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Influence of APS on the HDPCs' Proliferation and Osteogenic Differentiation in Vitro

Author: GuoZuo
Tutor: ChenHuiZhen;GaoZuo
School: Hebei Medical University
Course: Stomatology
Keywords: Angelica Polysaccharides (APS) Human Dental Pulp Cells(HDPCs) Cell Counting Kit-8(CCK-8) CollagenⅠ(Coll Ⅰ) AlkalinePhosphatase (ALP) osteocalcin (OC) mineralized nodules
CLC: R285.5
Type: Master's thesis
Year: 2014
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Abstract


Objective: The purpose of this experiment is to study the influence ofAngelica Polysaccharides (APS) on the Human Dental Pulp Cells’(HDPCs)proliferation and osteogenic differentiation.Methods:1Isolation and identification of HDPCs: Pulp samples were obtainedfrom healthy,caries-free impacted third molars or premolar for orthodonticpurposes, primary HDPCs were gained by the method of improvedexplant culture, and serial subcultivation in vitro was made. The slides of cellswere carried on with HDPCs which grow in the stage of logarithmic growthperiod (3rd to5th generation). Vimentin and keratin immuohistochemicalstaining were made by SABC method. They were observed under the invertedmicroscope.2The detection of HDPCs’ Proliferative activity: The logarithmic growthperiod(3rd to5th generation) HDPCs were inoculated separately in five96-well plates with the cell concentration of2×104cells/ml. Randomly theywere divided into five groups, four experimental groups and one control group.The experimental groups contains high glucose DMEM medium and1%fetalbovine serum(FBS), and the concentrations of APS in experimental groupswere different(50、100、200、400μmoL/l respectively). A column hole oneach plate without fluid was as blank control group, while in the control group,the high glucose DMEM medium with1%fetal bovine serum was added.Make sure each well contains200microliters of high glucose DMEM medium.The original liquid in96-well plates was abandoned respectively after3,6,12,24,48hours.110μl cck-8which is diluted by pure DMEM culture solution(The concentration ratio of DMEM to cck-8is10:1) were added into the plate.Then the solution was placed into the37℃incubator and continued culturingfor2hours. Zero setting was made on the blank control well, then the absorbance value for each well were measured on the microplate reader at thewavelength of450nm (A450).3The detection of HDPCs’ osteogenic differentiation ability:3.1The expression of CollagenⅠ (Coll Ⅰ): After being digested by the0.25%trypsin, HDPCs in logarithmic growth period (3rd to5th generation) wereseeded into25ml culture bottles with the cell concentration of2×104cells/ml.Each bottle had3ml culture fluid and the total number of bottles is eight.Randomly those eight groups were divided into four experimental groups andfour control groups. The cells were cleaned3times with PBS respectivelyafter3,6,12,24hours. After1ml of Trizol reagent was added, repeatedlypipetting the cells until they are completely dissolved. Then the liquid wascollected into zero-enzyme EP tubes and stored it in refrigerator at-80℃.Lastly, the primers of Coll Ι were synthesized and the expression of this genewas detected by Beijing SBS Genetech technology.3.2The activity expression of alkaline phosphatase (ALP): The HDPCs inlogarithmic growth period(3rd to5th generation) were seeded separately infive96-well plates with the cell concentration of2×104cells/ml. Randomly thefive groups were divided into four experimental groups and one control group.APS which is made by high glucose DMEM medium with1%Fetal BovineSerum(FBS) was added into experimental groups with respectively fourdifferent concentrations(50、100、200、400μmoL/l), A column hole on eachplate without fluid was as blank control group, while in the control group, thehigh glucose DMEM medium with1%fetal bovine serum was added. Eachwell contains200microliters of high glucose DMEM medium. The originalliquid in96-well plates was abandoned respectively after3,6,12,24,48hours.After cells were cleaned3times using PBS buffer and draining,50μl0.1%TritonX-100separately was added into each well, then overnight inrefrigerator at4℃. Nextday,100μl substrate per well according to theinstruction of the ALP kit was added. The solution was placed into the37℃incubator and cultured for30minutes, and then50μl0.2mol/L NaOH wasadded to stop the reaction. Zero setting was made on the blank control well, then the absorbance value for each well were measured on the microplatereader at the wavelength of450nm (A450).3.3The expression of osteocalcin (OC): After being digested by the0.25%trypsin, HDPCs in logarithmic growth period (3rd to5th generation) wereseeded into25ml culture bottles with the cell concentration of2×104cells/ml.Each bottle has3ml culture fluid and the total number of bottles is fourteen.Randomly the eight groups were divided into four experimental groups andfour control groups. The cells were cleaned3times with PBS respectivelyafter1,3,5,7,9,11,13days.1ml of Trizol reagent was added; repeatedly thecells were pipetted until they were completely dissolved. Then the liquid wascollected into zero-enzyme EP tubes and stored in refrigerator at-80℃. Lastly,the primers of OC were synthesized and the expression of this gene wasdetected by Beijing SBS Genetech technology.3.4The observation of mineralized nodules:After being digested by the0.25%trypsin, HDPCs in logarithmic growth period(3rd to5th generation) wereseeded into6-well plates with the cell concentration of2×104cells/ml. Theywere divided into non-induction group and induction group. Each group had5wells. The liquid was changed every other day. After7,14,28days, the cellswere cleaned three times with PBS. The cells were fixed with4%formaldehyde solution about10-15minutes, The cells three were cleanedtimes again with PBS, and then them were stained by1%Alizarin red for10minutes. At last, repeat washing the cells was done by distilled water until theliquid looked colorless. The change process were observed and recorded underthe inverted microscope.The single-factor variance analysis was used by using SPSS13.0statisticalsoftware and multiple comparisons was made by using the S-N-K method.Experimental data are presented in the form of mean±standard deviation,which P<0.05is considered to be statistically significant.Results:1The sources identification of the HDPCs in vitro: theimmunohistochemical staining shows that positive expression for vimentin, negative expression for cytokeratin. This result meets the experimentalrequirements.2The influence of APS on proliferation ability of HDPCs: compared withthe control group, in a certain concentration range, APS could promote theproliferation of pulp cells. The effect reached the peak after12hours ofreaction with the drug concentration of200μmoL/l.3APS promoted every period of osteogenic differentiation of HDPCs at acertain degree.Conclusion:APS plays a role in promoting both human dental pulp cell proliferationand osteogenic differentiation, and the effect is associated with the reactiontime and the drug concentration. It is proved that pulp cells are capable ofbone formation and pulp-dentin complex formation at a certain degree. Thisexperiment result shows that APS could promote HDPCs’ proliferation andosteogenic differentiation, and it can also play a positive role on oral medicineclinical application.

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