Conjoint Study Of MTT Method,Voltage Threshold Measurement Method And High Content Screening Technology For Effect Of Nanosilver On Toxicity And Electrical Excitability Of PC12 Cell Clusters

Silver nanoparticles?SNPs?are widely used in cosmetics,food packaging and medical fields due to its good antibacterial properties.Studies have shown that nanoparticles with a diameter of less than 35 nm can cross the blood-brain barrier or enter the brain through the olfactory bulb and accumulate,which has toxic effects on the central nervous system.Thus it is very important to understand the cytotoxicity of SNPs.This thesis mainly studied the changes of cell electrical excitability,and the mechanism of SNPs'influence on the electrical excitability of PC12 cells.In this thesis,the 20nm SNPs were prepared according to the previous method of our group.The effect of different concentrations of 20nm SNPs on PC12 cells?rat adrenal pheochromocytoma cells?on cell proliferation rate was evaluated by MTT method.The concentration and duration of SNPs which could lead to 1 class cytotoxicity were found out,Finally,high-content screening system was utilized to study the changes of ROS,mitochondrial membrane potential,intracellular Ca²⁺content,neurite length and area of PC12 cells.Combined with the cell proliferation and voltage threshold of PC12 cells,the effect of SNPs on PC12 cell toxicity and electrical excitability and the related mechanism were studied.The main works of this thesis are introduced as follows:?1?SNPs were prepared by reducing silver nitrate with sodium borohydride.The average diameter of the SNPs particles was 21.45±2.72 nm.The ultraviolet absorption peak of the SNPs measured by the microplate reader was 389 nm,and the concentration of the SNPs solution measured by plasma emission spectrometer were 1319 mg/L.?2?MTT method was used to evaluate the influence on cell proliferation 20nm,different concentrations?5,25,50,100 and 200?M?of SNPs were applied with different durations?0.5,0.75,1,4,8,12,24,48 and 72h?to PC12 cells.The results showed that after PC12 cells were treated with SNPs at at 5,25,50 and 100?M for 0.5,0.75,1,4,8,12,24,48 and 72h,all of the cytotoxicity grades were 0.After PC12 cells were treated with SNPs at 5,25,50,100?M for 48h and 72h,all of the cytotoxicity grade is 0.After PC12 cells were treated with SNPs at 200?M for.5,0.75,1,4,8,12 and 24h,all of the cytotoxicity grade is 0.While when the treatment duration were prolonged to 48h and 72h,SNPs produced grade 1 cytotoxicity to PC12 cells.?3?The threshold voltage of PC12 cells after different durations of 20-nm and 200-?M SNPs.were measured via the voltage threshold measurement method which was established by our group.The results showed that the standard threshold for PC12 cell clusters was 37.8±1.09mV.After the treatment of 200-?M SNPs for 0.5¹h,the threshold voltage of PC12 cells increased continuously.After 1h of SNPs treatment,the threshold voltage of PC12 cells increased to 96.4±2.19 mV.After 1⁴8h of 200-?M SNPs treatment,the threshold voltage of PC12 cells decreased continuously.After 48h of SNPs treatment,the threshold voltage of PC12 cells decreased to 23.2±1.92 mV.After treated with 200?M SNPs for 72 h,PC12 cells on MEA died,atrophied or rounded off from the electrode area.?4?High-content screening system was used to study the ROS level,intracellular Ca²⁺content,changes in neutite length and area,and mitochondrial membrane potential of PC12 cells after 0.5,0.75,1,4,8,12,24,48 and 72h treatment of 20-nm and 200-?M SNPs.The results of staining data showed that after 0.5¹h treatment of 200-?M SNPs,SNPs can reduce ROS content,increase the neurite length and area and mitochondrial membrane potential of PC12 cell,increase the threshold voltage.After 4⁷2h of SNPs treatment,SNPs reduced the neurite length and area and mitochondrial membrane potential,increase ROS content of PC12 cells,decrease the threshold voltage.