硒代蛋氨酸对肝细胞L02中硒蛋白表达的影响及丝氨酸的协同作用

目的观察硒代蛋氨酸对肝细胞L02硒蛋白表达的影响及丝氨酸的协同作用。方法培养L02细胞,分为硒代蛋氨酸组和丝氨酸+硒代蛋氨酸组。硒代蛋氨酸剂量设为0.001、0.01、0.1、1和10μmol/L,丝氨酸和硒代蛋氨酸按照2∶1摩尔比混合。作用于L02细胞48 h后,采用酶联免疫吸附试验(enzyme-linked immuno-sorbent assay,ELISA)双抗体夹心法检测细胞培养上清液中硒蛋白P和裂解液中谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPx1)浓度,采用免疫印迹法检测裂解液中硒蛋白P和GPx1表达水平。结果硒代蛋氨酸浓度为0.1和1μmol/L时,硒蛋白P和GPx1表达分别达到拐点,硒代蛋氨酸浓度为10μmol/L时,GPx1和硒蛋白P表达下降。硒代蛋氨酸剂量为0.001~10μmol/L时,丝氨酸对细胞内GPx1和硒蛋白P表达具有协同作用。结论硒代蛋氨酸作用于人正常肝细胞L02时,硒蛋白合成出现拐点,且丝氨酸对硒代蛋氨酸在肝细胞L02内表达GPx1和硒蛋白P具有协同作用。

Study of selenomethionine on the selenoproteins expression in hepatocyte L02 and the synergistic effect of serine

OBJECTIVE To observe the effect of selenomethionine(SeMet)on the selenoproteins expression in hepatocyte L02 and the synergistic effect of serine.METHODS The L02 cells were cultured and divided into SeMet group and Serine+SeMet group.SeMet dose was set as 0.001,0.01,0.1,1 and 10μmol/L.Serine and SeMet were mixed according to 2∶1 molar ratio(Serine∶SeMet=2∶1).The L02 cells were cultured for 48 h after SeMet and Serine added.Finally,the cell culture supernatants and homogenates were collected for the selenoprotein P(SEPP)and glutathione peroxidase 1(GPx1)concentrations detection by a double-antibody sandwich enzyme-linked immuno-sorbent assay(ELISA).The expression of SEPP and GPx1 in cell homogenates was detected by western blot(WB).RESULTS ELISA and WB results showed that GPx1 and SEPP expressions were dose dependent in a low SeMet concentration range,reached their inflection points when SeMet concentration was 1μmol/L and 0.1μmol/L respectively,and began to decrease when SeMet concentration was further increased to 10μmol/L.WB result showed that serine had a synergistic effect on GPx1 and SEPP expressions as SeMet concentration was 0.001μmol/L to 10μmol/L.CONCLUSION There was an inflection point in selenium protein synthesis when SeMet acted on normal hepatocyte(L02),the specific mechanism needs to be further explored.Secondly,serine had a synergistic effect on GPx1 and SEPP expressions of SeMet in hepatocyte(L02).