- 學位論文
在纖維母細胞靜默幹細胞因子對黑色素細胞中色素生成之影響
The influences of stem cell factor silenced in fibroblasts affect melanogenesis in melanocytes
摘要
黑色素生成為一複雜生理機制，許多的旁分泌因子牽涉於其過程中。在色素的生成中，皮膚內的角質細胞、纖維母細胞與黑色素細胞會藉由旁分泌系統的調節彼此形成交互作用進而調控黑色素細胞。幹細胞因子是一種由纖維母細胞所分泌的旁分泌因子，其受體為表現於黑色素細胞上的c-kit。當幹細胞因子結合上c-kit，將會啟動c-kit自體磷酸化及酪胺酸激酶的活化以引發下游訊息傳遞；許多的研究指出幹細胞因子在黑色素的生成中扮演著重要的角色。本研究將探討幹細胞因子的靜默是否會影響纖維母細胞所生產的旁分泌蛋白，並如何改變黑色素細胞之行為。由MTT試驗顯示，幹細胞因子的靜默並不會因此抑制纖維母細胞的增生。透過即時定量聚合酶鏈鎖反應結果指出，幹細胞因子靜默會誘導纖維母細胞內肝細胞生長因子、神經調節蛋白與促腎上腺皮質素釋放荷爾蒙基因表現上調；經過紫外線B的刺激，肝細胞生長因子、神經調節蛋白與促腎上腺皮質素釋放荷爾蒙之基因表現更高於穩定狀態下，尤其是肝細胞生長因子與幹細胞因子表現出高度之關聯性。除此之外，此研究亦發現了纖維母細胞會以自分泌的方式調節幹細胞因子的分泌。將纖維母細胞培養後之條件培養基收集後施予黑色素細胞，透過即時定量聚合酶鏈鎖反應分析，若給予經幹細胞因子靜默或紫外線B刺激後的纖維母細胞之培養基，色素細胞內將上調黑色素生合成相關之酪胺酸酶與pmel17基因表現；藉由黑色素含量測定結果顯示，施予同樣條件下之條件培養基會實質增加黑色素細胞內黑色素的含量。總結來說，幹細胞因子的靜默會造成纖維母細胞內旁分泌因子與黑色素細胞內色素生成的變化，且此改變會因UVB的刺激而有不同表現。
並列摘要
Melanogenesis is a complex physiological mechanism involving several paracrine factors. During melanogenesis, skin cells such as keratinocytes, fibroblasts, and melanocytes communicate with each other through various secreted paracrine regulators, thereby regulating the activity of melanocytes. Stem cell factor (SCF) is a paracrine factor produced by fibroblasts, and its receptor, c-kit, is expressed on melanocytes. Binding of SCF to c-kit activates autophosphorylation and tyrosine kinase to switch on its signal transmission. Several studies have indicated that SCF plays a crucial role in melanogenesis. In this study, we investigated whether SCF silencing influences other paracrine factors secreted by fibroblasts and how the changes in melanocytes affect melanogenesis. MTT assay showed that SCF inhibition did not suppress fibroblast proliferation. Quantitative real-time polymerase chain reaction (qPCR) results showed that SCF silencing induced increased mRNA expression of the paracrine factor genes hepatocyte growth factor (HGF), neuregulin (NRG) -1, and cotricotropin-releasing hormone (CRH). After UVB stimulation, the gene expressions of HGF, NRG, and CRH were higher than homeostasis; especially, HGF exhibited a highest correlation with SCF variation. We also detected fibroblasts regulated SCF in an autocrine manner. The conditioned medium obtained from fibroblast culture was used treat melanocytes. qPCR analysis showed that the melanogenesis-related genes tyrosinase and pmel17 were upregulated under treatment with conditioned medium from fibroblasts with SCF silencing and exposed to UVB treatment. Melanin content assay under the same condition showed that melanin quantities in the melanocytes were obviously increased. In conclusion, SCF silencing causes variation in both fibroblast paracrine factors and melanocyte on melanogenesis, and the difference in expression was observed after UVB exposure.