臺灣博碩士論文加值系統

中文摘要
Discoidin domain receptor 2 ( DDR2 ) 為酪氨酸激酶接受器家族之ㄧ員，可以與纖維型態的膠原蛋白結合而活化，主要分布在間質細胞中。
之前我們探討DDR2和硬骨細胞分化的關係，發現DDR2會在培養細胞誘導走向硬骨分化時表現量增加；而抑制DDR2的表現，則發現分化晚期鈣離子沉積明顯受抑制，因此認為DDR2在硬骨分化中扮演重要角色。本篇論文想進一步探討調控DDR2 mRNA表現之機制。
利用TF search軟體分析DDR2啟動子上 -1240區段，發現具有五個C/EBP binding sites。在誘導MG63細胞走向硬骨分化時，發現C/EBPa 表現 會在誘導的1、4天增加。進一步在SAOS2細胞中大量表現C/EBPa ，發現DDR2 mRNA的確會被C/EBPa 活化； Reporter assay也證實C/EBPa 可以活化DDR2啟動子 -1240區段。
接著我們利用不同DDR2啟動子的deletion constructs經由reporter assay方式找到C/EBPa的活化區位在DDR2啟動子 -1240 ~ -1033區段。這個區段具有兩個C/EBPa 可能結合序列 : C/EBP和CHOP-C，以site-directed mutagenesis方式將這兩個binding site作突變，發現到C/EBPa活化這兩個突變構築的能力都受抑制。EMSA實驗中也証實C/EBPa 會分別與這兩個binding sites結合。由於CHOP-C binding site為CHOP (C/EBPz) 和C/EBPa heterodimer結合的位置，以EMSA實驗發現C/EBPa 與兩序列結合的能力會被CHOP抑制。
综合以上實驗，我們認為C/EBPa 會經由結合DDR2啟動子上 -1240 ~ -1033區段的C/EBP和CHOP-C binding sites進一步促進DDR2的活化。

Abstract

Differentiation of osteoblasts is controlled by complex activities involving signal transduction and gene expression. The human discoidin domain receptor 2 (DDR2) belongs to receptor tyrosine kinase superfamily that is activated by fibril collagen. We have previously found that DDR2 overexpression activated the expression of osteoblast marker genes, whereas knock-down of DDR2 inhibited osteogenesis of MG63 osteoblast-like cells. In addition, the expression and activity of DDR2 was elevated during induced osteogenesis. In this study, we further examined the mechanisms by where DDR2 gene expression is regulated.
Sequence analysis revealed the presence of 5 consensus C/EBP binding sites on the -1240 ~ +70 of the DDR2 promoter. Western blot analysis indicated that levels of C/EBPa and b increased during induced osteoblast differentiation, suggesting a role of C/EBP in osteogenesis. Moreover, overexpression of C/EBPa up-regulated the mRNA expression of DDR2, and enhanced the luciferase activity driving by the -1240 ~ +70 region of the DDR2 promoter. Using promoter deletion constructs, we showed that -1240 ~ -1033 of the DDR2 promoter is responsible for the C/EBPa mediated activation. In addition to the C/EBP binding site, this region contains a CHOP-C consensus sequence that is also necessary for C/EBPa transactivation. Analysis by EMSA demonstrated that C/EBPa bound to both C/EBP and CHOP-C binding sites, suggesting that both sites may be important for C/EBPa-dependent regulation of DDR2 gene expression. Moreover, CHOP bound and dose-dependently inhibited the binding of C/EBPa to these binding sites.
Together these results suggest that C/EBPa binds to the C/EBP and CHOP-C sites on the -1240 ~ -1033 region of the DDR2 gene and activates its expression.

目錄
中文摘要……………………………………………………………….4
英文摘要……………………………………………………………….5
一、 序論………………………………………………………….6
二、 實驗材料與方法…………………………………………. 14
三、 實驗結果………………………………………………… .27
四、 實驗討論……………………………………………………34
五、 參考文獻……………………………………………………37
六、 圖表…………………………………………………………43
七、 附錄…………………………………………………………55

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