CAJ | 학술논문

本文旨在研究17β-雌二醇(17β-estradiol,E2)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E2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、1×10-8mol/L牛血清白蛋白(bovine serum albumin,BSA)对照组、1×10-8mol/L E2组、1×10-8mol/L E2-BSA组、1×10-8mol/L E2+无钙液组、1×10-8mol/L E2+5μg/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[Ca2+]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1×10-8mol/L E2作用前和作用后5 min、15 min、30min细胞内磷酸化磷脂酶C(phospholipase C,PLC)的变化.基质细胞[Ca2+]I的变化结果显示,在E2,E2-BSA和E2+无钙液组中[Ca2+];均明显升高,15 min达到高峰,随后下降回到基础值.但DMSO和BSA组中[Ca2+]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E2引起的[Ca2+]I升高.免疫荧光结果显示,加入1×10-8mol/L E2后,PLC的磷酸化水平升高,15 min达高峰(P<0.001),然后逐渐下降回到基础值.结果提示,E2对延迟着床小鼠子宫内膜基质细胞[Ca2+]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.
본문지재연구17β-자이순(17β-estradiol,E2)대연지착상기소서자궁내막기질세포내개진탕적영향화궤제,탐토E2대착상기자궁내막기질세포시부존재비기인조쾌속작용.실험적제일부분,소서분위6조,매조4지,즉0.1%이갑기아풍(dimethylsulfoxide,DMSO)대조조、1×10-8mol/L우혈청백단백(bovine serum albumin,BSA)대조조、1×10-8mol/L E2조、1×10-8mol/L E2-BSA조、1×10-8mol/L E2+무개액조、1×10-8mol/L E2+5μg/mL타막서분(tamoxifen)조.급성분리연지착상소서잉제7천자궁내막기질세포,응용격광공취초현미경기술실시검측각조기질세포[Ca2+]I적변화.실험적제이부분,분리7지연지착상소서잉제7천자궁내막기질세포,용면역형광법검측1×10-8mol/L E2작용전화작용후5 min、15 min、30min세포내린산화린지매C(phospholipase C,PLC)적변화.기질세포[Ca2+]I적변화결과현시,재E2,E2-BSA화E2+무개액조중[Ca2+];균명현승고,15 min체도고봉,수후하강회도기출치.단DMSO화BSA조중[Ca2+]I미견명현변화;가입전통자격소포장수체조단제tamoxifen불능억제E2인기적[Ca2+]I승고.면역형광결과현시,가입1×10-8mol/L E2후,PLC적린산화수평승고,15 min체고봉(P<0.001),연후축점하강회도기출치.결과제시,E2대연지착상소서자궁내막기질세포[Ca2+]I적변화구유쾌속조절효응,해작용가능시통과비기인조도경실현적,여린산화PLC신호도경유관.
To investigate the existence of nongenomic action of 17β-estradiol (E2) in the delayed implantation mouse endometrialstromal ceils, the changes in intraceilular calcium concentration ([Ca2+]I) and the upstream of calcium signal in vitro were detected. Theexperiment was composed of two parts. Firstly, the change in [Ca2+]I in endometrial stromal cells induced by E2 under differentconditions was detected. The mice were divided into 6 groups as follows: 0.1% dimethylsulfoxide (DMSO) control group, 1×10-8 mol/Lbovine serum albumin (BSA) control group, 1×10-8 mol/L E2 group, 1×10-8 mol/L E2 conjugated with BSA (E2-BSA) group, 1×10-8 mol/LE2 + calcium-free medium group, 1×10-8 mol/L E2 + 5μg/mL tamoxifen group, with 4 mice in each group. The endometrial tissue wasobtained from delayed implantation mice at pregnant day 7, and digested by incubation of tissue minces in Hank's balanced salts(HBSS, pH 7.2), which contained glucose (1 g/L), and eollagenase I (0.125%), for 1 h at 37 ℃. The stromal cells were preloaded with2.5 mmol/L Fluo-3/AM, a fluorescent probe of calcium, for 30 rain. A confocal laser scanning microscope, which fixed the wave lengthof excitation and emission at 488 nm and 526 nm, respectively, was used to detect the change in [Ca2+]I Secondly, the mechanism of E2effects in endometrial stromal cells was investigated. Immunofluorescent method was used to detect the change in phosphorylation ofphospholipase C (PLC) before and after the stromal cells were treated with E2 for 5 rain, 15 rain, and 30 rain. Seven delayedimplantation mice were used. The results were as follows. [Ca2+]I increased immediately and reached the maximum at 15 rain after the stromal cells were treated with 1×10-8 mol/L E2 and returned to the normal level at 30 min. In E2-BSA group and E2 + calcium-free medium group the same results were obtained as that in E2 group, but there was no increase of [Ca2+]I in DMSO and BSA groups. Tamoxifen, a traditional antagonist of estrogen receptor, did not inhibit the increase in [Ca2+]I induced by E2. Immunofluorescent results showed that the change in phosphorylated-PLC level had the same trend as [Ca2+]I after the cells were treated with E2. 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 [Ca2+]I induced by E2 in the endometrial stromal cells in delayed implantation mice is possibly carded out through a nongenomic pathway, and the transmembrane signal transduction is related to the phosphorylation of PLC in this process.