过氧化氢对蜕膜化小鼠子宫内膜基质细胞的影响

发布时间：2018-05-06 11:32

本文选题：氧化应激 + 过氧化氢　；参考：《医学研究生学报》2017年02期

【摘要】：目的植入前的胚胎对氧化应激敏感,低氧状态可能更有利于胚胎的生长及发育。文中旨在探讨过氧化氢(H_2O_2)对蜕膜化小鼠子宫内膜基质细胞(ESCs)的影响,构建蜕膜化小鼠ESCs氧化应激模型。方法采用酶解联合筛网过滤及差时贴壁方法分离培养原代小鼠ESCs。实验分为4组(n=8):空白对照组为含基础培养液的小鼠子宫内膜基质细胞(ESCs);蜕膜化组:基础培养液中加入10 nmol/L E2、1μmol/L P4体外诱导小鼠ESCs蜕膜化72 h;阴性对照组:小鼠ESCs蜕膜化诱导成功后,仅予基础培养液干预4 h;氧化应激组:小鼠ESCs蜕膜化诱导成功后,分别加入含50、100、150、200、250、300μmol/L H_2O_2的基础培养液继续干预4 h。免疫组化法检测Vimentin、Cytokeratin表达鉴定原代小鼠ESCs,RT-PCR检测d PRP mRNA的表达鉴定ESCs蜕膜化,CCK-8法检测H_2O_2干预后细胞生长和增殖活性,流式细胞仪检测细胞内ROS水平判断氧化应激程度。结果成功分离、培养和鉴定原代小鼠ESCs。镜下观察细胞呈梭形和多角形、放射状排列;细胞免疫荧光染色检测结果表明Vimentin表达(+)、Cytokeratin(-),原代小鼠ESCS纯度达(96.3±0.49)%。10 nmol/L E2、1μmol/L P4作用下,蜕膜化组蜕膜化标志物d PRP mRNA显著高于空白对照组(1.0010±0.0011 vs 0.000 2±0.0000,P0.01)。与阴性对照组抑制率[(1.000±0.000)%]比较,氧化应激组在H_2O_2浓度为50、100、150、200、250、300μmol/L作用下抑制率[(0.013±0.004、0.032±0.007、0.069±0.023、0.172±0.007、0.395±0.018、0.706±0.013)%],其中150、200、250、300μmol/L H_2O_2与阴性对照组比较差异有统计学意义(P0.05)。进一步评估50、100μmol/L H_2O_2干预后测得的细胞内氧化应激组ROS明显高于阴性对照组(27.77±4.20 vs 17.47±0.61,43.57±6.58 vs 17.47±0.61,P=0.001)。结论 50、100μmol/L H_2O_2不影响蜕膜化小鼠ESCs的生长、增殖,且随H_2O_2浓度增加,刺激细胞内ROS的产生增多,100μmol/L H_2O_2可作为蜕膜化小鼠ESCs氧化应激模型建立的合适刺激浓度。
[Abstract]:Objective preimplantation embryos are sensitive to oxidative stress and hypoxia may be more conducive to the growth and development of embryos. The purpose of this study was to investigate the effect of H _ 2O _ 2 on decidualized mouse endometrial stromal cells (ESCs) and to establish a decidualized mouse ESCs oxidative stress model. Methods Primary mice ESCs were isolated and cultured by enzymolysis combined with sieve filtration and delayed time adherence. The experiment was divided into 4 groups: blank control group: mouse endometrial stromal cells containing basic culture medium (ESCs); decidualization group: 10 nmol/L E2 + 1 渭 mol/L P4 was added to basic culture medium to induce ESCs decidualization in vitro for 72 h; negative control group: mouse ESCs slough After successful membrane induction, In the oxidative stress group, the mouse ESCs decidua was induced successfully, and then added with 50100150200250300 渭 mol/L H_2O_2 for 4 h. Expression of Vimentin Cytokeratin expression in Primary Mouse ESCs by RT-PCR; ESCs decidualized CCK-8 method was used to detect the cell growth and proliferation activity after H_2O_2 intervention; flow cytometry was used to determine the level of ROS in the cells to judge the degree of oxidative stress. Results the primary mouse ESCs were isolated, cultured and identified successfully. Under microscope, the cells were spindle-shaped and polygonal, and arranged radially, and the results of cell immunofluorescence staining showed that the expression of Vimentin was 96.3 卤0.49.10 nmol/L E _ 2N _ 1 渭 mol/L P _ 4, the purity of the primary mouse ESCS was 96.3 卤0.49%. D PRP mRNA of decidualization in decidualization group was significantly higher than that in control group (1.0010 卤0.0011 vs 0.000 2 卤0.0000 P 0.01). Compared with the negative control group, the inhibition rate of oxidative stress group was 50100150200250300 渭 mol/L at the concentration of 50100150200250300 渭 mol/L. The inhibition rate was 0.013 卤0.004 卤0.007 卤0.069 卤0.023 卤0.172 卤0.007 卤0.395 卤0.018 卤0.706 卤0.013% in the oxidative stress group, and there was a significant difference between the 150200250300 渭 mol/L H_2O_2 group and the negative control group (P 0.05). The ROS of the 50100 渭 mol/L H_2O_2 treated group was significantly higher than that of the negative control group (27.77 卤4.20 vs 17.47 卤0.61 卤43.57 卤6.58 vs 17.47 卤0.61). Conclusion 50100 渭 mol/L H_2O_2 does not affect the growth and proliferation of ESCs in decidualized mice, and with the increase of H_2O_2 concentration, the production of ROS in the cells is increased. 50100 渭 mol/L H_2O_2 can be used as a suitable stimulation concentration for ESCs oxidative stress model of decidualized mice.
【作者单位】：广西医科大学第一附属医院生殖中心;广西壮族自治区人民医院生殖中心;
【基金】：国家自然科学基金(81260096)
【分类号】：R711.74

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