Different Ways Of Particle Size Of Nanometer Alumina To Neuronal Cell Death

[Objective] Observe the ways of neuronal cell death which induced by different particle size ofnanometer alumina and find the main way preliminary.[Methods] 1. Investigate the dispersion of 10nm-alumina and 50nm-alumina particles afterultrasound in solution and situation of micron aluminum particles and nanometer carbonparticles into the cells. 1) prepare the 10nm-C of 10μm Al₂O₃, 50nm Al₂O₃, 10nm Al₂O₃solutionwhich concentration are 0.1Mm. 2) 50nm of Al₂O₃and 10nm of Al₂O₃stock solution wasultrasound and observe the dispersion at the electron microscope . 3) neuronal cell exposed to10nm-C,10μm Al₂O₃at final concentrations of 500μmM. 2. Observe the viability and deathways of the neural cell, 1) primary cultured neuronal cell, and treated with the control group, the10nm-C group, AlCl310μm-Al₂O₃group, 50nm-Al₂O₃group, 10nm-Al₂O₃group at finalconcentration is 0.5mM, cultured for 48h. 2) detect the cell viability. 3) observe the cells underthe microscope. 4) AO-EB double staining. 5) MDC staining. 3. Determine neuronal cell deathmode which induced by the nanometer aluminum, observe cell ultrastructure. 4. Apoptosis andnecrosis of detection, 1) using Annexin-V/7-AAD to detect the apoptosis rate and necrosis rate. 2)Detect the expression of apoptosis-related genes Caspase-3,cyt-c and necrosis associated genesRip1,NF-κB. 5. Detect autophagy, detection the expression of autophagy related gene ofLC3-II, atg-5. 6. Add zVAD-fmk, Nec-1 ,3-MA respectively after exposed, detect cell viabilityrespectively.[Results] 1. Investigate the dispersion of 10nm-alumina and 50nm-alumina particles afterultrasound in solution and situation of micron aluminum particles and nanometer carbonparticles into the cells, 1) nanometer alumina by ultrasonic mixing the dispersion did not changesignificantly, and consistent with experimental exposure requirements; 2) nanometer carbon andmicron alumina can into neural cell. 2. Observe the viability and death ways of the neural cell ,1) compared with control group , the cell viability of exposed groups were was significantlydecreased （P <0.05）, and with the alumina particle size decrease, the cell viability was decreased.2) in control group and AlCl3group, the cell size were homogeneous, the membrane integrity ,cytoplasm uniform, synaptic connections close, in 10nm-C group and 10μm Al₂O₃group, thenumber of cells reduced and cell cytoplasm condensed and membrane ruptured, but the cell bodywere basic integrity, synapse is short, but clearly visible. in 50nm-Al₂O₃groups, and 10nmAl₂O₃group , a small number of connections between cells interrupted, the coexistence of cellswelling and cell shrinkage, cell The body is incomplete, the synapses disappear, and 10nmAl₂O₃exposed groups, cell disintegration, fragmentation. 3) AO-EB staining showing that thenormal control group of nerve cells showed a uniform fluorescent green, more axons and dendrites, can not find apoptosis or necrosis; in 10nm-C group and 10μm Al₂O₃group,thenumber of cell reduced, chromatin aggregation, a small number of apoptotic and necrotic cells,in the group 50nm Al₂O₃and 10nm Al₂O₃group, some nuclei stained bright red or orange-redfluorescence, there was a lot of apoptotic and necrotic cells. 4) normal control group and AlCl3group, only to see a small amount of MDC-positive cells, and AV in each cell containing a smallnumber ; 10nm- group C and 10μm Al₂O₃group, the number of MDC-positive cells increasedsignificantly, in the each MDC-positive cell, the number of cells in AV is greater; 50nm-Al₂O₃groups, and 10nm-Al₂O₃group, cell number reduced, cell body rounded and larger. 3. Verifyneuronal cell death manners which is induced by the nano meter alumina:by observe theultrastructure of neural cell after exposure, can determine that manners of death includeapoptotic, necrosis and autophagy. 4.The detection of apoptosis and necrosis, 1) compared tothe control group, 10umAl₂O₃group, 50nmAl₂O₃group and 10nmAl₂O₃groups apoptosis ratewas significantly increased,（P <0.05）, with the nano-alumina particle size decreases, theapoptosis rate increased; 3) compared to the control group, the expression of apoptosis-relatedgenes Caspase-3,cyt-c and necrosis associated genes Rip1, NF-κB all has changed , （P<0.05）,and dependent particle size. 5. Autophagy detection: compared with the control group, treatedgroup the expression of autophagy related gene LC3II, ATG-5 were both increased（P<0.05）, andhas particle size dependence. 6. Determine the main manner of cell death preliminary, afterjoining 3MA, the injury cells have the highest degree of repairmen.[Conclusion] 1. 50nm-alumina and 10nm-alumina conform to experimental requirements afterultrasound. Nanometer and micrometer particles are able to enter the cells, carbon nano meter andmicron aluminum can be used as a control group. 2. Nanometer alumina can cause neuronalcell toxicity, and have particle size dependence. 3. Nanometer alumina induced neuronal celldeath manners include apoptosis, autophagy and necrosis. 4. We find that the main neuronalcell death manner which is induced by nanometer alumina is autophagy.