无电流斑电击死的实验研究
发布时间：2018-08-26 10:30
【摘要】： 研究背景 无电流斑的电击死在法医学实践中时常遇及，而传统判定电流损伤的主要依据是电流斑、皮肤金属化等特殊改变。如果无电流斑的存在，这类案件的法医学鉴定则相当困难。目前的研究表明，电场作用下，细胞膜可以发生可逆性穿孔，但电击死(伤)是否也会引起细胞膜穿孔?假如有穿孔，这种穿孔是否对电流作用或电场作用有相对特异性?目前尚无研究报道。另外，对于无电流斑的电击死，还必须对其电流路径进行推断。有研究表明，电流刺激可以引起刺激部位的骨骼肌基因表达，而非刺激部位则无此种改变。因此，本研究从骨骼肌的基因表达变化入手进行探讨。此外，法医实践中还必须确定是电击致死还是死后电击。电流作用可以引起骨骼肌与心肌的收缩，骨骼肌的收缩必然会引起肌小节长度变化，生前与死后骨骼肌活性不一样同，电击死与死后电击是否会在长度变化上存在差异?这是本研究的另一项内容。研究表明，骨骼肌的收缩可以引起某些基因的表达，既然电流刺激可以引起肌肉收缩，那么电击死引起的肌肉收缩是否会引起基因的表达?这种表达与死后电击有什么区别?由于电击导致死亡的时间甚短，蛋白水平可能来不及产生太大的变化，我们从mRNA水平上来研究这种变化与区别。 研究目的 探求无电流斑电击死的法医学鉴定要点，包括： ①寻找无电流斑电击死的诊断指标； ②探讨无电流斑电击死的电流路径； ③探讨电击死与死后电击的鉴别依据。 研究方法 第一部分：无电流斑电击死的形态学诊断指标研究 1．体外血细胞电击穿孔研究 取成人血液6例，每人取12ml，每例血液均分为6等份，每份2ml，分为非电击组，电击5s，10s，20s，30s，1min，共6组。220v交流电电击后，扫描电镜观察血细胞膜的变化。 2．无电流斑电击死与非电击死血管内皮与血细胞穿孔研究以及电击死人体主动脉与肺动脉内皮的扫描电镜观察 取新西兰45只兔，，随机分为9组，每组5只，即正常对照组、A1、A2、B1、B2，C、D、E、F组。正常对照组直接经耳缘静脉注入空气处死，另不做任何处理。建立六种动物模型，即A～F。A1、A2组用模型A；B1、B2组用模型B，分别以220V及110V电击致死。其余组分别用相对应的模型进行实验。 模型A：将兔的右前肢与左后肢分别用0.1M PBS浸湿，再缠上用0.1M PBS浸泡的湿纱布，将导线直接缠绕在纱布外层，接通220V或110V交流电，通电致死。 模型B：在长方形塑料容器两端分别固定两根导线作电极，并在容器内加少量水，水深约1.5cm，将兔置于塑料容器中，上方放置一塑料网盖(以防兔跳出)，接通220V或110V交流电，通电致死。 模型C：将实验兔从40米高空坠下致死。 模型D：将无水乙醇喷洒于实验体表，点燃致其死亡。 模型E：将亚硝酸钠2g溶于10ml蒸馏水，全部灌入兔胃，致其死亡。 模型F：将实验兔置入充满二氧化碳密闭容器中，使其窒息死亡。取兔血液、肺动脉和主动脉，用扫描电镜观察红细胞及内皮细胞形态学变化。 取5例法医实践中电击死亡者主动脉与肺动脉根部，同时用3例其他死亡原因的主动脉与肺动脉根部做对照组。用扫描电镜观察其内皮细胞形态变化。 第二部分无电流斑电击死电流通路的推断研究-----无电流斑电击死兔对称部位骨骼肌hsp70、c-fos、il-6mRNA表达差异 新西兰兔10只，随机分为电击死组与对照组2组，每组5只。对照组直接经耳缘静脉注入空气处死，另不做任何处理。电击死组：将兔的右前肢与左后肢分别用0.1MPBS浸湿，用蘸有0.1M PBS的纱布缠绕其上，通220V交流电致死，建立无电流斑电击死动物模型。分别取股四头肌及肱二头肌中段，用荧光定量RT-PCR技术检测其hsp70、c-fos、il-6mRNA表达。 第三部分：电击死与死后电击的鉴别研究 1．生前电击与死后电击骨骼肌与心肌hsp70、c-los、il-6mRNA的表达 新西兰兔15只，随机分为三组，即电击死组、死后电击组、对照组，每组5只。电击死组，将兔的右前肢与左后肢分别用0.1M PBS浸湿，再缠上用0.1M PBS浸泡的湿纱布，将导线直接绕于纱布外层，接通220V交流电，直至死亡；死后电击组，用耳缘静脉空气注射50ml致死，死后即刻用220V交流电的两极分别连接实验动物的左后肢与右前肢通电3分钟；对照组直接用耳缘静脉空气注射50ml处死，不电击；取各实验动物左后肢与右前肢的股四头肌与肱二头肌中份及心室肌，用荧光定量RT-PCR技术检测电击死与死后电击兔的骨骼肌及心肌hsp70、c-fos、il-6mRNA含量变化。 2．生前电击与死后电击骨骼肌肌小节的长度变化 12只兔随机分为4组，每组3只，分别为对照组Ⅰ、对照组Ⅱ、电击死组、死后电击组。对照组Ⅰ、Ⅱ与死后电击组兔分别从耳缘静脉注射空气50ml处死。对照组Ⅰ于处死后立即取材；而对照组Ⅱ则于死后24h取材。电击死组，先将兔的右前肢与左后肢分别用磷酸盐缓冲液浸湿，再缠上用磷酸盐缓冲液浸泡的湿纱布，将导线直接绕于纱布外层，接通220V交流电，直至其死亡；死后电击组先将动物从耳缘静脉注射空气50ml处死，然后按电击死组方法电击4min。取左后肢股四头肌中段，固定。用透射电镜观察骨骼肌肌小节长度变化，对所得结果进行统计学分析。 研究结果 第一部分：无电流斑电击死的形态学诊断指标研究 1．体外血细胞电击穿孔研究 电击后，即可以观察到细胞膜(红细胞与白细胞)均有穿孔，红细胞穿孔可为一个或多个，形状多为圆形或卵圆形，白细胞穿孔为多个，呈筛网状；穿孔细胞数随电击时间延长而增多。非电击组细胞则无穿孔，各类细胞形态如常。 2．无电流斑电击死兔血管内皮与血细胞穿孔以及电击死人体主动脉与肺动脉内皮的扫描电镜观察 电击死兔肺动脉与主动脉内皮细胞可见穿孔，而非电击死动物与正常对照组则未见穿孔现象。 5例电击死人体肺动脉与主动脉标本中，4例可见内皮细胞穿孔，1例内皮细胞脱失，但可见平滑肌细胞穿孔，而其他原因死亡者则无血管内皮穿孔现象。 