大鼠早期缺血心肌中Basigin mRNA的表达

目的检测Basigin mRNA在大鼠早期缺血心肌和非缺血区心肌中的表达差异及探讨其法医学意义。方法建立大鼠早期心肌缺血手术模型,分为EIM组、NIM组、假手术组以及空白对照组,采用real-time PCR方法检测大鼠心肌缺血后15min、30min、1h和2h Basigin mRNA的表达量。结果与NIM组、假手术组及空白组相比,EIM组BSG mRNA表达量在缺血15min时降低,而冠状动脉结扎1h后表达量降低到SO组的一半,此时具有统计学差异(P0.05),结扎2h后表达量恢复至SO组水平。NIM组、SO组和空白对照组之间表达量无统计学差异(P0.05)。结论心肌急性缺血2小时内BSG mRNA表达降低明显,之后升高,说明BSG参与早期缺血心肌自我保护病理生理过程,提示BSG可用于鉴定早期心肌缺血。     

Sudden cardiac death and myocardial ischemia indicators: a comparative study of four immunohistochemical markers

The postmortem diagnosis of acute myocardial infarction represents a current challenge for forensic pathologists, particularly when death occurs within minutes to a few hours after the ischemic insult. Among the adult population the single most important cause of sudden cardiac death (SCD) is the well-known atherosclerotic coronary artery disease, commonly asymptomatic or unrecognized. The recognition of early myocardial damage using routine hematoxylin and eosin (H&E) staining is possible only if death has occurred at least 6 hours after the onset of the ischemic injury. The usefulness of immunohistochemical markers to the diagnosis of early myocardial damage has been recently suggested because most of them can be visible even serologically as early as few minutes after the beginning of the symptoms. To evaluate the usefulness of plasma and cellular antigens, their distribution patterns have been studied among a group of 18 SCD cases in which a myocardial ischemia was strongly suspected. For the present study, 4 markers have been selected on the basis of their different diagnostic potential as follows: among the plasma markers the C5b-9 and fibronectin, among the cellular markers the myoglobin and cardiac troponin. The results show that only the study of multiple markers such as those selected can provide enough evidence of myocardial ischemia and/or necrosis, supporting the final diagnosis of SCD. No single immunohistochemical staining is ideal for diagnosing early myocardial ischemia but a set of markers can improve the ability of forensic pathologists to detect ischemic areas when no macroscopic or microscopic evidence of necrosis is available. However, the interpretation of data obtained in each individual cannot be isolated from the overall assessment of the factors (cardiopulmonary resuscitation and/or agonal artifacts) that can affect the expression of each marker.     

Recognition of Early Myocardial Infarction by Immunohistochemical Staining with Cardiac Troponin‐I and Complement C9*

Abstract: The diagnosis of early myocardial infarction (MI) after death, especially in the first few hours (c. 6 h) after the onset of MI, poses a challenge to the forensic pathologists. During this time, the damaged myocardium does not show grossly identifiable morphological changes and may not be recognized even with routine histological microscopic examination. However, the infarcted cardiac tissue releases certain chemicals that can be detected microscopically, two of these being cardiac troponin‐I (CT‐I) and complement C9 (C9). This study utilizes the importance of these two biomarkers immunohistochemically in an attempt to identify this early phase of MI. This study reveals that the early phase of MI of <6 h duration may be detected through immunohistochemical staining with CT‐I and C9. The ischemic/infarcted cardiac myofibers in the <6 h group display reduced/absent CT‐I staining as well as positive C9 staining.     

心肌肌钙蛋白T在家兔缺血心肌中的表达及其死后稳定性

目的 探寻心肌肌钙蛋白T（cTnT）在家免缺血心肌中的表达规律及其死后稳定性．并对其在法医学实践中诊断早期心肌缺血的应用价值进行评价。方法用冠状动脉结扎法建立家兔急性心肌缺血模型．应用免疫组织化学法、图像分析和统计学处理系统,检测死后cTnT表达情况,并对实验组和对照组cTnT的表达进行比较。结果缺血区心肌组织可见明显不规则灶状、片状cTnT缺失区,间质呈阴性表达。在4℃放置．随放置时间延长,正常和缺血心肌组织cTnT阳性表达均有减弱趋势,至14d时均完全缺失。但仅在4℃放置1～7d内,缺血心肌和正常心肌标本之间cTnT阳性反应具有显著性差异。结论cTnT的免疫组化表达用于认定死后4℃放置的尸体是否有死前心肌缺血时．宜在7d以内进行。     

