Rapamycin On Apoptotic Gene Expression In Human Lens Epithelial Cell And Cell Cycle In Rats

【Objective】To explore the effect of rapamycin on in vitro cultured human lens epithelial cell (LEC) apoptosis-related Bcl-2 and Bax gene protein expression andcell cycle to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.【Materials and Methods】1. The second and third passage cultured lens epithelial cells were collected for rapamycin inhibition test. MTT colorimetric assay: The cells were transferred into 96-well culture plates and divided into 6 groups, and each group was set to have four duplicate wells. The first two groups were set to be the negative control group containing only cells and culture medium rather than rapamycin, and the blank control group containing only culture medium rather than cells and rapamycin. The other four groups were added with the culture medium containing rapamycin of 20, 40, 60 and 80ng/mL. 24 h, 48 h and 72 h after action, OD values in wells were determined with MTT ELISA reader. 2. Flow cytometry (FCM): The first two control groups were set not to contain rapamycin, and the other four experimental groups were set to contain rapamycin of 20, 40, 60 and 80ng/mL. 48 h after action, the cell cycles of all groups were detected with flow cytometry. 3. Real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR): The first two control groups were set not to contain rapamycin, and the other four experimental groups were set to contain rapamycin of 20, 40, 60 and 80ng/mL. SYBR Green I staining was used to detect mRNA expression in cells Bcl-2 and Bax. 4. Western-Blot: The first two control groups wereset not to contain rapamycin, and the other four experimental groups were set to contain rapamycin of 20, 40, 60 and 80ng /mL. SDS-PAGE electrophoresis was used to detect Bcl-2 and Bax protein expression.【Results】1. MTT colorimetric assay: Rapamycin on inhibition of human lens epithelial cell proliferation was time-dependent and dose-dependent. 2. Flow cytometry for detection of cell cycles: Rapamycin can block the conversion of human lens epithelial cells from G1 phase to S phase to increase G1 phase cells and decrease S phase cells. 3. RFQ-PCR: Rapamycin can inhibit Bcl-2mRNA expression and enhance BaxmRNA expression, which showed it can induce human lens epithelial cell apoptosis. 4. Western-Blot: Rapamycin can inhibit Bcl-2 expression and enhance Bax expression, which was same as the results of RFQ-PCR.