H-FABP死后诊断早期心肌梗死的免疫组化实验性研究
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摘要
背景: SCD的研究是法医病理学重要的任务之一,而AMI的死后诊断是法医病理学的重点和难点。不足6h的心肌梗死死亡病例,其心肌在肉眼和光学显微镜下无特异性的改变,不使用特殊方法,难以对AMI引起的猝死作出早期的组织学诊断。在常规的组织学染色出现特征性的形态学改变以前,缺血心肌已经发生了一系列的病理生理变化。生物化学,组织化学、酶组织化学、荧光组织化学染色,电子显微镜技术都能显示心肌的早期损害,但是上述方法因操作过于复杂,缺乏特异性、稳定性,费用过高,以及易受到组织自溶、腐败等因素的影响而限制了它们在法医学中的应用。IHC作为一项快捷、简便、实用的方法,它可以显示缺血心肌细胞内蛋白质质和量的变化,从而为AMI死后诊断提供了一种可能。心脏型脂肪酸结合蛋白是一种小分子蛋白质((14一15kDa),且有严格的器官特异性,大量地存在于心肌细胞浆中,其作用是上调心肌细胞中的长链脂肪酸。具有分子量小、心肌细胞中含量高且为水溶性的特点,当心肌细胞缺血时,心脏型脂肪酸结合蛋白(H-FABP)很容易迅速地透过细胞膜而漏出到细胞间质,从而进入血液循环。大量的临床研究证实心肌缺血12小时内,用血液中脂肪酸结合蛋白的浓度诊断心肌梗死在特异性和敏感性方面都优于肌红蛋白,原因主要是脂肪酸结合蛋白的分子量较肌红蛋白(18kDa)小且有心脏特异性。因此心肌脂肪酸结合蛋白目前被世界卫生组织列为新推荐的诊断急性心肌梗死的早期指标之一。用于心肌缺血引起猝死的死后诊断的IHC指标能否用于法医病理学诊断,需要考虑其特异性和敏感性以外,还必须考虑组织自溶、腐败等因素对抗原的影响。因此用于心肌缺血引起猝死的死后诊断的各种IHC指标的死后稳定性及其在死后不同时间的表达变化规律是值得进一步研究。
目的:本研究利用结扎左侧冠状动脉前降支的方法建立动物心肌缺血模型,运用免疫组化的方法,探讨H-FABP在死后诊断早期心肌梗死的敏感性及死后稳定性,以期对其在法医学中诊断SCD的应用价值进行评价。
实验分为两部分:
一. H-FABP在缺血心肌中的表达及敏感性研究
方法:结扎大鼠左侧冠状动脉前降支的方法建立大鼠心肌缺血模型,检测H-FABP在大鼠心肌缺血不同时间的表达。45只健康SD大白鼠,重250-300克,雌雄不限,随机分为8个实验组和1个对照组,每组5只。对照组大白鼠除不结扎冠状动脉外,其它步骤与实验组完全相同。各实验组大白鼠分别于结扎后15分钟、30分钟、1小时、2小时、3小时、4小时、6小时、8小时后处死,对照组大白鼠于手术后0小时处死.取出心脏放入10%中性缓冲福尔马林液中固定24h。取材后作HE染色和H-FABP免疫组织化学染色,显微镜下观察两组标本的HE、IHC切片;并按IHC染色结果判定标准对IHC染色进行全面的观察、判断,采用图像分析技术,客观测量IHC切片中H-FABP的缺失面积和阳性面积。
结果:
1.常规HE染色:对照组及心肌缺血3h以内组,心肌细胞染色清晰、边界清楚、横纹存在,仅见有轻度心肌间质水肿。心肌缺血4h后缺血区可见少许心肌细胞轻度水肿,缺血区边缘有少许炎性细胞浸润、心肌嗜伊红性增强,未见明显的细胞坏死。心肌缺血8h可见心肌嗜伊红性明显增强,部分心肌细胞出现凝固性坏死,炎性细胞增多。
2.免疫组织化学染色:对照组心肌胞浆全部染成均匀的棕黄色,用苏木素衬染后,细胞核染成淡蓝色,心肌细胞间质及其他细胞成分不着色。实验组心肌细胞H-FABP在缺血心肌内的脱失表达具有明显的时间规律。缺血15分钟组,在心内膜下心肌、乳头肌可见散在或小灶性的部分H-FABP缺失。缺血30分钟组,开始出现脂肪酸结合蛋白的完全脱失,但仍然局限于心肌内膜下和乳头肌。缺血1小时和2小时组的心肌则出现斑片状缺失且波及到心肌中层,缺血3小时累及心肌外层。缺血4小时,心肌细胞则出现全层细胞的完全脱失并随着缺血时间的延长,脱失面积增大。H-FABP在心肌缺血不同时间的各组缺失面积经比较和统计学分析表明:实验组的H-FABP缺失面积与其各相应对照组比较均有显著性差异,H-FABP在缺血3小时内各组之间没有明显的差异性,而心肌缺血3小时与心肌缺血4小时的心肌细胞中H-FABP脱失面积仍有显著差异,说明H-FABP的敏感性主要体现在心肌缺血3h以前。
二. H-FABP在诊断早期心肌梗死的死后稳定性的实验性研究
方法:结扎左侧冠状动脉前降支的方法建立家兔心肌梗死3小时模型,检测H-FABP在死后不同时间的表达情况。20只健康新西兰大白兔,体重2-2.5kg,雌雄不限,随机分为实验组(B组n=10)和对照组(A组n=10)。实验组予以结扎左冠状动脉前降支造成心肌缺血持续3小时后断颈处死,取缺血区心肌;对照组除不结扎冠状动脉外,其它步骤与实验组完全相同。术后0时直接处死,取左心室心肌。两组标本在4℃放置,分别于死后1h、2h、4h、8h、16h、1d、2d、3d、4d、5d、6d、7d、10d、2w、取材作HE染色和H-FABP免疫组化染色。观察两组标本的HE切片;并按IHC染色结果判定标准对IHC染色进行全面的观察、判断,采用图像分析技术,客观测量IHC切片中H-FABP的缺失面积和阳性表达强度。
