Correlative Analysis on the Relationship between PMI and DNA Degradation of Cell Nucleus in Human Different Tissues

Summary： To determining the postmortem interval （PMI） through quantitative analysis of the DNA degradation of cell nucleus in human brain and spleen by using image analysis technique （IAT）. The brain and spleen tissues from 32 cadavers with known PMI were collected, subjected to cell smear every 1 h within the first 5-36 h after death, stained by Feulgen-Van＇s staining, Three indices reflecting DNA in brain cells （astrocytes） and splenic lymphocytes, including integral optical density （IOD）, average optical density （AOD）, average gray （AG） were measured by employing the mage analysis instrument. The results showed that IOD and AOD declined and AG increased with the prolongation of dead time within 5 36 h. A correlation between the PMI and gray parameters （IOD,AOD and AG） was identified and the corresponding regression equation was obtained. The parameters （IOD, AOD and AG） were proved to be effective quantitative indicators for accurate estimation of PMI within 5-36 h after death.     

Image Analysis for Degradation of DNA in Retinal Nuclei of Rat after Death

The changes of retinal nuclear DNA content in rats after death was detected and the rela-tionship between degradation of retinal nuclear DNA and postmortem interval (PMI) was analyzed. Ninety healthy adult SD rats, female, weighing 250±10 g, were randomly divided into 15 groups. At 20 ℃, the retinal cells were withdrawn every 2 h within 0 to 28 h after death and stained with Feul-gen-Vans. Index of density (ID), integral absorbance (IA) and average absorbance (AA) in retinal nu-cleus were analyzed by image analysis system. And the obtained data were subjected to linear regres-sion analysis by using SPSS12.0 software. The results showed that in retinal nucleus, AA and IA were gradually declined with the prolongation of PMI, while ID had an increased tendency. Within 28 h after PMI, the regression equations were as follows: YAA=-0.009XAA 0.590 (R2=0.949), YIA=-0.097XIA 18.903 (R2=0.968), YID=0.122XID 2.246 (R2=0.951). It was concluded that retinal nuclear DNA after death in rats was degraded gradually and had a good correlation with PMI.     

Spectrophotometric Determination of Trimethylamine-nitrogen in Cadaver Tissues for the Estimation of Late Postmortem Interval：A Pilot Study

To study the relationship between the late postmortem interval (PMI) and trimethyl-amine-nitrogen (TMA-N) in postmortem tissues of cadaver, TMA-N in muscles, livers and kidneys of rats was measured at different postmortem intervals (PMI) by using a modified spectrophotometric method. The results indicated that the detection sensitivity of TMA-N was 1 mg/L, and there was a good linear correlation between the value of absorbance (A value) and TMA-N at the concentration of 1-10 mg/L (R2=0.9991). Although TMA variation in muscles was different from that in inner organs during the time since death, TMA-N changes in cadaver tissues was positively correlated with PMI. During 2 to 7d since death, the best correlation between PMI and TMA-N concentration was found in muscles. With PMI as an independent variable, the cubic polynomial regression equation was y=-0.457x3 6.519x2-24.574x 27.207 (R2=0.969). During 3 to 8 days since death, PMI was best correlated with TMA-N concentration in inner organs. With PMI as the independent variable, the cubic polynomial regression equation was y=0.509x3-9.153x2 55.727x-95.819 (R2=0.953). It was concluded that TMA-N in tissues could be used as a new estimator for late PMI. The method used in this study offered advantages such as accuracy, sensitivity, little samples required and wide PMI estimation.     

Bioluminescent assay of microbial ATP in postmortem tissues for the estimation of postmortem interval

To study the relationship between changes of microbial ATP in four kinds of murine tis-sues and the postmortem interval （PMI）, healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentra-tion of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8–10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X3–0.166X2–0.666X＋13.412 （R2=0.989, P〈0.01）. In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X3–0.127X2–0.809X＋13.324 （R2=0.986, P〈0.01）. There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.     

