To Screen For The Pivotal Proteins Of Human Gingival Fibroblasts Under Mechanical Stress By ITRAQ Technology

Objective:(1)Three dimensional culture of human gingival fibroblasts(HGFs)was carried out by using PLGA scaffold to construct HGFs-PLGA mechanical loading model.(2)Using i TRAQ markers with two-dimensional liquid chromatography tandem mass spectrometry(2D-LC-MS/MS)quantitative analysis of three-dimensional culture and under the compressive stress of HGFs were studied by proteomics,screening of differentially expressed proteins in HGFs mechanical force.Methods:(1)HGFs cells were cultured and subcultured.(2)Using fourth generation of HGFs,the cell density of cell suspension of 1×105 /ml was prepared,and the 1ml suspension was inoculated on 1cm~2 PLGA scaffold.The growth of HGFs was observed by fluorescence microscope after 5 days of culture with HGFs-PLGA,and the proliferation activity was detected by CCK8 method.(3)PLGA-HGFs three-dimensional co culture model was established,and the compressive stress of the size of 25g/cm~2 was 4 days after the culture.The experimental group was divided into group A,B,C,applied the size of its25g/cm~2 compressive stress various action time: 24,48,72h;the control group was divided into A0,B0,C0 group,and,were cultured for 24,48 and 72h?(4)The total protein of HGFs was extracted and the protein concentration was determined by Bradford method.(5)i TRAQ quantitative proteomics technology and bioinfomatics were used to analyze the differential protein expression in HGFs.Results:(1)HGFs was successfully cultured and purified.(2)The results of electron microscopy showed that HGFs concentration was observed in different planes of PLGA scaffolds.CCK-8 detection showed that the cells were cultured in PLGA scaffolds for 3-5 days,the cells were in logarithmic growth phase,5-7days were in the plateau stage,which was consistent with the growth of cells.(3)The total protein and quantitative protein identified by mass spectrometry was2449 and 2438 respectively.GO analysis show the biological process,which indicates that the differentially expressed protein involved in metabolism and cell signal transduction in cells are positioned such that they are mainly attached to the organelle,cell membrane and extracellular matrix.Signal pathway enrichment analysis showed that the differentially expressed proteins were mainly involved in energy metabolism pathway and signal transduction pathway.Using i TRAQ technology to study the pressure force of differential expression protein HGFs,KEGG enrichment analysis showed that in the interaction of key nodes,there are as many as 20 kinds of proteins including TGF-,beta,cytochrome P450,glutathione-S-transferase,platelet-derived growth factor,collagen,matrix metalloproteinase 1,MAP kinase p38,IL-6,p65 transcription factor,transcription factor P50,fibronectin,alpha 2-macroglobulin,and plasminogen activator inhibitor etc.Conclusion :(1)PLGA scaffolds can successfully construct HGFs three-dimensional culture and mechanical loading model.(2)i TRAQ technique could be used to screen the differentially expressed proteins of HGFs under stress,and analyze the key proteins in the nodes of the protein interaction network,the ten main proteins are nuclear NF-k B p65,P50,MAP kinase P38,TGF-beta,matrix metalloproteinase inhibitor-1(TIMP-1),alpha 2macroglobulin,collagen,fibronectin,IL-6,platelet-derived growth factor receptor,and thrombospondin-1.