小鼠卵巢内质网应激模型的建立及诱导凋亡的研究

目的通过观察内质网应激(ERS)诱导剂衣霉素(TM)及ERS抑制剂牛磺熊去氧胆酸钠(TUDCA)对ERS相关标识性蛋白表达水平的影响,建立小鼠卵巢ERS模型。方法取4周龄昆明白小鼠卵巢,进行半卵巢培养。实验分组:新鲜对照组,不同浓度TM诱导组(1、5、10、20、30μg·mL-1),TUDCA+TM组、不同浓度TUDCA(1、2、5、10mM)+TM组。通过免疫组织化学技术观测培养1h卵巢内内质网应激伴侣蛋白GRP78、培养3h后ERS凋亡相关蛋白CHOP的表达水平;TUNEL法检测培养3、6、12、24、36、48h后卵巢细胞凋亡情况。结果与新鲜组相比,TM诱导1h后卵巢发生内质网应激反应,TM浓度为5-10μg·mL-1时GRP78表达水平达到最高(P<0.05),之后随TM浓度增加缓慢递减;TM诱导3h后卵巢内ERS凋亡相关蛋白CHOP表达水平在TM 10μg·mL-1处达到最高(P<0.05),凋亡细胞数随着培养时间的延长增加。TUDCA抑制ERS实验显示,随着TUDCA浓度的增大,10μg·mL-1TM诱导的GRP78蛋白表达呈递减趋势,其中TUDCA 10mM组GRP78表达水平基本恢复到新鲜组水平。结论 TM可诱导小鼠卵巢ERS,5μg·mL-1为诱导ERS保护作用浓度,而10μg·mL-1以上则产生ERS途径介导的凋亡。TUDCA可抑制TM诱导的卵巢ERS。

Modeling of Endoplasmic Reticulum Stress and Inducing of Apoptosis in Mouse Ovaries

Objective To establish endoplasmic reticulum stress(ERS) model through observing the effect that ERS inducer-TM and ERS inhibitor-TUDCA act on ERS-related marker proteins.Methods Ovaries were taken from 4-week female Kunming white mice and were cultured in half parts.The experiment groups were designed as: fresh control group,concentration gradient of TM group(1,5,10,20,30 μg·mL-1and TUDCA + TM group: TM positive control group,concentration gradient of TUDCA group(1,2,5,10mM).Protein expression level of GRP78 in 1h cultured ovaries and CHOP in 3h cultured ovaries were detected by immunohistochemistry.Cell apoptosis in ovaries within 48h was monitored by TUNEL.Results Compared with fresh group,ERS response after the ovaries induced by TM for 1h and GRP78 expression level was the highest when the the concentration of TM reached 5 - 10μg·mL-1(P <0.05),then slowly decreased as the concentration was decreasing.The expression level of apoptosis-related protein CHOP was the highest after ovaries induced by TM for 3h,then slowly decreased with the concentration decreasing.The number of apoptotic cells increased with cultured time passing.TUDCA inhibit ERS experiments showed that with the increasing concentration of TUDCA,the expression of GRP78 induced by 10μg·mL-1TM showed a decreasing trend,in which 10mM GRP78 expression levels recovered to the level of fresh group(P < 0.05).Conclusion TM can induce ERS response in mouse ovaries.5μg · mL-1is the concentration that induced ERS protective effect and the concentration above 10μg·mL-1will lead to apoptosis mediated by ERS pathway.TUDCA can inhibit TM-induced ERS in ovaries.