| Objective:1.To investigate the expression of CALR,CaN,NFAT3,p-NFAT3,and COL-I in human gingival fibroblasts(HGFs)under mechanical compression stress at different time points.2.To investigate the expression changes of CaN,NFAT3,p-NFAT3 and COL-I after silencing the expression of CALR,in order to explore the influence of CALR expression down-regulation on CaN/NFAT3 signaling pathway in HGFs,and study the mechanism of CALR in fibrosis transduction signaling pathway.Methods:1.HGFs was cultured and isolated by tissue culture method.After passage purification,cells from the 4th passages were inoculated on PLGA scaffolds to establish a three-dimensional HGFs-PLGA scaffold compound culture model.2.The weighted loading method was used to load the constructed compound culture model with a static pressure of 25g/cm~2,and it was divided into0h,6h,24h,48h,72h groups according to the time of afterburning,and the control group was 0h(unburned)group.The mRNA expression of CALR,CaN and COL-I were evaluated by RT-qPCR and the protein expression of CALR,CaN,NFAT3,p-NFAT3 and COL-I were evaluated by Western Blot.3.The shRNA-CALR lentiviral expression vector was constructed and transfected into the 4th generation HGFs to silence the expression of CALR in HGFs.CCK-8 method was used to detect the effect of lentiviral vector transfection on the proliferation ability of HGFs.After 72 hours of transfection,the expression of green fluorescence in cells was observed under a fluorescent microscope and the transfection efficiency was calculated,and the efficiency of CALR gene silencing was verified by RT-qPCR and Western Blot.4.After silencing the expression level of CALR in HGFs,the mRNA expression of CaN and COL-I were evaluated by RT-qPCR,and the protein expression of CaN,NFAT3,p-NFAT3 and COL-I were evaluated by Western Blot.Results:1.The HGFs were successfully isolated and cultured in vitro,and the construction of the HGFs-PLGA scaffold composite culture model was completed.2.When 25g/cm~2 static pressure was applied to the three-dimensional composite cultured HGFs,the expression of CALR,CaN and COL-I were rapidly up-regulated,and reached the peak at 24h,then decreased gradually,but the expression level was still higher than that of the unstressed group at 72h.On the contrary,the expression level of p-NFAT3 and the ratio of p-NFAT3/NFAT3 were decreased,and reached the lowest point at 24h,then increased gradually.3.After72 hours of transfection,the transfection efficiency of the CALR silent group(sh-CALR group)reached 89.486±0.025%,and the transfection efficiency of the negative control group(sh-NC group)reached 87.775±0.015%.There was no statistically significant difference(P>0.05).4.The results of cck-8 showed that OD values of the sh-CALR group,sh-NC group and blank control group(Control group)increased with the extension of culture time,and the OD values of the sh-CALR group,sh-NC group and Control group were not statistically significant on the same day.5.The expression levels of CaN and COL-I in the sh-CALR group were significantly lower than that in the sh-NC group and Control group,and the expression level of p-NFAT3 and the ratio of p-NFAT3/NFAT3 were significantly higher than that in the sh-NC group and Control group.Conclusions:1.When static pressure was applied to HGFs,the expression of CALR,CaN,dephosphorylated NFAT3 and COL-I were all up-regulated and gradually decreased after reaching the peak.2.After the CALR gene was silenced,CaN activity in HGFs was inhibited,which rapidly phosphorylated activated NFAT3 and simultaneously down-regulated the expression of COL-I.The possible mechanism was that when CALR gene was silenced,the downstream CaN/NFAT3 signaling pathway was inhibited,thus the synthesis of the COL-I was decreased.3.Under the stimulation of static pressure,CALR may influence the synthesis of COL-I by mediating the downstream CaN/NFAT3 signaling pathway,and participate in the regulation of the fibrosis process of the extracellular matrix. |
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