【作者】 郝禄贵；

【导师】 邓世雄； 赵新才；

【作者基本信息】 重庆医科大学 ， 法医学， 2007， 硕士

【摘要】 目的:应用改良彗星试验方法检测兔死后肝细胞核DNA的降解情况,研究死后细胞核DNA含量变化与死亡时间的关系,从而探讨死后细胞核DNA含量改变在早期死亡时间推断中的应用价值。方法:健康成年大白兔经断颈错位处死后,置于室温下,分别在处死后0h、3h、6h、9h、12h、15h、18h、21h、24h、27h、30h共11个时间点进行在体肝脏组织取材,并按相应时间段分组共有11个组别。应用改良彗星试验方法技术对各组样本进行电泳,染色,在荧光显微镜下对电泳图像进行观察、摄像,再用CASP图像分析软件对彗星图像尾部情况进行分析,采用尾长(TL)、尾矩(TM)两个参数作为主要的分析指标,最后应用SAS 9.0统计分析软件对图像分析所得数据进行统计学相关回归分析。结果:应用改良彗星试验共制得凝胶体528块,无一例损坏脱落,试验的繁琐程度得到明显的减轻、实验时间明显缩短。彗星试验显示:兔死后肝细胞核DNA经过单细胞凝胶电泳出现了明显的彗星图像,并随死亡时间的推移彗星图像变得越清晰明显,而后又逐渐变得模糊不清;最早出现的时间是在死后3h,其拖尾率达到98%,而在死后30h彗星图像就变得模糊不清,不能进行准确辨认。图像分析显示:在死后0-18h随死亡时间的延长,彗星尾部逐渐增长、增宽,表现尾部情况的TL、TM参数值逐渐增大,18h时达到高峰,而后在21-27h有所下降并在一定范围内波动,二参数值表现出相对的稳定性,而在30h则不能检测到彗星图像相关数据,表明彗星图像不存在。统计分析显示:在死后0-18h各组间TL、TM值存在显著性差异,而在21-27h各组间TL、TM值无显著性差别;TL、TM在死后3-18h与死亡时间紧密相关,相关系数分别达到0.99614、0.99077,p﹤0.01,回归分析二者与死亡时间在此时间段几乎成直线关系。结论:1.改良彗星试验使得实验更为成功,更能够快速、简便、灵敏、准确检测死后细胞核DNA的降解,结合图像分析软件大大提高了结果分析的精确性和敏感性,在早期死亡时间推断中有较高的应用价值。2.兔在死后3h肝细胞核DNA就已经开始发生降解,死后9-18h降解明显加速,而后出现一段相对的稳定期,最后溶解消失。3.尾长和尾矩良好地反映了DNA降解情况,是彗星试验研究死后DNA含量变化与死亡时间关系的很好的指标。4.死后细胞核DNA含量变化与死亡时间存在一定的线性关系,研究二者关系将在早期死亡时间推断中有重要的意义。5.应用改良彗星试验测定死后肝细胞核DNA含量变化可望成为早期死亡时间推断的更简便方法。6.图像分析软件为彗星试验结果分析提供了便利。更多还原

【Abstract】 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.更多还原