【作者】 杨莹；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 法医学， 2010， 硕士

【摘要】 目的:急性心肌梗死引起的猝死是最常见的猝死类型。死后常规病理学检查方法常检测不出明显的急性心肌缺血性病理改变,从而使冠心病猝死(sudden coronary death,SCD)案例的鉴定变得困难和复杂。已有研究表明急性心肌缺血可诱导大鼠心肌细胞中诱导型一氧化氮合酶(induce nitricoxide synthase,iNOS)表达增加,导致NO生成增多,NO可与O2ˉ反应形成过氧亚硝酸阴离子(peroxynitrite,ONOOˉ),ONOOˉ使蛋白质中的酪氨酸残基硝基化生成3-硝基酪氨酸(3-nitrotyrosine,NT)。免疫组织化学(immunohistochemistry,IHC)是一种便捷、实用的方法,可以检测缺血心肌细胞内蛋白质的变化,从而为急性心肌梗死的死后诊断提供了一种技术支持。本实验拟采用IHC技术,检测大鼠急性心肌缺血早期不同时间缺血区心肌iNOS和NT的表达,并研究死后变化和组织固定时间对iNOS及NT的抗原稳定性的影响,以期寻找敏感的、实用的IHC指标,用以辅助SCD的死后诊断。一. iNOS及NT在缺血心肌中的表达及敏感性研究方法:50只健康SD大鼠,重250~300克,雌雄不拘,随机分为假手术组(S组)和缺血组(A组)。以结扎左冠状动脉前降支(LAD)的方法建立大鼠急性心肌缺血模型;缺血组大鼠分别于结扎LAD后0.5h、1h、2h、3h、4h、5h及6h处死,假手术组大鼠只在LAD下穿过缝线而不结扎,分别于术后1h、3h及6h处死,其它步骤与缺血组相同。取材后制作石蜡切片,以HE染色方法观察大鼠心脏的组织病理改变,并用IHC方法测定心肌组织中iNOS及NT表达的动态变化;用SPSS13.0统计分析软件对实验数据进行统计学分析,以P<0.05为有显著性差异。结果:1特殊染色观察1.1 Evans Blue染色:S组大鼠心脏均呈蓝色;A组大鼠心脏左心室前壁及外侧壁心肌呈红色,余心肌呈蓝色。1.2 TTC染色:急性心肌缺血0.5h、1h及2h组心肌呈均一的砖红色,3h组左心室前、外侧壁心肌局部出现小面积苍白色区域,4h、5h及6h组左心室前、侧壁的苍白色区域增大,与周围砖红色区域对比明显,边界较清晰;心内膜呈砖红色。2 HE染色:假手术组心肌组织结构清晰,急性心肌缺血2h以内仅局部心肌细胞及间质轻度水肿,3~5h局部可见间质少量炎细胞浸润,偶见心肌纤维肿胀及波浪样变,未见明显的细胞坏死;6h局部少数心肌细胞嗜酸性增强及个别心肌细胞坏死。3 IHC染色:3.1缺血后不同时间点缺血心肌iNOS的表达情况:假手术组:心肌细胞胞浆中iNOS呈阴性表达;缺血组:急性心肌缺血0.5h后缺血区心肌细胞胞浆中iNOS呈弱阳性表达,1h后各组缺血区心肌细胞胞浆中iNOS表达强度随着缺血时间的延长逐渐增强,至缺血后3~6h心肌细胞及间质血管内皮细胞内iNOS呈强阳性表达,与假手术组相比具有显著差异(P<0.05)。3.2缺血后不同时间点缺血心肌NT的表达情况:假手术组:心肌细胞胞浆中NT呈阴性表达;缺血组:急性心肌缺血0.5h后缺血区心肌细胞胞浆中NT呈弱阳性表达,1h后各组缺血区心肌细胞内NT表达随时间的延长逐渐增强,6h时可见大范围的NT阳性表达,与假手术组相比具有显著差异(P<0.05)。二. iNOS及NT的抗原稳定性的实验性研究方法:120只健康SD大鼠,重250~300克,雌雄不拘,随机分为3℃组(D组)、室温(20℃)组(E组)和固定组(F组)三组。D组和E组各分为1d、3d、5d、7d及14d五个亚组,每亚组均分为缺血组和假手术对照组;E组分为3d、5d、7d及14d四个亚组,以第一部分的S3h组做假手术对照。各缺血组大鼠分别于结扎LAD后3h后处死,假手术组大鼠只在LAD下穿过缝线而不结扎,其它步骤与缺血组相同。D组和E组大鼠处死后分别于4℃及室温环境下放置1d、3d、5d、7d及14d后固定24h;F组大鼠处死后即刻取出心脏放入10%中性甲醛中分别固定3d、5d、7d及14d;取材后制作石蜡切片,以HE染色方法观察大鼠心脏的组织病理改变,并用IHC方法测定不同环境下心肌组织中iNOS及NT表达的动态变化;用SPSS13.0统计分析软件对实验数据进行统计学分析,以P<0.05为有显著性差异。结果:1 HE染色:1.1 4℃放置1d,心肌组织细胞结构清楚,未见明显自溶改变;缺血组心肌可见缺血早期的非特异性病理改变。放置3~5d,缺血心肌部分改变如心肌细胞浊肿、横纹模糊或消失与正常心肌死后变化相似,不易区分。放置7~14d,细胞结构模糊、排列松散,胞浆呈均质化伊红色,细胞核大部分消失,缺血心肌组织和细胞形态与对照组类似。1.2室温放置1d,心肌轻度自溶,局部胞浆溶解,胞核淡染,缺血组心肌仍可见局部少量炎细胞浸润等病理改变。3d局部可见蓝染菌团、核溶解,细胞结构模糊,胞浆灶性淡染;缺血组心肌部分改变与正常心肌死后改变不易区分。5~7d大部分心肌细胞核消失,细胞排列松散,仅组织轮廓尚存,14d细胞核完全消失,胞浆呈均质化伊红色;5~14d缺血心肌组织和细胞形态与对照组类似。1.3固定14d以内对心肌组织的HE染色结果没有明显影响。2 IHC染色:2.1 4℃保存对iNOS及NT稳定性的影响假手术组:心肌细胞胞浆中iNOS及NT始终呈阴性表达;缺血组:随着放置时间的延长缺血区iNOS及NT的表达的强度及范围均逐渐减弱,iNOS放置7d以内、NT放置5d以内的表达强度与假手术组相比具有显著差异(P<0.05);iNOS放置14d、NT放置7d时与假手术组相比无明显差异(P>0.05);2.2室温保存对iNOS及NT稳定性的影响假手术组:心肌细胞胞浆中iNOS及NT始终呈阴性表达;缺血组:随着放置时间的延长缺血区iNOS及NT的表达强度及范围均逐渐减弱,3d时iNOS及NT的阳性表达强度及范围已明显减弱,但与对假手术相比仍有差异(P<0.05),5~14d iNOS及NT表达强度与假手术组相比无明显差异(P>0.05);2.3固定时间对iNOS及NT稳定性的影响缺血区iNOS及NT表达强度随着固定时间的延长而缓慢减弱,至固定14d时与假手术组之间仍具有明显差异(P<0.05)。结论:iNOS及NT对大鼠急性心肌缺血具有较高的敏感性,急性心肌缺血0.5h后缺血区心肌细胞内即有阳性表达,表达强度随着缺血时间的延长逐渐增强。iNOS及NT均具有较好的抗原稳定性,iNOS可用于死后4℃放置7d以内、室温放置3d以内及固定14d以内的大鼠心脏标本;NT可用于死后4℃放置5d以内、室温放置3d以内及固定14d以内大鼠心脏标本。更多还原

