【作者】 李瑞利；

【导师】 张国忠；

【作者基本信息】 河北医科大学 ， 法医学， 2014， 硕士

【摘要】 目的：因电流作用导致人体死亡称电击死。电击死典型尸体征象有电流斑、电灼伤痕、电击花纹及皮肤金属化等。电流斑是诊断电击死的主要形态学依据，寻找并提取电流损伤的皮肤、镜检确认电流斑是诊断电击死的主要依据。但在实际案例中并非每一例电击死都存在电流斑，且死后电击也可以出现电流斑。目前缺乏电流斑电击死的诊断以及生前和死后电击的鉴别是法医病理学鉴定的难点，也是法医学亟待解决的问题，该类案件主要是结合案情、受害人群、触电现场及尸体检验等作出综合判断。而法医病理学尚缺乏较为特异性的形态学或其他方面的指标为诊断提供依据。本实验在建立大鼠电击死模型的基础上，应用HE染色、透射电镜及免疫组织化学染色方法，观察电击死引起心脏超微结构及热休克蛋白70（HSP70）、缺氧诱导因子-1α（HIF-1α）表达的变化，探讨其在电击死与死后电击中的不同改变，为电击死的法医病理学鉴定提供一定的诊断依据。方法：健康Sprague-Dawley(SD)大鼠56只，体重220g±10g，雌雄不限，适应环境喂养7天，随机分7组，每组8只。对照组、死后30分钟对照组、死后60分钟对照组：对照组采用1ml水合氯醛腹腔注射麻醉后仰卧位固定在鼠架上，将对侧的前后肢用脱毛膏做去毛处理，断头处死大鼠，开胸后取其心脏，局部取材部分切成（0.5-1mm3）小块2-3块，放入装有4%戊二醛的小瓶中，备行透射电镜观察。将剩余心脏部分及肢体局部皮肤放入4％的多聚甲醛中固定，制备石蜡切片，进行HE染色观察，应用免疫组织化学方法观察心脏HSP70、HIF-1α表达情况。死后30分钟对照组和死后60分钟对照组用同样方法处死大鼠，室温放置至相应时间点后取其心脏和皮肤，观察指标同对照组。电击死组：采用1ml水合氯醛腹腔注射麻醉后将大鼠仰卧位固定在鼠架上，将对侧的前后肢用脱毛膏做去毛处理，并连接于自制的电击设备，闭合电路后用220V电压电击大鼠2分钟致死，关闭电源后撤去电击设备后即刻取材，实验内容同对照组。死后电击组、死后30分钟电击组、死后60分钟电击组：采用1ml水合氯醛腹腔注射麻醉后仰卧位固定在鼠架上，断头处死大鼠，做去毛处理后，死后电击组即刻用220V电压电击大鼠2分钟，关闭电源后撤去电击设备即刻取材，实验内容同对照组。死后30分钟电击组与死后60分钟电击组分别于处死后30分钟、60分钟用220V家用电压电击大鼠2分钟，关闭电源后撤去电击设备即刻取材，实验内容同上。数据以“均数±标准差(Mean±SD)”表示，用SPSS16.0统计软件进行统计学分析，各组均数的比较行单因素方差分析(ANOVA)，用最小显著差法(least significant difference， LSD)作两两比较，以P<0.05为有显著统计学差异。结果：1电击大鼠的一般表现与电击处皮肤观察电击死组大鼠通电后出现强直性抖动，尾巴高高翘起，有淡黄色尿液排出，部分大鼠有大便排出，肢体电击处有烧灼表现；电流斑与接触导体走行一致，色呈灰黄，质较硬、干燥，中央凹陷，周围稍隆起，边缘钝圆，与周围组织分界清晰。死后电击组大鼠也出现强直性抖动，尿液及大便排出。电流斑同样与接触导体走行一致，质较硬、干燥，中央凹陷，与周围组织分界清楚。2HE染色结果2.1皮肤HE染色对照组HE染色光镜下观察见表皮细胞无异常，排列整齐，细胞和胞核无极性化。电击死组光镜下观察见表皮细胞融合变薄、致密、着色深，细胞呈极性化改变，以基底层的细胞最为明显，基底层细胞及细胞核染色深，纵向伸长并且严重扭曲变形。死后电击组光镜下观察见表皮细胞融合变薄，致密，染色较深，细胞间界限不清，表皮细胞胞浆均质化，表皮下空泡形成，基底层细胞及细胞核染色深，纵向伸长并且严重扭曲变形，有核流征表现。2.2心脏HE染色对照组的心肌组织结构清晰可见，心肌细胞大小一致，以心肌动静脉为中心呈索状整齐排列，死后30分钟对照组、死后60分钟对照组的心肌细胞染色仅见部分区域有水肿表现，局部嗜酸性变。电击死组心肌细胞间质有水肿表现，胞浆染色变淡，胞核染色变淡、增大，心肌层有断裂及收缩带坏死的改变，心肌细胞有嗜酸性变。死后电击组的心肌细胞有轻度的水肿，胞浆染色变淡，胞核染色变淡、增大，部分心肌细胞有嗜酸性变；死后30分钟电击组、死后60分钟电击组的心肌细胞染色仅见部分区域有水肿表现，局部嗜酸性变。死后电击各组心肌无断裂及收缩带坏死。3心脏透射电镜观察对照组电镜下观察发现肌纤维间质无异常，血管周围间质无水肿，血管内皮细胞吞饮小泡排列整齐，毛细血管周围无异常表现，结构清楚，Z线M线清晰、无断裂。电击死组电镜下观察发现肌原纤维断裂并且溶解消失，间质出现水肿，线粒体嵴和膜消失并溶解，肌纤维间质水肿，血管周围间质出现水肿，血管内皮细胞吞饮小泡排列密集，毛细血管周围水肿，结构模糊混乱，Z线M线模糊排列紊乱可见断裂。死后电击组电镜下观察发现细胞核边移，肌纤维间质水肿，线粒体嵴和膜消失并溶解，Z线M线模糊排列紊乱。4免疫组织化学结果HIF-1α蛋白在心肌细胞内的表达为胞浆着色，对照组、死后30分钟对照组、死亡60分钟对照组心肌细胞阳性染色较少，各组之间无明显差异。电击死组镜下观察心肌细胞的胞浆染色增强，与对照组比较有明显增高，具有统计学差异（P＜0.001)；死后电击组有阳性表达，与对照组比较具有统计学意义(P＜0.01)，死后30分钟电击组与死后60分钟电击组与对照组比较无明显差异。HSP70蛋白在心肌细胞的表达为胞浆着色，对照组、死后30分钟对照组、死后60分钟对照组偶见胞浆黄染出现，阳性染色并不明显，各组之间无明显差异。电击死组细胞黄色增强，与对照组比较有明显增高，具有统计学差异（P＜0.001）；死后电击组有阳性表达，与对照组比较具有统计学意义(P＜0.01)，死后30分钟电击组与死后60分钟电击组与对照组比较无明显差异。结论：生前电击和死后电击均可出现电流斑，所以其不能作为区分生前电击与死后电击的依据。而电击死心脏HSP70、HIF-1α蛋白的表达比死后不同时间再电击明显增多，所以其对电击死与死后电击的鉴别诊断具有一定的意义。更多还原

【Abstract】 Objective: Death as a result of current is called electrocution.electrocution body signs have current typical porphyry, electric bright scars,shock pattern and skin metallization, etc. Current spot is the mainmorphological basis for diagnosis of death electric shock death, therefore, tofind and extract the current damage skin, microscopy confirmed the currentspot are the key to the diagnosis of electric shock death. But not every case ofelectric shock to death in the practical case had current spot, and it also canappear after the death of shock current spot; At present the lack of current spotshock death and lifetime and after the shocks of identification is a key problemfor forensic pathology identification, is also a problem urgently to be solved inforensic medicine. At present this kind of case is mainly combined with thecase, vulnerable people, get an electric shock the scene and body inspectionetc. to make a comprehensive judgment. And forensic pathology is still lack ofa more specific morphology or other indicators provide the basis for diagnosis.This experiment on the basis of model rats shock death, using HE stainingand transmission electron microscope and immunohistochemical stainingmethod, observing the morphological change of heart tissue in theelectrocution and a shock after the death. Through the observation of HSP70,HIF-1α, explore the different change in electrocution and a shock after thedeath，Provide certain diagnostic significance for forensic identification.Methods:56health SD rats, the weight is220g±10g, male