The effect of OPTC-shRNA on the bovine retinal pigment epithelial cells and hyalocytes co-culture collagen gel contraction system

Abstract

To investigate the OPTC-shRNA inhibiting effect on the opticin expression by the bovine hyalocytes and retina pigment epithelial (RPE) cells co-culture collagen gel contraction system. Experimental study. The OPTC-shRNA expression vector was designed and transfected into bovine RPE cells cultured in vitro. The relative expression and the inhibition rate of the opticin protein were measured by Western blot on days 3, 5 and 7. An in vitro cells co-culture bovine type I collagen gel contraction assay was constructed consisting of the hyalocytes and RPE cells. Six groups were established in this experiments:OPTC-shRNA plasmid transfected RPE cells and hyalocytes (group A), empty plasmid transfected RPE cells and hyalocytes (group B), non-transfected RPE cells and hyalocytes (group C), non-transfected RPE cells (group D), only hyalocytes (group E), and no cells (group F). The collagen gel contractile activities of these groups were compared by One-way ANOVA, SNK-q tests and regression analysis;and the influence of the hyalocytes density variance on the collagen gel contraction in groups A, B and C were also analyzed. The OPTC-shRNA expression vector with significant inhibition effect was constructed and transfected into bovine RPE cells successfully. The results of Western blot analysis showed that the inhibitory rates on the opticin expression on days 3, 5 and 7 were (83.91 ± 2.88), (84.71 ± 4.27) and (82.85 ± 2.72)%, respectively. Furthermore, the differences among days 3, 5 and 7 were insignificant (F = 1.15, P > 0.05). On day 3, the gel contraction rates for the sub-groups with various hyalocytes densities (2×10(7), 1×10(8) and 5×10(8)/L) in groups A, B and C were: group A: (23.52 ± 2.08), (56.00 ± 1.02), (61.62 ± 1.73)%; group B: (16.56 ± 2.01), (36.41 ± 1.33), (49.56 ± 1.75)%; group C: (15.75 ± 1.37), (37.45 ± 1.14), (48.45 ± 1.97)%. The gel contraction rates for groups D and E were (12.18 ± 0.95)% and (10.95 ± 0.93)%, respectively; no gel contraction was observed in group F. Pairwise comparisons of the gel contraction rates were performed by SNK-q test among groups A, B and C for various hyalocyte densities. In the 2×10(7)/L cell density group, the differences between groups A and B or C were significant (q = 11.38, 2.72, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 1×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 8.83, 46.22, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 5×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 48.83, 46.22, respectively, P both < 0.05), the differences between groups B and C were insignificant (q = 1.74, P > 0.05). Pairwise comparisons of the sub-groups with different hyalocyte densities in groups A, B and C (comparisons of 2×10(7)/L and 1×10(8)/L, 2×10(7)/L and 5×10(8)/L, 2×10(7)/L and 2×10(7)/L, respectively), the differences were all significant (group A:q = -55.97, -65.66, -9.69, respectively; group B: q = -34.53, -57.41, -22.88, respectively; group C: q = -41.94, -63.19, -21.25, P all < 0.05). Furthermore, the regression analysis was performed between the hyalocyte density and the collage gel contraction rates in each group. The results showed that there was a positive correlation between the gel contraction rates of the co-culture collagen gel contraction system and its hyalocyte density (groups A, B, C: r = 0.919, 0.981, 0.937, respectively, P all < 0.05). Pairwise comparison of groups D and E, D and F, E and F by SNK-q test revealed q = 54.87, 49.33, 5.54, respectively, P all < 0.05. Opticin is capable of regulating the contraction of bovine hyalocytes and RPE cells co-culture collagen gel.