结果:
1.常规HE染色:缺血心肌组织可见细胞浊肿、水样变,横纹模糊或消失,胞浆灶性嗜伊红性增强、胞核缩小而染色加深,心肌纤维波浪样改变等心肌早期缺血的病理改变,未见典型心肌梗死改变。缺血心肌4℃放置3-4天,心肌结构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,缺乏特异性。放置7-14天,缺血心肌组织和细胞形态与对照组类似。
2.免疫组织化学染色:对照组心肌组织在4℃放置3d内, H-FABP在心肌细胞胞浆内呈强阳性表达,未见明显的脱失区;4℃放置4d心肌内膜下出现部分小片状缺失。缺血区心肌组织均可见不规则点、灶、片状H-FABP缺失区;部分区域心肌细胞H-FABP阳性表达强度减弱。连续HE染色对比发现H-FABP缺失区及阳性表达减弱区的心肌细胞胞浆嗜伊红性增强、细胞核缩小而染色加深。梗死区与非梗死交界区域的心肌细胞内H-FABP阳性表达强度减弱与周围阳性表达的非缺血区心肌分界清楚。H-FABP在在对照组及心肌梗死组不同时间的缺失面积两两比较和统计分析表明:4℃放置3d内H-FABP阳性表达强度无明显减弱,缺失面积无增加;随着两组标本在4℃放置时间的延长,H-FABP阳性表达强度和阳性反应面积有减弱趋势。死后放置4d以内H-FABP脱失面积与相应正常对照组有显著性差异(p<0.05),放置超过5d以上,其脱失面积与正常对照组无显著性差异(p>0.05)。
结论:
1.大鼠心肌缺血后15min即可出现H-FABP脱失,随着缺血时间的延长,心肌细胞内H-FABP的缺失面积增加,心肌缺血4小时达到高峰。H-FABP检测心肌缺血敏感性强,能明确显示缺血3小时内的缺失区。所以H-FABP可用于死后病理诊断急性心肌缺血。
2.H-FABP的稳定性一般,受自溶影响相对较小。死后4℃放置4天以内,兔缺血区心肌和正常心肌细胞H-FABP表达有显著性差异。故H-FABP是一种敏感性强,稳定性一般的免疫组化指标。H-FABP的免疫组织化学染色在心肌缺血的死后诊断中有一定的法医学价值,可用于死后4℃放置4天以内的尸体的早期心肌梗死的死后诊断。
Background: Study of SCD is one of the most important fields and the postmortem diagnosis of EMI is a focus as well as difficult problem in forensic pathology .It is unlikely to find special changes with either unaided eye or light microscope for myocardial infarction less than 6 hours. Without the use of special methods, it is impossible to get the histological diagnosis of EMI as a cause of sudden death. A series of pathophysiologic changes have been taken place in the ischemic myocardial before the appearance of characteristic morphology on the routine histology stains. Although early ischemic myocardial injury can be detected biochemically, histochemically, enzyme histochemically, fluorescence histochemically, electronmicroscopicly. These methods are limited in forensic practice,either because they are too complexed to be operated, or because they are too unspecific、instabile、expensive or easy to be influenced by autolysis and putrefaction of tissue . However immunohistochemical technology as a swift convenient and practical method can show the qualitative and quantitative changes of proteins in the ischemic myocardial cell, which make it possible to diagnose EMI postmortem.
