TGF- ß 2對人類牙髓細胞的影響及其訊息傳導機制

Effects of TGF-β2 on human dental pulp cells and its signaling

戴岑芳(Tseng-Fang Tai)

指導教授 ： 鄭景暉

共同指導教授 ： 藍萬烘(Wan-Hong Lan)

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01999

摘要

轉型生長因子（TGF-ßs）可能會調節人類牙髓細胞的生長、分化與功能。我們早期的研究發現TGF-ß會抑制牙髓細胞的生長及刺激膠原蛋白的合成。實驗目的：在本實驗中，我們測試了不同TGF-ß2的訊息傳導途徑對人類牙髓細胞分化所造成的影響。實驗方法：我們使用西方點墨法、免疫螢光染色、反轉錄鏈聚合酶反應(RT-PCR)、MTT測定、鹼性磷酸酶(ALP)染色與鹼性磷酸酶定量分析來觀察我們的發現。實驗結果：我們觀察到人類牙髓細胞主要表現TGF-ß1的 mRNA， TGF-ß2次之，與少量的TGF-ß3之 mRNA。而西方點墨法的結果則是發現加入TGF-ß2 (10 ng/ml)於牙髓細胞中會誘導 smad2/3，smad1/5/8與 ERK這些不同訊息傳導分子的活化;我們可用免疫螢光染色法觀察到smad3與smad1的表現從細胞質移位進入細胞核內。人類牙髓細胞經長滿五天後會表現強的鹼性磷酸酶活性，而TGF-ß2 (≧5 ng/ml)會減低牙髓細胞鹼性磷酸酶的活性(P < 0.05) 與mRNA的表現。若先將牙髓細胞以 SB431542 (TGF-ß之ALK-4, ALK-5 and ALK-7受體之抑制劑) 前處理，則結果會降低TGF-ß2 (5 ng/ml) 於細胞存活率、鹼性磷酸酶染色與鹼性磷酸酶 mRNA表現所造成的抑制作用; 此種現象在經 U0126前處理的牙髓細胞中則較不明顯。結論：從以上的實驗結果推論- TGF-ß2可能會藉著自體內泌與旁泌的作用來影響人類牙髓細胞活化不同的訊息傳導途徑。而這些不同途徑活化的結果可能會對牙髓的修復與牙本質的再生產生決定性的影響。

關鍵字

牙髓 ； 轉型生長因子 ； 細胞存活率 ； 訊息傳導 ； 鹼性磷酸酶

並列摘要

TGF-ßs (Transforming Growth Factor-ßs) may regulate the growth, differentiation and function of human dental pulp cells. Our prior study has found that TGF-ß inhibited pulp cells growth and stimulated collagen synthesis. Objectives: In this study, we tested the differential effects of TGF-ß2 signaling on differentiation of cultured human dental pulp cells. Methods: We used western blotting, immunofluorescent observation, RT-PCR, MTT assay, alkaline phosphatase (ALP) staining, alkaline phosphatase quantitative assay to test our findings. Result: We found that human dental pulp cells expressed mainly TGF-ß1, less TGF-ß2 and trace amount of TGF-ß3 mRNA. Exposure of pulp cells to TGF-ß2 (10 ng/ml) induced the phosphorylation of smad2/3, smad1/5/8 and ERK signaling pathways as analyzed by western blotting; TGF-ß2 also stimulated the nuclear translocation of smad3 and smad1 as observed by immunofluorescent microscope. Pulp cells expressed strong ALK-P activities in confluent culture for 5 days. Exposure to TGF-ß2 (≧ 5 ng/ml) decreased the ALP activity ( P < 0.05 ) and mRNA expression. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-ß ALK-4, ALK-5 and ALK-7 receptors) and to a lesser extent the U0126 (MEK1 inhibitor) prevented the inhibitory effect of TGF-ß2 (5 ng/ml) on viable cell number, ALP staining, as well as the ALP mRNA expression in dental pulp cells. Conclusion: These results suggest that TGF-ß2 may affect the biological activities of dental pulp cells via autocrine and paracrine fashion by activation the differential signal transduction pathways. These events can be crucial in pulpal repair and regeneration.

並列關鍵字

Pulp ； TGF-β ； cellular survival rate ； signal transduction ； alkaline phosphatase

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