VEGF在家兔缺血心肌中的表达及其死后稳定性研究

目的观察心肌中VEGF在死后一定时间的稳定性及表达的变化规律，评价其对死后诊断机体早期心肌缺血的作用。方法用冠状动脉结扎法建立家兔急性心肌缺血模型，应用SP法染色、图像分析和统计学处理系统，检测缺血心肌细胞内VEGF阳性表达面积和强度，并对VEGF在死后不同时间的稳定性进行比较。结果VEGF在缺血心肌组织中，见灶性或成片心肌细胞强阳性表达，部分细胞呈阴性或弱阳性表达，血管内皮及平滑肌细胞表达弱阳性，间质表达阴性；在4℃放置，随时间延长，缺血心肌组织VEGF阳性表达有减弱趋势，至10d时缺血心肌组织仍可见VEGF在少许心肌细胞胞浆内呈弱阳性表达。在4℃放置10d以内，缺血心肌和正常心肌标本之间VEGF阳性表达具有极显著性差异。结论VEGF能耐受一定程度自溶及腐败的影响，其免疫组化检测对于判断死后4℃放置10d以内的尸体是否出现过心肌缺血具有一定意义。     

实验性早期心肌缺血补体C5的免疫组织化学研究

建立大鼠急性心肌缺血模型。应用免疫组织化学方法和图像分析技术 ,对急性心肌缺血早期不同缺血时间心肌细胞内补体C5的变化进行研究。结果发现 :C5在心肌缺血 15min即可出现阳性反应 ,并随缺血时间的延长 ,其反应面积逐步增大。心肌细胞内C5阳性是早期心肌缺血的敏感指标 ,免疫组织化学染色法检测心肌细胞内C5的变化可望成为早期心肌缺血死后诊断的一种有意义的手段     

心肌梗死6项免疫组化指标的死后稳定性比较

目的　探讨用于早期心肌梗死死后诊断的维连接蛋白 (Fn)、纤维蛋白原 (Fg)、补体 (C5 )、肌红蛋白(Mb)、肌动蛋白 (HHF3 5 )、结蛋白 (Dm )等 6项免疫组化指标在死后不同时间的稳定性。方法　应用免疫组织化学法、图像分析和统计学处理系统 ,检测缺血心肌细胞内Mb、HHF3 5、Dm的缺失面积和Fn、Fg、C5的阳性反应面积 ,并对 6项免疫组化指标在死后不同时间的稳定性进行比较。结果　正常心肌组织 4℃放置 1～ 2d ,Dm、HHF3 5、Mb染色均匀 ,未见明显缺失 ;放置 3d以上 ,即可见Dm、HHF3 5、Mb明显缺失 ,且随放置时间延长 ,缺失面积逐步增大。缺血心肌组织随放置时间延长 ,Dm、HHF3 5、Mb的缺失面积逐步增大 ,Fg、C5、Fn阳性反应面积逐渐减少 ;放置 14d以上 ,Fg呈阴性反应 ;放置 2 1d以上 ,C5呈阴性反应 ;放置 2 8d ,Fn仍呈阳性反应。正常心肌组织放置不同时间 ,均未见Fg、C5、Fn阳性反应。图像分析结果显示阳性反应面积逐步减少。结论　Dm、HHF3 5、Mb的稳定性最差 ,易受死后自溶的影响 ,只适用于新鲜尸体 (死后 1～ 2d) ;Fg次之 ,可用于死后 4℃放置 7d的尸体 ;C5较好 ,可用于死后 4℃放置 14d的尸体 ;Fn稳定性最好 ,可用于死后 4℃放置 2 8d的尸体。     

Detection of apoptosis in ischemic heart. Usefulness in the diagnosis of early myocardial injury

Myocardial samples of hearts with histologic findings of acute myocardial infarction (group A), sudden coronary deaths without histologic changes (group B), and chronic ischemic heart disease (group C) were analyzed to investigate the appearance of apoptosis in acute and chronic ischemic cardiac disorders. This analysis involved the morphologic detection of DNA strand breaks in myocyte nuclei by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and the biochemical determination of DNA laddering in the myocardium using archival formalin-fixed, paraffin-embedded tissue sections of human myocardium. The authors demonstrated that apoptosis of myocardial cells could occur after ischemic myocardial cell injury. In all documented cases of acute myocardial infarction (group A), the infarcted area included extensive presence of both apoptosis and necrosis. In the tissue bordering on and away from the obviously infarcted areas, positive nuclei were intermingled with nonstained normal myocytes. The number of positive nuclei decreased with the distance from the infarction foci. In group B, myocardial samples showed focal or diffuse nuclear positivity of varying degrees for apoptosis, confirming the presence of myocardial ischemic cell death, whereas the histologic diagnosis remained inconclusive. This finding suggests that apoptosis could be used as a marker for acute ischemic injury. In group C, stained nuclei were dispersed with intermingled normal cardiomyocytes.     

