Potent antitumor efficacy of human dental pulp stem cells armed with YSCH-01 oncolytic adenovirus.

BACKGROUND: Systemic administration of oncolytic adenovirus for cancer therapy is still a challenge. Mesenchymal stem cells as cell carriers have gained increasing attention in drug delivery due to their excellent tumor tropism, immunosuppressive modulatory effects, and paracrine effects. However, the potential of human dental pulp stem cells (hDPSCs) loaded with oncolytic adenovirus for cancer biotherapy has not been investigated yet. METHODS: The stemness of hDPSCs was characterized by FACS analysis and Alizarin red staining, Oil Red O staining, and immunofluorescence assays. The biological fitness of hDPSCs loaded with oncolytic adenovirus YSCH-01 was confirmed by virus infection with different dosages and cell viability CCK-8 assays. Additionally, the expression of CAR receptor in hDPSCs was detected by qPCR assay. Tumor tropism of hDPSC loaded with YSCH-01 in vitro and in vivo was investigated by Transwell assays and living tumor-bearing mice imaging technology and immunohistochemistry, Panoramic scanning of frozen section slices assay analysis. Furthermore, the antitumor efficacy was observed through the different routes of YSCH-01/hPDSCs administration in SW780 and SCC152 xenograft models. The direct tumor cell-killing effect of YSCH-01/hDPSCs in the co-culture system was studied, and the supernatant of YSCH-01/hDPSCs inhibited cell growth was further analyzed by CCK-8 assays. RESULTS: hDPSCs were found to be susceptible to infection by a novel oncolytic adenovirus named YSCH-01 and were capable of transporting this virus to tumor sites at 1000 VP/cell infectious dosage in vitro and in vivo. Moreover, it was discovered that intraperitoneal injection of hDPSCs loaded with oncolytic adenovirus YSCH-01 exhibited potential anti-tumor effects in both SW780 and SCC152 xenograft models. The crucial role played by the supernatant secretome derived from hDPSCs loaded with YSCH-01 significantly exerted a specific anti-tumor effect without toxicity for normal cells, in both an active oncolytic virus and an exogenous protein-independent manner. Furthermore, the use of hDPSCs as a cell carrier significantly reduced the required dosage of virus delivery in vivo compared to other methods. CONCLUSIONS: These findings highlight the promising clinical potential of hDPSCs as a novel cell carrier in the field of oncolytic virus-based anti-cancer therapy.

[Effect of GLUD1 on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells].

PURPOSE: To investigate the effect of glutamate dehydrogenase 1 (GLUD1) on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were isolated by tissue-explant method in vitro, and shGLUD1 lentivirus was transfected to knock down the expression of GLUD1. RT-PCR and Western blot were performed to detect the expression of GLUD1. CCK8 assay was used to evaluate cell proliferation. After culture with osteogenic inducing medium for 14 days, alizarin red staining was used to detect the formation of mineralization nodules, and RT-PCR and immunofluorescence staining were performed to detect the expression of Runx2 and OCN, respectively. The data were analyzed with SPSS 20.0 software package. RESULTS: The expression of GLUD1 was significantly increased in hDPSCs after osteogenic induction compared with the control. After transfection with shGLUD1 lentivirus, GLUD1 expression was significantly decreased (P<0.05). Compared with the control group, mineralization nodule formation was significantly decreased in shGLUD1 group after osteogenic induction. The expression of OCN (late-staged markers for osteogenic differentiation) were significantly decreased both in mRNA and protein levels, while the expression of Runx2 (early-staged markers for osteoblast differentiation) was up-regulated. CONCLUSIONS: shGLUD1 inhibits the proliferation, mineralization and the late stage of osteogenic differentiation of hDPSCs in vitro. GLUD1 may play an important role in osteogenic differentiation of hDPSCs.

Local Inflammation Alters MMP-2 and MMP-9 Gelatinase Expression Associated with the Severity of Nifedipine-Induced Gingival Overgrowth: a Rat Model Study.