Heart-type fatty acid binding protein (H-FABP) is a low molecular weight protein (14-15kDa) with special tissue distribution, which is abundant in the cytoplasm of myocardial cells. It may play an important role in the uptake and oxygenation of long-chain fatty acids in cardiac myocyte. As myocardial cell membrane damaged by ischemia, H-FABP leaks to the extracellular space and enters the blood circulation very easily and quickly due to its small size and water soluble. It has been demonstrated that H-FABP is more sensitive and specific than myoglobin (Mb) for clinical detection of AMI within 12 hours after the onset of symptoms by quantifying its plasma concentration, because its molecule is smaller than that of Mb (18kDa) and it presents mainly in cardiomyocyte. So it is proposed as an early indicator of AMI by World Health Organization recently. Whether an immunohistochemical marker can be used in forensic practice or not depends on not only its specificity and sensitivity, but also the influence of autolysis and putrefaction of tissue on antigen . At present, what needs studying further is postmortem stability of immunohistochemical markers used in forensic practice and the regularity of its expression alteration in different postmortem intervals.
Objective: The present study used an animal model of early myocardial ischemia by ligating the left anterior descending branch of coronary artery of animals. In order to explore its postmortem sensitivity and stability , we detected the expression of immunohistochemical markers of H-FABP in myocardium at different interval. The applicability Value of the new immunohistochemical marker was evaluated for diagnosis of SCD in forensic practice.
This study included two parts:
1. Expression of heart-type fatty acid-binding protein in myocardial ischaemic and study on its sensitivity. Methods: The present study used an animal model of myocardial ischemia by ligating the left anterior descending branch of coronary artery of rats. we detected the expression of immunohistochemical markers H-FABP in myocardium at different interval . Briefly, 45 SD rats weighting 250-350g of either sex, were randomly distributed into8 study groups and 1 control group. Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation.
The study group ratswere killed at 15min, 30min, l h, 2h, 3h,4h, 6h,8h after ligation and the control rats killed after operation. Then all hearts were dissected and fixed in 10% formalin over 24h. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. Then treated with HE staining and H-FABP immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the intensity of positive reaction and the depletion area of H-FABP in each sample of the two groups.
Results:
1. HE staining: The control group and myocardial ischemia group within 3h were stained clearly, with distinct cross striation and oval, dense nuclei. Mild cellular swelling or/and interstitial edema, increased eosinophilia were found in ischaemic zones after 4h’s period of ischaemia. No typical necrotic myocardial cells and inflammatory cells were found in all groups. In 8h myocardial ischemia that myocardial eosinophilia has noticeably increased, and some of myocardial cells ischemic necrosis, inflammatory cells increased.
2. IHC staining: In control group, cytoplasm of all cardiomyocytes were stained homogeneous brown (positive for H-FABP), after counterstaining with hematoxylin, the nuclei stained light blue. No positive expression of H-FABP was found in interstitial tissue or other cell component. In study groups, the depletion of H-FABP from cardiomyocytes in infarcted areas at varying post-infarction intervals exhibited dentical pattern. Briefly, partial depletion occurred in a few disseminated cells in the subendo cardial layer and papillary muscles in 15min group. In 30min group, diminished or absent immuno staining cells appeared, but still located in subendo cardial layer and papillary muscles. Patchy depletion happened in lh group, extended to the middle myocardium in 2h group and extended to the outer myocardium in 3h group. By 4h after ligation completely depletion involving almost all myocardium which was distinct from normal cell started and then the depletion area increased with the prolongation of infarcted time. The comparison and statistical analysis between the depletion areas of H-FABP in various ischemic time group show: the depletion area of H-FABP in every study group is significant different from that in their relative control group; there is no significant difference between the depletion area of H-FABP within 3hours; but the depletion area of H-FABP in 3h group is significant different from that in 4h group. The sensitivity of H-FABP manifested mainly in the 3h prior myocardial ischemia.
