臺灣博碩士論文加值系統

摘　要
　　Fibronectin (Fn) 在調控細胞附著、移動及成骨細胞的成熟中扮演了一個非常重要的角色，而fibronectin的纖維固化也包含在礦質化過程當中，為了去了解protein kinases對於fibronectin間質纖維合成的調控作用，我們在成骨細胞 (Osteoblasts) 研究fibronectin的合成及聚合情形。Osteoblasts內生性的釋放fibronectin作用會隨時間延長而有增加的趨勢，給予TPA及forskolin都能增加fibronectin的合成，然而，TPA能增加內生性或直接加入soluble fibronectin的作纖維聚合作用，但是forskolin卻相反的抑制這作用，此外PKC的抑制劑H7及Ro 318220及 Go 6976也有相同的抑制作用。這使我們了解PKC及PKA對於動態的fibronectin纖維固化過程有不同的調節作用。而在osteoblasts之中TPA所活化的PKC為PKC a 及 b 二種型式。在之前的報導有指出 a5b1 integrin與fibronectin的纖維化有相關連性，利用免疫螢光染色法或流式細胞分析儀都可測出TPA及forskolin分別增加和減少a5 integrin的clustering。但是分別使用西方點墨法去測量TPA及forskolin對於a5 integrin蛋白質層次都沒有發現明顯的變化，而b1 integrin的蛋白質及mRNA層次也沒有產生變化。這結果建議了TPA及forskolin影響了a5b1 integrin相互的親和力。而只有forskolin會促進CREB的磷酸化TPA並沒有此作用。
總結此篇的論文可以得：對於fibronectin的纖維化，PKC有加強的作用，相反的PKA則有抑制作用，而對於fibronectin的合成及細胞外的聚合作用，PKA有不同的調控情況。

ABSTRACT
Fibronectin (Fn) plays an important role in the regulation of adhesion, migration and maturation of osteoblasts. Fibronectin fibrillogensis may be involved in the bone mineralization process. To elucidate the regulatory role of protein kinases in the formation of fibrillar fibronectin matrix, we examined the fibronectin synthesis and assembly in cultured osteoblasts. The assembly of endogenously released fibronectin underneath osteoblasts proceeded in a time-dependent way. Both TPA and forskolin increased the synthesis of fibronectin. However, the extracellular assembly of fibronectin fibril from both endogenously released or exogenously applied soluble fibronectin is increased by TPA but decreased by forskolin. PKC inhibitors, H7 、Ro 318220 and Go 6976, inhibited fibronectin fibrillogenesis. These results indicate that the dynamic of fibronectin fibrillogenesis is differentially regulated by PKC and PKA activation. Both PKC a and PKC b are involved in the action of TPA in osteoblasts. It has been reported that a5b1 integrin is related to fibronectin fibrillogenesis. Immunocytochemistry and cytoflowmetry showed that TPA and forskolin increased and inhibited the clustering of a5 integrin, respectively. Both TPA and forskolin did not affect protein level of a5 integrin. The Western blotting and RT-PCR showed that both protein and mRNA levels of b1 integrin were also not affected by TPA and forskolin. These results suggest that TPA and forskolin may affect the avidity of a5b1 integrin. CREB phosphorylation is involved in the action of forskolin but not that of TPA.
Our results suggest that PKC activation enhanced fibronectin fibrillogenesis, whereas, PKA activation inhibited extracellular fibronectin fibrillogenesis in primary cultured osteoblasts. Intracellular fibronectin synthesis and extracellular fibronectin assembly may be differentially regulated by the activation of PKA.

目　錄
英文摘要…………………………..……………….1
中文摘要………………………………….………..3
緒論……………………………………….………..5
實驗材料與方法…………………………………..15
結果………………………………………………..24
討論………………………………………………..55
參考文獻………………………………….……….62

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