心肌病猝死者心肌连接蛋白43的免疫组化染色观察

目的探讨心肌病猝死者心肌连接蛋白43(Cx43)染色变化及其与猝死的关系。方法运用免疫组化和图像分析技术,分2组(A和B组)检测20例心肌病猝死者心室肌的Cx43染色情况;并与14例非心肌病猝死者(C组)的检测结果对照。结果扩张型心肌病(DCM)猝死组(A组,11例)心肌Cx43染色明显减弱,阳性着色斑点大小不等、深浅不一、分布不均,有的呈散在颗粒状;其它类型的心肌病猝死组(B组,9例)亦见类似变化;非心肌病猝死的对照组(C组,14例)未见明显变化。定量检测并经统计分析发现,Cx43蛋白染色阳性的面积,A组与B组和C组的差异有非常显著性意义(P<0.01),B组与C组的差异无显著性意义(P>0.05);而平均光密度各组之间的差异无显著性意义(P>0.05)。结论心肌病猝死者心肌Cx43免疫组化染色明显减弱,尤以扩张型心肌病明显;心肌病猝死者心肌Cx43变化可能与其猝死有一定关系。     

冠心病猝死的病理学研究——附128例尸检分析

本文报道128例冠心病猝死的法医病理学研究结果。其中冠脉病变4级63例,3级26例,2级29例。3级以上病变者斑块分布多较广泛。各支病变中以左前降支最常见。并发新鲜血栓形成者18例,斑块内出血17例,急性心肌梗死仅2例。36例冠脉斑块有炎性细胞浸润。56例见心肌间质纤维化或小灶疤痕形成。指出虽然我国冠心病发病率较低,但仍是猝死最常见的原因,尤以中壮年男性多见;多数病例无明显诱因而于睡眠中猝死。对冠心病猝死发生的特点、冠脉和心肌病变及病理诊断等进行了分析讨论。     

冠心病猝死100例小冠状动脉法医病理形态学观察

1案例资料1·1一般资料本组100例猝死均为1982~2003年,丹东地区例(60%);Ⅳ狭窄者30例(30%);其组织学改变主要是内皮细胞增生、肿胀,肌性肥厚,腔内积血,微血栓形成,管腔狭窄呈花环状、乳头状、同心圆状等变化。(4)心肌病变:本组100例中均有心肌断裂,其中有陈旧性心肌梗塞者30例,心肌充血者6例,心肌微血栓形成者4例。上述心肌病变均系心缺血性病变;对照组冠状动脉、小冠状动脉及心肌未见明显改变。2讨论(1)病理诊断小冠状动脉狭窄不仅发生于冠状动脉粥样硬化性心脏病,据报道,约有17种疾病可伴有小动脉狭窄病变,因此,小冠状动脉狭窄并非是特异…     

冠心病猝死心肌肌动蛋白缺失的免疫组织化学定量研究

应用免疫组织化学LSAB法和图像处理系统，对德国慕尼黑大学法医学研究所1994年尸检冠心病猝死和对照组病例（各15例）的心肌肌动蛋白缺失情况进行定量研究。发现冠心病猝死病例心肌纤维内有明显的肌动蛋白缺失，是大小不一，多发、散在分布；对照组无或仅局限性小灶状缺失。将检测结果定量分析比较，其差异非常显著，说明重度冠状动脉粥样硬化患者是否冠心病猝死，其心肌肌动蛋白缺失的范围和程度有明显差异，冠心病猝死和对照组病例心肌肌动蛋白缺失的差异具有诊断意义，而形态定量分析能较客观准确地反映心肌纤维内肌动蛋白缺失量的变化。     

冠心病心肌线粒体DNA5.0kb缺失的检测

目的探讨冠心病心肌线粒体DNA5.0kb缺失的检测。方法120例心脏的左、右心室肌各1份,分为正常对照组、病例相关组和病例组,每组40例80个样本。用断裂点连接PCR分别扩增样本含线粒体DNA5.0kb缺失的片段,巢式PCR检测含5.0kb缺失片段的扩增产物的准确性,半定量PCR对该产物进行定量,聚丙稀酰胺凝胶电泳检测PCR产物。结果在对照片段扩增成功的样本中,正常对照组未检出线粒体DNA5.0kb缺失;病例相关组检出2例,占6.07%(2/33);病例组检出29例,占85.29%(29/34);左、右心室肌线粒体DNA5.0kb缺失量分别为0.0015%~0.7813%和0.0008%~0.3906%。病例组与其他两组缺失率经χ2检验,其差异具有极显著性意义(P<0.001);左、右心室肌的缺失量经t检验,其差异具有极显著性意义(P<0.001)。结论冠心病心肌多有线粒体DNA5.0kb缺失,缺血明显的区域其缺失量也较高。     

