第五生長分化因子對人類牙髓細胞的影響及其訊息傳導機制

Effects of Growth/Differentiation Factor-5 on human dental pulp cell and its signaling

曾惠君(Hui-Chun Tseng)

指導教授 ： 鄭景暉

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00675

摘要

實驗目的: 第五生長分化因子 (Growth/Differentiation factor-5, GDF-5) 與許多組織的發育與修復有很大的相關性，如軟骨、韌帶、椎間盤、骨頭與皮膚。本篇研究注重於第五生長分化因子對於人類牙髓細胞在形態表現、細胞增生與生存能力、細胞分化及膠原蛋白生成的影響，並進一步探討第五生長因子於細胞增生與分化相關的訊息傳導路徑。
實驗方法: 使用不同濃度的第五生長分化因子刺激人類牙髓細胞，在某些實驗中則先加入特定訊息抑制劑做前處理以探索相關訊息傳導路徑。利用光學顯微鏡觀察細胞形態；以MTT測定牙髓細胞之存活能力；利用鹼性磷酸酶染色與定量分析來檢測細胞分化程度；以膠原蛋白定量分析來做膠原蛋白之定量；使用反轉錄鏈聚合反應印證細胞分裂相關基因的表現；另外利用免疫螢光抗體技術觀察單核及雙核細胞的比例。
實驗結果: 第五生長分化因子可增加人類牙髓細胞的存活能力；影響與細胞增生相關基因 (促進Cyclin B1的表現，抑制p21的表現)；提高雙核細胞的比例。SB431542 (ALK-4/5/7之特殊訊息抑制劑) 可些微逆轉第五生長分化因子所誘發的細胞增生現象，而Noggin (骨形成蛋白質之拮抗劑) 及U0126 (ERK之抑制劑) 則無法。在細胞分化方面，在第五生長分化因子作用下，鹼性磷酸酶的染色程度及定量表現會有顯著下降的趨勢；使用之抑制劑，包含Dorsomorphin (ALK-2/3/6之特殊訊息抑制劑)、SB431542、U0126和SB203580 (p38 kinase之抑制劑)，都無法改善第五生長分化因子所誘發的細胞分化抑制現象。在細胞間質生成方面，第五生長分化因子對於膠原蛋白的含量沒有顯著影響。
結論: 人類牙髓細胞受到第五生長分化因子的刺激下，會促進細胞增生、抑制細胞分化，但對膠原蛋白之形成無影響。在細胞增生方面的訊息傳導路徑可能與ALK-4/5/7部分相關，但在細胞分化方面則尚未找到相關之訊息傳導路徑。

關鍵字

ALK-4/5/7 ； 細胞增生及分化 ； 人類牙髓細胞 ； 第五生長分化因子

並列摘要

Aim: Growth/Differentiation factor-5 (GDF-5) is a multifunctional protein and close related to the development and repair of multiple tissues, including cartilage, tendon, intervertebral disk, bone and skin. The purpose of our study is to investigate whether GDF-5 influences the morphological changes, cell proliferation and viability, cell differentiation and collagen formation of human dental pulp cells in vitro within the period of 5 or 10 days. Furthermore, the related signal transduction pathways were also been evaluated.
Materials and Methods: Primary-cultured human dental pulp cells were treated with different concentration of GDF-5 (0-500 ng/ml). In some experiments, cells were pretreated with different specific signaling inhibitors 30 minutes before adding GDF-5 for investigating the signaling of GDF-5. Morphology of pulp cells was observed under light microscopy (100X). Cell proliferation was evaluated by MTT assay. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining and ALP quantitative assay. Changes in mRNA expression of cell mitosis-related genes (Cyclin B1, CDC2, CDC25C and p21) were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Besides, immunofluorescence assay was used to observe the percentages of binuclear and mononuclear cells.
Results: Human dental pulp cells were spindle with extended cellular processes with/without GDF-5 treatment. Under the treatment by various concentrations of GDF-5: cell viability was up-regulated significantly in dose-dependent manner; Cylin B1 mRNA expression was stimulated, but the expression of p21 mRNA was inhibited; the percentage of binuclear cells was increased. In the inhibitory experiment of MTT assay, SB431542 (ALK-4/5/7 specific inhibitor) could slightly prevent the GDF-5-induced declination in cell proliferation. In cell differentiation: GDF-5 declined the ALP activity of human dental pulp cells; four inhibitors, including Dorsomorphin (ALK-2/3/6 specicic inhibitor)、SB431542、U0126 and SB203580 (p38 inhiibitor) could not reverse the effect of GDF-5 on ALP activity. GDF-5 did not affect the collagen content significantly.
Conclusion: GDF-5 demonstrated proliferative property in human dental pulp cells, but had inhibitory effect on cell differentiation and no effect on collagen formation. Signal transduction of GDF-5 in human dental pulp cells is a complex system. GDF-5 could induce cell proliferation through the activation of ALK-4/5/7 partially, but the signal pathway of GDF-5-induced cell differentiation is still unknown. These events are crucial in the mechanism of pulpal repair and regeneration.

並列關鍵字

ALK-4/5/7 ； cell proliferation and differentiation ； human dental pulp cells ； GDF-5

參考文獻

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