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<p><b>OBJECTIVE</b>To investigate the effect of small interfering RNA (siRNA)-mediated inhibition of tumor necrosis factor receptor associated factor 6(TRAF6) gene on murine odontoblast-like cell line, MDPC-23 cell and the effect of TRAF6 on MDPC-23 cell proliferation.</p><p><b>METHODS</b>The vectors expressing siRNA against TRAF6 were constructed and introduced into MDPC-23 cell with lipofectin , and the cell line with stable expression of siRNA of TRAF6 was obtained by G418 screening and colony culture. Reverse transcription-PCR (RT-PCR) and Western blotting were performed to detect the expression of TRAF6. The proliferation of transfected MDPC-23 cell was investigated through methabenzthiazuron (MTT) and flow cytometry (FCM) assay.</p><p><b>RESULTS</b>The positive single colony was screened out, and was found to express siRNA against TRAF6 effectively because both TRAF6 mRNA and protein relative expression were significantly decreased in the experimental group (pSUPER-TRAF6siRNA: mRNA 0.163 ± 0.008, protein 0.215 ± 0.006) compared with controls (pSUPER: mRNA 0.778 ± 0.017, protein 0.964 ± 0.007 (P < 0.001). The A value of treated pSUPER-TRAF6siRNA cells (3 d: 0.46 ± 0.03, 5 d: 1.35 ± 0.06) was increased compared with controls (P<0.01). The result of proliferation index (PrI) was also increased compared with controls [pSUPER- TRAF6siRNA: (24.1 ± 2.2)%; pSUPER (11.2 ± 1.0)%; control (10.5 ± 0.7)%, P < 0.01].</p><p><b>CONCLUSIONS</b>The transcription and expression of TRAF6 gene were inhibited. The proliferation ability was increased in MDPC-23 cells by the constructed pSUPER-TRAF6siRNA vector. It may further influence the formation and repair of dentin, and may be involved in the regulation of normal tooth eruption and process of dentin repair after injury.</p>
Subject(s)
Animals , Mice , Cell Line, Tumor , Cell Proliferation , Flow Cytometry , Gene Silencing , Genetic Vectors , Odontoblasts , Cell Biology , RNA, Messenger , RNA, Small Interfering , TNF Receptor-Associated Factor 6 , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate more deeply the function and mechanism of DSPP during tooth development.</p><p><b>METHODS</b>Explants of tooth germs from embryonic 17th day mice were divided into two groups. In the control experiment, explants were cultured in agarose semi-solid medium under serum-free and chemically defined conditions, while explants in the other group were cultured with 30 mumol/L, 15 bp antisense oligodeoxynucleotide targeted to DSPP mRNA. After 10 ds, the explants were examined by transmission electron microscope. The width of dentin matrix at the tip of the cusps were then measured and statistically analyzed with Student t-test.</p><p><b>RESULTS</b>Ultrastructure analyses showed that large cisternae of the rough endoplasmic reticulum (RER) existed in the odontoblasts at the tip of the cusps of antisense-treated explants and the average thickness of dentin matrix (2.5 microns) was thinner compared to the control ones (3 microns, P < 0.001). In addition, the collagen fibers in extracellular matrix were disorganized.</p><p><b>CONCLUSION</b>These findings indicated that DSPP played an important role in keeping tooth normal development, as well as in dentin mineralization by maintaining odontoblasts' secreting ability and controlling fiber structure and orientation.</p>
Subject(s)
Animals , Mice , Embryo, Mammalian , Extracellular Matrix Proteins , Mice, Inbred BALB C , Microscopy, Electron , Molar , Oligoribonucleotides, Antisense , Pharmacology , Phosphoproteins , Protein Precursors , Pharmacology , Sialoglycoproteins , Tooth GermABSTRACT
Objective:To study the function of tumor necrosis factor r eceptor-associated factor 6(TRAF6) in the development of rat dental germ. Methods:After specimen preparation of every stage of rat developing d ental germs, immunohistochemical staining of TRAF6 was carried out. Resu lts:Positive expression of TRAF 6 was observed in thickening stage of de ntal lamina, peripheral dental lamina of bud stage, outer and inner enamal epith elium, stellate reticulum,dental papilla cells and dentinoblasts in early bell s tage, ameloblasts, dentinoblast,intermediate layer, stellate reticulum and denta l papilla cells in late bell stage, and dentinoblasts and ameloblasts in the ger m 7 days after birth. Conclusions:TRAF6 may be a signal transduc tion factor which modulates the proliferation and differentiation of developing epithelial and mesenchymal cells in the development of the dental germ.