大鼠心肌急性缺血再灌注NF-кB P65和caspase-3的表达

目的观察大鼠急性心肌缺血再灌注后NF-кBP65和caspase-3在心肌中表达的规律,为心肌急性缺血早期死亡的法医学鉴定提供科学证据。方法运用免疫组化和图像分析技术观察66只SD大鼠心肌缺血再灌注后NF-кBP65和caspase-3蛋白在不同实验组中的表达规律。结果心肌缺血组缺血区域心肌组织缺血5min就可见NF-кBP65和caspase-3蛋白表达,并随缺血时间延长表达逐渐增强;而非缺血区域心肌组织仅见低水平表达或不表达;但两种蛋白(两者)并非在缺血区域心肌同一部位表达,也不是在心肌细胞内的同一部位表达;caspase-3主要在心肌细胞浆表达,而кBP65既在心肌细胞浆表达,也在心肌细胞核中表达。心肌缺血再灌注组(在)心肌缺血5min~1h内,再灌注与否并不影响这两种蛋白的表达。正常对照组未见这两种蛋白表达。结论心肌缺血再灌注时,心肌组织中有NF-кBP65和caspase-3蛋白的表达,这两种蛋白在心肌中的表达可视作诊断早期心肌缺血的参考指标。     

急性心肌缺血再灌注HSP70及c-Fos的免疫组织化学研究

运用免疫组织化学方法 ,观察心肌缺血后不同时间再灌注HSP70及Fos蛋白在心肌组织不同区域表达的规律 ,为心肌缺血 /再灌注损伤所致心性猝死的法医学诊断提供形态学依据。结扎大鼠左冠状动脉前降支建立急性心肌缺血 /再灌注模型 ,取缺血区域 (左冠脉前降支供血区 )及非缺血区 (右心室 )心肌组织。结果显示 :对照组正常心肌组织无Fos蛋白及HSP70表达。Fos蛋白在心肌缺血 15min再灌注 0 5h后即在缺血区域表达 ,随着再灌注时间延长其表达增强 ;非缺血区域 1h始有表达 ,缺血区域表达明显强于非缺血区域。HSP70在心肌缺血区域再灌注 1h后始有表达 ,随着再灌注时间延长其表达增强 ;非缺血区域心肌 2h始有表达 ,且明显弱于缺血区域。同时发现再灌注早期HSP70及Fos均先于心肌内层表达 ,随着时间延长其表达向心肌外层扩展。Fos在再灌注 0 5h时主要在心肌内层表达 ,1h时已扩展到心肌全层 ,4h时其心肌外层表达明显强于心肌内层。HSP70在再灌注 1、 2h时主要在心肌内层表达 ,4、 6h时表达扩展至全层心肌。心肌缺血 /再灌注早期不同时间HSP70及Fos表达有不同的区域性及强度 ,此可为心肌缺血 /再灌注的早期诊断提供一项新的辅助诊断依据     

抗肌动蛋白单克隆抗体在早期心肌缺血诊断中的应用

应用抗肌动蛋白单克隆抗体（HHF35），对10例正常心肌、6例已知心肌梗死、18例可疑早期心肌缺血组织进行石蜡切片免疫组化染色。结果：正常心肌阳性，梗死心肌阴性，18例可疑病例中14例呈不同程度弱阳性或阴性。表明心肌发生了缺血性改变，对比清晰，特异性强，对心肌缺血诊断有明显的应用价值。     

大鼠早期心肌缺血纤维蛋白原与肌红蛋白的免疫组织化学对比性研究

按Selye法结扎Wistar大鼠冠状动脉左前降支，复制早期心肌缺血（EMI）模型。根据心肌缺血不同时间（15min、30min、1h、2h、3h）分5个实验组，另设1个对照组；每组取心尖部及其邻近的心肌组织制成石蜡切片，进行HE、链霉系合素生物素过氧化物酶（SAB）染色。结果表明：早期心肌缺血30min组，缺血区心肌即出现肌红蛋白（Mb）缺失和纤维蛋白原（Fg）染色增强。随着缺血时间延长，Mb缺失或Fg染色增强范围扩大，程度加重，从心内膜下向外层心肌发展呈“波浪式推进现象”，二者在心肌缺血过程中的变化规律相同，灵敏度相似，但Fg易受血液污染而致其可靠性较Mb差。在注意血液污染的前提下，SABC－Fg技术为诊断EMI提供了新的途径。     

