Effects Of TGF-β₁ On Collagen Construct Of Bovine Keratocytes In Pellet 3-D Culture Model

Background Destructive construction of collagen fibers is a critical regulator during many diseases development, including corneal scarring. To deeply discuss the Molecular mechanism of corneal stroma scarring, we have been studying on the construction of collagen fibers. That maybe play a central role of scar-free healing in corneal wounding and blindness prevention. During the corneal wound healing, fibrotic extracellular matrix （ECM） is the main pathological change. Collagen fibers abnormal formation is the major procedure of the abnormal deposition of ECM. Three-demensional （3-D） culture provides an effective way to investigate the construction of the ECM compared with two-dimensional （2-D） model. In our previous research, we successfully developed a 3-D model Pellet as a vitro system of bovine corneal stroma ECM fibrosis, in which Transforming growth factor-β₁（TGF-βi） induced bovine keratocytes express the mRNA and protein of a-smooth muscle actin （α-SMA）、Collagen Ⅰ（Col Ⅰ）、Collagen Ⅲ（Col Ⅲ）、fibronectin （FN） . This study is to apply TGF-β₁ to this 3-D system, then evaluate the effects of TGF-β₁ on collagen synthesis and construction, in order to approach the mechanism of corneal stroma scarring.Objective To evaluate the effects of TGF-β₁ inducing bovine keratocytes on collagen synthesis （ expression and ratio of collagen ） and collagen construciont （ diameter and space aligment of collagen fibrils ） in Pellet 3-D culture model.Methods The primary bovine keratocytes were cultured in Pellet 3-D culture model in vitro. According to different concentrations of TGF-β₁, two groups were included: Group low concentration （0.5 ng/ml TGF-β₁） and Group high concentration （ 1.0 ng/ml TGF-β₁） . After 2 weeks, 3 weeks and 4 weeks cultured, the protein expression of col Ⅰ and Col Ⅲ were evaluted by indirect immunofluorescence. At the time points of 2 weeks and 4 weeks, the transmission electron microscopy was used to observe morphology, trend and space alignment of collagen fibrils cultured 2 weeks or 4 weeks. Image Pro Plus 6.0 software was used to analyze the protein expression of col Ⅰ and Col Ⅲ,and measure the diameter of collagen fibrils at 4 weeks.Results （1 ） With the increase of concentration of TGF-β₁ or stimulation time, the protein expression of Col Ⅰ and Col Ⅲ were upregulated. These showed upregulation of Col Ⅰ and Col Ⅲ had a trend of time and dose dependent. （2）As time goes on, the ratio of Col Ⅲ and Col Ⅰ were down-regulated in both two groups. Then at 4 weeks, the ratio was significantly lower than that at 2 weeks. At the same point, cultures treated with high concentration of TGF-β₁ showed a down-regulation as compared to Group low concentration. （3）The transmission electron microscopy showed that the collagen fibrils accumulated markly, and the aligment was obviously more irregular in Group high concentration compared with Group low concentration.（4）At 4 weeks, collagen fibril diameters in Group high concentration significantly increased than those in the lower group.Conclusion In Pellet model, 0.5 ng/ml or 1.0 ng/ml TGF-β₁ can promote the bovine keratocytes synthesize the Col Ⅰ and Col Ⅲ, and leads to the imbalance ratios of Col Ⅰ and Col Ⅲ formation. These variation collagen formation showed a trend of time and concentration dependent. The higher concentration of the TGF-β₁ regulated the bigger diameter and the more disorderly allignment of the collagen fibrils. The longer cultured, the same effects showed. TGF-β₁ influences the synthesis and construction of the collagen in the Pellet 3-D system.