人线粒体DNA序列分析在法医学中的应用研究及其进展

综述人线粒体DNA(m tDNA)序列分析在法医学种属鉴别、个体识别,以及个体年龄推断中的应用研究及其进展,展望对m tDNA异质性的研究及建立人m tDNA数据库,并具有重要的法医学实践意义。     

染色毛干mtDNA不同提取方法的研究

目的为染色毛发线粒体DNA选择最佳的提取方法。方法在5个染发个体中各取5根头发,采用Chelex-100法、有机法、磁珠法、H2O2结合Chelex-100法及Chelex-100结合M icrocon100法等5种不同的提取方法提取染色毛发m tDNA,利用2%琼脂糖凝胶电泳检测扩增产物。结果Chelex-100结合M icrocon100方法提取染色毛发扩增m tDNA效果较好,其余方法无扩增产物。毛发的不同部位对扩增结果无影响。结论染色毛发采用恰当的提取方法可以提取到m tDNA模板,为法医日常检案提供帮助。     

用dHPLC技术检测线粒体DNA编码区单核苷酸多态性

目的研究线粒体DNA(m tDNA)编码区单核苷酸多态性,建立检测m tDNA编码区单核苷酸多态性(SNP)的变性高效液相色谱(dHPLC)方法。方法设计针对线粒体DNA编码区nt10287-10679及nt8507-8805引物,应用dHPLC技术检测其序列多态性。结果100例中国汉族无关个体中,m tDNA nt10287-10679检出13个SNP位点,13种单倍型,基因多样性(H)为70.79%,偶合概率(P)为29.92%;m tDNA nt8507-8805检出10个SNP位点,12种单倍型,H为70.42%,P为30.28%;两段序列联合起来共检出23个SNP位点,23种单倍型,H为84.14%,P为16.70%。结论所建立的dHPLC方法可用于快速、准确地检测m tDNA编码区序列多态性;m tDNA编码区多态性位点作为m tDNA控制区多态性位点的补充,联合应用可以提高m tDNA的个体识别能力。     

毛干mtDNA单倍型母系家系排查法侦破命案

笔者在实践工作中遇到利用受害者指尖粘附微量毛干线粒体DNA(m tDNA)测序的多态信息,最终成功侦破一起命案,现报道如下。1材料与方法1.1简要案件及样本DNA的提取2008年9月某市发生一命案,现场提取死者(杨某,女)指尖粘附约1cm长毛干及胸部、膝关节上毛发各1份;杨某丈夫(王某)和杨某血痕,编号1~5。按母系遗传关系排查的杨、金、何、刘姓嫌疑人血痕各1份,编号6~9。参照张纯斌等人[1]的方法提取毛干m tDNA;有机法提取血痕m tDNA。1.2巢式PCR扩增m tDNA高变区I(HVRI)15996-16401区域长片段的PCR扩增,上、下游引物序列为L15996:5-′CTCCAC-CATTAGCACCCAAAGC-3;′H16401:5-′TGTTTCACG-GAGGATGGTG-3′。15μL反应体系包括含镁离子10×PCR缓冲液1.5μL,5mmol/L dNTP1μL,1μmol/L正反向引物各1μL,0·2μL(5U/μL)TaqDNA聚合酶,0·5μLDNA(50ng/μL),余量用水补足。PCR反应条件:95°C变性2m in,94°C变性20s,58°C退火20m in,72°C...     

冠状动脉内弹力膜缺损与冠心病猝死的关系

目的探讨冠状动脉内弹力膜(IEL)病变与冠心病猝死(SCD)的关系。方法从本单位近5年尸检案例中挑选60例病例和心脏标本,分为SCD组、对照组A和B组。每例分别在冠状动脉左前降支近段取材,常规制片,HE和双重组合特殊染色,显微镜下观察冠状动脉IEL,并用图像分析技术对特殊染色的IEL进行形态定量分析。结果60例中,SCD组(男性24例,女性6例,年龄30~83岁)的冠状动脉IEL缺失明显(缺失率=41.64%),未缺失的IEL失去原有弯曲度而变平直;对照组A(冠心病非猝死者,男性13例、女性2例,年龄41~80岁)的冠状动脉IEL缺失程度较SCD组稍轻(缺失率=30.53%);对照组B(无冠心病的其他死者,男性8例,女性7例,年龄17~54岁)的冠状动脉IEL仅有轻度缺失(缺失率=15.67%),多保持原有波浪状结构。统计学分析显示,SCD组与对照组、SCD组与对照组B、对照组A与对照组B之间IEL缺损率的差异均具有极显著性意义;SCD组与对照组A在IEL缺损率上的差异有显著性意义。结论SCD与其冠状动脉IEL缺损率的大小密切相关。     