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Objective:To determine whether the immortalized human odo nt oblast-like cell line hTERT-hOd-l has transformed phenotype. Methods: The tumorigenicity, anchorage-independent growth, serum dependence and contact inhibition of the immortalized human odontoblast-like cell line hTERT- hOd-l were observed during continuous culture in vitro. Results: The cells showed to be nontumorigenic in nude mice, and to have no anchorag e-independent growth. The cells maintained the normal serum dependence and cont act inhibition. Conclusion:hTERT-hOd-l is a normal cell line w ithout obvious transformed phenotype.
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Objective:To study the effects of parathyroid hormone (PTH) on BMP3 expression of cultured human dental papilla mesenchymal cells (DPMCs). Methods:DPMCs were obtained by cell culture, BMP3 mRNA expression was studied by in situ hybridization. BMP3 gene expression of human DPMCs after exposure to 33.3 nmol/L PTH for 5 days were measured. The cells cultured in the medium without PTH served as control.Results: Stronger BMP3 positive signals were observed in PTH treated cells than that in the control cells.Conclusion:PTH stimulats BMP3 systhesis of cultured DPMCs.
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Objective:To determine the effects of parathyroid hormone(PTH) upon dentinogenesis in rat tooth. Methods:15 SD rats aged 42 d were evenly divided into 3 groups.In the groups of control,PTT and PTX,shamoperatoin,parathyroid autotransplantation and parathyroidectomy were conducted respectively.After operation serum Ca 2+ was weekly measured with methylthymol method.30 d after operation dentinogenesis was observed with HE staining and light microscopy.Results:30 d after operation,serum Ca 2+ concentration in PTX group was lower than that in control and PTT groups(P
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Objective: To evaluate the function of dentin sialophosphoprotein (DSPP) during mouse teeth development. Methods:Expression of DSPP mRNA in the first molar of Balb/c mice at different developmental stages was detected by in situ hybridization. Resullts: DSPP mRNA in odontoblasts was detected in the samples at middle bell stage and ran through the later stages. In addition, it was detected in ameloblasts as well as in preameloblasts. Conclusion: DSPP expresses with temporospatial characters. It may play an important role in tooth morphogenesis
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Objective: To study the role of Fas/FasL signal in pulpitis. Methods: The expression of Fas/FasL in rat normal and inflammatory dental pulp was observed by immunohistochemical method and the expression of fas mRNA was studied by in situ hybridization. Results: A few of Fas and few of FasL positive cells were observed in normal dental pulp, while in inflammatory dental pulp many polymorphonuelear neutrophyl leukocytes(PMNs) and some pulp cells in the inflammatory area were stained positive. Conclusion: The apoptosis in dental pulp induced by Fas/FasL may play a role in pulpitis.
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Objective: To study the effects of parathyroid hormone(PTH) on the calcification characters of cultured human dental papilla mesenchymal(DPM) cells.Methods: DPM cells were cultured up to 35 days in two groups. The control group was cultured in DMEM supplemented with 15% FCS,50 ?g/ml ascorbic acid and 10 mmol/L ?-glycerophosphate. The cells in experimental group were cultured in above mentionned medium containing 33.3 nmol/L of PTH. The medium was changed every 3 or 4 days. Osteocalcin secretion of the cells was measured by radioimmunoassay.Results: Addition of TPH in medium caused a significant increase of osteocalcin secretion from 21 to 35 days culture (P