α-硫辛酸对氧化应激下大鼠睾丸支持细胞保护作用的实验研究
本文选题:α-硫辛酸 + 支持细胞 ; 参考:《重庆医科大学》2012年硕士论文
【摘要】:目的:探讨α-硫辛酸对氧化应激下大鼠睾丸支持细胞的氧化损伤是否具有保护作用,并通过细胞骨架蛋白表达角度研究α-硫辛酸对氧化应激下大鼠睾丸支持细胞的保护作用途径及相关的机制。 方法:取大鼠睾丸进行支持细胞原代培养,,并通过细胞形态学进行细胞鉴定。将各种浓度的H2O2(0,50,100,200,400,800,1600μmol/L)诱导体外培养的大鼠睾丸支持细胞氧化损伤,并通过CCK-8法检测睾丸支持细胞的活性及DCFH-DA荧光探针法检测细胞内活性氧水平进行睾丸支持细胞氧化损伤模型的鉴定。将原代培养大鼠睾丸支持细胞按以下四组进行处理:对照组,即正常细胞培养,不加任何处理因素;α-LA组,仅加入含终浓度200μmol/Lα-LA的培养基;H2O2组,即加入含终浓度400μmol/LH2O2的培养基;α-LA+H2O2组,即预先加入含终浓度200μmol/Lα-LA孵育4h后再加入含终浓度400μmol/LH2O2的培养基。各组处理12h后进行实验检测。采用CCK-8法检测各组睾丸支持细胞的活性及DCFH-DA荧光探针法检测各组睾丸支持细胞内活性氧水平;酶活性检测试剂盒检测抗氧化酶活性;免疫组织化学法、蛋白质免疫印迹法检测波形蛋白及微管蛋白的表达。 结果:大鼠睾丸支持细胞经分离、接种后,细胞贴壁生长,3至4天后细胞呈现单层融合状态生长。H2O2损伤组较正常对照组,细胞活力有所降低,并随H2O2浓度的升高,细胞活力降低明显,同时,细胞内ROS水平上升,H2O2作用于细胞致氧化损伤的适合浓度为400μmol/L。与对照组比较,H2O2刺激后支持细胞存活率下降,具有统计学意义(P0.05);细胞内ROS水平上升(P0.05);总抗氧化能力、超氧化物歧化酶、过氧化氢酶和谷胱甘肽还原酶的活性均降低(P0.05);波形蛋白及微管蛋白的表达也明显降低(P0.05),而α-LA干预后可提高细胞存活率,降低细胞内ROS水平,提高抗氧化酶的活性,并提高波形蛋白及微管蛋白的表达。 结论:氧化应激可导致大鼠睾丸支持细胞波形蛋白及微管蛋白表达降低,使细胞存活率下降;α-LA通过清除细胞内ROS,提高抗氧化酶的活性及波形蛋白和微管蛋白的表达而对氧化应激的支持细胞起保护作用。
[Abstract]:Objective: to investigate the protective effect of 伪 -lipoic acid on oxidative injury of testicular Sertoli cells in rats under oxidative stress. The protective effects of 伪 -lipoic acid on rat testicular Sertoli cells under oxidative stress and its related mechanism were studied by cytoskeleton protein expression. Methods: Sertoli cells were cultured from rat testis and identified by cell morphology. Oxidative damage of rat testicular Sertoli cells was induced by different concentrations of H _ 2O _ 2: 50100200400800c1600 渭 mol / L in vitro. The activity of testicular Sertoli cells was detected by CCK-8 and the reactive oxygen species was detected by DCFH-DA fluorescence probe method to identify the oxidative injury model of testicular Sertoli cells. Primary cultured rat testicular Sertoli cells were treated in the following four groups: control group, that is, normal cell culture without any additional factors, 伪 -LA group, which was treated with H _ 2O _ 2 medium containing final concentration of 200 渭 mol/L 伪 -LA, that is, medium containing final concentration of 400 渭 mol/LH2O2, 伪 -LA H2O2 group, The medium containing final concentration of 200 渭 mol/L 伪 -LA was added for 4 h and then added to the medium containing final concentration of 400 渭 mol/LH2O2. After 12 hours of treatment, experimental examination was carried out in each group. The activity of testicular Sertoli cells in each group was detected by CCK-8 method and reactive oxygen species in Sertoli cells was detected by DCFH-DA fluorescence probe method, the antioxidant enzyme activity was detected by enzyme activity test kit, and the activity of antioxidant enzyme was detected by immunohistochemical method. The expression of vimentin and tubulin was detected by Western blot. Results: rat testicular Sertoli cells were isolated and inoculated. After 3 to 4 days of adherent growth, the cells showed monolayer fusion growth. H2O2 injury group was significantly lower than the normal control group, and the cell activity decreased with the increase of H2O2 concentration. At the same time, the level of ROS in cells was increased. The suitable concentration of H2O2 was 400 渭 mol 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) for oxidative damage induced by H _ 2O _ 2. Compared with the control group, the survival rate of Sertoli cells decreased after stimulation with H _ 2O _ 2, which had statistical significance (P 0.05), the level of ROS in cells increased (P 0.05), the total antioxidant capacity, superoxide dismutase (SOD), total antioxidation ability, superoxide dismutase, The activities of catalase and glutathione reductase both decreased, vimentin and tubulin expression also decreased significantly. 伪 -LA increased cell survival rate, decreased intracellular ROS level, and increased antioxidant enzyme activity. The expression of vimentin and tubulin was increased. Conclusion: oxidative stress can decrease the expression of vimentin and tubulin in rat testicular Sertoli cells. 伪 -LA can protect the supporting cells from oxidative stress by scavenging intracellular ROSs and increasing the activity of antioxidant enzymes and the expression of vimentin and tubulin.
【学位授予单位】:重庆医科大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R363
【共引文献】
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