Effects of 3-aminobenzamide on expressions of poly（ADP ribose） polymerase and apoptosis inducing factor in cardiomyocytes of rats with acute myocardial infarction

Background Poly（ADP-ribose） polymerase （PARP） plays an important role in cell survival and death. However, the mechanisms involved are not fully understood. Therefore, we investigated the effect of inhibition of PARP on acute myocardial infarction （AMI） at different time points in rats. Methods AMI was induced in rats by ligating the left anterior descending coronary artery. One group received 3-aminobenzamide （3-AB, a kind of PARP inhibitor） （30 mg/kg） by intraperitoneal injection. The changes of ultramicrostructure of cardiocytes in infarction region were noted, PARP cleavage was measured by Western blotting, and expressions of protein of PARP and apoptosis inducing factor （AIF） were measured by immunohistochemical staining after treatment with 3-AB for 2 hours, 4 hours, 6 hours, 1 week, 4 weeks and 8 weeks. Results Few damages to the ultramicrostructure of cardiocytes were observed after treatment with 3-AB. PARP cleavage was detected as early as 4 hours and markedly increased by 6 hours following AMI without 3-AB, but was not found until 6 hours following AMI treated with 3-AB. There were significant differences between 3-AB and AMI groups at the same time points. The expression of PARP was observed gradually increased, but that of AIF was suppressed for 6 hours after treatment of 3-AB, compared with AMI groups in positive cells at the same time points. There was significantly less cleavage of PARP and more PARP expression in 3-AB treated group compared with AMI and control groups at all matched time points. Conclusions Our results suggest that 3-AB inhibits degradation of PARP, increases the expression of PARP protein, and suppresses the expression of AIF protein. Inhibition of PARP activity may protect cardiocytes in rats with AMI and reduce apoptosis.     

Significance of Clinicopathology in Quantitative Measurement of DNA Content in Hepatocellular Carcinoma

DNA content of 38 hepatocellular carcinomas (HCC) was quantitativelydetermined by means of image analysis technology.The results showed that DNA contentin HCC was correlated well with the tumor size,capsule breaking,cancerous thrombusforming and clinical prognosis,that is,the higher DNA content was,the more malignanceappeared,but not with the clinical staging and pathological grading of HCC.66.7% ofDNA diploid/near diploid HCC were less than or equal to 3 cm in diameter,whereas 75%of DNA aneuploid HCC were more than 3cm in diameter (P<0.05),suggesting that HCC at3cm in size may be an important period when the change of biological characteristics wouldoccur.This study indicates that the quantitative analysis of DNA content can provide avaluable supplement to morphology in predicting the biological features and objective basesfor assessing clinical prognosis of HCC.     

Polymorphism of the DNA repair gene XPA and susceptibility to lung cancer

Objective： To study the relationship between one polymorphism in the promoter of the DNA repair gene XPA and the susceptibility to lung cancer. Methods： Genotypes were determined by the PCR-restriction fragment length polymorphism （PCR-RFLP） method in 310 histologically-confirmed lung cancer cases and 341 age and sex frequency-matched cancer-free controls. Results： The XPA A23G genotype frequencies were 27.1% （AA）, 42.9% （AG）, and 30.0% （GG） in case patients and21.1% （AA）, 5218% （AG）, and 26.1% （C-G） in control subjects. Multivariate logistic regression analysis revealed that individuals carrying at least one 23G variant allele （AG ＋ GG genotypes） had a significantly decreased risk for lung cancer （adjusted OR = 0.66; 95 % CI = 0.44- 0.98） compared with the wild-type genotype （23AA）. Stratified analysis showed that the protective effect was more evident in subjects with a family history of cancer. Conclusion： These results suggest that the XPA A23G polymorphism may have a role in lung cancer susceptibility in this study population.     

Characteristic changes of DNA stemlines during hepatocarcinogenesis in rats.

