The Effect Of ETS2 On Osteogenetic Differentiation Of Human Periodontal Ligament Stem Cells(PDLSCs)

BackgroundPeriodontitis is a common disease resulting in loss of tooth of adults.Specifically,it is an inflammatory destructive disease caused by many factors?bacterial plaque,dental calculus and traumatic occlusion?and occurring in peridentium.The main clinical features of periodontitis include formation of periodontal pocket,at tachment loss,and absorption and destruction of alveolar bone.Periodontitis is mainly found to occur in adults aged above 35.Besides,the prevalence rate is closely related to physical condition.Presently,the main clinical treatment method is symptoma tic treatment with a purpose of controlling periodontitis development.Nevertheless,there is no effective treatment method to cure alveolar bone defect caused by periodontitis.Thus,studying the regeneration of periodontal tissues,especially the mechani sm of alveolar bone regeneration,is of great significance for the treatment of periodontitis.Periodontal ligament stem cells?PDLSCs?are stem cells of MSC source,has potential of disintegrating into alveolar bone cell,periodontal ligament cell and ondontoblast,and participates in regulating functional homeostasis of periodontal cell.In the event that periodontal diseases cause physiological dysfunction or cellular damage of periodontal cells,stem cells in a static state will be activated to repai r and reconstruct the damaged peridentium.Thus,PDLSCs are regarded as important seed cell for periodontal repair and regeneration.As stem cell technology research deepened in recent years,tissue engineering,cell therapy and genetic engineering technol ogy based on PDLSCs provide a new direction for generation of periodontal defect.ETS2?ETS Proto-Oncogene 2?are the most important constituent parts of ETS transcription factors,which are located at chromosome 21 of human body.The protein coded by it is a phosphorylated protein dependent on Ca2+,and participates in regulating cell proliferation,differentiation and apoptosis.Most of the previous researches on ETS factors are related to tumorigenesis and tumor progression.According to some research results,ETS protein also can participate in regulating the formation of cartilage and bone.For purpose of this study,adult teeth were used for isolating and culturing primary hPDLSCs;detecting the expression of ETS2 and osteogenesis-related gene in the process of in vitro osteogenesis of hPDLSCs;and detecting the osteogenic capability of hPDLSCs and expression change of osteogenesis gene via disturbing ETS2 expression,so as to determine the function of ETS2 in the process of hPDLSCs osteogenesis and provide a new target spot for regulating and controlling hPDLSCs osteogenic differentiation.Method1.hPDLSCs were isolated from healthy adult permanent teeth by combining tissue block with the enzymatic digestion technique,the cell morphology was observed under microscope vision,and cell purification was conducted with the limiting dilution method;2.MSC marker was detected with flow cytometry to identify cell source,and its multi-directional differentiation and potency were detected with the three-line differentiation induction method;3.hPDLSCs were randomized into a control group and a osteogenesis group,and the two groups were detected for the expression of ETS2,ALP,BMP2,OCN,OPN,and RUNX2 on the 3^rd,7^th and 14^th days;4.hPDLSCs were randomized into a control group?non-transfected?,a GFP group?transfected with LV-GFP?,and an shETS2 group?transfected with LV-shETS2?,and all the groups were detected for the expression of gene and protein with Western blot and QPCR in 72 hours of steogenic induction;5.hPDLSCs were randomized into a control group?non-transfected?,a GFP group?transfected with LV-GFP?,and an shETS2 group?transfected with LV-shETS2?,and all the groups were dyed with alizarin red,scanned and photographed,dissolved in ce tyl pyridinium chloride and measured OD value with 540 nm in 14 days of osteogenic induction.Result1.Primary PDLCs were obtained by combining tissue block with the enzymatic digestion technique,and hPDLSCs of a stable source were obtained by cloning purification with the limiting dilution method;2.Detections with flow cytometry show that the cell surface markers of CD44 and CD29 are positive,and CD34 and CD45 are negative,indicating that the cells obtained by means of primary culture are of MSC source;the result of alizarin red dyeing under osteogenic induction shows that a lot of mineralized nodules have been dyed red,the result of oil red O dyeing under adipogenic induction shows the formation of lipid droplet,and the result of dyeing with hematoxylin and Alcian blue under chondrogenic induction shows that karyon has been dyed purple,and extracellular mucopolysaccharide has been dyed blue;3.In the process of in vitro osteogenic differentiation of hPDLSCs,the expression of ETS2 mRNA of the osteogenesis group increased on 3d,7d,and 14d comparing with the control group?P<0.05?;the expression of ALP,BMP2 and RUNX2 mRNA largely increased on 7d,and 14d comparing with the control group(P<0.05;the expression of OCN and OPN mRNA largely increased on 14d comparing with the control group?P<0.05?;4.After LV-shETS2 transfection of cells,the expression of OCN,OPN,ALP mRNA and protein of the shETS2 group reduced comparing with the GFP group?P<0.05?;5.After LV-shETS2 transfection of cells,the osteogenic differentiation capacity of the shETS2 group on the 14^th day of osteogenic induction reduced comparing with the GFP group?P<0.05?.Conclusion1.In this experiment,purification of primary periodontal ligament cells are conducted successfully with the tissue block enzymatic digestion technique and limiting dilution method,the cells are identified to be of MSC source with flow cytometry and have strong capacity of osteogenic,adipogenic and chondrogenic differentiation;2.In the process of in vitro osteogenic differentiation of hPDLSCs,the expression of ETS2 and osteogenesis genes?ALP,OPN,and OCN?mRNA increase;3.After LV-shETS2 transfection of cells,the osteogenic differentiation capacity of hPDLSCs declines,and the expression of osteogenesis genes?ALP,OPN,and OCN?reduces;It was confirmed that ETS2 can interfere with osteogenic differentiation of human periodontal ligament stem cells.