雌二醇及丙酸睾酮对H2O2诱导泪腺上皮细胞凋亡的抑制作用

背景性激素在干眼症的发病过程中起着重要的作用，尤其是对泪腺的功能起到非常重要的调控作用，但其对泪腺上皮细胞凋亡作用的机制尚不完全明确。目的探讨雌二醇及丙酸睾酮对H2O2诱导的兔泪腺上皮细胞的凋亡作用。方法分离2～3月龄雄性普通级新西兰兔的泪腺组织，用组织块培养法体外培养兔泪腺上皮细胞，用细胞角蛋白（CK）抗体对培养的细胞进行免疫细胞化学鉴定。取对数生长期的细胞，以5×10^-5个／ml接种于96孔板培养44h后，培养基中分别加入1×10^-5、1×10^-6、1×10^-7、1×10^-8mol／L的雌二醇或丙酸睾酮培养24h，再加入1×10^-4 mol／L H2O2培养1h诱导细胞凋亡，仅用H2O2作用的细胞作为凋亡对照组，常规培养的细胞作为空白对照组。MTT比色法检测各组细胞570nm处泪腺上皮细胞活性的吸光度（A570）值，流式细胞仪Annexin V／PI双染法检测各组细胞的凋亡情况。结果培养的细胞呈不规则多边形，其中80％以上的细胞对CK呈阳性反应。MTT比色法检测表明，与空白对照组比较，凋亡对照组、不同浓度雌二醇组和丙酸睾酮组的A。值均明显降低，差异均有统计学意义（P〈0．01），1×10^-5、1×10^-6、1×10^-7mol／L雌二醇组和1×10^-6mol／L丙酸睾酮组的A570值均明显高于凋亡对照组，差异均有统计学意义（P〈0．01）。与相应浓度丙酸睾酮组相比，雌二醇组A570值均明显增高，差异均有统计学意义（P〈0．01）。与凋亡对照组比较，丙酸睾酮组和雌二醇组早期及晚期细胞凋亡率均明显下降，差异均有统计学意义（P〈0．01，P〈0．05）；与丙酸睾酮组相比，雌二醇组早期及晚期的细胞凋亡率明显下降，差异均有统计学意义（P〈0．01，P〈0．05）。结论雌二醇及丙酸睾酮可抑制H2O2诱导的泪腺上皮细胞的凋亡，雌二醇的抑制作用强于丙酸睾酮。雌二醇对泪腺上皮细胞凋亡的抑制作用呈一定的剂量依赖性。

The effect of estradiol and testosterone on the apoptosis of lacrimal gland cell induced by H2O2

Background The sex hormones plays an important role in the incidence of dry eye,especially for the regulation of function.However,the effects of sex hormones on lacrimal gland epithelial cells are below understand.Objective This study was to investgate the effects of estradiol and testosterone on the apoptosis of lacrimal gland cells induced by H2O2.Methods The lacrimal gland tissue was obtained from 2- or 3-month-old clean male New Zealand rabbits and the lacrimal gland epithelial cells were cultured in vitro using esplant culture method.The cells were identified by pan cytokeratin antibodies with immunocytochemistry.lacrimal gland epithelial cells were incubated in the 96 well plate at the density of 5 × l04 cells/ml for 44 hours.Estradiol or testosterone with the concentrations of 1 × 10-5,1 × 10-6,1 × 10-7,1 × 10-8 mol/L were added into the medium for 24 hours respectively and 1× 10-4 mol/L H2O2 treated the cells for 1 hour to induce the apoptosis in experimental groups.The cells treated by only 1 × 10-4 mol/L H2O2 were used as apoptotic control group,and the cells cultured by regular method were used as blank control group.The cell viability in different groups was detected using MTT at 570 nm ( A570 ),and the apoptotic rates of the cells were assayed using Annexin V/PI double staining.This use and maintain of experimental animals followed the Regulation for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The cultured cells showed the irregular polygon in shape,and about 80％ cells was positive response for cytokeratin.MTT assay showed that the lower A570 values were detected in the H2O2-induced group,various concentrations of estradiol or testosterone groups compared with blank control group (P＜0.01 ).The A570 values in 1 × 10-5,1 × 10-6,1 × 10-7 mol/L estradiol groups or 1 × 10-6 mol/L testosterone group were significantly higher than ones of H2 O2-induced group (P＜0.01 ).Compared with corresponding concentrations of testosterone groups,the A570values in various concentrations of estradiol groups were elevated( P＜0.01 ).The apoptosis rates at the early and later phase were significantly declined in both estradiol group and testosterone group in comparison with H2 O2-induced group (P ＜ 0.01,P＜ 0.05 ),and those in estradiol group were lower than the testosterone group( P＜0.01,P＜0.05 ).Conclusions Estradiol and testosterone suppress the apoptosis of lacrimal gland cells induced by H2O2,and the stronger effect is found in estrogen.The inhibition of estrogen on lacrimal gland cell apoptosis show a dose-dependent manner to some extent.