大鼠尸体软组织生物力学及脏器量化病理学时序性变化的实验研究

发布时间：2018-07-04 17:52

本文选题：死亡时间 + 软组织时序性变化　；参考：《汕头大学》2008年硕士论文

【摘要】： 背景与目的早在三百多年前被誉为欧洲法医学之父的意大利医生Paul明确指出:死亡时间(Postmortem interval,PMI)的推断是法医学鉴定首先要解决的问题。准确的死亡时间的推断在很大程度上为侦察工作提供线索、划定侦察范围、确定嫌疑犯或凶犯有无作案时间、或者确定案件性质、阐明作案过程均有价值。近几十年来,国内外法医工作在死亡时间的推断方面使用生物学、化学、物理学、生物化学、生物物理学、免疫组织化学,分子生物学等方法取得可喜呈果。但鉴于死亡后尸体受多种因素的影响,包括自身因素和环境因素等,尤其是自溶和腐败的影响,迄今,还没有找到一种方法对完全推断PMI具有相同程度的敏感性。因此,准确的死亡时间的推断尚需综合多种手段和方法进行综合评估。对于死后早期时间的推断,目前的推断方法较多;而死后较长时间尤其是腐败尸体死亡时间的评估方法则相对较少,推断的误差也相对较大,且至今尚缺乏精确而简便的方法,因此死亡时间的评估尤其是死后较长时间的评估至今还是法医学的难题之一。为此,本实验将生物力学理论和技术应用于PMI的推断,探讨了尸体软组织的生物力学性状的时序性变化与死亡时间的关系,进而将法医生物力学应用到死亡时间的推断,使法医生物力学(forensic biomechanism)的应用进一步得到扩展和延伸。同时,本实验运用用计算机图像数字分析(Computer digital image analysis)技术,对肝细胞HE染色下进行病理学图像的体视学分析与量化处理,并将其应用于法医病理学PMI的推断,为病理学的发展趋势——量化病理学在法医学的应用提供了新的思路,并进一步结合尸体主要脏器的常规病理图片观察,探讨其与PMI的关系。材料与方法清洁级健康雄性SD大鼠80只,3～3.5月龄,体重250～300g,用脱颈法处死后,立即置于恒温箱内,分0、6、12、18、24、30、36、42、48、60、72、96、120、144、168、192h时间组,提取胸部皮肤、腹壁肌肉、胸主动脉、小肠和大肠进行生物力学指标检测,每组5只,检测极限载荷、应力、应变等软组织生物力学指标。同时,提取全脑、最大叶肝脏、全心、左肺最大叶、左肾进行HE切片染色和体视学观察,观察组织细胞的自溶和腐败的形态学变化;并对肝细胞进行病理图像分析和量化处理,检测肝细胞的核面积、核浆比、核浆光密度比和核异形指数,探讨其与PMI的关系。结果 1大体及镜下改变: 大体改变:死后尸体主要经历了尸僵、尸绿、毛发脱落、腹部膨胀至“巨人观”,软化塌陷的过程,腹腔内脏器逐步液化。镜下改变:脑、心、肺、肝、肾等实质器官组织形态随PMI呈一定规律性变化,脑自溶相对较快,48h即难以取出,心脏自溶速率较慢,96h仍可见心肌纤维。各组织细胞均经历嗜碱性逐渐减弱、嗜酸性逐渐增强的过程,细胞核染色质由逐渐聚集到最后突破核膜溶解。 2软组织生物力学性状皮肤具有最大的极限载荷,平均为11.67N;小肠的极限载荷最小,平均仅为1.37N。皮肤、小肠死后18h内均呈较明显下降趋势(Y_(PMI)=-1.26X_(ML)+31.03,r=0.985和Y_(PMI)=-27.5X_(ML)+53.5,r=0.956)。皮肤18～36h转呈上升趋势,36h后呈缓慢下降趋势,但相邻各点差别无统计意义。小肠18h后无明显变化趋势。肌肉与大肠的规律较为相似,24h内均呈较明显下降趋势,相邻各点均有统计意义(Y_(PMI)=-5.18X_(ML)+42.639,r=0.992和Y_(PMI)=-7.83X_(ML)+58.1,r=0.988)。之后,变化趋势不明显,称之为“平台期”。其中,肌肉24～96h呈平台期,之后缓慢下降,但96～168h相邻各点差别具统计意义(Y_(PMI)=-23.812X_(ML)173.91,r=0.97);大肠24～72h呈平台期,之后缓慢下降(Y_(PMI)=-44.05X_(ML)+231.7,r=0.981)。血管死后变化趋势不明显。血管的应变最大,平均为4.12,但变化趋势无明显的统计学意义。其他组织24h内均呈明显下降趋势,下降速率较快。24h后肌肉与小肠变化趋势不明显;皮肤24～72h呈平台期,72h后呈现缓慢下降趋势,72～96h无统计意义,96小时后下降趋势具有统计意义;大肠与皮肤较为相似,24～72h呈现平台期,72～192h呈逐渐下降趋势(Y_(PMI)=-333X_(应变)+297.7,r=0.991),120h后各时间点的差别具有统计意义。各组织的最大应力改变趋势与极限载荷趋势较为一致,血管仍无明显规律,其他组织在24h内下降趋势较明显。24h后,除小肠未见明显趋势外,均有下降趋势,但较24h内缓慢。 3肝细胞病理图像分析的量化指标肝细胞核面积变化不稳定,随PMI的变化趋势不明显。核浆比总体呈逐渐下降趋势,其中,0h～36h显著(P＜0.05)。36h～60h呈缓慢上升趋势,但二者差别无统计意义(P＞0.05)。60h～72h下降明显,有统计意义(P＜0.05)。核浆光密度比总体呈先上升后下降趋势,36h内呈逐渐上升趋势,36h达峰值,相邻点间差别具有统计意义(P＜0.05):24h与36h差别明显(P＜0.01)。36h～72h呈逐渐下降趋势,相邻点时间点差别均有统计意义。异形指数72小时内呈逐渐上升趋势(Y_(PMI)=512.09X_(ID)-699.5,r=0.982)。结论 1、PMI与各器官组织形态学的时序性变化之间存在较明显的相关性。各脏器的自溶和腐败速率不同,因此在各时段的PMI中各组织脏器存在不同的镜下特点,根据个组织脏器的镜下特点,可大约估算PMI,并为日常法医病理检案鉴别生前弥漫性病变与死后自溶腐败性变化提供了参考依据。 2死后尸体软组织皮肤、肌肉、大肠、小肠的极限载荷、应力、应变随PMI的延长呈逐渐下降趋势,且各组织在死后不同时间的下降速率不同,使得各组织在推断PMI的“窗口期”不同,并且在一定程度存在互补性。提示软组织生物力学指标和方法可以作为推断死亡时间的一种简便、客观、量化的检测手段。血管无明显规律,说明血管的极限载荷及应力、应变不适合推断PMI。 3将病理学图像量化分析技术应用于PMI得推断,证实了在HE染色下肝细胞的核浆比、核浆光密度比、异形指数能较好的反映肝细胞的自溶程度,其中,核浆光密度比呈现先上升后下降趋势,核浆比和异形指数呈现线性下降趋势,而核面积与其自溶程度无明显的相关性。提示组织细胞自身比较的相对性指标,可以避免不同制片、染色效果的影响,更客观地反应病变情况和分级标准,为量化病理学提供了新的思路。
[Abstract]:Background and purpose
