| The induction of odontoblast differentiation is the key event during dental pulp repair and tooth germ development, studies on which are a highlight of endodontics. The Signaling initiation of odontoblast differentiation involves many growth factors, especially TGF- B . TGF-B can not only induce functional differentiation of odontoblasts, but also modulate their synthesis and secretion of dentine matrix. But the molecular mechanisms of TGF- B Signaling as well as its regulation of gene expression in odontoblasts and dental pulp cells are still unknown.In 1996, a new gene, termed Smad2, was cloned from a human kidney cDNA library. Functional analysis demonstrated that Smad2 was the downstream effector of TGF- B induced signaling. So far, Smadl~9 have been cloned, called Smads family. Among this family, Smad2 and Smad3 are the specific mediators of TGF- B Signaling,which can transduce signal into nucleus and regulate target gene expression after phosphorylated by type I receptor for TGF- P . A study has indicated the expression of Smad2 in human dental pulp cells, but there are no reports about the functions of Smad2/3 in TGF- P modulating dental pulp repair and tooth development.In order to gain insight into the molecular mechanisms of TGF- 3 Signaling for regulating odontoblast differentiation and dentinogenesis, the authors sought to characterize the roles of Smad2/3 in TGF- ?1 Signaling, investigate the modulation of Smad2/3 expression by TGF-B 1 in cultured human dental pulp cells, and detect the expression of Smad2/3 as well as its change in human normal > carious and inflammatory pulp tissues and during tooth development in this study.1. A observation of Smad2/3 transducing TGF- 0 1 signal in cultured human dental pulp cells with laser scanning confocal microscopyThe HDPC of passage 5 were selected for this experiment. Cells in experimental groups were treated with 5ng/ml TGF- ?1 or 200ng/ml BMP2 for different time periods, i.e., 15min, 30min, Ih, 2h and control group without treatment. All specimens were submitted to irnmunofluorescent staining for Smad2/3, then precisely observed and photoed with LSCM. The results showed the immunofluorescence in cells treated with TGF- ?1 remained decreasing in cytoplasm but increasing in nucleus within 2h, forming a trend that Smad2/3 translocated into nucleus from cytoplasm, but this phenomenon wasn't found in cells treated with BMP2 and control group.2. In vitro effect of TGF- P 1 on Smad2/3 protein expression in human dental pulp cellsIn this study, the HDPC in passage 5 were grouped into experimental and control groups. Cells in experimental group were incubated with 5ng/ml TGF- ?1 for 6h, 12h, 24h, 48h leading up to simultaneous harvest. Total protein was isolated from cells in each group respectively and modulation of Smad2/3 protein expression by TGF- P 1 was evaluated by Western blot analyses. It was found that compared with control group, the expression level of Smad3 wasn't affected by treatment with TGF- P 1 for 6h and 12h, but after 24h it decreased markedly and kept droping by 48h. In contrast, the protein expression of Smad2 remained unchanged after TGF- ?1 treatment.3. The immunolocalization of Smad2/3 in human dental pulp tissues This study was undertaken to detect the expression and localization of Smad2/3 in human normak carious and inflammatory pulp tissues by immunohistochemical staining. The results demonstrated that the odontoblasts of normal and carious teeth were Smad2/3 strongly stained, weaker staining for Smad2/3 was observed in the underlying cell rich layer and pulp core, and strong specific staining was detected in inflammatory cells at the sites of pulp inflammation. The expression of Smad2 was stronger than that of Smad3 in all specimens .4. The expression and functions of Smad2/3 during human tooth germ developmentImmunohistochemical technique was utilized in this experiment to observe the temporal and spatial expression of Smad2/3 at different stages of human tooth germ development.
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