雌激素受体β在卵巢透明细胞癌细胞增殖中的作用

Effects of estrogen receptor β on proliferation of ovarian clear cell adenocarcinoma ES-2 in vitro and in vivo

目的了解导入外源性人类全长雌激素受体(ER)β cDNA 后,卵巢透明细胞癌细胞系Es-2生长情况和细胞周期的变化。方法将带有 ERβ cDNA 的质粒 pRSV-ERβ和对照空质粒 pRSV分别转染 ES-2细胞(分别命名为 ES-pRSV-ERβ、ES-pRSV 细胞),RT-PCR 和蛋白印迹法检测 ERβmRNA 和蛋白的表达情况,四甲基偶氮唑蓝法、流式细胞仪监测 ES-2、ES-pRSV、ES-pRSV-ERβ细胞在雌激素作用下的体外增殖能力、细胞周期的变化,并观察 ES-2、ES-pRSV、ES-pRSV-ERβ细胞接种于裸鼠皮下的成瘤和种植瘤的生长情况,分别将裸鼠命名为 ES-2、ES-pRSV 和 ES-pRSV-ERβ组。结果ES-pRSV-ERβ细胞稳定表达 ERβ mRNA 和蛋白,而 ES-pRSV 和未转染的 ES-2细胞均未检测出 ERβmRNA 和蛋白的表达条带。ES-pRSV-ERβ细胞在体外的增殖速度明显落后于未转染的 ES-2和 ES-pRSV 细胞,雌激素作用7d 后,未转染的 ES-2细胞的吸光度值为0.78±0.05,ES-pRSV 细胞为0.81±0.06,两者比较,差异无统计学意义(P>0.05);而 ES-pRSV-ERβ细胞的吸光度值为0.53±0.07,明显低于 ES-pRSV 和未转染的 ES-2细胞,差异有统计学意义(P<0.01)。裸鼠接种第24天,ES-pRSV-ERβ组的种植瘤平均体积为(2868±879)mm^3,低于 ES-2组的(3603±724)mm^3和 ES-pRSV 组的(3913±624)mm^3(P<0.05)。未转染的 ES-2、ES-pRSV、ES-pRSV-ERβ细胞 S 期细胞所占比例分别为(37±9)%、(39±10)%、(20±5)%,ES-pRSV-ERβ细胞分别与未转染的 ES-2、ES-pRSV 细胞比较,差异有统计学意义(P<0.05)。结论稳定表达 ERβ的 ES-2细胞体内、体外增殖能力明显减弱,ERβ以雌激素依赖方式使 ES-2细胞周期中 S 期细胞比例减少,提示 ERβ可能有肿瘤抑制作用。

Objective To investigate the involvement of estrogen receptor （ER） β in the proliferation of ovarian clear cell adenocarcinoma by restoring ERβ expression in a cell line ES-2. Methods A plasmid with full length ERβ cDNA, pRSV-ERβ and its negative vector control pRSV were introduced into ES-2. The cells transfected were named according to the plasmids： ES-pRSV, ES-pRSV-ERβ. RT-PCR and western blot were used to detect the expression of ERβ in ES-2, ES-pRSV and ES-pRSV-ERβ cells. The growth actiyities of cells in vitro were detected by methyl thiazolyl tetrazolium （MTT） assay, and in vivo growth in nude mice was also observed. Flow cytometry was performed to show the change of cell cycles. Results The ES-pRSV-ERβ cells were identified with ERβ mRNA and protein expression. The growth activities of ES-pRSV-ERβ were inhibited in vitro. In MTT analysis, the values of ES-2, ES-pRSV, and ES- pRSV-ERβ cells were 0. 78 ±0.05, 0. 81 ±0.06, and 0. 53 ±0. 07 （the third was lower compared with the former two, P 〈0.01 ）. In vivo, the volume of transplants of ES-pRSV-ERβ cells in mice, （2868 ± 879） mm^3 was smaller than that in ES-2, （3603 ± 724）mm^3, and in ES-pRSV, （3913 ± 624）mm^3（P 〈 0. 05 ）. The S phase ratios of the cell cycle of ES-2, ES-pRSV, and ES-pRSV-ERβ were （ 37 ＋ 9 ） % , （ 39 ±10）%, and （20 ±5）% （P 〈0.05）. Conclusions ERβ may play an important role in the proliferation, and DNA synthesis of ES-2. The evidence indicates ERβ may be an inhibitor in the initiation and development of ovarian clear cell adenocarcinoma.