不同死后时间组织中吗啡免疫组化检测的初步研究

目的检测不同死后时间组织中的吗啡。方法应用抗吗啡兔血和SABC法对不同死后时间大鼠肝肾组织中吗啡进行了检测。结果初步发现死后27h仍可在肝肾组织普通石蜡切片上检出阳性染色。结论免疫组化技术可以用以检测死后组织中的毒物。     

不同死后时间组织中吗啡免疫组化检测的初步研究

目的 检测不同死后时间组织中的吗啡。方法 应用抗吗啡兔血和ＳＡＢＣ法对不同死后时间大鼠肝肾组织中吗啡进行了检测。结果 初步发现死后２７ｈ仍可在肝肾细胞普通石蜡切片上检出阳性染色。结论 免疫组化技术可以检测死后组织中的毒物。     

亚急性黄药子中毒的实验病理学研究

目的 研究黄药子对小鼠毒性作用的病理变化及其毒作用机制。方法 按1/4LD_(50),1/10LD_(50),1/30LD_(50)剂量给ICR小鼠连续灌服200％的黄药子水煎剂,观察中毒小鼠各主要器官的组织学与超微结构变化,酶组织化学检查及血清生化检测。结果 中毒小鼠肝细胞脂肪变性,糖原增多,严重者可见灶性坏死;肾小管上皮细胞变性、坏死。通过酶组织化学方法发现肝脏SDH酶和G-6-P酶活性下降,血清ALT和BUN升高。结论 黄药子毒作用的主要靶器官或靶组织为肝和肾。毒作用机理是对线粒体、内质网等膜性结构的破坏导致酶活性的降低,影响代谢。     

短柄乌头急性中毒——乌头碱在大鼠体内分布的研究

本文用高效液相色谱法检测了大白鼠短柄乌头急性中毒后(LD_(50)剂量灌胃),乌头碱在心、肝、肾、血、脑内的含量分布。结果表明;体内乌头碱含量甚微或检不出。30例大鼠LD_(50)剂量灌胃后,16例2h内中毒死亡。其肝、肾内的乌头碱检出率明显高于心、血、脑,且中毒表现明显。另14例于2h整处死,其肝、肾、血中检出较高。16例2h内死亡组肝中乌头碱含量分析提示乌头碱在体内代谢很快。此结果为法医工作中乌头碱中毒案件调查、检材提取、毒物分析结果的评价提供了一定的依据。     

不同染色组织切片及不同组织固定方法对检测STR基因座的影响

目的探讨不同染色组织切片及不同组织固定方法对DNA检验的影响。方法采用Chelex100法及浓缩纯化方法提取DNA,PCR扩增后310型遗传分析仪检测。结果福尔马林固定4天以内或70%乙醇、无水乙醇分别固定1年及15年的HE染色切片可以检见Amel及9个STR基因座。PTAH等5种特殊染色未能检出相应基因座DNA谱带。结论70%乙醇或无水乙醇固定组织,石腊包埋组织及HE染色切片可以作为DNA?STR检验鉴定的样本材料。     

缢死大鼠肝肾细胞内HIF-1α免疫组化染色观察

目的探讨缺氧诱导因子1α(HIF-1α)在窒息死亡鉴定中的意义。方法制作大鼠缢死后0、2、6、24h的窒息死模型,以相应时间段断颈处死大鼠为对照,用免疫组织化学ABC法结合图像分析观测肝和肾组织中的HIF-1α表达情况,并对其结果进行统计分析。结果除0h段外,HIF-1α免疫组化阳性染色可见于窒息组和对照组的各时段大鼠,主要位于肝细胞、肾近曲小管和远曲小管上皮细胞。死后6h内的肝组织HIF-1α免疫组化染色显示窒息组与对照组差别明显(P<0.05),24h后则无明显差别(P>0.05)。肾脏窒息组与对照组差别明显(P<0.05)。结论观测HIF-1α在死后6h内肝脏或24h内肾脏中的表达状态,对机械性窒息的鉴定有一定的法医学意义。     

