| The symptom of brain damage from acute anoxia is a common manifestation of acute carbon monoxide ( CO) poisoning. Researchers have paid close attention to the brain damage mechanism resulted from acute CO poisoning. Recent studies have shown the patterns of neuron death involve apoptosis and necrosis. Recently, biologists have recog-nized that apoptosis regulation is as complicated as cellular prolifera-tion. Apoptosis needs to initiate associated genes. It has been known that oncogene bcl - 2 and tumor suppressor gene p53 are the important apoptosis - associated genes. The function of bcl - 2 is to inhibit ap-optosis while the function of p53 is to accelerate apoptosis. In this study, we utilized flow cytometry to detect the expression of apoptosis - associated protein bcl - 2 and p53 in cerebral cortex of rats in a se-ries of designed time points after acute CO poisoning. The purpose was to explore the mechanism of brain damage after CO intoxication.Meterials and methods1. Experiment animal and group: twenty - five healthy Wistar rats weighing 180 ~220g without restraining sex were randomly divid-ed into 5 groups. There were five rats in each group, which was named as the time point, i. e. prompt, 1 hour, 24 hours and72 hoursafter exposure to CO as well as control group.2. Experiment reagents and equipments: CO gas , bcl -2 mouse monoclonal IgG, p53 rabbit polyclonal IgG, Fluorescein - conjugated Goat Anti - Moue IgG, Fluorescein - conjugated Goat Anti - Rabbit IgG and other reagents made in china. Flow cytometer, homeothermia centrifuger, blood gas analyzer, light microscope,CO quick detection tube (type A) and 02 detection tube (type B).3. CO poisoning model; The rats were put into the airtight bottle whose capacity is 5L and inhaled 2000 ~3000ppm CO for one hour to be acute CO poisoned. We detected the concentration of CO and 02 every twenty minutes in order to maitain their concentration constant.4. Sample collection; The blood of each poisoned rat was ob-tained from postbulbar venous plexus immediatly after exposure to CO in order to measure COHb level. Then the rat was anesthetized with 10% chloral hydrate by intraperitoneal injection and we separated cer-ebral cortex from cerebrum. Mono - cell suspension was prepared from the cerebral cortex of each rat. The cells were suspended in stain -application fluid for conservation after fixation with 1% paraformalde-hyde.5. Staining: Cell suspension was centrifuged and the supernatant was throw away. We added l0μl bcl ? mouse monoclonal IgG and p53 rabbit polyclonal IgG into cells respectively (1:10, control tubes were not added with this antibody) , incubating 30 minutes at room temperature, then we added 20μl goat anti - mouse IgG - FITC and goat anti - rabbit IgG - FITC ( 1 : 50 ) into cells after the cells were washed twice with stain - application fluid, incubating 30 minutes in dark condition at 0 ~4℃. The cells were washed twice with PBS and were conserved in the enviroment without light at 0 ~ 4℃ , waiting forbeing detected.6. Detection; The cell with the expression of bcl -2 or p53 pro-tein would send out fluorescence stimulated by argon ion laser. Fluo-rescence intencity value represented the expression level. Computer could draw diagram about the relation between cell number and fluo-rescence intencity. The MFI of positive tube minus that of control tube was the MFI representing the true expression of bcl -2 or p53 protein.7. Statistical analysis; The MFI was expressed as mean ?SE. Satistical significance was determined by Analysis of Variance (ANOV) , q test and correlation analysis. The level of significance was taken as P <0.05.Results1. The changes of the expression of bcl - 2 protein in cerebral cortex; There was marked increase in the expression at 1 hour after the exposure, which was significantly different from the other four groups (P <0.01) , then the expression decreased at 72 hours but it was still higher than control group.2. The changes... |
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