彗星试验检测大鼠牙髓细胞DNA降解推断死亡时间的研究

Study on DNA degradation of the rat skeletal muscle by Comet assay for estimation of postmortem interval

目的在特定条件下,用彗星试验技术,动态观察SD大鼠死后6～36h牙髓细胞DNA降解规律,探讨推断死亡时间的一种方法。方法模拟广州地区近5年秋季10月份的平均气温25.1℃,建立SD大鼠死亡模型。分别于死后6、12、24、36、48h提取大鼠牙髓组织进行彗星试验,荧光显微成像系统采集图像,同时获取多个分析参数(Comet4.0),并进行统计学处理。结果在死后6～36h,SD大鼠牙髓细胞DNA的碎片随着死后间隔时间的延长而增加;尾长在死后各时间点依次呈明显的上升趋势;Oliver尾矩和尾DNA随着死亡时间的延长,也均有一定的上升趋势。结论本研究提示细胞DNA的降解随着死后间隔时间的延长而增加;尾长随着死后间隔时间的延长在各时间点呈明显的上升趋势,用于推断死亡时间优于Oliver尾矩和尾DNA。

Objective To investigate nuclear DNA degradation of the rats, Spregue-Dawley dental pulp cells by Comet assay from 6 h to 36 h during postmortem interval for estimation of death time. Methods Imitated environment was build according to climate temperature of average 25.1℃ in October late five years in Guangzhou. Eighteen female Spregue-Dawley rats were killed by cervical dislocation and put into imitated environment for 6, 12, 24, 36, 48 h, respectively. Dental pulps were sampled at each time point. the nuclear DNA degradation was observed by comet assay. The comet images were captured by HITACHI KP-M1E/K-S10 CCD camera and Meteor Ⅱ image grabber. Kinetic Komet 4.0 software was used to analyze images. The results were analysed by statistics. Results The nuclear DNA of the rat's dental pulp cells by comet assay appeared as a single round or genomic DNA in the early period, the length and density of the comet tail increased with the postmortem interval period, which was an increase of DNA fragments. The tail length of comet shows a significant increase in turn from 6 h to 36 h during postmortem interval. It showed the increases that Oliver tail moment and the tail DNA of comet from 6 h to 36 h during postmortem interval, too. Conclusion This study indicates an increase of degradation of nuclear DNA of the rat's dental pulp cells with postmortem interval in 6～36 h postmortem period. It is shown that using the change trend of tail length to estimate postmortem interval better than Oliver tail moment and tail DNA.