DNA stemlines of hepatocellular carcinoma (HCC) model of adult Wistar rats established by diethylnitrosamine were quantitatively measured by flow cytometry. The cancer-inducing course was divided into four stages, eg, precirrhosis stage, cirrhosis stage, early cancer stage and cancer progression stage. The advantageous DNA stemline of hepatocytes of normal adult Wistar rats was tetraploid (4C). It was from the cirrhosis stage that atavistic proliferation of hepatocytes with diploid (2C) DNA stemline started and replaced 4C hepatocytes, resulting in a new advantageous population. The features of early cancer stage were formation of HCC with 2C DNA stemline, whereas during the cancer progression stage, HCC cells with aneuploid (AN) DNA stemline presented the advantageous growth. The study clearly shows the change pattern of DNA stemline during the course of hepatocarcinogenesis from 4C-2C-AN, which is believed to be the biokinetic mechanism of the development and progression of HCC.

     

死后5～36 h人脑细胞DNA降解含量的图像分析

目的通过对死后5～36 h脑细胞降解后残存DNA含量的定量分析来推断其早期死亡时间.方法选取32例已知死亡时间的人体脑组织,在死后5～36 h内每小时进行细胞学涂片、Feulgen-Vans染色,采用图像分析技术检测反映脑细胞核DNA含量的平均光密度、积分光密度、平均灰度等灰度参数,分析脑细胞残存DNA的含量与其对应的死亡时间的变化规律.结果在5～36 h内平均光密度、积分光密度随死亡时间的延长而逐渐减小、平均灰度逐渐增大,且其均值均与死亡时间显著性相关,并得出对应的回归方程.结论反映人脑细胞DNA含量改变的灰度参数均与死亡时间有明显的相关性,是推断早期人体死亡时间的有效的定量指标.     

应用单细胞凝胶电泳检测大鼠死后脾细胞核DNA降解与死亡时间的关系

目的:探讨应用单细胞凝胶电泳技术(SCGE)检测大鼠死后脾细胞核DNA降解与死亡时间的一般规律,为早期死亡时间的推断提供新的方法。方法:建立大鼠死亡模型,在死后27h内,每隔3h取脾组织样本进行单细胞凝胶电泳,用共聚焦显微镜摄取彗星图像,应用彗星图像分析软件(IMI1.0)进行图像分析,并作统计学分析。结果:大鼠死后,脾细胞在电泳图像上出现了明显的彗星形拖尾,其尾长(TL)、尾矩(TM)在一定的时间范围内(0～15h)随死亡时间的延长而逐渐增大,二者均与死亡时间(PMI)呈现一定的相关回归关系。结果:单细胞凝胶电泳技术可应用于早期死亡时间的推断;彗星图像分析软件为SCGE的结果分析提供了便利。     

死后小鼠肾组织细胞核DNA变化规律的研究

目的:研究DNA降解变化与死亡时间的关系,为法医学推断死亡时间提供一种比较准确可靠的新方法。方法:应用单细胞凝胶电泳(SCGE)技术结合荧光显微镜和专业的计算机图像分析技术,测定111只小鼠在死后72h内不同时间点小鼠肾组织细胞核头半径、尾长度、头DNA含量比例、尾DNA含量比例、尾矩、Olive矩、头面积、尾面积8项参数的变化值。结果:在个体死亡72h内,测定的8项参数指标中尾DNA含量比例、彗星尾长、尾矩、Olive矩、尾面积都呈增加趋势,头半径、头DNA含量比例、头面积均呈下降趋势。上述参数均与死亡时间具有高度的相关性。并将每个参数的测量值进行了多项式运算,获得了更能体现DNA降解趋势的二项式回归方程和多元回归方程,均具有高度的统计学意义(P均<0.01)。结论:应用本研究提供的72h内肾组织组织DNA变化与死亡时间之间呈线性关系的各组回归方程,为法医学推断死后经过时间提供了一种新的、客观的、精确的方法和参考依据。     

人死亡后特定条件下骨髓细胞核DNA降解与死后时间间隔推断

目的：探讨人死亡后骨髓细胞核DNA含量变化与晚期死后时间间隔关系.方法：离体人胸骨置于特定条件下,随时间延长分别取材制成病理切片,应用DNA高度特异性染色方法Feulgen改良法结合计算机图像分析技术对死后不同时间胸骨骨髓细胞核DNA含量变化进行分析.结果：人死亡后1～10d,Feulgen染色阳性产物随死亡时间延长逐渐减少,胸骨骨髓细胞核DNA含量随死亡时间延长呈规律下降,DNA染色逐渐变浅淡,至死后10d已基本观察不到.结论：人死亡后骨髓细胞核DNA含量随死亡时间延长呈下降趋势,两者之间存在线性关系.     

