| Background:Extracellular matrix (ECM) fibrosis leads to corneal scaring during the process of cornea wound healing. TGF-P 1 is known to mediate overproduce of ECM components. We developed a three-dimensional (3D) model of corneal stroma in vitro previously. The hypothesis of this study is to apply TGF-β1 to the 3D model then a corneal stroma ECM fibrosis 3D model generated.Objective:To construct a candidate corneal stroma ECM fibrosis 3D model in vitro induced by TGF-β1.Methods:Constructed 3D culture models Pellet by bovine keratocytes. The Pellets were cultured in three medium conditions while three groups respectively:Group control, DMEM/F12+10% Fetal Bovine Serum(basic medium); Group T1 low concentration(basic medium+0.5ng/ml TGF-β1); Group T1 high concentration (basic medium+lng/ml TGF-β1). After 2 weeks cultured, Calcein AM/PI staining was carried out to assay the cell viability. The mRNA and protein expression of a-smooth muscle actin (aSMA), type Ⅰ collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ) were evaluated by real-time PCR and Western Blot respectively after 48 hours, 1 week and 2 weeks cultured. The results were statistically analyzed. Results:(1) Calcein AM/PI staining showed that most of cells were alive in the Pellets after 2 weeks cultured. (2) Either all of the three genes’ mRNA expression in Group T1 low concentration or in Group T1 high concentration was significantly higher than that in the Group control in each time points. The mRNA synthesis rate of Col Ⅲ was significantly higher than that in Col Ⅰ. (3) The protein expression of aSMA, Col Ⅰ and Col Ⅲ were detected by Western Blot only in the two groups containing TGF-β1 at the point time of 2 weeks, while not detected in the control Group and all the groups at the other time points.Conclusions:0.5ng/ml or lng/ml TGFβ1 can induce bovine keratocytes Pellet expresses the mRNA and protein of aSMA, Col Ⅰ and Col Ⅲ. It can be a candidate 3D model for corneal stroma ECM fibrosis. |
PDF Full Text Request