【摘要】 目的:探讨双酚AF （BPAF）对子宫内膜基质细胞（hESCs）衰老的影响,并阐明雷帕霉素（Rapa）对BPAF诱导的hESCs衰老的抑制作用。方法:体外培养hESCs,采用0、7.5、15.0、30.0、60.0和120.0μmol·L^-1 BPAF处理hESCs。采用MTT法检测不同浓度BPAF作用后hESCs存活率。将hESCs分为对照组（正常培养基）、BPAF组(加入含30.0μmol·L^-1 BPAF培养基)和Rapa组(加入含30.0μmol·L^-1 BPAF及100.0 nmol·L^-1 Rapa培养基)。采用衰老相关β-半乳糖苷酶（SA-β-gal）染色法检测各组细胞中SA-β-gal阳性细胞百分率,流式细胞术检测各组细胞周期百分率,实时荧光定量PCR （RT-qPCR）法检测各组细胞中p16、p21和p53 mRNA表达水平,Western blotting法检测各组细胞中衰老相关蛋白p16、p21和p53及自噬相关蛋白p62、微管相关蛋白1轻链3型蛋白Ⅱ（LC3-Ⅱ）和微管相关蛋白1轻链3型蛋白Ⅰ（LC3-Ⅰ）表达水平。结果:MTT法,当BPAF浓度为30.0μmol·L^-1时,细胞存活率达75%,后续实验选用30.0 0μmol·L^-1 BPAF诱导细胞衰老。与对照组比较,BPAF组SA-β-gal阳性细胞百分率明显升高（P<0.01）,G₁期细胞百分率明显升高（P<0.01）,细胞中p16、p21和p53 mRNA及蛋白表达水平均明显升高（P<0.01）。与BPAF组比较,Rapa组SA-β-gal阳性细胞百分率明显降低（P<0.01）,G₁期细胞百分率明显降低（P<0.01）,细胞中p16、p21和p53 mRNA及蛋白表达水平均明显降低（P<0.01）,自噬相关p62蛋白表达水平明显降低（P<0.01）,LC3-Ⅱ/LC3-Ⅰ比值明显升高（P<0.01）。结论:高浓度BPAF可抑制hESCs增殖并诱导细胞衰老,Rapa可抑制BPAF诱导的细胞衰老,其作用机制可能与细胞自噬激活有关。更多还原

【Abstract】 Objective:To investigate the effect of bisphenol AF（BPAF） on the senescence in the human endometrial stromal cells（hESCs）,and to elucidate the inhibitory effect of rapamycin（Rapa） on the BPAFinduced senescent hESCs.Methods:The hESCs were cultured in vitro and treated with 0,7.5,15.0,30.0,60.0,and 120.0 μmol·L^-1 BPAF.MTT assay was used to detect the cell survival rates of the hESCs after treated with different concentrations of BPAF.The cells were divided into control group（normal medium）,BPAF group(medium containing 30.0 μmol·L^-1 BPAF),and Rapa group(medium containing both 30.0 μmol·L^-1 BPAF and 100.0 nmol·L^-1 Rapa).Senescence-associated β-galactosidase（SA-β-gal） staining was used to assess the percentages of SA-β-gal positive cells in various groups;flow cytometry was used to analyze the percentages of cells at different cell cycles in various groups;real-time fluorescence quantitative PCR（RT-qPCR） method was used to assess the expression levels of p 16, p21and p53 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of senescence-related proteins（p16,p21 and p53）,autophagy-related proteins p62,microtubuleassociated protein 1-light chain 3（LC3）-Ⅱ and LC3-Ⅰ in the cells in various groups.Results:The MTT assay results showed that the cell survival rate was 75% when the BPAF concentration was 30.0 μmol·L^-1;therefore,this concentration was selected for subsequent senescence induction experiments.Compared with control group,the percentage of SA-β-gal positive cells in BPAF group was significantly increased（P<0.01）,the percentage of the cells at G₁ phase was significantly increased（P<0.01）,and the expression levels of p16,p21, and p53 mRNA and proteins were significantly increased（P<0.01）.Compared with BPAF group,the percentage of SA-β-gal positive cells in Rapa group was significantly decreased（P<0.01）,the percentage of the cells at G₁ phase was significantly decreased（P<0.01）,the expression levels of p16,p21,and p53 mRNA and proteins in the cells were significantly decreased（P<0.01）,and the expression level of the autophagy-related protein p62 in the cells was significantly decreased（P<0.01）while the LC3-Ⅱ/LC3-Ⅰ ratio was significantly increased（P<0.01）.Conclusion:High concentrations of BPAF can inhibit the proliferation of the hESCs and induce cellular senescence.Rapa can suppress BPAF-induced cellular senescence,and its mechanism may be related with the activation of autophagy.更多还原