Nifedipine-induced gingival overgrowth (NIGO) is characterized by cell proliferation and extracellular matrix (ECM) component accumulation in gingival connective tissues, with varying degrees of inflammation and fibrosis. Impaired collagen and ECM homeostasis may be among the underlying molecular mechanisms that lead to the fibrotic changes that occur in drug-induced gingival overgrowth (DIGO). Because matrix metalloproteinases (MMPs) play vital roles in regulating collagen and ECM metabolism, many studies have been performed to reveal the relationship between MMPs and DIGO. It is thought that the gelatinases MMP-2 and MMP-9, both type IV collagenases, are involved in the development of tissue inflammation and organ fibrosis. However, the few studies regarding gelatinase expression in DIGO are controversial. Recent studies have demonstrated the inhibitory effect of cyclosporine A (CsA) on gelatinase expression and/or activity; however, similar changes have yet to be detected in Nif-treated gingival tissues. In this study, we verified that Nif treatment could lead to gingival overgrowth in rats and that gingival inflammation played a pro-proliferative role in NIGO development. Additionally, we examined the temporal expression of gelatinases on days 0, 7, 14, 21, 30, and 40 during NIGO development. The aim was to investigate whether MMP-2 and MMP-9 played significant roles in regulating NIGO development and progression. MMP-2 gene expression was not altered by Nif treatment alone but was significantly inhibited by Nif treatment for 30 days in the presence of local inflammation. However, no significant alterations in MMP-2 protein expression were detected in the Nif-treated gingival tissue, regardless of the presence or absence of local inflammation. Moreover, Nif treatment could lead to transient and significant increases in MMP-9 gene and protein expression levels in the presence of local inflammation. In particular, active MMP-9 expression increased significantly in the gingival tissue that received the combined effect of Nif and ligation treatment; besides, a temporal, but not significant, change was also observed in the gingival tissue that received Nif treatment alone. Taken together, these results provided evidence that temporal changes in MMP-2 and MMP-9 expression occurred during NIGO development. Nif treatment accompanied by local inflammation regulated MMP-2 and MMP-9 expression, primarily MMP-9, which was most likely associated with NIGO severity. Thus, MMP-9 is a potential contributing factor in the process of NIGO development.

Expression of dentine sialophosphoprotein in mouse nasal cartilage.

OBJECTIVE: Dentine sialophosphoprotein (DSPP) was initially thought to be unique for dentine formation during tooth development, whilst recent reports have shown a much broader expression pattern such as in bone, periodontium and inner ear. Our goal was to explore its expression and potential impact during early nasal cartilage formation in comparison with tooth development. STUDY DESIGN: We investigated DSPP expression in the nasal cartilage by immunohistochemistry and in situ hybridisation. We also cloned a 719bp partial DSPP cDNA from nasal cartilage and analysed its homology to the published mouse DSPP cDNA sequence. In addition, quantitative RT-PCR was undertaken to compare the expression pattern of DSPP in nasal cartilage and tooth germs during embryonic development. RESULTS: The expression of DSPP in mouse nasal chondrocytes was detected using in situ hybridisation and immunohistochemical staining. The quantitative RT-PCR data showed that expression levels of DSPP in nasal cartilage are similar to that of tooth: low at E18, and increased during development with the peak level at P3. Furthermore, DSPP levels in nasal cartilage are lower than tooth but higher than bone. CONCLUSION: DSPP is expressed in nasal cartilage, and a similar temporal expression pattern in cartilage and tooth indicates the potential importance of DSPP during development.

[Three-dimensional finite element analysis of effect of root canal taper and post on tooth stress distribution].

OBJECTIVE: To evaluate the effect of root canal taper and post on tooth stress distribution. METHODS: Three-dimensional finite element models of human mandibular first molar with root canals prepared with 35# K file, ProTaper and Profile were established. The tooth were restored with fiber-resin, stainless steel and silver amalgam posts respectively. A vertical load on tooth occlusal surface was simulated. Marc software was used to analyze and calculate the stress distributions in the tooth restored with three kinds of different root canal posts, especially the in the cervical part and root. RESULTS: Different tapered root canals had no obvious influence on stress distribution in all three different posts. Stress distribution of stainless steel post located at the cervical and middle part of distal root, the highest Von-Mises stress was about 45 MPa. Stress distribution of silver amalgam post located at the orifice of root canal and pulp fundus, the highest Von-Mises stress was about 16 MPa. Stress distribution of fiber-resin post had no obvious stress concentration. CONCLUSIONS: Fiber-resin post is the most ideal root canal post. Stainless steel post causes remarkable stress concentration in the root, which may raise the possibility of root fracture.

