Animals Healthy male SPF SD rats,weighing 200±20 g(n=12),were obtained from the Animal Laboratory Center,Southern Medical University[license No.SCXK (Guangdong,China) 2016-0041].All experimental animals were fed and followed-up in the animal room of the Medical Research Center of the Third Affiliated Hospital of Southern Medical University. The experimental protocols were approved by the Animal Ethics Committee of Southern Medical University.
Diabetes Induction The rats were randomly divided into a non-diabetes mellitus(NDM) group and diabetes mellitus(DM) group. After adaptive feeding for 3 days, the DM group(n=6) was injected intraperitoneally with 10% streptozotocin(STZ) at a dose of 60mg/kg body weight,STZ was dissolved in a citric acid-sodium citrate buffer solution of 0.1mmol/L and pH of 4.4, which was currently used, protected from light and placed on ice. The NDM group(n=6) received the same dose of citric acid-sodium citrate buffer. As DM model rats,animals with blood sugar ≥ 16.7 mmol/L were used for tail venous blood tests. All animals were euthanized at 3 months by intraperitoneal injection of pentobarbital sodium, and their blood samples and retinas were harvested for protein preparation, and the eye balls were removed for paraffin sections.
Major Reagents STZ, citric acid, and sodium citrate (Sigma-Aldrich, St. Louis, MO, USA), anti-VEGF mouse monoclonal antibody (Abcam, Cambridge, MA, USA), anti-PLK4 rabbit polyclonal antibody (Proteintech, Rosemount, IL, USA), anti-PS6 rabbit monoclonal antibody (Ser235/236; Cell Signaling Technology, Danvers, MA, USA), anti-S6 rabbit polyclonal antibody (Cell Signaling Technology), anti β-tubulin mouse monoclonal antibody (Beijing Kangwei Century Biotechnology, Beijing, China), anti-mouse secondary antibody and anti-rabbit secondary antibody (Beijing Ruikang Biotechnology, Beijing, China), Endothelial Cell Medium (ECM; Sciencell, Carlsbad, CA, USA), and rapamycin (APExBIO; Boston, MA, USA) were used in this study.
Cell Culture HRCECs were from Guangzhou Jennio Biotechnology (Guangzhou, China) and were cultured in media supplemented with 1% endothelial cell growth supplement1% penicillin/streptomycin solution, and 5% fetal bovine serum (FBS) at 37°C, and were incubated in a humidified incubator with 5% CO2. When HRCECs reached 80%–90% confluence, they were digested with 0.25% trypsin without EDTA and passed at a ratio of 1:2. The concentration of glucose in the ECM was 1 g/L (5.5 mmol/L), which was similar to the normal blood glucose concentration of the human body. HRCECs were cultured in the same normal glucose concentration as the control group (NG group). D-(+)- glucose was added to the ECM , and the final concentration of glucose was adjusted to 4.5 g/L (25 mmol/L) to simulate the diabetic microenvironment of the human body, which was used to develop a HRCEC HG model as the HG group[10]. The final concentration of rapamycin (inhibitor of the mTORC1 pathway) in HG-ECM used to treat HRCECs was 50 nM (HG + rapamycin). Cells treated under different conditions were placed in a constant temperature incubator for culturing, and the medium was changed every day for subsequent protein extractions.
Transfection The siTSC1 (sense, 5'-CCAAAUCUCAGCCCGCUUUTT-3' and antisense, 5'-AAAGCGGGCUGAGAUUUGGTT-3') were purchased from Sangon Biotech (Shanghai, China). They were separately transfected into HRCECs using Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Transwell Assay After HRCECs were treated with different interventions for 48 h, 1 × 105 cells/mL were diluted with serum-free ECM and transferred into the upper chamber of a Transwell insert (Corning, Corning, NY, USA). ECM containing 5% FBS was added to the lower chamber. After 24 h of incubation in a CO2 incubator at 37°C, the non-migrating cells were gently removed from the upper chamber. Cells that had migrated through the membrane were fixed with 4% paraformaldehyde for 20 min and stained with a 0.5% Crystal Violet solution for 10 min. The migrated cells were imaged using an inverted optical microscope, and five fields of view were randomly selected to count cell numbers.
The 5-ethynyl-2´-deoxyuridine (EdU) Assay HRCECs were inoculated into 15 mm glass-bottomed dishes. After becoming adherent and reaching 60%–70% confluency after 6 h, they were separately treated with different interventions. After 48 h of treatment, the cell proliferative capacity was assayed with a kFluor488-EdU assay kit (KeyGen, Nanjing, China) according to the manufacturer’s instructions. Samples were incubated with 50 µM EdU working solution for 2 h. The cells were then imaged using a fluorescence microscope. Five fields of view were randomly selected to calculate the positive rate.
Statistical Method SPSS statistical software for Windows, version 19.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. The experimental data are expressed as the average number ± standard deviation (x ± s). Each test sample was provided with two secondary wells. The t-test was used for comparison between two groups. The difference was statistically significant with a value of p < 0.05.