Influence Of17β-E₂on Biological Behaviour Of HDPCs Cultured In Vitro

Objective: The aim of the experiment is to study the effects of17β-E₂onproliferation activity, alkaline phosphates’（ALP）activity, mineralization andthe cell cycle of human dental pulp cells （HDPCs） in vitro, to investigate theeffects and the action mechanism of17β-E₂at different concentrations onbiological behaviour of HDPCs, and to provide the basis for using17β-E₂inthe dental pulp tissue repair and regeneration as a new type of revulsive.Methods: Pulp tissue was removed immediately from healthy younghumen’ impacted third molars or premolar for orthodontic treatment. Thedental pulp tissue was removed under the asepsis condition and culturedaccording to the tissue explant-collagenase digestion method. When the cellsgrew out from the explants and reached80%of the culture wells’ area, theywere separated from trypsin. Then multiplicity cells which could be used instudy were obtained. The fourth generation human dental pulp cells wereseeded for the identification of cell source by vimentin and keratinimmunohisto-chemical stains and observed by inverted microscopy. Thefourth generation human dental pulp cells in logarithmic phase were digestedand made into cell suspension with0.25%of trypsin. Then the cell suspensionwas planted in five96-well microplates with10⁴cells/ml and150μl for eachwell. After24h, the cells, whose supernatant fluid was abandoned, wereattached in good condition and were cleaned3times by PBS buffer, then thePBS buffer was blotted. The cells were divided into the experimental group,the control group and the blank control group, and six holes were selected ineach group. The experimental group: in the corresponding cell holes,150μl17beta estradiol whose terminal concentration respectively was10^-9,10^-8,10^-7,10^-6,10^-5mol/L was added.17beta estradiol of the terminal concentrations wasmade with phenol red free high sugar DMEM nutrient solution containing2% activated carbon adsorption PBS. The control group: in the corresponding cellholes, only150μl phenol red free high sugar DMEM nutrient solutioncontaining2%activated carbon adsorption PBS was added. The blank controlgroup: in the corresponding cell free holes,150μl phenol red free high sugarDMEM nutrient solution containing2%activated carbon adsorption PBS wasadded.The impacts of17β-E₂on HDPCs’ proliferation were detected by MTTmethod: took out of a96-well microplate at48h and72h respectively, thenadded20μl5mg/ml MTT into the measured holes. After4h, supernatant fluidwas abandoned and150μl dimethyl sulfoxide （DMSO） was added into eachhole, was shaked for5minute and the OD values were tested by microplatereader in492nm.The impacts of17β-E₂on HDPCs’ cell cycle were detected by flowcytometry: choose the group of10^-9,10^-8,10^-7,10^-6,10^-5mol/L17β-E₂as thetreatment group to determinate the cell cycle by flow cytometry analysisinstrument, following the operation of the cell cycle kit.The impacts of17β-E₂on HDPCs’ ALP activity were tested: took out ofa96-well microplates at7d and14d respectively, abandoned supernatant fluidand cleaned3times by PBS buffer, then blotted the PBS buffer, added50μl0.1%TritonX-100, and took it into4℃refrigerator overnight at last. If nocomplete cell structure was observed, shaked it and added alkalinephosphatase substrate under the instructions of the alkaline phosphatase kit.Finally,0.2mol/L NaOH was added into each well to stop the reaction andtested the OD values by microplate reader in410nm.The impacts of17β-E₂on HDPCs’ mineralization: The fourth generationhuman dental pulp cells in logarithmic phase were planted in6-wellmicroplates with2×10⁴cells/ml then were divided into not induced group andthe mineralized induced group which was induced by10^-8mol/L17β-E₂. Eachgroup owned5complex wells. Remove a cell climb piece from not inducedgroup and the mineralized induced group respectively in7,14and28days,flushed cells3times with PBS, fix it10¹5min with4%formaldehyde solution, then wash it3times with PBS, dyed10min with1%alizarin red,and finally clean it with distilled water until the absorption liquid becamecolorless, then was observed it by inverted phase contrast microscope.By using the SPSS13.0statistics software, one-way analysis of variancewas compared among groups, and S-N-K test for comparison between twogroups. Data were expressed as mean±se. The difference was statisticallysignificant （p<0.05）.Results:1The HPDLCs cultured in vitro were stained positive for vimentin andnegative for cytokeratin. It met the experiment requirements.2In contrast with the control group,17β-E₂could promote human dentalpulp cells proliferation, ALP activity, the mineralization activity and the cellcycle in a certain range of concentration.17β-E₂in concentrations of10^-8mol/L made the most obvious effect.Conclusion:1The HDPCs were successfully cultivated by the method of tissueexplant-collagenase digestion. The growth of cells were vigorous and formeda uniformity of fibroblasts. In a word, the result met up with the requirementof the experiment.2The17β-E₂could stimulate the growth and proliferation of humandental pulp cell in vitro, raise ALP activity, and promote the deposition ofbone mineralized substances and the cell cycle. The result reveals that17β-E₂induced differentiate from the HDPCs into odontoblast.