A Preliminary Study On The Mechanism Of Mechanotransduction Of Integrin ?5 And ?1 Of HGFs On PLGA Scaffolds

Objective:To investigate the effects of compressive force and Integrin?5?1 inhibited on expression of Integrin?5 and?1,focal adhesion kinase(FAK),phospho-FAK(p-FAK)and Collagen-?(COL-?)of human gingival fibroblasts(HGFs)cultured on PLGA-collagen scaffold,and to study the mechanism of Integrin?5?1 on the mechanotransduction of HGFs.Methods:1.HGFs were cultured by the method of tissue adhering and plated on the PLGA scaffolds from the 4th to 6th generation.2.Using loading force applied compressive force of 25g/cm~2 to the PLGA-HGFs.The mRNA and protein expression of Integrin?5?1,FAK,p-FAK and COL-?of HGFs were detected by RT-qPCR and Western blot after 0,3,6,12,24,48 hours.3.Various concentrations(0,10nM,100nM,1?M,10?M,100?M,1mM)of Integrin?5?1 inhibitor(ATN-161)were apllied to the HGFs cultured with PLGA scaffold.The proliferation of HGFs was detected by CCK8 after 0,24,48,72 hours.And then various concentrations(0,10nM,100nM,1?M,10?M)of ATN-161 were apllied to the PLGA-HGFs.The mRNA expression of Integrin?5?1 was detected by RT-qPCR after 48 hours.4.The ATN-161(concentrations of 1?M)individual or the ATN-161(1?M)with compressive force of 25g/cm~2was apllied to the PLGA-HGFs.And the mRNA and protein expression of Integrin?5,Integrin?1,FAK(p-FAK)and COL-?of HGFs were detected by RT-qPCR and Western blot after 0,3,6,12,24,48 hours.Results:1.HGFs were successfully cultured by the method of tissue adhering.The cells from the 4th to 6th generation were pure and stable in shape and plated on the PLGA scaffolds for the next experiments.2.The mRNA and protein expressions of Integrin?5?1,FAK,p-FAK and COL-?of HGFs under compressive force were all up-regulated.The mRNA and protein expressions of Integrin?5?1 and FAK reached the highest level at 3-6h and decreased slowly after 6h.The protein expression of p-FAK reached the peak at 12h,and decreased gradually to the normal level after 12h.The mRNA and protein expressions of COL-?were both increased,with the highest expression level at 24h,and decreased slowly after 24h.Compared with integrin?5?1 and FAK,COL-?showed slower response to the compressive force.3.As the concentration less than or equal to 10?M,the ATN-161 had no effect on the proliferation of HGFs for 72h.As the concentration over 10?M,the ATN-161 had negative effect on the proliferation of HGFs.And it increased by time.The ATN-161 had the most significant inhibiting effect on integrin?5?1 at concentration of 1?M.4.The mRNA and protein expressions of Integrin?5?1,FAK,p-FAK and COL-?of HGFs with ATN-161(0.1?M)were all decreased.The mRNA expressions of Integrin?5?1,FAK and COL-?of HGFs with ATN-161 and compressive force were slightly increased at 3h and decreased after 3h.The protein expression of integrin?5?1,FAK and COL-?were also decreased generally.The protein expression of COL-?increased significantly at 3-6h and decreased gradually after 6h.Conclusions:The three-dimensional culture and mechanical loading model of compressive force were successfully established.The compressive force can promote the mRNA and protein expression of Integrin?5?1,FAK,p-FAK and COL-?of HGFs,indicating that they might play important roles in the mechanotransduction and extracellular matrix fibrosis of HGFs.HGFs accept mechanical signal by Integrin?5?1,regulate the expression and phosphorylation of FAK,and affect the synthesis of COL-?finally.