Protective Effects Of Vitamin C On The Cultured Primary Mouse Type Ⅱ Alveolar Epithelial Cells From Oxidative Injury In Vitro

Objective:This experiment use ROS as the stimulating factor,selected primary mouse typeⅡ alveolar epithelial cells in vitro for the study,established the separation methods and constructed its oxidative injury model,and research the protective effect of vitamin C on the culture AT-Ⅱ from oxidative injury in vitro.Provide an experimental basis for the study of lung disease and to provide experimental evidence for the establishment of vitamin C in respiratory disease drug development.Materials and methods:1.The isolate,culture and identification ways of the primary mouse type Ⅱ alveolar epithelial cells.2.Establish primary mouse type Ⅱ alveolar epithelial cells of oxidative injury model:AT-Ⅱcells were treated with different concentrations of H₂O₂（0.5mmol/L,1mmol/L,1.5mmol/L）and assayed at the different time points（6h,12h,24h）by CCK-8,at the same time,combine morphological observation with Hoechst staining to ensure the best time points and concentrations.Finally,confirmed the oxidative injury model production conditions of primary mouse AT-Ⅱ cells.3.Study the protective effect of vitamin C on primary mouse AT-Ⅱ cells following oxidative injury by H₂O₂ in virto.Experiment divided into normal group（epithelial cells in serum-free medium group）,H₂O₂ injury group（1mmol/L,6h）,each dose of vitamin C protection group（25μmol/L,50μmol/L,75μmol/L）.Cell viability was evaluated by CCK-8 and the level of MDA and SOD was measured with antioxidation testing kit.Results:Through this experiment,the optimum condition was determined,that the manufacture of oxidative injury model in primary mouse AT-Ⅱ cells.The best condition was that the concentration of H₂O₂ was 1mmol/L and treating time was 6h.2.Normal group and each doses of vitamin C protection group,protein content,SOD activity was higher than H₂O₂ injury model group（P<0.05）;MDA content in injury group than the normal group and each dose of vitamin C protection group（P<0.05）.Conclusion:1.Successfully creative the way to separate the primary mouse AT-Ⅱ cells;2.The injury model were made successfully in primary mouse AT-Ⅱ cells;3.Vitamin C could reduce the oxidative damage induced by H₂O₂,but not with its concentration dose correlation.