摘要
死后间隔时间(postmortem interval，PMI)或称死亡时间推断(estimation of time since death，ETSD)是法医病理学实际检案及研究工作的重点和难点之一。长期以来，国内外的学者们已采用多种技术与方法尝试PMI的推断，尤其是早期PMI的推断，如利用死后各种尸体现象、肌肉超生反应、玻璃体液钾离子浓度、死后血液的生化变化等，但其准确性常受到外界各种因素的影响，往往误差较大。至目前为止，尚无公认的简易实用、准确可靠的推断方法，尤其是中晚期PMI的推断方法更不尽人意。因此，本课题研究大鼠死后皮下肌肉组织电阻抗幅值和相位角的变化规律，以探讨生物电阻抗推断中晚期PMI的价值，试图发展一种简单方便、准确可靠的推断中晚期PMI的新方法；选择前期研究尚未用过的分析指标——headDNA％，应用单细胞凝胶电泳技术检测大鼠死后脑、骨髓细胞核DNA降解随PMI的变化规律，以探索单细胞凝胶电泳技术推断早期PMI的新参数。
第一部分大鼠死后皮下肌肉组织电阻抗幅值和相位角变化推断死后间隔时间的实验研究
【目的】研究大鼠死后皮下肌肉组织电阻抗幅值和相位角的变化规律，探讨生物电阻抗技术推断中晚期PMI的价值，试图发展一种简单方便、准确可靠的推断中晚期PMI的新方法。同时从肌肉组织形态学的角度初步分析了电阻抗幅值变化趋势产生的机制。
【方法】取Wistar健康成年大鼠20只，分成4个实验组，每组5只，分别对应于四个不同的测量温度(9±1℃、14±1℃、16±1℃、19±1℃)。用颈椎脱臼的方式处死动物，将电极插入大腿部肌肉并固定。迅速将动物置于设定温度的恒温培养箱或冰箱中，利用四电极阻抗测量系统测量大鼠死后皮下肌肉组织电阻抗幅值和相位角。自死后0h开始，每隔3h记录一次电阻抗数据(夜间关机时除外)和环境温度。当实验时间足够长时(阻抗下降到原始阻抗的70％左右或15天以上)便可停止实验。再用Excel对所得的离散阻抗数据进行简单的均值、方差分析，用MATLAB对阻抗数据进行相关分析和直线拟合并作图、计算相关系数。
取前述4只实验大鼠(1只为死前对照组，3只为死后实验组)，分别于死后12h、24h和72h在大鼠电阻抗测量部位皮下肌肉组织取材、常规制片、HE染色后，在光镜下观察组织形态学变化，并与其电阻抗的变化进行对比分析。
【结果】
1．所有死亡大鼠皮下肌肉组织的电阻抗幅值都呈现出相似的先升后降的变化趋势，且中、长期的下降部分有一定的线性变化趋势。死亡初期(PMI=1～4天)电阻抗幅值迅速上升，达峰值后随着时间的推移，呈线性下降趋势(PMI=4～15天)，最终趋于平坦。
2．所有死亡大鼠皮下肌肉组织的电阻抗相位角没有相似的变化规律，其变化杂乱无章。
3．相对电阻抗可以一定程度上消去不同大鼠由于初值的差异造成的特异性，而且同一温度组不同大鼠之间的离散性较绝对电阻抗小。
4．温度越高绝对电阻抗或相对电阻抗上升得越快，下降得亦更快；温度越低绝对电阻抗或相对电阻抗上升得越缓慢，下降得更缓慢。绝对电阻抗或相对电阻抗下降段斜率亦与温度的变化呈正相关关系。
5．各温度组下降段斜率与PMI的相关系数均大于0.97，表明各温度组下降段与PMI之间的相关性很高，因此，有可能利用这一线性关系推断中、晚期PMI。
6．大鼠死前核未见改变，肌束横纹清晰；死后12h，表层少数肌束肿胀，肌丝多数呈细颗粒状或匀质状，横纹消失；死后72h，较多见肌纤维肿胀、颗粒样变、横纹消失。随着PMI推移逐步产生细胞膜自溶裂解过程，组织的导电性增加，因而表现为电阻抗不断下降的结果。
【结论】
1．大鼠死后皮下肌肉组织电阻抗幅值随时间的推移呈现先上升后下降的变化趋势，并且下降部分的电阻抗幅值变化和PMI之间存在线性关系。在测量环境相似的条件下，电阻抗变化也相似。
2．环境温度是影响电阻抗幅值最主要的参数，且环境温度越高其变化速度越快。因此，电阻抗的测量还要注意其适用的温度范围，温度过高和过低都不适合使用。
3．测量电阻抗幅值时，将绝对电阻抗转换成相对电阻抗，更能反映电阻抗变化的客观规律。
4．电阻抗下降段斜率与温度的变化呈正相关关系，且在不同环境温度时，下降段斜率与PMI的相关性都较高。如果找出下降段起点、峰值、斜率这三者之间的确定关系，就可利用下降段的线性关系为推断中晚期PMI提供一定的判断依据。
5．由于电阻抗相位角受环境因素的影响很大，目前还不能用于PMI的推断。
6．通过对电阻抗变化趋势产生的机制从组织形态变化方面进行的初步探讨，得出了和细胞生物学理论解释相一致的结果。
7．相对于其它PMI推断技术而言，电阻抗法具有测量简单方便、准确可靠的优点，但是鉴于目前的检测水平和环境因素的限制，能否应用于实际情况，发展成为一种新型的PMI推断技术，还需要做进一步的研究和探讨。
第二部分单细胞凝胶电泳检测大鼠死后脑、骨髓细胞核DNA降解推断死后间隔时间的研究
【目的】应用单细胞凝胶电泳技术(Single-cell gel electrophoresis，SCGE)直接检测大鼠死后不同温度下脑、骨髓细胞核DNA降解的情况，选择既往研究尚未用过的参数headDNA％分析不同温度下DNA降解的程度随PMI变化的规律，建立两者的回归方程，并与标准指标尾长(TL)、Oliver尾距(TM)进行对比，以期为该方法推断早期PMI探索新的参数。
【方法】以颈髓离断法处死大鼠，保存在温度分别控制在10℃±1℃或20℃±1℃的实验环境下，在大鼠死后0～40h内，每隔4h，分别对脑组织和骨髓取材，并制作单细胞悬液；然后进行单细胞凝胶电泳；在荧光显微镜下观察并拍照。所摄图像用CASP0.22彗星图像分析软件进行检测，选择既往研究尚未用过的参数headDNA％分析不同温度下DNA降解的程度随PMI变化的规律，用SAS8.0和SPSS12.0分析软件进行统计学分析，建立两者的回归方程，并与标准指标尾长(TL)、Oliver尾距(TM)的分析结果进行对比。同时，依据建立的方程和headDNA％估计值95％可信区间计算公式反推PMI范围的计算公式。
【结果】
