Systematically Transplanted Human Gingiva-derived Mesenchymal Stem Cells Regulate Lipid Metabolism And Inflammation In Periodontitis Mice With Hyperlipidemia

OBJECT: The gingival mesenchymal stem cells(GMSCs)were transplanted into periodontitis mice with hyperlipidemia via the tail vein to explore the role of GMSCs in the regulation of lipid metabolism and inflammation and provide new ideas for clinical treatment of periodontitis with hyperlipidemia and other systemic diseases.METHODS: 20 8-week-old male C57BL/6J mice were assigned as group A,40 Apo E-/-mice with the same gene background were randomly divided into groups B and C,with 20 mice in each group.Mice were all fed on a high fat diet(HFD).The body weight of these animals was recorded every week.Fasting blood samples were taken from the angular vein at 0,4,8,9,10,12 weeks after HFD,serum lipid levels including triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL),and low density lipoprotein(LDL)were determined using an autoanalyzer.The serum interleukin(IL)-6,IL-10,and tumor necrosis factor(TNF)-α levels were determined by ELISA.A 5-0 silk ligature was placed on both maxillary second molars in 12-week-old Apo E-/-mice,then 2of every group were sacrificed after ligation for 4 weeks.Alveolar bones with molars were harvested,and periodontitis were examined using stereomicroscope by measuring distances from the cementoenamel junction(CEJ)to the alveolar bone crest(ABC).The GMSCs labeled with GFP were injected via tail vein into Group C and α-MEM medium was injected into Group B as control,while group A were not given any treatment as the empty group.6 of every group were sacrificed at 1,2 and 4 weeks after the transplantation.Liver samples were collected to measure the m RNA levels of PPARα and SREBP-1c by RT-PCR.Alveolar bones with molars were harvested.Left alveolar were examined using stereomicroscope by measuring distances from the CEJ to ABC.Hematoxylin and Eosin(HE)staining were performed to evaluate periodontitis in the right alveolar bones,then the right alveolar bones were performed with DAPI staining and immunohistochemical analysis and then monitored by a fluorescence microscope to observe the migration and differentiation of GMSCs.RESULTS:1 GMSCs were isolated from gingival tissue using enzyme digestion and cells formed clonogenic cell clusters.GMSCs were found to differentiate to adipocytes and osteoblast under specific condition.And flow cytometry analysis showed that GMSCs were positive for CD73、CD90、CD105 and STRO-1 and negative for CD45 and CD31.The expression rate of GFP in infected GMSCs was over 90%.2 After a HFD for 4 weeks and 4-week placement of ligatures,Apo E-/-mice demonstrated dramatic increases in serum lipid levels and periodontal damage.Aftertransplantation for 1 and 2 weeks,a large amount of GFP+ fibroblast-like cells were detected in the periodontitis area by fluorescence microscope observation and immunohistochemical analysis.At 4 weeks after the transplantation,GFP+ osteoblasts were also found in the newly formed alveolar.HE staining revealed that alveolar bone heights of group C were significantly higher than group B,and the infiltration of inflammatory cells in the gingival margin of group C were significantly lower than group B at 4 weeks after injection.Morever,distances from CEJ to ABC in group C were significantly less than group B(P<0.05).At 1,2 and 4 weeks after injection,GMSCs resulted in a significant decrease in TC,TG,LDL,IL-6 and TNF-α,and a significant increase in HDL and IL-10 in group C as compared to group B(P<0.05).At the 1st week after cell transplantation,compared with group B,PPAR-αm RNA expression in liver of group C increased significantly,while the levels of SREBP-1cm RNA reduced significantly.At the 2nd week,the m RNA levels of PPAR-α and SREBP-1c reduced significantly in group C than group B.However at the 4th week,compared with group B PPAR-αm RNA and SREBP-1c m RNA expression in liver of group C increased significantly(P<0.05).CONCLUSION: 1 GMSCs could be easily isolated from gingival by enzyme digestion and showed stem cell properties.The transfected GMSCs could express GFP stably with the cell passage increases.2 An experimental hyperlipidemia model with periodontitis in mice is successfully established by ligation in Apo E-/-mice.This method is economical and time saving,and worthy of more general application.Systematically transplanted GMSCs could regulate lipid metabolism and inflammation in periodontitis mice with hyperlipidemia,and home to periodontitis site to attenuate periodontitis.