Effects Of Smad4 On The Expression Of Caspase-3 And Bcl-2 In Human Gingival Fibroblasts Cultured On 3D PLGA Scaffold Induced By Compressive Force

Objective:1.To establish a three-dimensional culture and gravity loading model of human gingival fibroblasts in vitro,and to explore the effect of Smad4 on the proliferation of HGFs under compression pressure.2.To investigate the effect of Smad4 on the expression of Caspase-3 and Bcl-2 in HGFs under compression pressure.To preliminarily study the mechanism of Smad4 in human gingival proliferation and apoptosis signaling pathway under compression force.Methods:1.HGFs primary cells were obtained by tissue culture and identified.HGFs were purified to passage 4-6 and inoculated on PLGA scaffolds to establish a three-dimensional culture model of HGFs-PLGA.2.The HGFs-PLGA was loaded with 25 g/cm ~2 by weight loading method.According to the loading time,the HGFs-PLGA was divided into 12 h,24 h,48 h and 72 h groups,and the control group was 0 h(no loading)group.CCK-8 method was used for detecting proliferation activity of cells in each group,and the expressions of Smad4,Caspase-3 and Bcl-2 were detected by RT-qPCR and Western Blot.3.To construct a model of Smad4 overexpression lentivirus transfection and transfect the fourth generation of HGFs to overexpress Smad4in HGFs.RT-qPCR and Western Blot were used to verify the overexpression.4.Using CCK-8 technology to detect the effect of overexpression of Smad4 on the proliferation of HGFs under compression pressure.5.The effects of Smad4overexpression on the expression of Caspase-3 and Bcl-2 mRNA and protein under compression pressure were detected by RT-qPCR and Western Blot.Results:1.The results of CCK-8 showed:(1)Compression force promoted the proliferation of HGFs and reached the peak at 24 hours(P<0.05),then decreased.(2)OD value of blank control group?transfection group and blank carrier group increased with the prolongation of culture time;OD value of transfection group was lower than that of blank carrier and blank control group from the third day(P<0.05).(3)Compared with the empty vector group and the blank control group,the OD values of the overexpression group were significantly decreased in the 0h group and the 24h group(P<0.05).2.The results of RT-qPCR and Western Blot showed:(1)With the prolongation of the exertion time,the gene and protein expression of Smad4?Caspase-3 in HGFs decreased,and reached the lowest level at 24 hours,then increased gradually;on the contrary,the expression of Bcl-2 gene and protein increased with the prolongation of the exertion time,and reached the peak at 24 hours,and then decreased gradually.(2)After 72 hours of transfection,the transfection expression efficiency of the overexpression group was 90.091±0.085%,and the efficiency of the empty vector group was 89.346±0.022%.(3)After Smad4overexpression,the expression of Smad4 and Caspase-3 mRNA and protein were up-regulated and the expression of Bcl-2 was decreased in Smad4overexpression group(P<0.05).There was significant difference between 0h group and 24h group(P<0.05).Conclusion:1.Compression pressure promoted the proliferation of HGFs in a time-dependent manner.The proliferative ability increased with the prolongation of time,reaching the peak at 24h and then declining.2.The proliferation of HGFs under compression pressure was negatively correlated with the expression of Smad4.That was,with the prolongation of the exertion time,the gene and protein expression of Smad4 in HGFs decreased,and reached the lowest level at 24 hours,then increased gradually 3.Compression pressure inhibited the expression of Smad4 and Caspase-3 and promoted the expression of Bcl-2.All three were time-dependent:the strongest at 24h,and this promotion or inhibition was weakened over time.4.Under compression pressure,overexpression of Smad4 could inhibit the proliferation of HGFs.Moreover,lentiviral transfection itself had no effect on the proliferation of HGFs in a short period of time(three days).However,as the transfection time was prolonged(three days later),the lentivirus itself inhibited the proliferation of HGFs.5.Under compression pressure,Smad4 could regulate the expression of Caspase-3and Bcl-2 in HGFs.That was,Smad4 could promote the expression of Caspase-3 and inhibit the expression of Bcl-2.