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

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.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

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)

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