Effects Of Compressive Stress And Calreticulin On Expression Of TGF-?1 In Human Gingival Fibroblasts

Objective:1.To investigate the expression of calreticulin(CRT)and transforming growth factor-?1(TGF-?1)in human gingival fibroblasts(HGFs)under mechanical compression stress.2.To investigate the effect on the expression of TGF-?1 after knockdown HGFs CRT expression,to analyze the effect of CRT on the expression of TGF-?1 in HGFs under mechanical compressive stress,in order to study the mechanism of CRT in fibrosis transduction pathway.Methods:1.HGFs were cultured by tissue culture method.After passage,cells from the 4th to 6th passages were inoculated on PLGA scaffolds to establish a composite culture model of HGFs-PLGA scaffolds to construct a three-dimensional culture model.2.The force of 25g/cm~2 was loaded on the composite culture model by the gravity loading method and divided into groups of 0h,6h,24h,48h and 72h according to the time of exertion.The control group was 0h group.The expression of CRT and TGF-?1 genes in each group was evaluated by real-time quantitative PCR(RT-qPCR).The expression of CRT and TGF-?1 protein in each group was evaluated by Western Blot.3.The rAdE5-CRT 1p2-shRNA was constructed,and the 4th passage of HGFs was transfected with recombinant adenovirus to silence the expression of CRT in HGFs.The effect of adenovirus transfection on the proliferation of HGFs was detected by CCK-8 assay.72 hours after transfection,the transfection efficiency was observed by fluorescence microscope,and the silencing efficiency was detected by RT-qPCR and Western Blot.4.The expression of TGF-?1 mRNA and protein after silenced HGFs CRT expression was detected.Results:1.HGFs were successfully cultured by tissue culture method to complete the construction of HGFs-PLGA scaffold composite culture model.2.With the prolongation of the exertion time,the gene and proteinexpression of CRT and TGF-?1 in HGFs increased,and reached the peak at 24h,then decreased gradually.3.After 72 hours of transfection,the transfection efficiency of the Ad-shRNA group was 88.101±0.058%,and the Ad-HK group was88.359±0.022%.The mRNA and protein expression of CRT and TGF-?1 mRNA and protein in the Ad-sh RNA group was significantly lower than that in the blank control group.4.CCK8 results showed that the OD values of the transfection group and the blank control group increased with time,and the OD values of the transfection group and the blank group on the same day were not statistically different.5.The expression of CRT and TGF-?1 mRNA and protein in the Ad-shRNA group was significantly lower than that in the blank control group.Conclusion:1.Mechanical compressive stress can promote the expression of CRT and TGF-?1 in human gingival fibroblasts.With the prolongation of the exertion time,the expression level of both increased and reached the peak and then decreased.2.CRT plays an important role in the generation of TGF-?1 in HGFs.