Purpose To study the possibility of prevention of proliferative vitreoretinopathy(PVR) by transduction of exogenous gene in vivo. Methods PVR model of rabbits was induced by intravitreal injection of fibroblasts.? galactosidase (lacZ) gene as a reporter gene was transfered into the vitreous of PVR model eyes mediated by retroviral vector, and the expression of the gene in eye tissues was determined. Gene transfection was done on the 6th day after fibroblasts injection,and the dosage of intravitreal injection of reporter gene was 0.1ml PLXSN/lacZ serum free supernatant (1.1?10 6 cfu/ml). Results lacZ gene expression was seen in proliferative membranes after gene transfection, and the expression was located maily at the surface of PVR membrane. The reporter gene expression lasted at least more than 30 days. No expression was found in retinal tissues. Conclusions Retrovirus mediated gene can be directionally transducted in PVR membrane,and might possess the feasibility of gene therapy for PVR. [

Objective To investigate the effects of intravitreal injection of matrix metalloproteinase-3 (MMP-3) on the vitreoretinal adhesion and the vitreous gelatin. Methods Twenty-four pigmented rabbits were randomly divided into 3 experimental groups (group A, B, and C)and one control group with 6 rabbits (12 eyes) in each. Different concentrations of 0.1 ml MMP-3 (5,10, 20 ng in group A, B, and C, respectively) and equivalent dose of balanced salt solution were intravitreally injected to the rabbits, respectively. Clinical examinations (such as gross observation, slit-lamp biomicroscopy, indirect fundus ophthalmoscopy ), electroretinography (ERG) and fundus fluorecein angiography (FFA) were taken before and after injection. Results One week after injection, posterior vitreous detachment (PVD) and focal vitreous liquefaction were recognized clinically for the first time in 1 eye in group B. By the end of this study, clinically detected PVD developed in 1 eye in group A, 3 eyes in group B, but the synchisis developed slowly, and no liquefaction or PVD occurred in control group. As for the histological examination, partial PVD was observed in 1 eye in group A and 3 eyes in group B 60 minutes after injection. All of the eyes in group A and B showed partial PVD 1 week after injection, and the area of PVD enlarged in contrast with before. Complete PVD were recognized in 1 eye in group A and 3 eyes in group B 15 weeks after injection, and the cleavage was narrow and limited. In group C, inflammatory cell infiltration in the inner layer of retina, destruction of retinal structure, and fluorescein leakage at late phase was found in the early period after injection. Conclusions MMP-3 is effective in disrupting the adhesion of the posterior hyaloid to the inner limiting membrane leading to PVD, and helpful to some extent in producing vitreous liquefaction. The dose of 10 ng MMP-3 is safe and effective for the rabbits eyes, which may be used as an promising assistant of vitreous surgery.

Background Hypoxia is an important cause resulting in many retinal diseases,such as retinal edema,diabetic retinopathy,proliferative retinopathy and so on.ObjectiveThis study is to investigate the effects of hypoxia on the expression of AQP-4 in Müller cells in vitro.MethodsMüller cells were isolated from New Zealand white rabbits and primarily cultured in DMEM containing 20% fetal bovine serum by the explant culture method.The cells were identified by immunostaining for the glial fibrillary acidic protein(GFAP).Generation 2 of cells was cultivated with the chemical hypoxia inducer,CoCl_2,for 24 hours in the hypoxic group and only with DMEM in the control group.The expression of the AQP-4 protein in Müller cells was detected by immunocytochemistry.The expression of AQP-4 mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).ResultsAbout 90% of Müller cells(generation 2) presented a positive immunoreactivity for GFAP,showing a brown staining in the cytoplasm.Cultured cells displayed the presence of intermediate filaments,microvillus and various cellular organs.The Integralabsorbance of the AQP-4 protein in Müller cells was markedly increased 24 hours after incubation with CoCl_2 in comparison with the control group (t=6.74,P＜0.05).The expression level of AQP-4 mRNA in Müller cells was significantly enhanced 24 hours after incubation with CoCl_2 in comparison with the control group (t=21.79,P＜0.05). ConclusionHypoxia enchances the expression of AQP-4 in Müller cells and further increases fluid accumulation in the retina.These results suggest that Müller cells play an important role in the formation of retinal edema in diabetic retinopathy or proliferative retinopathy.

