不同粒径纳米氧化铝致神经细胞死亡方式的研究

发布时间：2019-06-11 12:25

【摘要】：【目的】通过体外实验研究不同粒径纳米氧化铝诱导神经细胞死亡的方式，并且初步探讨其主要的死亡方式。【方法】1.观查10nm氧化铝和50nm氧化铝粒子在溶液中的分散情况以及微米铝颗粒和纳米碳颗粒进入细胞的情况：1）将50nm Al2O3，10nm Al2O3储存液超声10分钟，在电镜下观察其分散情况；2）10nm-C，10μmAl2O3染毒细胞，终浓度为500μmM，观察颗粒进入细胞的情况。2.纳米氧化铝对神经细胞活力及死亡方式的形态学观察：1）原代培养神经细胞，染毒，设空白对照组，10nm-C组，AlCl3铝，10μmAl2O3组，50nmAl2O3组，10nmAl2O3组，染毒浓度为0.5mM继续培养48h；2）检测细胞活力；3）显微镜下观察细胞；4）AO-EB双染，观察细胞凋亡与坏死；5）MDC染色观察细胞自噬。3.透射电镜确定纳米氧化铝诱导神经细胞的死亡方式：1）细胞培养，，染毒；2）收集细胞，制作电镜标本；3）电镜下观察细胞超微结构。4.细胞凋亡与坏死的检测：1）Annexin-V/7-AAD双染法检测神凋亡率与坏死率；2）分别检测凋亡相关基因Caspase-3,Cyt-c和坏死相关基因Rip1,Nf-κB的表达情况。5.自噬的检测：1）检测自噬小体的发生情况；2）检测自噬相关基因LC3-Ⅱ和Atg-5的表达。6.染毒后分别加入凋亡特异性抑制剂zVAD-fmk，坏死特异性抑制剂Nec-1，自噬特异性抑制剂3-MA，分别检测细胞活力。【结果】1.观查10nm氧化铝和50nm纳米氧化铝粒子超声后在溶液中的分散情况以及微米铝颗粒和纳米碳颗粒进入细胞情况：1）纳米氧化铝经超声混匀后静置前后纳米颗粒的形状及分散程度没有明显变化，符合实验染毒要求；2）纳米碳和微米氧化铝颗粒均可以进入神经细胞。2.纳米氧化铝对神经细胞活力及死亡方式的形态学观察：1）染毒细胞活力均有明显下降（P0.05），且随着氧化铝粒径的减小，细胞活力逐渐降低。2）镜下观察正常对照组和AlCl3组的细胞大小形态均一,胞膜完整,胞质丰满,细胞连接丰富；10nm-C组和10μm Al2O3组,细胞减少，胞间连接减少甚至消失,胞膜破裂、胞质浓缩,但细胞体基本完整，突触变短，但清晰可见；50nmAl2O3组和10nmAl2O3组细胞数目少,细胞间连接中断,细胞肿胀和细胞皱缩并存，细胞体不完整，突触消失，且10nmAl2O3染毒组，细胞崩解，碎裂。3）AO-EB染色神经细胞，可见正常对照组神经细胞呈绿色的均匀荧光，有较多的轴突和树突,没有凋亡或坏死的细胞，10nm-C组和10μm Al2O3组神经细胞数量减少，染色质聚集，固缩呈较浓的黄绿色，看到少量的凋亡和坏死细胞，在50nm Al2O3组、10nm Al2O3组有些神经细胞的胞核染成鲜红色或桔红色荧光，并且胞浆也染成淡红色，部分细胞结构不清或溶解，胞膜破裂，细胞崩解，大量的凋亡和坏死细胞。4）正常对照组和AlCl3组，仅见到少量的MDC阳性细胞，并且每个细胞中含的AV数量很少；10nm-C组和10μmAl_2O_3组，MDC阳性细胞数明显增加，每个细胞中AV数量也较多；50nmAl_2O_3组和10nmAl_2O_3组细胞数目少,胞体变圆，变大，MDC阳性细胞布满视野，且充溢着大量AV。3.电子显微镜下验证纳米氧化铝诱导神经细胞的死亡方式：通过对染毒后神经细胞超微结构的观察可以确定其死亡方式有凋亡，坏死和自噬。4.细胞凋亡与坏死的检测：1）空白组相比，10umAl_2O_3组、50nmAl_2O_3组和10nmAl_2O_3组的细胞凋亡率与坏死率显著增加，具有统计学意义（P0.05），且随着纳米氧化铝粒径的减小，凋亡率与坏死率有递增的趋势。2）与空白组比，染毒组的凋亡相关基因Caspase-3,Cyt-c,坏死相关基因Rip1,Nf-κB表达均有变化，具有统计学意义，且具有粒径依赖性。5.自噬的检测：1）与空白对照组相比，10umAl_2O_3组、50nm Al_2O_3组和10nm Al_2O_3组的细胞自噬小泡数量增加，平均荧光强度峰值向右偏移（P0.05），且随着纳米氧化铝粒径的减小，自噬率有递增的趋势。2）与对照组相比，染毒组自噬相关基因LC3-Ⅱ,ATG-5表达均增高（P0.05），且具有粒径依赖性。6.细胞主要死亡方式的初步测定：加入3MA后，细胞损伤后修复程度最高。【结论】1.纳米氧化铝使神经细胞活力下降；2.纳米氧化致神经细胞死亡方式有凋亡坏死和自噬；3.纳米氧化铝致神经细胞死亡程度具有粒径依赖性，随着其粒径的减小，其损伤程度加重；4.初步认定纳米氧化铝致神经细胞死亡的主要方式为自噬。
[Abstract]:[Objective] To study the way to induce the death of nerve cells with different particle size of nano-alumina by in vitro experiment, and to probe into the main methods of death. [Method] 1. To observe the dispersion of 10 nm alumina and 50 nm alumina particles in solution and the condition of micro-aluminum particles and nano-carbon particles to enter the cell:1) The 50 nm Al2O3 and 10 nm Al2O3 storage solution were sonicated for 10 minutes, and the dispersion was observed under the electron microscope;2)10 nm-C,10. m 1) detecting the occurrence of autophagy bodies;2) detecting the expression of autophagy related genes LC3-II and Atg-5. .