| Aim(1) We established vitro model of lipid accumulation by exposed oleic acid in Hep G2 cells to simulate fat accumulation state of nonalcoholic fatty liver disease(NAFLD), and observed the effects of ethyl hexyl ester phthalate(DEHP) on the model and investigated the potential molecular mechanism.(2) We established a rapid, convenient, high sensitivity of method to detect DEHP in aqueous solution by using surface enhanced Raman scattering(SERS) technology.MethodWe established vitro model of lipid accumulation by exposed 0.5m M oleic acid in Hep G2 cells, and evaluated the cell model by Oil red O staining and ADRP immunohistochemistry method; the activity of hepatic SOD and the levels of hepatic MDA and TG were measured according to commercial analysis kits. Western blot detected the expression of intracellular peroxisome proliferator-activated receptor γ(PPAR-γ).We synthesized nanosize silver particles of uniform size by using the method of microwave assisted, prepared silver basement membrane by spin coating method, and detected extremely low concentration of DEHP in aqueous solution by surface enhanced Raman spectroscopy.Results 1. The effects of DEHP on the Hep G2 cells induced by oleic acid 1.1 The establishment of cell modelThe Oil red O staining results showed: the cell edge was clear in control group, rare coloring were observed in the cell; there were many red lipid droplets around the inner regions of cell membrane in model group, make whole cell "signet ring shaped. Above results suggested that hepatocyte model of lipid accumulation was successfully established. 1.2 The effects of oleic acid on the expression of ADRP in the Hep G2 cellsThe immunohistochemistry results showed that compared with control group, the expression of ADRP significantly increased in model group, suggested that oleic acid induced the increase of ADRP expression in the Hep G2 cells. It further supported that the model of lipid accumulation was successfully established. 1.3 The effects of DEHP on the lipid droplets in model groupThe Oil red O staining results showed: compared with model group, lipid droplets in cells increased significantly after DEHP treatment; the TG content detection showed: compared with control group, the content of TG significantly increased in model group, and further increased after DEHP treatment. The results have statistical significance. 1.4 The effects of DEHP on the activity of hepatic SOD and the levels of hepatic MDACompared with control group, the activity of hepatic SOD decreased significantly in model group, the results have statistical significance; compared with model group, the activity of hepatic SOD decreased significantly in DEHP(5m M) group, the results have statistical significance. The results suggested that DEHP decreased the SOD enzyme activity in the Hep G2 cells induced by oleic acid and reduced the cellular oxidation resistance.Compared with control group, the level of hepatic MDA increased significantly in model group, the results have statistical significance; compared with model group, the level of hepatic MDA increased significantly in DEHP(5m M) group, the results have statistical significance. The results suggested that DEHP aggravated the level of lipid peroxidation in the Hep G2 cells. 2. The establishment of methods to detect DEHP in aqueous solution by SERS 2.1 The Characterization of silver nanoparticlesWe observed silver nanoparticles prepared through sodium citrate hydrothermal reduction method, microwave method and improved microwave amino acid reduction method with the transmission electron microscope. The results showed that silver nanoparticles prepared through former two methods had not uniform particle size, however, silver particles obtained from improved microwave amino acid reduction method not only had good morphology, uniform particle size and substantial production, which can meet the needs of the follow-up study. 2.2 The detection results of DEHP by SERSDEHP had obvious characteristics peaks of Raman in 1729, 1600, 1578, 1439, 1393, 1293, 1161, 1121, 1080, 1038, 956, 891, 842, 648, 396 cm-1. The surface enhanced spectroscopy and Raman spectroscopy of DEHP silver basement membrane in the main wave number position is basically similar. The basement membrane assembled by microwave assisted silver nanoparticles significantly enhanced Raman spectra of DEHP, and the detection limitation of DEHP content can reach 10-9 mol/L.Conclusion 1. DEHP aggravated lipid accumulation in the model which exposed oleic acid in Hep G2 cells. Its mechanism may be related that DEHP increased oxidative stress and lipid peroxidation and the expression of PPAR-γ. 2. Nano silver basement membrane which prepared with microwave assisted by spin coating detected low concentration of DEHP, the detection limit can reach 10-9 mol/L. The method is of high sensitivity, quick and has wide market application prospect. |
