Mechanism Of Interaction Between Periodontal Stem Cells And CD3~+T Cells In Inflammatory Microenvironment

Periodontal membrane stem cells(PDLSCs)are ideal seed cells for periodontal tissue regeneration.However,under the action of chronic inflammatory microenvironment,the regeneration ability of PDLSCs decreases,which leads to irreversible periodontal tissue destruction,alveolar bone resorption and tooth loosening.The occurrence of chronic inflammation is the body’s response to infection and/or other triggers,which can produce a large number of inflammatory factors,directly or indirectly lead to tissue damage,and interfere with the process of tissue regeneration and repair,which is a complex biological process.Inflammatory cytokines,especially TNFa,can activate a series of downstream cascades,leading to increased inflammation and tissue destruction.It can lead to chronic inflammatory disease.Such as arthritis,periodontitis and so on.Under the action of initial factors such as plaque and calculus,chronic inflammation and damage are formed in the periodontal tissue due to excessive local immune response,and then progressive alveolar bone resorption,tooth loosening and other symptoms occur.Under the action of chronic periodontitis microenvironment,the osteogenic differentiation ability of PDLSCs is reduced,and the ability of repair and regeneration is weakened.This is the root cause of infeasible reverse alveolar bone resorption in periodontitis.Immune cells also play an important role in this process.T cells are highly expressed in the inflammatory infiltrates of periodontitis.CD3 molecules are expressed on the surface of all T cells.CD3+ T cells are the main effector cells of the adaptive immune system,which are antigen-specific and the core of homologous immune memory.We know that in chronic periodontitis,innate and adaptive immune disorders of periodontal tissues.The immunomodulatory capacity of PDLSCs is also decreased under the influence of chronic microenvironment.The classical Wnt pathway plays an important role in embryo development and homeostasis maintenance.The classical Wnt/β-catenin pathway plays an important role in osteogenesis and osteoclast balance during the process of osteogenic differentiation.TNFα can activate-catenin to play a role in bone resorption and bone homeostasis in chronic inflammation.Classical Wnt/β-catenin plays an important role in the alveolar bone resorption and reconstruction in periodontitis.The Wnt pathway can also affect innate and adaptive immunity.At present,mesenchymal stem cells(MSCs)can inhibit the proliferation of activated lymphocytes and have been widely studied in many inflammatory and autoimmune diseases.However,the mechanism of decreased immunomodulatory capacity of PDLSCs in periodontitis microenvironment,the role of Wnt pathway in immune and osteogenic differentiation,and the role of CD3 T cells in this process have not been fully elucidated.The purpose and direction of this study is to study the mechanism of PDLSCs’ interaction with CD3+T cells,and to explore the key molecules of PDLSCs’ immunomodulation in inflammatory microenvironment,which is of great significance for the clinical application of PDLSCs.This topic is divided into the following four parts.Part Ⅰ:The effect of Inflammatory microenvironment on the biological function of PDLSCsMethods:1.Collect the extracted premolars and intelligent teeth due to orthodontics,scrape the periodontal membrane of the middle 1/3 of the root,and culture HPDLSCs by limited dilution method,and obtain the inflammatory PDLSCs(IPDLSCs)by using the inflammatory microenvironment constructed by lOng/mL TNFα,and conduct clone formation,multi-directional differentiation ability,cell cycle and apoptosis detection,etc.2.After mRNA of PDLSCs group and PDLSCs group were extracted and reversely transcribed into cDNA,gene expressions of Runx2,ALP and TNF were detected by qPCR.Results:1.HPDLSCs and IPDLSCs are of long spindle shape with full inclusion slurry.2.The clone of IPDLSCs resulted in more single colony cells than HPDLSCs,and the clone was formed earlier.However,compared with PDLSCs,the lipogenic and osteogenic abilities were significantly decreased,and the proliferation ability was enhanced.Part ii:The effect of inflammatory microenvironment on the immunomodulatory ability of PDLSCsMethods:1.Fresh human peripheral blood and sterile PBS1:1 were diluted and slowly added into the human peripheral blood mononuclear lymphocyte separation solution.Density gradient centrifugation method was used to obtain the white membrane of human peripheral blood,namely,human peripheral blood mononuclear cells.CD3e magnetic beads were used to incubate the human peripheral blood white membrane,and the CD3+T cells were isolated by the upper magnetic separator.2.Human HPDLSCs,IPDLSCs and CD3+T cells were