第二部分无电流斑电击死电流通路的推断研究----无电流斑电击死免对称部位骨骼肌hsp70、c-fos、il-6mRNA表达差异 电击死组四肢骨骼肌il-6、hsp70、c-fosmRNA的水平均较对照组高，但通电肢il-6、hsp70、c-fosmRNA的水平远大于非通电肢。 第三部分：电击死与死后电击的鉴别研究 1．生前电击与死后电击骨骼肌与心肌hsp70、c-fos、il-6mRNA的表达 电击死兔的通电肢体骨骼肌及心肌hsp70、c-fos、il-6mRNA表达水平显著升高；而死后即刻电击肢体骨骼肌及心肌hsp70、c-fos、il-6mRNA水平仅轻微升高，表达微弱。 2．生前电击与死后电击骨骼肌肌小节的长度变化 生前电击与死后电击骨骼肌肌小节均缩短，但生前电击缩短的程度更甚，死后电击组与正常对照组相比，仅轻微缩短；对照组Ⅱ死后24小时肌小节显著伸长。 结论 1．交流电流可以引起构成电路的细胞穿孔； 2．电流或电场引起的细胞膜穿孔具有相对特异性； 3．主动脉与肺动脉根部内皮细胞穿孔可作为无电流斑电击死的诊断指标之一。 4．对称部位骨骼肌il-6mRNA、hsp70mRNA、c-fosmRNA的含量变化可以作为无电流斑电击死电流通路推断的一个指标。 5．骨骼肌il-6mRNA、hsp70mRNA、c-fosmRNA的含量变化和骨骼肌肌小节长度变化可以作为鉴别电击死与死后电击的一个指标。
[Abstract]:Research background
Electric shock death without electric spot is often encountered in forensic practice, but the traditional judgment of electric current injury is mainly based on special changes such as electric spot and metallization of skin. Is it possible to cause cell membrane perforation if there are perforations? Is the perforation relatively specific to current action or electric field action? There are no reports yet. In addition, it is necessary to deduce the current path of electrocution without electric plaque. Studies have shown that electrical stimulation can induce the skeletal muscle base at the stimulating site. In addition, in forensic practice, it is also necessary to determine whether electric shock is lethal or postmortem. Electric current can cause contraction of skeletal muscle and myocardium, and contraction of skeletal muscle will inevitably lead to changes in the length of sarcomeres before and after life. This is another aspect of the study. Studies have shown that contraction of skeletal muscles can cause the expression of certain genes. Since electrical stimulation can cause muscle contraction, does the contraction of muscle caused by electrical stimulation cause the base? What's the difference between this expression and postmortem shock? Because of the short duration of death caused by electric shock, the protein level may not have changed much. We study this change and difference at the mRNA level.
research objective
The key points of forensic identification for electric shock without electric current spot are as follows:
(1) to find the diagnostic index of electric shock without current spots.
(2) to explore the current path of electric current without spot.
(3) to explore the difference between electrocution and postmortem shock.
research method
Part I: morphological diagnostic indexes of electroshock without current spots.
1. study on perforation of blood cells in vitro
