【摘要】 胚胎早期死亡及流产严重影响母牛的繁殖性能，造成极大的经济损失。本研究利用不同浓度双氧水（H₂O₂）处理子宫内膜细胞6 h,通过流式细胞仪、酶标仪分别进行细胞内活性氧（ROS）水平、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性以及还原型谷胱甘肽（GSH）与氧化型谷胱甘肽（GSSG）比值检测，建立子宫内膜细胞氧化应激模型。在此基础上，利用流式细胞仪检测细胞凋亡和生长周期，利用荧光定量RT-PCR和Western blot检测Fas/FasL信号通路关键基因的mRNA和蛋白表达水平，并利用慢病毒介导的RNA干扰（RNAi）抑制子宫内膜细胞中Fas基因表达，进一步验证Fas/FasL凋亡通路是否参与氧化应激诱导的子宫内膜细胞凋亡。所有试验都重复3次以上。结果发现，利用200μmol·L^-1 H₂O₂处理子宫内膜细胞6 h,细胞内ROS阳性水平急剧增加（P<0.05）,SOD和CAT活性以及GSH与GSSG比值都显著降低（P<0.05）,说明H₂O₂处理对子宫内膜细胞造成氧化应激损伤。在此处理条件下，生长周期中S期比例显著降低（P<0.05）,凋亡比例显著提高（P<0.05）;Fas/FasL凋亡通路中关键基因Fas、Caspase 8和Caspase 3的mRNA表达水平显著升高（P<0.05）;培养液中FasL浓度以及Fas、Caspase 8和Caspase 3蛋白表达水平显著升高（P<0.05）。进一步研究发现，抑制Fas基因表达在一定程度上降低了氧化应激诱导的细胞凋亡比例以及Fas/FasL凋亡通路中关键基因的表达水平。总之，本研究结果表明，氧化应激通过激活Fas/FasL信号通路促进奶牛子宫内膜细胞凋亡，这为解释氧化应激对子宫内膜细胞的不利影响提供了新的见解。更多还原

【Abstract】 Early embryonic death and abortion in dairy cows impair the reproductive performance and cause great economic losses. In this study, endometrial cells were treated with different concentrations of hydrogen peroxide（H₂O₂） for 6 h. The levels of intracellular reactive oxygen species（ROS） were measured by flow cytometry. The activities of superoxide dismutase（SOD） and catalase（CAT）, and the ratio of reduced glutathione（GSH） to oxidized glutathione（GSSG） were measured by microplate reader, which were to establish an oxidative stress model in endometrial cells. Then apoptosis and cell cycle were detected by flow cytometry, mRNA and protein expression of key genes in Fas/FasL signaling pathway were detected by fluorescent quantitative RT-PCR and Western blot, and lentivirus-mediated RNA interference（RNAi） was employed to inhibit Fas expression in endometrial cells to further verify whether the oxidative stress-induced apoptosis is mediated through Fas/FasL signaling pathway. All experiments were repeated more than three times. Results are as follows: Treatment of endometrial cells with 200 μmol·L^-1 H₂O₂ for 6 h resulted in an increase in the amount of intracellular ROS（P<0.05）, a significant decrease in the activities of SOD and CAT（P<0.05）, and the ratio of GSH to GSSG was significantly reduced（P<0.05）, which indicated that H₂O₂ caused oxidative stress damage in endometrial cells. Compared with control group, the proportion of S phase in cell cycle was significantly reduced（P<0.05）, and the proportion of apoptosis was significantly increased（P<0.05）. The levels of mRNA expressions of Fas, Caspase 8 and Caspase 3 in Fas/FasL signaling pathway significantly increased（P<0.05）; The concentration of FasL in the cultured medium significantly increased（P<0.05） and the levels of protein expression of Fas, Caspase 8 and Caspase 3 significantly increased（P<0.05）. Further studies have found that interfering with Fas gene expression partly reduced the proportion of apoptosis and the levels of protein expression of key genes in Fas/FasL signaling pathway induced by oxidative stress. In conclusion, these results demonstrated that oxidative stress-induced apoptosis in bovine endometrial epithelial cells is mediated through Fas/FasL signaling pathway, which may provide new insights into the adverse effects of endometrial cells in response to oxidative stress.更多还原