2.Study on the postmortem stability of heart fatty-acid-binding protein for the diagnosis of the early myocardial infraction
Methods:
The present study used an 3h animal model of myocardial ischemia by ligating the left anterior descending branch of coronary artery of rabbits. We detected the expression of immunohistochemical markers H-FABP in myocardium at different postmortem interval. Briefly, 20 healthy adult rabbits weighting 2-2.5kg of either sex, were randomly distributed into study groups and control group. Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation. The study group rabbits were killed at 3h after ligation and the control rabbits killed were sacrificed directly without ligation. The myocardium was fixed after 1h, 2h、4h、8h、16h、1d、2d、3d、4d、5d、6d、7d、10d and 14d of postmortem stored at 4℃. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. Then treated with HE staining and H-FABP immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the intensity of positive reaction and the depletion area in each sample of the two groups stored at 4℃for different time intervals after death.
Results:
1. HE staining: There some pathological changes of early myocardial ischemia such as cellular cloudy swelling, hydropic degeneration, blur or disappearance of transverse striation, hypereosinophilia, nucleus contract with deeper staining, waviness of muscle fiber in ischemic myocardium, no definite indicators of myocardial infraction. During 3-4 days, the cellular cloudy swelling blur or disapearanse of transverse striation in ischemic myocardium are similar to control group’s changes. While samples stored at 4℃for 7-14d after death, the two groups experienced similar changes in morphous of tissue and in ischemic myocardial cell were similar to control group’s.
2. IHC staining: Myocardial tissue in the control group 4℃for 3 d, H-FABP in myocardial cells of a strong expression, and there was no obvious loss of District 4℃for 4 d endometrial myocardial under some small pieces missing. It showed irregularity punctiform, focus, patch depletion regions with ununiform distribution in ischemic myocardial cytoplasm, and the positive reaction was weakened in partial regions. Compared with the serial paraffin section stained with HE, the endochylema of cadiocytes showed fortified eosnophilia, nucleus contract with deep staining. There is a clear boundary between the normal positive expression of H-FABP in normal myocardium and the weakened positive expression of H-FABP at the junction of infracted zone and non-infracted myocardium. The comparison and statistical analysis between the depletion areas of H-FABP in various postmortem interval group show: While samples stored at 4℃for 3 days after death, the intensity of positive expression without significant weakened and the depletion areas without increased of H-FABP in two groups. The results of image analysis indicate that the intensity of positive reaction trends weaker as well as bigger in the depletion area along with prolongation of postmortem interval. Stored at 4℃for 4 days after death, there were significant differences in the depletion areas of H-FABP between the ischemic groups and the control groups(p<0.05). After 5 day , the depletion area of H-FABP in every study group is not significant different from that in their relative control group(p>0.05).
Conclusion:
1. There can be loss of H-FABP in myocardial ischemia cells in rats after 15 minutes, and with the ischemia intervals prolonged, the deletion areas increased gradually of myocardial cells. The deletion areas of H-FABP reached a peak at four hours of myocardial ischemia. H-FABP is a sensitive detection of myocardial ischemia, and can be detected the deletion area in the ischemic or infracted myocardial cells during 3 hours.
2. The stability of H-FABP was relatively small influenced of autolysis. While stored at 4℃within four days, the depletion areas of H-FABP in ischemic myocardial of rabbit is significant different from that in the normal myocardial. So it can be conclude that the immunohistochemical detection of H-FABP can be used in corpse stored at 4℃for 4d or less than 4d after death, and is helpful for postmortem diagnosis of AMI.
