| Objective: The aim of the experiment is to Investigate the effects ofdifferent concentrations of naringin on proliferation activity, differentiationactivity, mineralization, cell cycle and alkaline phosphates’（ALP）activity ofcultured human dental pulp in vitro.Methods: The freshly extracted, healthy, complete, no caries, no crackedpremolar or wisdom due to orthodontic or impacted cause were chose toremove pulp tissue under the sterile conditions and the tissue enzymaticmethod was used to obtain human dental pulp cells and primary culture wascarried on. The cell morphology and growth were observed under the invertedmicroscope after the cells growing. The cells passaging when the cellsoccupied80%confluence bottom of the hole and make the4th generationlogarithmic phase cells in human pulp to crawl on slides when cells passagedsuccessfully and identification of cell sources was done with vimentin andcytokeratin immunohistochemistry of SABC method. The logarithmic phaseof the fourth generation of human dental pulp were digested with0.25%trypsin and made cell suspension which were seeded in six96-well cultureplate of the concentration of2×104cells/ml, each plate out of a row overtra-ining hole without fluid, the remaining amount of liquid in each well to200μland incubator. After the adherent cells were in good condition, the supernatantwas discarded and washed with PBS buffer three times, and they were madedry. The cells were randomly divided into five experimental groups, a controlgroup and a blank control group, five holes were selected in each group.Experimental group:200μl naringin which terminal concentration respectivelywas10-9,10-8,10-7,10-6,10-5mol/L was added in the corresponding cell holes.Naringin of the terminal concentrations was made with the standard high sugarDMEM nutrient solution containing15%activated carbon adsorption FBS. The control group: only200μl the standard high sugar DMEM nutrientsolution containing15%activated carbon adsorption FBS was added in thecorresponding cell holes. The blank control group: only200μl the standardhigh sugar DMEM nutrient solution containing15%activated carbon adsorp-tion FBS was added in the corresponding cell free holes.The impacts of naringin on HDPCs’ proliferation were detected by MTTmethod: a96-well microplate was taken at24h,48h and72h respectively, then20μl5mg/ml MTT was added into the measured holes. After4h, supernatantfluid was abandoned and150μl dimethyl sulfoxide (DMSO) was added intoeach hole, was shaked for5minute and the OD values were tested by micro-plate reader in492nm.The impacts of naringin on HDPCs’ proliferation were detected byCCK-8method: a96-well microplate was taken at24h,48h and72hrespectively, then supernatant fluid was abandoned and10μl CCK-8wasadded in each holes and them were put in an incubator incubating for twohours. The absorbance was measured at450nm using a plate reader.The impacts of naringin on HDPCs’ cell cycle were detected by flowcytometry: the standard high sugar DMEM nutrient solution containing15%activated carbon adsorption FBS of10-7mol/L was chosen as a experimentalgroup, the standard high sugar DMEM nutrient solution containing15%activated carbon adsorption FBS as the control group for cell-cycle tests.Analysis was made in according to kit instructions of the cell cycleflowingcytometry.The impacts of naringin on HDPCs’ ALP activity were tested: a96-wellmicroplates at24h,48h,72h were taken respectively, supernatant fluid wasabandoned and cleaned3times by PBS buffer, then50μl0.1%TritonX-100was added, and took it into4℃refrigerator overnight at last. If no completecell structure was observed, shaked and alkaline phosphatase substrate wasadded under the instructions of the alkaline phosphatase kit. Finally,0.2mol/LNaOH was added into each well to stop the reaction and the OD values weretested by microplate reader in410nm. The impacts of naringin on HDPCs’ mineralization: The fourth gener-ation human dental pulp cells in logarithmic phase were planted in6-wellmicroplates with2×104cells/ml. They were divided into a control group andexperimental group (the10-7mol/L concentration of naringin), each group havethree holes. After being cultured14d and28d, stained with Alizarin red andthe Mineralized nodules were observed.The impacts of naringin on expression of dentin sialophosphoproteinsmRNA of HDPCs: The fourth generation human dental pulp cells in logari-thmic phase were planted in6-well microplates with2×104cells/ml. Theywere divided into a control group and experimental group(the10-7mol/L conc-entration of naringin), each group have three holes. After being cultured24h,48h and72h, the cells were washed three times by PBS. Trizol reagent1mlwas added and pipetted repeatedly to completely dissolve. At last the cellswere collected to the no enzyme EP tubes, stored in-80℃refrigerator. Accor-ding to kit instructions, total RNA was extracted and the expression of DSPPgene was detected.By using the SPSS13.0statistics software, one-way analysis of variancewas made among groups, and S-N-K test for comparison between two groups.Data were expressed as mean±se. P<0.05is considered to be statisticallysignificant.Results:1Immunohistochemical staining showed that pulp cells stain waspositive for the anti-vimentin, negative for cytokeratin staining, which theresults confirmed the cells come from mesoderm between mesenchymal cellsand without epithelial cells confounding. The cells conformed the claim of theexperiment.2Compared with the control group, naringin could promote humandental pulp cell proliferation and cell cycle progression and improve itsalkaline phosphatase activity, and promote dental pulp cells to odontoblastdifferentiation, and enhance people mineralization of dental pulp cells in acertain range of concentrations. naringin in concentrations of10-7mol/L had the most obvious effect.Conclusion: Naringin could promote cultured human dental pulp cellproliferation and promote cell cycle progression; naringin could enhance theactivity of alkaline phosphatase, differentiation and mineralization of humandental pulp cells. |
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