| Objective:Bevacizumab for intravitreal injection in the treatment of various ocular neovascular diseases has become a new focus,the reports (1,2,3) that visual function in patients improved with varying degrees have published known to the world .But for its long-term effect, scholars whether domestic or foreign have not carried out a large number of long-term and prospective randomized clinical study. When a drug used in local, we should understand their target sites in different organizations and the potential toxic effects. The Bevacizumab on cultured porcine retinal pigment epithelial cell, less clinical and basic research whether domestic or foreign .In this study,we cultured porcine RPE cells in vitro to observe the Bevacizumab's potential effects.Methods:1 Primary Culture of Procine RPE Cells1.1 Primary Porcine RPE was harvested with trypsin digestion.Cell morphology and characterization were assessed by phase contrast microscopy. The cells were identified with transmission electron microscopy and inlnlunofluorescence.1.2 By MTT assay to detect cell proliferative activity, with the concentration of 2.0-2.5 mg/m1,Bevacizuman inhibited the growth of RPE cells obviously in a dose—dependent manner compared with the control(P< O.O5);1.3 PRE cells indentified by transmission electron microscope and immunofluorescence.2 Toxic Effect Of Bevacizuman On The Cultured Porcine Retinal Pigment Epithelium Cells In Vitro2.1 The culture RPEs are plantated in 96-pore culture plate.Adding bevacizumab at the concentration of 0, 0.125, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5mg/ml to the cell in 96-pore culture plate,after 12, 24, 48, 72hours,we measure the absorbency using MTT colorimetric method.2.2 Adding bevacizumab at the concentration of 2.0 and 2.5mg/ml to the third generation of RPE cells,after 72 hours,we measured cellular cycle and apoptosis by flow cytometry (FCM ).2.3 Adding bevacizumab at the concentration of 2.0mg/ml to the third generation of RPE cells,after 72 hours,we observed the morphological and ultrastructural changes of the cells by phase contrast microscopy and transmission electron microscopy(TEM).3 Effects Of Bevacizumab On Function Of Cultured Retinal Pigment Epithelium Cells In Vitro3.1 Fluorescent Labeling of Isolated ROS, Preparation Of CytochromeIsolated ROS were pelleted in Hanks's balanced salt solution, resuspended in serum-free culture medium and transferred to a1.5-ml microcentrifuge tube for fluorescent staining.The FITC-stained ROS were resuspended in growth medium,and diluted to a final concentration of 107/ml.The growth medium containing the pigment sgranulations was obtained and resuspended in growth medium,and stored at -20℃.3.2 Fluorescent Labeling of Lysosomes in Cultured RPE CellsThe cultured RPEs are plantated in 24-pore culture plate.Adding bevacizumab at the concentration of 0,0.25,1.0,2.0,2.5mg/ml to the cell in 96-pore culture plate,after 12,24,48,72hours,maintained for 36 hours in growth medium containing SR at a final concentration of 40ug/ml.3.3 Determine the kinetics of phagosome-lysosome fusion in phagocytosing RPE cellsConfluent cultures of SR-stained Long Evans RPE cells were overlaid with FITC-ROS (107/ml), and incubated at 37°C,after 24 hours,washed with a stream of culture medium .The living RPE cells were examined by fluorescence microscopy.3.4 The Kinetics In non-specific Phagocytosis Of RPE CellsThe 7th generation of RPE cells are plantated in 96-pore culture plate. Adding bevacizumab at the concentration of 0, 0.25, 1.0, 2.0, 2.5mg/ml to the cell in 96-pore culture plate,after 12, 24, 48, 72hours,we added the pigments granulations into the 96-pore culture plate. The quality of the pigment granulations phagocytized in cells were ana1yzed with Enzyme-linked immunosorbent Detector.3.5 Migration Of RPE CellsThe third generation of RPE cells are plantated in 24-pore culture plate. Adding bevacizumab at the concentration of 0, 0.25, 1.0, 2.0, 2.5mg/ml to the cell in -pore culture plate,after 12, 24, 48, 72hours,we added the pigments granulations into the 96-pore culture plate. Wound healing model was used to count the number of cells that had entered the denudate area in RPE migration treated with Bevacizumab after 48h.Result:1 This Primary culture resulted in cells with well-preserved morphology,some cells grew in colony.The cells morphology was irregular,but they could be identified shape of sexangle when reaching confluence,The cells were vigorous for further passage. The result of immunofluorescence were positive of the antigen of antl一keratin.2 With the concentration of 2.0-2.5 mg/m1,Bevacizuman inhibited the growth of RPE cells obviously in a dose—dependent manner compared with the control(P< 0.05).3 The cells in S phase treated with Bevacizuman decreased compared with the control(P< 0.05),but the apoptosis rate was not different from the control.4 The results of transmission electron microscopy showed asymmetrically distributed cellular chromatin was,cavitated cytoplasm ,and even some necrotic cells.5 The phagocytosis tests indicated that Bevacizumab has no effect on the function of cultured RPE cells whatever its concentration or working time .6 With the concentration of 2.0-2.5 mg/m1,Bevacizumab inhibited the migrationt of RPE cells obviously in a dose—dependent manner compared with the control(P< 0.05). Conclusion:1 These experiments establish and maximize the culture of primary porcine RPE cells,and the cells maintain active proliferation,and positive to anti一keratin antigen by imxnunofluoreseence. These experiments get the similar results using the same methods with human RPE cells culture,but porcine RPE cells are easier to obtained and convenience to operate,and can be used to do some research work.2 The results of toxic effect of bevacizumab on cultured porcine RPE cells show that bevacizumab,with the concentration of 2.0mg/ml, demonstrate the impact on RPE cell proliferation.3 Only the higher concentration of bevacizumab inhibite the migrationt of RPE cells.Low-dose bevacuzumab proved relatively safe.
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