Effects Of Vesicles Of Emulsified Silicone Oil On Phagocytosis, Proliferation And Migration Of Human Retinal Pigment Epithelial Cells In Vitro

PurposeAt present, silicone oil is widely used to treat complicated rhegmatogenous retinal detachment, especially for proliferative vitreoretinopathy (PVR). But there are many complications if silicone oil was filled in eye for a period of long time, most of which are associated with emulsification of silicone oil. It is reported that periretinal membranes were found in the patients with emulsified silicone oil filled in the eye, and RPE cells had proliferated; and it was hypothesized that emulsification of silicone oil might stimulate periretinal membrane formation and PVR. But there are not many related researches in detail. This experiment aimed to: (1) Preparation of vesicles of emulsified silicone oil by a novel membrane emulsification technique in order to use it in the following experiments. (2) To assess the effects of vesicles of emulsified silicone oil and FCS on the proliferation and migration of human RPE cells,and the ability of RPE cells to phagocytize the vesicle of emulsified silicone oil in vitro.Methods1. Micro-pore filter membranes with 0.2μm and 0.45μm pores were employed in this study. Vesicles of emulsified silicone oil were produced by extrusion of oil through a micro-pore filter membrane under certain pressure, dispersing in PBS. The vesicles of emulsified silicone oil were observed under a light microscope, and the average diameter of silicone oil droplets was measured.2. Phagocytosis of RPE: Vesicles of emulsified silicone oil were labelled and monitored with phthalocyanine blue dye. Cultured human RPE cells were incubated with labelled silicone oil vesicles and with or without serum for 24 h. The numbers of the vesicles engulfed in the human RPE cells were counted under a microscope.3. AgNORs test: Cultured human RPE cells were incubated with silicone oil vesicles and with or without serum for 24 h. The number of AgNORs of human RPE nucleus was measured to assess the effects of vesicles of emulsified silicone oil and FCS on the proliferation of human RPE cells.4. Migration test: We used an in vitro wound healing model by denuding cells growing on a cover glass. Cultured human RPE cells were incubated with silicone oil vesicles and with or without serum for 72 h. The number of cells that had entered the denuded area was counted.Results1. The mean vesicle diameter was 2.33±0.618μm and 4.63±0.833μm through micro-pore filter membranes with 0.2μm and 0.45μm pore under 3 ATM, respectively.2. The mean vesicle diameter was 4.25±0.77μm through micro-pore filter membrane with 0.45μm pore under one ATM. Vesicles of emulsified silicone oil were labelled and monitored with phthalocyanine blue dye. Vesicles of silicone oil had a dose-dependent influence on phagocytosis of human RPE cells. The more the concentration of the vesicles, the more phagocytosis the cells did. The average number of vesicles of silicone oil phagocytized in the cells was 35.93±3.28 with 50.0 mL/L of vesicles of emulsified silicone oil; while in 100mL/L FCS, the number was 40.27±4.34(P<0.05).3. Vesicles of emulsified silicone oil had an influence on human RPE cell proliferation. There was significantly difference in treated groups and controls in the average number of AgNORs of human RPE nucleus (P<0.01). But there was no significantly difference among treated groups (P>0.1).The average number of AgNORs of human RPE nucleus was 2.8±0.63 with 12.5mL/L of vesicles of emulsified silicone oil (1.6±0.60 in controls; P<0.01). In 100mL/L of FCS, the number was 4.3±0.92 (2.3±0.65 in controls; P<0.01).4. Higher concentration of vesicles of emulsified silicone oil could inhibit human RPE cell migration in a concentration-dependent manner. The average number of cells that had entered the denuded area was 10.6±3.03 with 50.0mL/L of vesicles of emulsified silicone oil (69.9±9.88 in controls; P<0.001).While in 100mL/L of FCS, the number was 13.2±3.79 (91.0±12.99 in controls; P<0.001).Conclusion1. The unique advantage of preparing vesicles of emulsified silicone oil by micro-pore filter emulsification technique was that the size of silicone oil droplets can be controlled accurately and uniformly. This study demonstrated that preparation of vesicles of emulsified silicone oil by a micro-pore filter membrane with 0.45μm pore could be suitable for cell study in vitro. 2. Vesicles of emulsified silicone oil had an influence on human RPE cell phagocytosis in a concentration-dependent manner, and stimulated the proliferation of human RPE cells. But our study showed that even emulsified silicone oil with higher concentration could inhibit the migration of human RPE cells.Innovation1. In this study, we first report that uniform-sized vesicles of silicone oil could be prepared by a micro-pore filter membrane emulsification technique.2. We first established a method to label and monitor vesicles of emulsified silicone oil with phthalocyanine blue dye. The number of the vesicles engulfed in the human RPE cells was counted to assess the influence of vesicles of silicone oil on the phagocytosis of human RPE cells. We observed the effects of vesicles of emulsified silicone oil on the proliferation and migration of human RPE cells.