[Maxillary second molar with five root canals: a case report].

Root canals have a lot of variations in maxillary second molar and most molar have only three root canals. The emergence of 4 root canals in maxillary second molar with two palatal root canals is especially rare. A case of 5 root canals in maxillary second molar with two palatal root canals and two mesiobuccal root canals was successfully treated and reported in this article.

[Survey of root canal curvature of anterior teeth from Chuang population].

OBJECTIVE: To investigate the root canal curvature of permanent anterior teeth from Chuang population. METHODS: 245 anterior teeth from Chuang population were collected and examined by X-ray radiography both from labiolingual and mesiodistal directions. For 218 type I anterior teeth, degree of root canal curvature, radius of curvature and length of the curved part of root canal were measured by a special electronic vernier caliper according to Schneider's and Schäfer's method and the data obtained were analyzed. RESULTS: Root canals of anterior teeth from Chuang population were mainly of type I. The number of type II, III, IV were about 13 in mandibular central and 12 in mandibular lateral incisors. The incidence of curvature in maxillary central incisors, lateral incisors, canines and mandibular central incisors, lateral incisors, canines were 40%, 80%, 77%, 65%, 66%, 73% in mesiodistal directions, 62%, 69%, 70%, 62%, 41%, 61% in labiolingual directions respectively. The most curvature was moderate and happened in apical third. The heaviest curvature occurred in maxillary canines in mesiodistal direction and mandibular canines in labiolingual direction. The shortest radius and length of curvature occurred in maxillary lateral incisors. CONCLUSION: Root canal curvature of anterior tooth in Guangxi Chuang population is complex. The incidence of type II, III, IV is high in mandibular incisors.

Root canal morphology of permanent maxillary teeth in the Han nationality in Chinese Guanzhong area: a new modified root canal staining technique.

INTRODUCTION: The purpose of this study was to investigate the canal morphology of 504 maxillary permanent teeth of subjects of Han nationality in Chinese Guanzhong area. METHODS: Maxillary permanent teeth were randomly collected in Guanzhong area. After regular preparation, the teeth were immersed into ink without preparing access cavities and then put into hyperbaric oxygen chamber (0.6 Mpa) for 2 hours to let the ink penetrate into root canal from apical foramen, apical deltas and foramen of lateral canals under stable positive pressure. After demineralization and clearing, the following observations were made: (1) number of root canals, (2) root canal configuration by using Vertucci's classification, (3) presence of lateral canals, and (4) frequency of apical deltas. RESULTS: All the teeth were well-stained, and the fine details were well-revealed. Apical deltas (12.2%-83.3%) and lateral canals (13.7%-68.8%) could be frequently found in all types of maxillary teeth. Most of central incisors (95.8%), lateral incisors (91.4%), and canines (75.4%) displayed type I canal configuration, whereas most of first premolars (87.3%) and second premolars (72.3%) possessed 2 canals with type II, IV, or VI canal configuration. The majority of distobuccal roots and palatal roots of first molars (88.9%, 97.8%), second molars (92.0%, 94.0%), and third molars (87.5%, 91.6%) possessed type I canal configuration. The prevalence of mesiobuccal roots with type I configuration was 66.7% in maxillary first molars, 82% in second molars, and 62.5% in third molars. CONCLUSIONS: The modified technique of canal staining can effectively reveal detailed root canal system. The canal configuration of maxillary teeth in subjects of Han nationality in Chinese Guanzhong area is consistent with previous reports in other races.

[Root canal treatment of mandibular first premolar with 3 root canals: a case].

The occurrence of three canals in mandibular premolars is very rare. This article reported and discussed the treatment of a mandibular first premolar with 3 root canals, specially in aspect of root-detecting and root-shaping.

[Effect of cyclic strain on cell morphology, viability and proliferation of human dental pulp cells in vitro].

