| Objective: The aim of this study was to investigate the influence ofAngiotensinⅡon the proliferation activity,cell cycle and migration of humandental pulp cells (HDPCs) in vitro. The tissue explant-collagenase digestionmethod was used to obtain a large number of human dental pulp cells andestabish stable model of human dental pulp cells.Methods:1Sound human third molars or premolars freshly extracted fororthodontic reason was collected (with the patients’ infromed consent) Thedental pulp tissue was gently removed under the asepsis condition andcultured according to the tissue explant-collagenase digestion method. Cellularmorphology was observed under an inverted phase microscope. When cellsemigrated from the edge of tissue blocks and reached80%confluency,adigestion procedure with0.25%of trypsin was followed by passage culture ata proportion of1:1or1:3. The4th generation human dental pulp cells wereseeded for the identification of cell source by vimentin and keratinimmunohisto-chemical stains and observed by inverted microscopy.2The fourth generation human dental pulp cells in logarithmic phasewere digested and made into cell suspension with0.25%of trypsin. Then thecell suspension was planted in five96-well microplates with2×104cells ml-1and200μl for each well. After24h, the cells, whose supernatant fluid wasabandoned, were attached in good condition and were cleaned3times byphosphate-buffered saline (PBS), then the PBS buffer was blotted. The cellswere divided into the experimental groups, the control group and six holeswere selected in each group. The experimental group: in the correspondingcell holes,200μl AngiotensinⅡwhose terminal concentration respectively was10-9,10-8,10-7,10-6,10-5mol/L was added. AngiotensinⅡ of the terminal concentrations was made with DMEM containing5%FBS. The control group:in the corresponding cell holes, only200μl DMEM containing5%FBS wasadded. Cells were incubated at37℃in5%CO2. Took out of a96-wellmicroplate at24h,48h and72h respectively, WST-8reagentswere added toeach well.Incubated for4h at37°C,absorbance readings at a wavelength of450nm were taken with a spectrophotometer.3The4th generation human dental pulp cells in logarithmic phase weredigested with0.25%of trypsin and removed from the culture plate using PBSbuffer.Centrifugating for5min in1000r/min, the supernatant was abandoned,and cells were suspended in10%serum containing medium at a density of2×104cells ml-1, and3ml of media was seeded into culture flasks. The cellswere divided into the experimental group and the control group three flaskswere selected in each group. Cells were incubated for24h at37℃in5%CO2.Supernatant fluid was abandoned, cells were attached in good condition andwere cleaned3times by phosphate-buffered saline (PBS), then the PBSbuffer was blotted. The cells were divided into the experimental group and thecontrol group three flasks were selected in each group. The experimentalgroup: in the corresponding cell flasks,3ml AngiotensinⅡwhose terminalconcentration respectively was10-7mol/L was added. AngiotensinⅡ of theterminal concentrations was made with DMEM containing5%FBS. Thecontrol group: in the corresponding cell flasks, only3ml DMEM containing5%FBS was added. At37℃in5%CO2,cells were Incubated for48h.Collected the cells and fixed it with70%ethanol.determinate the cellcycle by flow cytometry analysis instrument, following the operation of thecell cycle kit.4The migration assay. The4th generation human dental pulp cells inlogarithmic phase were digested with0.25%of trypsin and removed from theculture plate using PBS buffer and suspended in10%serum containingmedium at a density of1×106cells ml-1,and200μl of media was seeded into theupper chambers of a24-well chemotaxis chamber. They were separated fromthe lower wells by an8μm pore-size polycarbonate membrane filter. Cells were incubated for24h at37℃in5%CO2humidified condition.when observedcells atteching in good condition, supernatant fluid was abandoned.1%serumcontaining medium was added in the upper compartments.Cells wereincubated for24h at37℃in5%CO2humidified condition. Supernatant fluidwas abandoned.After being washed in DMEM for3times. The cells weredivided into the experimental group, the control group and three holes wereselected in each group.The upper chambers of both experimental groups andcontrol group were filled with100μl serum-free DMEM. In the lowerchambers of the experimental groups: in the corresponding cell holes,600μlAngiotensinⅡwhose terminal concentration respectively was10-9,10-8,10-7,10-6,10-5mol/L was added.AngiotensinⅡ of the terminal concentrations wasmade with serum-free DMEM. In the lower chambers of the control group: inthe corresponding cell holes, only600μl serum-free DMEM was added. Thecells were incubated at37°C for24hours. The cells that had not migratedthrough the barrier were removed with a cell scraper, and then the migratedcells were stained with Crystal Violet staining solution.The number ofmigrated cells in each well was counted in5random high-power fie.Results:1Under inverted phase contrast microscope, the obtained dental pulpcells were found to be primarily typical long-shuttled appearance,well-rounded cell body, uniform cytoplasm, round or oval nucleus, and clearnucleolus. The dental pulp cells were stained positive for vimentin andnegative for cytokeratin. It met the histopathological characteristics of humandental pulp cells.2The concertions ranged from10-9to10-5mol L-1, AngiotensinⅡpromoted cell proliferation in a concentration-dependent manner comparedwith the control. The cell cycle was promoted by Angiotensin Ⅱ in theconcentration of10-7mol L-1（P<0.05）.3The results from the cell migration assay indicated that AngiotensinⅡcould induce migration of human dental pulp cells across the polycarbonatemembrane. The chemotactic effect of Angiotensin Ⅱ was observed in a dose-dependent manner ranging from ranged from10-9to10-5mol L-（1P<0.05）.Conclusion:1The HDPCs were successfully cultivated by the method of tissueexplant-collagenase digestion.The results of morphology and cell sourceidentification keep consistent with the HDPCs.2The data support that AngiotensinⅡcould stimulate the growth andproliferation of human dental pulp cell in vitro and act as a chemotactic factorfacilitating migration. AngiotensinⅡcould play a crucial role in dental pulptissue repair by promoting the proliferation and inducing migration dental pulpcell. |
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