Cathepsin-L在缺血心肌中的表达

目的检测cathepsin-L(CTSL)在大鼠早期缺血心肌中的表达变化,并探讨其法医学意义。方法建立大鼠早期心肌缺血模型,设置早期缺血组(early ischemic myocardium,EIM)、非缺血组(non-ischemic myocardium,NIM)、假手术组以及空白对照组,采用real-time PCR方法检测大鼠缺血后15min,30min,1h和2h CTSL mRNA的表达量,并进行组内和组间比较。收集心源性猝死者缺血心肌和机械性损伤致死者正常心肌样本,采用免疫组化染色方法观察心肌组织中CTSL蛋白表达。结果 EIM组CTSL mRNA表达量与NIM组、假手术组及空白组相比,在15min时无统计学意义(P>0.05),在30min、1h和2h时分别升高1.4、3.1和4.5倍(P<0.05);其余3组间差异无统计学意义(P>0.05)。观察10例冠脉狭窄程度Ⅲ～Ⅳ级的心源性猝死(SCD)者缺血心肌,CTSL表达增强。结论大鼠心肌缺血后早期即可检测到CTSL mRNA的升高,并在SCD冠脉狭窄死者缺血心肌中高表达,提示CTSL可作为心肌缺血与SCD的参考指标。     

早期心肌缺血猝死心肌Ⅰ型胶原的表达

目的探讨Ⅰ型胶原蛋白在心肌早期缺血死后诊断中的法医学价值。方法将收集的案例心肌蜡块分为3组：正常对照组、早期心肌缺血组、心肌梗死组,用免疫组织化学技术（S-P法）,观察猝死心肌内Ⅰ型胶原的表达情况,对其结果进行半定量分析。结果早期心肌缺血组和心肌梗死组心肌细胞质以及细胞核染色阳性,且早期心肌缺血组与心肌梗死组表达强度相近,说明早期心肌缺血组在胶原网结构方面已经发生与心肌梗死相同的改变。结论Ⅰ型胶原可以作为早期心肌缺血的一个诊断指标。     

Cytoskeleton immunohistochemical study of early ischemic myocardium

Previous studies on cytoskeletal changes of in vitro and in vivo animal models of ischemic myocardium have suggested the possibility of using alterations in cytoskeleton proteins as an early marker for the post-mortem diagnosis of myocardial ischemia in cases of sudden death due to coronary artery disease (CAD). In the present study, using the technique of ABC-immunohistochemistry, we examine the changes of three cytoskeletal proteins: vinculin, desmin and α-actinin in human myocardial samples taken from 14 cases of CAD sudden death and 13 cases of non-CAD death. Results of these examinations are compared with immunohistochemical changes of myoglobin and histochemical staining of hematoxylin and eosin and phosphotungstic acid, and Masson trichrome. Patchy and extensive loss of the three cytoskeletal proteins was demonstrated in the myocardium of victims who died 1 h or later following the onset of symptoms of ischemic myocardium. The pattern of cytoskeleton change is equivocal in the cases of CAD who died less than 1 h after the onset of symptoms and of the cases of non-CAD. In these cases, no significant histological change was observed. With less non-specific background changes and stronger positive staining, immunohistochemical staining of the three cytoskeletal proteins is more reliable than myoglobin, which has attracted the attention of many pathologists searching for anatomic evidence of ischemic myocardium in coronary artery disease.     

实验性早期心肌缺血结蛋白的免疫组织化学研究

目的　探讨急性心肌缺血早期不同缺血时间心肌细胞内结蛋白的变化 ,为早期心肌梗死死后诊断提供新的方法。方法　建立大鼠急性心肌缺血模型 ,用免疫组织化学S -P法研究心肌细胞内结蛋白的缺失情况。结果　正常心肌细胞结蛋白呈棕褐色阳性反应 ,急性心肌缺血 15min ,结蛋白即可在缺血区心肌呈小灶性缺染 ,随缺血时间延长 ,其缺染范围逐步扩大。结论　心肌细胞结蛋白的缺失是早期心肌缺血的极为敏感的指标 ,免疫组织化学染色法检测心肌细胞结蛋白的变化可望成为早期心肌缺血死后诊断的一种有意义的手段。     

