Effects Of IL-17 On The Expression Of Rankl And Opg In Human Periodontal Ligament Fibroblasts Cells And On The Formation Of Osteoclast Like Cells

Ojective:Root resorption is common complication caused by orthodontic treatment,the IL-17 may be involved in the occurrence and development of osteoclast in the orthodontic root resorption process.In this study,we established the periodontal ligament fibroblasts model,and explored the biological effects of IL-17 on HPDLF,detected the expression of OPG and RANKL in HPDLF stimulated by IL-17.We alse established a co-culture model,observed the morphology and function of osteoclasts to explore the role and mechanism of IL-17 on root resorption during orthodontic treatment.Methods:1.The periodontal ligament tissues from the roots were prepared forseparation and cultivation.Inverted phase-contrast microscope was used to observe the cell growth situation.In order to establish HPDLF cell line,immunocytochemistry was used to identify the source of the cultured cells,An MTT assay was used to draw the growth curve of HPDLF.2.The 3-6 passage of HPDLF was plated onto 96-well plastic plates,they were subjected to 0?5?10?20?40?60?80ng/ml of IL-17 for 24h,The MTT assay was performed to assess the changes in cell proliferative activity after application of IL-17.3.After HPDLF were subjected to 0?5?10?20?40ng/ml of IL-17 for 24h,and HPDLF were subjected to 20ng/ml of IL-17 for 0?12?24?48?72h,the mRNA and protein expression of OPG and RANKL by HPDLF were detected by real-time PCR and ELISA.4.The HPDLF and PBMC were co-cultured for 14d to observe the morphology and numbers of osteoclasts ability by TRAP staining.Results:1.The cultured cells were spindle,radial or spiral growth;and had stable biological character.Combined with material positions,the cells,which were identified from the mesoderm cells,can be identified as HPDLF.2.The result of MTT showed as follows:The OD value of all the groups was higher than control group except the concentration of 80ng/ml group(P<0.05).The peak of OD value after application of the concentration was observed at 40ng/ml group.3.The results of RT-PCR and ELISA were consistent.Comparing with control group,the mRNA and protein expression of RANKL were significantly increased in HPDLF after treating IL-17 for 24h,whereas that of OPG were decreased,especially in 20ng/ml group and 48h group(P<0.05).4.Under the co-culture system,the mononuclear cells can generate positive TRAP multinucleated cells,the number of TRAP positive multinucleated cells in IL-17(+)group was more then in IL-17(-)group,the difference were statistically significant(P<0.05).Conclusion:1.By using tissue-explant method for primary cells,the HPDLF cell line was successfully established.The 4-6 generations grew fast and had stable biological character.2.IL-17 could induce HPDLF up-regulating the expression of RANKL and down-regulating the expression of OPG.3.PBMC can differentiate into TRAP(+)multi-nucleate OLC when co-cultured with HPDLF.4.IL-17 may be involved in the formation of OCL by inducing OPG/RANKL expression in HPDLF.