孕激素对子宫内膜癌Ishikawa细胞中claudin-4基因表达的调节作用  

Regulation of claudin-4 gene expression in endometrial adenocarcinoma Ishikawa cell line by progesterone

作　　者：金妍[1] 冯翠平[1] 王雅楠[1] 林华[1] 李华军[1] 潘晓玉[1] 

机构地区：[1]中日友好医院妇产科,北京100029

出　　处：《中华妇产科杂志》2012年第5期368-372,共5页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金（30901599）

摘　　要：目的探讨孕激素对子宫内膜癌细胞系Ishikawa细胞中claudin4基因表达的调节作用。方法用浓度分别为2．5、5、10、15、20mg／L的甲地孕酮培养Ishikawa细胞24、48、72h，四甲基偶氮唑蓝（MTT）比色法检测Ishikawa细胞生长的抑制率，选出抑制率最接近50％时甲地孕酮的最佳处理浓度和时间，用于以下实验。以15mg／L（最佳处理浓度）的甲地孕酮处理Ishikawa细胞72h（最佳处理时间），透射电镜观察细胞形态的变化，流式细胞仪检测细胞凋亡率，实时定量PCR技术测定细胞中claudin-4mRNA的表达水平，蛋白印迹法检测细胞中claudin-4蛋白的表达水平，细胞免疫荧光染色观察细胞中claudin4蛋白表达部位的变化。结果15mg／L的甲地孕酮处理72h时，Ishikawa细胞生长的抑制率达50％。甲地孕酮处理后，透射电镜下观察，部分Ishikawa细胞明显肿胀，内质网扩张，线粒体数目减少、肿胀呈空泡化，核分裂象少见，严重者见胞质内细胞器消失，膜结构消失；少数Ishikawa细胞表面形成伪足样突起，细胞核溶解，凋亡小体形成。甲地孕酮处理前、后，Ishikawa细胞凋亡率分别为（0．076±0．024）％、（3．934±0．816）％，两者比较，差异有统计学意义（P〈0．05）；Ishikawa细胞中claudin-4mRNA的表达水平分别为0．64±0．20和0．47±0．15，claudin4蛋白的表达水平分别为0．94±0．18和0．62±0．15，甲地孕酮处理前、后claudin-4mRNA和蛋白质的表达水平分别比较，差异均有统计学意义（P〈0．05）；Ishikawa细胞免疫荧光染色，甲地孕酮处理后，claudin-4蛋白的表达部位由细胞膜进入细胞质和细胞核。结论孕激素可以抑制Ishikawa细胞的生长，其作用机制可能与claudin-4基因表达的降低及细胞凋亡有关。Ishikawa细胞膜表达的claudin-4蛋白整合人细胞内可能与孕激素抑制Ishikawa细胞的生长有关。Objective To explore the adenocarcinoma cell lines by progesterone. Methods regulation of claudin-4 expression in endometrial Ishikawa cells were treated with various concentrations of megestrol acetate （MA：2, 5, 10, 15, 20 mg/L）. After cultured for 24, 48 and 72 hours, cells growth were measured by methyl thiazolyl tetrazolium （MTT）. The group of Ishikawa cells incubated with MA at the 50% inhibitory concentration （ IC50 ） was selected for cell apoptosis assay by using transmission electron microscopy and flow cytometry method. Real-time PCR and western blot were used for detecting the mRNA and protein expression levels of elaudin-4. The localization of clandin-4 was examined by immunofluorescent staining. Results The inhibitory effects of megestrol acetate on the growth of Ishikawa cells were dose- dependent and time-dependent. IC50 of MA on Ishikawa ceils was 15 mg/L after incubated for 72 hours. After MA treatment, Ishikawa cells showed shrinkage, nuclear ehromatin condensation, fractures of nuclear membrane and endoplasmie retieulum expansion, even round apoptotic bodies were found. The apoptosis rate of cells before MA treatment was （0. 076 ±0. 024）% , and the rate was （3. 934 ±0. 816）% by MA treated for 72 hours, in which there were signieant difference （ P 〈 0. 05 ）. The relative quantification of elaudin-4 mRNA and protein of the cells before MA treatment were 0. 64± 0. 20 and 0. 94 ± 0. 18, while they were 0. 47 ±0. 15 and 0. 62 ±0. 15 after MA treated. The expression of claudin-4 was significantly decreased after MA treatment （ P 〈 0. 05 ）. The localization of elaudin-4 transferred from eytomembrane to cytoplasm and nucleus after MA treatment. Conclusions MA could inhibite the growth of Ishikawa cells, in which the mechanism may be decrease the expression of elaudin-4 and the apoptosis of cells. The distribution change of elaudin-4 may be related to the anti-cancer effect of progesterone.

关 键 词：子宫内膜肿瘤 孕激素类 细胞系 肿瘤 CLAUDINS 

分 类 号：R737.33[医药卫生—肿瘤]