应用酶组织化学方法鉴定死亡时间的初步探讨

利用7例颅脑外伤死亡的健康青年尸体,在死后48h,环境温度18～24℃,空气相对湿度83～92％和实验湿度54～64％的条件下,检测肝脏、肾脏酶活性的变化。实验结果表明,肝脏乳酸脱氢酶(LDH)和L-苹果酸脱氢酶(MDH),随着死亡时间的延长,活性逐渐减低,48h近于阴性;而肾脏上述二种酶活性则在死亡后6h和24h出现高峰,36h开始下降;肝脏的酸性磷酸酶(ACP)亦于死后6h和24h出现高峰,36h开始下降。而肾脏此种酶在死后18～24h,有增高趋势。笔者认为上述酶活性的规律性变化有助于死亡时间的推断。应用二种以上酶活性的变化特点,能够较准确地判断死亡时间。     

大鼠皮肤挫伤后Beclin1和LC3的表达

目的 研究大鼠皮肤损伤后Beclin1、LC3的表达,观察皮肤损伤中的自噬现象,并探讨其在损伤诊断及损伤经历时间推断的意义.方法 运用免疫组化及图像分析技术研究大鼠皮肤挫伤后、损伤不同时间段后Beclin1、LC3表达的变化.结果 大鼠正常皮肤表皮细胞Beclin1、LC3低表达,大鼠皮肤挫伤6h后局部表皮细胞Beclin1、LC3表达开始增多,挫伤3、5和7d呈强表达.结论 皮肤损伤后局部细胞Beclin1、LC3表达增多;其表达与损伤经历时间具有相关性;细胞自噬增强是对损伤的反应.     

FN-EIIIA肽段表达与皮肤损伤时间推断研究

目的 在mRNA研究的基础上,研究大鼠皮肤挫伤后含EIIIA□段的Fn蛋白水平表达与损伤时间关系。方法片段特异性单克隆抗体免疫组织化学染色。结果伤后6h表皮细胞、毛囊上皮细胞等出现棕黄色阳性染色颗粒,并随损伤时间延长染色加深。结论Fn-EIIIA片段检测可用于法医学生前伤确认和损伤时间推断。     

中介素对大鼠急性心肌缺血损伤的作用及机制

目的 探讨中介素(intermedin,IMD)对大鼠急性心肌缺血损伤的作用及机制,为研究心源性猝死机制提供思路.方法 72只健康雄性大鼠随机分为对照组、缺血组、IMD干预组.酶化学法检测心血中乳酸脱氢酶(1actate dehvdrogenase,LDH)、丙二醛(malondialdehyde,MDA)、超氧化物歧...     

Fibronectin EIIIA splicing variant: a useful contribution to forensic wounding interval estimation

A fibronectin splicing variant, Fn containing an extra type III domain (EIIIA in rat, ED1 or EDA in human) has recently attracted more attention because of its sensitive response in injury of adult tissue. The characteristic that this form is absent in adult tissue while it is commonly expressed in fetal tissues and injured adult tissue is useful in estimation of injury interval in forensic science. The regulation of fibronectin splicing was studied by immunohistochemistry by using monoclonal antibody to EIIIA on sections from paraffin embedded rat skin sample after contusion. The results indicated that epidermic cells and hair follicle epithelium of rat skin were found positive staining at 6h after injury, and the color became darker with prolonged wounding time. Based on the above result, we invented a type of test paper assayed for EIIIA-Fn splicing variant by using immune colloidal gold technique. After dipping one end of test paper in the supernatant fluid of tissue homogenates of injury skin for few minutes, detecting line could be found. It showed that all experimental injured samples revealed positive staining; the darkness of positive staining was dependent on the injury time, while control normal skin cannot found positive staining. It is concluded that there is a close relationship between the expression of EIIIA-Fn splicing variant and wounding interval, and that the gold-labeled test paper can be useful in distinguishing ante- and post-mortem injury, and in estimation of wounding interval in forensic science.     