Immunocytochemical diagnosis of early myocardial ischaemic/hypoxic damage

The sensitive and reliable dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure (B. Jasani et al., Virchows Arch (Pathol. Anat.), 406 (1985) 441-448) was used to study the distribution of immunoperoxidase staining seen with antibodies to seven protein markers in post-mortem heart tissue. This was obtained from 12 cases with macroscopic myocardial infarction and 17 cases without myocardial infarction (10 with and 7 without significant coronary artery atherosclerosis). The immunostaining patterns were compared with the appearances seen in adjacent sections stained by the routine haematoxylin and eosin (H & E) and phosphotungstic acid haematoxylin (PTAH) methods and a method previously recommended for the detection of early myocardial infarction, the haematoxylin basic fuchsin picric acid (HBFP) stain. Loss of immunostaining with an antibody to myoglobin was found to be a reliable and more objective marker of both early and established myocardial infarction compared with the histological stains. Antibodies to myosin, caeruloplasmin, C-reactive protein and pre-albumin gave similar but less reliable results, whilst those to complement factor C3b and alpha-1 anti-trypsin gave the least reliable results for early myocardial ischaemic/hypoxic damage. The immunocytochemical results are considered sufficiently encouraging to extend the work to a large number of sudden death cases in order to establish a new, more reliable approach to the detection of histologically latent ischaemic/hypoxic damage in the myocardium.     

Immunostaining by complement C9: a tool for early diagnosis of myocardial infarction and application in forensic medicine

Before the first 12 hours, diagnosis of early myocardial infarctions is always difficult for forensic pathologists. We tested complement C9 expression in 121 formalin-fixed and paraffin-embedded heart samples by an immunohistochemical procedure. The heart specimens were separated into four groups: 33 cases in group 1 with typical ischemic damages histologically located, 20 cases in group 2 with death related to myocardial infarction on the basis of ischemic presentation on electrocardiogram but no obvious histological ischemic damage, 35 cases in group 3 with severe coronary disease without cause of death found at the autopsy, and 33 cases in group 4 without sign of myocardial infarction and without coronary disease. In the first group, all 33 heart samples showed a well-defined C9 expression in the necrotic areas. The second group in 17 of 20 cases showed positive areas for C9 expression. In the other three heart specimens, only few stained cells were observed whereas the painful symptoms had begun less than 1 h before death. The third group showed C9 immunopositive areas in six of 35 cases, few stained cells in 8 cases, and no C9 deposition in the 21 other cases. The last group showed no staining area. To avoid nonspecific C9 staining due to tissue autolysis, we studied C9 expression during a controlled putrefactive process in four cases included in group 1; staining was found only in infarcted myocardial areas, and was observed up to ten days. Specificity of C9 expression was evaluated to be 100% [89.4 to 100%] and sensitivity to be 85% [62.11 to 96.79%]. In conclusion, evaluation of immunohistochemical expression of C9 appears to be a highly sensitive and specific marker of early myocardial infarction, useful in forensic medicine if survival is more than 1 h after the beginning of myocyte damage.     

Primary study of vascular endothelial growth factor immunohistochemical staining in the diagnosis of early acute myocardial ischemia

The expression of vascular endothelial growth factor (VEGF) in the model of rat early acute myocardial ischemia was studied by Strept-Avidin-Biotin-Peroxidase Complex (SABC) immunohistochemical staining. After ligating the anterior descending branch of the left coronary artery, an initial rapid (30min) positive expression of VEGF in myocardial ischemic areas was observed, the intensity of positive expression of VEGF increased with the continuation of myocardial ischemia. After 5h infarction, the strongly positive myocytes of SABC-VEGF staining were predominantly limited to perimyocardial infarction areas. No positive expression of VEGF was found in the control group. These findings suggested that SABC-VEGF method could give a sensitive, specific, simple and objective morphologic evidence to the diagnosis of sudden cardiac death caused by acute early myocardial ischemia.     

大鼠心肌早期缺血内皮素的表达

为探讨心肌缺血后内皮素在心肌组织内表达的变化。用免疫组化SABC法研究内皮素的表达情况。结果发现缺血60min后 ,缺血区心肌细胞呈局灶性阳性 ;缺血120min后 ,血管平滑肌、内皮细胞及心肌细胞浆均呈阳性着色。对照和缺血30min组心肌组织未见有阳性反应。上述各组HE染色未见明显病理改变。