【作者】 何文喜； 牛忠英； 赵守亮； Anthony J．Smith；

【Author】 HE Wen-xi, NIU Zhong-ying, ZHAU Shou-liang, Anthony J. Smith. (Department of Conservative Dentistry, School of Dentistry, The Fourth Military Medical University. Xi’an 710032, Shanxi Province ,China;Oral Biology, School of Dentistry, University of Birmingham. B4 6NN, UK)

【机构】 第四军医大学口腔医学院牙体牙髓病科； Oral Biology,School of Dentistry,University of Birmingham.B4 6NN,UK；

【摘要】 目的:研究牙本质基质蛋白提取物(dentin matrix proteins,DMPs)对牙本质涎磷蛋白基因表达的影响,探讨DMPs调控 DSPP基因表达的信号途径。方法:培养成牙本质细胞样细胞MDPC-23,细胞计数观察DMPs对MDPC-23细胞增殖作用的浓度效应。半定量PCR观察DMPs对MDPC-23细胞内DSPP基因表达的时间效应。瞬时转染观察DMPs对含DSPP基因启动子不同片段的荧光素酶报告基因载体活性的影响。共转染Smad2和Smad3突变体(Smad2 C和Smad3 C)观察Smad蛋白对DMPs调控DSPP基因启动子活性的影响。用Erk(extracellular signal-regulated kinas)特异性抑制剂PD98059和p38 MAPK特异性抑制剂SB203580观察Erk和p38信号途径在DMPs调控DSPP基因启动子活性中的作用。结果:细胞计数结果表明1-10 μg／ml DMPs促进细胞增殖,100-1000 μg／ml DMPs抑制细胞增殖。DMP迅速诱导DSPP基因mRNA表达,3h到达最高峰,以后逐渐下降。10 μg／ml DMPs作用3h,显著诱导Pdspp-2525～+54bp启动子活性,但是对Pdspp-1243～+54bp启动子活性无明显影响。共转染 Smad3(C则显著增强DMPs对Pdspp-2525～+54bp启动子的诱导作用,而Smad2 C则对DMPs作用无明显影响。PD98059显著抑制DMPs诱导Pdspp-2525～+54bp启动子活性,而SB203580对DMPs作用无显著影响。结论:DMPs显著增强MDPC-23细胞内DSPP 基因表达,其调控作用位点可能位于-2525 bp和-1243 bp之间。ERK1／2信号途径可能参与DMPs对DSPP基因表达的调控。更多还原

【Abstract】 PURPOSE: To investigate the effect of dentin matrix proteins (DMPs) on dentin sialophosphoprotein (DSPP) expression and to explore the possible signaling pathway involved in DSPP transcriptional regulation by DMPs. METHODS: The dose-dependent effect of DMPs on proliferation of MDPC-23 was detected by cell counting. The time-dependent effect of DMPs on DSPP expression was evaluated by semi-quantitative PCR. The role of DMPs on transcriptional regulation of DSPP gene was investigated by transient transfection with promoter-luciferase reporter gene constructs. The effect of Smad proteins transcriptional regulation of DSPP gene promoter by DMPs was observed in cotransfection experiments using Smad2 and Smad3 dominant negative mutant, Smad2 C and Smad3 C.The roles of Erk and p38 pathways in mediating transcriptional regulation of DSPP gene promoter by DMPs were determined using specific inhibitors such as PD98059 and SB203580. respectively. RESULTS: The results showed that DMPs significantly increased cell proliferation among 1-10μg/ml, whereas decreased cell proliferation among 100-1000 μg/ml. DSPP mRNA was rapidly induced by DMPs, with a maximal peak of expression after 3 hrs stimulation, and reduced gradually to low level thereafter. DMPs markedly increased the luciferase activity of Pdspp-2525～+54bp, however, only minimal effects on luciferase activity of Pdspp-1243～+54bp. Transfection of Smad3 C potently increased the role of DMPs on luciferase activity of Pdspp-2525-+54bp, while Smad2 C only have little effect on role of DMPs. PD98059 diminished significantly up-regulatory role of DMPs on luciferase activity of Pdspp-2525-+54bp, whereas SB203580 had no distinct effect on up-regulatory role of DMPs. CONCLUSION: These data suggested that DMPs significantly enhanced DSPP gene expression. DMPs response element was maybe located in the region between -2525bp and -1243 bp. Erk appeared to be involved in the transcriptional regulation of DMPs on DSPP gene expression.更多还原