ROS在硫酸镍致大鼠Leydig细胞睾酮合成障碍中的作用
发布时间：2018-08-30 13:38
【摘要】：目的:通过体外培养大鼠睾丸间质细胞(Leydig细胞),观察硫酸镍(NiSO_4)致大鼠Leydig细胞活性氧(ROS)水平及睾酮合成酶mRNA和蛋白表达水平的变化,探讨ROS在镍致大鼠Leydig细胞睾酮合成障碍中的作用。方法:(1)采用0.25%胶原酶消化、Percoll连续梯度离心法和体外贴壁培养提取和纯化Leydig细胞,并通过3β-HSD(3β-羟类固醇脱氢酶)对纯化后的细胞进行纯度鉴定。(2)细胞处理取处于对数生长期的大鼠Leydig细胞,给予不同浓度(0、250、500和1000μmol/L)NiSO_4及1 IU/ml人绒毛膜促性腺激素(hCG)分别处理0、6、12和24 h;干预实验细胞处理取对数生长期Leydig细胞,分别经活性氧抑制剂NAC和TEMPO提前干预1 h后,再行1000μmol/L NiSO_4处理24 h。(3)用酶联免疫吸附法(ELISA)检测培养上清液中睾酮浓度。(4)采用实时荧光定量PCR(RT-qPCR)和Western blot技术分别检测Leydig细胞类固醇合成快速调节蛋白(StAR)、细胞色素P450胆固醇侧链裂解酶(CYP11A1)、类固醇17α-羟化酶(CYP17A1)、3β-HSD和17β-羟类固醇脱氢酶(17β-HSD)的mRNA和蛋白表达水平。(5)采用DCFH-DA探针法检测大鼠Leydig细胞内ROS水平。结果:(1)3β-HSD染色法鉴定结果显示,Percoll纯化及细胞贴壁生长24h后,可得到纯度较高的Leydig细胞。(2)ELISA法检测结果显示:与0 h组比较,hCG+0μmol/L NiSO_4处理Leydig细胞12 h和24 h后,培养液中睾酮浓度均明显升高(P0.05)。与0μmol/L NiSO_4组(对照组)比较,hCG+500μmol/L NiSO_4处理Leydig细胞24 h,以及hCG+1000μmol/L NiSO_4处理细胞12和24h后,细胞培养液中睾酮浓度均显著降低(P0.05)。RT-qPCR结果显示:与对照组比较,500μmol/L NiSO_4处理Leydig细胞24 h后,睾酮合成酶CYP11A1和17β-HSD mRNA表达水平均下降(P0.05)。与对照组比较,1000μmol/L NiSO_4处理Leydig细胞24 h后,睾酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD mRNA表达水平均下降(P0.05)。Western Blot结果显示:与对照组比较,1000μmol/L NiSO_4处理Leydig细胞24 h后,睾酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD的蛋白表达水平均下降(P0.05)。(3)DCFH-DA探针法检测结果显示:与对照组比较,1000μmol/L NiSO_4处理Leydig细胞24 h(NiSO_4组),荧光显微镜下可见细胞中ROS水平明显升高。与NiSO_4组比较,经5 mmol/L NAC(NAC组)和1 mmol/L TEMPO(TEMPO组)干预后,Leydig细胞中ROS水平明显下降(P0.05)。ELISA法检测结果显示:与对照组比较,hCG+1000μmol/L NiSO_4处理细胞24 h后,细胞培养液中的睾酮浓度显著降低(P0.05)。与NiSO_4组比较,NAC组和TEMPO组Leydig细胞培养液中的睾酮浓度则显著升高(P0.05)。RT-qPCR和Western Blot分析结果显示:与对照组比较,NiSO_4组Leydig细胞睾酮合成酶StAR、CYP11A1、CYP17A1、3β-HSD和17β-HSD的mRNA和蛋白表达水平均明显降低(P0.05)。与NiSO_4组比较,NAC组细胞上述基因mRNA和蛋白表达水平均升高(P0.05),而TEMPO组除CYP17A1和17β-HSD外,其他蛋白表达水平也显著升高(P0.05)。结论:NiSO_4可致大鼠Leydig细胞睾酮合成障碍,这可能与其诱导Leydig细胞ROS大量生成有关。NiSO_4可降低大鼠Leydig细胞睾酮分泌和下调睾酮合成酶相关基因和蛋白表达水平,且前述变化均可被活性氧抑制剂NAC和TEMPO所抑制,表明ROS在NiSO_4诱导的大鼠Leydig细胞睾酮合成障碍中发挥重要作用。
[Abstract]:AIM: To observe the changes of reactive oxygen species (ROS) and testosterone synthase mRNA and protein expression in rat Leydig cells induced by nickel sulfate (NiSO_4), and to explore the role of ROS in nickel-induced testosterone dyssynthesis in rat Leydig cells. Leydig cells were extracted and purified by gradient centrifugation and adherent culture in vitro, and purified by 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). (2) Rat Leydig cells in logarithmic growth phase were treated with NiSO_4 and 1 IU/ml human chorionic gonadotropin (hCG) at different concentrations (0,250,500 and 1000 micromol/L). Leydig cells were treated for 0, 6, 12, and 24 hours respectively. After 1 hour of intervention with active oxygen inhibitor NAC and TEMPO, the cells were treated with 1 000 micromol/L NiSO_4 for 24 hours. (3) Testosterone concentration in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). (4) Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used. The levels of mRNA and protein expression of steroid synthesis rapid regulator protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1), steroid 17 alpha hydroxylase (CYP17A1), 3 beta-HSD and 17 beta-hydroxysteroid dehydrogenase (17beta-HSD) in Leydig cells of rats were detected by DCFH-DA probe. (2) The results of ELISA showed that the concentration of testosterone in Leydig cells treated with hCG+0 micromol/L