In order to investigate the application of par plana vitrectomy in the treatment of combined rhegmatogenous retinal detachment (RRD) and choroidal detachment (CD), 12 eyes of 12 cases of combined RRD and CD were retrospectively analyzed. Twelve eyes of 12 cases were subjected to par plana vitrectomy. Six mm infusion channels were used. Pars plana vitrectomy, membrane peeling and internal fluid－gas exchange with encircling scleral buckle placement were performed in a standard fashion. One patient received injection of silicone oil. Hormones were routinely administered pre- and post-operation. The results showed that the intraocular pressure was rapidly reconstructed in the 12 eyes of 12 cases, the fluid in the subchoroidal cavity was drained via the three sclera incisions. The detached choroidea replaced. No other sclera incision was needed to drain the fluid in the subchoroidal cavivity. The follow－up after operation lasted 2 to 16 months. The 12eyes were replaced anatomically. No postoperative proliferation of vitreous body and retina was induced. It was suggested that par plana vitrectomy was the first choice in the treatment of CD combined with RCD.

Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells. Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamine TM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 ?mol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcription factor E2F,and inhibit the proliferation of HRPE cells.

Small hairpin RNA (shRNA) was used to silence the HIF1alpha gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2, to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1alpha mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1alpha was interfered in RPE cultured under hypoxia (induced by 150 micromol/L CoCl2). RT-PCR was employed to detect the expression of HIF1alpha and TIMP1. The expression levels of HIF1alpha and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1alpha mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1alpha gene silencing in RPE was 83.4%. Western blotting revealed that the expression levels of HIF1alpha protein was dramatically dropped. In addition. RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9%, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1alpha mRNA could effectively silence the HIF1alpha gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.
Cell Hypoxia ; Cells, Cultured ; Gene Silencing ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Pigment Epithelium of Eye/cytology ; Pigment Epithelium of Eye/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Small Interfering/*pharmacology ; Retina/cytology ; Retina/*metabolism ; Tissue Inhibitor of Metalloproteinase-1/*biosynthesis ; Tissue Inhibitor of Metalloproteinase-1/genetics

Objective To investigate proliferating cell nuclear antigen (PCNA) gene expression in retinal pigment epithelium (RPE) cells and inhibition of antisense oligonucleotides(AS OND) encoding PCNA mRNA to gene expression and proliferation of RPE cells, so as to search for new genetic therapy way for pro1iferative vitreoretinopathy (PVR). Methods (1) Rabbit RPE cells cultured in vitro were detected for PCNA expression by streptoavidin biotin enzyme complex (SABC) immunohistochemistry at several times. (2) The liposome mediated synthetic antisense oligodeoxynucleotides (AS ODN) and sense oligodeoxynucleotides (S ODN) encoding PCNA were delivered to the RPE cells at different concentrations, then PCNA expresstion were detected by immunohistochemistry. (3) Exposed to different concentrations of AS ODN and S ODN, growth activity and suppressive rate of RPE cells were measured by methyl thiazolyl tetrazolium (MTT) methods. Results (1) PCNA were expressed in RPE cells, culmination in 48 hours of culture. (2) PCNA expression were markedly suppressed in the RPE cells treated with 0.28 and 1.12 ?mol/L PCNA AS ODN . (3) 0.28 ?mol/L and 1.12 ?mol/L PCNA AS ODN significantly inhibited proliferative activity of RPE cells in a dose dependent manner, the arrest rates of cellular growth reached 53% and 81% respectively. Conclusion AS ODN complementary to PCNA mRNA at some concentration can sequence specifically suppress PCNA expression in RPE cells and cellular proliferative activity, and show potential application to further experimental study for PVR genetic medication.

Objective To explore the expression and activation of transcription factor E2F1 in cultured human retinal pigment epithelium (RPE) cells. Methods Cultured human RPE cells were divided into two groups after synchronization: one was cultured in Dulbecco′s modified Eagle′s medium (DMEM) without serum; the other was cultured in DMEM supplemented with 20% serum of newborn calf. The expressions of E2F1 protein in two groups were detected by Western blot analysis. The E2F1 DNA binding activities were measured by gel mobility shift assay(EMSA). Results E2F1 protein of 60 000 molecular weight was detected in the nuclear extract of human RPE cells, and serum stimulation could increase its expression( P

Objective To observe the therapeutic effect of poly tetrahydrofurfuryl-co-lactic acid (copolymer C 4) as the biodegradable vitreous substitutes on rhegmatogenous retinal detachment. Methods Vitreoretinal surgery with copolymer C 4 tamponades was performed on 32 pigmented rabbits (64 eyes) with rhegmatogenous retinal detachment. The rate of reattached retina and the post-operative complications were observed. Results Three months after the operation, reattached retina was found in 96.4%, glaucoma in 5.5%, cataract in 10.9%, and copolymer emulsion in 10.2% of all the eyes. Conclusion copolymer C 4 may withstand the retinal tear effectively for 3 months, and can be a valuable vitreous substitutes.