内质网应激在氟致神经干细胞凋亡中的作用

发布时间：2018-04-12 19:31

  本文选题：氟化钠 + 神经干细胞　； 参考：《华中科技大学》2013年硕士论文

【摘要】：氟神经毒性近年来受到广泛关注，但其对神经干细胞（neuralstemcells，NSCs）的直接毒性尚未见报道。本研究以小鼠来源神经干细胞株-C17.2NSCs和原代大鼠胎鼠皮层NSCs为研究对象，研究氟化钠（sodiumfluoride，NaF）对NSCs存活率、氧化应激及凋亡的影响，并检测内质网应激（endoplasmicreticulumstress，ERS）相关蛋白的表达水平以探讨ERS在NaF诱导NSCs凋亡中的作用，为研究氟的神经毒性作用机制提供理论依据。 第一部分内质网应激在氟致C17.2神经干细胞凋亡中的作用研究 目的：探讨不同剂量NaF对C17.2NSCs存活率、氧化应激、凋亡、Caspase-3以及ERS相关蛋白GRP78、IRE1和CHOP表达水平的影响。 方法：体外培养的C17.2NSCs分别用预设浓度10、30、60和80mg/LNaF染毒24h后，采用MTT法测定存活率；经10、30、60mg/LNaF染毒24h后检测ROS、MDA水平和SOD活力、凋亡细胞核形态改变、Caspase-3表达水平以及ERS相关蛋白GRP78、IRE1和CHOP的表达水平。 结果：与对照组相比较，，30、60和80mg/LNaF染毒组存活率明显下降（P0.05），80mg/LNaF染毒组存活率低于50%；10、30、60mg/LNaF染毒组ROS水平明显升高（P0.05）；SOD活力显著降低（P0.05）；30和60mg/LNaF染毒组MDA水平、凋亡率明显增高（P0.05）；60mg/LNaF染毒组Caspase-3表达水平显著降低（P0.05）；30和60mg/L染毒组IRE1、CHOP的表达水平明显升高（P0.05）。 结论：一定剂量的NaF可抑制C17.2NSCs的增殖，诱导氧化应激的产生，并介导其凋亡的发生，同时内质网凋亡通路可能参与了氟对C17.2NSCs的损伤过程。 第二部分氟对原代大鼠胎鼠皮层神经干细胞存活率的影响 目的：探讨NaF对大鼠胎鼠皮层NSCs存活率的影响，确定后续实验染毒剂量。 方法：采用DMEM/F12培养液对原代大鼠胎鼠皮层NSCs进行培养，NSCs特异性标志物巢蛋白（Nestin）对其进行鉴定；经预设浓度40、80、120、200mg/LNaF染毒24h后，用MTT法测定细胞存活率。 结果：分离培养的细胞Nestin表达丰富；与对照组相比，各NaF染毒组细胞存活率均下降，差异具有统计学意义（P0.05）。 结论：本实验所分离培养的细胞为NSCs；氟可抑制原代培养大鼠胎鼠皮层NSCs增殖。
[Abstract]:Fluorine neurotoxicity has been widely concerned in recent years, but its direct toxicity to neural stem cells (NSCs) has not been reported.The effects of sodium fluoride (NAF) on the survival rate, oxidative stress and apoptosis of neural stem cell line C17.2NSCs derived from mice and primary rat fetal cortex NSCs were studied.The expression level of endoplasmic reticulum stress (ERS) related protein was detected to investigate the role of ERS in the apoptosis of NSCs induced by NaF, and to provide a theoretical basis for studying the neurotoxic mechanism of fluoride.The role of endoplasmic reticulum stress in fluorine-induced apoptosis of C17.2 neural stem cellsAim: to investigate the effects of different doses of NaF on the expression of C17.2NSCs survival, oxidative stress, apoptotic Caspase-3 and ERS associated protein GRP78 IRE1 and CHOP.Methods: the survival rate of C17.2NSCs cultured in vitro was determined by MTT method after exposure to 1030 mg / L NAF for 24 hours, and the level of MDA and the activity of SOD were detected after exposed to 1030 mg / L NAF for 24 hours.The expression level of Caspase-3 and the expression of ERS associated protein GRP78, IRE1 and CHOP in apoptotic nuclei.The expression of Caspase-3 in 60 mg / L NAF group significantly decreased the expression level of IRE1 / CHOP in P0.05 and 60mg/L groups, and increased the expression level of IRE1 / CHOP in P0.05 / L NAF group.Conclusion: a certain dose of NaF can inhibit the proliferation of C17.2NSCs, induce oxidative stress, and mediate the apoptosis of C17.2NSCs. The endoplasmic reticulum apoptosis pathway may be involved in the process of C17.2NSCs injury induced by fluoride.Effect of fluoride on survival rate of primary rat fetal cortical neural stem cellsAim: to investigate the effect of NaF on the survival rate of rat fetal cortical NSCs and determine the dose of the following experiment.Methods: Nestin, a specific marker of nestin, was identified in primary rat fetal cortex NSCs by DMEM/F12 culture medium, and the cell survival rate was determined by MTT assay after exposure to 40 80120 mg / L NAF for 24 h.Results: compared with the control group, the cell survival rate of the cells exposed to NaF was lower than that of the control group, and the difference was statistically significant (P 0.05).Conclusion: the cells isolated and cultured in this experiment are NSCs and fluoride can inhibit the proliferation of NSCs in primary cultured rat fetal cortex.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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