利多卡因在蛛网膜下腔和静脉注射致死犬体内的死后分布

目的比较利多卡因在蛛网膜下腔和静脉注射致死犬体内的死后分布特点。方法犬12只,其中6只经蛛网膜下腔,另6只经股静脉匀速注入利多卡因(5×15mg/kg)致死,迅速解剖动物,取大脑、侧脑室脑脊液、腰段脊髓腔脑脊液、不同脊髓节段(颈髓、胸髓、腰髓、骶髓),心、肺、肝、脾、肾、胆汁、尿、心血、周围血、注射部位肌肉和注射部位20 cm以外肌肉等脏器组织和体液,用气质联用法定性,气相色谱法定量检测其中利多卡因含量。结果蛛网膜下腔注射致死犬体内利多卡因的含量由高到低顺序依次为腰段脊髓腔脑脊液、骶段脊髓、胸段脊髓、侧脑室脑脊液、腰段脊髓、颈段脊髓、肺、肾、注射部位肌肉、心、大脑、脾、心血、肝、周围血、胆汁、注射部位20 cm以外的肌肉、尿;静脉注射致死犬体内利多卡因的含量由高到低顺序依次为肾、心、肺、脾、大脑、肝、周围血、胆汁、心血、颈段脊髓、胸段脑脊液、注射部位肌肉、腰段脊髓、注射部位20 cm以外的肌肉、侧脑室脑脊液、尿、腰段脊髓腔脑脊液、骶段脊髓。结论蛛网膜下腔注射致死犬背侧脊髓液中利多卡因含量最高,静脉注射致死犬肾脏利多卡因含量最高,此分布特征可为利多卡因麻醉意外法医学鉴定中入体途径的判定提供参考。     

HE染色切片组织的STR检测及法医学应用

目的建立并优化HE染色切片组织STR分型方法,评估HE染色切片组织的STR分型有效性。方法用QIAgen法提取2例尸检人体的心、肝、肺、肠等8种组织的HE染色切片DNA,用TaqMan荧光探针法通过荧光域值循环数(Ct值)测定获得各提取液中的DNA质量浓度,以阳性内对照(internal posi-tive control,IPC)监测PCR过程中的抑制水平。再用Identifiler试剂盒扩增,在3100-Avant上进行STR片段分析。结果在本研究建立优化的STR分型技术条件下,8种人体组织的HE染色切片DNA提取液质量浓度均可达到1ng/μL以上,其IPC的Ct值提示无PCR抑制剂。HE染色切片组织的STR分型有效性与石蜡包埋组织相当,其DNA随时间延续而缓慢降解。结论在一定保存时限内,HE染色切片的DNA质和量可以满足STR分型检测需要,但受残余甲醛固定剂的影响,HE染色切片的分型成功率随保存时间延长而降低。     

远离电击部位电流通路上骨骼肌的病理学研究

研究电损伤时骨骼肌中肌红蛋白(Mb)和纤维连接蛋白(Fn)的异常分布情况。采用大鼠模型和人体电击死标本,对电流通路上骨骼肌组织进行肌红蛋白、纤维连接蛋白免疫组化(LSAB)检测。结果显示:电流通路上肌细胞呈现深浅不同竹节样改变,免疫组化法见Mb脱失,Fn呈阳性,呈蟒蛇状纹理。电流通路上骨骼肌竹节样和蟒蛇纹理改变对无典型电流斑法医学鉴定有实用价值。     

甲拌磷在大鼠体内的死后分布研究

目的建立甲拌磷灌胃染毒致死的大鼠动物模型,建立生物检材中甲拌磷的气相色谱和气相色谱质谱联用检测方法 ,观察甲拌磷在3种剂量染毒大鼠体内的死后分布特点。方法大鼠2LD50、4LD50或8LD50甲拌磷灌胃染毒,死后立即解剖,采集心、肝、脾、肺、肾、脑、肌肉、睾丸、心血和胃组织,GC/MS、GC/FPD法定性定量检测各组织和心血中甲拌磷。结果大鼠2LD50、4LD50和8LD50甲拌磷染毒后31±3min、19±4min和11±6min死亡。气相色谱和气相色谱质谱联用法均可检到甲拌磷。染毒死亡大鼠体内甲拌磷的含量由高到低顺序依次为：2LD50组：胃组织〉肝〉脾〉肾〉肺〉脑〉睾丸〉肌肉〉心〉心血。4LD50组：胃组织〉肝〉肺〉脾〉肾〉睾丸〉肌肉〉脑〉心〉心血。8LD50组：胃组织〉肝〉肾〉脾〉肺〉心〉肌肉〉睾丸〉心血〉脑。结论甲拌磷在大鼠体内死后分布不均匀。胃组织中含量最高,其次是肝、脾、肺和肾,脑、肌肉和睾丸含量最低。甲拌磷的灌胃染毒致死动物模型、气相色谱和气相色谱质谱联用方法及死后分布规律可应用于甲拌磷中毒死亡案件的法医学鉴定和法医毒物动力学研究。     

