雌、孕激素通过激活磷酸化信号传导通路调控卵巢上皮性癌细胞nm23-H1蛋白的表达  

Influence of estrogen and progestin on nm23-H1 expression in epithelial ovarian cancer cell lines via activation of phosphorylation signaling

作　　者：华克勤[1] 尧良清[1] 曹琦[1] 黄妍[1] 赵宇清[1] 丰有吉[1] 

机构地区：[1]复旦大学附属妇产科医院妇科,上海200011

出　　处：《中华妇产科杂志》2006年第11期756-761,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：上海市医学重点学科建设资助项目(05-Ⅲ016);上海市卫生局重点研究资助项目(2005Z.001)

摘　　要：目的探讨雌、孕激素激活磷酸化信号传导通路后,对卵巢上皮性癌(卵巢癌)细胞转移抑制基因nm23-H1蛋白表达的影响。方法采用不同浓度的17β雌二醇(雌激素组)及醋酸甲羟孕酮(孕激素组)分别作用于卵巢透明细胞癌细胞株ES-2细胞,以二甲基亚砜作为对照(对照组)。采用划痕实验观察各组细胞迁移能力的变化;采用穿膜小室(Transwell小室)体外侵袭实验观察各组细胞侵袭能力的变化;采用蛋白印迹法检测各组细胞nm23-H1蛋白表达及蛋白激酶B(AKT)蛋白、磷酸化蛋白激酶B(pAKT)蛋白表达的变化及其与时间、剂量的相关性;采用RNA干扰技术观察转染AKT小分子干扰RNA(siRNA)质粒后,各组细胞nm23-H1蛋白表达的变化及其侵袭能力的变化。结果划痕实验24h时,各组ES-2细胞均向划痕处弥合,划痕间距雌激素组为(1·39±0·08)mm,孕激素组为(1·96±0·07)mm,对照组为(1·65±0·12)mm;雌激素组明显小于对照组(P=0·029),而孕激素组明显大于对照组(P=0·014)。Transwell小室培养12h时,穿过微孔膜的细胞雌激素组为(119±13)个,孕激素组为(78±8)个,对照组为(92±16)个;雌激素组明显多于对照组(P=0·015),而孕激素组明显少于对照组(P=0·006)。蛋白印迹法检测结果显示,17β雌二醇降低ES-2细胞nm23-H1蛋白表达的作用与剂量和时间呈明显负相关关系(P=0·020,P=0·001),醋酸甲羟孕酮增加ES-2细胞nm23-H1蛋白表达的作用与剂量和时间呈明显正相关关系(P=0·003,P=0·002);17β雌二醇增加ES-2细胞pAKT蛋白表达的作用与剂量和时间呈明显正相关关系(P=0·001,P=0·007),醋酸甲羟孕酮降低ES-2细胞pAKT蛋白表达的作用与剂量和时间呈明显负相关关系(P=0·012,P=0·039);而17β雌二醇和醋酸甲羟孕酮对ES-2细胞AKT蛋白的表达均无明显影响(P>0·05)。AKTsiRNA干扰后,减弱了17β雌二醇对ES-2细胞nm23-H1蛋白表达的降调节作用(P=0·076)和促进ES-2细胞穿过Transwell小室的能Objective To assess the estrogen and progestin＇s effect on protein expression of metastasis repression gene nm23-H1 via regulation of phosphorylation signaling in epithelial ovarian cancer cell line ES-2. Methods Ovarian clear cell adenocarcinom cell line ES-2 was treated by different doses of 17β-estradiol（estrogen）, medroxyprogestogen（progestin） and dimethyl sulfoxide （ control group）, and then the following experiments were conducted. （ 1 ） Change of the cell migration capacity after treatment with estrogen and progestin for 24 and 48 hours was measured by in vitro wound healing assay. （2） Transwell experiments were used to detect the ability of cell invasion, which was also used to inhibit the phosphorylation of protein kinase B （AKT） pathway with estrogen and progestin. （ 3 ） Change of nm23-H1, AKT and phosphorylated protein kinase B （pAKT） protein level of ES-2 cells after treated with estrogen and progestin was detected by western blot. （4） Cells were transfeeted with the small interfering RNA（siRNA） expression vector targeting AKT. The efficiency of cells transfeeted was calculated according to the number of cells with green fluorescent produced by cells transfeeted. Change of nm23-H1 expression was assessed. Results ES-2 cells treated with estrogen, progestin or vehicle all migrated into the wound surface after 24 and 48 hours. The migration of the cells treated with estrogen was （ 1.39 ± 0. 08） ram, significantly elevated （ P = 0. 029）, and that of the cells treated with progestin was （ 1.96 ± 0. 07 ） ram, significantly decreased compared with cells treated with vehicle （P =0. 014）. The cells were cultured in transwell after 24 hours. The invasion of the cells treated with estrogen was 119 ± 13, significantly elevated（ P =0. 015） , and that of the cells treated with progestin was 78 ± 8, significantly decreased compared with cells treated with vehicle （P = 0. 006）. Western blot results showed that 17β-estradiol decreased nm23-H1 exp

关 键 词：卵巢肿瘤 腺癌 透明细胞 雌激素类 孕激素类 转录因子 蛋白激酶类 

分 类 号：R737.31[医药卫生—肿瘤]