胰岛素和睾酮对Ishikawa细胞葡萄糖转运蛋白4表达的影响

The effects of insulin and testosterone on the expression of GLUT4 in Ishikawa cell

目的探讨胰岛素（INS）和睾酮（T）对多囊卵巢综合征（PCOS）子宫内膜腺上皮细胞生长的影响和葡萄糖转运蛋白4（GLUT4）表达的调节机制。方法体外培养Ishikawa细胞，予不同浓度INS（90、60、30、3、0．3U／L）或T（10^-2、10^-4、10^-5、10^-6、10^-7mmol／ml）刺激Ishikawa细胞48h，MTT法检测INS、T对Ishikawa细胞生长的作用；免疫细胞化学检测GLUT4蛋白在Ishikawa细胞定位表达；分别以30U／LINS和10^-5mmol／mlT刺激Ishikawa细胞24和48h，逆转录聚合酶链反应（RT-PCR）方法测定INS和T对Ishikawa细胞GLUT4 mRNA表达的影响。结果（1）不同浓度的INS均可促进Ishikawa细胞的生长，随着INs浓度的增加，INS促进Ishikawa细胞生长作用越强，INs浓度自0．3～30U／L时，Ishikawa细胞生长依次加强，与对照组相比均有显著性差异（P〈0．01）。INS浓度达60、90U／L时，细胞生长状况与INS浓度为30U／L相似。不同浓度的T均可抑制Ishikawa细胞的生长，随着T浓度的增加，T抑制Ishikawa细胞生长作用越明显。T浓度自10^-7、10^-6、10^-5mmol／ml，Ishikawa细胞生长依次减弱，与对照组相比均有显著性差异（P〈O．01，P〈0．05），T浓度达10^-4、10^-2mmol／ml时，细胞生长抑制状况与T浓度10^-5mg／ml相似。（2）GLUT4蛋白，定位表达于Ishikawa细胞的细胞浆内。（3）Ishikawa细胞中GLUT4 mRNA表达，在INS组和T组均较对照组减弱（P〈0．01，P〈0．05），INS组比T组减弱更明显（P〈0．05），且INS和T作用24和48h GLUT4 mRNA表达无显著性差异（P〉0．05）。结论不同浓度INS和T均可影响Ishikawa细胞生长，并降低GLUT4 mRNA的表达，推测PCOS高胰岛素、高雄激素血症的病理生理特性有可能影响子宫内膜的代谢过程，与子宫内膜的病变相关。

Objectives： To investigate the possible roles of insulin and testosterone in the growth of endometrial glandular epithelial cell of polycystic ovarian syndrome （PCOS） and explore their regulatory mechanism for the expression of glucose transporter 4 （GLUT4）. Methods： Cultured Ishikawa cells were stimulated with exogenous insulin at different concentrations （90,60,30,3,0.3 U/L） or testosterone （10^-3 ,10^-4 ,10^-5 , 10^-6 ,10^-7mmol/ml） for 48 h, and the growth of Ishikawa cells was detected by MTT and the expression of GLUT4 protein in Ishikawa cells detected by immunocytochemistry. The expression of GLUT4 mRNA in Ishikawa cells was detected by RT-PCR after the cultured cells were treated with insulin （30 U/L） or testosterone （10^-5mmol/ml） for 24 h and 48 h, respectively. Cultured Ishikawa cells without any treatment were as control. Results. （1） Insulin at the concentrations of 0.3 to 30 U/L can stimulate Ishikawa cells to grow in a dose-response manner, while the effect of insulin at the concentration of 60 or 90 IU/L was the same as that of 30 U/L. Testosterone can inhibit cell growth in a dose-response manner at the concentrations of 10^-7, 10^-6, 10^-5mmol/ml, with the effect at the concentration of over 10 5mmol/ml same as that of 10 mmol/ml. （2） The expression of GLUT4 in Ishikawa cell was proved by immunocytochemistry and located in intracytoplasm of Ishikawa cell. （3） RT-PCR method demonstrated the decreased GLUT4 mRNA expression in Ishikawa cell after exposure to insulin 30 U/L or testerone 10 5mmol/ml for 24 hours and 48 hours （P〈0.01 or P〈0.05）. The expression of GLUT4 mRNA in insulin group was significantly weaker than that of testosterone group （P〈0.05）. There was no significant difference in the expression of GLUT4 mRNA in Ishikawa cell between 24 hours and 48 hours of treatment with insulin or testosterone （P〉0.05）. Conclusions： Insulin and testosterone can affect the growth of Ishikawa cells and decrease the expression of GLUT4 mRNA in Ishikawa cells. It is supposed that hyperinsulinism and hyperandrogenism in PCOS maybe affect endometrial metabolism and relate to endometrial pathological changes.