急性吗啡中毒大鼠主要器官内吗啡的免疫组化定位研究

一次静脉注射１２．５ｍｇ／ｋｇ。ｂｗ的盐酸吗啡染毒雄性Ｓ－Ｄ大鼠，２小时后处死，取其脑、肾、肝、肺、心组织以２％戊二醛和４％多聚甲醛混合液固定后，常规石蜡切片。运用抗吗啡抗血清及ＳＡＢＣ技术染色。结果显示上述组织切片有不同程度的阳性染色，阳性着色主要见于肾髓质部分肾小管上皮细胞，肝脏中央静脉周围的肝细胞、肺泡上皮细胞及肺内小支气管粘膜上皮细胞、中脑部分神经细胞、室管膜细胞、心肌细胞．以及各器官小血管及毛细血管内皮细胞胞浆、血浆及肾小管腔内尿液。     

Histochemical demonstration of phenobarbital by immunocytochemistry

A method for the demonstration of the topographical distribution of phenobarbital at the cellular level in various tissues was established. Mice that had been exposed to various doses of phenobarbital by intraperitoneal injection were killed, and their tissues were fixed with 0.1 M phosphate buffer solution (pH 7.4) containing paraformaldehyde and glutaraldehyde. Thereafter, paraffin and frozen sections were made and stained by the indirect immunoperoxidase method using antisera obtained from commercial sources and used for the immunochemical assay of the blood level of phenobarbital in clinical medicine. A specific positive reaction was observed solely in testing the intoxicated tissues, and this reaction was inhibited when phenobarbital was added to the antisera. The minimal sensitivity of the positive reaction, which can be discerned by observing the stained slides macroscopically, was in the range of 10 mg/kg. Thus, the diagnosis of phenobarbital intoxication in the forensic autopsy can be made by immunohistochemistry. A positive reaction was found in various tissue cells, including nerve cells, myelin sheaths, glia cells, hepatocytes, cells of the alveolar and bronchial wall, epithelial cells of the distal part of the renal tubules, and so forth. Endothelial cells of the capillaries in all tissues gave a strong positive reaction. The immunocytochemical electron microscopy of the hepatocytes revealed that the positive reaction in the cytoplasm was located solely in the intraluminal space of the smooth endoplasmic reticulum. These results indicate some interesting aspects of the pharmacokinetics of phenobarbital in vivo. It is expected that the antisera, which are used widely for the assay of the blood concentration of various drugs (phenobarbital, amphetamines, morphine, and so forth), may be regarded as excellent reagents for immunocytochemistry. This clearly indicates that morphological evidence in toxicology, which had so far remained obscure, can be easily obtained by applying these antisera against various drugs.     

Species identification from dried snake venom

Illegal trade in snake parts has increased enormously. In spite of strict protection under wildlife act, a large number of snakes are being killed ruthlessly in India for venom and skin. Here, an interesting case involving confiscation of crystallized dried snake venom and subsequent DNA-based species identification is reported. The analysis using the universal primers for cytochrome b region of the mitochondrial DNA revealed that the venom was extracted from an Indian cobra (Naja naja). On the basis of this report, the forwarding authority booked a case in the court of law against the accused for illegal hunting of an endangered venomous snake and smuggling of snake venom. This approach thus has immense potential for rapid identification of snake species facing endangerment because of illegal trade. This is also the first report of DNA isolation from dried snake venom for species identification.     

电击伤最佳病理取材部位的初步研究

目的探讨日常低电压电击后机体电流通路上极向化等病变规律和最佳病理取材部位。方法SD大鼠随机分为电击组和对照组,建立左前爪-右后爪路径220V电击大鼠模型,取电极接触处皮肤、四肢血管、神经和周边组织及腹腔大血管,常规HE染色病理组织学图像分析。结果电极接触处皮肤基底细胞(skinbasalcell,SBC)核明显极向化改变,长/短径比值与对照组比较差异高度显著,P<0.001;电流通路上腕和踝部血管及腹主动脉内皮、平滑肌细胞核的极向化与对照组比较,差异显著,P<0.05,其中腕和踝部病理改变最明显;周围骨骼肌细胞核的长/短径比值无明显规律性。结论在电击死中最常见的“手-足通路”,腕、踝部血管可能为病理取材的最佳部位。同时,由于腕和踝部距离手脚掌较远,直接接触热源和化学试剂等其他可能引起“极向化”改变的因素机会少,因此,腕和踝部血管及其周边组织的极向化改变更具有电损伤的特异性。     

蛇毒检测方法的实验研究

目的建立系统的蛇毒检测方法。方法通过对3种常见蛇毒样品检测,确定用ELISA法检测蛇毒的最适条件。结果本研究所建立的ELISA蛇毒检测法灵敏度为3.9ng/ml;交叉实验结果显示,该方法特异性高,只有个别蛇毒存在交叉反应;样本从采集到检出时间在1h40min之内;动物实验显示,半致死量中毒家兔的血液蛇毒检测在中毒后48h内有效,中毒死亡家兔的血液蛇毒检测在死后72h内有效。结论所建立方法灵敏、准确、快速、简便,是一种可靠的蛇毒检测法。     

