氧化应激诱导神经干细胞自噬发生的实验研究

发布时间：2018-02-13 11:35

  本文关键词： 神经干细胞 氧化应激 自噬　出处：《吉林大学》2012年硕士论文　论文类型：学位论文

【摘要】：神经干细胞（neural stem cells，NSCs）是当今生命科学研究的热点之一。NSCs治疗应用主要体现在：细胞移植治疗、细胞替代和基因治疗及其自我修复治疗。随着NSCs研究的深入，发现NSCs移植后在炎症反应、氧化应激较重的微环境下NSCs的存活率均较低。 实验目的：观察NSCs在氧化应激状态下是否有自噬的发生，为研究神经干细胞在氧化应激状态下的生存机制提供新的依据。 实验方法：分离、培养Wistar孕鼠的神经干细胞，对生长良好的神经干细胞球进行Nestin免疫细胞化学染色。应用免疫细胞化学技术对神经干细胞分化情况进行鉴定。应用MTT法检测不同浓度H_2O_2对NSCs生存率的影响，计算神经干细胞的生存率。通过吖啶橙(AO)染色观察细胞内酸性自吞噬空泡的积聚。应用透射电子显微镜对比观察各组NSCs超微结构的变化。通过Western blot技术检测各组LC3蛋白的表达情况。 实验结果：我们成功的分离、培养了NSCs；MTT比色法结果显示，不同剂量H_2O_2作用于NSCs12h或24h后，随着H_2O_2浓度的增加，细胞生存率逐渐降低，并呈剂量时间相关性，而0.5mM、1mM H_2O_2组与对照组相比具有统计学意义。与对照组相比，0.5mM H_2O_2组AO染色结果可见红色荧光阳性结果显著增强，细胞酸性自吞噬空泡产生明显增多。0.5mM H_2O_2作用于NSCs12h后，应用透射电子显微镜检测自吞噬空泡的形成。结果显示，对照组NSCs表现为正常细胞形态，细胞核呈规则圆形。0.5mM H_2O_2作用后细胞体积变大，，细胞膨胀，有明显的自吞噬空泡生成。0.5mM H_2O_2作用于NSCs12h后，通过Western Blot检测LC3蛋白表达。结果表明，与对照组相比，0.5mM H_2O_2作用后LC3-Ⅱ蛋白表达明显增强。结论：氧化应激可以通过一些通路来诱导神经干细胞自噬的发生，这为探讨神经干细胞生存与死亡机制的研究提供了新的理论依据。
[Abstract]:Neural stem cells (NSCs) is one of the hotspots in life sciences. The therapeutic applications of NSCs are mainly reflected in cell transplantation, cell substitution, gene therapy and self-repair therapy. It was found that the survival rate of NSCs was lower in microenvironment with severe oxidative stress and inflammatory response after NSCs transplantation. Objective: to observe whether autophagy occurs in NSCs under oxidative stress, and to provide a new basis for studying the survival mechanism of neural stem cells under oxidative stress. Methods: neural stem cells of Wistar pregnant mice were isolated and cultured. The differentiation of neural stem cells was identified by immunocytochemistry. The effect of different concentrations of H _ 2O _ 2 on the survival rate of NSCs was detected by MTT method. The survival rate of neural stem cells was calculated. Acridine orange (AOO) staining was used to observe the accumulation of acridine autophagocytic vacuoles. The ultrastructural changes of NSCs were observed by transmission electron microscopy (TEM). The ultrastructure of NSCs was detected by Western blot technique. Expression of LC3 protein. The results showed that the cell survival rate decreased with the increase of H _ 2O _ 2 concentration in NSCs12h or 24 h after different doses of H _ (2) O _ (2) were treated with NSCs12h or 24 h, and the cell survival rate decreased with the increase of H _ 2O _ (2) concentration, and the cell survival rate decreased with the increase of H _ 2O _ 2 concentration. In comparison with the control group, the AO staining results of 0.5 mm M H _ 2O _ 2 group and 0.5 mm M H _ 2O _ 2 group showed that the red fluorescence positive results were significantly increased, and the vacuoles produced by acid-induced autophagocytosis increased significantly. 0.5 mm H _ 2O _ 2 significantly increased the effect of NSCs12h. The formation of autophagocytic vacuoles was detected by transmission electron microscope. The results showed that the NSCs of the control group showed normal cell morphology, the nucleus of the control group was regular round, 0.5mm H _ 2O _ 2, the cell volume became larger and the cells expanded. The expression of LC3 protein was detected by Western Blot, and the expression of LC3 protein was detected by Western Blot. Compared with the control group, the expression of LC3- 鈪

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