Study Of The Effects Of Arsenic Trioxide On Human Retinal Pigment Epithelial Cells Cultured In Vitro

Proliferative vitreoretinopathy (PVR) is a disease which often happened after rhegmatogenous retinal detachment ,retinal reattachment surgery and ocular trauma, it caused tractional detachment of retina resulted by the growth and contraction of cellular membranes within the vitreous cavity and on both retinal surfaces. PVR is not only a severe complication of rhegmatogenous retinal detachment and the most common cause of failure in the retinal reattachment surgery but also a common refractory ocular disease to grow blind.The firm mechanism of PVR was unknown, but its basic pathogenesis has been realized. A great deal of studies show PVR is a intraocular exaggerated wound-healing process and a multi-element disease with reciprocite of several cells and factors. The excessive proliferation of RPE cells is main pathology of PVR. RPE cells were seen almost in all PVR membrane and it can transform different shape and possess multifunction ,such as secreting various factors transforming macrophage, synthesizing glial, proliferating in stimulated by growth factors.Despite the improvement of surgical reattachment rates with vitrectomy which can relieve the retinal traction and make it reattachment, the modern vitrectomy surgery was unable to prevent the cell proliferation and PVR membrane formation. So there was certain postoperative relapse rate. For increasing the surgical success rate, recently many studies have tried to use medicine as adjuvant treatment.The close relation of PVR and RPE cells has been confirmed, so the PVR treatment almost was begun from RPE cells by inhibiting RPE cells proliferation,migration, and fibro-membrane contraction, as well as inducing cells apoptosis. Medication of PVR is always main research in ophthalmology, therefore to seek for new preferable medicine for the proliferation and apoptosis of RPE cells plays a key role in preventing PVR and decreasing the postoperative relapse rate.Arsenic trioxide is effective element of Chinese traditional medicine arsenic which has been used to treat acute primyelocytic leukemia(APL) in clinic and achieved significant effect. As anti-tumor medication, Arsenic trioxide mainly induced apoptosis of tumor cells with changing cellular chromosome structure, restraining cellular sulfhydrase activity, changing cellular chromosome penetration, disturbing cellular metabolize. In recent years, there are more and more researchers to study the effect of arsenic trioxide on tumor cells. The experiments show arsenic trioxide can induce apoptosis of excessively proliferative normal cells, such as vascular smooth muscle cell and vascular endothelium cell, as well as many diverse tumor cells. This shows very wide clinical prospect. The research has not been seen to prevent PVR with arsenic trioxide in country and abroad. This study firstly investigates the effects of arsenic trioxide on proliferation and apoptosis of human RPE cells cultured in vitro, in addition, we also observe the medical sensitivity of RPE cells in different growth state. This study may provide next research in arsenic trioxide and preventing PVR in ocular local application with experimental data.Materials and methods(1) Culture of Human RPE: RPE cells were isolated from donor eyes using general method as follow. A circumferential incision was made in the sclera 8mm posterior to the limbus. The vitreous and the neuroretina were excised under a dissecting microscope, then the RPE was exposed, the eye cup was washed with D-Hanks solution 2-3 times. Trypsin(0.25%)-EDTA(0.02%) solution was added and the eye cup was incubated for 30~60min at 37 ℃. The RPE cells were then separated with a fire-blown Pasteur pipette under a dissecting microscope. DMEM medium supplemented with 20% fetal bovine serum (FBS) was added to end digesting. The cells were centrifugal for 5 minutes at 1000rpm and the solution was abandoned. The isolated RPE cells werecultured with DMEM medium supplemented with 20% FBS in 25ml plant bottle, the culture bottles were incubated in a humidified 5% CO2 atmosphere. The medium was changed 4 days later. After reaching confluence, the RPE cells were diluted 1:3, and plated for subculture with 10% FBS DMEM medium. All tested cells lines were in the 3rd-6th passages. Immunocytochemical staining could be used to distinguish the pigment epithelium from melanocytes and fibroblasts. Primary antibodies is a monoclonal mouse antibody to human cytokeratin.(2) Experimental drug :Arsenious Acid Injection( AS2O3): Production of Harbin YiDa Pharmaceutical Co.,Ltd.(3) The observation of morphology of human RPE cell: Observe the morphology of human RPE cell under inverse microscope and record with camera.