Paul, a Italy doctor known as the father of European forensic science more than 300 years ago, made it clear that the Postmortem interval (PMI) is the first problem to be solved in forensic identification. The exact time of death provides a line to the reconnaissance work to a large extent, delineate the scope of the reconnaissance, and determine the suspect or the murderer. No case time, or the nature of the case, is of value. In recent decades, the forensic work at home and abroad has achieved good results in the use of biological, chemical, physical, biophysics, biophysics, immuno histochemistry, molecular biology and other methods to infer the time of death. But in view of the many causes of death after death. The effects of elements, including their own and environmental factors, especially the effects of autolysis and corruption, have not yet been found to have the same degree of sensitivity to the complete inference of PMI. Therefore, a comprehensive assessment of the exact time of death is required by a comprehensive variety of means and methods. There are more methods to infer, but the longer time after death, especially the dead time of decayed corpse, is relatively small, and the error is relatively large, and there is still a lack of accurate and simple methods. Therefore, the assessment of the time of death, especially the long time after death, is still one of the difficult problems in forensic medicine.
Therefore, this experiment applies the theory and technology of biomechanics to the inference of PMI, discusses the relationship between the temporal changes of the biomechanical properties of cadaver tissues and the time of death, and then applies the forensic biomechanics to the inference of the time of death to further expand and extend the application of the forensic biomechanics (Forensic biomechanism). At the same time, this experiment uses Computer digital image analysis technology to analyze and quantify the pathological image of liver cells under HE staining, and apply it to the inference of PMI in forensic pathology, which provides a new trend of pathology in the development of Pathology - the application of quantitative pathology in forensic medicine. In addition, the relationship between PMI and the pathological pictures of the main organs of the corpse was further observed.
Materials and methods
80 healthy male SD rats, 3 to 3.5 months old, weight 250 ~ 300g, were put to death in a constant temperature box and divided into 0,6,12,18,24,30,36,42,48,60,72,96120144168192h time group. The chest skin, abdominal wall muscles, the thoracic aorta, the small intestine and the large intestine were detected by the biomechanical indexes of 5 rats in each group. The limit load was detected. Stress, strain and other soft tissue biomechanical indexes. At the same time, the whole brain, the most lobar liver, the whole heart, the largest left lung leaf, the left kidney were stained with HE section and stereological observation, the morphological changes of autolysis and corruption of the tissue cells were observed, and the pathological image of the liver cells was analyzed and quantified, and the nuclear area, the ratio of nuclear plasma and the nucleus were detected. The relationship between the optical density ratio and the nuclear heteromorphic index was discussed.
