The Role Of Nf-¦Êb Signaling Pathway In The Tensile Stress-mediated Apoptosis In Fibroblasts

Objective This study first constructed a fibroblast model of mechanical stimulation in vitro, and applied different periodic tensile stress to cells to observe the time and rule of cell apoptosis by Hochest,Flow Cytometry,RT-PCR. To study the expression and activity change of NF-κB in fibroblast under the periodic tensile stress by Western-blot.Identify the relationship among NF-κB,periodic tensile stress and apoptosis of fibroblast. Thus explaining the specific mechanism of role of NF-κB in fibroblasts apoptosis under periodic tensile stress to provide rationale for the treatment of periodontal disease.Methods The experimental object was hPDLFs in vitro culture. The size of the force that was applied on the hPDLFs was expressed by the Tensile strain rate of the silicagel membrane on the bottom of the petri dishes. Cyclic tensile stress was applied on the hPDLFs respectively for Oh,6h,12h and 24h, The Flexcell Strain Unit FX-4000T, was used to expose hPDLFs to an equiaxial cyclic stretch of 15% magnitude and a frequency of 1/6HZ, a frequency of 10 cycles/min, each cycle including the 3-s stretch/3-s relaxation. Hochest, flow cytometry analysis and RT-PCR were used to observe the proliferation of the hPDLFs, the activity of phospho-NF-κB were detected by western blotting. This time makes the hPDLFs prior to impose cell-specific inhibitor PDTC, and then imposed on the same tensile stress, and then we detected the changes of cell proliferation and the expression of phosphor-NF-κBp65. The experimental data were analyzed used SPSS 18.0 Statistical Package.Results 1.The hPDLFs can grow in the cell culture plate under the force of 15%,1/6HZ,and the growth state changed significantly with the time of the force,which meet the demand of mechanical stimulation model in vitro.2. The results of Hochest assay, flow cytometry analysis and RT-PCR showed that the cyclical tensile stress of 15%,1/6HZ applied in this experiment can induce hPDLFs apoptosis.3. With the extension of time, the apoptosis of hPDLFs also increased, and reached the peak in 12h, and then declined but still higher than the group without tensile stress.4. The results of western blotting showed that phosphor-p65 were expressed in all groups, reaching the peak in 6h, then declining to the lowest in 12h,after that rising again.5. Cells imposed on PDTC before imposing cyclical tensile stress, the results showed that apoptosis was markedly affected, and the phosphor-p65 expression declined apparently. Conclusions 1. This study constructed a stable mechanical stimulation model hPDLFs in vitro, and provide a basis for the follow-up research of the hPDFLs which can meet the requirements of the present study of cell mechanics.2. Cyclical tensile stretch of 15%,1/6HZ may induce the apoptosis of hPDLFs.3. The apoptosis of HPDLF has positively correlation with the time of cyclical tensile stress in a certain range.4. The expression of phosphor-p65 has negative correlation with the apoptosis of hPDLFs, suggesting NF-κB is one of the mechanisms of hPDLFs apoptosis induced by the cyclical tensile stretch.