改良彗星试验检测兔死后肝细胞核DNA含量变化及其法医学意义
发布时间：2018-11-10 22:58
【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㩳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。
[Abstract]:Objective: to detect the degradation of DNA in the liver nucleus of rabbits after death by modified comet assay, and to study the relationship between the changes of nuclear DNA content and the time of death after death. To explore the application value of nuclear DNA content change in early death time estimation. Methods: the healthy adult white rabbits were killed by cervical dislocations and placed at room temperature. The liver tissue samples were collected from the liver in vivo at 30 hours after the death at 1h, 3h, 6h, 9h, 12h, 15h, 18h, 21h, 21h, 24h, 27h, 30h, respectively. According to the corresponding time groups, there are 11 groups. The modified comet assay technique was applied to the electrophoresis and staining of each group of samples. The electrophoretic images were observed under fluorescence microscope, the image was photographed, and the tail of comet image was analyzed by CASP image analysis software, and the tail length (TL), was used. The two parameters of tail moment (TM) are used as the main analysis indexes. Finally, the statistical correlation regression analysis of the image analysis data is carried out by using the SAS 9.0 statistical analysis software. Results: a total of 528 gel bodies were prepared by modified comet assay, none of them was damaged and shedding, the tedious degree of the experiment was obviously reduced, and the experimental time was shortened obviously. Comet assay showed that the DNA of the liver nucleus appeared obvious comet image by single cell gel electrophoresis after death, and the comet image became clearer and more obvious with the passage of death time, and then became blurred gradually. The earliest time was 3 hours after death, and the tail trailing rate reached 98%, but the comet image became blurred and could not be accurately identified 30 hours after death. Image analysis showed that the tail of comet gradually increased and widened with the prolongation of death time from 0-18 h after death, the TL,TM parameter of tail increased gradually, and reached the peak at 18 h. Then it decreased at 21-27 h and fluctuated in a certain range. The two parameter values showed relative stability, but the relative data of comet images could not be detected at 30 h, indicating that the comet images did not exist. Statistical analysis showed that there was significant difference in TL,TM between groups at 0-18 h after death, but no significant difference in TL,TM between groups at 21-27 h. TL,TM was closely related to the time of death from 3 to 18 hours after death, and the correlation coefficient was 0.99614 ~ 0.9907p0. 01, respectively. The regression analysis showed that there was almost a linear relationship between the time of death and the time of death. Conclusion: 1. The improved comet assay makes the experiment more successful, and it is more rapid, simple, sensitive and accurate in detecting the degradation of nuclear DNA in postmortem nuclei, and the accuracy and sensitivity of the result analysis are greatly improved by combining with image analysis software. It has high application value in early death time inference. 2. The degradation of DNA in the liver nuclei of rabbits began to occur 3 hours after death, and accelerated obviously from 9 to 18 hours after death, followed by a relatively stable period, and finally dissolved and disappeared. 3. The tail length and tail moment well reflect the degradation of DNA, which is a good index to study the relationship between the changes of DNA content and the time of death in comet assay. 4. There is a linear relationship between the changes of nuclear DNA content and the time of death after death, and the study of the relationship between them will be of great significance in the estimation of early death time. 5. The modified comet assay is expected to be a simple method for estimating the early death time of liver nuclei. 6. Image analysis software provides convenience for the analysis of comet test results.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919
本文编号：2323806
[Abstract]:Objective: to detect the degradation of DNA in the liver nucleus of rabbits after death by modified comet assay, and to study the relationship between the changes of nuclear DNA content and the time of death after death. To explore the application value of nuclear DNA content change in early death time estimation. Methods: the healthy adult white rabbits were killed by cervical dislocations and placed at room temperature. The liver tissue samples were collected from the liver in vivo at 30 hours after the death at 1h, 3h, 6h, 9h, 12h, 15h, 18h, 21h, 21h, 24h, 27h, 30h, respectively. According to the corresponding time groups, there are 11 groups. The modified comet assay technique was applied to the electrophoresis and staining of each group of samples. The electrophoretic images were observed under fluorescence microscope, the image was photographed, and the tail of comet image was analyzed by CASP image analysis software, and the tail length (TL), was used. The two parameters of tail moment (TM) are used as the main analysis indexes. Finally, the statistical correlation regression analysis of the image analysis data is carried out by using the SAS 9.0 statistical analysis software. Results: a total of 528 gel bodies were prepared by modified comet assay, none of them was damaged and shedding, the tedious degree of the experiment was obviously reduced, and the experimental time was shortened obviously. Comet assay showed that the DNA of the liver nucleus appeared obvious comet image by single cell gel electrophoresis after death, and the comet image became clearer and more obvious with the passage of death time, and then became blurred gradually. The earliest time was 3 hours after death, and the tail trailing rate reached 98%, but the comet image became blurred and could not be accurately identified 30 hours after death. Image analysis showed that the tail of comet gradually increased and widened with the prolongation of death time from 0-18 h after death, the TL,TM parameter of tail increased gradually, and reached the peak at 18 h. Then it decreased at 21-27 h and fluctuated in a certain range. The two parameter values showed relative stability, but the relative data of comet images could not be detected at 30 h, indicating that the comet images did not exist. Statistical analysis showed that there was significant difference in TL,TM between groups at 0-18 h after death, but no significant difference in TL,TM between groups at 21-27 h. TL,TM was closely related to the time of death from 3 to 18 hours after death, and the correlation coefficient was 0.99614 ~ 0.9907p0. 01, respectively. The regression analysis showed that there was almost a linear relationship between the time of death and the time of death. Conclusion: 1. The improved comet assay makes the experiment more successful, and it is more rapid, simple, sensitive and accurate in detecting the degradation of nuclear DNA in postmortem nuclei, and the accuracy and sensitivity of the result analysis are greatly improved by combining with image analysis software. It has high application value in early death time inference. 2. The degradation of DNA in the liver nuclei of rabbits began to occur 3 hours after death, and accelerated obviously from 9 to 18 hours after death, followed by a relatively stable period, and finally dissolved and disappeared. 3. The tail length and tail moment well reflect the degradation of DNA, which is a good index to study the relationship between the changes of DNA content and the time of death in comet assay. 4. There is a linear relationship between the changes of nuclear DNA content and the time of death after death, and the study of the relationship between them will be of great significance in the estimation of early death time. 5. The modified comet assay is expected to be a simple method for estimating the early death time of liver nuclei. 6. Image analysis software provides convenience for the analysis of comet test results.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919
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