| Objective:The liver cell nucleus DNA degeneration situation of rabbit after death were tested by using improved comet assay, to study the relationship between the cell nucleus DNA content change and postmortem interval, and to find the application value of DNA degeneration situation in estimation of early time of death, to provide a new method and technology and science basis for deducing the time since death on forensic practice work.Method:The necks of adult active and well big rabbits were breaken and dislocated, then put the dead animals in the room. The materials were drawn from livers of the dead rabbits in vivo at 3-hours- interval within 30 hours after death. The was carried on using comet assay improved to the samples of liver, then dyed, the electrophoresis images were observed and captured under fluorescence microscope. The comet images were analyzed by CASP image analysis software, tail length(TL) and tail moment(TM) were the analysis parameters. The data obtained by image analysis was analysed using SAS 9.0 satistical analysis software.Result:The comet assay improved was successful, and reduced the experimental tedious degree , the effective of experiment was upgraded . The experiment demonstrated that comet images of cell nucleus DNA of rabbits'livers appeared after elecrophoresis, in 0-18h of PMI comet images became lamproser and the comet rear part grew and widened gradually along with the death time passage, then became cloudiness slowly, finally comet images couldn't be identified accurately. The most early time of comet images appearance was at 3h after death. The image analysis shew that the parameter numerus of TL and TM increased gradually and achieved the peak at 18h after death, then appeared dropping and in 21-27h undulated incertain scope, finally the numerical data couldn't be examinated at 30h of PMI. The statistical analysis demonstrated that in 0-18h of PMI the values of TL and TM appeared significant difference among different death time period, in 3-18h of PMI the correlation coefficient of PMI and the two respectively achived 0.99614 and 0.99077, p﹤0.01, by regression analysis the two parameters with the death time became the linear relation nearly; but in 21-27h of PMI non- significant difference. Conclusion:1.The comet assay can detect cell nucleus DNA damage fast , conveniently, sensitively, accurately. This method and technology improved would make experiment be done well furthermore and the result analysis accuracy and sensitivity would be upgrade greatly with image ananlysis software. Come assay has high application value in estimation of early time of death.2. After death, cell nucleus DNA of liver at 3h of PMI stars to degrade because of autolysis, and in 9-18h the DNA degradation speeds up obviously, then appears section of relative stabilization periods, finally dissolves vanishing.3. The tail length and tail moment had reflected good the DNA degradation situation, the two are very good targets to study cell nuleus DNA content change after death using comet assay.4.There are certain curve relations between cell nucleus DNA content change and death time, studying the two relationships will be vital significance on the early death time inference.5.The postmortem DNA fragmentation observed by using comet assay hopes into a more convenient method and technology in deducing early time of death.6.The image analysis software provides the convenience for the comet assay result analysis. |
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