1．大鼠死后，脑细胞、骨髓细胞在电泳图上出现了不同程度的彗星形拖尾；
2．大鼠死后早期PMI内，不论在10℃还是20℃下，脑、骨髓细胞核HeadDNA％的下降与PMI之间存在着良好的线性关系，建立了四个线性回归方程，其确定系数均较高，可用于早期PMI推断；
3．死后细胞核HeadDNA％的下降受环境温度、组织种类的影响；
4．建立了由已知HeadDNA％值反推PMI范围的计算公式：如脑组织在20℃下，据某已知HeadDNA％值反推PMI范围的计算公式：
timel=(86.83-DNA_(已知))／1.9634
time2=(99.27-DNA_(已知))／1.9634
5．选用的参数HeadDNA％较前期研究惯用的标准指标TL、TM与PMI的线性回归方程拟合较理想；
6．脑组织、骨髓组织分别用三种参数与PMI拟合的线性回归分析，前者的确定系数较后者高。
【结论】
1．单细胞凝胶电泳技术可应用与早期PMI的推断；
2．环境温度和组织差异明显影响死后核DNA的降解；
3．新参数HeadDNA％较前期研究惯用的标准指标TL、TM推断PMI的价值更高，但其可靠性有待于今后进一步的研究，尤其是人体材料研究的验证；
4．由已知HeadDNA％值可反推PMI的范围；
5．脑组织是较骨髓组织更为理想的备选检材。
Estimating the postmortem interval is one of the important and difficult points in the practical case detecting and research work in forensic pathology. For a long time, scholars from domestic and abroad tried to deduce PMI, especially early deduction, by applying lots of techniques and methods, such as various phenomena of posthumous cadaver, muscular supravital reaction, K~+ concentration of vitreous body liquid, biochemical changes in blood of posthumous and so on. However, the accuracy is often influenced by various factors outside, the errors are usually large. Till now, there is still no public received simple, practical, accurate and reliable deduction way, specifically for the middle and late PMI deduction. Therefore, we investigate the role of the magnitude and phase angle changes of musculature impedance in rat cadavers, in order to evaluate the significance of middle and late PMI estimation by Bio-impedance measurement, and try to develop a simple and accurate new method. We selected an unused index — HeadDNA% to detect the role of nuclear DNA degradation of both bone marrow and brain in rat cadavers followed by PMI by applying the single-cell gel electrophoresis, and to develop a new parameter of estimating early PMI by single-cell gel electrophoresis. Part I
Research on estimating of the postmortem interval by the magnitude and phase angle changes of musculature impedance
in rat cadavers
[Objective] To investigate the role of the magnitude and phase angle changes of musculature impedance in rat cadavers, to evaluate the significance of middle and late PMI estimation by Bio-impedance measurement, and try to develop a simple and accurate new methods to estimate the middle and late PMI. Meanwhile, we analyze the mechanism of the trend of impedance magnitude changes generated in terms of muscular morphology.
