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
|
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。
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.
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。
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.
引文
[1]丛峰松主编.生物化学实验[M].上海:上海交通大学出版社,2005,47-48.
[2]李凡,刘永茂主编.基础医学实验教程[M].北京:高等教育出版社,2002,204-206.
[3]Tice RR, Agurell E, Anderson D,etal. Single cell gel/comet assay: guideline for in vitro and in vivo genetic toxicology testing[J].Environ Mol Mutagen,2000,35(3):206-221.
[4]Singh NP, McCoy MT, Tice RR,etal. A single technique for quantification of low level of DNA damage in individual[J].EXP Cell Res,1988,175(1): 184 -191.
[5]Bowden RD, Buckwalter MR, McBride JF,etal. Tail profile:a more accurate system for analyzing DNA damage using the Comet assay[J]. Mutat Res,2003, 537:1.
[6]Adler CP, Beckhove P. Postmortem DNA changes in heart muscle[J]. Beitr Pathol,1971,142(3):306-320.
[7]Atmadja DS, Tatsuno Y, Ueno Y, etal. The effect of extraction method. The kind of organ samples and the examination delay on the DNA yield and tying[J]. Kobe J Med Sci,1995,41(6):197-211.
[8]高建军,丰盛梅,金慰芳等.碱性单细胞凝胶电泳铺胶技术的改进[J].复旦学报(医学版),2004,31(3):311-312.
[9]Claus Henssge. The estimation of the tine since death in the early postmortem period[M]. London,The Bath Press,Avon, 1995,32-33.
[10]Brown A, Marshall T. Body temperature as a means of estimation thetime of death[J]. Forensic Sci Int,1974,4:125-133.
[11]Brzezinski PM, Godlewski A. Assessment of potassium and sodium ion concentrations in the vitreous humour of swine isolated eyeballs after organism death[J]. Rocz Akad Med Bialymst,2004,49(suppl 1):161-163.
[12]Munoz JI, Suarez PJM, Otero XL, etal. A new perspective in the estimation of postmortem interval(PMI)based on vitreous[J]. J Forensic Sci,2001,46 (2):209-214.
[13]Munoz Barus JI, Suare-Penaranda J, Otero XL, etal. Improved estimation of postmortem interval based on differential behaviour of vitreous potassium and hypoxantine in death by hanging[J]. Forensic Sci Int,2002,125(1):67-74.
[14]赵子琴主编.法医病理学[M].-3 版.北京:人民卫生出版社,2004,47-50,71.
[15]Giround F, Montmasson MP. Revaluation of optimal Feulgen reaction for automated cytology,influences offixatives[J]. Annal Quant Cytol Histol,1989,11:87-93.
[16]Schalte EKW. Standardization of Feulgen reaction for absorption DNA image cytometry :a review[J]. Annal Cell Pathol,1991,3:167-174.
[17]Fataoka A, Kubota M, Wakazono Y,etal. Association of high molecular weight DNA fragmentation with apoptotic or nonapoptotic cell death induced by calcium ionophore[J]. FEBS Letl,1995,364:264.
[18]祝家镇主编.法医病理学[M].-2 版.北京:人民卫生出版社,1999:43-46.
[19]莫耀南主编.法医学司法鉴定[M].郑州:郑州大学出版社,2003:12-16.
[20]齐凤英,许树棠,刘汝俊,等.死后不同时间细胞 DNA 含量分析[J].中国法医学杂志,1989,4(4):213-215.
[21]Cina SJ. Flow cytometric evaluation of DNA degradation:A predictor of postmortem interval?[J]. Am J Forensic Med Pathol,1994,15(1):300-304.
[22]Di Nunno NR, Costantinides F, Bernasconi P,etal. Is flow cytometric evaluation of DNA degradation areliable method toinvestigation the early postmortem period?[J]. Am Forensic Med Pathol,1998,19(1):50-53.
[23]刘良,林乐泉,刘艳,等.大鼠脾细胞 DNA 含量与早期死亡时间关系的图像分析研究[J].法医学杂志,2000,16(1):8-9.
[24]林乐泉,刘良,邓伟年,等.大鼠肝细胞 DNA 含量与早期死亡时间关系的图像分析研究[J].法医学杂志,2000,16(2):68-69.
[25]Ostling O, Johanson KJ. Microelectrophoretic study of radiation induced DNA damage in individual mammalian cells[J]. Biochphy Res Commun,1984,123:291-298.
