Objective:To express human dentin sialoprotein (hDSP) gene in COS-7 cells. Methods:hDSP gene was subcloned into mammalian expression vector pcDNA3. The recombined plasmids were transfected into COS-7 cells using lipofectamune PLUS TM kit for transient expression. Western blot analysis and immunohistochemical staining were used to examine the gene products. Results:The constructed vectors were confirmed by digestion with restriction enzyme. An immuno-reaction positive band with relative molecular mass of 60 000 was found by Western blot analysis in culture supernatant and cytoplasms of COS-7 cells. Immunohistochemical staining showed strong positive particles in the cytoplasms. Conclution:hDSP gene can be expressed in COS-7 cells.

Objective:To analyse the nucleotide polymorphism of dentin sialoprotein(DSP) coden region in Chinese people. Methods:The DSP segments were amplified by PCR;single-stranded conformation polymorphism(SSCP) and DNA sequencing were employed to detect the nucleotide polymorphisms in DSP coden region. Results:Three single nucleotide polymorphisms(SNP) were found in DSP coden region,two were same sense SNPs resulting in no change of amino acid product,and one was missense SNP resulting in change of asparagine and aspartic acid. Conclusion:There are some SNPs existing in DSP coden region in Chinese people.

Objective: To study the mechanism of differentiation of rat ectomesenchymal cells to odontoblasts. Methods: Ectomesenchymal cells were cultured in three-dimension culture model using collagen gel as frame, and the change of phenotype of ectomesenchymal cells were observed and detected by phase-contrast microscopy and immunohistochemistry after the cells had been treated by 10 ng/ml of bFGF or/and 100 ng/ml IGF-1. Results: 4 days after treatment by bFGF and IGF-1, the cells appeared to be odontoblast-like cells aligned parallelly and polarized with long cytoplasmic processes attached to one end of the cell body.The cells were positive for DSP expression. However, the cells were DSP negative and aligned disorderly in other groups. Conclusion: Ectomesenchymal cells can be induced to differentiate to odontoblast-like cells in three-dimension culture model with the treatment by bFGF and IGF-1.

Objective: To induce human dental mesenchymal cells to differentiate into odontoblasts in vitro.Methods:The cultured human dental mesenchymal cells were induced in two-dimensional culture model by bFGF(10 ng/ml)+IGF-1(100 ng/ml) or TGF-?1(5 ng/ml) for 4-7 d. Cell growth and morphology after induction were observed. The expression of human DSP protein and DSPP mRNA were detected by immunofluorescent staining and RT-PCR. Mineralization capability of the induced cells was evaluated using Von Kossa staining. Results:In both bFGF+IGF and TGF-?1 groups 20%-30% of the induced cells showed long single process.DSP protein and DSPP mRNA were observed in the induced cells.Mineralized nodules were found in the induction cultures.Conclusion: bFGF+IGF-1 or TGF-?1 can induce dental mesenchymal cells to differentiate into odontoblasts.

Objective:To construct a suppression subtractive library of suppression-related genes from c serotype Streptococcus mutans (S.mutans). Methods:After being isolated from virulent and avirulent strain of S. mutans respectively, the intact and high-pure genomic DNAs were digested with restriction enzyme AluⅠ. The digested DNA of the avirulent strain ligated with an adaptor was used as tester DNA, and that of the virulent strain as driver DNA. Then the suppression subtractive hybridization(SSH) was carried out, the efficiency of ligation and subtraction were detected respectively. The subtracted fragments were inserted into vector pCR2.1 using T/A cloning kit and transformed into E. coli TOP10F' competent cells. The white colonies were selected to construct the suppression subtractive library. Results: Through electrophoresis of AluⅠ-digested DNAs, a smear ranged from 0.1 to 2.0 kb was observed. The ligation efficiency of tester DNA with adaptor was at least higher than 25 percent. The subtraction efficiency confirmed the success in enrichment of differential genes between virulent and avirulent strain of S. mutans. In the subtracted group, the appearance time of the 23S rRNA gene in both tester and driver DNA was later than that in the unsubtracted group by twelve cycles. It suggested that suppression subtractive hybridization happened indeed. Then the subtracted fragments were cloned and the suppression-related gene library between virulent and avirulent strain of S. mutans was constructed. Conclusions:The suppression subtracted library of suppression-related genes has been constructed.

