Effects Of Compressive Force On ERK1/2-c-Fos Pathway And COL-Ⅰ Of Human Gingival Fibroblasts Cultured On PLGA Scaffold

Objective:1.To investigate the effects of compressive force on the expression of extracellular regulated protein kinase(ERK1/2),p-ERK1/2,c-Fos and Collagen-Ⅰof human gingival fibroblasts(HGFs)cultured on PLGA scaffold.2.To investigate the effects of compressive force on the expression of extracellular regulated protein kinase(ERK1/2),p-ERK1/2,c-Fos and Collagen-Ⅰof human gingival fibroblasts(HGFs)cultured on PLGA scaffold under the action of a specific inhibitor of ERK1/2 and further to study the signal transduction mechanism of ERK1/2-c-Fos pathway and the mechanism related to human gingival fibrosis.Methods:1.In this experiment,HGFs was primary cultured by using tissue adhering method.The HGFs from 4th to 6th generation were plated on the PLGA scaffold to construct the 3-dimention culture model.2.The gravity loading advice was placed on the PLGA-HGFs complex so that HGFs were subjected to the mechanical force of 25 g/cm~2.In this experiments,RT-q PCR and Western blot were used to detect the m RNA and protein expression of ERK1/2、p-ERK1/2、c-Fos、and COL-Ⅰof HGFs after 0,3,6,12,24,48 hours respectively.3.The specific inhibitor of ERK1/2(PD98059)was applied to ERK1/2-c-Fos signaling pathway to construct the pathway inhibition model of HGFs.The inhibitor concentrations were 0,2.5,5,10,20,40,and 80μM.The technique of CCK-8 was used to detect the proliferation activity of HGFs treated with different concentrations of PD98059and the detection times were 0,24th,48th and 72th h,respectively.The PD98059 with non-cytotoxic and effective concentration(0,2.5,5,10,20μM)was applied to HGFs for 48 h,then the expression of ERK1/2 m RNA was detected by using RT-q PCR.4.The PD98059(concentration of 10μM)and the compressive force of 25g/cm2 were applied to the PLGA-HGFs complex model for 0,3,6,12,24,48 hours,respectively.RT-q PCR and Western blot technique was applied to detect the expression of m RNA and protein of ERK1/2,p-ERK1/2,c-Fos and COL-Ⅰ.Results:1.In this experiment,the primary HGFs were successfully cultured by using tissue adhering method,and the growth was normal and the character was stable after purification.The 4-6th generation of HGFs was taken as the research object and plated on the PLGA scaffolds for the next experiments.2.Apply a compressive force of 25g/cm~2 to the HGFs on the PLGA for 0,3,6,12,24,and 48 hours,respectively.The m RNA and protein expression level of ERK1/2,c-Fos and COL-Ⅰin HGFs were up-regulated.The m RNA and protein expression levels of ERK1/2 are both up-regulated under the compressive force and reached the highest expression levels within 0-12h while reached peak at 12h,then gradually decreased to the normal level.The protein expression level of phosphorylating ERK1/2 was up-regulated within 0-3h while reached peak at 3h,then gradually decreased to the normal level.The m RNA and protein expression levels of c-Fos were both up-regulated within 0-3h while reached peak at 3h,then gradually decreased to the normal lecel.The m RNA and protein expression levels of COL-Ⅰwere both up-regulated within 0-24h while reached peak at 24h,then gradually decreased to the normal level.Compared with the expression time of ERK1/2,p-ERK1/2 and c-Fos,the expression time of COL-Ⅰwas slower.3.There is no significant effect on the proliferation activity of HGFs cultured when the concentration of PD98059 was less than or equal to 20μM.As the concentration was over 20μM,PD98059began to affect the proliferation activity of HGFs.The inhibitory effect was gradually improved as the culture time increased.In addition,different concentrations of PD98059 had different specific inhibitory effects on ERK1/2.The result showed that PD98059 had the most significant inhibitory effect as ERK1/2 was at a concentration of 10μM.4.After adding PD98059 at a concentration of 10μM to the experimental group,it was found that the m RNA and protein expression of ERK1/2,p-ERK1/2,c-Fos and COL-Ⅰof HGFs in the group without compressive force were all decreased,and the minimum value was lower than normal.The m RNA and protein expression levels of ERK1/2,p-ERK1/2 and c-Fos of HGFs in the group under compressive force were gradually increased within 0-3h while reached peak at 3h,and then decreased to below normal level.The m RNA and protein expression levels of COL-Ⅰgradually were gradually increased within 0-6h while reached peak at 6,and then decreased to below normal level.The expression of ERK1/2,p-ERK1/2,c-Fos and COL-Ⅰin the experimental group under the combined action of PD98059and compressive force was higher than that of the control group treated with PD98059 only.Conclusions:1.In this experiment,the three-dimensional culture model of PLGA-HGFs was successfully established.2.The compressive force can promote the m RNA and protein expression of ERK1/2,p-ERK1/2,c-Fos and COL-Ⅰ of HGFs,and the ERK1/2-c-Fos pathway was involved in mechanical signal in HGFs.3.Under the effect of mechanical static pressure,HGFs accept the mechanical signal by ERK1/2 and initiate phosphorylation of ERK1/2,which regulates of the expression of c-Fos,and then affects the synthesis of COL-Ⅰ.3.PD98059 can inhibit the ERK1/2 and the optimal inhibitory concentration is 10μM.4.The ERK1/2-c-Fos pathway was involved in the regulaition of the expression of COL-Ⅰ in HGFs and the PD98059 can inhibit the expression of COL-Ⅰ.