目的:本研究利用结扎左侧冠状动脉前降支的方法建立动物心肌缺血模型,运用免疫组化的方法,探讨H-FABP在死后诊断早期心肌梗死的敏感性及死后稳定性,以期对其在法医学中诊断SCD的应用价值进行评价。
实验分为两部分:
一. H-FABP在缺血心肌中的表达及敏感性研究
方法:结扎大鼠左侧冠状动脉前降支的方法建立大鼠心肌缺血模型,检测H-FABP在大鼠心肌缺血不同时间的表达。45只健康SD大白鼠,重250-300克,雌雄不限,随机分为8个实验组和1个对照组,每组5只。对照组大白鼠除不结扎冠状动脉外,其它步骤与实验组完全相同。各实验组大白鼠分别于结扎后15分钟、30分钟、1小时、2小时、3小时、4小时、6小时、8小时后处死,对照组大白鼠于手术后0小时处死.取出心脏放入10%中性缓冲福尔马林液中固定24h。取材后作HE染色和H-FABP免疫组织化学染色,显微镜下观察两组标本的HE、IHC切片;并按IHC染色结果判定标准对IHC染色进行全面的观察、判断,采用图像分析技术,客观测量IHC切片中H-FABP的缺失面积和阳性面积。
结果:
1.常规HE染色:对照组及心肌缺血3h以内组,心肌细胞染色清晰、边界清楚、横纹存在,仅见有轻度心肌间质水肿。心肌缺血4h后缺血区可见少许心肌细胞轻度水肿,缺血区边缘有少许炎性细胞浸润、心肌嗜伊红性增强,未见明显的细胞坏死。心肌缺血8h可见心肌嗜伊红性明显增强,部分心肌细胞出现凝固性坏死,炎性细胞增多。
2.免疫组织化学染色:对照组心肌胞浆全部染成均匀的棕黄色,用苏木素衬染后,细胞核染成淡蓝色,心肌细胞间质及其他细胞成分不着色。实验组心肌细胞H-FABP在缺血心肌内的脱失表达具有明显的时间规律。缺血15分钟组,在心内膜下心肌、乳头肌可见散在或小灶性的部分H-FABP缺失。缺血30分钟组,开始出现脂肪酸结合蛋白的完全脱失,但仍然局限于心肌内膜下和乳头肌。缺血1小时和2小时组的心肌则出现斑片状缺失且波及到心肌中层,缺血3小时累及心肌外层。缺血4小时,心肌细胞则出现全层细胞的完全脱失并随着缺血时间的延长,脱失面积增大。H-FABP在心肌缺血不同时间的各组缺失面积经比较和统计学分析表明:实验组的H-FABP缺失面积与其各相应对照组比较均有显著性差异,H-FABP在缺血3小时内各组之间没有明显的差异性,而心肌缺血3小时与心肌缺血4小时的心肌细胞中H-FABP脱失面积仍有显著差异,说明H-FABP的敏感性主要体现在心肌缺血3h以前。
二. H-FABP在诊断早期心肌梗死的死后稳定性的实验性研究
方法:结扎左侧冠状动脉前降支的方法建立家兔心肌梗死3小时模型,检测H-FABP在死后不同时间的表达情况。20只健康新西兰大白兔,体重2-2.5kg,雌雄不限,随机分为实验组(B组n=10)和对照组(A组n=10)。实验组予以结扎左冠状动脉前降支造成心肌缺血持续3小时后断颈处死,取缺血区心肌;对照组除不结扎冠状动脉外,其它步骤与实验组完全相同。术后0时直接处死,取左心室心肌。两组标本在4℃放置,分别于死后1h、2h、4h、8h、16h、1d、2d、3d、4d、5d、6d、7d、10d、2w、取材作HE染色和H-FABP免疫组化染色。观察两组标本的HE切片;并按IHC染色结果判定标准对IHC染色进行全面的观察、判断,采用图像分析技术,客观测量IHC切片中H-FABP的缺失面积和阳性表达强度。
结果:
1.常规HE染色:缺血心肌组织可见细胞浊肿、水样变,横纹模糊或消失,胞浆灶性嗜伊红性增强、胞核缩小而染色加深,心肌纤维波浪样改变等心肌早期缺血的病理改变,未见典型心肌梗死改变。缺血心肌4℃放置3-4天,心肌结构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,缺乏特异性。放置7-14天,缺血心肌组织和细胞形态与对照组类似。
2.免疫组织化学染色:对照组心肌组织在4℃放置3d内, H-FABP在心肌细胞胞浆内呈强阳性表达,未见明显的脱失区;4℃放置4d心肌内膜下出现部分小片状缺失。缺血区心肌组织均可见不规则点、灶、片状H-FABP缺失区;部分区域心肌细胞H-FABP阳性表达强度减弱。连续HE染色对比发现H-FABP缺失区及阳性表达减弱区的心肌细胞胞浆嗜伊红性增强、细胞核缩小而染色加深。梗死区与非梗死交界区域的心肌细胞内H-FABP阳性表达强度减弱与周围阳性表达的非缺血区心肌分界清楚。H-FABP在在对照组及心肌梗死组不同时间的缺失面积两两比较和统计分析表明:4℃放置3d内H-FABP阳性表达强度无明显减弱,缺失面积无增加;随着两组标本在4℃放置时间的延长,H-FABP阳性表达强度和阳性反应面积有减弱趋势。死后放置4d以内H-FABP脱失面积与相应正常对照组有显著性差异(p<0.05),放置超过5d以上,其脱失面积与正常对照组无显著性差异(p>0.05)。
结论:
1.大鼠心肌缺血后15min即可出现H-FABP脱失,随着缺血时间的延长,心肌细胞内H-FABP的缺失面积增加,心肌缺血4小时达到高峰。H-FABP检测心肌缺血敏感性强,能明确显示缺血3小时内的缺失区。所以H-FABP可用于死后病理诊断急性心肌缺血。
2.H-FABP的稳定性一般,受自溶影响相对较小。死后4℃放置4天以内,兔缺血区心肌和正常心肌细胞H-FABP表达有显著性差异。故H-FABP是一种敏感性强,稳定性一般的免疫组化指标。H-FABP的免疫组织化学染色在心肌缺血的死后诊断中有一定的法医学价值,可用于死后4℃放置4天以内的尸体的早期心肌梗死的死后诊断。