NiSO_4 for 12 h and 24 h was significantly higher than that of the control group (P 0.05). The levels of testosterone synthase CYP11A1 and 17 beta-HSD mRNA expression in cultured Leydig cells treated with CG+500 micromol/L NiSO_4 for 24 hours and hCG+1000 micromol/L NiSO_4 for 12 and 24 hours were significantly decreased (P 0.05). Compared with the control group, the expression levels of testosterone synthase StAR, CYP11A1, CYP17A1, 3-beta-HSD and 17-beta-HSD mRNA in Leydig cells treated with 1 000 micromol/L NiSO_4 for 24 hours decreased (P 0.05). The Western Blot results showed that, compared with the control group, the eggs of testosterone synthase StAR, CYP11A1, CYP17A1, 3-beta-HSD and 17-HSD were treated with 1 000 micromol/L NiSO_4 for 24 hours. The results of DCFH-DA probe assay showed that compared with the control group, the ROS level of Leydig cells treated with 1 000 micromol/L NiSO_4 for 24 hours (NiSO_4 group) was significantly increased under fluorescence microscope. Compared with NiSO_4 group, the ROS level of Leydig cells was significantly increased after 5 mmol/L NAC (NAC group) and 1 mmol/L TEMPO (TEMPO group) intervention. The results of ELISA showed that the concentration of testosterone in the cell culture medium decreased significantly (P 0.05) after 24 hours of treatment with hCG + 1000 micromol / L NiSO_4 compared with the control group. Compared with NiSO_4 group, the concentration of testosterone in the Leydig cell culture medium of NAC group and TEMPO group increased significantly (P 0.05). RT-q PCR and Western Blot analysis showed that the concentration of testosterone in the cell culture medium was significantly higher (P 0.05). Compared with the control group, the levels of mRNA and protein expression of testosterone synthase StAR, CYP11A1, CYP17A1, 3beta-HSD and 17beta-HSD were significantly decreased in NiSO_4 group (P 0.05). Compared with NiSO_4 group, the levels of mRNA and protein expression of these genes in NAC group were increased (P 0.05), while those of TEMPO group were significantly increased except CYP17A1 and 17beta-HSD. CONCLUSION: NiSO_4 may induce the production of ROS in rat Leydig cells. NiSO_4 can decrease the secretion of testosterone and down-regulate the expression of testosterone synthase-related genes and proteins in rat Leydig cells, and these changes can be inhibited by the inhibitors of reactive oxygen species NAC and TEMPO. Ming ROS plays an important role in NiSO_4 induced Leydig cell testosterone synthesis disorder in rats.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2213237
[Abstract]:AIM: To observe the changes of reactive oxygen species (ROS) and testosterone synthase mRNA and protein expression in rat Leydig cells induced by nickel sulfate (NiSO_4), and to explore the role of ROS in nickel-induced testosterone dyssynthesis in rat Leydig cells. Leydig cells were extracted and purified by gradient centrifugation and adherent culture in vitro, and purified by 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). (2) Rat Leydig cells in logarithmic growth phase were treated with NiSO_4 and 1 IU/ml human chorionic gonadotropin (hCG) at different concentrations (0,250,500 and 1000 micromol/L). Leydig cells were treated for 0, 6, 12, and 24 hours respectively. After 1 hour of intervention