环磷酸腺苷对人羊膜上皮细胞水通道蛋白8表达及分布的影响  

Relationship between cyclic adenosine monophosphate and auqaporin 8 expression and distribution in human amnion-derived cells

作　　者：李莉[1,2] 漆洪波[1] 

机构地区：[1]重庆医科大学附属第一医院妇产科,400016 [2]重庆市妇幼保健院产科,400013

出　　处：《中华妇产科杂志》2008年第9期685-689,共5页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金（30400616）

摘　　要：目的探讨环磷酸腺苷（cAMP）对人羊膜上皮细胞水通道蛋白8（AQP8）表达及分布的影响。方法培养人羊膜上皮细胞WISH细胞株，随机分为实验组和对照组。对照组给予PRMI1640培养液常规培养。实验组将8-溴腺苷-3，5’环磷酸腺苷（8-Br—cAMP）加入培养液中，使其在细胞培养液中浓度分别为10、20、40、100、200、400μmol／L，不同浓度分别培养2、4、8、16、24和48h时取样。各浓度重复培养6瓶。采用蛋白印迹法定量检测WISH细胞AQP8的蛋白表达；采用RT—PCR技术半定量检测WISH细胞AQP8的mRNA表达。采用免疫荧光细胞化学法检测WISH细胞上AQP8的分布。结果对照组WISH细胞AQP8蛋白表达水平为0．0284-0．003，AQP8mRNA表达水平为0．0274-0．003。实验组WISH细胞随着培养液中8-Br—cAMP浓度的升高，AQP8蛋白表达水平明显升高。培养液中8-Br．cAMP浓度为20、40、100、200、400μmol／L时，其蛋白表达水平分别为0．062±0．007、0．063±0．006、0．125±0．008、0．145±0．010、0．1004-0．008，培养液中8-Br-cAMP浓度为200μmol／L，培养8h时，AQP8蛋白水平开始升高，24h时达到峰值，升高约5倍。随着8．Br—cAMP浓度的升高，AQP8mRNA水平明显升高，培养液中8-Br—cAMP浓度为20、dO、100、200、400μmol／L时，其mRNA表达水平分别为0．065-1-0．007、0．069±0．006、0．130±0．008、0．1504-0．010、0．1104-0．009，同样在200μmol／L时达到峰值；8-Br—cAMP浓度为200gmol／L，培养2h时AQP8mRNA水平开始升高，培养8h时达到峰值，16h时开始下降，24h时降到8-Br—cAMP培养前的水平。8-Br．cAMP浓度为200μmol／L培养24h时，细胞内的荧光减弱而细胞膜的荧光明显增强。结论cAMP可能是调控羊膜上皮细胞AQPS表达及分布的重要介质。Objective To study the effect of cyclic adenosine monophosphate on aquaporin 8 （AQP8） expression and distribution in human amnion-derived WISH cells. Methods Human amnion- derived WISH cells were cultured. The cells were divided into control group and study group at random. The study group was established by exposure to various concentrations of 8-Br-cAMP. Western analysis was used to quantify AQP8 expression levels. RT-PCR was used to quantify AQP8 mRNA expression levels. Immunofluorescence was used to determine the localization of AQP8 in WISH cells. Results With increase of cAMP, AQP8 mRNA and protein expressions significantly increased in WISH cells in vitro. When the concentration of cAMP reached 200 μmol/L, AQP8 mRNA and protein expressions were highest. Incubation with cAMP （200μmol/L） for 2 hours resulted in a 2-fold increase in AQP8 mRNA level, and incubation for 8 hours resulted in a peak. After incubation for 16 hours AQP8 mRNA level began to descend, and after 24 hours it decreased to baseline. Incubation with cAMP for 8 hours AQP8 protein level began to increase. Incubation for 24 hours resulted in a peak in AQP8 protein level, and after incubation for 48 hours it began to decline. By immunofluoreseence microscopy after incubation with cAMP （200 μmol/L） cells, AQP8 labeling in plasma membrane was enhanced and intracellular AQP8 labeling was decreased. Conclusion The cAMP triggers translocation of AQP8 from cytosol to the plasma membrane via vesicle-transporting related protein instead of AQP8 itself, cAMP may upregulate the transcription of target gene protein kinase A. The cAMP may be the critical regulatory medium of AQP8 in WISH cells.

关 键 词：羊膜 上皮细胞 水孔蛋白类 环AMP 

分 类 号：R686[医药卫生—骨科学]