摘要
目的观察米非司酮对人羊膜上皮细胞水通道蛋白8(AQP8)表达的影响,探讨药物调控羊膜上皮细胞AQP8表达的可行性。方法将体外培养的人羊膜上皮WISH细胞分为不同浓度米非司酮作用48h组和单一浓度米非司酮作用不同时间组,前者培养基中分别加入终浓度为0.1、1、5、10、15μmol/L的米非司酮,培养48h;后者培养基中加入终浓度为10μmol/L的米非司酮,分别培养6、12、24、36、48h,同时2组均设加入含0.01%二甲基亚砜(DMSO)完全培养液的对照组。采用免疫荧光细胞化学方法检测10μmol/L米非司酮作用48h后细胞AQP8蛋白的分布;RT-PCR和蛋白质印迹技术分别检测不同浓度米非司酮作用组和米非司酮作用不同时间组各组细胞AQP8mRNA和蛋白的表达水平。结果免疫荧光细胞化学检测显示,米非司酮作用后WISH细胞细胞质内及细胞膜上AQP8蛋白黄绿色荧光信号较对照组均明显减弱。在不同浓度米非司酮作用组中,除0.1μmol/L组外,其余各组细胞的AQP8 mRNA和蛋白表达水平分别与对照组相比均明显减弱(均P<0.01),而0.1、1、5、10μmol/L组细胞的AQP8 mRNA表达水平和1、5、10μmol/L组细胞的AQP8蛋白表达水平分别与15μmol/L组比较,差异均有统计学意义(均P<0.05)。在米非司酮作用不同时间组中,除6h组外,其余各组细胞的AQP8 mRNA和蛋白表达水平分别与对照组相比均明显减弱(均P<0.01),而6、12、24、36h组细胞的AQP8 mRNA和蛋白表达水平分别与48h组比较,差异均有统计学意义(均P<0.05)。结论米非司酮对人羊膜上皮细胞AQP8 mRNA和蛋白的表达具有抑制作用。
Objective To observe the effects of mifepristone on aquaporin8 expression in the human amniotic epithelial cells, so as to explore the feasibility of using drugs to control the expression of AQP8. Methods The human amniotic epithelial WISH cells cultured in vitro were divided into the different concentrations of mifepristone treating for 48 h group and a single concentration of rnifepristone treating for different hours group. The former culture mediums were added to a final concentrations of 0.1, 1, 5, 10, 15 μmol/L mifepristone respectively to culture for 48 h, and the latter culture mediums were added to a final concentration of 10 μmol/L mifepristone to culture for 6, 12, 24, 36, 48 h respectively, while two groups included the control group that was containing 0.01% dimethyl sulfoxide （DMSO） complete culture medium. Immunofluorescence was used to detect the distribution of AQP8 protein in WISH cells treated with 10 μmol/L mifepristone for 48 h. RT-PCR and Western blot were used to detect the AQP8 mRNA and protein expression levels in WISH cells of the different concentrations of mifepristone treating group and the different times of mifepristone treating group.Results Immunofluorescence showed that after treated by mifepristone, the yellow-green fluorescence signals of AQP8 protein within the cytoplasm and membrane of WISH cells were significantly reduced compared with the control group. In the different concentrations of mifepristone treating group, in addition to 0.1 μmol/L group, the expression levels of AQP8 mRNA and protein in WISH cells of the other groups compared with the control group were significantly decreased （all P 〈 0.01）, while the expression levels of AQP8 mRNA in WISH cells of the 0.1, 1, 5, 10 μmol/L groups and the expression levels of AQP8 in WISH cells of 1, 5, 10 μmol/L groups separately compared with 15 μmol/L group, the differences were statistically significant （all P 〈 0.05）. In the different times of mifepristone treating group, except for 6 h group, the expression levels of AQP8 mRNA and protein in WISH cells of the other groups compared with the control group were significantly decreased （all P 〈 0.01）, while the expression levels of AQP8 mRNA and protein in WISH ceils of the 6, 12, 24, 36 h groups separately compared with 48 h group, the differences were statistically significant （all P 〈 0.05）. Conclusion Mifepristone could decrease the expressions ofAQP8 mRNA and protein in the human amniotic epithelial cells.
出处
《中国医药生物技术》
CSCD
2009年第6期411-416,共6页
Chinese Medicinal Biotechnology
基金
国家自然科学基金(30400616
30672244)
重庆市人口计生委科研基金(2004-65)