Common mitochondrial DNA deletion associated with sudden natural death in adults

One of the most frequent causes of death in developed countries is sudden natural death (SND), which is the most common indication for medico-legal autopsies. Cardiac diseases are frequently detected among SND. Mitochondrial DNA (mtDNA) is easily damaged by reactive oxygen species, and it may cause dysfunction in tissues, leading to early events in cardiovascular disease. A specific mtDNA deletion of 4977 bp is associated to aging, myocardial dysfunction, and bioenergetic deficit. The potential link between mtDNA damage and SND has not been investigated before. Our aim was to evaluate the accumulation of the common mtDNA4977-deletion in cardiac muscle samples from autopsies of SND in adults (n = 14) in comparison to control samples from unnatural deaths (n = 12). Serial dilution-polymerase chain reaction method was performed to estimate the proportion of the total mtDNA harboring the mtDNA4977-deletion. Coefficient variation intra-assay was 8%, and inter-assay was 12%. MtDNA4977-deletion percentage was higher in samples obtained from victims of SND than in those from subjects who died of unnatural causes (p < 0.05). No differences in mtDNA4977-deletion were found between SND victims 39-51 years old, and no correlation was found between these samples and age, r = 0.30, p = 0.29 while it was significant among control samples, r = 0.68, p < 0.05. The association between mtDNA4977 deletion with SND victims might offer a tool to provide additional information to clarify complex SND investigations.     

Myocardial Cx43 expression in the cases of sudden death due to dilated cardiomyopathy

PURPOSE: Probing into myocardial connexin (Cx) 43 expression in the cases of sudden death due to dilated cardiomyopathy (DCM) and relationship between Cx43 expression and sudden death. METHOD: Myocardial Cx43 was detected with immunohistochemical staining in the cases of 11 sudden death caused by DCM and 14 cases of control group who died of violent reasons and other diseases, which were autopsied in our department from 1997 to 2003. RESULT: Of 11 cases of DCM, there were 10 men and 1 woman with ranging in age from 7 to 49 years old (x (37.8) years old for 9 adult cases). Of 14 cases in the control group, there were 10 men and 4 women with ranging in age from 11 to 53 years old (x (29.9) years old for 11 adult cases). Myocardial Cx43 expression was obviously decreased in DCM group. Positive dyeing spots were different in size, distribution, color and disparity, some of them were distributed in the form of particle. Obvious change had not been observed in the cases of control group or with only slight changes in coloring degree and expressive area. The quantitative data showed that there was significant difference between two groups (p=0.0075) about Cx43 expressive area, but there was no difference between the left and right ventricles (p>0.05) in each group itself. And there was not difference between the two groups about average optical density of expression. CONCLUSION: Myocardial Cx43 expression is obviously reduced in the patients with DCM who die suddenly. The alteration of quantity and distribution of myocardial Cx43 expression is probably related to sudden death of the patients with DCM.     

Estimation of age at death based on quantitation of the 4977-bp deletion of human mitochondrial DNA in skeletal muscle

The 4977-bp deletion in human mitochondrial DNA (mtDNA) is known to accumulate in various tissues with age. Since this deletion in mtDNA correlates closest with age in muscle tissue, iliopsoas muscle tissue was taken at autopsy from 50 persons aged 24-97 years to determine whether age at death can be estimated based on the amount of the 4977-bp deletion in skeletal muscle. Total DNA (nuclear and mtDNA) was extracted from 100 mg tissue and the 4977-bp deletion quantified using a kinetic polymerase chain reaction (PCR) followed by visualization of the products on silver stained polyacrylamide gels. The amount of the 4977-bp deletion of mtDNA ranged from 0.00049% to 0.14% depending on age, with a correlation coefficient of r = 0.83 (P = 0.0001). In forensic practice this method can aid in the estimation of age at death with a relatively wide confidence interval, thus enabling a discrimination between young and elderly persons in the identification of human remains based solely on skeletal muscle.     

