TGF-β₃Reduces Bovine Corneal Stromal Extra Cellular Matrix Fibrosis:In A Three-Dimensional Culture Model In Vitro

Background:Transforming growth factor-β₁（TGF-β₁）can promote the hyperplasia of keratocytes in the repair of corneal injury,resulting in the change of the phenotype of keratocytes and the synthesis of extra cellular matrix（ECM）.However,TGF-β₃ can promote ECM degradation and inhibit the proliferation of keratocytes,it was considered to be the Cytokines that anti-scarring.The low concentration of TGF-β₃ was applied to the research of human keratocyte in three-dimensional culture,It was found that it could inhibit and reverse the synthesis of light fibrosis ECM,but when the concentration of TGF-β₃ and TGF-β₁ was greater than 0.5ng/ml,The cellular structure would be constricted and The cells could not be cultivated for a long time.In previous study,we established the three-dimensional culture model of corneal stromal ECM fibrosis in vitro mediated by TGF-β₁,The effects of different concentration of TGF-β₁ on corneal stromal ECM fibrosis were observed.so far,The effect of high concentration of TGF-β₃ on the synthesis and construction of fibrosis ECM in the repair of corneal injury has not been elucidated.Objective:To evaluate the effect of TGF-β₃ on the synthesis and construction of bovine cornea fibrosis ECM in vitro.Methods:established Pellets derived from bovine keratocytes in a three-dimensional culture model in vitro,The bovine keratocytes were cultured with this culture system.the experiment was divided into three groups,respectively,basal culture medium group（named T0）,（0.5 ng/ml TGF-β₁+basal culture medium）stimulated for 1 week and then Changed to basal culture medium group（named T1）,（0.5 ng/ml TGF-β₁+basal culture medium）stimulated for 1 week and then Changed to（0.5 ng/ml TGF-β₃+basal culture medium）group（named T3）.Each group was cultivated for 3 weeks,and the same method was used to test the culture products of each group at the same time point.The morphology of keratocytes were observed by inverted microscope and The growth of Pellets were observed by naked eye.observed the cell structural by HE staining.After Pellets were cultured for 48h,1 week,2weeks,3 weeks,Calcein-AM/PI staining were used to test the activity of keratocytes,and RT-PCR was used to detect the expression ofα-SMA（α-smooth muscle actin）,FN（Fibronectin）,ColⅠ（collagenⅠ）,ColⅢ（collagenⅢ）,LUM（Lumican）,KERA（Keratocan）at mRNA level.after Pellets were cultivated for 3 weeks,Immunofluorescence staining was used to detect the expression and localization ofα-SMA,FN,ColⅠ,ColⅢprotein.The diameter and spatial arrangement of collagen fibers were observed by transmission electron microscope.Image Pro Plus 6.0 was used to measure experiment results and statistical analysis was performed on the results.Results:The adherent keratocytes were intact and in flat-shuttle shape.Cells in Pellet clustered after adding TGF-β₃,they could be cultivated for a long time.HE staining showed all Pellets had red and light colored collagen fiber、Some normal and necrotic cells.Calcein-AM/PI staining showed that with the extension of culture time,the died cells in Pellets were gradually increased,But there are still plenty of viable cells.After Pellets were cultured for 48h and 1week,the expression ofα-SMA,FN,ColⅢmRNA in T1 and T3 were higher than T0.After Pellets were cultured for 2 weeks and 3 weeks,the expression ofα-SMA,FN,ColⅢmRNA and the ratio of ColⅢ/ColⅠin T3 were obvious lower than T1（P<0.05）.LUM and KERA mRNA were positively expressed in three groups when Pellets were cultured for 48h,1 week,2 weeks,3 weeks and at 2 weeks and 3 weeks,they expressed higher in T0 than T1 and T3,There was no significant different between T1 and T3.After Pellets were cultured for 3weeks,Immunofluorescence showed they all expressedα-SMA、FN、COLⅠ、ColⅢprotein,the expression ofα-SMA,FN,ColⅢand the ratio of ColⅢ/ColⅠin T3 were obvious lower than T1（P<0.05）.TEM showed that collagen fibrils diameter was more uniform in T3 than in T1,and the arrangement of collagen fibers was More tidy in T3 than in T1,T3 was Close to T0.Conclusions:TGF-β₃ can reduce bovine corneal stromal ECM fibrosis:in a 3D culture model with temporal dynamic distribution of TGF-β1 in vitro.