Adult blood samples were taken from 6 patients, 12 ml each. Each patient was divided into 6 equal parts, 2 ml each. They were divided into 6 groups: non-electric shock group, 5 s, 10 s, 20 s, 30 s, 1 min, altogether.
2. Study of vascular endothelium and blood cell perforation by non-electric plaque electroporation and non-electric plaque electroporation and scanning electron microscopic observation of human aorta and pulmonary artery endothelium by electroporation
Forty-five New Zealand rabbits were randomly divided into 9 groups, 5 rabbits in each group, namely normal control group, A1, A2, B1, B2, C, D, E, F group. The normal control group was injected air directly into the ear vein without any treatment. The model should be tested.
Model A: The rabbit's right forelimb and left hind limb were soaked with 0.1M PBS, then wrapped with 0.1M PBS soaked wet gauze, the wire was wound directly in the gauze outer layer, connected with 220V or 110V alternating current, electrified to death.
Model B: Two wires were fixed at both ends of the rectangular plastic container as electrodes, and a small amount of water was added to the container. The water depth was about 1.5 cm. The rabbits were placed in the plastic container. A plastic net cover was placed above the container (to prevent the rabbits from jumping out) and 220V or 110V alternating current was switched on.
Model C: the experimental rabbit fell from 40 meters high to death.
Model D: spraying anhydrous ethanol on the body surface and igniting it to death.
Model E: dissolve sodium nitrite 2G in 10ml distilled water and pour into the rabbit stomach, resulting in death.
Model F: The rabbits were placed in a sealed container filled with carbon dioxide and asphyxiated to death. The morphological changes of red blood cells and endothelial cells were observed by scanning electron microscopy.
The morphological changes of endothelial cells were observed by scanning electron microscopy (SEM) in the aorta and pulmonary artery roots of 5 patients who died of electric shock.
The second part is the deduction of dead current pathway in non-spot electrocution--the difference of hsp70, c-fos, IL-6 mRNA expression in skeletal muscle of rabbits killed by non-spot electrocution
Ten New Zealand rabbits were randomly divided into two groups: the electric shock group and the control group, with 5 rabbits in each group. The expression of hsp70, c-fos and IL-6 mRNA in quadriceps femoris and biceps brachii were detected by fluorescence quantitative RT-PCR.
