The Mechanical Behaviors Of The Scleral Fibroblasts And The Relationships Between The Scleral Fibroblasts, The Retinal Pigment Epithelial Cells And Some Cytokines In Experimental Myopia

Objective:To determine the cell proliferation and the growth period of anterior and posterior retinal pigment epithelial (RPE) cells and scleral fibroblast (SF) cells in cultured guinea pig model of lens-induced myopia (LIM), and to determine the expression changes of MMP-2, TGF-β2and bFGF in RPE and SF cells.To determine the impact of TGF-β2on the cell proliferation of anterior and posterior scleral fibroblast cells in cultured guinea pig and the impact of TGF-P2on the expression of MMP-2, TβRII and bFGF in scleral fibroblast cells; and also to study the biomechanical characteristics changes. Some conclusions were valuable for revealing the pathological mechanism and the treatment of myopia.Methods:Divide30two-week-old guinea pigs into three groups,10of each group. The guinea pigs were randomly lens-induced monocularly. The unilateral eye was treated by defocus method as the experimental eye, and induced by a spherical lens of-10.00D to build the myopia model. The other eye in each animal was untreated as self-control (SC) eye.5two-week-old guinea pigs were randomly closed as normal-control (NC). Each group was raised for6,15and30days separately. Before and after the experiment, the refraction was measured using retinoscopy and the axis oculi lengths of binoculus were determined by A-scan ultrasonography.The retinal pigment epithelial cells of each group were cultured and passed continually for2generations. The cultured cells were verified to be retinal pigment epithelial cells using immunocytochemical method. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior retinal pigment epithelial cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.The scleral fibroblasts of each group were cultured and passed continually for2generations. The cultured cells were verified to be scleral fibroblasts using immunocytochemistry method. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior scleral fibroblast cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.The scleral fibroblasts of each group were purified with the tissue explant method and passed continually for2generations. The cultured cells were verified to be scleral fibroblasts using immunocytochemistry method. Add TGF-β2of different mass concentrations (0,1,10, and100ng/ml) to serum-free DMEM to react for24hours. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior scleral fibroblast cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.Results:1The expressions of TGF-β2, bFGF, MMP-2in cultured guinea pig anterior and posterior retinal pigment epithelial cells and scleral fibroblasts in lens-induced myopia shown by the results of Immunocytochemistry, Real-Time PCR Analysis and Western blot method:1.1The expression of TGF-β2was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2in posterior cells was higher than the in anterior cells (P>0.05) on the6th,15th and30th day. There was a significant difference of the expression of TGF-β2between model eyes and control eyes (P<0.05).1.2The expression of bFGF was lower in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of bFGF in anterior cells was almost the same with the expression in posterior cells on the6th,15th and30th day (P>0.05).1.3The expression of MMP-2was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2in posterior cells was higher than the expression in anterior cells (P>0.05) on the6th,15th and30th day. There was a significant difference of the expression of HGF between model eyes and control eyes (P<0.05).2The effect of TGF-β2on the expressions of MMP-2, TβR II and bFGF (mRNA, protein)2.1The expression of MMP-2was located in cytoplasm, and it was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2becomes gradually higher as the concentration of TGF-β2increases.2.2The expression of TPR II was located in cytoplasm and nuclear membrane, and it was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TβRⅡ becomes gradually higher as the concentration of TGF-β2increases.2.3The expression of bFGF was located in cytoplasm and nuclear membrane, and it was lower in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of bFGF does not have a significant change as the concentration of TGF-β2increases.3The observation of growth periodThe ratio of G1/G0phrase of anterior and posterior retinal pigment epithelial cells increased after6-day and15-day inductions separately (P<0.05), while the ratio in S period of anterior and posterior retinal pigment epithelial cells decreased significantly (P<0.05) after6-day and15-day inductions separately.4The observation of ultrastructureAfter the anterior and posterior retinal pigment epithelial cells and scleral fibroblast cells were induced for6days, it was observed that the endoplasmic reticulum was dilated and some organelles decreased, such as endoplasmic reticulum and ribosome. The proliferation of the anterior retinal pigment epithelial cells is reduced after a15-day induction, same results with those after a6-day induction.5The mechanical behaviors of the anterior part of LIM group and the statistical analysis5.1In comparison of the mechanical behaviors of sclerotic desmocytes between LIM group and SC group, it was found that, whether on equilibrium Young’s modulus or on cellular apparent viscosity, all the anterior parts of LIM group were longer than those of SC group on the30th day, which was significant statistically (P<0.05); and all the extreme posterior parts of LIM group were larger than SC group on the15th and the30th day, which was significant statistically too (P<0.05).5.2The effect of TGF-β on the mechanical behaviors of the posterior parts of LIM and SC group and the statistical analysis Compared with the fellow eyes and normal control eyes, the equilibrium modulus and apparent viscosity in model eyes were significantly higher (P<0.05). After treatment of TGF-β2, the equilibrium modulus and apparent viscosity in the model group and fellow eyes were positively correlated to the concentrations of TGF-β2(r=0.743, r=0.533, r=0.654, r=0.576, P<0.05). Following the addition of1ng/ml TGF-β2and10ng/ml TGF-β2, the equilibrium modulus and apparent viscosity of scleral fibroblasts were significantly reduced in model eyes compared with fellow eyes (P<0.05). No significant difference was found in the equilibrium modulus and apparent viscosity of scleral fibroblasts between model eyes and fellow eyes after treatment with100ng/ml TGF-β2(P>0.05).Conclusions:1LIM could up-regulate the expressions of TGF-p2and MMP2in both RPE and scleral fibroblasts cells, while the bFGF expression was down-regulated.2LIM and TGF-β2could inhibit the proliferation of RPE and scleral fibroblasts cells.3TGF-β2could up-regulated the expression of MMP-2and TβR Ⅱ and remodel the ultrastructure of RPE and scleral fibroblasts cells, but has no effects on bFGF expression.4LIM could increase the equilibrium modulus and apparent viscosity of scleral fibroblasts cells, and there was a difference between the anterior and the posterior part of sclera cells.5Light concentration of TGF-β2could decrease the equilibrium modulus and apparent viscosity of scleral fibroblasts cells.