Proteomic Analysis Of Dentin Under Acid Etching And The Effect Of FAM20C On The Biological Properties Of Human Dentine Pulp Stem Cells

Objectives:This project aimd to use Label-free and quantification proteomic analysis to screen out the differentially expressed dentin proteins under acid etching conditions,and to further clarify the effect of FAM20C on the proliferation,migration and differentiation of human dental pulp stem cells.Methods:With the informed consent of the patients,100 complete and caries-free third molars were collected,and the patients were treated in the Department of Stomatology,920 Hospital of the Joint Logistics Support Force.The extracted third molar was sected for dentin(about 1mm thick),and the experimental group(35%phosphoric acid gel for 10s)and the control group were set.The total proteins were extracted from the dentin samples according to the groups.The proteins were hydrolyzed into peptides by trypsin,and then the peptides were analyzed by liquid chromatography-mass spectrometry.Quantitative proteomics software package MaxQuant was used to retrieve the obtained secondary mass spectrometry data and screen for differential proteins.Screening criteria were set as follows:differential multiple ?1.5 times and P value ?0.05.Bioinformatics analysis was performed on the screened differential proteins,and their biological functions were analyzed through gene ontology.KEGG pathway database was used for pathway enrichment analysis to find the significant enrichment pathways of different proteins.After comparing the interaction between the differentially expressed proteins and the STRING protein network database,the interaction relationship of the differentially expressed proteins was obtained.Combined with literature out after pickling protein expression significantly raised the golgi apparatus of casein kinase(FAM20C),through cloning determined by MTT,cell,scratches and Transwell FAM20C experimental observation of different concentration of human Dental pulp stem cells(human Dental pulp stem cells,HDPSCs))and their ability to proliferate and migrate;The effect of FAM20C on odontogenic/osteogenic differentiation of hDPSCs was detected by Alizarin red staining,RT-qPCR and Western Blot.Results:The protein quantification method is accurate and good,the difference is small,and the repeatability is good.The distribution of the length of the peptide identified by the mass spectrum meets the quality control requirements;the mass spectrometry of the two groups of protein samples obtained a total of 2090903.0 secondary spectra.Among them,there are 30296.0 valid and usable maps,and a total of 2756.0 peptides were identified,including 2014.0 specific peptides.The experiment identified a total of 418.0 proteins,of which 266.0 can be quantified;we use differential expression with a threshold greater than 1.5 times as a significantly up-regulated protein,and less than 1/1.5 as a significantly down-regulated protein,and 46 proteins are up-regulated,56 proteins down-regulated;3.102 proteins screened by 1.5-fold difference multiples for GO secondary annotation classification,subcellular structure positioning,COG/KOG functional classification,GO enrichment analysis,KEGG pathway enrichment results,protein interaction Based on a series of technical analysis such as the Internet,the expressions of related proteins in this experiment have significant differences including Gas6,FAM20C,insulin-like growth factor binding protein,and osteogenic differentiation-related proteins.By co-cultivating hDPSCs with different concentrations of FAM20C,it was found that as the concentration of FAM20C protein increased,their proliferation and migration ability increased;FAM20C at 1000ng/ml could significantly promote the differentiation of hDPSCs into odontogenesis/osteogenesis.Conclusions:This experiment uses Label-free quantitative proteomics techniques to reveal the protein composition of dentin under acid etching conditions.A total of 416 proteins were identified,among which 102 differentially expressed proteins were identified,including 46 down-regulated proteins and 56 up-regulated proteins.The significantly upregulated FAM20C protein after dentin etching can promote the proliferation,migration and odontogenic/osteogenic differentiation of hDPSCs.