Result
1 general and microscopic changes:
General change: after death, the corpse mainly experienced rigor mortem, corpse green, hair loss, abdominal swelling to "giant view", softening collapse process, abdominal viscera gradually liquefaction.
The morphological changes under the microscope: brain, heart, lung, liver, kidney and other parenchyma tissues are changed regularly with PMI, the brain autolysis is relatively fast, the brain autolysis is relatively fast, 48h is difficult to take out, the rate of cardiac autolysis is slower, 96h still can be seen in the myocardium. All tissues and cells have experienced gradually weakened basophilia and gradually increasing eosinophilic activity, and the chromatin chromatin of the nucleus gradually gathered to the most Then break through the nuclear membrane dissolving.
2 biomechanical properties of soft tissue
The maximum limit load of the skin was 11.67N, the minimum load of the small intestine was the smallest, the average of the skin was 1.37N., and the 18h of the small intestine was obviously decreased after the death of the small intestine (Y_ (PMI) =-1.26X_ (ML) +31.03, r=0.985 and Y_ (PMI) =-27.5X_. There was no statistical significance. There was no obvious change trend after 18h in small intestine. The regularity of muscle and large intestine was more similar, 24h was obviously decreased, and the adjacent points had statistical significance (Y_ (PMI) =-5.18X_ (ML) +42.639, r=0.992 and Y_ (PMI) =-7.83X_ (ML)). After that, the change trend was not obvious, which was called "platform period". Among them, muscle 24~9 6h showed a plateau period and then slowed down slowly, but the difference between 96 to 168h was not statistically significant (Y_ (PMI) =-23.812X_ (ML) 173.91, r=0.97), and the 24 ~ 72h of the large intestine was on the platform stage, and then decreased slowly (Y_ (PMI) =-44.05X_ (ML)).
The vascular strain was maximum, average 4.12, but there was no significant statistical significance in the change trend. Other tissues were obviously decreasing in 24h. The trend of muscle and small intestine was not obvious after.24h, and the skin was in platform period from 24 to 72h. After 72h, there was a slow decline trend, and there was no statistical significance from 72 to 96h. After 96 hours, the decline trend was unified. Significance: the large intestine was similar to the skin, and the 24 ~ 72h showed a platform period, and the 72 ~ 192h decreased gradually (Y_ (PMI) =-333X_ (strain) +297.7, r=0.991). The difference of each time point after 120h was statistically significant.
The trend of maximum stress change in each tissue is consistent with the limit load trend, and there is no obvious rule in the blood vessels. After the decrease of other tissues in 24h, the trend is more obvious than that in the small intestine, but it has a downward trend, but it is slower than that in 24h.
Quantitative index of pathological image analysis of 3 liver cells
The change trend of nuclear area of liver was not stable, with the change trend of PMI, the ratio of nuclear plasma ratio decreased gradually. Among them, 0h to 36h (P < 0.05).36h to 60H showed a slow rising trend, but there was no statistical significance (P > 0.05),.60h to 72h decreased significantly (P < 0.05). The nuclear plasma light density increased first and then decreased. Trend, 36h was gradually rising, 36h reached peak value, and the difference between adjacent points had statistical significance (P < 0.05): the difference between 24h and 36h was obvious (P < 0.01).36h to 72h gradually declined, and the time point difference between adjacent points had statistical significance. The abnormal shape index was gradually upward trend within 72 hours (Y_ (PMI) =512.09X_ (ID)).
conclusion
1, there is a significant correlation between PMI and the temporal changes in the morphological changes of the organs. The autolysis and corruption rate of each organ are different. Therefore, there are different characteristics of the organs in each organ in each period of PMI. According to the characteristics of the viscera, the PMI can be estimated. It provides a reference for sex lesion and postmortem autolysis and rotten change.
2 after death, the ultimate load, stress and strain of the soft tissue of the soft tissue, the stress and the small intestine are gradually decreasing with the extension of PMI, and the decrease rate of each tissue at different time after death is different, which makes each organization deduce the "window period" of the PMI and complementarity to a certain extent. It suggests the biomechanical indexes of the soft tissue and the prescription. The method can be used as a simple, objective and quantitative detection method to infer the time of death. There is no obvious rule of blood vessel, which shows the ultimate load and stress of blood vessel, and the strain is not suitable for inference PMI..
3 the quantitative analysis technique of pathological image was applied to PMI to be inferred. It was proved that the ratio of nucleo plasma to nuclear plasma, the ratio of light density of the nucleo plasma, and the heteromorphic index can better reflect the degree of autolysis of liver cells in HE staining. There is no obvious correlation between the degree of autolysis. It is suggested that the relative indexes of the tissue cell's own comparison can avoid the influence of different filmmaking and dyeing effect, and reflect the pathological conditions and grading standards more objectively, and provide a new way of thinking for the quantification of pathology.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：D919

【引证文献】