PURPOSE: To investigate the effect of mechanical strain on cell morphology, viability and proliferation of human dental pulp cells in vitro. METHODS: Human dental pulp cells were cultured and subjected to 2% or 8% strain with Flexcell Tension Plus System for 0.5 hour, 12 hours and 24 hours, respectively, and then the cell morphology, viability and proliferation were examined by phase contrast microscope, trypan-blue staining and MTT method. The results were analyzed by one-way ANOVA with SPSS16.0 software package. RESULTS: Cells were stretched and aligned perpendicular to the direction of the force applied with obvious pole formation under the tested condition. The viability and proliferation of the cells subjected 2% or 8% strain were significantly higher than that of untreated cells, which reached the peak at 12 hours. The proliferation of the cells increased after loading strain which was significantly higher than that in the control by 2% stain subjected for 24 hours (P<0.01). CONCLUSIONS: Cyclic strain could affect morphology, viability and proliferation of in vitro cultured human dental pulp cells in a magnitude/time dependent manner.

[Effect of composite restoration on the reinforcement of teeth].

OBJECTIVE: To determine the effect of composite restoration on reinforcement of weakened tooth structure and the possible mechanism. METHODS: Sixty freshly extracted non-carious maxillary premolars were collected and divided into 6 groups with 10 specimen in each group. MOD cavities (buccolingual width: 2.8 to 3.2 mm; palatal cusp width: 2.0 mm; cusp height: 5.0 mm) were prepared individually. Group 1 was prepared and not restored (control). The other 5 groups were restored with silver amalgam alloy (group 2), Z250 without bonding (group 3), F2000 (group 4), Z250 (group 5) and Z350 nanocomposite (group 6) (3M ESPE) respectively. The fracture resistance of the tested teeth was determined by applying a vertical splitting load through a specially shaped steel rod at a crosshead speed of 1 mm/min. The data were analyzed by ANOVA. RESULTS: The average fracture resistance of the 6 groups was: (245.29 +/- 39.49) N (group 1), (255.09 +/- 42.14) N (group 2), (267.34 +/- 31.56) N (group 3), (293.90 +/- 33.42) N (group 4), (337.81 +/- 32.63) N (group 5) and (349.08 +/- 32.93) N (group 6). There was no significant difference between the group 1, group 2 and group 3. The fracture resistance of group 4, group 5 and group 6 was higher than that of group 1 and group 2 (P < 0.05). Significant difference was noted between group 5 and group 3 (P < 0.01). The fracture resistance of group 4 was much lower than that of group 5 and group 6 (P < 0.01). No significant difference was found between group 5 and group 6. CONCLUSIONS: The use of composite increased the fracture resistance of the tooth with an MOD restoration. This effect was related to the adhesive force, polymerization shrinkage stress and the elastic modulus of the composite.

[Influence of soft-start irradiation on polymerization shrinkage stress of universal hybrid composite resins].

OBJECTIVE: To compare the difference between soft-start curing mode and standard curing mode in polymerization shrinkage stress of universal hybrid composite resins and to study effect of the soft-start curing mode on the decrease of shrinkage stress. METHODS: Three universal hybrid resins (A: Charisma, B: TPH Spectrum, and C: Esthet-X) were respectively filled in cavities (4 mm in diameter) of epoxide resin disks, 16 specimens of each. Off them, eight of the specimens for each composite resin were exposed using soft-start mode and the other eight using standard mode. Polymerization contraction stress was calculated during 48 h after curing with photo-elastic stress analysis. RESULTS: Three composite resins cured with soft-start mode showed the same trend of shrinkage stress changing as that with standard curing mode and values of polymerization shrinkage stress at 24 h after curing were (3.80 +/- 0.31) MPa, (3.21 +/- 0.40) MPa, and (2.84 +/- 0.22) MPa respectively for A, B and C composite resins. The corresponding figures for the composites with standard curing mode were (4.19 +/- 0.24) MPa, (3.69 +/- 0.33) MPa, and (3.14 +/- 0.28) MPa. Three composite resins cured with soft-start mode had significantly lower polymerization shrinkage stress compared with standard curing mode at 24 h after curing (P < 0.05). CONCLUSIONS: Using soft-start curing mold can reduce, to some extent, the polymerization shrinkage stress of universal hybrid composite resins.

[Transcriptional regulation of dentin sialophosphoprotein by c-Jun/c-Fos].

