FK506 對於乙型轉型生長因子（TGF-β1）刺激人類培養纖維母細胞增生、移動與膠原蛋白生成調控之影響

The effects of FK506 on proliferation，migration and collagen synthesis in human cultured fibroblasts stimulated with TGF-β1

王于禎(Yu-Chen Wang)

指導教授 ： 吳慶軒

高雄醫學大學/健康科學院/醫學檢驗生物技術學研究所/碩士(2010年)

摘要

蟹足腫是一種過度增生的疤痕組織，其特徵為纖維母細胞過度增生和細胞外基質，特別是膠原蛋白 (collagen) 之堆積。過去研究結果顯示乙型轉型生長因子 (TGF-β1) 與過度疤痕生成及纖維化疾病有關。FK506是一種巨環類免疫抑制劑，臨床上常被用於器官移植後之抗排斥藥物。FK506會與FK binding protein 12 (FKBP12)結合而抑制calcineurin之活性。過去研究顯示FK506會阻止FKBP12與乙型轉型分子第一型接受器結合對乙型轉型分子的訊息傳遞造成影響。在in vivo和in vitro實驗均指出FK506能夠抑制TGF-β1所促進之傷口癒合作用。因此本論文將探討FK506對於TGF-β1作用人類培養纖維母細胞在增生、移動與膠原蛋白生成等之影響。研究結果顯示FK506可以抑制TGF-β1所促進之細胞增生。刮傷試驗 (wound scrath assay) 之結果顯示，TGF-β1會促進人類培養纖維母細胞之移動，而加入FK506則可以抑制由TGF-β1所誘導促進之細胞移動。其次，我們的研究結果顯示TGF-β1抑制人類正常纖維母細胞matrix metalloproteinase 2 (MMP-2)、matrix metalloproteinase 9 (MMP-9)、α-smooth muscle actin (α-SMA)、fibronectin、collagen mRNA 之表現，均能夠因為加入FK506而增加表現。此外，研究結果顯示TGF-β1能夠促進Smad3與pro-COL1A1蛋白並抑制Smad7蛋白之表現，加入FK506則能夠反轉其表現。加入FK506可以抑制由TGF-β1刺激增加phosphorylated JNK (p-JNK) 與phosphorylated p38 (pp38) 之表現。總結以上結果得知，TGF-β1作用人類培養正常纖維母細胞所造成之增生、移動與膠原蛋白生成之增加能夠藉由加入FK506而受到抑制。

關鍵字

乙型轉型生長因子 ； 纖維母細胞 ； 膠原蛋白

並列摘要

Keloids are considered to be a result of altered wound healing with excessive scar tissue formation which extends beyond the area of the initial wound and does not regress spontaneously. Keloids are characterized by fibroblastic proliferation and accumulation of extracellular matrix (ECM), especially collagen. Transforming growth factor β1 (TGF-ß1) has been reported to be associated with formation of hypertrophic scar and fibrotic diseases. FK506 is an immunosuppressive macrolide which is used to prevent the rejection of organ transplantation. FK506 can bind to FK binding protein 12 (FKBP12). The FK506/FKBP12 complex recruits and thereby inactivates the activity of calcineurin. FK506 is reported to prevent the binding of FKBP12 to TGF-β type I receptor and affects TGF-β signaling. Previous studies revealed that FK506 inhibited the enhanced effects of TGF-β1 on wound healing both in vitro and in vivo. Our study set out to investigate the effects of FK506 on proliferation，migration and collagen synthesis in human cultured fibroblasts stimulated with TGF-β1. Our results showed that FK506 inhibited the increased fibroblast proliferation stimulated by TGF-ß1.The results from wound scratch assay revealed that TGF-ß1 enhanced the migration of fibroblasts. The motility of fibroblasts stimulated by TGF-ß1 was abrogated by addition of FK506. Furthermore, the decrease in matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), α-smooth muscle actin (α-SMA), fibronectin, and collagen mRNA expressions were abrogated by FK506 treatment. The increase in Smad3 and pro-COL1A1 and the decrease in Smad7 expressions by TGF-ß1 stimulation were reversed by FK506 treatment. The inductions of JNK phosphorylation (p-JNK) as well as p38 phosphorylation (pp38) stimulated by TGF-ß1 were inhibited by FK506. Taken together, our results demonstrated that FK506 inhibited the enhanced effects of TGF-ß1 on proliferation, migration, and collagen synthesis in cultured human fibroblasts.

並列關鍵字

FK506 ； TGF-β1 ； collagen ； fibroblast

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