with active oxygen inhibitor NAC and TEMPO, the cells were treated with 1 000 micromol/L NiSO_4 for 24 hours. (3) Testosterone concentration in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). (4) Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot were used. The levels of mRNA and protein expression of steroid synthesis rapid regulator protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1), steroid 17 alpha hydroxylase (CYP17A1), 3 beta-HSD and 17 beta-hydroxysteroid dehydrogenase (17beta-HSD) in Leydig cells of rats were detected by DCFH-DA probe. (2) The results of ELISA showed that the concentration of testosterone in Leydig cells treated with hCG+0 micromol/L NiSO_4 for 12 h and 24 h was significantly higher than that of the control group (P 0.05). The levels of testosterone synthase CYP11A1 and 17 beta-HSD mRNA expression in cultured Leydig cells treated with CG+500 micromol/L NiSO_4 for 24 hours and hCG+1000 micromol/L NiSO_4 for 12 and 24 hours were significantly decreased (P 0.05). Compared with the control group, the expression levels of testosterone synthase StAR, CYP11A1, CYP17A1, 3-beta-HSD and 17-beta-HSD mRNA in Leydig cells treated with 1 000 micromol/L NiSO_4 for 24 hours decreased (P 0.05). The Western Blot results showed that, compared with the control group, the eggs of testosterone synthase StAR, CYP11A1, CYP17A1, 3-beta-HSD and 17-HSD were treated with 1 000 micromol/L NiSO_4 for 24 hours. The results of DCFH-DA probe assay showed that compared with the control group, the ROS level of Leydig cells treated with 1 000 micromol/L NiSO_4 for 24 hours (NiSO_4 group) was significantly increased under fluorescence microscope. Compared with NiSO_4 group, the ROS level of Leydig cells was significantly increased after 5 mmol/L NAC (NAC group) and 1 mmol/L TEMPO (TEMPO group) intervention. The results of ELISA showed that the concentration of testosterone in the cell culture medium decreased significantly (P 0.05) after 24 hours of treatment with hCG + 1000 micromol / L NiSO_4 compared with the control group. Compared with NiSO_4 group, the concentration of testosterone in the Leydig cell culture medium of NAC group and TEMPO group increased significantly (P 0.05). RT-q PCR and Western Blot analysis showed that the concentration of testosterone in the cell culture medium was significantly higher (P 0.05). Compared with the control group, the levels of mRNA and protein expression of testosterone synthase StAR, CYP11A1, CYP17A1, 3beta-HSD and 17beta-HSD were significantly decreased in NiSO_4 group (P 0.05). Compared with NiSO_4 group, the levels of mRNA and protein expression of these genes in NAC group were increased (P 0.05), while those of TEMPO group were significantly increased except CYP17A1 and 17beta-HSD. CONCLUSION: NiSO_4 may induce the production of ROS in rat Leydig cells. NiSO_4 can decrease the secretion of testosterone and down-regulate the expression of testosterone synthase-related genes and proteins in rat Leydig cells, and these changes can be inhibited by the inhibitors of reactive oxygen species NAC and TEMPO. Ming ROS plays an important role in NiSO_4 induced Leydig cell testosterone synthesis disorder in rats.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
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