Effects Of Low-dose Of TGF-β₁ On Maintaining Bovine Corneal Stromal Cell Growth And Retarding Extra Cellular Matrix Fibrosis In A Three-dimensional Culture Model

Background:Transforming growth factor-β₁（TGF-β₁）plays an important role in corneal wound healing.The effects on the synthesis of extra cellular matrix（ECM）varies under different concentrations of TGF-β₁.Previous studies focused on the effects of high concentration（2ng/ml-10ng/ml）of TGF-β₁ on keratocytes in two-dimentional cultures.Our previous studies not only successfully established the three-dimensional culture model of bovine corneal stromal cells in vitro,but also stimulated bovine corneal stromal cells with 0.5ng/ ml-1ng / mlTGF-β₁,and observed its effect on ECM synthesis.The effect of low concentration of TGF-β₁ on the synthesis of ECM in the 3-D model in keratocytes remains unclear.Objective:To investigate the growth of Pellet（a three-dimensional model of corneal stroma cells in vitro）and its ECM synthesis under a lowconcentration of TGF-β₁ based on the three-dimensional culture model of bovine corneal stromal cells in vitro.Methods:Establish Pellets derived fresh bovine keratocytes,with culture medium containing 0.25ng/ml TGF-β₁+5% FBS and 0.50ng/ml TGF-β₁+5% FBS,respectively.Calcein-AM/propidium（Calcein-AM/PI）staining was used to assay the cell viability.HE staining was employed to observe cell structures.Real-time PCR was applied to analyze the transcription and expression ofα-smooth muscle actin（α-SMA）,fibronectin（FN）,type Ⅰcollagen（Col Ⅰ）,type Ⅲ collagen（Col Ⅲ）at the mRNA level and to detect the expression of genes of Lumican（LUM）and Keratocan（KERA）.Immunocytochemistry staining and Western-Blot were used to detect the expressions of α-SMA、FN、ColⅠ、ColⅢ at the protein level.Results : Cells in Pellet clustered during culture for 48 hours,1week,2weeks and 3 weeks.The mortality of cells increased with time,but no statistical difference between two groups（P=0.887）.HE staining results show that the mass red-dyed collagen fibers were observed in two groups,and most cells possessed complete structures.The ratio of Col Ⅲ /Col Ⅰ in 0.25ng/ml TGF-β₁+5% FBS group was lower than that in 0.50ng/ml TGF-β₁+5% FBS group in both mRNA and protein level（PmRNA=0.038,Pprotein=0.032）.The expression of LUM and KERA were detected in pellet at different time points.The expression of LUM in 0.25ng/ml TGF-β₁+5% FBS group increased with time（P=0.006）.While in 0.50ng/ml TGF-β₁+5% FBS group,the expression ofLUM peaked at 1 week,but declined at 2 weeks（P﹤0.001）.The expression of KERA in two groups were all peaked at 1 week,but declined at 2 weeks（P0.25ng/ml TGF-β₁ group=0.004,P0.50ng/ml TGF-β₁ group =0.020）.The expressions ofα-SMA,FN and Col Ⅲ at the protein level in 0.25ng/ml TGF-β₁+5% FBS group were lower than those in 0.50ng/ml TGF-β₁+5% FBS group（Pα-SMA=0.010,PFN=0.040,PColⅢ﹤0.001）,but the expression of ColⅠ at the protein level in0.25ng/ml TGF-β₁+5% FBS group was higher than that in 0.50ng/ml TGF-β₁+5% FBS group（PColⅠ=0.016）.Conclusions : Reducing the concentration of TGF-β₁ in Pellet can also maintain the normal growth of keratocytes and synthesize ECM.Ratio of ECM components stimulated by low-concentration TGF-β₁ was different from that of high concentration of TGF-β₁.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β₁,implying a scarless expression.