【Abstract】 Objective: The cases of sudden coronary death (SCD) caused by acute myocardial infarction(AMI) often occur in the forensic medicine practice. The morphplogical changes of cardiac tissue caused by ischemic injury were so limited that normal phathological examination could not detect manifest myocardial damage. Therefore the assessment of SCD became difficult and complex. It has been demonstrated that AMI could induce nitricoxide synthase(iNOS) expression in myocardia cell, resulted in the increase of NO; peroxynitrite formed from NO and O2ˉ, could led to nitration of protein tyrosine residues producting 3-nitrotyrosine (NT). Immunohistochemical technology, as a convenient and practical method, which can detect the changes of proteins in the ischemic myocardial cells, may be a possible method for the postmortem identification of AMI. In this study, we detected the expression of iNOS and NT in myocardium using IHC at different time after early myocardial ischemia, and observed the effect of postmortem changes and fixation time on the expression of iNOS and NT, in order to search an utility, sensitiveness immunohistochemical markers for assist postmortem diagnosis of SCD.1. Expression and sensitiveness of iNOS and NT in myocardium at different time after early myocardial ischemiaMethods: 50 SD rats weighting 250~300g of either sex, were randomly distributed into Sham group and ischema group. The rat model of early aute myocardial ischemia was established by ligating the left anterior descending branch of coronary artery. The ischemia group rats were killed at 0.5h, lh, 2h, 3h, 4h, 5h and 6h after ligation. Sham group rats underwent the same surgical protocol as the ischemia group except LAD ligation, and were killed at lh, 3h, and 6h after operation. Then all hearts were dissected and fixed, the tissue blocks were taken and embedded in paraffin and the tissue sections were prepared according to routine histological procedures. Then histological sections were dyed with HE staining, and the expression of iNOS and NT were detected by IHC. The data were analyzed using SPSS13.0 statistical program. A level of P<0.05 was considered as statistical significance.Results:1 Specific stain1.1 Stain of Evans Blue: Myocardium of ischemic area was unstained presenting red; in contrast, myocardium of non-ischemic area was stained with Evans Blue presenting blue.1.2 TTC staining: 0.5h, 1h and 2h groups, all around the myocardium presented red; 3h group, small pale area of left ventricle(LV); manifest pale area of LV compared obviously to other area during 4~6h.2 HE staining: Myocardium of control group and myocardial ischemia group within 2h was stained clearly, with very mild cellular swelling or/and interstitial edema in ischemic myocardium. During 3~5h after myocardial ischemia there were manipulus inflammatory cells in the matrix and cardiac wavy muscle fibers. In the 6h group which a few myocardial acidophiliar was reinforcing, and a few of myocardium necrosis.3 