苯甲酸雌二醇对离体子宫内膜腺上皮细胞中热休克蛋白70及90表达的影响

Effect of estradiol benzoate on the heat shock protein 70, 90 expressions in endometrial glandular epithelial cell

目的探讨苯甲酸雌二醇对离体子宫内膜腺上皮细胞中热休克蛋白(HSP)70、90表达的影响。方法采用酶消化法,行原代子宫内膜腺上皮细胞培养。将培养的子宫内膜腺上皮细胞分为对照组(加入正常培养液)、苯甲酸雌二醇组、苯甲酸雌二醇+雌激素拮抗剂ICI182780组和ICI182780组,各组细胞分别作用6、12、18和24h。四甲基偶氮唑蓝还原法测定不同浓度的苯甲酸雌二醇及ICI182780对离体子宫内膜腺上皮细胞生长的影响,免疫组化法及图像分析法测定苯甲酸雌二醇和ICI182780对腺上皮细胞中HSP70、90表达的影响。结果苯甲酸雌二醇可刺激腺上皮细胞生长,细胞增殖率随着作用时间的延长和药物浓度升高而升高,浓度为10-9、10-8、10-7、10-6mol/L的苯甲酸雌二醇作用24h时,平均细胞增殖率分别为(170±9)%、(207±11)%、(231±12)%、(257±10)%,均明显高于上述各浓度苯甲酸雌二醇作用6h时的细胞增殖率[(117±13)%、(129±10)%、(146±10)%、(176±6)%],差异有统计学意义(P<0.05);浓度为10-9、10-8、10-7、10-6mol/L的苯甲酸雌二醇作用24h时,苯甲酸雌二醇+ICI182780组与苯甲酸雌二醇组比较,细胞平均增殖率均明显降低,分别为(137±4)%、(145±10)%、(151±9)%、(167±3)%,差异有统计学意义(P<0.05)。苯甲酸雌二醇可刺激细胞中HSP90的表达,这种刺激作用同样存在药物浓度和作用时间依赖性,苯甲酸雌二醇浓度为10-9、10-8、10-7、10-6mol/L,作用24h时HSP90表达的相对不透光度分别为(64.8±10.7)%、(75.9±12.6)%、(80.4±8.2)%、(83.2±7.6)%,明显高于相同浓度、相同作用时间时对照组的(28.0±3.3)%、(29.0±5.6)%、(29.0±5.0)%和(30.0±6.4)%,差异也有统计学意义(P<0.05);浓度为10-9、10-8、10-7、10-6mol/L的苯甲酸雌二醇作用24h时,苯甲酸雌二醇+ICI182780组与苯甲酸雌二醇组比较,腺上皮细胞中HSP90表达明显降低,分别为(28.2±2.1)%、(29.7±3.2)%、(35.0±4.7)%、(34.7±6.5)%。ICI182780不影响雌二醇对腺上皮细胞中HSP70表达的抑制作用。结论苯甲酸雌二醇对子宫内膜腺上皮细胞增殖及HSP90表达的影响是雌激素受体依赖性的,HSP90表达可能有助于苯甲酸雌二醇刺激腺上皮细胞增殖;苯甲酸雌二醇对HSP70表达的抑制作用是非雌激素受体依赖性的,HSP70表达可能对腺上皮细胞的损伤有保护作用。

Objective To explore the effect of estradiol benzoate on the expressions of heat shock protein （HSP）70, 90 in endometrial glandular epithelial cell. Methods Normal endometrial glandular epithelial cells were isolated, and cultured by enzymolysis method and identified by electron microscopy and immunohistochemical analysis. The normal endometrial glandular epithelial cells were treated with culture medium only, estradiol benzoate （10^-9, 10^-8, 10^-7 or 10^-6 mol /L）, estradiol benzoate（ 10^-9, 10^-8, 10^-7 or 10^-6 mol/L） and antiestrogen ICI 182780（ fulvestrant,faslodex, 1 g, mol/L）and ICI 182780 only for 6, 12, 18, 24 hours respectively. The dose- and time-related effect of estradiol benzoate and ICI 182780 on the cell growth was measured by mononuclear cell direct cytotoxicity assay （methyl thiazolyl tetrazolium assay） , and that on the expression of HSP70 and HSP90 in normal endometrial glandular epithelial cell in vitro was measured by immunohistochemical analysis and computerized image analysis system. Results Estradiol benzoate stimulated cell growth in a time- and dose-dependent manner and the effect was attenuated by the antiestrogen ICI 182780. The average cell growth rates of 10^-9, 10^-8, 10^-7, 10^-6 mol/L estradiol benzoate for 24 hours were（170 ±9）%,（207 ± 11）%,（231 ±12）%,（257 ± 10）%, which were significantly higher than those of 6 hours [ （ 117 ± 13 ） %, （ 129 ± 10） %, （ 146 ± 10） %, （ 176 ± 6） %, P〈0.05] and that those estradiol ± ICI 182780 for 24 hours [（137 ±4）%, （145 ±10）%, （151 ± 9）%, （167 ± 3）%, P 〈 0. 05 ]. Estradiol benzoate treatment resulted in a time- and dose-dependent increase of the expression of HSP90 and decrease of HSP70 in human endometrial glandular epithelial cell. The expression of HSP90 in 10 -9, 10^-s, 10 -7, 10^-6 mol/L estradiol benzoate for 24 hours were（64. 8 ± 10.7 ） %, （75.9 ± 12.6 ） %, （ 80. 4 ±8. 2 ） %, （ 83.2 ± 7.6） %, which were significantly higher than that of the control [（28.0±3.3）%, （29.0±5.6）%, （29.0 ±5.0）%, （30.0 ±6.4）%, P〈0.05] and that of estradiol benzoate ± ICI 182780 for 24 hours [ （28.2 ±2. 1 ） %, （29. 7 ± 3.2 ） %, （ 35.0 ± 4. 7 ） %, （34.7 ±6.5）%, P 〈0.05]. The expression of HSP70 in 10^-9, 10^-8, 10^-7, 10^-6 mol /L estradiol benzoate for 24 hours were （ 38. 4 ± 5.7 ） %, （ 35.3 ± 4. 9 ） %, （ 32. 5 ± 3.1 ） %, （ 25.3 ± 6. 2 ） %, which were significantly lower than that of the control. The estradiol benzoate-induced effects on HSP70 levels were not attenuated by the antiestrogen ICI 182780. Conclusions The effects of estradiol benzoate on the cell growth and HSP90 expression are estrogen receptor-dependent and the expression of HSP90 may be beneficial to the cell growth. The effect of estradiol on the expression of HSP70 is estrogen receptor-independent and the expression of HSP70 may protect the cell from damage.