and femaleunlimited, Fed7days to adapt to the environment, randomly divided into7groups, each group of8. Preparation of the model with a homemade electricshock devices.Control group, the30min after death control group, the60min after deathcontrol group, control group adopts after intraperitoneal injection of1ml chloralhydrate anesthesia supine fixed in rats on the shelf, will be on the sideof the front and hind legs do unhairing process using hair removal creams,beheaded kill rats, take its heart after thoracotomy, locally based part cut into0.5-1mm3(after) small2-3pieces, into a small bottle of4%glutaraldehyde,line for transmission electron microscope. The heart of the remaining localskin and body fixed in4%paraformaldehyde, the preparation of paraffinsection, HE staining observation, immunohistochemical method to observe theheart HSP70, HIF-1alpha expression. the30min after death control group andthe60min after death control group used the same method to be put to death inthe rats.Placed at room temperature to the corresponding time points after takeits heart and skin, observe with the control group.The electrical shock group：Anesthetized rats by intraperitoneal injectionof1ml chloral hydrate, then the rat in supine position fixed in rat frame,Willbe on the side of the front and hind legs using hair removal creams to woolprocessing connected self-made electric equipment，After a closed circuit witha220V voltage shock in rats2minutes to kill.After turning off the power andretreat the shock device immediately draw materials. The experiment contentwas the same as the control group.The0min after death electrical shock group, the30min after deathelectrical shock group and the60min after death electrical shock group:decapitated the rats after anesthesia by1ml intraperitoneal injection of chloralhydrate and fixed them on rat shelves supinely, do unhairing process. Shockedthe rats in the group of electric shock instantly with a220V household voltagefor two minutes, fixed them on rat shelves supinely after turned off the powerand removed Electrical equipment, the experiment contents such as drawmaterials instantly reference to the control group. Shocked the rats in theother two groups with a220V household voltage for two minutes, fixed themon rat shelves supinely after turned off the power and removed Electricalequipment, the experiment contents such as draw materials instantly referenceto above method.With “SD” indicates the data, statistical analysis was performed using SPSS16.0statistical software, using one-way ANOVA compared the mean ofeach group，for pairwise comparisons using the least significant differencemethod (least significant difference, LSD). With p<0.05indicated there werestatistically significant differences.Results:1The basic performance of shock rats and shock observation of the skinShock after death group of rats live ankylosing jitter, high upwarp tail, apale yellow urine, some rats have defecate eduction, limbs had burn shock；Current spot in accordance with line contact conductor walk, color showssallowness, qualitative hard, dry, central sag, slightly uplift, around the edgeof obtuse, with clear boundary between the surrounding tissue. Shock afterdeath group of rats also appear ankylosing jitter, urine and stool.Current spotalso agree with line contact conductor walk, qualitative hard, dry, central sag,with a clear demarcation between the surrounding tissue.2The result of HE staining2.1The change of HE staining in skin pathologyControl group of HE staining, we found that epidermis cells showednormally, arranged in neat order and no fracture, meanwhile, the staining ofcytoplasm and nucleus were normally and uniform under the light microscopyobservation.The electrical shock groups were observed under light thinning of theskin cell fusion, dense, deeply staining, cell polarity is changed to change, ismost evident in basal layer cells, basal layer cells and nuclei are deep,longitudinal elongation and serious distortionThe0min after death electrical shock group were observed under lightsee thin skin cell fusion, dense, dark stain, unclear boundaries between