肝豆灵对高铜负荷小鼠神经干细胞活性氧水平及核因子E2相关因子2表达的影响(1)
摘要：目的 观察肝豆灵对高铜负荷小鼠神经干细胞内活性氧(ROS)水平及核因子E2相关因子2(Nrf2) mRNA及蛋白表达的影响。方法 体外培养高铜负荷小鼠神经干细胞模型。实验小鼠随机分为空白对照组、模型组和肝豆灵低、中、高剂量组，各给药组给予相应浓度肝豆灵药物血清灌胃。采用MTT法检测神经干细胞增殖水平，流氏细胞仪检测细胞内ROS水平，qRT-PCR检测Nrf2基金表达，Western blot检测细胞内Nrf2蛋白表达。结果 与空白对照组比较，模型组小鼠神经干细胞增殖率显著下降，ROS水平明显升高，Nrf2基因、蛋白表达明显降低(P<0.01)；与模型组比较，肝豆灵各剂量组小鼠神经干细胞增殖率显著升高，ROS水平明显降低，Nrf2基因、蛋白表达明显升高(P<0.05，P<0.01)。结论 肝豆灵通过降低高铜负荷小鼠ROS含量、上调Nrf2的表达，从而促进小鼠神经干细胞的增殖。
关键词：肝豆灵；高铜负荷；体外培养；神经干细胞；活性氧；核因子E2相关因子2；小鼠
DOI：10.3969/j.issn.1005-5304.2018.07.013
中图分类号：R285.5 文献标识码：A 文章编号：1005-5304(2018)07-0053-04
Abstract： Objective To observe the effects of Gandouling on reactive oxygen species (ROS) level， the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein of neural stem cells of the mice cultured in high concentration copper. Methods The model of neural stem cells of the mice was cultured in vitro with high concentration copper. The experimental rats were randomly divided into blank control group， model group， and Gandouling low-， medium-， and high-dose groups. Each medication group was given relevant concentration of Gandouling serum for gavage. The MTT was adopted to test proliferation level on neural stem cells； flow cytometer was used to examine the change of ROS level in cells； qPCR was used to measure the expression of Nrf2 mRNA； Western blot was used to measure the change of the level of protein Nrf2 in cells. Results Compared with the blank control group， the proliferation rate of neural stem cells was significantly decreased， ROS levels were significantly increased， and Nrf2 gene and protein expression was significantly decreased (P<0.01). Compared with the model group， neural stem cells proliferation rate was significantly increased， ROS levels were significantly reduced， and Nrf2 gene and protein expression was significantly increased (P<0.05， P<0.01). Conclusion Gandouling can promote the proliferation of neural stem cells in mice by reducing ROS content in high copper-loaded mice and up-regulating Nrf2 expression.
Keywords： Gandouling； high copper load； cultured in vitro； neural stem cells； reactive oxygen species； nuclear factor erythroid 2-related factor 2； mice
Wilson病是一种ATP7B基因缺陷所致的铜代谢障碍疾病，患者不能清除体内多余的铜，导致铜在肝脏等组织中沉积，甚至可能引起严重的中枢损伤。铜是一种强氧化剂，它可以激活自由基的活性，进而促进细胞内活性氧(reactive oxygen species，ROS)的产生，细胞内铜过载，继而诱发的氧化应激，是大部分神经变性疾病致病的共同特征与机理。氧化应激产生的ROS从不同途径损伤了各种大分子的生理功能，并攻击特定脑组织区域的神经细胞，导致细胞发生凋亡，直至疾病发生。而对此类疾病，降低ROS引起的损害是治疗的关键，核因子E2相关因子2(nuclear factor erythroid 2-related factor 2，Nrf2)，作为一类对抗氧化应激和保护細胞生存的转录因子，成为神经干细胞(neural stem cell，NSC)损伤修复的潜在机制之一。在Wilson病研究过程中，研究人员发现，神经干细胞(NSC)是高铜负荷的重要靶点之一，NSC的增殖受损将严重影响脑部神经细胞的再生能力，肝豆灵能抵抗高铜导致的NSC生长阻滞，发挥神经保护作用[1]。然而，作为肝豆灵重要靶点的NSC中发生分子变化机制不明，给精确解析肝豆灵的作用机理及开发新的药物靶点造成障碍。因此，本实验制备药物血清，体外建立及高铜培养NSC，观察肝豆灵对小鼠NSC的ROS和Nrf2的影响，探讨其对高铜诱导的NSC损伤小鼠的保护作用及其分子机制。, http://www.100md.com(董婷 杨文明 吴明彩 蒋怀周 黄鹏 匡春俊 张娟 韩辉)
