The Effects Of LRP5 Gene Silencing On RANKL And OPG Expression In Human Periodontal Ligament Fibroblasts Under Static Prssure

Objective:In this study,we constructed a human periodontal ligament fibroblasts(HPDLF)static stress loading model.After lentiviral-mediated silencing of LRP5 gene in HPDLF to affect the transmission of Wnt pathway,we observed the expression of OPG and RANKL in HPDLF.Changes to further clarify whether the Wnt signaling pathway further regulates alveolar bone remodeling by affecting the RANKL/OPG ratio to explore possible mechanisms of alveolar bone remodeling during orthodontic tooth movement.Methods:1.Scratch the 1/3 periodontal ligament tissue in the root of healthy premolars removed for orthodontic treatment,and culture the primary HPDLF in vitro by tissue block culture,using immunocytochemistry(ICC)SP method.The cultured cells are subjected to source identification to provide experimental cells for subsequent processing.The LRP5 protein in human periodontal ligament fibroblasts and human gingival fibroblasts(provided by other research groups in this laboratory)was detected by immunocytochemistry SP method and RT-PCR,and confirmed in periodontal tissue cells.Express and observe its expression characteristics.2.The HPDLF with good growth in the third to fifth generation was inoculated into a six-well plate to establish an in vitro static pressure model of HPDLF.HPDLF was loaded under static pressure of 0,1,2,3,4 g/cm2 for 24 hours,and the expression of LRP5 protein in different groups of cells under different static pressure was detected by Western blot(WB).Real-time quantitative PCR(RT-PCR)was used to detect the expression of LRP5 mRNA in HPDLF at 2g/cm2 static pressure for 0,12,24,48 hours.3.Recombinant lentiviral shRNA-LRP5 virus was transfected into HPDLF cells,and five groups of MOI=15?25?55?75?100 were set up.The inverted blank microscope was used to observe the blank group(untransfected group)and the transfection negative control.The expression of green fluorescent protein in the group and sh RNA-LRP5 group was determined to determine the optimal MOI value;HPDLF cells were transfected with the best MOI value of shRNA-LRP5 virus,and Cell Counting Kit-8(CCK-8)was used.The method was used to detect the effect of sh RNA-LRP5 virus on the proliferation of HPDLF cells at this time.The OD value of sh RNA-LRP5 group decreased significantly4.shRNA-LRP5 was transfected into HPDLF cells for 48 hours,and after screening with ampicillin,RP-PCR was used to detect LRP5 gene expression and WB was used to detect LRP5 protein expression,and the silencing efficiency of shRNA-LRP5 virus transfected HPDLF cells was detected.5.After silenced LRP5 in HPDLF,the expression of OPG and RANKL?p-GSK-3(3 gene was detected by RT-PCR under static pressure,the expression of OPG protein was detected by ELISA,and the expression of RANKL and p-GSK-3? protein was detected by WB.The experimental group:blank control group,negative transfection group,shRNA-LRP5 transfection Dyeing groupResults:1.The cultured cells were long fusiform and grew radially;after passage,the cells were evenly distributed and the growth state was stable.The cells identified are derived from mesenchymal cells and are not epithelial-derived cells.The cells can be identified as HPDLF by combining the extracted parts.Immunocytochemical detection showed that LRP5 was positively expressed in periodontal fibroblasts,but not in gingival fibroblasts.2.The results of RT-PCR and ELISA showed that LRP5 was expressed after 0,12,24,48 hours of 0-4 g/cm2 static pressure loading,and LRP5 in HPDLF cells after 24 hours of action at 2 g/cm2.The expression level was the most obvious,and the difference between the groups was statistically significant(P<0.05).3.The virus was transfected with HPDLF for 48 hours,and the cells were in good condition.Under the microscope,HPDLF expressing green fluorescent protein was more than 90%of the total cells.The effect of shRNA-LRP5 virus transfection on HPDLF cell proliferation was detected by CCK-8 method.There was no significant difference in OD values between shRNA-LRP5 group,negative transfection and blank group.4.q RT-RCR and Western blot analysis showed that compared with the blank group,the silencing efficiency of shRNA-LRP5 virus transfected HPDLF cells reached more than 70%,P<0.05,and the expression level of negative transfection group did not decrease significantly.The lentiviral vector was successfully constructed to silence the LRP5 gene in human periodontal ligament fibroblasts,and the expression of LRP5 in cells was highly silent.5.The expressions of RANKL and p-GSK-3? were detected by RT-PCR and WB under static pressure.The results showed that the expression of RANKL was lower in the blank group and the empty-loaded group,and there was no significant difference between them(P>0.05),compared with the former two,the expression of RANKL in shRNA-LRP5 transfection group was significantly up-regulated,the difference was statistically significant(P<0.05).The expression of p-GSK-3(3 in the blank group and the empty-loading group was higher than that in the shRNA-LRP5 transfection group,and the difference was statistically significant(P<0.05).The expressions of OPG gene and protein were detected by RT-PCR and ELISA.The results showed that the OPG of the blank group and the empty-loaded group were significantly different(P>0.05),compared with the former two,shRNA.The expression of OPG in the-LRP5 transfection group was significantly lower,and the difference was statistically significant(P<0.05).Conclusion:1.LRP5 is abundantly expressed in human periodontal ligament fibroblasts.In addition,the expression of LRP5 in human periodontal ligament fibroblasts changes with different static pressures,and the amount of change can be time-continuous,excessive static pressure.It can inhibit the expression of LRP5,and it is speculated that LRP5 may be involved in the reconstruction of alveolar bone during orthodontic tooth movement.2.LRP5 gene silencing under static pressure can down-regulate OPG expression in human periodontal ligament fibroblasts,but has no significant effect on RANKL expression,This indicates that LRP5 participates in the reconstruction process of orthodontic tooth movement alveolar bone,and achieves bone mass regulation by controlling the expression levels of RANKL and OPG to affect the osseointegration and osteoclast-related signaling molecules,and this process is likely to have Wnt/--catenin signaling pathway mediates participation.