Distributions of genetic markers in United States populations: II. Isoenzyme systems

All published and unpublished population frequency data that could be located for U.S. populations is tabulated and presented for the isoenzyme systems phosphoglucomutase, esterase D, adenylate kinase, acid phosphatase, glyoxalase I, adenosine deaminase, 6-phosphogluconate dehydrogenase, glutamic-pyruvic transaminase, carbonic anhydrase II, and glucose-6-phosphate dehydrogenase. Results obtained by combining data for comparable racial/ethnic groups are also presented. The results obtained with combined data may give better information on frequencies for the U.S. population at large than is obtainable from studies conducted in restricted geographic areas.     

Fatal familial haemolytic anaemia

A 19-year-old man, mentally handicapped but physically well, died within 2 days of onset of an acute episode of gastrointestinal upset and "haematuria." Autopsy and microscopy suggested a haemolytic episode and family studies showed haemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency, which was almost certainly present in the deceased.     

大鼠皮肤挫裂创细胞间粘附分子-1的表达

目的 观察大鼠皮肤钝器伤后细胞间粘附分子-1(ICAM-1)表达的变化规律。方法 应用免疫组化方法,观察大鼠皮肤钝器伤后1h、3h、6h、12h、1d、3d、5d、7d、9d ICAM-1的表达情况,并用MIAS图像分析系统进行图像分析。结果 正常对照组大鼠皮肤有低水平ICAM-1表达,钝器伤后ICAM-1表达增加,伤后7d达峰值,伤后9d开始下降。结论 皮肤钝器伤可诱导ICAM-1表达增加,其时序性变化可望作为一种客观指标用于法医学损伤时间的推断。     

大鼠皮肤钝器伤血管内皮生长因子表达变化的研究

目的观察大鼠皮肤钝器伤后VEGF表达的变化规律。方法应用免疫组化技术观察大鼠皮肤钝器伤后不同时间(1h、3h、6h、12h、1d、3d、5d、7d、9d)VEGF的表达情况,并用MIAS图像分析系统进行图象分析。 结果正常对照组大鼠皮肤有低水平VEGF表达,钝器伤后VEGF表达逐渐增加,于伤后7d达峰值,伤后9d开始下降。 结论皮肤钝器伤可诱导VEGF表达增加,其时序性变化可望作为一种客观指标用于法医学损伤时间的推断。     

兔急性心肌缺血脱氢酶电镜—细胞化学变化的动态观察

为了研究急性心肌缺血脱氢酶变化的规律性，本文应用电镜酶细胞化学技术，通过结扎５０只家兔冠状动脉左前降支，观察心肌缺血后０．２５、０．５、１、１．５、２、４、６、１２、２４、３６、７２小时ＳＤＨ和ＬＤＨ的变化。结果显示：心肌缺血０．５小时后，ＳＤＨ和ＬＤＨ活性开始下降。同时伴有致密体的形成和超微结构的改变。随着缺血时间的延长，酶活性下降更加明显，最后消失。     

家兔非热低压电击伤血清LDH和HBDH活性的动态变化

目的研究家兔非热低压电击伤后不同时相乳酸脱氢酶（lactate dehydrogenase，LDH）和羟丁酸脱氧酶（hydroxybutyrate dehydrogenase，HBDH）活性的变化，为临床和法医学非热电击伤提供诊断依据。方法健康新西兰大白兔40只，随机分为对照组和电击伤后7个时间点的实验组，每组5只。实验各组家兔用220V 50Hz交流电电击2S后自由进食，按照预定时间点麻醉后抽取心室血5mL，使用自动生化仪（type Selectra-2）测定血清LDH、HBDH酶活性。对照组家兔不电击，其他处理同实验组。结果实验组家兔目标血清酶活性发生了动态变化，有一定规律性，LDH活性在电击后4h，12h，1d，2d，3d明显升高（4h，12h，1dP〈0．01；2d，3dP〈0．05）；HBDH活性在电击后2d、3d明显升高（P〈0．05）；HBDH／LDH比值在2h、4h、12h与对照组比较显著降低（P〈0．01）。结论动态监测LDH、HBDH活性，可为非热电击损伤的诊断、损伤时间推断提供理论依据。     

Diagnostic role of organ-specific isoforms of lactate dehydrogenase in blood plasma

Changes in plasm isoenzymic spectrum of lactate dehydrogenase (LDG), a glycolytic enzyme, were studied in death of cardiovascular diseases, ethanol poisoning and a mechanical injury. The tests used electrophoresis in 1% solution of agarose. Isoenzymic composition differed between the groups with acute coronary failure, chronic coronary heart disease, alcohol cardiomyopathy and a mechanical injury. Ethanol poisoning caused two types of characteristic shifts in LDG isoenzymic spectrum. Nomograms were designed for practical application in forensic medical expertise of sudden death.     

