Effect Of TGF-?1 On Smurf2 And COL1A1 Production By Human HFSFs Cells

Objective TGF-?1/Smad signaling pathway is one of the important signaling pathways in scleral remodeling,and Smurf2 plays a pivotal role in TGF-?1/Smad signaling pathway.In this experiment,the expression of Smurf2 in HFSFs cells and the changes of Smurf2 and COL1A1 levels in HFSFs cells treated with TGF-?1 were investigated.Methods Recovery of HFSFs cells and then passage them steadily,Smurf2 protein was detected by immunohistochemistry;HFSFs cells were treated with different concentrations of TGF-?1(0 ng/ml,1 ng/ml,5 ng/ml,10 ng/ml)for 24 h;HFSFs cells were co-cultured with TGF-?1(10ng/ml)for 1h,6h,12 h and 24 h.Then,the expression levels of COL1A1 m RNA and Smurf2 m RNA were detected by real-time fluorescence quantification and protein levels were detected by Western blot(Western-Blot)method.Results 1.Smurf2 protein expressed in HFSFs assayed by Immunohistochemistry: The positive expression of Smurf2 protein in HFSFs cells was observed by confocal microscopy.2.Effects of different concentrations of TGF-beta 1 on HFSFs cells The expression of Smurf2 m RNA was up-regulated in the 5 ng/ml and 10 ng/ml TGF-?1 intervention groups((P=0.002?0.028)).Smurf2 protein expression was increased in the 1 ng/ml and 5 ng/ml TGF-?1 intervention groups(P=0.011?0.037).The expression of COL1A1 m RNA and protein was significantly increased in the 10 ng/ml TGF-?1 intervention group(P<0.01).3.The effect of TGF-beta 1 on the level of Smurf2 and COL1A1 in HFSFs cell: Co-cultured HFSFs cells with 10ng/ml TGF-beta 1 for 1h,6h,12 h and 24 h,the expression of COL1A1 mRNA was significantly increased in Group 24h(P=0.033)?The expression of COL1A1 protein was significantly increased in Group 6h(P=0.003).The expression of COL1A1 protein was decreased at 12 h(P=0.121).Group 24 h return to a level similar to basal expression(P=0.017).The expression of Smurf2 m RNA and protein increased firstly and then decreased.The highest level of Smurf2 m RNA and protein was observed at 6h after co-culture(P=0.024?0.044).At 24 h,the level of Smurf2 m RNA showed no difference when compared with 0h(P=0.750,>0.05).Conclusions 1.Smurf2 protein is expressed in HFSFs cells.2.The early stage of intervention of ten ng/ml TGF-?1 promotes the expression of Smurf2 m RNA and protein,which can effectively induce the synthesis of type I collagen in HFSFs cells.3.Smurf2 protein may be involved in regulating HFSFs TGF-beta 1 / Smad signaling pathways in cells of regulation.