过氧化氢对小鼠睾丸支持细胞损伤机制的代谢组学研究

发布时间：2018-11-20 13:13

【摘要】：目的:应用基于气相色谱联用质谱(GC-MS)的代谢组学技术研究小鼠睾丸支持细胞氧化应激损伤机制。方法:以过氧化氢(H_2O_2)干预小鼠睾丸支持细胞TM4细胞,建立氧化损伤细胞病理模型。分别采用MTT方法检测存活率,流式细胞仪分析细胞凋亡率,荧光分光光度计检测细胞内活性氧(ROS)水平;同时通过GCMS检测H_2O_2干预后各组细胞内源性代谢物的指纹图谱。结果:成功建立H_2O_2损伤模型,600μmol/L H_2O_2干预2 h后细胞存活率降至50%左右;低(100μmol/L)、中(300μmol/L)和高(600μmol/L)H_2O_2剂量组总凋亡率分别为(19.45±0.53)%、(20.12±0.58)%和(37.13±0.35)%,均显著高于不加H_2O_2的空白对照组[(10.28±0.35)%,P均0.05];中、高H_2O_2剂量组ROS水平[(1.27±0.10)%、(2.07±0.09)%]也显著高于对照组[(1.00±0.08)%,P0.05、P0.01];代谢组学结果显示:与对照组相比,高剂量组细胞内氨基酸类、糖类等代谢水平均出现明显变化,其中缬氨酸、正缬氨酸、亮氨酸、谷氨酸、阿拉伯糖、果糖、5-羟色胺和胆固醇等在内的15种代谢物差异显著(VIP1,P0.05)。结论:睾丸支持细胞在H_2O_2干预后产生的损伤或凋亡与细胞内的氨基酸代谢、糖代谢和能量代谢密切相关。
[Abstract]:Aim: to study the mechanism of oxidative stress in mouse testicular Sertoli cells by gas chromatography-mass spectrometry (GC-MS). Methods: TM4 cells of mouse testicular Sertoli cells were treated with hydrogen peroxide (H_2O_2), and the pathological model of oxidative injury cells was established. The survival rate was detected by MTT, the apoptosis rate was analyzed by flow cytometry, and the level of reactive oxygen species (ROS) was detected by fluorescence spectrophotometer. At the same time, GCMS was used to detect the fingerprint of endogenous metabolites in each group after H_2O_2 intervention. Results: the H_2O_2 injury model was successfully established, and the cell survival rate decreased to about 50% after the treatment of 600 渭 mol/L H_2O_2 for 2 h. The total apoptotic rates were (19.45 卤0.53)%, (20.12 卤0.58)% and (37.13 卤0.35)% in low (100 渭 mol/L), middle (300 渭 mol/L) and high (600 渭 mol/L) H_2O_2 groups, respectively. It was significantly higher than the control group without H_2O_2 [(10.28 卤0.35)%, P 0.05]. The level of ROS in the high H_2O_2 group [(1.27 卤0.10)%, (2.07 卤0.09)%] was significantly higher than that in the control group [(1.00 卤0.08)%, P 0.05 P0.01]. Compared with the control group, the metabolic levels of amino acids and carbohydrates in high dose groups were significantly changed, including valine, orthvaline, leucine, glutamic acid, arabinose, fructose, etc. There were significant differences (VIP1,P0.05) in 15 metabolites including serotonin and cholesterol. Conclusion: the injury or apoptosis of testicular Sertoli cells after H_2O_2 intervention is closely related to the metabolism of amino acids, glucose and energy.
【作者单位】：南京工业大学药学院;南京大学医学院附属金陵医院/南京军区南京总医院男科;
【基金】：国家自然科学基金(81373478,81370750) 2015年江苏高校优秀科技创新团队计划~~
【分类号】：R698.2

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