ABSTRACT
Fluoride can be widely ingested from the environment, and its excessive intake could result in adverse effects. Dental fluorosis is an early sign of fluoride toxicity which can cause esthetic and functional problems. Though apoptosis in ameloblasts is one of the potential mechanisms, the specific signal cascade is in-conclusive. High-throughput sequencing and molecular biological techniques were used in this study to explore the underlying pathogenesis of dental fluorosis, for its prevention and treatment. A fluorosis cell model was established. Viability and apoptosis rate of mouse ameloblast-derived cell line (LS8 cells) was measured using cell counting kit-8 (CCK-8) assay and flow cytometry analysis. Cells were harvested with or without 2-mM sodium fluoride (NaF) stimulation for high-throughput sequencing. Based on the sequencing data, subcellular structures, endoplasmic reticulum stress (ERS), and apoptosis related biomarkers were verified using transmission electron microscopy, quantitative real-time polymerase chain reaction, and Western blotting techniques. Expression of ERS markers, apoptosis related proteins, and enamel formation enzymes were detected using Western blotting after addition of 4-phenylbutyrate (4-PBA). NaF-inhibited LS8 cells displayed time- and dose- dependent viability. Additionally, apoptosis and morphological changes were observed. RNA-sequencing data showed that protein processing in endoplasmic reticulum was obviously affected. ERS and apoptosis were induced by excessive NaF. Downregulation of kallikrein-related peptidase 4 (KLK4) was also observed. Inhibition of ERS by 4-PBA rescued the apoptotic and functional protein changes in cells. Excessive fluoride induces apoptosis by activating ERS, which is mediated by GRP-78/PERK/CHOP signaling. Key proteinase is present in maturation-stage enamel; KLK4 was also affected by fluoride, but rescued by 4-PBA. This study presents a possibility for therapeutic strategies for dental fluorosis, while further exploration is required.
Subject(s)
Butylamines , Fluorides , Fluorosis, Dental , Mice , Animals , Fluorides/pharmacology , Fluorides/metabolism , Ameloblasts , Fluorosis, Dental/metabolism , Endoplasmic Reticulum Chaperone BiP , Sodium Fluoride/pharmacology , Apoptosis , Endoplasmic Reticulum StressABSTRACT
OBJECTIVE: This study aimed to evaluate the influence of age on the pulpal blood flow (PBF) of immature maxillary incisors of maxillary incisors, which was detected by laser Doppler flowmetry (LDF). METHODS: LDF was used to detect the PBF value of maxillary central and lateral incisors of a child group (aged 7-13 years old) and a positive control group (aged 18-25 years old), as well as the central incisor of a negative control group (the central incisor had undergone endodontic treatment). We then compared the features of PBF in all groups with the influence of gender and position on PBF. The relation of maxillary central incisor and lateral incisor, age, and maxillary incisor were analyzed. RESULTS: The PBF value of the negative control group was (2.08±0.73) PU. The PBF values in the positive control group in central and lateral incisors were (8.49±1.88) and (7.52±1.82) PU. In the child group, PBF values in central incisors and lateral incisors were (11.31±2.21) and (12.18±2.65) PU. A significant difference was observed between different groups and between central and lateral incisors (P<0.01). Meanwhile, no significant difference was found in the PBF values between the right and the left parts in both males and females (P>0.05). Age had a linearity negative correlation with the PBF value of incisors in the child group. A linear negative correlation existed between the age and PBF of central and lateral incisors (r=-0.310 and r=-0.510, respectively) (P<â©0.01). CONCLUSIONS: PBF value decreased with increased age in children aged 7-13 years old.
Subject(s)
Dental Pulp , Incisor , Tooth Avulsion , Adolescent , Adult , Child , Dental Pulp/blood supply , Dental Pulp/diagnostic imaging , Female , Humans , Laser-Doppler Flowmetry , Male , Maxilla , Young AdultABSTRACT
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.
ABSTRACT
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 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
ABSTRACT
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.
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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.