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Objective:To culture and characterize the deciduous tooth pulp stem cells(DTPSCs)of Beagle dog.Methods:DTPSCs were cultured using enzyme tissue block method from Beagle dog aged 6 weeks.The cells were purified by cloning culture,identified by immunohistochemistry and flowcytometry.The biological characteristics were studied by CCK-8 assay,osteogenetic induction,li-pogenic induction and dentinogenic induction assays.Results:Beagle stem cells from deciduous tooth pulp were obtained,the cell colony formation rate was 32%.The cells were STRO-1 and CD146 positive,CD14,CD45 and CD86 negative.After multiple induc-tion culture the cells were positive for alizarin red staining,oil red staining,ALP expression and DSSPP expression.Conclusion:The deciduous tooth pulp stem cells of Beagle dog have multilineage differentiation abilities.
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Objective:To investigate the adhesion,proliferation and differentiation of the stem cells from human exfoliated deciduous teeth(SHEDs)on 3 different types of hydroxyapatite(HA)composite scaffold materials.Methods:Pulp cells from human exfoliated de-ciduous teeth were harvested from impacted deciduous teeth by enzyme digestion,expanded and cultured.Cells were verified by immuno-histochemical methods and in vitro differentiation test.Then the cells were cultured on HA/beta tricalcium phosphate (HA/TCP),HA/collagen (HA/COL)and HA/poly-ethylene propylene lactide (HA/PLGA)scaffold respectively.Adhesion rate was examined at hour 4,6,8 and 10 of culture,proliferation was observed by MTT assay on day 1,4,7 and 10 of culture,respectively.The osteogenic dif-ferentiation was studied by alkaline phosphatase(ALP)test,Von Kossa staining and calcium content measur.Results:The attachment of SHEDs was significantly lower on the HA/COL than on the other 2 scaffolds(P <0.05).The ALP activity,mineralization and calci-um content were the highest on HA/PLGA,and the last on HA/COL(P <0.05).Conclusion:HA/PLGA scaffold is more effective in the promotion of the proliferation,attachment and differentiation of SHEDs than HA/TCP and HA/COL scaffolds.
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Objective:To synthesize autoinducer-2 by the clone and prokaryotic expression of Streptococcus mutans(S.mutans)UAl59 luxS gene and to observe the influence factors.Methods:The expression vector pET21 a(+)-luxS of S.mutans UAl59 was transformed into Escheriehia coli BL2l(DE3).The S-ribosylhomocysteinase(Luxs)expression was induced by IPTG.The His tag fusion protein was isolated by Ni-chelating column and identified by Western blotting.Finally the protein was renatured by dialysis method.S-ribosylhomo-cysteine (SAH)was catalyzed by s-adenosylhomocysteine nucleosidas (Pfs)and LuxS,and then AI-2 was syntheszed.The AI-2 activi-ty was examined by luminescence of Vibrio harveyi BB1 70 when the concentration of LuxS protein or pH(4 -1 2)or the concentration of sodium fluoride was changed in reaction mixes of AI-2 synthesis.Results:Compared with the control group,with the increase of LuxS protein concentration,the relative activity of in vitro synthesized AI-2 increased gradually(P <0.001 ).When pH was between 6 -1 0, the relative activity of AI-2 were the highest,beyond the range of pH,the relative activity of AI-2 decreased(P <0.001 ).When a final concentration of sodium fluoride was more than 0.3%,the luminescence values decreased(P <0.05).Conclusion:LuxS fusion protein can promote the production of AI-2.Optimum pH for AI-2 biosynthesis in vitro must be between 6-1 0.Biosynthesis of AI-2 is inhibited by sodium fluoride with final concentration of more than 0.3%.
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Objective:To evaluate the effect of gtfB specific antisense phosphorothioate-modified oligodeoxyribonucleotides(PS-ODNs) on S.mutans sucrose-dependent adherence.Methods:Antisense oligodeoxyribonucleotide targeted to gtfB sequence 709~726 bp(PS-ODN1) and 3 479~3 497 bp(PS-ODN2) were synthesized.Natural genetic transformation of S.mutans with PS-ODN1 and PS-ODN2 was respectively performed.Adhesion of S.mutans to saliva coated hydroxyapatite was examined by crystal violet staining and destain with absolute ethanol.The absorbance at 620 nm was measured by plate reader(the absorbance value derived from the wells without sucrose was used as background and was subtracted).Results:The adhesion ability of the strains treated with antisense PS-ODN was significantly lower than that of the control(P
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0.05).Conclusion:So-fastTM can greatly improve the penetration of PS-ODN into S.mutans and can be used as appropriate delivery system of PS-ODN for S.mutans. No matter what approaches were adopted, the uptake rate reached the maximum at 5 ?mol/L and 2 h-exposure. The penetration can not be enhanced by increasing the PS-ODN concentration and the transformation time.
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Objective:To identify metastasis-associated genes in salivary gland adenoid cystic carcinoma(ACC).Methods:Salivary gland adenoid cytic carcinoma cell line ACC-2 and its highly metastatic ACC-M cells were used to screen the metastasis-related genes in ACC by microarray technology.Two fluorescent cDNA probes labeled with Cy3 and Cy5 dyes,were prepared from the mRNA samples of ACC-2 and ACC-M cells by reverse transcription method.The two color probes were then mixed and hybridized on the cDNA chip constructed by double dots of 1152 human genes,and scanned at two wave lengths.Differentialy expressed genes of the two cell lines were analyzed using computer.Then seven of the differently expressed genes were further validated by RT-PCR technique.Results:Of the 1,152 known genes and expressed sequence tags,26 showed significantly different expression level(minimum 2 fold) between the two cell lines.Among the 26 genes,19 were up regulated(with ratio more than 2) and 7 were down(with ratio less than 1/2).The results of RT-PCR analysis for 7 differently expressed genes were coincident with those of microarray assay.Conclusions:Down regulation of LIFR,LCP1,DPEP1 and ABLIM1,and up regulation of DCC,MMP1 and CNTN2 may be related to the highly metastatic potential of ACC-M cell line.