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 銘傳生物科技學系 指導教授：陳秀儀 教授 學生：凌錦瑩

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.

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

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

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

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

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

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

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-P 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)

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

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

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

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

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

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.

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

The End