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Ruehl et.al.,(2005) J.Biol.Chem. 385367-38543

The Elongated First Fibronectin TypeIII Domain of Collagen XIV Is An Inducer Of Quiescence And Differentiation In Fibroblasts And Preadipocytes. Ruehl et.al.,(2005) J.Biol.Chem. 385367-38543. 銘傳生物科技學系. 學生：凌錦瑩. 指導教授：陳秀儀 教授. Introduction.

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Ruehl et.al.,(2005) J.Biol.Chem. 385367-38543

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  1. The Elongated First Fibronectin TypeIII Domain of Collagen XIV Is An Inducer Of Quiescence And Differentiation In Fibroblasts And Preadipocytes Ruehl et.al.,(2005) J.Biol.Chem. 385367-38543 銘傳生物科技學系 學生：凌錦瑩 指導教授：陳秀儀 教授

  2. Introduction ＊Collagen XIV (CXIV) belongs to group of fibril associated collagen. • CXIV is produced by fibroblasts and myofibroblasts. Non-collagendomain Fibronectin III domain • CXIV is a homotrimeric protein of about 650 KDa. • Each chain is 210KDa and consists of two COL1 and COL2 and three non-collagen domain.

  3. Motivation 因為CXIV只出現在well differentiation tissue裡 是否CXIV具有誘導分化的機制???

  4. Materials and Method • Cell lines: 3T3 cell(CCL-92) 3T3-L1 preadipocytes (CL-173) Human fibroblast (HF) • -thymidine uptake assay: 利用 -thymidine incorporated into DNA 來觀察de novo DNA合成情形 • 利用GST將CXIV的片斷純化出來

  5. Coated with Collagens，1h 37℃ 加入3T3、HF培養，20h 加入 -thymidine，4h NaOH lyse cell 利用beta-scintillation counter偵測放射線量

  6. Result 3μg/well CI為control ＊大致上減少30~40％ De novo DNA synthesis Fig.2.A

  7. 以GST為control Q29-P145有明顯anti-proliferation P478-V580刺激 de novo DNA合成 Fig.2.B

  8. 1. 先在培養盤分別coated上 1μg/well CI與CXIV 2.個別加入1μg/well soluble 片段與GST Soluble Q29-P154在 3T3 cell培養有較少 de novo DNA synthesis Fig.3.

  9. 3T3-L1 1.左邊培養盤分別coated上1μg/well CI、CXIV、Q29-P154 2. 右邊只加入3μg/well soluble Q29-P154 3T3-L1對溶入方式的Q29-P154 有減少de novo DNA synthesis的效果 ㄧ旦serum濃度增加會使CXIV抑制 de novo DNA合成量更明顯 (1μg/well) Fig.4.

  10. 3T3 cell 培養在6 well-tissue culture Plate 實驗組加入30μg/well CXIV 利用flow cytometry偵Apoptosis 細胞量 細胞apoptosis量並無明顯增加 Fig.5

  11. HF Coated 1μ/well CXIV、片段 比較serum 0.5％和調整至 10％後de novo DNA 合成量 Fig.5

  12. 3T3 cell Coated 30μg/well CXIV 分化並形成環狀 Fig.6

  13. 3T3-L1 與具有growth factor 的Hormone cocktail 比起來： 1.soluble Q29-P145 可誘導11%細胞分化 2.CXIV 誘導23%細胞分化 Fig.7 3μg/well To demonstrate fat or lipids in fresh tissue sections. Treatment 9天後 以Oil Red O染色 Lipids ----------------------------- red Nuclei ---------------------------- pale blue

  14. 3T3-L1 培養 5天後做 Western-blot Lipid droplet synthesis 會有Gult4 protein表現 增加現象。 coated soluble • Gult4: Glucose transporter 4 Fig.8

  15. Conclusion • CXIV inhibits fibroblast proliferation. • The elongated first FN-III domain harbors the anti-proliferative site in CXIV. • Soluble Q29~P154 enhances the anti-proliferative effect of immobilized native CXIV. • Effect of native CXIV and soluble CXIV Q29~P154 on DNA synthesis of 3T3-L1 preadipocytes. • Anti-proliferative effect of CXIV are not due to toxicity or apoptosis. • Immobilized CXIV induces 3T3 embryonic fibroblasts differentiation.

  16. Application It may open a way to development CXIV analogs to healing wound.

  17. The End

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