本文選題：氟化鈉 + 神經(jīng)干細胞　； 參考：《華中科技大學》2013年碩士論文

【摘要】：氟神經(jīng)毒性近年來受到廣泛關注，但其對神經(jīng)干細胞（neuralstemcells，NSCs）的直接毒性尚未見報道。本研究以小鼠來源神經(jīng)干細胞株-C17.2NSCs和原代大鼠胎鼠皮層NSCs為研究對象，研究氟化鈉（sodiumfluoride，NaF）對NSCs存活率、氧化應激及凋亡的影響，并檢測內質網(wǎng)應激（endoplasmicreticulumstress，ERS）相關蛋白的表達水平以探討ERS在NaF誘導NSCs凋亡中的作用，為研究氟的神經(jīng)毒性作用機制提供理論依據(jù)。 第一部分內質網(wǎng)應激在氟致C17.2神經(jīng)干細胞凋亡中的作用研究 目的：探討不同劑量NaF對C17.2NSCs存活率、氧化應激、凋亡、Caspase-3以及ERS相關蛋白GRP78、IRE1和CHOP表達水平的影響。 方法：體外培養(yǎng)的C17.2NSCs分別用預設濃度10、30、60和80mg/LNaF染毒24h后，采用MTT法測定存活率；經(jīng)10、30、60mg/LNaF染毒24h后檢測ROS、MDA水平和SOD活力、凋亡細胞核形態(tài)改變、Caspase-3表達水平以及ERS相關蛋白GRP78、IRE1和CHOP的表達水平。 結果：與對照組相比較，，30、60和80mg/LNaF染毒組存活率明顯下降（P0.05），80mg/LNaF染毒組存活率低于50%；10、30、60mg/LNaF染毒組ROS水平明顯升高（P0.05）；SOD活力顯著降低（P0.05）；30和60mg/LNaF染毒組MDA水平、凋亡率明顯增高（P0.05）；60mg/LNaF染毒組Caspase-3表達水平顯著降低（P0.05）；30和60mg/L染毒組IRE1、CHOP的表達水平明顯升高（P0.05）。 結論：一定劑量的NaF可抑制C17.2NSCs的增殖，誘導氧化應激的產生，并介導其凋亡的發(fā)生，同時內質網(wǎng)凋亡通路可能參與了氟對C17.2NSCs的損傷過程。 第二部分氟對原代大鼠胎鼠皮層神經(jīng)干細胞存活率的影響 目的：探討NaF對大鼠胎鼠皮層NSCs存活率的影響，確定后續(xù)實驗染毒劑量。 方法：采用DMEM/F12培養(yǎng)液對原代大鼠胎鼠皮層NSCs進行培養(yǎng)，NSCs特異性標志物巢蛋白（Nestin）對其進行鑒定；經(jīng)預設濃度40、80、120、200mg/LNaF染毒24h后，用MTT法測定細胞存活率。 結果：分離培養(yǎng)的細胞Nestin表達豐富；與對照組相比，各NaF染毒組細胞存活率均下降，差異具有統(tǒng)計學意義（P0.05）。 結論：本實驗所分離培養(yǎng)的細胞為NSCs；氟可抑制原代培養(yǎng)大鼠胎鼠皮層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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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