蛋白質。結果顯示，89個蛋白質具有明顯表現量的差異而37個蛋白質在半胱氨酸殘基上具有巰基的變化。在所有鑑定到的蛋白質當中，包括參與細胞內蛋白質的摺疊 (例如：NPM)、氧化還原調控 (例如：Prxns)、核苷酸生和成、生長調控 (例如：PHB) 和細胞轉移 (例如：cofilin-1) 有關的蛋白質在紫外光照射後其表現量明顯上升，而參與蛋白質的生合成和蛋白質裂解 (例如：PIAS 1) 其表現量明顯下降。除此之外，一些細胞骨架、代謝和訊息傳遞相關的蛋白質具有明顯巰基的變化。這些經由紫外光照射後具有表現量差異且具有巰基變化的蛋白質進一步利用西方點墨法、酵素連結免疫分析法及免疫沉澱法做驗證，並且討論這些蛋白質在紫外光所引起的皮膚癌其早期形成所扮演的角色。總結來說，本實驗鑑定到許多細胞內經由紫外光調控的蛋白質和具有氧化還原變化的蛋白質是，這些蛋白質可以當作藥物設計的目標分子，進而避免由紫外光所引發的癌症及皮膚老化。

PART Π. 透明質酸在鹼傷害的角膜細胞中所扮演的保護作用

透明質酸 (hyaluronic acid, HA) 是一種高分子量的糖胺也是胞外基質不可或缺的成分，並參與細胞的增生、遷移和傷口癒合等許多功能。角膜位於眼睛的最外層，是最容易受到家用清潔劑及眼藥水添加物等鹼性物質傷害的部位。許多文獻指出，在動物實驗中，透明質酸有助於鹼傷害的角膜細胞傷口癒合的作用，但是其體外實驗及分子作用機制尚未有人研究。因此，此實驗的主要目的是利用體外實驗的研究方式，探討透明質酸在鹼傷害的角膜細胞中的保護作用。我們將人類角膜上皮細胞株 (HCE-2) 處理氫氧化鈉及不同濃度低分子量 (100 千道耳吞) 與高分子量 (1000 千道耳吞) 的透明質酸，並利用各種功能性分析和蛋白質體學方式探討透明質酸所引發的細胞反應。實驗結果顯示，透明質酸確實有促進角膜細胞存活及傷口癒合的能力，而且在經由鹼處理角膜細胞後，高分子量的透明質酸明顯降低鹼對細胞的傷害，並經由細胞骨架的調控，促進細胞的遷移跟傷口癒合。此外，二維差異電泳結合基質輔助雷射脫附游離飛行式質譜儀 (MALDI-TOF-MS) 的實驗結果發現，高分子量的透明質酸保護角膜細胞免於鹼的傷害且促進細胞傷口癒合的作用機制可能是經由調控細胞內蛋白質的生合成、裂解、運輸和細胞遷移路徑。總結來說，本實驗證明高分子量的透明質酸能有效的促進鹼傷害後的角膜上皮細胞癒合，並提出一個透明質酸促進角膜細胞修復的可能機制。

PART Ι. Proteomic analysis of UVB-induced protein expression and thiol-reactivity alterations in skin fibroblasts using lysine- and cysteine-labeling two-dimensional differential gel electrophoresis

UVB is the most energetic and DNA-damaging of the Ultraviolet (UV) irradiation that reaches the earth surface. Many previous reports suggested that exposure to the UVB irradiation causes skin pathologies like photoaging and skin cancer due to the direct damage on DNA molecules and the generation of reactive oxygen species (ROS) which interfere downstream signaling cascades via post-translational modifications of cysteine-residues in target proteins resulting in losing their biological functions. However, the detailed molecular and biochemical mechanisms that lead to skin cancer by UVB are yet to be clarified. In this study, we treated normal skin fibroblast cells (CCD-966SK) with various doses of UVB and monitored the protein expression and thiol-reactivity changes with lysine- and cysteine-labeling 2-D DIGE and MALDI-TOF MS. Our approaches revealed that 89 and 37 identified proteins showed significant changes in protein expression and thiol-reactivity of cysteine residues, respectively. Many identified proteins involving in protein folding (NPM), redox-regulation (Prxns), nucleotide biosynthesis, growth regulation (PHB) and cell migration (cofilin-1) are up-regulated under UVB irradiation. In contrast, proteins responsible for biosynthesis and protein degradation (PIAS 1) are down-regulated. In addition, proteins involving cytoskeleton, metabolism and signal transduction were shown to be altered in their thiol-reactivity. Furthermore, the UVB-induced protein expression and thiol-reactivity changes were validated with western blotting, ELISA and immunoprecipitation as well as discussed their roles on the early event of UVB-induced skin cancer formation. To sum up, we have identified numerous cellular proteins and redox-regulated proteins which are modulated by UVB irradiation. These identified proteins might play important roles on the early stages of skin cancer formation induced by UVB irradiation and might be potential targets for the rational design of drug to prevent UVB-induced tumorigenesis and potoaging.

PART Π. Hyaluronic acid-dependent protection on alkali-damaged corneal cells

Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan served as an essential extracellular matrix component and involved in many biological functions including cell proliferation, migration and wound healing. Cornea locates at the outer surface of eye, which suffers more alkali-injuries from household detergents or eye drop augmentations. Many reports revealed that hualuronic acid promote the wound healing of alkali-damaged corneal cells in animal models but the in vitro experiments and detail mechanisms were not to be clarified. The aim of this study is to in vitro investigate the protective process of hualuronic acid in alkali-damaged corneal cells. Accordingly, human corneal epithelial cell lines were treated with NaOH and two different molecular weights hyaluronic acid (100 kDa and 1000 kDa) in various concentrations. Many functional assays and proteomic analysis were performed to investigate the cell responses to hyaluronic acid treatment. Our study suggested that hyaluronic acid indeed stimulates corneal cell viability and wound healing ability. After alkali-damage, high molecular weight hyaluronic acid significantly reduced alkali-induced cell damage and enhances cell migration and wound healing which were modulated by cytoskeleton in the immune fluorescent data. Moreover, 2-D DIGE coupled with MALDI-TOF-MS and MALDI-TOF/TOF-MS analysis showed that high molecular weight hyaluronic acid protects corneal cell against alkali-injuries and stimulates cell wound healing might be due to the modulation of multiple cellular pathways including protein biosynthesis, degradation, trafficking and cell migration. To sum up, we demonstrate that high molecular weight hyaluronic acid facilitates corneal epithelial wound healing after alkali-injuries and suggest the possible mechanisms of hyaluronic acid-induced repair in corneal cells.

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