[26]Collins A,Dusinska M,Franklin M,etal.Comet assay in human biomonitoring studies:reliability,validation,and application[J].Environ Mol Mutagen.1997,30(2):139-146.
[27]Kassie F,Parzefall W,Knasmuller S.Single cell gel electrophoresis assay:a new technique for human biomonitoring studies[J].Mutation Research,2000,463(1):13-31.
[28]Olive PL. Cell proliferation as a requirement for the development of the contact effect in Chinese hamster v79 spheroids[J]. Radit Res,1989, 117:79-92.
[29]Gedik CM,Ewen SWB,Collins AR.Single cell gel eletrophoresis applied to the analysis of UV-C damage and it’s repair[J].Int J Radiat Biol,1992,62: 313-320.
[30]何远,闫平,胡家伟,等.单细胞凝胶电泳检测大鼠死后肝细胞核 DNA 降解[J].中国法医学杂志,2005,20(6):325-328.
[31]涂彬,孔祥麟,阎祖康,等.大鼠死后肝细胞核 DNA 的组织化学定量研究.法医学杂志,1994,10(4):149.
[1]郭景元.现代法医学[M].第 1 版.北京:科学出版社,2000.90-92.
[2]莫耀南.法医司法鉴定[M].第 1 版.郑州:郑州大学出版社,2003.12-16.
[3]Adler CP,Beckhove P. Postmortem DNA changes in heart muscle[J].Beitr Pathol,1971,142(3): 306-320.
[4]Kiliovska MS,Charkarov EL. Changes in the DNA content in the differential-densitometric characteristics of the deoxyribonucleo- proteins in the cell nucleus in postmortem autolysis[J]. Dukl Dolg Akad Nank, 1973, 26(8):1121-1123.
[5]Avtandilov GG,Viter VI,Gorden ES. Dynamics DNA content in the liver ,skeletalmusicles and myocardium of the rat in the postmortem period(a microstrophotometric study)[J]. Biull Eksp Biol Med,1979,87(6): 620-622.
[6]Bar W,Kratzer A,Machler M, et al. Postmortem stability of DNA.Forensic Sci Int,1988,39(1):59-70.
[7]Perry WL 3rd,Bass WM,Riggsby WS, et al. The autodegration of dexoxyribonucleic acid(DNA) in human rib bone and its relation to the time interval since death.J Forensic Sci,1988Jan,33(1):144-153
[8]Cina SJ. Flow cytometric evaluation of DNA degradation:A predictor of postmortem interval?[J].Am J Forensic Med Patho,1994,15(4):300-304.
[9]Atmadja DS,Tatsuno Y,Ueno Y, et al. The effect of extraction method .The kind of organ samples and the examination delay on the DNA yield and tying. Kobe J Med Sci,1995,41(6):197-211.
[10]Petito CK,Roberts B. Eflect of postmortem interval on in situ end-labeling of DNA oligonucleosomes[J]. J Neuropathol Exp Neurol,1995, 54(6):761-765.
[11]Di Nunno NR, Costantinides F, Bernasconi P, et al. Is flow cytometric evaluation of DNA degradation areliable method to investigation the early postmortem period?[J]. Am J Forensic Med Pathol,1998,19(1):50-53.
[12]Di Nunno NR, Costantinides F, Melato M. Determination of the time of the death in a homicide-suicide case using flow cytometry[J]. Am J Forensic Med Pathol,1999,20(3):228-231.
[13]Di Nunno NR, Costantinides F, Cina SJ, et al. What is the best sample for determining the early postmortem period by on-the-spot flow cytometry analysis?[J]. Am J Forensic Med Pathol, 2002,23(2):173-180.
[14]Johnson LA,Ferris JA. Analysis of postmortem DNA degradation by single gelectrophoresis[J]. Forensi Sci Int,2002,126(1):43-47.
[15]Shu X, Liu Y, Ren L, et al. Correlative analysis on the relation between PMI and DNA degradation of cell nucleus in human different tissue[J]. J Huazhong Univ Sci Tecnolog Med Sci, 2005,25(4):423-426.
[16]Boy SC, Bernitz H, Van Heerden WF. Flow cytometric evaluation of postmortempulp DNA degradation[J]. Am J Forensic Med Pathol,2003,24(2):123-127.
[17]王伟平,龙仁,熊平,等.牙髓细胞平均 DNA 含量与 PMI 相关性的研究.美国中华临床医学杂志,2005,7(3):189-190,194.
[18]罗光华,陈玉川,成建定,等.DNA 降解与腐败尸体死亡时间的相关性.法医学杂志,2006,22(1): 7-9.
