Cytotoxicity Of Triamcinolone Acetonide On Human Retinal Pigment Epithelial Cells In Vitro

Objective: To study the influence of triamcinolone acetonide (TA) on HRPE cell growth, proliferation and cell cycle, and to observe the morphological and ultrastructural changes of HRPE cells induced by TA, which can provide some evidences on the safety of TA intravitreal injection.Methods: 1. Normal HRPE cells were cultured in vitro, and were identified by observing the morphous and cytochemical staining of keratin; 2. Different TA(0.4mg/ml, 0.2mg/ml, 0.1 mg/ml, 0.05 mg/ml, 0.025mg/ml) added to the HRPE cultures on day 0 and then subsequently on day 1,3,5 and 7. The amount of cell proliferations with or without TA treatment was determined using MTT assay. All samples were read in triplicate. And the changes of cell cycle treated by 0.3mg/ml TA were measured with Flow Cytometry (FCM) analysis on day 3; 3. Observe the morphological and ultrastructural changes by using inverted phase-contrast microscope and transmission electron microscope (TEM) respectively.Results: Normal HRPE growth curve was similar to an "S" type, which contained the adaptative phase in first 2 days, then entered into logarithmicgrowth phase, and finally into platform phase at about the 7th day. TA at a dosage ranging from 0.4 to 0.025mg/ml caused a significant reduction in HRPE cells proliferation that had been exposed to it for more than 1 day in a dose-dependent manner. FCM revealed that the cells treated with 0.3mg/ml TA compared to the control cells were increased 11.7% in Gi phase and decreased 10.6% in S phase (PO.05). Under light microscope, TA-treated cells were less than the control group, and their shapes were irregular with many prominences and a great deal of cellular vacuoles, and even some debris. Meanwhile, under the TEM cell nucleuses were still obviously, but chromatin was maldistributed and plasm was cavitated. Also we can find some necrosis cells with maldistributed chromatin, ruptured membrane and degenerated organelles. All these changes indicated the cytotoxicity of TA on HRPE.Conclusion: TA had an effective inhibition on HRPE cellular proliferation in a dose-dependent manner; TA inhibited the karyomitosis by decreasing cells in S phase, which eventually inhibited the proliferation. This study first compared the morphological and ultrastructural features between the TA-treated group and the control group. In the TA-treated group, the cell morphology altered, the cell organelles disappeared and even some necrosis cells could be observed. So 0.3mg/ml TA was actually cytotoxic to cultured HRPE cells in vitro.