[Methods ]Divided 20 healthy adult Wistar rats into 4 groups, 5 each correlated to 4 different detecting temperature(9±1℃、 14±1℃、 16±1℃、 19±1℃). Rats were sacrificed by cervical dislocation, and inserted the electrode into leg muscles and fixed it. Rats were put in the homeothermia incubator or refrigerator with the fixed temperature, and the magnitude and phase angle changes of musculature impedance in rat cadavers were measured by applying a Bio-impedance measurement system based on four-electrode method. The impedance and the environment temperature were recorded once for every 3 hours, since Oh of death. The experiment stopped when the time was long enough (impedance decreased to around 70% of the original one or over 15 days). The simple means and variance were analyzed based on the discrete data by excel method. The data were analyzed, the line was fitted, the graph was made , and the related coefficient was calculated.
Pick out the 4 same rats (one as control before death, 3 as control after death), draw the materials from the leg subdermal muscular tissues in the site of electrical impedance measured at 12h, 24h and 72h, respectively. The tissues were fixed, sectioned, HE stained regularly, and the morphological changes were observed under microscope, and changes of impedance were comparatively analyzed. [Results]
1. All endermic musculature impedance magnitude of rat cadavers presented the similar change, first rose, later decreased, and the downgrade part of middle and late phase shown some linear change tendency. The impedance magnitude at early stage of death(PMI = 1～ 4 day) upgraded quickly, and presented linearly downgraded tendency (PMI=4～15 day) after reached the peak value with the time passed, and finally to platform.
2. All phase angle of endermic musculature impedance showed no similar regular change, the changes of which were in a great mess.
3. The relative impedance could eliminate the specificity caused by different rats, and the dispersion from different rats at the same temperature was lower than that of absolute impedance.
4. The higher the temperature, the faster it rose and faster it descended for the absolute or relative impedance; the lower the temperature, the slower it rose and slower it descended for the absolute or relative impedance. The slope rate of downgrade part of the absolute or relative impedance positively related with changes of temperature.
5. Relative coefficients of the slope rate of downgrade part from each group at different temperature with PMI were all over 0.97, which showed that the dependability was quite high. So PMI at middle or late stage was supposed to estimate by use of this point.