The third part: the distinction between electrocution and postmortem shock.
1. expression of HSP70, c-los and il-6mRNA in skeletal muscle and myocardium before and after electroporation
Fifteen New Zealand rabbits were randomly divided into three groups: electrocution group, postmortem electrocution group and control group, with 5 rabbits in each group. The rats in the control group were killed by air injection of 50 ml without electric shock, and the quadriceps femoris, biceps brachii and ventricular muscles of the left hind limb and right forelimb were taken from the left hind limb and right forelimb of the experimental animals, and the left hind limb and biceps brachii muscle were connected with 220 V AC poles for 3 minutes immediately after death. The changes of HSP70, c-fos and il-6mRNA contents in skeletal muscle and myocardium of electrocuted rabbits after electrocution were observed.
2. the length of skeletal muscle segment of electric shock and postmortem shock
Twelve rabbits were randomly divided into four groups: control group I, control group II, electrocution group and postmortem electrocution group. Control group I, II and postmortem electrocution group were executed by intravenous injection of air 50 ml from ear margin. Control group I was executed immediately after execution, while control group II was executed 24 hours after death. The hind limbs were soaked in phosphate buffer solution, then wrapped in wet gauze soaked in phosphate buffer solution. The wire was wound directly around the outer layer of the gauze and connected with 220V AC until death. After death, the animals in the electroshock group were killed by injecting air 50 ml from the ear vein, and then electrocuted for 4 minutes according to the electrocution method. The transmission electron microscope was used to observe the change of skeletal muscle segment length, and the results were statistically analyzed.
Research results
Part I: morphological diagnostic indexes of electroshock without current spots.
1. study on perforation of blood cells in vitro
After electric shock, the perforation of cell membrane (red blood cells and white blood cells) can be observed. The perforation of red blood cells can be one or more, most of which are round or oval in shape, and the perforation of white blood cells is multiple, and the number of perforated cells increases with the duration of electric shock.
2. Scanning electron microscopic observation of rabbit vascular endothelium and blood cell perforation and human aorta and pulmonary artery endothelium killed by electric shock without electric spot
Pulmonary artery and aortic endothelial cells were perforated in electrocuted rabbits, but not in electrocuted rabbits and normal control group.
Of the 5 electrocuted pulmonary artery and aorta specimens, endothelial cell perforation was seen in 4 cases, endothelial cell loss in 1 case, but smooth muscle cell perforation was seen, while no vascular endothelial perforation was found in other causes of death.
The second part is the deduction of the dead current pathway in non-spot electrocution--the difference of the expression of hsp70, c-fos, IL-6 mRNA in skeletal muscle of non-symmetrical sites after non-spot electrocution
The levels of il-6, HSP70 and c-fos mRNA in skeletal muscle of the electrocuted limbs were higher than those of the control group, but the levels of il-6, HSP70 and c-fos mRNA in the electrocuted limbs were much higher than those in the non-electrocuted limbs.
The third part: the distinction between electrocution and postmortem shock.
1. expression of HSP70, c-fos and il-6mRNA in skeletal muscle and myocardium before and after electroporation
The expression levels of hsp70, c-fos and IL-6 mRNA in skeletal muscle and myocardium of rabbits were significantly increased after electric shock, while the expression levels of hsp70, c-fos and IL-6 mRNA in skeletal muscle and myocardium of rabbits were slightly increased after electric shock.
2. the length of skeletal muscle segment of electric shock and postmortem shock
The skeletal muscle segments were shortened before and after death, but the degree of shortening was worse. Compared with the normal control group, the skeletal muscle segments were slightly shortened in the postmortem shock group.
conclusion
1. alternating current can cause cell perforation in the circuit.
2. cell membrane perforation induced by electric current or electric field is relatively specific.
3. Endothelial cell perforation of aorta and pulmonary artery root can be used as one of the diagnostic criteria of electric shock death without electric spot.
4. The changes of interleukin-6 mRNA, HSP70 mRNA and c-fos mRNA in skeletal muscle of symmetrical site can be used as an index for deducing the dead current pathway of non-spot electric shock.