大鼠急性心肌缺血后肌浆网RyR2 mRNA表达变化

目的 研究大鼠急性心肌缺血后心肌肌浆网兰尼碱受体蛋白2(ryanodine receptor 2,RyR2)mRNA表达的变化.方法 将SD大鼠分为正常对照组、心肌缺血组和缺血性猝死组.采用腹腔注射垂体后叶素的方法复制大鼠急性心肌缺血和猝死模型,对心肌进行半定量荧光RT-PCR检测,观察RyR2 mRNA表达水平的变化.结果 与正常对照组相比,不同时间和不同程度的急性心肌缺血后心肌肌浆网RyR2 mRNA表达均显著降低(P<0.05).结论 心肌缺血性损伤可诱导心肌钙调控蛋白RyR2 mRNA表达下调.     

心肌缺血猝死心肌中高迁移率族蛋白B-1的表达研究

目的探讨高迁移率族蛋白B-1(High Mobility Group Box protein 1,HMGB1)在心肌缺血猝死后诊断中的法医学价值。方法收集不同案例心肌蜡块分为疑似早期心肌缺血猝死组(早期梗死组)20例、心肌梗死猝死组(心肌梗死组)15例、冠心病非心源性猝死组(对照组1)10例和正常心肌组(对照组2)10例,应用免疫组织化学二步法染色,观察心肌胞核和胞浆中HMGB1表达,用ImagePro Plus 6.0软件计算HMGB1表达的平均光密度,用SPSS 13.0对表达进行数据统计分析。结果 HMGB1在四组心肌细胞胞核中表达均呈阳性;早期梗死组和心肌梗死组胞浆均呈阳性表达,对照组1和对照组2胞浆呈阴性。各组平均光密度分别为0.3031±0.0557、0.3195±0.0523、0.0252±0.0030、0.0207±0.0029,早期梗死组和心肌梗死组的阳性反应与两个对照组相比存在显著差异(P﹤0.01)。结论 HMGB1可作为早期心肌缺血猝死的一个辅助诊断指标。     

Quantification of mitochondrial DNA in human blood cells using an automated detection system

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.     

缺血修饰白蛋白在急性缺血性心脏病中的法医学应用

目的探讨心包液中缺血修饰白蛋白(ischemia modified albumin,IMA)水平在心脏性猝死诊断中的应用价值及法医学意义。方法应用白蛋白-钴离子结合法检测急性缺血性心脏病组(36例)、急性心肌梗死组(6例)、心肌病组(4例)、对照组(15例)死者的心包液中IMA水平,比较各组之间IMA水平的差异。通过ROC曲线获得最佳IMA水平的截断值以及区分急性缺血性心脏病和对照组的敏感度和特异度。结果急性缺血性心脏病组心包液中IMA水平高于对照组(P0.05);而与急性心肌梗死组、心肌病组心包液中IMA水平比较,差异均无统计学意义(P0.05)。应用ROC曲线分析得出识别急性心肌缺血的IMA的截断值为40.65 U/m L,其诊断急性心肌缺血的敏感度为60.0%,特异度为80.5%。结论心包液中IMA有望作为诊断急性心肌缺血的参考指标,为心脏性猝死的法医学诊断提供客观依据。     

过敏性和冠心病猝死者心肌组织中肥大细胞类胰蛋白酶、脑利钠肽的表达

目的探讨肥大细胞类胰蛋白酶、脑利钠肽(brain natriuretic peptide,BNP)在过敏性猝死和冠心病猝死鉴别诊断中的意义。方法选取山西医科大学法医病理学教研室2010—2015年尸检案例心肌标本共30例,分为颅脑损伤致死组、过敏性猝死组、冠心病猝死组,每组各10例。采用免疫荧光染色和Western印迹法分析各组心肌组织肥大细胞类胰蛋白酶和BNP的表达。结果过敏性猝死组、冠心病猝死组心肌组织内肥大细胞类胰蛋白酶免疫荧光染色均出现阳性染色;三组间两两比较,表达差异均具有统计学意义(P0.05)。冠心病猝死组心肌组织内BNP的表达量高于过敏性猝死组、颅脑损伤致死组(P0.05),过敏性猝死组与颅脑损伤致死组之间差异无统计学意义(P0.05)。结论联合检测心肌组织内肥大细胞类胰蛋白酶、BNP有望为过敏性猝死和冠心病猝死的法医学鉴别诊断提供帮助。