| The odontoblasts are one kind of typical polarized cells, which stay at the outer pallof the dental pulp and appearance as high column cells. It holds an apical side which cansecret dentin matrix and a bottom side which stays in the dental pulp. The odontoblastsexperience from the unpolarized cells to the polarized cells during the developmentprocess, which goes with the mature of odontoblasts and the secretion of dentin. Duringthe process of cell apical-bottom polarization, the construction of cell junctions and thelocation of polarization protein complexes are two necessary cell biological behaviors.Cell junctions work as the arch structure between the neighbor cells, which can stop theout substances from penetrating. As the separating belt, they divide the cell membrane intoapical side and bottom side. The construction of cell junctions, which is the preconditionof cell polarization, also provides arch structure for the location of polarization proteincomplexs. The genesis of cell polarization complexes and their location in the specificarea of cell membrane are the molecule foundation for cell polarization. The locations of polarized proteins in cell membrane decide the functions of the related areas. At the sametime, the construction of cell junctions and the location of polarization molecules can alsoaffect the distribution of organelles in cell body. The cell nucleus locals at the bottom ofcell body in mature odontoblasts, while the ER(endocytoplasmic reticulum) and the Golgibody face the secretion side—the apical side. The genesis of this kind of cell form is themorphological foundation of cell polarized secretion. This study confirmed theexpressions of some proteins which were close related with the construction of theodontoblast polarization and made some acknowledgements of their functions, whichmight provide some foundations for the related studies.1. The expressions of some proteins related with polarization in odontoblastsAfter the extraction of total RNA and total protein from the mouse dental pulp andOLCs, the mRNA expression of cdc42，ZO-1，occludin，claudin-1and E-cadherin weretested by PCR and the protein expression of cdc42was tested by western blot. After thefixation，dehydration，permeation of OLCs, the location of cdc42in OLCs was detected bycell Immunofluorescence. After the fixation，dehydration，paraffin，embedding，sectionof mouse at prenatal14d，16d，18d and postnatal2w, the expression of cdc42during thedevelopment of dental germ was detected by organ Immunofluorescence. The resultsshowed that the mRNA of cdc42，ZO-1，occludin，claudin-1，E-cadherin and the proteinof cdc42were expressed in both the mouse dental pulp and OLCs. Cdc42expressed atboth the cell membrane and cell body, and it showed at a higher level around the cellnucleus than other parts of the cell. During the development of dental germ, cdc42participated in the genesis of odontoblast polarization.2. The construction of cell junctions of odontoblasts and its relation with the LPSAfter the OLCs were cultured with α-MEM in transwell for2w, the cell spread wasdetected by SEM and the construction of cell junctions were tested by TEM. OLCs weretreated by different concentrations of LPS (10ng/ml,100ng/ml and1000ng/ml). Cellscultured with LPS and PDTC were taken as control. The cell junction protein E-cadherinwas tested by Western blot and the mRNA expression of ZO-1, claudin-1，occludin and E-cadherin was detected by real-time PCR at different time point(0.5h,1h,2h and6h). Theresults showed that the cell junctions formed between the neighbour OLCs. The mRNAexpressions of claudin-1, ZO-1, occludin and E-cadherin decreased significantly in cellstreated by LPS with a time-dependent effect but no dose-dependent effect. The expressionof E-cadherin at protein level showed a time-dependent decrease with increasing time ofLPS, while the NF-κB inhibitor PDTC inhibited this effect.3.The relation between cdc42and odontogenic cellsAfter the OLCs were cultured to80%confluence, the groups changed bymineralization induction medium， α-MEM with10ng/ml LPS and mineralizationinduction medium with10ng/ml LPS respectively were cultured to the planning time, theexpression change of cdc42was tested by western blot. The groups changed bymineralization induction medium and mineralization induction medium with cdc42inhibitor ML141were cultured to the planning time, the mineralization nodus was stainingand analyzed by alizarin red (AZR). HDPSCs was cultured by mineralization inductionmedium with different concentration of LPS or ML141for planning time and themineralization nodus was staining and analyzed by alizarin red. In the western blot results,the expression of cdc42showed no significant change in OLCs treated by mineralizationinduction medium but an increase with increasing time of LPS. The AZR staining showedthat the mineralization nodus formation of OLCs were positive correlation with time andthe mineralization nodus formation of HDPSCs is positive correlation with theconcentration of LPS, while both of them are negative correlation with the concentrationof ML141. |
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