5. The changes of interleukin-6 mRNA, HSP70 mRNA and c-fos mRNA in skeletal muscle and the length of skeletal muscle segments can be used as an index to differentiate electric shock from postmortem electric shock.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：D919
本文编号：2204591
[Abstract]:Research background
Electric shock death without electric spot is often encountered in forensic practice, but the traditional judgment of electric current injury is mainly based on special changes such as electric spot and metallization of skin. Is it possible to cause cell membrane perforation if there are perforations? Is the perforation relatively specific to current action or electric field action? There are no reports yet. In addition, it is necessary to deduce the current path of electrocution without electric plaque. Studies have shown that electrical stimulation can induce the skeletal muscle base at the stimulating site. In addition, in forensic practice, it is also necessary to determine whether electric shock is lethal or postmortem. Electric current can cause contraction of skeletal muscle and myocardium, and contraction of skeletal muscle will inevitably lead to changes in the length of sarcomeres before and after life. This is another aspect of the study. Studies have shown that contraction of skeletal muscles can cause the expression of certain genes. Since electrical stimulation can cause muscle contraction, does the contraction of muscle caused by electrical stimulation cause the base? What's the difference between this expression and postmortem shock? Because of the short duration of death caused by electric shock, the protein level may not have changed much. We study this change and difference at the mRNA level.
research objective
The key points of forensic identification for electric shock without electric current spot are as follows:
(1) to find the diagnostic index of electric shock without current spots.
(2) to explore the current path of electric current without spot.
(3) to explore the difference between electrocution and postmortem shock.
research method
Part I: morphological diagnostic indexes of electroshock without current spots.
1. study on perforation of blood cells in vitro
Adult blood samples were taken from 6 patients, 12 ml each. Each patient was divided into 6 equal parts, 2 ml each. They were divided into 6 groups: non-electric shock group, 5 s, 10 s, 20 s, 30 s, 1 min, altogether.
2. Study of vascular endothelium and blood cell perforation by non-electric plaque electroporation and non-electric plaque electroporation and scanning electron microscopic observation of human aorta and pulmonary artery endothelium by electroporation
Forty-five New Zealand rabbits were randomly divided into 9 groups, 5 rabbits in each group, namely normal control group, A1, A2, B1, B2, C, D, E, F group. The normal control group was injected air directly into the ear vein without any treatment. The model should be tested.
Model A: The rabbit's right forelimb and left hind limb were soaked with 0.1M PBS, then wrapped with 0.1M PBS soaked wet gauze, the wire was wound directly in the gauze outer layer, connected with 220V or 110V alternating current, electrified to death.
Model B: Two wires were fixed at both ends of the rectangular plastic container as electrodes, and a small amount of water was added to the container. The water depth was about 1.5 cm. The rabbits were placed in the plastic container. A plastic net cover was placed above the container (to prevent the rabbits from jumping out) and 220V or 110V alternating current was switched on.
Model C: the experimental rabbit fell from 40 meters high to death.
Model D: spraying anhydrous ethanol on the body surface and igniting it to death.
Model E: dissolve sodium nitrite 2G in 10ml distilled water and pour into the rabbit stomach, resulting in death.
Model F: The rabbits were placed in a sealed container filled with carbon dioxide and asphyxiated to death. The morphological changes of red blood cells and endothelial cells were observed by scanning electron microscopy.
The morphological changes of endothelial cells were observed by scanning electron microscopy (SEM) in the aorta and pulmonary artery roots of 5 patients who died of electric shock.
The second part is the deduction of dead current pathway in non-spot electrocution--the difference of hsp70, c-fos, IL-6 mRNA expression in skeletal muscle of rabbits killed by non-spot electrocution
Ten New Zealand rabbits were randomly divided into two groups: the electric shock group and the control group, with 5 rabbits in each group. The expression of hsp70, c-fos and IL-6 mRNA in quadriceps femoris and biceps brachii were detected by fluorescence quantitative RT-PCR.
The third part: the distinction between electrocution and postmortem shock.
