- 學位論文
FK506 抑制TGF-β1所誘導之纖維母細胞增生、移動和膠原蛋白合成：治療蟹足腫之前瞻性研究
The inhibitory effect of FK506 on TGF-β1 induced fibroblast proliferation, migration and collagen synthesis: implications of therapeutic approach for keloid
摘要
傷口癒合為一種動態平衡的生理狀態及一系列複雜相互作用的過程。傷口癒合過程包含止血期、發炎期、增生期及成熟期。不正常之傷口癒合常造成許多疾病的發生。蟹足腫就是一種不正常之傷口癒合所造成的皮膚疾病，其致病原因可能是因為傷口癒合的過程中，不正常的發炎反應及過多的細胞外基質(extracellular matrix metalloproteinase ; ECM) 堆積，而使得癒合之疤痕超過原有傷口的邊緣，且不會隨著時間自發性地消退，然而蟹足腫確實之致病原因至今仍未完全解明。過去研究發現乙型轉型生長因子 (Transforming growth factor β1；TGF-β1) 會促使細胞生長和細胞外基質過度表現，且與增生性疤痕堆積及纖維化疾病有密切相關。 FK506 是一種新型外用的免疫調節劑，臨床上，FK506常被用於器官移植後之免疫抑制功能。此外，FK506也常被用於治療許多發炎性或非發炎性之皮膚疾病。最近研究文獻指出，FK506對於乙型轉型生長因子（TGF-β1）所促進的傷口癒合作用具有抑制之效果，因此我們推測FK506可能對於蟹足腫這類因為乙型轉型生長因子 （TGF-β1）過度表現所造成的纖維化疾病具有療效，因此本研究擬探討FK506對於乙型轉型生長因子（TGF-β1）所促使人類蟹足腫纖維母細胞增生、移動及膠原蛋白合成之影響，也進一步探討可能參與之訊息傳遞路徑。 研究結果顯示，FK506可顯著地抑制乙型轉型生長因子 （TGF-β1）促進人類蟹足腫纖維母細胞的增生與移動。其次，乙型轉型生長因子（TGF-β1）對於人類蟹足腫纖維母細胞之基質金屬蛋白 (Matrix metalloproteinase , MMPs) 中MMP-1、MMP-2、MMP-8、MMP-9、MMP-13 mRNA表現有抑制現象，而對於基質金屬蛋白內生性抑制劑 (Tissue inhibitors of metalloproteinases ; TIMPs) 中TIMP-2、Type I collagen、α-Smooth musle actin (α-SMA) mRNA之表現則有增加之情形，然而這些mRNA之表現均能夠因為加入 FK506而反轉其表現。此外，本研究結果顯示乙型轉型生長因子（TGF-β1）會促使其結合器I (TGF-β1 receptor I, TβRI) 、結合器 II (TGF-β1 receptor II, TβRII)、Smad2、Smad3、Smad4、Smad2/3及Smad2/3磷酸複合物之形成及pro-COL1A1蛋白增加並抑制Smad7蛋白之表現，而加入FK506後會抑制TβRI、TβRII、Smad2、Smad3、Smad2/3、phosphorylated Smad2/3 及pro-COL1A1之表現，而Smad7蛋白則有增加之趨勢。另外，乙型轉型生長因子（TGF-β1）也會增加Mitogen activated protein kinase (MAPKs)中JNK與p38 磷酸化的表現，而加入FK506後能夠抑制JNK及p38 磷酸化。 綜合本研究結果，FK506對於乙型轉型生長因子（TGF-β1）所促進之蟹足腫纖維母細胞增生、移動及膠原蛋白合成作用具有抑制的效果。希望本研究成果能夠提供未來FK506應用於臨床上治療蟹足腫之理論基礎。
並列摘要
Wound healing process is a complex and carefully regulated physiologic response to traumatic skin injury, composed of four phases including hematostasis, inflammation, proliferation, and maturation. Abnormal wound healing process often results in many diseases. Keloids are considered to be a skin disease resulted from abnormal wound healing after injury. Keloids are characterized by overgrowth of scar tissue beyond the borders of the original wound and do not regress spontaneously. Several lines of evidences have revealed that transforming growth factor β1 (TGF-β1) stimulates the cell proliferation as well as the synthesis of extracellular matrixes (ECMs) and possesses strong associations with the formation of hypertrophic scar and fibrotic diseases. FK506 is a novel topical immunomodulator. Clinically, FK506 is used as a immunosuppressor to prevent the rejection of organ transplantation. In addition, FK506 is also often used to treat many inflammatory and non-inflammatory skin diseases. Recently, FK506 has been reported to inhibit the enhancing effects of TGF-β1 on wound healing. Therefore, we speculated that FK506 may have therapeutic effects on keloids which are caused by the overexpression of TGF-β1. In this study, we set out to investigate the effects of FK506 on TGF-β1-induced cell proliferation, migration, and collagen synthesis in keloid fibroblasts. Furthermore, the possible regulatory pathways involved were also investigated. Our results showed that FK506 significantly inhibited the TGF-β1-induced proliferation and migration in keloid fibroblasts. The downregulations of matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 9 (MMP-9), and matrix metalloproteinase 13 (MMP-13) mRNA and upregulations of tissue inhibitors of metalloproteinase 2 (TIMP-2), type I collagen, and α-smooth muscle actin (α-SMA) mRNA after TGF-β1 treatment in keloid fibroblasts were noticed. However, addition of FK506 could reverse the expressions of those aforementioned mRNA in keloid fibroblasts treated by TGF-β1. In addition, our results indicated that FK506 abrogated the increased expressions of TGF-β1 receptor I (TβRI), TGF-β1 receptor II (TβRII), Smad2, Smad3, Smad4, Smad2/3 complex, phosphorylated Smad2/3 complex, and pro-collagen 1A1 stimulated by TGF-β1 in keloid fibroblasts. The decreased expression of Smad7, an inhibitory Smad protein acted as a negative feedback regulator, stimulated by TGF-β1 was slightly increased after addition of FK506. The expressions of phosphorylated JNK and phosphorylated p38 enhanced by TGF-β1 treatment were inhibited by FK506. Taken together, our results suggested that FK506 demonstrates an inhibitory effect on TGF-β1-induced cell proliferation, migration, and collagen synthesis in keloid fibroblast. We hope that our study will provide a solid theoretical basis for application of FK506 in treating keloid.