Fn—EⅢA金标单克隆抗体试纸条的试制和初步应用

目的开发可用于法医病理学损伤时间推断临案的金标Fn-EⅢA单克隆抗体试纸条.方法选取两种敏感、特异的片段特异性Fn-EⅢA单克隆抗体,ELISA方法进行效价观察,标记胶体金后分别采用渗滤法及层析法检测其效果,抽提阳性对照中目标蛋白进行初步测评,并以大鼠皮肤损伤模型进行验证.结果两种单克隆抗体均可用于Fn-EⅢA肽段的金标法检测,以IST9搭配Fn多抗效果最佳;正常皮肤组织样本中试纸条检测线不显色,损伤3h后显色,随损伤时间延长,染色逐渐加深.结论该试纸条可靠性、可重复性以及可操作性均佳,可望在法医学损伤时间推断有广阔的应用.     

HSP70表达与脑挫伤经过时间的关系

目的观察大鼠脑挫伤后不同时间内HSP70蛋白表达的变化关系,探讨其与脑损伤时间的关系。方法应用免疫组织化学染色观察自由落体撞击大鼠脑挫伤后HSP70蛋白在伤后不同时间(0.5h、6h、12h、24h、3d、7d、14d、28d)表达情况。结果0.5h伤侧皮质挫伤灶周围HSP70阳性细胞表达开始增强,12h达高峰,24h降至较低,3d又再次升高,以后逐渐下降,28d恢复至正常对照组水平。结论HSP70免疫组化染色可以作为法医学推断早期脑损伤时间的敏感性指标之一;HSP70可作为判断脑损伤是否存在及区分生前和死后脑损伤的重要标志。     

人脑挫伤后bFGF和COX2免疫组化染色观察

目的　观察人脑损伤后bFGF和COX2的动态变化规律 ,建立推断脑挫伤经过时间新方法。方法　应用免疫组织化学染色和图像分析技术 ,对 3 5例重度颅脑损伤案例的脑挫伤灶周边区bFGF和COX2的表达进行观察检测。结果　bFGF阳性细胞在即刻死亡组表达强度最大 ,伤后 12h急剧下降 (P <0 0 1) ,以后逐渐缓慢下降 ;COX2阳性细胞表达于伤后 12h急剧升高 (P <0 0 1) ,伤后 1d达峰值 ,以后逐渐下降 ,在伤后 11d消失。结论　bFGF和COX2的免疫组织化学染色可作为推断人脑挫伤经过时间的参考指标。     

Identification and determination of age of needle tracks in human skin (author's transl)]

Examination of enzymic activities and histochemical reactions on needle pricks can be used to determine the age of micro-injuries with sufficient exactness. Well preserved skin shows patterns of enzymes which also appear with other skin injuries and which were described in detail especially by Raekallio (1970, 1972). In autolytical changes of the skin the adenosinetriphosphatase-reaction is still reliable and can be successfully estimated. If one has sufficient experience it is possible to fairly exactly estimate the age of an injury within the first hour after the injury by preparation of the adenosinetriphosphatase-reaction. If the times of injections are known one frequently succeeds in determining the time of death by exactly determining the age of the injury. In legal expertises the histochemical reactions can rightly be regarded as useful indications of a certain age of the injury. But they do not allow an exact estimate "beyond a reasonable doubt".     

