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.
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
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.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.