(4) The experiment of medical sensitivity in vitro (MTT colorimetric assay): The RPE cells were detached by trypsin-EDTA solution and plated into 96-well plates with DMEM medium supplemented with 10% FBS at a density of 4~5× 104 dells per well. After 24 hours in serum group or reaching confluence in serum-free group, the medium was replaced by DMEM medium supplemented with 10% FBS and DMEM medium respectively, and arsenic trioxide at various concentrations (0% 1.56% 3.12% 6.25% 12.5% 25% 50μmol/L) was added to the culture medium. Each concentration of the drug for each time period was tested in triplicate wells. Then the cells in serum group were cultured for 24h, 48h, 72h, in serum-free group for 48h. MTT solution(5mg/ml) was added to the culture medium 20//1 per well. After 4 hours culture, the culture medium was removed and each well was added into 150^1 dimethyl sulphoxide(DMSO), the plates were vibrated for 15 minutes, the absorbency was obtained at 490nm wavelength in enzyme-immunity examination. The experiment was repeated two times.(5 ) The assay of cell cycle and apoptotic rate (Flow cytometry): The RPE cells collected as above method were plated into 50ml plant bottles, the medium was replaced by DMEM medium supplemented with 10% FBS after 24h , and arsenic trioxide was added to the medium at various concentrations (0, 1.56, 3.12, 6.25, 12.5, 25μmol/L), after 24 hours or 48hours culture, the cells collected were washed two times with phosphate balanced solution (PBS). 70% cold grain alcohol was added to fix the sample.The samples were kept at 4℃ for more than 12 hours and the fixing solution was removed with PBS. PI dying solution was added to dye at 4℃ for 30 minutes. Filtrating the sample with 500-hole cuprum net, the cellular status and apoptotic rate were tested with flow cytometry.(6) Statistical analysis: One-way analysis of variance (ANOVA) was used for analysis of all results.rusults1. Culture and identify of human RPE cell: The RPE cells in the primary culture were polygonal or spindle shaped cells with full of pigmentation in cellular plasm. The cellular nuclear was round and transparence. The pigment granules were gradually diluted after growth. Immuocytochemical study shows the RPE cells were stained with brown-yellow in cellular plasm, the positive rate was 100%.2. Effects of As2O3 on the morphology of human RPE cell: With inverse microscope, we can see the human RPE cells in control group grew in adhibition manner almost same in shape and volume, few suspending cells, the cells in serum group treated with arsenic trioxide(<25μmol/L) presented such changes: volume of some cells enlarged, abnormal modality, increasing suspending cells. The effect was enhanced with concentration. After 48 hours at 25μmol/L treatment, there were such changes: the density of RPE cells markedly minished, the cellular shape round, cellular volume smaller, intact cell membrane, cytoplasmic and nuclear condensation consistent with apoptosis. when the drug concentration was more than 50μmol/L, the cells were almost dead. The cells in serum-free group were markedly suspending when exposed in more than 25μmol/L concentration and the volume of some cells was enlarged, no cellular apoptotic features.3. The experiment of medical sensitivity in vitro: The results of MTT revealed the inhibition of arsenic trioxide on human RPE cells was different in different dose and time. The drug concentration of statistic significance compared with control group respectively was: 6.25μmol/L at 24 hours(P=0.018) and 48 hours(P<0.001), while 1.56μmol/L at 72 hours(P<0.001). The inhibition rate increased in a concentration andtime-dependent manner. The inhibition was significant at 12.5μmol/L in serum-free group after 48 hours treatment.4. The assay of cell cycle and apoptotic rate: Flow cytometry analysis indicated changes of cell cycle distribution in human RPE cell, including a S phase arrest after 1.56 -12.5μmol/L arsenic trioxide treatment, especially significant at 6.25μmol/L, and G2/M phase arrest at 25μmol/L. The sub-G0/G1 apoptotic cell population increased in concentration-dependent manner. These changes were not markedly related to treatment time.conclusion1. Arsenic trioxide can inhibit the proliferation of human RPE cells cultured in vitro in a concentration and time-dependent manner over certain range.2. Arsenic trioxide can induce marked morphologic changes consistent with apoptosis and sub-G0/G1 apoptotic cell peak in human RPE cells cultured, and change cell cycle with cell cycle arrest.3. The culture human RPE cells in different growth state were differently sensitive to arsenic trioxide. In certain drug concentration range, arsenic trioxide can inhibit proliferation of human RPE in speedy growing status more significant than in slow growing status.