关键词：肝豆灵；高铜负荷；体外培养；神经干细胞；活性氧；核因子E2相关因子2；小鼠
DOI：10.3969/j.issn.1005-5304.2018.07.013
中图分类号：R285.5 文献标识码：A 文章编号：1005-5304(2018)07-0053-04
Abstract： Objective To observe the effects of Gandouling on reactive oxygen species (ROS) level， the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and protein of neural stem cells of the mice cultured in high concentration copper. Methods The model of neural stem cells of the mice was cultured in vitro with high concentration copper. The experimental rats were randomly divided into blank control group， model group， and Gandouling low-， medium-， and high-dose groups. Each medication group was given relevant concentration of Gandouling serum for gavage. The MTT was adopted to test proliferation level on neural stem cells； flow cytometer was used to examine the change of ROS level in cells； qPCR was used to measure the expression of Nrf2 mRNA； Western blot was used to measure the change of the level of protein Nrf2 in cells. Results Compared with the blank control group， the proliferation rate of neural stem cells was significantly decreased， ROS levels were significantly increased， and Nrf2 gene and protein expression was significantly decreased (P<0.01). Compared with the model group， neural stem cells proliferation rate was significantly increased， ROS levels were significantly reduced， and Nrf2 gene and protein expression was significantly increased (P<0.05， P<0.01). Conclusion Gandouling can promote the proliferation of neural stem cells in mice by reducing ROS content in high copper-loaded mice and up-regulating Nrf2 expression.
Keywords： Gandouling； high copper load； cultured in vitro； neural stem cells； reactive oxygen species； nuclear factor erythroid 2-related factor 2； mice
Wilson病是一种ATP7B基因缺陷所致的铜代谢障碍疾病，患者不能清除体内多余的铜，导致铜在肝脏等组织中沉积，甚至可能引起严重的中枢损伤。铜是一种强氧化剂，它可以激活自由基的活性，进而促进细胞内活性氧(reactive oxygen species，ROS)的产生，细胞内铜过载，继而诱发的氧化应激，是大部分神经变性疾病致病的共同特征与机理。氧化应激产生的ROS从不同途径损伤了各种大分子的生理功能，并攻击特定脑组织区域的神经细胞，导致细胞发生凋亡，直至疾病发生。而对此类疾病，降低ROS引起的损害是治疗的关键，核因子E2相关因子2(nuclear factor erythroid 2-related factor 2，Nrf2)，作为一类对抗氧化应激和保护細胞生存的转录因子，成为神经干细胞(neural stem cell，NSC)损伤修复的潜在机制之一。在Wilson病研究过程中，研究人员发现，神经干细胞(NSC)是高铜负荷的重要靶点之一，NSC的增殖受损将严重影响脑部神经细胞的再生能力，肝豆灵能抵抗高铜导致的NSC生长阻滞，发挥神经保护作用[1]。然而，作为肝豆灵重要靶点的NSC中发生分子变化机制不明，给精确解析肝豆灵的作用机理及开发新的药物靶点造成障碍。因此，本实验制备药物血清，体外建立及高铜培养NSC，观察肝豆灵对小鼠NSC的ROS和Nrf2的影响，探讨其对高铜诱导的NSC损伤小鼠的保护作用及其分子机制。, http://www.100md.com(董婷 杨文明 吴明彩 蒋怀周 黄鹏 匡春俊 张娟 韩辉)