Immunohistochemical in ischemic myocardium:3.1 The change of the expression of iNOS at different time points after myocardial ischemia: In sham group there was little expression of iNOS in myocardium. In the ischemia groups, there was weakly positive expression of iNOS in the cardiac cytoplasm at 0.5h after myocardial ischemia, and the intensity of positive expression was reinforcing significantly along with ischemic time; there was strong positive expression in ischemic myocardium during 3~6h, having obvious difference compared to sham group’s (P<0.05).3.2 The change of the expression of NT at different time points after myocardial ischemia: In sham group there was little expression of NT. In the ischemic groups, there was weakly positive expression of NT in the cardiac cytoplasm at 0.5h after myocardial ischemia, and the intensity of positive expression was reinforcing with ischemic time, having obvious difference compared to sham group’s (P<0.05). .2.Study on the antigen stability of iNOS and NTMethods: 120 healthy adult rats weighting 250~300g of either sex, were randomly distributed into 4℃group, room temperature group and fixation group. 4℃group and room temperature group were distributed into 1d, 3d, 5d, 7d and 14d groups, which include ischemic group and sham group; fixation time group was distributed into 3d, 5d, 7d and 14d groups, using the S3h group in part one as control. The ischemic group rabbits were killed at 3h after ligation. Sham group rats underwent the same surgical protocol as the ischemic group except LAD ligation. The myocardium of 4℃groups and room temperature groups was fixed for 24h after 1d, 3d, 5d, 7d and 14d of postmortem stored at 4℃and room-temperature respectively. The myocardium of fixation time group was fixed in 10% neutral formalin immediately after killed for 3d, 5d, 7d and 14d. The tissue blocks were taken and embedded in paraffin and the tissue sections were prepared according to routine histological procedures. Then histological sections were dyed with HE staining, and the expression of iNOS and NT were detected by IHC. Observe the changes of ischemic myocardia as well as the expression of iNOS and NT stored at the same environment for different time intervals after death. The data were analyzed using SPSS13.0 statistical program. A level of P<0.05 was considered as statistical significance.Results:1.HE staining:Stored at 4℃: There were some pathological changes of early myocardial ischemia in ischemic myocardium, no definite indicators of myocardial infraction. At 3~5d, the cellular cloudy swelling blur or disapearanse of transverse striation in ischemic myocardium are similar to sham group’s changes. While samples stored at 4℃for 7~14d after death, the two groups experienced similar changes in morphous of tissue.Stored at room-temperature: For 1d the autolysis is not obviously with partly plasmolysis and light-stained nucleus in both control and ischemic