[2]李凡,刘永茂主编.基础医学实验教程[M].北京:高等教育出版社,2002,204-206.
[3]Tice RR, Agurell E, Anderson D,etal. Single cell gel/comet assay: guideline for in vitro and in vivo genetic toxicology testing[J].Environ Mol Mutagen,2000,35(3):206-221.
[4]Singh NP, McCoy MT, Tice RR,etal. A single technique for quantification of low level of DNA damage in individual[J].EXP Cell Res,1988,175(1): 184 -191.
[5]Bowden RD, Buckwalter MR, McBride JF,etal. Tail profile:a more accurate system for analyzing DNA damage using the Comet assay[J]. Mutat Res,2003, 537:1.
[6]Adler CP, Beckhove P. Postmortem DNA changes in heart muscle[J]. Beitr Pathol,1971,142(3):306-320.
[7]Atmadja DS, Tatsuno Y, Ueno Y, etal. The effect of extraction method. The kind of organ samples and the examination delay on the DNA yield and tying[J]. Kobe J Med Sci,1995,41(6):197-211.
[8]高建军,丰盛梅,金慰芳等.碱性单细胞凝胶电泳铺胶技术的改进[J].复旦学报(医学版),2004,31(3):311-312.
[9]Claus Henssge. The estimation of the tine since death in the early postmortem period[M]. London,The Bath Press,Avon, 1995,32-33.
[10]Brown A, Marshall T. Body temperature as a means of estimation thetime of death[J]. Forensic Sci Int,1974,4:125-133.
[11]Brzezinski PM, Godlewski A. Assessment of potassium and sodium ion concentrations in the vitreous humour of swine isolated eyeballs after organism death[J]. Rocz Akad Med Bialymst,2004,49(suppl 1):161-163.
[12]Munoz JI, Suarez PJM, Otero XL, etal. A new perspective in the estimation of postmortem interval(PMI)based on vitreous[J]. J Forensic Sci,2001,46 (2):209-214.
[13]Munoz Barus JI, Suare-Penaranda J, Otero XL, etal. Improved estimation of postmortem interval based on differential behaviour of vitreous potassium and hypoxantine in death by hanging[J]. Forensic Sci Int,2002,125(1):67-74.
[14]赵子琴主编.法医病理学[M].-3 版.北京:人民卫生出版社,2004,47-50,71.
[15]Giround F, Montmasson MP. Revaluation of optimal Feulgen reaction for automated cytology,influences offixatives[J]. Annal Quant Cytol Histol,1989,11:87-93.
[16]Schalte EKW. Standardization of Feulgen reaction for absorption DNA image cytometry :a review[J]. Annal Cell Pathol,1991,3:167-174.
[17]Fataoka A, Kubota M, Wakazono Y,etal. Association of high molecular weight DNA fragmentation with apoptotic or nonapoptotic cell death induced by calcium ionophore[J]. FEBS Letl,1995,364:264.
[18]祝家镇主编.法医病理学[M].-2 版.北京:人民卫生出版社,1999:43-46.
[19]莫耀南主编.法医学司法鉴定[M].郑州:郑州大学出版社,2003:12-16.
[20]齐凤英,许树棠,刘汝俊,等.死后不同时间细胞 DNA 含量分析[J].中国法医学杂志,1989,4(4):213-215.
[21]Cina SJ. Flow cytometric evaluation of DNA degradation:A predictor of postmortem interval?[J]. Am J Forensic Med Pathol,1994,15(1):300-304.
[22]Di Nunno NR, Costantinides F, Bernasconi P,etal. Is flow cytometric evaluation of DNA degradation areliable method toinvestigation the early postmortem period?[J]. Am Forensic Med Pathol,1998,19(1):50-53.
[23]刘良,林乐泉,刘艳,等.大鼠脾细胞 DNA 含量与早期死亡时间关系的图像分析研究[J].法医学杂志,2000,16(1):8-9.
[24]林乐泉,刘良,邓伟年,等.大鼠肝细胞 DNA 含量与早期死亡时间关系的图像分析研究[J].法医学杂志,2000,16(2):68-69.
[25]Ostling O, Johanson KJ. Microelectrophoretic study of radiation induced DNA damage in individual mammalian cells[J]. Biochphy Res Commun,1984,123:291-298.
[26]Collins A,Dusinska M,Franklin M,etal.Comet assay in human biomonitoring studies:reliability,validation,and application[J].Environ Mol Mutagen.1997,30(2):139-146.
