纳米氧化铝致神经元细胞线粒体自噬的研究

发布时间：2018-04-25 16:10

本文选题：纳米氧化铝 + 自噬　；参考：《山西医科大学》2014年硕士论文

【摘要】：【目的】通过体外培养Wistar乳鼠皮质神经元细胞，研究纳米氧化铝对神经元细胞线粒体自噬的影响，初步探讨线粒体自噬在纳米氧化铝致神经元细胞损伤中的作用。【方法】1.原代培养Wistar乳鼠皮质神经元细胞，对其进行纯度鉴定及细胞活力测定：（1）免疫组化法检测原代培养的神经元细胞纯度；（2）13nm氧化铝0.5mmol/L染毒0、12、24和48h后，LDH法检测培养液上清中乳酸脱氢酶（LDH）活性。2.纳米氧化铝对神经元细胞线粒体的损伤：（1）流式细胞仪检测染毒不同时间后细胞内线粒体膜电位（MMP）、活性氧（ROS）的变化情况；（2）透射电镜（TEM）观察纳米氧化铝对神经元细胞线粒体超微结构的影响。3.纳米氧化铝致神经元细胞自噬的检测：（1）单丹酰戊二胺(MDC)荧光染色观察染毒不同时间后细胞内自噬小体的形成；（2）Western blot检测自噬相关蛋白Beclin1、微管相关蛋白轻链3（LC3）Ⅱ/Ⅰ的表达；（3）自噬最明显组加自噬抑制剂Spautin-1后自噬的变化情况。4.纳米氧化铝致神经元细胞线粒体自噬的检测及自噬抑制剂Spautin-1对神经元细胞凋亡率的影响：（1）TEM观察染毒后细胞内自噬及线粒体自噬的超微结构；（2）溶酶体荧光探针Lysotracker和线粒体荧光探针Mitotracker共定位观察溶酶体与线粒体的融合情况；（3）加抑制剂Spautin-1后，流式细胞术检测细胞凋亡率的变化。【结果】1.神经元纯度及纳米氧化铝对其活力的影响：（1）神经元细胞经免疫组化实验纯度鉴定95%以上属于神经元细胞；（2）与正常对照组相比，纳米氧化铝0.5mmol/L染毒12和24h后上清液中LDH活性均明显增加（P0.05）,但随着染毒时间的延长LDH活性呈下降趋势，染毒48h下降到接近正常对照组水平。2.纳米氧化铝对神经元细胞线粒体的损伤：（1）流式检测活性氧结果显示，染毒12h和24h组细胞内活性氧水平均高于正常对照组，且具有统计学意义（P0.01），细胞内活性氧水平呈上升趋势，而在48h时有下降趋势，且接近正常对照组水平；线粒体膜电位结果显示，随着染毒时间的延长，细胞内线粒体膜电位呈下降趋势，且均具有统计学意义（P0.01）；（2）细胞超微结构显示，纳米氧化铝组线粒体分布不均匀并且出现变形、肿胀及空泡化，且随着染毒时间的延长，，其损伤程度越严重。3.纳米氧化铝对神经元细胞自噬的影响：（1）MDC对神经元细胞进行荧光染色，可见正常对照组细胞几乎没有MDC荧光颗粒，而纳米氧化铝组有明显的MDC荧光颗粒且随着染毒时间的延长，MDC阳性荧光颗粒增加，加自噬抑制后MDC荧光颗粒减少；（2）与正常对照组相比，纳米氧化铝染毒不同时间后Beclin1蛋白表达水平呈上升趋势且均具有统计学意义（P0.05）。 LC3Ⅱ/Ⅰ蛋白表达水平随着染毒时间的延长呈上升趋势且与正常对照组相比均有统计学意义（P0.05）。加自噬抑制剂后Beclin1和LC3Ⅱ/Ⅰ表达水平均下降且有统计学意义（P0.05）。4.纳米氧化铝对神经元细胞线粒体自噬的影响及自噬抑制剂Spautin-1对神经元细胞凋亡率的影响：（1）电镜图显示，正常对照组细胞器结构完整，没有双层膜包裹线粒体结构，而纳米氧化铝组受损的线粒体被双层膜包裹形成线粒体自噬体的结构；（2）荧光共定位结果显示对照组酸性溶酶体较少，仅见很少的线粒体与溶酶体融合的现象，而纳米氧化铝染毒组可见线粒体与溶酶体融合，且随着染毒时间的延长，融合现象越来越明显；（3）纳米氧化铝可使神经元细胞凋亡率增加且具有统计学意义（P0.01），加入自噬抑制剂Spautin-1后凋亡率有所上升且具有统计学意义（P0.01），而抑制剂本身对细胞凋亡率没有影响。【结论】1.纳米氧化铝可致神经元细胞线粒体损伤； 2.纳米氧化铝可引起神经元细胞发生自噬； 3.受损的线粒体可能通过自噬（即线粒体自噬）作用清除，自噬可能通过降低细胞凋亡而对细胞起到一定的保护作用。
[Abstract]:[Objective] to study the effect of nano alumina on mitochondrial autophagy of neuronal cells by culture of Wistar rat cortical neurons in vitro, and to explore the role of mitochondrial autophagy in the neuronal cell injury induced by nano alumina.
[Methods] 1. primary cultured cortical neurons of Wistar rats were cultured, and their purity and cell viability were determined. (1) the purity of primary cultured neurons was detected by immunohistochemistry. (2) after 13nm alumina 0.5mmol/L was infected with 0,12,24 and 48h, LDH method was used to detect the lactic dehydrogenase (LDH) activity.2. nano alumina in the supernatant of the culture liquid. Damage of mitochondria in the cells: (1) flow cytometry was used to detect the mitochondrial membrane potential (MMP) and active oxygen (ROS) after exposure to different time. (2) transmission electron microscopy (TEM) observation of the effect of nano alumina on the ultrastructure of mitochondria of neuronal cells: detection of autophagy induced by.3. Nano-Al2O3 cells: (1) Dan Danxian Amyl two amine (MDC) fluorescence staining was used to observe the formation of autophagic corpuscles in cells after different time; (2) Western blot was used to detect autophagy related protein Beclin1, microtubule related protein light chain 3 (LC3) II / I; (3) autophagy was the most significant group with autophagy after the autophagy of autophagy,.4. nano alumina induced mitochondria in neuronal mitochondria Autophagy detection and the effect of autophagy inhibitor Spautin-1 on the apoptosis rate of neuron cells: (1) TEM observed the ultrastructure of autophagy and mitochondrial autophagy in the cells after exposure; (2) the fusion of lysosome and mitochondria was observed by the lysosomal fluorescent probe Lysotracker and the mitochondrial fluorescence probe Mitotracker; (3) adding inhibitor Spautin After -1, the apoptosis rate was detected by flow cytometry.