[Results] 1. The dispersion of the 10 nm alumina and 50 nm nano-alumina particles in the solution and the entry of the micro-aluminum particles and the nano-carbon particles into the cell:1) The shape and the dispersion degree of the nano-alumina particles and the nano-carbon particles were not significantly changed after the ultrasonic mixing of the nano-alumina, and it was in accordance with the requirements of experimental exposure;2) Both the nano-carbon and the micron-sized alumina particles can enter the nerve cell.2) The morphology of the nano-alumina on the vitality of the nerve cells and the mode of death:1) The cell viability and the mode of death of the cells were observed:1) The cells were intact, the cytoplasm is full, the cell connection is rich, the cell membrane is broken, the cytoplasm is concentrated, but the cell body is basically intact, the synapse is short, but the cell body is clearly visible; the number of cells in the 50-nm-Al2O3 group and the 10-nnAl2O3 group is small, the intercell connection is interrupted, and the cell is swollen. In the normal control group and in the AlCl3 group, only a small amount of MDC positive cells were observed, and the number of AV in each cell was very small; the number of the AV in each cell was small; the number of AV in each cell was much higher; the number of cells in each cell was much higher; the number of cells in each cell was increased; the number of cells in the 50 nm Al _ 2O _ 3 group and the 10 nm Al _ 2O _ 3 group was small, the number of the cells in each cell was increased, and the MDC positive cells were filled with the field of view, and a large number of AV.3.3. The apoptosis, necrosis and autophagy of the cells were determined by the observation of the ultrastructure of the nerve cells after exposure. High.[Conclusion] 1. Nano-alumina has decreased the vitality of the nerve cells,2. The death of the nerve cells induced by nano-oxide has the effects of apoptosis and autophagy.3. The degree of cell death caused by the nano-alumina has a particle size dependence, and the degree of damage to the nerve cells is increased with the decrease of its particle size;4. The primary way of the preliminary determination of the death of the nerve cells caused by the nano-alumina is:
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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