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 指導教授：陳秀儀 教授 學生：凌錦瑩

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

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

4. Materials and Method - thymidine uptake assay: 利用 -thymidine incorporated into DNA Cell lines: 3T3 cell(CCL-92) 3T3-L1 preadipocytes (CL-173) Human fibroblast (HF) 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 NaOH lyse cell 加入 -thymidine ， 4h 利用 beta-scintillation counter 偵測放射線量

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

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

8. Soluble Q29-P154 在 3T3 cell 培養有較少 de novo DNA synthesis Fig.3. 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

9. Fig.4. 3T3-L1 對溶入方式的 Q29-P154 有減少 de novo DNA synthesis 的效果 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 ㄧ旦 serum 濃度增加會使 CXIV 抑制 de novo DNA 合成量更明顯 (1μg/well)

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

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

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

13. Fig.7 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 3T3-L1 Treatment 9 天後 以 Oil Red O 染色 Lipids ----------------------------- red Nuclei ---------------------------- pale blue To demonstrate fat or lipids in fresh tissue sections. 3μg/well

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

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