cells,skin cells cytoplasm homogenization, skin cavity formation, basal layer cellsand nuclei are deep, longitudinal elongation and serious distortion, nuclearflow performance.2.2The changes of HE staining in cardiac pathologyControl group of myocardial tissue structure is clearly visible, myocardial cell size is consistent, is for the center with myocardial arteriovenous funicularlined up, after the death of30minutes,60minutes after the death ofmyocardial cell dyeing only part of the area is edema, eosinophilic changeperformance gradually.The electrical shock group of myocardial cells in interstitial edema,cytoplasm staining, lighter nuclei and increase, myocardial layer fractureperformance, part of the myocardial cells with eosinophilic change.The0min after death electrical shock group of heart muscle cells withmild edema, cytoplasm staining, lighter nuclei and increase, part of themyocardial cells with eosinophilic change; The30min after death electricalshock group and the60min after death electrical shock group of myocardialcell dyeing only part of the area is edema, partial eosinophilic change. Afterthe death of an electric shock each myocardial no obvious fracture.3The results of heart by electron microscopyThe control group were observed by electron microscopy there was noabnormality in fibromuscular stroma, and no edema in perivascularinterstitial,the vascular endothelium cells pinocytosis vesicles were in goodorder, no significant changes around capillaries, structure was clear,the line ofZ and M were clearly visible without fracture.The electrical shock group was observed by electron microscopy thatmyofibril was broken and dissolved to disappear; mitochondrial ridges andmembrane was dissolved to disappear, the dropsy observed in interstitial,fibromuscular, perivascular interstitial and surrounding capillaries; thevascular endothelium cells pinocytosis vesicles were densely arranged,surrounding capillaries structure fuzzy and chaos, the line of Z and M wasfuzzy derangement visible with fracture.The0min after death electrical shock group was observed that nuclearwas side shift；muscle fiber was showed dropsy; mitochondrial ridges andmembrane dissolved to disappear;the line of Z and M was fuzzy derangementvisible with fracture by electron microscopy. 4The results of immunohistochemistryHIF-1alpha protein expression in myocardial cells as the cytoplasmstaining, the control group, the30min after death control group, the60minafter death control group myocardial cell death dyeing is less, no significantdifferences between groups. the electrical shock group and the microscopicobservation of myocardial cell cytoplasm staining enhancement, increasedsignificantly compared with controls, with statistical difference (P<0.001);The0min after death electrical shock group has positive expression, comparedwith the control group with statistical significance (P<0.01), The30min afterdeath electrical shock group,the60min after death electrical shock group andthe control group was no significant difference.HSP70protein expression in myocardial cells of cytoplasm staining, thecontrol group, the30min after death control group, the60min after deathcontrol group accidental cytoplasm yellow dye, positive staining is notobvious, no significant differences between groups. the electrical shock groupenhanced cell death group yellow, increased significantly compared withcontrols, with statistical difference (P <0.001); The0min after death electricalshock group has positive expression, compared with the control group withstatistical significance (P<0.01), The30min after death electrical shockgroup,the60min after death electrical shock group and the control group wasno significant difference.Conclusions:Lifetime shock and after shocks are current spot, so its not as a basis todistinguish the lifetime shock and after shocks. Cardiac death and shock theexpression of HSP70and HIF-1alpha protein than electric shockssignificantly increased again after the death of a different time, so the electricshock after the death of death and the differential diagnosis of electric shockhas a certain significance.更多还原