臺灣博碩士論文加值系統

紫外線的曝曬是促使皮膚老化以及皮膚癌的原因之一。許多研究已經證明表皮細胞在紫外線的曝曬下，會活化細胞外訊息調節激酶（ERK）、c-Jun氨基末端激酶（JNK）、p38等分裂原活化蛋白激酶（MAPK）的磷酸化表現，更進一步會造成第二型環氧化酶（COX-2）的表現增加，導致皮膚發炎並造成皮膚癌形成。而紫外線Ｂ的刺激會降低第三型水通道蛋白（AQP-3）在皮膚的表現，並影響水分的傳輸，造成皮膚乾裂。黃酮類的白楊素（chrysin）已被證實可以抗氧化，並抑制細胞發炎以及腫瘤的生長。此外，有研究指出白楊素可以降低紫外線Ａ對人類真皮纖維母細胞所引起的傷害。然而，較少研究是探討白楊素對抗紫外線Ｂ的保護效果。因此，我們利用人類表皮細胞株，探討白楊素對抗紫外線Ｂ的保護作用，以及抗紫外線Ａ與抗紫外線Ｂ之相關作用機轉。結果顯示，表皮細胞在白楊素前處理後，可對抗紫外線的傷害，提高細胞的存活率。進一步探討其作用機轉，發現白楊素對於紫外線所活化的分裂原活化蛋白激酶表現，具有抑制其磷酸化的作用，例如p38與JNK。而且白楊素也會減少表皮細胞在紫外線刺激後所產生的第二型環氧化酶表現。此外，表皮細胞在白楊素前處理下，也可減少紫外線Ｂ所引起的細胞凋亡。另一方面，白楊素也能回復表皮細胞在紫外線Ｂ刺激後所減少的第三型水通道蛋白表現。由上述結果顯示，白楊素可預防紫外線Ａ與紫外線Ｂ對皮膚造成的傷害，因此有助於改善紫外線在皮膚造成的損傷。

UV irradiation is one cause of skin aging and skin cancer. Numerous studies have demonstrated UV exposure result in phosphorylation of extracellular signal-regulated kinases (ERK), p38 kinase, and c-Jun NH2-terminal kinases (JNK) mitogen-activated protein kinases (MAPK) in epidermal keratinocytes. Moreover, UV exposure can induce cyclooxygenase-2 (COX-2) expression and lead to skin inflammation and further cause of skin cancer. What is more, UVB induces aquaporin-3 (AQP-3) down-regulation in human skin keratinocytes, influences water transporting, and causes the dehydration of skin. Chrysin, a flavone, has been proved its efficacy on antioxidant, anti-inflammatory and anti-tumor proliferation. In addition, chrysin has been demonstrated its photoprotective effect in UVA-irradiated human dermal fibroblast. However, little attention has been paid on the protective effect against UVB-induced injury in keratinocytes. Therefore, in this study we investigate underlying protective effect of chrysin on UV-induced damage on HaCaT keratinocytes. We found that chrysin significantly increased cell viability and attenuated the phosphorylation of MAPK on HaCaT keratinocytes after exposure to UVA or UVB. Furthermore, chrysin could decrease the COX-2 expression and reduce cell apoptosis after UV exposure. Chrysin also reversed UVB-induced down-regulation of AQP-3. These data demonstrate that chrysin can prevent UV-induced damage and may be beneficial in the prevention of UV-induced skin injury.

目 錄
中文摘要……………………………………………………………………………IX
英文摘要……………………………………………………………………………X
第一章 緒言………………………………………………………………………1
第二章 文獻回顧…………………………………………………………………4
第一節　皮膚的構造……………………………………………………………4
第二節　紫外線造成皮膚細胞的傷害…………………………………………7
第三節　紫外線影響細胞內的訊息傳遞路徑…………………………………13
第四節　第二型環氧化酶的角色…………………………………………………17
第五節　第三型水通道蛋白對於皮膚的作用……………………………………18
一、水通道蛋白的分類…………………………………………………18
二、水通道蛋白的功能…………………………………………………18
三、第三型水通道蛋白在皮膚的功能…………………………………19
四、第三型水通道蛋白的作用機轉……………………………………19
第六節　白楊素之藥理作用………………………………………………………21
第三章　研究動機與目的……………………………………………………………23
第四章　材料與方法…………………………………………………………………24
第一節 材料………………………………………………………………………24
第二節 儀器………………………………………………………………………25
第三節　方法………………………………………………………………………26
一、細胞培養………………………………………………………………26
二、初代表皮細胞培養……………………………………………………26
三、細胞存活率測試（MTT assay）……………………………………27
四、流式細胞儀檢測………………………………………………………28
五、西方墨點法分析………………………………………………………30
六、細胞核蛋白分離方法…………………………………………………32
七、細胞實驗研究方法……………………………………………………34
八、動物體外試驗…………………………………………………………34
九、動物體內試驗…………………………………………………………35
十、統計方法………………………………………………………………37
第五章 實驗結果……………………………………………………………………39
第一節 細胞實驗結果……………………………………………………………39
一、白楊素預防紫外線Ｂ對表皮細胞株的傷害………………………………39
（一）白楊素對於表皮細胞的抗紫外線效果…………………………………36
（二）白楊素增加初級培養之表皮細胞在紫外線B照射後的細胞存活率……40
（三）白楊素對於紫外線Ｂ誘發H2O2產生的抑制情況………………………40
（四) 抑制分裂原活化蛋白激酶之磷酸化……………………………………41
（五）發炎相關機轉之探討……………………………………………………41
（六）第三型水通道蛋白表現…………………………………………………42
（七）對於細胞凋亡的影響……………………………………………………42
二、白楊素對於紫外線Ａ的保護效果……………………………………………44
（一）白楊素對於表皮細胞株之抗紫外線A效果………………………………44
（二）對於紫外線Ａ誘發H2O2產生的抑制情況………………………………44
（三) 抑制紫外線Ａ活化分裂原活化蛋白激酶路徑之探討…………………44
（四）發炎相關機轉之探討……………………………………………………45
三、白楊素對於癌細胞之抗增生效果…………………………………………46
第二節 動物實驗結果……………………………………………………………47
一、離體經皮吸收實驗……………………………………………………………47
二、活體皮內藥物含量測定………………………………………………………48
三、安全性…………………………………………………………………………49
第六章　討論……………………………………………………………………………50
第七章　結論……………………………………………………………………………53
第八章　圖表……………………………………………………………………………55
參考文獻…………………………………………………………………………………85

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