ABSTRACT
Objective To explore the role of forkhead box F2 (FoxF2) in the extracellular matrix of trabecular meshwork.Methods The cultured human trabecular meshwork cells (HTMCs) were divided into Scramble control group and FoxF2 small hairpin RNA (shRNA) group,then FoxF2 shRNA,the FoxF2 restructuring interference carrier was built,HTMCs were infected with FoxF2 shRNA lentivirus.Western blot assay was used to detect the expression of FoxF2 protein and extracellular matrix.Furthermore,Transwell counting experiment was used to analyze the migration ability of HTMCs.Results The cultured HTMCs grew well and showed a long spindle shape.The growth status of HTMCs was well,and their morphological characteristics were consistent with the HTMCs in vivo.The relative expression level of FoxF2 protein in the FoxF2 shRNA group was lower than that in the Scramble control group,with a significant difference between them (0.72 ± 0.02 vs.1.27 ± 0.05;t =16.68,P ＜ 0.01).The relative expression level of fibronectin (FN),collagen type Ⅰ (COL Ⅰ) and α-smooth muscle actin (α-SMA) were 0.43±0.03,0.53 ±0.08 and O.86±0.15 in the FoxF2 shRNA group,and 0.87±0.04,1.66±0.06 and 1.73 ±0.13 in the Scramble control group,respectively,the relative expression levels of FN,COL Ⅰ and α-SMA in the FoxF2 shRNA group were significantly lower than those in the Scramble control group (t =15.08,18.81,7.50,all at P＜0.01).The migration number of HTMCs in the FoxF2 shRNA group was significantly lower than that in the Scramble control group (117.30±11.41 vs.251.00±10.37;t =8.72,P＜0.01).Conclusions The FoxF2 shRNA lentivirus are successfully constructed,which can decrease the expression of FoxF2 in HTMCs.Low expression of FoxF2 can reduce the expression level of extracellular matrix protein in HTMCs and inhibit the migration ability of HTMCs.
ABSTRACT
Objective To investigate the effect of estrogen on the fibrosis process of intrauterine adhesions and the expression of forkhead box F2 (FoxF2).Methods Primary human endometrial stromal cells (HESCs) were obtained by separation with 0.2％ collagenase Ⅰ digestion-mesh filtration-differential adherence,and identified by immunocytochemistry.HESCs affected with 10ng/ml transforming growth factor β1 (TGF-β1) for 48 hours.HESCs in model group were affected with 0,10-6,10-8,10-10 and 10-12mol/L estrogen,the expressions of smooth muscle actin alpha (α-SMA),Collagen I (COL Ⅰ) and FoxF2 were detected by quantitative PCR (qPCR) and Western blotting.Results HESCs with high purity and good activity were obtained by using 0.2％ collagenase Ⅰ digestion-mesh filtration-differential adherence separation method.Immunocytochemistry showed positive vimentin and negative cytokeratin 18 in HESCs.The results of qPCR and Western blotting showed that the mRNA and protein expression levels of α-SMA,COL Ⅰ and FoxF2 were higher in model group than in control group (P＜0.05),the model was built successfully.qPCR revealed that the mRNA expression levels ofα-SMA,COL Ⅰ and FoxF2 were significantly lower in 10-6,10-8 and 10-10mol/L estrogen groups than in model group (P＞0.05 in 10-10mol/L estrogen group,P＜0.05 in other groups),while in 10-12mol/L estradiol group,the expression levels of FoxF2 mRNA significantly decreased (P＜0.05),and of α-SMA and COL Ⅰ mRNA increased,but no significant difference were found (P＞0.05).Compared with the model group,the protein expression levels of α-SMA,COL Ⅰ and FoxF2 in 10-6,10-8 and 10 10mol/L estrogen groups decreased,but no significant difference was found (P＜0.05),while in 10-12mol/L estradiol group,the expression levels ofα-SMA protein increased (P＞0.05),and of COL Ⅰ and FoxF2 proteins decreased (P＜0.05).Conclusions The expression of FoxF2 in intrauterine adhesions is increased.Estrogen can reverse the fibrosis process of intrauterine adhesions in a certain range and inhibit the expression of FoxF2.