[results] the purity of 1. neurons and the effect of nano alumina on its vitality: (1) more than 95% of neuron cells were identified as neuron cells by immunohistochemistry. (2) the activity of LDH in the supernatant after 12 and 24h was significantly increased (P0.05) compared with the normal control group (P0.05), but with the prolongation of the time of exposure L The activity of DH decreased and the 48h decreased to the normal control group, which was close to the normal control group of.2. nano alumina. (1) the flow detection reactive oxygen species showed that the active oxygen water in the cells of the 12h and 24h groups was higher than the normal control group, and it was statistically significant (P0.01), and the intracellular active oxygen level was rising. The potential was decreased at 48h and was close to the normal control group, and the mitochondrial membrane potential showed that the mitochondrial membrane potential decreased with the prolongation of the exposure time, and all had statistical significance (P0.01). (2) the ultrastructure of the cells showed that the mitochondria in the nanomai Al2O3 group were unevenly distributed and swelled. And cavitation, and with the prolongation of time, the more serious the damage was, the effect of.3. nano alumina on the autophagy of neuron cells: (1) MDC was stained with fluorescence of neuron cells, and there were almost no MDC fluorescent particles in the normal control group, while the nano alumina group had obvious MDC fluorescent particles and with the prolongation of the time of exposure. MDC positive fluorescent particles increased and MDC fluorescent particles decreased after autophagic inhibition. (2) compared with the normal control group, the expression level of Beclin1 protein showed an upward trend and was statistically significant (P0.05). The expression level of LC3 II / I protein increased with the prolongation of the time of exposure and was compared with normal control. The results were statistically significant (P0.05). The expression levels of Beclin1 and LC3 II / I were decreased and the effects of.4. nano alumina on mitochondrial autophagy and the effect of autophagy inhibitor Spautin-1 on the apoptosis rate of neuron cells were statistically significant (P0.05): (1) a normal control group of organelles The structure of mitochondria was complete without a double layer membrane, and the damaged mitochondria in the nano alumina group were encapsulated by the double layer membrane to form the mitochondria autophagosome structure. (2) the fluorescence co localization results showed that the acid lysosomes were less in the control group, only a few mitochondria were fused with the lysosomes, and the nano alumina dyeing group could see the mitochondria. Fusion with lysosomes, and with the prolongation of exposure time, the fusion phenomenon is becoming more and more obvious. (3) nano alumina can increase the apoptosis rate of neuron cells and have statistical significance (P0.01). After adding autophagy inhibitor Spautin-1, the apoptosis rate has increased and has statistical meaning (P0.01), but the inhibitor itself has no shadow on the cell apoptosis rate. Ringing.
[Conclusion] 1. nano alumina can cause mitochondrial damage in neurons.
2. nano alumina can induce autophagy in neurons.
3. the damaged mitochondria may be eliminated by autophagy (mitochondrial autophagy). Autophagy may protect cells from apoptosis.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R994

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