组织细胞DNA含量变化与死亡时间的相关性研究

目的研究人死后各器官组织细胞DNA降解变化规律,探讨其在推断死亡时间方面的应用价值,为推断死亡时间提供科学依据。方法已知死亡时间人尸体的心、肝、脾、肾,按死后6,12,24和48h四个时间段取材,制成石蜡切片,经Feulgen染色并图像分析;同时将各器官组织按不同时间段取材,制成单细胞悬液,应用流式细胞仪检测DNA含量。结果(1)人体各器官组织细胞经FCM检测,脾细胞核DNA随死亡时间延长呈现较好的下降梯度,而心、肝、肾,则由于自溶较快,死后6h细胞核DNA含量已很低,其变化情况与死亡时间的相关性不佳;(2)各器官组织细胞核DNA染色强度指数均随死亡时间的延长而下降。结论(1)机体死亡后,各器官组织细胞核DNA含量随死亡时间延长逐渐减少,具有一定的变化规律,其中以脾细胞核DNA含量的变化趋势与死亡时间最具相关性,肝、肾次之,而心最差;(2)方法学分析,图像分析技术(IAT)与流式细胞术(FCM)两种检测方法在研究组织细胞DNA含量变化方面,各有利弊,可互补不足。     

常规冷冻对大鼠尸体组织形态学的影响

目的观察分析死后不同时间冷冻大鼠尸体主要内脏组织细胞形态变化规律。方法 SD大鼠20只,分为尸僵前冷冻组、尸僵期冷冻组、尸僵缓解冷冻组及对照组（各5只）,颈椎脱臼处死大鼠,实验组大鼠在室温25℃±5℃、相对湿度80%±5%分别放置0h、6h、24h后-20℃±2℃冰箱冷冻72h,解剖提取脑、心、肺、肝、肾,常规切片HE染色,显微图像体视学测量细胞外间隙的面密度。结果各实验组内脏组织间质疏松增宽,冰晶裂隙沿组织间隙或肝血窦分布、周边细胞固缩,红细胞冻溶、血管周围浆液淤积,以肝组织变化最明显。细胞外间隙的面密度：对照组明显小于实验组（P〈0.01）;脑尸僵前组较尸僵组大（P〈0.05）;心、肾尸僵前组和尸僵组无显著差异（P〉0.05）,均较尸僵缓解组大（P〈0.01）;肝各实验组组间比较均无显著差异（P〉0.05）。结论冷冻不同尸僵状态下内脏组织细胞有不同的形态结构,可作为区别和判断生前和死后组织细胞形态改变的参考。     

Immunofluorescence detection of drugs in postmortem tissues: a new technique with potential for assessment of drug influence in cause of death

This report describes a new technique, immunofluorescence, for the detection and possible characterization of drug content in postmortem tissues. By using antisera generated against a drug-protein conjugate, the stabilization of tissue-sequestered drug is accomplished by incubation of fresh frozen sections of tissue with dilute solutions of rabbit anti-drug antibodies. Secondary incubation with a fluorescence-labeled anti-rabbit immunoglobulin labels these points of sequestration. Tissue sections so stained are examined by fluorescence microscopy. In studies with rats given graded doses of morphine sulfate, there were discernible differences in tissue binding of morphine in brain sections from animals treated "therapeutically," fatally, and chronically. Extension of these studies to human autopsy material is anticipated and potential problems are discussed. This technique offers the forensic toxicologist the potential for evaluating the drug content of tissues in situ.     

The Effect of Decalcifying Solutions on Hemosiderin Staining

Abstract: To determine whether routine decalcification may reduce the amount of stainable iron that is visible on tissue sections, samples of liver and lung tissue with excessive iron stores were placed in three standard decalcifying solutions (i) formic acid [33%], formaldehyde [4%], and NaCl [0.85%]; (ii) formic acid [30%], formaldehyde [4%], and water; and (iii) nitric acid [5%] for 24, 48, 72, and 96 h. After exposure to the decalcifying solutions, the tissues were stained with Perls stain. The slides were examined blind and the intensity of iron staining was scored semiquantitatively from 0 to 3+. The trend in all samples over the course of the experiment (96 h) was for reduction in the intensity of hemosiderin staining. As the amount of stainable hemosiderin in tissues may be significantly altered by decalcification, the absence of hemosiderin in tissues adjacent to a fracture site does not necessarily indicate that the injury was acute.