Objective:To explore avirulent strain-specific new genes or new functions of already known genes from Streptococcus mutans of serotype c.Methods:Twenty-six DNA fragments unique to avirulent strain of Streptococcus mutans were sequenced.The sequences of these presumptive avirulence DNA fragments were subjected to homology search through BLASTn and BLASTx in public database,and their putative biological functions were analyzed.Results:The size of the DNA fragments ranged from 113 bp to 776 bp.The average G+C content was 38.27%,similar to that of the gene-codingsequences in Streptococcus mutans strain UA159 whose genome sequences were just completed.Seven clones were picked repeatedly.Of the nineteen DNA fragments,eight potentially represented new DNA fragments were registered in GenBank.The remaining DNA fragments showed high homology to known genes of Streptococcus mutans strain UA159.Conclusions: The gene analysis and identification supply the groundwork for further study of gene functions of the avirulent strain of Streptococcus mutans serotype c.

Present study investigated the effect of endotoxin from Bacteroides melaninogenicus ATCC 25845 on release of colony-stimulating factor (CSF)in mice. The bone marrow cells were cultured in semisolid agar medium,the number of colonies was as a level index of CSF. The results showed that as much as 0.1?g endotoxin could induce the release of CSF,moreover, The level of CSF increased with dose of endotoxin untill 50 ?g. The colony-stimulatin factor level of B. melaninogenicus endotoxin was 66.6?8.5(CFU-C). This endotoxin showed significant effect on bone marrow cells of mice.

Objective: To investigate the effect of leukemia inhibitory factor (LIF) on the of proliferation and differentiation of ectomesenchymal cells of mandibular process in Balb/c fetal mice . Methods: Ectomesenchymal cells from the E12.5 mice mandibular process were cultured in DMEM/F12 with 10 6u/L LIF (experimental group) or without LIF (control). The proliferation effect was detected by MTT assay, Brdu test and flow cytometry. Immunohistochemistry were used to identify the differentiation state. Results: By day 7 the A value of the experimental group was 0.38?0.03,that of the control 0.30?0.02 (P

Objective: To investigate the expression of platelet-derived growth factor receptor (PDGF-R) in periapical granuloma.Methods:Immunohistochemical staining was conducted on prepared specimens of 15 cases of human periapical granuloma. Results:The expression of PDGF receptor ? was detected in fibroblasts,capillary endothelial cells,plasmacytes and macrophage cells in all the 15 cases of human periapical granuloma.Conclusion:These results suggested that these cells are the target cells of PDGF, and PDGF may play an important role in the lesion.

BACKGROUND: It remains unclear whether ectomesenchymal cells also derived from neural crest stem cell in mammals.OBJECTIVE: To understand the specific markers and differentiation directions of maxillofacial and mandibular processes progenitor cells,and to explore the source and differentiation phenotype of ectomesenchymal stem cells.METHODS: The expression and changes of expression profiles of rat ectomesenchymal cells at E9.5,E10.5,E11.5,and E12.5days were observed by immunohistochemistry and flow cytometry.RESULTS AND CONCLUSION: The progenitors expressed multi-lineage markers,including neural system and several rnesenchymal tissue types,importantly the facts that molecule profiles were changed with time prolonged,suggesting these progenitors were in active differentiating stage,so they were stem like cells or contain stem like cells.Moreover,small populations(2%-3%)of CD57 and P75 phenotypes were detected by flow cytornetry,suggesting that ectomesenchymal stem cells were derived from neural crest,which maintained a quantitative stabilization though it is gradually differentiate after localization.