| Objectives:At present,the mechanism of the occurrence and development of pulpitis is not completely clear,but the clinical treatment of pulpitis is mainly root canal therapy,and the prognosis of treatment still has the risk of tooth splitting and reinfection after treatment.it is of great clinical significance to explore the pathogenesis of pulpitis from the perspective of biological health.Bacterial infection is an important factor leading to reversible or irreversible injury of dental pulp.When the dental pulp is invaded by bacterial virulence factors,it will stimulate its defense response,when the degree of inflammatory reaction is mild,the dental pulp can repair the pulp injury thro μg h the formation of reparative dentin;if the degree of inflammatory reaction continues to increase,dental pulp tissue will be necrotic and even lead to periapical tissue damage.Dental pulp stem cells which exist in dental pulp stem cell niche play an important role in the process of dental pulp injury.Dental pulp stem cells are a kind of mesenchymal stem cells with multiple differentiation potentials.a series of changes will take place in the biological characteristics of dental pulp stem cells in the inflammatory microenvironment.by secreting relevant bioactive molecules to regulate cell proliferation,differentiation,apoptosis and other important signal pathways,so as to complete the life activities of the body or the outcome of dental pulp disease.Therefore,it is of great value to study the difference of biological signal molecule expression of dental pulp stem cells in inflammatory microenvironment.Proteomics technology has significant advantages in the study of protein expression,which can be used to analyze the protein abundance,post-translational modification and protein-protein interaction of tissue cells on a large scale at the protein expression level.it brings new research ideas for the exploration of the law of occurrence and development of diseases,drug research and development,niche region of odontogenic stem cells and so on in the field of stomatology.Labelfree(LFQ)is a proteomic analysis technology based on unlabeled quantitative strategy and quantitative qualitative protein by using protein signal strength and peptide map counting.Now it has become the core method to obtain quantitative data from proteomic data based on mass spectrometry.In this study,human dental pulp stem cell(h DPCs)was stimulated by Porphyromonas gingivalis lipopolysaccharide(Pg-LPS)to simulate the inflammatory reaction of dental pulp stem cells in the state of dental pulp inflammation.Labelfree proteomics was used to detect the protein expression of dental pulp stem cells in inflammatory state and normal state,and the differential protein expression in the two states was qualitatively and quantitatively analyzed by bioinformatics technology.Methods:Choose healthy orthodontic teeth and teeth,extract human pulp stem cells and separate culture in sterile states.The surface antigens of stem cells were identified by flow cytometry.Then human dental pulp stem cells were treated with 10 μg /ml Pg-LPS for 24 hours to induce inflammation.The expression of inflammatory factors such as interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected by real-time fluorescence quantitative polymerase chain reaction.Labelfree proteomic technique was used to identify and screen the differentially expressed proteins in dental pulp stem cells under normal and Pg-LPS induced inflammation,and the differential proteins were analyzed by bioinformatics data such as KEGG pathway,GO functional enrichment analysis and PPI protein interaction network.Results:1.The primary human dental pulp stem cells grow irregularly and radially around the tissue mass and grow in colonies.After passage,the cells are arranged in bundles and grow in a whirlpool.2.The results of CCK-8 detection showed that there was no significant difference in the proliferation activity of dental pulp stem cells treated with 0-20 μg/ml Pg-LPS for 1-7 days.3.The results of flow cytometry showed high expression of specific mesenchymal surface antigen.CD90: 89.45% of PE tags;CD105: 99.68% of PE tags.Low expression of hematopoietic antigens: CD45:0.49%marked by PE.CD34:0.62%marked by PE.The results were consistent with the characteristics of mesenchymal stem cells.4.PCR results showed that the m RNA expression of pro-inflammatory cytokines IL-1β and IL-6,TNF-α increased significantly after LPS induction for 24 h.5.Using label free proteomics detection technology to detect the protein expression of dental pulp stem cells in the inflammatory state and in the normal state,a total of 772 proteins were detected,and there were 61 differential proteins,of which 50 protein molecules were in the inflammatory state The expression of dental pulp stem cells was increased,and the expression of 11 protein molecules was down-regulated.The analysis of these differential proteins by bioinformatics proved that in the inflammatory state of dental pulp,the significantly different proteins are mainly distributed in exosomes and cell membranes,and are mainly involved The biological pathway is the energy metabolism pathway,and the main molecular function is binding activity.Conclusion:In summary,Pg-LPS can induce an inflammatory response in human dental pulp stem cells.This experiment explored the differential expression of the proteome of human dental pulp stem cells in the LPS-induced inflammatory microenvironment,in order to explore the relevant mechanisms of the occurrence and development of dental pulp inflammation.The research has laid a certain theoretical foundation. |
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