子宫内膜癌细胞雌激素受体亚型的调控和功能的初步研究

目的初步建立子宫内膜癌雌激素受体（ER）亚型α或β弱表达的细胞模型,研究雌激素及他莫昔芬（TAM）与ER亚型的关系。方法分别设计针对ERα和ERβ的反义寡脱氧核苷酸（ODN）、正义ODN、错义ODN,并转染子宫内膜癌细胞株Ishikawa,将Ishikawa细胞分为4组,即未转染组、反义ODN组、正义ODN组及错义ODN组。蛋白印迹法测定转染后细胞ERα和ERβ蛋白的表达;四甲基偶氮唑蓝（MTT）法检测转染ODN后,17β雌二醇和TAM对Ishikawa细胞生长的影响。结果（1）分别针对ERα及ERβ的反义ODN可以选择性下调转染的Ishikawa细胞中ERα及ERβ蛋白的表达（分别下调45％和54％）。（2）17β雌二醇及TAM可以促进未转染组Ishikawa细胞的生长。转染ERα反义ODN后,可抑制17β雌二醇及TAM对Ishikawa细胞的促生长作用,以转染后的24、48及72h为著,反义ODN组各时间点分别与未转染、正义ODN、错义ODN组比较,差异均有统计学意义（P〈0．05）。（3）转染ERβ反义ODN后,17β雌二醇对Ishikawa细胞的促生长作用的改变不明显。转染ERβ反义ODN可抑制TAM对Ishikawa细胞的促生长作用,以转染后72h为著,反义ODN组分别与未转染、正义ODN、错义ODN组比较,差异有统计学意义（P〈0．05）。结论（1）反义核酸技术能够特异性地有效下调Ishikawa细胞中ERα和ERβ蛋白的表达,其可作为子宫内膜癌细胞中ER调控的一种有效的实验手段。（2）17β雌二醇促进子宫内膜癌细胞生长的作用主要通过ERβ介导;ERβ和ERβ均参与TAM促进子宫内膜癌细胞生长的作用。

Modulation and function of estrogen receptor isoforms alpha and beta in endometrial carcinoma cells

OBJECTIVE: To explore an efficient way to modulate the expression of estrogen receptor (ER) alpha and beta, and to build up a model of endometrial cancer cell expressing predominantly one isoform of ER and to verify the roles of ER alpha and beta in the tumorigenesis of endometrial cancer associated with estrogen and tamoxifen (TAM). METHODS: A series of oligodeoxyribonucleotides (ODN) against alpha or beta regions of ER alpha or beta were synthesized and tested in human endometrial cancer cell lines (Ishikawa) that express functional ER alpha and beta. The expressions of two ER isoforms were detected by western blot using specific antibodies. Then we studied the change of Ishikawa proliferation in response to 17beta-estradiol and TAM under the influence of antisense ODN. RESULTS: (1) Transfection with antisense ODN directed against the ERalpha and ERbeta could significantly inhibit target protein production. (2) 17beta-estradiol could increase the proliferation of Ishikawa cells, but they lost the ability to proliferate in response to 17beta-estradiol after transfected with ERalpha antisense ODN especially at hours 24, 48 and 72 (P < 0.05). There was no obvious change in cell numbers of Ishikawa in response to 17beta-estradiol which were transfected with ERbeta antisense ODN. (3) TAM could also increase the growth of Ishikawa cells. After transfected with ERalpha antisense ODN, the cells lost the ability to proliferate in response to TAM at hours 24, 48 and 72 (P < 0.05). The inhibition was also seen in Ishikawa cells transfected with ERbeta antisense ODN at hour 72. CONCLUSIONS: (1) Antisense ODN directed against the ERalpha or ERbeta can inhibit the expression of ERalpha or ERbeta effectively. (2) ERalpha may be the primary receptor in the proliferation of Ishikawa cells in response to 17beta-estradiol. Both ERalpha and ERbeta are involved in agonist impact of TAM on endometrial cancer cells.