RESUMO
Background Proliferative vitreoretinopathy (PVR) is one of the major causes of retinal detachment surgery failure.Based on proteomic studies of PVR vitreous,the insulin-like growth factor binding protein-6 (IGFBP-6) protein was specifically expressed in the vitreous and serum of PVR patients.Furthermore,its expression level is higher in the vitreous and serum in severe PVR patients than that in mild PVR patients.Objective This experiment was to detect the expression of IGFBP-6 in a PVR rat model.Methods Seventy 7-week old male SPF Wistar rats were included and were randomized into the PVR model group and control group.A mixture of RPE-J cell suspension(5 μl) and platelet-rich plasma (5 μl) was intravitreally injected in the left eyes of adult Wistar rats to establish the PVR model,and normal saline solution was administered in the same way in the control group.The rat eyes were clinically examined 1 week,2,3 and 4 weeks after injection,and PVR was graded based on the criteria of Francine.The animals were sacrificed after 1 week,2,4 or 8 weeks for the preparation of retinal sections and liver extraction.Expression levels of IGFBP-6 mRNA in the rat retina and liver were assayed by real-time Q-PCR.The expression of IGFBP-6 protein in the rat serum and vitreous was detected by ELISA.The use of animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results Purified IGFBP-6 RNA was extracted from the liver and retina of Wistar rat and quantified by real-time Q-PCR.The expression level of IGFBP-6 mRNA in retina was (3.79± 1.33) × 10-4 in the PVR model rats,showing a significant decline in comparison with the control rats with a level of(8.32±2.96) × 10 4,4 weeks after injection (t =3.42,P＜0.01).The expression of IGFBP-6 mRNA in the 4th week was significantly lower than that of 1 week,2 or 8 weeks after the establishment of the PVR model(P＜0.05).No significant difference was found in the IGFBP-6 mRNA level in the liver between the PVR group and control group(27.60± 14.01 × 10 4 vs.25.01 ± 12.04 ×10-4,respectively),as well as among the different time points(P＞0.05).IGFBP-6 mRNA content in the retina was significantly reduced in grades 1,2 or 3 of the PVR groups compared with the control group(P＞0.05),but there was no significant difference among the different grades of PVR groups (P＞0.05).Concentrations of IGFBP-6 protein in grades 1,2 and 3 of the PVR model group were (221.00 ± 19.32),(229.63 ± 18.89) and (225.70 ± 26.71) μg/L,with a significant elevation in comparison with (173.25 ±21.11) μg/L of the control group (t =2.14,P＜0.05).However,there was no significant change among the different grades of PVR groups(t=1.24,1.46,P＞0.05).The concentrations of IGFBP-6 protein in the vitreous and serum were higher in PVR rat samples (vitreous:225.44±19.36 μg/L;serum:108.48 ± 15.78 μg/L) than in control rats (vitreous:173.25 ± 21.11 μg/L,serum:95.96 ±17.40 μg/L)(P＜0.05).Conclusions The concentrations of IGFBP-6 protein in the vitreous and serum increase in PVR rats.The results indicate that the increased IGFBP-6 in the vitreous might be a localized autocrine secretion of the eye.
RESUMO
Background Our previous study demonstrated that kallikrein-kinin is a special protein in vitreous of the eye with proliferative vitreoretinopathy(PVR),and the expression intensity of kallikrein-kinin showed the positive correlation with the grade of PVR.Objective This study was to further explore whether kallikrein-kinin participate in the formation of PVR.Methods Rat retinal pigment epithelial cell line(RPE-J cells) was cultured in DMEM containing 4% fetal bovine serum and then prepared into suspension by PBS with the cells density of 2.5×108 cells/ml.Platelet-rich plasma was prepared by PBS with the platelet 2.5×108 /ml.RPE cell suspension(4μl) and platelet-rich plasma(6μl) was intravitreally injected in the left eyes of 30 clean Wistar rats to establish the PVR models,and 10μl sterile pyrogen-free normal saline solution was used in the same way in other matched rats as controls.The PVR was graded on Francine's criteria in 1 day,3,7,14,21,28 days after injection under the slit lamp.The serum,vitreous and retina were obtained in 28 days after injection to assess the expression of bradykinin using Western blot.The histopathology examination of rat retina was performed in the 28th day after injection.This experimental procedure followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Typical PVR was seen in 25 models with the successful rate 89.3% at 28 days after injection.PVR 1,2,3 grades were respectively exhibited in 7,14,28 days under the slit lamp.Infiltration of inflammatory cells and migration of RPE cells were found in the 7th day.In the 14th day after injection,RPE cells transformed into fibroblasts and retinal detachment occurred after that.Western blot analysis revealed that bradykinin was detected in vitreous,serum and retinal samples of rats in experimental and control rats,but the expression intensity was higher in the rats of model groups.Conclusion Intravitreal co-injection of RPE cells and platelet-rich plasma can effectively induce a model of PVR in Wistar rat.The kallikrein-kinin system probably takes part in the onset of PVR.