[27]Kassie F,Parzefall W,Knasmuller S.Single cell gel electrophoresis assay:a new technique for human biomonitoring studies[J].Mutation Research,2000,463(1):13-31.
[28]Olive PL. Cell proliferation as a requirement for the development of the contact effect in Chinese hamster v79 spheroids[J]. Radit Res,1989, 117:79-92.
[29]Gedik CM,Ewen SWB,Collins AR.Single cell gel eletrophoresis applied to the analysis of UV-C damage and it’s repair[J].Int J Radiat Biol,1992,62: 313-320.
[30]何远,闫平,胡家伟,等.单细胞凝胶电泳检测大鼠死后肝细胞核 DNA 降解[J].中国法医学杂志,2005,20(6):325-328.
[31]涂彬,孔祥麟,阎祖康,等.大鼠死后肝细胞核 DNA 的组织化学定量研究.法医学杂志,1994,10(4):149.
[1]郭景元.现代法医学[M].第 1 版.北京:科学出版社,2000.90-92.
[2]莫耀南.法医司法鉴定[M].第 1 版.郑州:郑州大学出版社,2003.12-16.
[3]Adler CP,Beckhove P. Postmortem DNA changes in heart muscle[J].Beitr Pathol,1971,142(3): 306-320.
[4]Kiliovska MS,Charkarov EL. Changes in the DNA content in the differential-densitometric characteristics of the deoxyribonucleo- proteins in the cell nucleus in postmortem autolysis[J]. Dukl Dolg Akad Nank, 1973, 26(8):1121-1123.
[5]Avtandilov GG,Viter VI,Gorden ES. Dynamics DNA content in the liver ,skeletalmusicles and myocardium of the rat in the postmortem period(a microstrophotometric study)[J]. Biull Eksp Biol Med,1979,87(6): 620-622.
[6]Bar W,Kratzer A,Machler M, et al. Postmortem stability of DNA.Forensic Sci Int,1988,39(1):59-70.
[7]Perry WL 3rd,Bass WM,Riggsby WS, et al. The autodegration of dexoxyribonucleic acid(DNA) in human rib bone and its relation to the time interval since death.J Forensic Sci,1988Jan,33(1):144-153
[8]Cina SJ. Flow cytometric evaluation of DNA degradation:A predictor of postmortem interval?[J].Am J Forensic Med Patho,1994,15(4):300-304.
[9]Atmadja DS,Tatsuno Y,Ueno Y, et al. The effect of extraction method .The kind of organ samples and the examination delay on the DNA yield and tying. Kobe J Med Sci,1995,41(6):197-211.
[10]Petito CK,Roberts B. Eflect of postmortem interval on in situ end-labeling of DNA oligonucleosomes[J]. J Neuropathol Exp Neurol,1995, 54(6):761-765.
[11]Di Nunno NR, Costantinides F, Bernasconi P, et al. Is flow cytometric evaluation of DNA degradation areliable method to investigation the early postmortem period?[J]. Am J Forensic Med Pathol,1998,19(1):50-53.
[12]Di Nunno NR, Costantinides F, Melato M. Determination of the time of the death in a homicide-suicide case using flow cytometry[J]. Am J Forensic Med Pathol,1999,20(3):228-231.
[13]Di Nunno NR, Costantinides F, Cina SJ, et al. What is the best sample for determining the early postmortem period by on-the-spot flow cytometry analysis?[J]. Am J Forensic Med Pathol, 2002,23(2):173-180.
[14]Johnson LA,Ferris JA. Analysis of postmortem DNA degradation by single gelectrophoresis[J]. Forensi Sci Int,2002,126(1):43-47.
[15]Shu X, Liu Y, Ren L, et al. Correlative analysis on the relation between PMI and DNA degradation of cell nucleus in human different tissue[J]. J Huazhong Univ Sci Tecnolog Med Sci, 2005,25(4):423-426.
[16]Boy SC, Bernitz H, Van Heerden WF. Flow cytometric evaluation of postmortempulp DNA degradation[J]. Am J Forensic Med Pathol,2003,24(2):123-127.
[17]王伟平,龙仁,熊平,等.牙髓细胞平均 DNA 含量与 PMI 相关性的研究.美国中华临床医学杂志,2005,7(3):189-190,194.
[18]罗光华,陈玉川,成建定,等.DNA 降解与腐败尸体死亡时间的相关性.法医学杂志,2006,22(1): 7-9.