臺灣博碩士論文加值系統

　　人體皮膚表面覆蓋著一層由皮脂腺所分泌的皮脂薄膜，其成分包括triglyceride、wax esters、squalene及少量的cholesterol ester。皮脂由皮脂腺分泌出來到皮膚表面的過程之中，細菌會分解部分的triglyceride而形成free fatty acid和少量的mono-和di-glycerides。皮脂在生理上具潤滑及抗菌功能，但文獻上對於皮脂在皮膚障壁功能所扮演的角色鮮有記載，曾有報告指出皮脂會增加皮膚滲透性，依電子繞射結果顯示皮脂改變角質層脂肪的晶形排列，使其由orthorhombic轉為hexagonal。本實驗室近期在人體上的研究結果顯示，塗抹人類皮脂於前臂內側兩小時後，親水性藥物穿透角質層的滲透性增加約3.5倍，但是對於親脂性藥品沒有影響。而在額頭部位去除皮脂後，則造成親水性藥物小幅但有意義的下降。而臨床上易伴隨有脂漏的相關疾病，其所觀察到的表皮障壁功能及表皮角質層結構異常，是否與皮脂分泌過多導致的表皮障壁破壞相關還有待進一步證實。
　　近來拉曼光譜運用於生物醫學方面，其光譜波段即代表結構中化學鍵振動的特徵。由於拉曼光譜是屬於非侵入性的且樣品的前處理過程簡單，可提供正常及病態皮膚其相對的分子結構資訊。如合併共軛焦顯微鏡，更可提供皮膚結構空間上的解析。
　　本研究的目的在於以無毛鼠的動物模式，來探討長時間給予人類皮脂及其個別成分後，它們對皮膚障壁及結構上的影響。研究結果顯示，每天持續給予人類皮脂會造成無毛鼠皮膚表面增生形成過多皮屑，經皮水分散失於一週內增加至3-4倍，而表皮含水量則減少至正常的三分之一，之後無明顯改變。停止皮脂給予後，皮膚障壁功能可在五天內逐漸恢復正常；顯微染色方面的觀察顯示於給予皮脂後表皮增生及角質層增厚且鬆散，細胞間隙脂質結構中，脂肪分子分佈異常，電子顯微鏡觀察亦顯示角質細胞間脂肪，由平行之膜狀結構變為散亂之非定形結構，免疫染色顯示發炎因子IL-1α、IL-1β表現隨表皮障壁破壞的程度而呈現平行的增加。另外塗抹不飽和脂肪酸亦造成皮膚障壁功能的降低，表皮增生及角質層細胞間隙脂質結構的改變，因而我們推測影響皮膚障壁功能及結構的可能是皮脂中的不飽和脂肪酸。
　　拉曼光譜分析利用皮膚內生的phe強度的改變評估持續給予皮脂五天後的皮膚，亦顯示了皮膚厚度的增加，與顯微染色觀察到的結果一致。皮膚內脂肪分子trans排列的波峰位置之波數增加，顯示脂肪分子亂度增加，但僅侷限於皮膚最表層。分析蛋白質分子之CH2 scissoring及amide I α-helix結構並無明顯變化。然而受限於儀器之配備，無法明確定位光譜吸收之皮膚組織位置，影響了拉曼光譜分析在皮膚結構空間解析上之準確性。
　　以上結果為探討皮脂影響表皮障壁功能及結構之研究，將有助於之後進一步探討與脂漏有關疾病如脂漏性皮膚炎、頭皮屑、痤瘡等相關疾病之機轉研究。