OBJECTIVE: To investigate the role of c-Jun and c-Fos as transcriptional factors in regulation of dentin sialophosphoprotein (DSPP) gene by a promoter-luciferase reporter gene construct in odontoblast cell line MDPC-23. METHODS: Endogenous c-Jun or c-Fos protein was determined by immunocytochemistry. The role of c-Jun or c-Fos in transcription of DSPP was investigated in co-transfection experiments using promoter-luciferase reporter gene construct containing the sequence between -791 bp and +54 bp of mouse DSPP gene. RESULTS: c-Jun and c-Fos was expressed by MDPC-23 cells, and located in the nucleus of MDPC-23 cells. Overexpression of c-Jun or c-Fos significantly inhibited luciferase activity of DSPP promoter. CONCLUSION: These findings suggest c-Jun and c-Fos down-regulated the transcription of DSPP gene as a transcriptional factor in odontoblast.

[The effect of core binding factor alpha1 on transcriptional regulation of mouse dentin sialophosphoprotein].

PURPOSE: To investigate the effect of core binding factor alpha1 (cbfalpha1) on transcriptional regulation of mouse dentin sialophosphoprotein (DSPP) gene. METHODS: The MDPC-23 cells and the segment of nt -2475bp to +53bp were chosen. After co-transfected, the MDPC-23 cells were measured for luciferase activity using the dual luciferase reporter assay system. The results were analyzed by SPSS10.0 software package. RESULTS: In MDPC-23 cells, the luciferase activity was significantly low in the group of co-transfected with pGL3-Enhancer-2.6K and pcDNA3-cbalpha1 than the group of pGL3-Enhancer-2.6K and pcDNA3 (P < 0.01). CONCLUSION: Cbfalpha1 can reduce the activity of DSPP promoter including the segment of nt-2475bp to +53bp in MDPC-23 cells. This suggests that cbfalpha1 can transcriptionally regulate the expression of DSPP. Supported by National Natural Science Foundation of China (Grant No.30271418).

Smad protein mediated transforming growth factor beta1 induction of apoptosis in the MDPC-23 odontoblast-like cell line.

OBJECTIVE: The function of apoptosis and its regulation in odontoblasts remain unclear. In this study, we characterize the possible role of transforming growth factor (TGF)-beta 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-beta1-induced apoptosis in odontoblasts. METHODS: Annexin V/propidium iodide staining, cell Death Detection ELISA and DNA ladder were used to examine the effect of TGF-beta1 on apoptosis in a mouse odontoblast-like cell line, MDPC-23. Stable cell clones expressing Smad2 or Smad3 dominant negative mutants, or wild-type Smad7 were constructed to investigate the role of Smad proteins in the mediation of apoptosis by TGF-beta1 in MDPC-23 cells. The TGF-beta1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-beta-responsive reporter gene, p3TP-Lux. RESULTS: TGF-beta1 can induce apoptotic cell death in MDPC-23 cells in a dose-dependent manner. Transfection of dominant negative mutant forms of Smad2 or Smad3 blocked TGF-beta1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-beta1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-beta1-induced transcriptional activity. CONCLUSION: These results suggest that Smad proteins are involved in TGF-beta1-induced apoptosis of odontoblast cells.

[Role of Smad signaling in transcription of Smad7 gene mediated by TGF-beta1 in odontoblast cell line MDPC-23].

PURPOSE: To investigate the role of Smad signaling in transcription of Smad7 gene mediated by TGF-beta1 in odontoblast cell line MDPC-23, and to explore the molecular mechanism of Smad7 gene expression mediated by TGF-beta1 at the transcriptional level. METHODS: Smad function and its role in transcription of Smad7 were investigated in cotransfection experiments using Smad7 promoter-luciferase reporter construct containing the sequence between -408 bp and +112 bp of mouse Smad7 gene. The data were analysed by one-way ANOVA. RESULTS: When the Smad7 promoter-luciferase reporter gene construct was expressed in MDPC-23 cells, its transcriptional activity was significantly induced by TGF-beta1 treatment, whereas not by BMP-2 treatment. Overexpression of Smad1, 2, 4, or 5 had no effect on transcriptional activity of Smad7 promoter. Overexpression of Smad3 markedly promoted transcriptional activity of Smad7 promoter, whereas co-transfection of Smad3 and Smad4 doubled the effect of Smad3. Overexpression of Smad3 dominant negative mutant or Smad3 antisense cDNA (AS-Smad3) significantly inhibited transcriptional activity of Smad7 promoter induced by TGF-beta1. CONCLUSION: TGF-beta1 regulated transcription of Smad7 gene through association of Smad3 and Smad4 in MDPC-23 cells.

