Protective Effect Of Sodium Selenite On The Oxidative Damage To Yanbian Dairy Goat Mammary Gland Epithelial Cells Induced By Hydrogen Peroxide

Objective:In this experiment, Yanbian dairy goat mammary epithelial cells as experimental material, determine the most appropriate conditions on Yanbian dairy goat mammary epithelial cells in vitro culture. The Yanbian dairy goat mammary epithelial cells were stimulated by exogenous hydrogen peroxide under oxidative stress, and then adding a certain amount of sodium selenite to exert its protective effects on mammary epithelial cells and to determine the best concentration of sodium selenite on mammary epithelial cells.Method:(1) Respectively using tissue culture method and Type I collagenase/hyaluronic acid digestion method to establish Yanbian dairy goat mammary epithelial cells, and the cells were purified. Followed by detection of the purified Yanbian dairy goat mammary gland epithelial cell skeleton protein keratin expression;(2) Yanbian cultured dairy goat mammary epithelial cells after passaging experiments, hydrogen peroxide with DMEM/F12culture medium were diluted to5,50,100,200,500uM. The DMEM/F12medium was as a control group. By hydrogen peroxide treatment after4h, MTT method was used to study the effect of hydrogen peroxide on activity of mammary epithelial cells, the staining method of Hoechst33342/PI was detected the apoptosis of mammary epithelial cells, and the method of DCFH-DA was detected the ROS level;(3) The Yanbian dairy goat mammary epithelial cells were divided into three groups:control group (group1), hydrogen peroxide injury group (group2), sodium selenite protection group. The method of MTT for detecting cell viability, Hoechst33342/PI staining and the method of Giemsa staining were observed the apoptotic cells, and the method of DHE was to detect the ROS level.Result:(1) Using the method of tissue culture, the result showed that5to8days began to appear a small amount of mammary epithelial cells were polygonal or ovoid. Using the Type I collagenase/hyaluronic acid digestion after3h, the cells were collected almost the epithelial cells. By immunofluorescence staining showed that the mammary epithelial cells were presented positive expression;(2) MTT activity assay showed that there were significant differences between the treated group and the control group (P<0.05). After the treatment of5,50.100μM H2O2for4h, the cell viability were:91.21%,77.50%,54.48%, and200.500μM H2O2only were35.53%,27.79%. Hoechst33342/PI staining results showed that mammary epithelial cells inducing by H2O2can reduce the cell viability. The staining of DCFH-DA showed that the intracellular ROS levels were significantly increased after H2O2treatment;(3) Compared with the control group2, the concentration of sodium selenite with0.25,0.5,1.0μg/mL can enhance the mammary epithelial cell viability, reducing the number of apoptotic cells and the ROS levels. With0.5μg/mL sodium selenite treatment group is the most significant. These were showed that0.5μg/mL of sodium selenite can reduce oxidative damage induced by hydrogen peroxide in Yanbian dairy goat mammary gland epithelial cells, increase cell viability, reduce the apoptosis and the ROS level.Conclusion:(1) In the primary culture using the method of Type I collagenase/hyaluronic acid, the resulted that the the epithelial cells had a larger proporation. After2-3purification, we can get the purified mammary epithelial cells;(2) With keratin monoclonal antibodies on cultured cells, the results were positive, and this were proved the characteristics of the acinar cells;(3) The addition of H2O2resulted in decreased cell viability, cell apoptosis or necrosis, and rinsing of intracellular ROS levels. The results indicated that H2O2as a matter of mammary epithelial cells induced oxidative damage model can simulate the in vivo environment;(4) After treatment with100μmol/L H2O2role for4h, resulting in about50%cell death, necrosis of small proportions, ROS levels are relatively low.On this concentration, the cell also has the possibility to restore its function. So in order to establish a model of oxidative damage,we selected the concentration of100μmol/L H2O2on Yanbian dairy goat mammary epithelial cells were treated for4h;(5)0.5u g/mL SS can enhance the cells ability to resist oxidative stress, thereby reducing the degree of oxidative damage to cells and increase cell viability;(6) The results of Hoechst33342/PI staining and Giemsa staining showed that the addition of SS can reduce the rate of apoptosis and necrosis, so that SS plays a protective role of corresponding to DGMECs;(7)0.5μg/mL SS can significantly reduce the intracellular levels of ROS.