group’s, and there were some pathological changes of early myocardial ischemia in ischemic myocardium. For 3d there were blue-stained cenobium, hypochromatosis, cellularity unclear and hyalomitome light-stained in part myocardial cell; while some changes in ischemic myocardium are similar to sham group’s changes. While samples stored at room-temperature for 5~7d nuclei almost disappeared, cell spreaded looseing with outline of tissue kept only; the two groups experienced similar changes in morphous of tissue; For stored 14d after death the two groups experienced similar changes in morphous of tissue and in ischemic myocardial cell with nuclei disappeared and hyalomitome homogenized.Fixation: there were not obviously changes of HE staining for different fixation time.2 Immunochemistry in ischemic myocardium:2.1 The effect of stabilitu to iNOS and NT stored at 4℃:Sham group: there was little positive expression in myocardium.Ischemia group: intensity and extent of positive expression to iNOS and NDT in ischemic myocardial cell were decreasing along with stored time. The positive expression to iNOS of 7d and NT of 5d was decreasing obviously, but having obvious difference compared to sham group’s(P<0.05). While iNOS stored for 14d and NT stored for 7d after death, there was little positive expression, having no difference compared to sham group’s (P>0.05).2.2 The effect of stability to iNOS and NT stored at room temperature:Sham group: there was little positive expression in myocardium.Ischemia group: intensity and extent of positive expression to iNOS and NT in ischemic myocardial cell were decreasing along with stored time; at 3d after death the positive expression was decreasing obviously, but have difference compared to sham group’s(P<0.05). For 5d-14d there was little positive expression having no difference compared to sham group’s (P>0.05).2.3 The effect of fixation time to iNOS and NT stability:The positive expression of iNOS and NT decreased slowly in ischemic myocardial cell along with fixation time. At 14d there was still obvious difference compared to sham group’s (P<0.05).Conclusions: iNOS and NT were sensitivity to early rat’s AMI, and appeared positive expression 0.5h after AMI; the intensity of positive expression to iNOS and NT was increasing with ischemic time. The antigen stability of iNOS and NT was relatively small influenced of autolysis and fixation time; it can be conclude that the immunohistochemical detection of iNOS can be used in myocardia of rat stored at 4℃for 7d or less than 7d after death, stored at room temperature for 3d or less than 3d after death and fixed for 14d or less than 14d after death. It also can be conclude that the immunohistochemical detection of NT can be used in myocardia of rat stored at 4℃for 5d or less than 5d after death, stored at room temperature for 3d or less than 3d after death and fixed for 14d or less than 14d after death.更多还原