Study Of The Inhibiting Effects Of Simvastatin On Bovine Retinal Pigment Epithelium Cells Cultured In Vitro

Proliferative vitreoretinopathy (PVR) is a disease which happens after rhegmatogenous retinal detachment or retinal reattachment surgery and ocular trauma, and causes retinal detachment because of proliferation and contraction of cellular membranes in the vitreous cavity and on either the inner or outer retinal surface. It causes not only the secondary retinal detachment, but also causes vision lose.Although the mechanism of PVR wasn't known clearly, its pathogenesis has been realized. The procedures of PVR can be divided into three stages: inflammation, cell proliferation and scaring. Retinal pigment epithelial cells (RPE) and retinal glial cells proliferate and migrate from retinal pigment epithelium layer to the vitreous cavity, they contract and cause retinal detachment finally. RPE cells play an important role in PVR, so many studies have tried to use medicine to prevent PVR by inhibiting RPE cells proliferation and migration.Simvastatin is a competititive inhibitor of the 3- hydroxyl-3- methyl- glutaryl-coenzyme A reductase (HMG-CoA R). It has been utilized in the treatment of human hypercholesterolemia for two decades, with good results and limited side effects. Recently, many researches demonstrated that the competititive inhibitor of HMG-CoA R inhibit proliferation of many kinds of cells. Our study is to observe the effect of simvastatin on proliferation of bovine RPE cells. This study also provides a new method on preventing PVR with simvastatin.Materials and methods1. RPE cell culture: RPE cells were isolated from bovine eyes using general method as follow: A circumferental incision was made in the sclera 5mm posterior to the limbus. The vitreous and the neuroretina were excised to expose RPE cell layer. The eye cup was washed with PBS solution for 3 times, and added with trypsin (0.25%) solution, and then the eye cup was incubated for 30-60 minutes at 37℃. The RPE cells were deprived and the RPE cells suspension was made, and DMEM medium (high glucose) with 20% fetal bovine serum (FBS) was added to terminate digesting, and then the RPE cells suspension was centrifugal for 5 minutes at 1000rpm. The supernatant was aspirated and cells were resupended in DMEM medium (high glucose) with 20% FBS. Then RPE cells were planted into 25ml culture flask and incubated at 37℃, 5% CO₂, 95% air. The medium was changed 3 days later. The RPE cells were subcultured after confluence was achieved. Immunocytochemical staining was used to distinguish the retinal pigment epithelial cells from melanocytes and fibroblasts. Primary antibody is a monoclonal mouse antibody to human cytokeratin.2. Experimental drug: Simvastatin.3. The observation of morphology of RPE cells: The morphology of RPE cells was observed under inverse microscope and recorded with camera.4. The experiment of medical sensitivity in vitro--MTT colorimetric assay: In this study RPE cells from the third to the sixth subculture were used. The RPE cells were plated into 96-well plates at a density of 4～5×10⁴/ml cells per well. After 24 hours, cells were divided into control and experimental groups. In control group, cells were cultured with DMEM medium contained 10% FBS. In experimental group, cells were added with simvastatin, the concentration of simvastatin was respectively 1μmol/L, 5μmol/L, 10μmol/L, 50μmol/L, and cells were cultured for 24h, 48h, 72h. Each concentration for each time period was tested in triplicate wells. MTT solution was added into the culture medium at 20μl (5mg/ml) per well. Cultured for 4 hours, the culture medium was removed and DMSO was added into each well at 150μl. After 15 minutes, the absorbency was obtained at 490nm wavelength in enzyme-immunity examination, and this experiment was repeated for two times.5. Statistical analysis: one-way analysis of variance (ANOVA) was used for analysis of all results.Results1. The observation of morphology and identification of RPE cell: The RPE cells in the primary culture were polygonal or spindle shaped cells full of pigment in cellular plasma, and the round and transparent nucleus could be seen. The pigment granlin were gradually diluted when the cells grew. Immunocytochemical staining study showed the RPE cells were stained with brown-yellow in plasma.2. Effect of simvastatin on the morphology of RPE cell: In control group, we could see the RPE cells grew almost in the same shape and volume as before and few cells suspended under inverse microscope. In experimental group, the RPE cells tested with simvastatin appeared changes as follow: the densities of RPE cells diminished, the cellular volume was smaller, the suspended cells increased.3. MTT results: The results of MTT revealed the inhibition of simvastatin on RPE cells was different in different doses and times. The drug concentrations of statistic significance compared with control group respectively were: 5μmol/L at 24h and 48h, and 1μmol/L at 72h. The inhibition rate increased in a concentration and the inhibition appeared in dose-time-dependent manner.ConclusionSimvastatin can inhibit the proliferation of bovine retinal pigment epithelial cells cultured in vitro in certain range, and the inhibition appears in dose-dependent manner. Simvastatin may be a new method for preventing proliferative vitreo- retinopathy.