6. No change of nucleus was observed before death, the transverse striation of muscular bundles was clear; 10h after death, a few muscular bundles in surface layer were swelled, most of myofilament presented particle shape or homogenous shape, the transverse striation disappeared; 72h later, more muscular fiber swelled, particular like change, and the transverse striation disappeared. Self autopepsia splitting gradually developed with PMI passed, the tissue conductibility increased, consequently showed that the impedance continuously decreased
[Conclusion]
1. The endermic musculature impedance magnitude showed first upgraded then downgraded tendency with time passed in rats after death, there was linear relation between changes of the impedance magnitude in upgraded part with PMI. The changes of impedance were similar under the similar measurement circumstance.
2. The environmental temperature is the main parameter which affects the impedance magnitude, and the higher the temperature the faster the changes. Thus, the measurement of impedance should pay attention to the optimal scale of temperature, both too high and too low temperatures are unavailable.
3. Transfering the absolute impedance to relative one better reflects the objective law of changes of impedance when the magnitude of impedance is measured.
4. The slope rate of impedance on downgraded part positively correlated with changes of temperature, and which highly related with PMI at different temperature. If the definite relation of the beginning point, peak value and slope rate on the upgraded part was determined, the linear relation on the downgraded part could be used to provide the evidence for estimating the middle and late stage of PMI.
5. Since phase angle of impedance is strongly influenced by circumstance, which can't be used to estimate PMI.
6. The result consistent with the theory of cellular biology is drawn from the preliminary study on the mechanism of the tendency of impedance magnitude changes generated in terms of muscular morphology.
7. Compared with other PMI estimating techniques, impedance possess the advantages of easy to measure, curacy and reliability. However, with the limit of techniques and environmental factors, whether it is available in practical settings, or develops to a new PMI estimating technique should be further studied. Part II
Research on estimating of the postmortem interval by determining nuclear DNA degradation of both bone marrow and brain in rat cadavers with single-cell gel electrophoresis
[Objective] To detect directly nuclear DNA degradation of both bone marrow and brain in rat cadavers at different temperature by applying the single cell gel electrophoresis (SCGE), analyze the amount of DNA degradation followed by changes of PMI by selecting an unused index — HeadDNA%, to establish the regression function of both and compare with standard index TailLength(TL), Oliver TailMoment (TM) and be expected to develop a new parameter of estimating early PMI.
[Methods] Rats were sacrificed by cervical dislocation, put in the experimental environment with the fixed temperature at 10℃±1℃ or 20℃±1℃. The brain and bone marrow were taken out for every 4h, during 0～40h after death, and single cell suspension was prepared; Then single cell gel electrophoresis was carried out and observed under fluorescence microscope and photographed. The pictures were analyzed by CASP1.22 comet graph essaying software, and an unused index-head DNA% parameter analysis was used to essay the role of DNA degradation with changes of PMI, Statistic analysis was conducted with SAS 8.0 and SPSS 12.0 software. Set up regression function of both and the results were compared with standard index TailLength(TL), Oliver TailMoment (TM). Meanwhile, the calculating function for PMI was inferred reversely based on the established function and headDNA% value within 95% confidence interval.
[Results]
1. Different degree of comet like tail was found in the electrophoresis pictures of brain cells and bone marrow cells in rats after death;
2. The good linear relationship existed between decreasing of nuclear HeadDNA% in both brain and bone marrow with PMI during the early PMI, whatever at 10℃ or 20℃; four linear regression function was set up, their coefficients were high and could be used to estimate early PMI;
3. The downgraded nuclear HeadDNA% was influenced with environment temperature and tissue kinds after death;
4. The calculating function was established to deduce reversely PMI by a known HeadDNA%;
5. HeadDNA% was better in fitting by linear regression function than the standard common used TL、 TM and PMI in early study;
6. The linear regression analysis fitted by using three parameters with PMI of brain tissues and bone marrow, the definite coefficients of former was higher.
[Conclusion]
1. SCGE can be used to estimate early PMI;
2. The environment temperature and tissue disparity significantly affect nuclear DNA degradation after death; ;
3. The new parameter HeadDNA% is more valuable for PMI estimation than the common used standard parameter TL, TM in early study. The reliability needs further investigation, especially for testing the human materials;
4. The known HeadDNA% can be used to estimate reversely PMI scale;
5. Brain tissues are more ideal as candidate materials than bone marrow.
引文
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