　　The human skin surface is covered with a fluid film of sebum, secreted by sebaceous glands associated with hair follicles. Human sebum is primarily composed of triglycerides, wax esters, squalene and small amounts of cholesterol esters. During passage of sebum through the hair canal, bacterial enzymes hydrolyze some of the glycerides, so that the lipid mixture reaching the skin surface contains free fatty acids and small proportions of mono-and di-glycerides. Studies on the role of sebum in the barrier function have been scarce. It has been demonstrated that sebum may increase skin permeability. Electron diffraction studies revealed that, the sebaceous lipid may interact with human SC lipid, resulting in a shift in lattice packing pattern from orthorhombic to hexagonal. Our previous studies demonstrated that sebum supplement increased the SC permeability of hydrophilic molecules at the forearm, but not that of lipophilic molecules. Sebum removal at the forehead has small, but significant effect on the SC permeability of hydrophilic molecules.
　　Raman spectroscopy has been applied in biomedical studies with the advantage of non-invasivity and simplify of sample preparation. When combined with confocal microscopy, the technique can be permit the collection of detailed information about molecular composition of the skin with high spatial resolution.
　　The objectives of the studies were to demonstrate in mouse model, the chronic effects of sebum and its components on skin barrier function an morphology. The results demonstrated that daily treatment of human sebum on the flank skin of hairless mice gradually increased the transepidermal water loss (TEWL) which reached a maximal on the 6th day (18.0±3.0 g/m2/h). On the other hand, skin hydration was decreased from 94.8±6.5 a.u. to 33.5±6.0 a.u. in 4 days. Both TEWL and skin hydration have gradually returned to their normal values after cessation of sebum treatment.
Effects of sebum on skin barrier morphology were examined using H&E, Nile red, safranin staining, and Ruthenium electron microscopy. Daily application of sebum on hairless mouse skin induced epidermal proliferation and abnormal neutral lipid distribution in the stratum corneum. Electron microscopic examinations have revealed alterations in intercellular membrane structures as a result of sebum treatment. Immunochemistry staining revealed extensive expression of cytokines - IL-1α and IL-β in the epidermis.
　　Using Phe intensity as the internal marker, confocal Raman spectra demonstrated the increase in the thickness of epidermis due to sebum treatment, in accord with the results from microscopic examinations. The band position of “trans” from lipids in Raman spectra were observed to shift to high wave numbers, at the utmost skin surface, indicating the change in lipid disorder. However, no changes in the peak positions of CH2 scissoring and amide I α-helix originating from protein molecule were found. The accuracy of the spatial resolution was compromised by the inadequacy of instrument setup current.
　　Application of the single lipid component – palmitoleic acid, an unsaturated fatty acid, induced skin barrier disruption, epidermal hyperplasia and abnormal lipid distribution in the SC, similar to sebum treatment, but not the application of triglycerides (triolein and tripalmitin). The effects of sebum on the skin barrier are mainly attributed to its unsaturated fatty acid compon- ents.
　　The studies provide insights into the pathophysiology of skin abnormal- ity related to excess sebum production.

中文摘要……………………………………………………………………i
英文摘要…………………………………………………………………iv
誌謝………………………………………………………………………vii
目錄…………………………………………………………………………iix
表目錄………………………………………………………………………xi
圖目錄……………………………………………………………………xiii
縮寫表…………………………………………………………………xvii
第一章　文獻回顧……………………………………………………1
第一節　人體皮膚的結構與功能………………………………… 1
第二節　皮脂之組成、分泌及功能………………………………11
第三節　皮脂對皮膚障壁的影響…………………………………15
第四節　不飽和脂肪酸對皮膚障壁功能的影響…………………16
第五節　拉曼光譜之原理與應用…………………………………18
第二章　研究目的…………………………………………………24
第三章　研究材料及儀器…………………………………………26
第一節　實驗動物…………………………………………………26
第二節　研究材料…………………………………………………26
第三節　實驗儀器…………………………………………………29
第四節　分析軟體…………………………………………………30
第四章　研究設計…………………………………………………31
第一節　皮脂之收集與分析………………………………………31
第二節 動物實驗……………………………………………………33
1.　人類皮脂的添加…………………………………………………33
2.　皮脂所含脂肪成分的添加………………………………………34
第三節　皮脂對皮膚障壁功能的影響………………………………35
第四節　皮脂對於皮膚組織學上的影響……………………………37
1.　光學顯微鏡的樣品處理法………………………………………37
2.　Hematoxyline and Eosin染色…………………………………38
3.　Safranin 染色……………………………………………………39
4.　Nile red 染色……………………………………………………39
5.　Ruthenium tetroxide染色………………………………………40
6.　免疫組織化學染色…………………………………………………40
第五節　皮膚拉曼光譜分析技術………………………………………42
1.　共軛焦拉曼光譜儀器…………………………………………42
2.　皮膚檢品製備流程……………………………………………42
3.　光譜掃描條件…………………………………………………43
4.　光譜分析方法…………………………………………………44
第五章　研究結果………………………………………………………49
　　第一節 皮脂之收集與分析…………………………………………49
　　第二節 人體皮脂對皮膚障壁功能的影響…………………………52
　　第三節 皮脂對於皮膚組織學上的影響……………………………58
第四節　皮脂對皮膚拉曼光譜的影響……………………………………70
　　第五節 單一脂肪成分對皮膚的影響………………………………87
1.　不同脂肪的添加對皮膚障壁功能的影響……………………87
2.　不飽和脂肪酸Palmitoleic acid的影響……………………92
第六章　討論………………………………………………………………99
第一節　皮脂成分含量分析………………………………………99
第二節　皮脂對皮膚障壁功能的影響……………………………100
第三節　皮脂對於皮膚組織學上的影響…………………………101
第四節　皮脂對皮膚拉曼光譜的影響……………………………104
第五節　單一成分脂肪酸對於皮膚的影響………………………109
第六節　結論……………………………………………………………111

參考文獻…………………………………………………………………113

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