Cell Culture and Treatment
ARPE-19 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in dulbecco’s modification of eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 UL/mL penicillin, and streptomycin in a 37°C, 5% CO2 incubator. The cells were sub-cultured every 2 d and treated with 0.25% trypsin. Because diabetes was characterized by hyperglycemia and hyperlipidemia, high glucose (HG) was used to induce DR cell model in ARPE-19 cells. Cells were grouped into four groups namely control, shRNA against SIRT1 (sh-SIRT1), CMS, and CMS + sh-SIRT1. Cells were inoculated to a 6 cm medium at a density of 2 × 105 and put into an incubator of 5% CO2 at 37°C until cells grew to 70% - 80%. On the day before transfection, cells were trypsinized and then dissociated into single cell suspension with culture medium. Then single cell suspension was inoculated to a 6-well plate at a density of 2 × 105 cells per well. After that, 10 μL of shRNA was mixed with Opti-MEM to a total volume of 250 μL for 5 min at room temperature, which then incubated with an equal volume of liposome for 20 min at room temperature to form a shRNA-liposome complex. Next, the complex was added into the 6-well plate at a density of 0.5 nL per well and mixed gently. Then each well was added with 1.5 mL serum-free DMEM medium and cultured at 37°C in 5% CO2 for 6 h. After that, medium was renewed with a normal serum-containing DMEM medium and continued to culture with 5% CO2 at 37°C for a proper period of time .