[Immortalized human odontoblast-like cell line expressed dentin extracellular matrix in vitro].

OBJECTIVE: To determine whether dentin matrix proteins were expressed by the human odontoblast-like cell line hTERT-hOd-1 in vitro. METHODS: Collagen type I, bone sialoprotein (BSP), dentin matrix protein 1 (DMP1) and the marker for odontoblast, dentin sialophosphoprotein (DSPP) and dentin sialoprotein (DSP) were detected in these cells by immunohistochemistry, RT-PCR and in situ hybridization. During being cultured in mineralizing medium for 5 weeks, the secretion of OC and activity of ALP were measured once a week. RESULTS: DSPP, DMP1, BSP and collagen type I were expressed in hTERT-hOd-1 either at mRNA or protein level. Under the induction of mineralizing medium, the cells showed higher activity of ALP and increased secretion of OC. CONCLUSION: hTERT-hOd-1 expressed dentin extracellular matrix in vitro, which means the cell line has the potential of mineralization.

[Bone morphogenetic protein-2-induced alpha 2 (I) collagen expression in odontoblastic MDPC-23 cells mediated by Smad proteins].

OBJECTIVE: To characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23. METHODS: Endogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct. RESULTS: MDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant. CONCLUSIONS: Smad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

TGF-beta activated Smad signalling leads to a Smad3-mediated down-regulation of DSPP in an odontoblast cell line.

OBJECTIVE: Transforming growth factor-beta (TGF-beta) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-beta have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-beta. In this study, we characterise the role of Smad proteins as mediators of TGF-beta in a mouse odontoblast cell line MDPC-23. METHODS: Transcription of Smads was detected by RT-PCR. The change of intracellular location of Smad proteins treated by TGF-beta1 was evaluated immunocytochemically. Smad function and its role in transcription of dentin sialophosphoprotein (DSPP) were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: MDPC-23 cells expressed Smad2, Smad3 and Smad4 mRNA. Endogenous Smad2, Smad3 and Smad4 rapidly translocated from the cytoplasm into the nucleus in response to TGF-beta1. The activity of the TGF-beta-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-beta1 treatment. Over-expression of wild-type Smad3 promoted TGF-beta1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-beta1 also inhibited the activity of DSPP promoter luciferase reporter construct containing the sequence between -791 bp and +54 bp of the mouse DSPP gene. Over-expression of wild-type Smad3 potentiate the inhibitory effect of TGF-beta1 on transcriptional regulation of DSPP, while dominant negative Smad3 decreased the effect. In contrast to Smad3, wild-type Smad2 or its dominant negative mutant had little effect on TGF-beta1 regulation of the promoter activity of DSPP. CONCLUSIONS: Smad2, Smad3 and Smad4 are present and activated by TGF-beta1 in MDPC-23 cells. The Smad pathway is functional in these cells and Smad3 appears to be involved in down-regulation of DSPP by TGF-beta1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.

[Cloning and sequencing of the upstream of mouse dentin sialophosphoprotein promoter].

OBJECTIVE: To clone and sequence the upstream of mouse dentin sialophosphoprotein promoter. METHODS: Genomic DNA was obtained from Balb/c mouse blood. The upstream of mouse dentin sialophosphoprotein promoter segments was obtained by PCR. Then the segments were inserted into T-vector. The plasmids were identified by digestion with the restriction enzyme analysis. The positive clone was sequenced and compared with Genebank. RESULTS: The upstream of mouse dentin sialophosphoprotein promoter was divided into three sequences and three different target segments with 997 bp, 1004 bp and 674 bp in length were obtained. After identified, sequenced and compared with Genebank, the sequences of the segments were consistent with those displayed on Genebank by 99%. CONCLUSION: The clone of the upstream of mouse dentin sialophosphoprotein promoter was successful. This work will help to study the regulation of DSPP expression.