一步法RT-PCR检测大鼠死后脑HGPRT mRNA的降解

目的:探讨大鼠死后不同时间次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)mRNA的降解情况与死亡时间(PMI)的关系。方法:将36只大鼠断颈处死后置于20℃温度控制系统内,利用一步法荧光标记RT-PCR技术检测大鼠脑管家基因HGPRT mRNA在死后即刻至8d的相对表达量的变化。结果:在死后即刻至7d大鼠脑组织均可检测到HGPRT mRNA,其扩增产物呈下降趋势。结论:一步法RT-PCR技术检测大鼠脑组织HGPRT mRNA,在死后的降解呈现出一定的规律性,可为PMI推断提供一种新的指标。     

大鼠肾HGPRT mRNA降解与死亡时间关系的研究

〗目的:探讨大鼠死后肾次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)mRNA降解情况与死亡时间(PMI)的关系。方法:40只Wistar大鼠断颈处死,置于20℃温控系统内,利用一步法荧光标记RT-PCR技术检测大鼠肾管家基因HGPRTmRNA在死后即刻至36 h降解情况。结果:在死后即刻至32 h大鼠肾组织均可检测到HGPRTmR-NA,且其扩增产物呈规律性下降趋势。结论:大鼠死亡后肾HGPRTmRNA降解与PMI负相关,可为PMI推断提供一种新的观测指标。     

单细胞凝胶电泳技术研究兔牙髓细胞核DNA降解的时间效应

目的研究DNA降解变化与死亡时间关系,探讨兔死后牙髓细胞核DNA的降解机理及寻找灵敏、实用的DNA降解监测指标,为死亡时间的法医学研究提供一种新的方法和技术。方法利用快速、灵敏的检测细胞DNA损伤的单细胞凝胶电泳(SCG)技术结合荧光显微镜和专业的计算机图像分析技术,以"彗星样"细胞出现率(计数一定量细胞其中彗星样细胞所占的比例)、头半径、尾长度、头/尾DNA含量比例、Oliver矩、尾矩、头面积和尾面积来评价死后兔牙髓细胞核DNA的降解程度。结果在个体死亡72h内,测定的8项参数指标中尾DNA含量比例、彗星尾长、尾矩、Olive矩、尾面积都呈增加趋势,头半径,头DNA含量比例,头面积均呈下降趋势。上述参数均与死亡时间具有高度的相关性。并将每个参数的测量值进行了多项式运算,获得了更能体现DNA降解趋势的二项式回归方程(P<0.001),具有显著的统计学意义。结论应用单细胞凝胶电泳技术,监测机体死后牙髓细胞核DNA降解变化,将会成为推断死亡时间精确、客观的新方法。     

大鼠脑挫伤DNA含量变化与损伤时间的关系

目的观察大鼠脑挫伤后DNA含量的时序变化规律。方法建立自由落体打击大鼠脑挫伤动物模型，采用Feulgen’s DNA染色方法，结合图像分析技术。结果损伤侧大脑皮质浅层和海马区平均积分光密度（IOD）变化呈：24h内迅速下降，24—96h有缓慢上升趋势；伤后24h内上述两区域平均积分光密度均与损伤时间呈线性关系，R^2分别为0．668和0．615。可导出直线回归方程。结论采用Feulgen’s DNA染色法结合图像分析技术观察伤后DNA含量变化，可以应用于脑损伤时间推断的研究，探索推断脑损伤时间的新方法。