鹼性成纖維細胞生長因子對牙髓之增生、移動、分化及細胞間質生成的影響

實驗目的：鹼性成纖維細胞生長因子（basic fibroblast growth factor, bFGF）功能包括促進傷口癒合、血管新生、黑色素形成、硬骨及軟骨的生成等…。本篇研究著重於鹼性成纖維細胞生長因子對於人類牙髓細胞在形態表現、細胞增生及生存能力、及細胞移動、細胞分化、細胞間質生成的影響，進而了解bFGF 在牙髓組織修復與重建所扮演的角色。實驗方法：使用不同濃度的鹼性成纖維細胞生長因子（bFGF）刺激人類牙髓細胞。在不同的時間點下，以光學顯微鏡觀察細胞型態及移動情況。利用MTT 測定牙髓細胞的存活能力；以鹼性磷酸酶染色檢測細胞分化程度、以Sircol Collgen assay 做膠原蛋白定量。另外使用反轉錄鏈聚合反應（RT-PCR）印證相關基因的表現。實驗結果：鹼性成纖維細胞生長因子（bFGF）會增加細胞數目；人類牙髓細胞在較高濃度的bFGF 刺激下，細胞的存活能力有兩倍至三倍的提升。而與細胞增生相關的基因（Cyclin B1, CDC2, CDC25c）表現會因應bFGF 濃度而增加。bFGF 亦促進牙髓細胞的傷口癒合及細胞移動速度。然而，Rho 和ROCK 基因的表現最高峰會落在bFGF 濃度為10 ng/ml 之上；a-sma 則因應bFGF 濃度而遞減。在細胞分化方面，bFGF 會降低鹼性磷酸酶的染色程度。在細胞間質生成方面，bFGF 對於膠原蛋白的含量沒有顯著影響；而相關基因（TIMP2, MMP2）則因應bFGF 刺激而下降。結論：人類牙髓細胞受到短期bFGF 刺激後（五天內），細胞增生、移動、傷口癒合能力會因應生長因子而有所增進。然而，bFGF 會影響細胞分化及膠原蛋白的形成。

關鍵字

牙髓細胞；鹼性成纖維細胞生長因子；細胞增生及移動

並列摘要

Aim: Basic fibroblast growth factor (bFGF) is multifunctional protein with a wide variety of effects. The functions of bFGF include wound healing, angiogenesis, melanogenesis, development of bone and cartilage, and so on. The purpose of our study is to investigate whether basic fibroblast growth factor influences the morphological changes, cell proliferation and viability, cell differentiation, and extracellular matrix formation of human dental pulp cells in vitro within the period of 5 days. Materials and Methods: Primary-cultured human dental pulp cells were treated with different concentrations of bFGF (0, 1, 10, 50, 200 ng/ml). Morphology of pulp cells and cell migration were observed under light microscopy (40X). Cell proliferation was evaluated by MTT assay. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining. Changes in mRNA expression of ALKP, Runx2, Cyclin B1, CDC2, CDC25c, p21, Rho, ROCK, a-sma, collagen type I, TIMP2, and MMP2 were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Results: Dental pulp cells are spindle with extended cellular processes with/without bFGF treatment. The numbers of dental pulp cells without any bFGF treatment were much lower than those under the treatment of various concentrations of bFGF. Cell viability was two to three fold increased in higher concentrations of bFGF (50, 200 ng/ml). bFGF increased Cyclin B1, CDC2, and CDC25C mRNA gene expressions dose-dependently. Migrating rates were higher in the groups of higher concentrations of bFGF than those in the groups of lower concentrations. Rho and ROCK gene expressions were at peak at 10 ng/ml of bFGF. Nevertheless, α-sma mRNA gene expression of pulp cells declined in dose-dependent manner. bFGF downregulated ALPactivity of human dental pulp cells but did not affect significantly RUNX2 mRNA expression. bFGF did not affect significantly collagen content . Gene expression of MMP2 and TIMP2 were down-regulated by bFGF treatment. Conclusion: Human dental pulp cells incubated with basic fibroblast growth factor demonstrated proliferative and migratory properties during the early stage of wound repairing (within 5 days). Meanwhile, bFGF had inverse effects on cell differentiation and extracellular matrix formation.

並列關鍵字

dental pulp cells ； bFGF ； cell proliferation and migration

參考文獻

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鹼性成纖維細胞生長因子對牙髓之增生、移動、分化及細胞間質生成的影響

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