1. expression of HSP70, c-los and il-6mRNA in skeletal muscle and myocardium before and after electroporation
Fifteen New Zealand rabbits were randomly divided into three groups: electrocution group, postmortem electrocution group and control group, with 5 rabbits in each group. The rats in the control group were killed by air injection of 50 ml without electric shock, and the quadriceps femoris, biceps brachii and ventricular muscles of the left hind limb and right forelimb were taken from the left hind limb and right forelimb of the experimental animals, and the left hind limb and biceps brachii muscle were connected with 220 V AC poles for 3 minutes immediately after death. The changes of HSP70, c-fos and il-6mRNA contents in skeletal muscle and myocardium of electrocuted rabbits after electrocution were observed.
2. the length of skeletal muscle segment of electric shock and postmortem shock
Twelve rabbits were randomly divided into four groups: control group I, control group II, electrocution group and postmortem electrocution group. Control group I, II and postmortem electrocution group were executed by intravenous injection of air 50 ml from ear margin. Control group I was executed immediately after execution, while control group II was executed 24 hours after death. The hind limbs were soaked in phosphate buffer solution, then wrapped in wet gauze soaked in phosphate buffer solution. The wire was wound directly around the outer layer of the gauze and connected with 220V AC until death. After death, the animals in the electroshock group were killed by injecting air 50 ml from the ear vein, and then electrocuted for 4 minutes according to the electrocution method. The transmission electron microscope was used to observe the change of skeletal muscle segment length, and the results were statistically analyzed.
Research results
Part I: morphological diagnostic indexes of electroshock without current spots.
1. study on perforation of blood cells in vitro
After electric shock, the perforation of cell membrane (red blood cells and white blood cells) can be observed. The perforation of red blood cells can be one or more, most of which are round or oval in shape, and the perforation of white blood cells is multiple, and the number of perforated cells increases with the duration of electric shock.
2. Scanning electron microscopic observation of rabbit vascular endothelium and blood cell perforation and human aorta and pulmonary artery endothelium killed by electric shock without electric spot
Pulmonary artery and aortic endothelial cells were perforated in electrocuted rabbits, but not in electrocuted rabbits and normal control group.
Of the 5 electrocuted pulmonary artery and aorta specimens, endothelial cell perforation was seen in 4 cases, endothelial cell loss in 1 case, but smooth muscle cell perforation was seen, while no vascular endothelial perforation was found in other causes of death.
The second part is the deduction of the dead current pathway in non-spot electrocution--the difference of the expression of hsp70, c-fos, IL-6 mRNA in skeletal muscle of non-symmetrical sites after non-spot electrocution
The levels of il-6, HSP70 and c-fos mRNA in skeletal muscle of the electrocuted limbs were higher than those of the control group, but the levels of il-6, HSP70 and c-fos mRNA in the electrocuted limbs were much higher than those in the non-electrocuted limbs.
The third part: the distinction between electrocution and postmortem shock.
1. expression of HSP70, c-fos and il-6mRNA in skeletal muscle and myocardium before and after electroporation
The expression levels of hsp70, c-fos and IL-6 mRNA in skeletal muscle and myocardium of rabbits were significantly increased after electric shock, while the expression levels of hsp70, c-fos and IL-6 mRNA in skeletal muscle and myocardium of rabbits were slightly increased after electric shock.
2. the length of skeletal muscle segment of electric shock and postmortem shock
The skeletal muscle segments were shortened before and after death, but the degree of shortening was worse. Compared with the normal control group, the skeletal muscle segments were slightly shortened in the postmortem shock group.
conclusion
1. alternating current can cause cell perforation in the circuit.
2. cell membrane perforation induced by electric current or electric field is relatively specific.
3. Endothelial cell perforation of aorta and pulmonary artery root can be used as one of the diagnostic criteria of electric shock death without electric spot.
4. The changes of interleukin-6 mRNA, HSP70 mRNA and c-fos mRNA in skeletal muscle of symmetrical site can be used as an index for deducing the dead current pathway of non-spot electric shock.
5. The changes of interleukin-6 mRNA, HSP70 mRNA and c-fos mRNA in skeletal muscle and the length of skeletal muscle segments can be used as an index to differentiate electric shock from postmortem electric shock.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：D919
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