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.

[Study of cbfalpha1 on the expression of extracellular matrix proteins in dental papilla cells induced by BMP-2 in vitro].

PURPOSE: To evaluate the function of cbfalpha1 on BMP-2 signaling to extracellular matrix proteins in dental papilla cells in vitro. METHODS: RT-PCR and Western blot were performed to detect the expression of ALP, OC, ON, OPN, BSP, DMP-1 and DSPP in cultured dental papilla cells induced by 200ng/mL BMP-2 and/or down-regulated by cbfalpha1 antisense technology, the results were analysed with SPSS 11.0 software package. RESULTS: We found that the amount of ALP and OC and the expression of OPN, BSP and ON were upregulated significantly after the cells were treated with BMP-2. After transfected with antisense cbfalpha1, the cells downregulated the expression of ALP, OC, OPN and BSP significantly(P<0.01). CONCLUSIONS: As a kind of transcription factor, cbfalpha1 could be an important tache in the BMP-2 signal networks controlling cells differentiation and mineralization.

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

OBJECTIVE: To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). METHODS: HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. RESULTS: Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. CONCLUSION: The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

[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).

[Generation of transgenic mouse of dentin sialoprotein and transgene expression analysis].

PURPOSE: To generate the transgenic mouse model of DSP and perform transgene expression analysis by RT-PCR. METHODS: Plasmid pcDNA3.1-CX was constructed by substituting promoter cbeta-actin for CMV promoter of pcDNA3.1, and the ultimate transgenic vector, pcDNA3.1-CX-dsp, was constructed by cloning DSP coding sequence into pcDNA3.1-CX. The pcDNA3.1-CX-dsp plasmid was linearized and microinjected into the male pronucleus of the zygotes. The tail DNA of pups was tested by PCR and Southern blot. A member of F1 generation of one positive mouse was used to perform transgene expression analysis by RT-PCR. RESULTS: 717 embryos were implanted to 29 recipient pseudopregnant mice, 4 of the 67 pups carrying the transgene. Expression of DSP was detected in a member of F1 generation of one positive mouse by RT-PCR. CONCLUSION: Founders of the DSP transgenic mouse were obtained successfully, and the expression of DSP was primarily confirmed.

[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.

[Generation of dspp-LacZ transgenic mouse founders].

PURPOSE: To establish a transgenic mouse founders in which the expression of LacZ was directed by a dentin sialophosphoprotein-specific promoter. METHODS: The DSPP-specific promoter was obtained by PCR and confirmed by sequencing, and the transgenic plasmid, pTN-DPM-LacZ, was constructed by subcloning the DSPP-specific promoter and LacZ-encoding sequence into one vector. The linearized transgenic plasmid was microinjected into the male pronucleus of the zygotes, and the microinjected zygotes were implanted to recipient pseudopregnant mice. The tail DNA of 4 week-pups was tested by PCR. RESULTS: 503 embryos were implanted to 20 recipient pseudopregnant mice, 12 of the 89 pups carrying the transgene. The establishment of the dspp-LacZ transgenic mouse line is under progress. CONCLUSION: 12 founders of the dspp-LacZ transgenic mice, in which the expression profile of LacZ should be the same as that of DSPP, were obtained by microinjection successfully, and the mouse line which is being established could be used as a good tool to investigate the exact expression profile of DSPP in the future.

[IRF6 gene mutation analysis in a van Der Woude syndrome family in Henan province].

PURPOSE: To investigate IRF6 gene mutation in a van Der Woude syndrome (VWS) family in Henan province. METHODS: PCR and DNA sequencing was employed to detect the mutation of IRF6.Secondary construction transformation analysis was performed using PIX-Protein Identification software. RESULTS: A CGC>TGC(r.279c-->t) transversion of IRF6 was identified in condon 6, showing complete segregation with the disease phenotypes and was resulting in changes of the secondary constructure of IRF6. CONCLUSION: VWS is caused by mutations in IRF6 gene, and IRF6 is closely related to the development of lip, palate and tooth.

[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.

[Cbfa1 induces the expression of the mineral-related proteins in human dental papilla cells].

OBJECTIVE: To explicit whether the expression of the mineral-related proteins is regulated by cbfa1 in human dental papilla cells. METHODS: Human dental papilla cells were cultured in vitro and transfected with pcDNA3-cbfa1 recombinant plasmids. After selected with G418 sulfate, a cell clone named PC-3, which could stably express the cbfa1 mRNA and protein, was proved by PCR and western blot. Then the amount of ALP and OC and the expression of OPN, BSP, ON, DMP1, DSP and DSPP were detected by immunohistochemistry, Western blot and PCR methods. RESULTS: We established the human dental papilla cells model PC-3 which could stably express the cbfa1 mRNA and protein. Compared with normal cells, a lot of mineral-related proteins such as ALP, OC, OPN, BSP, ON, DMP1 were upregulated in PC-3 cells. CONCLUSIONS: In human dental papilla cells, cbfa1 can induce the expression of most mineral-related genes and proteins. It may implicate that cbfa1 must play a key role during tooth development and mineralization.