co-cultured at the following ratios:1:10,1:20,1:50 and 1:100 for 48h.RNA from PDLSCs in each group was extracted and reversely transcribed into cDNA.3.After the RNA of CD3+T cells was extracted and reversely transcribed into cDNA,the expression level of cyclin CCND1 mRNA was detected by qPCR.Results:1.Clear stratification of human peripheral blood can be obtained by density gradient centrifugation,from top to bottom:diluted plasma,mononuclear cells,granulocytes and erythrocytes.CD3+T cells were isolated from CD3+T cells by immunomagnetic bead separation method.2.When the co-culture ratio was 1:20 and 1:50,mRNA expression levels of key genes of Wnt pathway β-catenin and LEF1 in the HPDLSCs group were decreased.Interestingly,the expression level of TCF4 mRNA increased at 1:20,but decreased at 1:50.At 1:20,mRNA expression levels of p-catenin and LEF1 in IPDLSCs group increased,while gene expression level decreased at 1:50.The expression level of TCF4 decreased at 1:20 and 1:50.3.HPDLSCs and IPDLSCs showed significant inhibitory effects on the proliferation of CD3 T.cells,especially at 1:20 and 1:50.4.At the co-culture ratio of 1:20,the mRNA expression level of CCND1 in CD3+T cells co-cultured with HPDLSCs and IPDLSCs decreased,but the expression level of the latter was still higher than that of the former.Part iii:Influence of CD3+T cells on osteoblastic differentiation of HPDLSCs and IPDLSCsMethods:1.HPDLSCs,IPDLSCs and CD3 T cells were co-cultured at a ratio of 1:20,and tested 48 hours later.2.Expressions of TNFa mRNA,osteoblast gene Runx2 mRNA,and classical Wnt pathway gene β-catenin mRNA were detected in HPDLSCs and IPDLSCs.Results:1.The expression level of Runx2 mRNA increased,but decreased compared with that of PDLSCs mRNA in the control group under osteogenic induction.2.At the co-culture ratio of 1:20,the expression level of β-catenin mRNA in the HPDLSCs group was significantly reduced after co-culture.The expression level ofβ-catenin mRNA in IPDLSCs group was increased.Part iv:Regulatory mechanism of Wnt pathwayMethods:1.PDLSCs,IPDLSCs and CD3 T cells were co-cultured at a ratio of 1:20.After 48h,the expressions of PDLSCs and IPDLSCs inflammatory factor TNFa mRNA,osteogenic gene Runx2,ALP mRNA and classical Wnt pathway β-catenin mRNA were detected,respectively.2.Detection of Wnt5a protein expression of HPDLSCs and IPDLSCs.Results:1.After co-culture with CD3 T cells,the expression level of TNF mRNA in IPDLSCs group was decreased.The expression level of TNFa mRNA was increased in both the HPDLSCs group and the IPDLSCs group,and the latter increased significantly.However,after co-culture with CD3 T cells,the expression level of TNFa mRNA in IPDLSCs group was significantly decreased.2.After co-culture with CD3 T cells,the expression level of TCF4 mRNA was increased by about 1.1 times after empty transfection of HPDLSCs,while the expression level of TCF4 mRNA was reduced by 2.5 times after co-culture with CD3 T cells after empty transfection of IPDLSCs.The expression levels of TCF4 mRNA in both HPDLSCs group and IPDLSCs group were increased by 2.4 times and 2.6 times respectively.However,after co-culture with CD3+ T cells,the expression.level of β-catenin overexpressed IPDLSCs TCF4 mRNA was significantly reduced.The HPDLSCs group was significantly improved.3.After co-culture with CD3 T cells,the expression level of TNFa mRNA in IPDLSCs group was decreased.The expression level of TNFa mRNA was increased in both the Si RNA β-catenin HPDLSCs group and the IPDLSCs group.However,after co-culture with CD3 T cells,the expression level of TNFa mRNA was decreased in both HPDLSCs group and IPDLSCs group,and the latter was significantlydecreased.4.Compared with PDLSCs,Wnt5a mRNA and protein expression levels of IPDLSCs were increased.Conclusion:1.In this study,HPDLSCs were successfully obtained from healthy periodontal tissues,and IPDLSCs were obtained by constructing an in vitro inflammatory microenvironment.It was found that the osteogenic ability of PDLSCs was decreased and the proliferation ability was increased in the inflammatory microenvironment.2.In this study,CD3+ T cells were successfully obtained from human peripheral blood albumen by Immunomagnetic bead method,and co-cultured with different proportions of CD3 T cells(1:10,1:20,1:50,1:100)in HPDLSCs,IPDLSCs,and CD3 T cells.It was found that the immunomodulatory ability of PDLSCs was decreased under the action of inflammatory microenvironment.3.The classical Wnt pathway may play an important role in the osteogenesis,proliferation and immune regulation of stem cells.4.Wnt5a may play an important role in the occurrence and development of periodontitis.Inhibiting the expression of Wnt5a may reduce the occurrence and severity of periodontitis.Wnt5a and p-catenin may cooperate to regulate the osteogenesis and bone homeostasis.