The Preliminary Study Of The Lethal Effect Of Oxygen Atoms On Rats' C6 Glioma Cells

Objective: To observe the influence of the rats, C6 glioma cells vitality under different inffect time and different concentration of the H₂O₂ , to explore the lethal effect and the possible effect mode of oxygen atoms on glioma cells.Method:1 Take the C6 rat glioma cells as the experimental object, make its, growth curve by MTT method and to understand its growth characteristics .2 Cell coulture Placed the rat's C6 glioma cells in the RPMI-1640 medium which containing 10% FBS and 100U/ml double-antibody, cultured them in the environment which has 37oC and 5% CO2. Waiting until the train occupies the 90% of the bottom, preparing the cell suspension and counting, adjusting the cell concentration of about 104/ml, to seed them in the 96 well culture block board.3 Divided the experimental groupsTo divide them into 5min group, 10min group and 15min group according to intervene time, and each time group was divided into normal control group, 1% H₂O₂ group, 2% H₂O₂ group, 3% H₂O₂ group, 4% H₂O₂ group and blank control group. Each group with 30 holes.Add 100μl medium in the blank control groups, hole,add 100μl cell suspension in the rest of the holes, cultured them in the environment which has 37oC and 5% CO2 for 4 hours.4 Measure the cells, vitalityFirst, to observe cells, morphological before add the MTT and the dyeing before add the MTT of the each group by the inverted fluorescence microscope, determined C6 cells vitality initially. To intervente groups by reservation.Washing each well 3 times with culture medium carefully after interventioned scheduled time. Add culture medium in each well 200μl. Culturing them for 4 hours ,to observe and photographe them by the inverted fluorescence microscope. Add 15μl 5mg/ml MTT solution to each well, cultured them in the 37oC, 5% CO2 environment for 4 hours, to observe and photographe them by the inverted fluorescence microscope. Drawing each wells, medium carefully, to add 150μl DMSO to each well (DMSO), oscillated them 10 minutes by micro oscillator, measured each holes, absorbance (OD) at 570nm by microplate reader. All above experiments were repeated three times.5 The rats C6 glioma cells, ultrastructural changes after the treatment of H₂O₂Each group was separately with 0.125% trypsin +0.01% EDTA digested and prepare for cell suspension, to centrifuge by 1500rpm for 10 minitues, the supernatant was discarded, leaving the cells on the bottom of tube, plus 2.5% glutaraldehyde for 4 hours, to wash it with pH 7.13 0.11 molPL PBS buffer and overnight, we should gradually and slowly dehydration, immersion and embedding it by Epon81, ultrathin section, uranyl acetate (Pb) double staining,to observe cells, ultrastructural changes by Hitachi H-7000 transmission electron microscope .6 Statistical analysisTake OD values of each group as mean±Standard Deviation (x±s)(Tab.2), Analysis each group,s data by factorial experimental design ANOVA (F test). Analysis pairwise comparison between groups by SNK test methods and take p <0.05 as significant difference.Results:1 Make the growth curve by MTT methodTo draw growth curve with culture time as the horizontal axis, the average OD value as the vertical axis. Curve shows the C6 cells was logarithmic and plateau, the growth curve has good repeatability.2 The vitality change of C6 cells in experimentTo observe cells by the inverted fluorescence microscope , the gap between cells increased, cell bodies became round,the contact between cells lost, some cells floating ,cells lose their normal growth patterns in experiment contrast to the normal growth cells. After adding MTT , the staining was not significant and some don't even staining in the intervention group contrast to the normal control group, the normal control group were staining significantly. Prompt the vitality of the intervention group cell lost .3 Statistical analysisThe analysis data of OD are in the table two(Tab.2). Analysis each group's data by factorial experimental design ANOVA (F test), p <0.05,there has a significant difference between the groups.Pairwise comparison between groups using SNK-q test method, the normal control group and the rest of the group's p <0.05, there has a significant difference;between the rest of the group, p> 0.05, they don,t have significant difference.Conclusion:1 C6 cell growth is stability, which can meet the requirement of experiment.2 The cell of the intervention group loss normal cell morphology, does not stained by MTT , cell viability decreased or lost compared with the control group .3 The effect on C6 cells was no significant difference between the groups which were interventioned by H₂O₂ and the blank control groups, can make the C6 cell viability completely lost.There has significant difference between the normal control group and the other groups.4 In this experiment,each concentration of H₂O₂ has killing effect on C6 cells, and its mechanism is mainely in the cytotoxicity.