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17#beta#--雌二醇对延迟着床期小鼠子宫内膜基质细胞内钙的快速调节效应

王强, 岳利民, 张金虎, 田吉梅, 何亚萍

四川大学华西基础医学与法医学院生理学教研室.四川,成都 610041

摘要

该文旨在研究17#beta#--雌二醇(17#beta#--estradiol,E_(2))对延迟着床期小鼠子宫内膜基质细胞内钙振荡的影响和机制,探讨E_(2)对着床期子宫内膜基质细胞是否存在非基因组快速作用。实验的第一部分,小鼠分为6组,每组4只,即0.1%二甲基亚砜(dimethylsulfoxide,DMSO)对照组、1×10~(-8)mol/L牛血清白蛋白(bovine serum albumin,BSA)对照组、1×10~(-8)mol/L E_(2)组、1×10~(-8)mol/L E_(2)--BSA组、1×10~(-8)mol/L E_(2)+无钙液组、1×10~(-8)mol/L E_(2)+5#mu#g/mL他莫西芬(tamoxifen)组。急性分离延迟着床小鼠孕第7天子宫内膜基质细胞,应用激光共聚焦显微镜技术实时检测各组基质细胞[Ca~(2+)]_(i)的变化。实验的第二部分,分离7只延迟着床小鼠孕第7天子宫内膜基质细胞,用免疫荧光法检测1×10~(-8)mol/L E_(2)作用前和作用后5min、15min、30min细胞内磷酸化磷脂酶C(phospholipase C,PLC)的变化。基质细胞[Ca~(2+)]_(i)的变化结果显示,在E_(2),E_(2)--BSA和E_(2)+无钙液组中[Ca~(2+)]_(i)均明显升高,15min达到高峰，随后下降回到基础值。但DMSO和BSA组中[Ca~(2+)]_(i)未见明显变化；加入传统雌激素胞浆受体阻断剂tamoxifen不能抑制E_(2)引起的[Ca~(2+)]_(i)升高。免疫荧光结果显示，加入1×10~(-8) mol/L E_(2)后，PLC的磷酸化水平升高，15 min达高峰({sl P}<0.001)，然后逐渐下降回到基础值。结果提示，E_(2)对延迟着床小鼠子宫内膜基质细胞内[Ca~(2+)]_(i)的变化具有快速调节效应，该作用可能是通过非基因组途径实现的，与磷酸化PLC信号途径有关。

关键词： 雌激素; 磷脂酶C; 钙; 基质细胞

Nongenomic action and mechanism of 17#beta#--estradiol in cytosolic calcium concentration in delayed implantation mouse endometrial stromal cells

Wang Qiang, Yue Limin, Zhang Jinhu, Tian Jimei, He Yaping

Department of Physiology,West China School of Preclinical and Forensic Medicine,Sichuan University.Chengdu 610041,Sichuan

Abstract

To investigate the existence of nongenomic action of 17#beta#-estradiol (E_(2)) in the delayed implantation mouse endometrial stromal cells, the change of intracellular calcium concentration ([Ca~(2+)]_(i)) and the upstream of calcium signal in vitro were detected. The experiment was composed of two parts. Firstly, the change of [Ca~(2+)]_(i) in endometrial stromal cells induced by E_(2) under different conditions was detected. The mice were divided into 6 groups as follows: 0.1% dimethylsulfoxide (DMSO) control group, 1×10~(-8) mol/L bovine serum albumin (BSA) control group, 1×10~(-8) mol/L E_(2) group, 1×10~(-8) mol/L E_(2) conjugated with BSA (E_(2)-BSA) group, 1×10~(-8) mol/L E_(2) + calcium-free medium group, 5 #mu#g/mL tamoxifen + 1×10~(-8) mol/L E_(2) group, with 4 mice in each group. The endometrial tissue was obtained from delayed implantation mice at pregnant day 7, and digested by incubation of tissue minces in Hank’s balanced salts (HBSS, pH7.2), which contained glucose (1 g/L), collagenase I (0.125%), for 1 h at 37 oC. The stromal cells were preloaded with 2.5 mmol/L Fluo-3/AM, a fluorescent probe of calcium for 30 min. A confocal laser scanning microscope, which fixed the wave length of excitation and emission light at 488 nm and 526 nm, respectively, was used to detect the change of [Ca~(2+)]_(i). Secondly, the mechanism of E_(2) effects in endometrial stromal cells was investigated. Immunofluorescent method was used to detect the change in phosphorylation of phospholipase C (PLC) after the stromal cells were treated with E_(2) for 5 min, 15 min, and 30 min. Seven delayed implantation mice were used. The results were as follows: the [Ca~(2+)]_(i) concentration increased immediately and reached the maximum at 15 min after the stromal cells were treated with 1×10~(-8) mol/L E_(2) and returned to the normal level at 30 min. In E_(2)-BSA group and E_(2) + calcium-free medium group the same results were obtained as that in E_(2) group, but no increase of [Ca~(2+)]_(i) in DMSO group and BSA group. Tamoxifen, an antagonist of traditional estrogen receptor, did not inhibit the increase of [Ca~(2+)]_(i) induced by E_(2). Immunofluorescent results showed that the change in phosphor-PLC level had the same time-dependent result of [Ca~(2+)]_(i) after the cells were treated with E_(2). Compared with that in the control group, the immunofluorescent intensity increased at the beginning and achieved the maximum at 15 min (P<0.001), then declined to the normal level at 30 min. These results suggest that the rapid response of [Ca~(2+)]_(i) induced by E_(2) in the endometrial stromal cells of delayed implantation mice is possibly carried out through a nongenomic pathway, and that the transmembrane signal transduction is related to the phosphorylation of PLC in this process.

Key words: Estrogen;Phospholipase C;Calcium;stromal cells

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引用本文：

王强, 岳利民, 张金虎, 田吉梅, 何亚萍. 17#beta#--雌二醇对延迟着床期小鼠子宫内膜基质细胞内钙的快速调节效应[J]. 生理学报 2008; 60 (2): 169-174.

Wang Qiang, Yue Limin, Zhang Jinhu, Tian Jimei, He Yaping. Nongenomic action and mechanism of 17#beta#--estradiol in cytosolic calcium concentration in delayed implantation mouse endometrial stromal cells. Acta Physiol Sin 2008; 60 (2): 169-174 (in Chinese with English abstract).