重伤家兔体内Cu及代谢酶变化与MOF及死亡关系

目的探讨兔重伤后机体内铜（Cu）含量及其代谢酶铜锌超氧化物歧化酶（Cu/Zn-SOD）、细胞色素c氧化酶（CCO）活性变化与继发性多器官衰竭（MOF）及死亡的关系。方法参照国际通用的创伤程度量化评分标准（ISS）,建立致MOF的家兔重伤模型,检测伤后不同时段血清及组织Cu含量与Cu/Zn-SOD和CCO的活性,分析其变化规律与继发MOF及死亡的关系。结果①重伤组家兔（包括死亡组家兔）血清Cu含量在伤后12h显著下降,36h后逐步回复;脑、心、肺组织中Cu含量伤后下降,肝脏组织Cu含量于伤后12h显著升高,36h后回落。伤后死亡组家兔脑、心、肺、肝Cu含量均较对照组降低,其中脑、肝组织Cu含量下降显著。②重伤组家兔血清Cu/Zn-SOD活性伤后12h下降3,6h之后呈升高的趋势,肝脏组织中的Cu/Zn-SOD活性伤后持续显著升高。③重伤后家兔（包括死亡组家兔）大脑皮质、心、肝CCO活性变化不明显,但脑干组织CCO活性显著升高。伤后死亡组家兔脑、心、肺、肝等器官的大体及组织学呈MOF改变。结论严重创伤可引发兔血清和组织Cu水平及Cu/Zn-SOD、CCO活性变化,这些变化可能与继发多器官衰竭和死亡相关。     

Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1beta,IL-6, TNF-alpha) in human skin wounds

The proinflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) hold important functions in the early and late courses of inflammation, trauma and wound healing. In the present study, human skin wounds due to sharp force (n = 105) were collected during surgery and autopsy. The wound age mainly varied from several minutes to 5 h, some specimens aged up to 6 weeks. Control specimens from uninjured skin were available in each case. After preparation of cryostat sections, immunohistochemistry was performed according to the APAAP technique, using monoclonal and polyclonal antibodies. The results were evaluated semiquantitatively. All markers were weakly expressed in normal human skin constitutively. However, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands. IL-1beta and IL-6 showed enhanced expression after 15 and 20 min at the earliest (increase of epidermal reactivity). After 30-60 and 60-90 min, respectively, marked expression was observed with these markers. Similar alterations were detectable with TNF-alpha after 15 and 60-90 min. The reactivity of all three markers persisted over several hours, then decreased to basal levels again and sometimes reappeared after days and in granulation tissue. Leukocytes reacting with IL-1beta and IL-6 appeared after approximately 2 h. CONCLUSION: proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early post-traumatic interval prior to leukocyte reaction. Autolysis did not play a role in the samples investigated (postmortem interval up to 8 days). Problems could sometimes rise from constitutive expression. Therefore, it is recommended to examine control samples from the same individual and to compare the reactivity with wound specimens.     

EIIIA＋纤维连接蛋白在大鼠皮肤切创的表达

目的：探讨大鼠皮肤切创纤维连接蛋白EIIIA片段的表达与损伤时间的关系。方法取48只成年SD大鼠并切割大鼠背部皮肤致伤。于伤后即刻、0.5h、1h、2h、3h、4h、6h、8h共8个时间段分别处死大鼠,取其创伤周围皮肤,利用免疫组织化学方法和Western印迹法检测EIIIA＋纤维连接蛋白,观察其表达水平与损伤时间的关系。结果伤后即刻未出现EIIIA＋纤维连接蛋白的表达,伤后0.5 h,皮肤基底细胞开始出现阳性表达,随损伤时间的延长,阳性着色持续增强。 Western印迹法检测发现,所测得的相对光密度值随损伤时间的延长而逐渐增高。结论 EIIIA片段表达变化可以作为法医学早期损伤时间的推断指标。     

不同损伤时间大鼠皮肤切创 Fos表达研究

目的研究皮肤切创在伤后不同时间段 Fos的表达 . 方法在大鼠皮肤切创模型石蜡切片上进行 Fos免疫组化染色 . 结果伤后 10min, 表达量开始增高 , 伤后 3h达高峰 , 以后又逐渐降低 , 至损伤 1d后 , 表达量与对照组无显著性差别 . 结论 Fos为皮肤伤后的敏感指标 , 但需结合其他指标综合评价 .