- 學位論文
探討維生素C對於鈣羥磷灰石促進膠原蛋白合成之研究
The study of vitamin C on calcium hydroxyapatite-induced collagen synthesis
摘要
紫外線 (Ultraviolet；UV) 是導致皮膚光老化 (Photoaging) 的主要原因。長期暴露在UV下，會使皮膚膠原蛋白降解 (Collagen degradation) ，導致皺紋生成 (Wrinkles generation) 及皮膚免疫力下降，嚴重更可能造成皮膚病變。近來研究指出光老化過程中，UVB會刺激纖維母細胞 (Fibroblast) 分泌基質金屬蛋白酶 (Matrix metalloproteinase；MMPs) 造成膠原蛋白的降解。因此，如何抑制膠原蛋白降解使皺紋減少生成成為重要的議題。鈣羥磷灰石 (微晶瓷Radiesse；Calcium Hydroxylapatite；CaHA) 是一種生物相容性的材料，用在牙科及骨科醫療上已使用數十年。目前也常使用於醫療美容，微晶瓷有填補功能，可改善皮膚凹陷、不飽滿的情況。微晶瓷亦可刺激纖維母細胞，使自體膠原蛋白增生，改善皺紋。維生素C (Vitamin C；Ascorbic acid) 是靈長類動物必需的營養素，也是一種抗氧化劑，過去研究也發現能促進膠原蛋白的生成。因此，本研究利用UVB照射裸鼠與人類真皮纖維母細胞WS-1細胞 (Human dermal fibroblast WS-1 cells) ，分析微晶瓷與維生素C對抗UVB導致光老化的分子機轉。 本研究利用動物模式及細胞模式，探討維生素C 對於微晶瓷促進膠原蛋白合成之功效，並分析其分子機制。在動物實驗中，使用BALB裸鼠，在背部注入微晶瓷並每日餵食1 %與2 %的維生素C並照射UVB (1.2 kJ/m2) ，每週3次，為期12週，犧牲後，取其血液及皮膚進行分析。實驗結果顯示，在血液生化數值方面，單獨餵食2 %維生素C組中的CHO (Total cholesterol) 、TG (Triglyceride) 的表現量顯著低於正常組。施打微晶瓷組與正常組相比並不影響血液生化值。在組織膠原蛋白染色結果發現，照射UVB使膠原蛋白層減少，但照射UVB+維生素C組、UVB+微晶瓷組及UVB+維生素C+微晶瓷組都可以使膠原蛋白層增加。再以西方墨點法分析皮膚組織膠原蛋白蛋白也有相同結果。再詳細分析分子機制發現，上述三組與單獨照射UVB組相比，結果皆能透過活化TIMP-1/2來抑制MMP-1/8/13 (collagenases) 及MMP-2/9 (gelatinases)的活性表現，使MMPs維持在不活化態的Pro-MMPs，因而增加膠原蛋白的含量。在細胞實驗中，以西方墨點法分析。照射UVB同樣能誘發WS-1細胞中的MMP-1/8/13 (collagenases) 及MMP-2/9 (gelatinases) 的活性表現，因而使Pro-MMPs的表現量減少，而在照射UVB下給與維生素C、微晶瓷或共同給予下皆能增加細胞內的Pro-MMP-1/2/8/9/13及前膠原蛋白 (pro-collagen) 的含量。接著分析調控MMPs的細胞核內轉錄因子之表現，結果發現在照射UVB下給與維生素C、微晶瓷或共同給予下皆能抑制核內c-Fos、c-Jun和NF-κB的表現。綜合實驗結果顯示，施打微晶磁並服用維生素C可有效抵抗UVB照射導致皮膚光老化現象。
並列摘要
Ultraviolet (UV) irradiation is one of the main factors of skin photoaging. Long-term exposure to the UV leads to skin collagen degradation and generate wrinkles in the skin. Recently, it has been shown that excessive matrix degradation was performed by UVB-induced matrix metalloproteinases (MMPs) secreted by fibroblasts cells during skin photoaging. Calcium hydroxylapatite (CaHA, also known as Radiesse), a biocompatible material, stimulates fibroblast cells to synthesis collagen, improving formation of wrinkles. Vitamin C (ascorbic acid), an essential nutrient and an antioxidant for human, promotes collagen production. In this study, we evaluated the effects of CaHA combined vitamin C on MMPs expressions in hairless mice and human dermal fibroblast WS-1 cells exposed to UVB irradiation. In vivo study, mice were injected CaHA and irradiated with UVB (1.2 kJ/m2) for 3 times a week, and than fed vitamin C (1 % and 2 %) daily for 12 weeks. Results showed that serum levels of total cholesterol (CHO) and triglyceride (TG) in 2% vitamin C group were decreased comparing to control. Compared with control group, CaHA had no effect on the blood biochemical parameters. Using Masson’s trichrome stain for collagen fibers, it was demonstrated that UVB irradiation could reduced collagen production in mice skin, while an increase in collagen was observed in three groups of UVB + 2 % vitamin C, UVB + CaHA, and UVB + 2 % vitamin C + CaHA. The molecular data also shown the protein levels of pro-MMP-1/8/13 (collagenases) and pro-MMP-2/9 (gelatinases) were increased in three groups compareing to UVB group. In vitro study, Western blot showed that UVB inhibited the protein levels of pro-MMP-1/2/8/9/13 and pro-collagen in WS-1 cells. In the presence of UVB, vitamin C alone, CaHA alone, or combination of two agents increased the protein levels of pro-MMP-1/2/8/9/13 and collagen. The down-regulation of MMPs appeared to be via the inactivation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-