Background: Study of SCD is one of the most important fields and the postmortem diagnosis of EMI is a focus as well as difficult problem in forensic pathology .It is unlikely to find special changes with either unaided eye or light microscope for myocardial infarction less than 6 hours. Without the use of special methods, it is impossible to get the histological diagnosis of EMI as a cause of sudden death. A series of pathophysiologic changes have been taken place in the ischemic myocardial before the appearance of characteristic morphology on the routine histology stains. Although early ischemic myocardial injury can be detected biochemically, histochemically, enzyme histochemically, fluorescence histochemically, electronmicroscopicly. These methods are limited in forensic practice,either because they are too complexed to be operated, or because they are too unspecific、instabile、expensive or easy to be influenced by autolysis and putrefaction of tissue . However immunohistochemical technology as a swift convenient and practical method can show the qualitative and quantitative changes of proteins in the ischemic myocardial cell, which make it possible to diagnose EMI postmortem.
Heart-type fatty acid binding protein (H-FABP) is a low molecular weight protein (14-15kDa) with special tissue distribution, which is abundant in the cytoplasm of myocardial cells. It may play an important role in the uptake and oxygenation of long-chain fatty acids in cardiac myocyte. As myocardial cell membrane damaged by ischemia, H-FABP leaks to the extracellular space and enters the blood circulation very easily and quickly due to its small size and water soluble. It has been demonstrated that H-FABP is more sensitive and specific than myoglobin (Mb) for clinical detection of AMI within 12 hours after the onset of symptoms by quantifying its plasma concentration, because its molecule is smaller than that of Mb (18kDa) and it presents mainly in cardiomyocyte. So it is proposed as an early indicator of AMI by World Health Organization recently. Whether an immunohistochemical marker can be used in forensic practice or not depends on not only its specificity and sensitivity, but also the influence of autolysis and putrefaction of tissue on antigen . At present, what needs studying further is postmortem stability of immunohistochemical markers used in forensic practice and the regularity of its expression alteration in different postmortem intervals.
Objective: The present study used an animal model of early myocardial ischemia by ligating the left anterior descending branch of coronary artery of animals. In order to explore its postmortem sensitivity and stability , we detected the expression of immunohistochemical markers of H-FABP in myocardium at different interval. The applicability Value of the new immunohistochemical marker was evaluated for diagnosis of SCD in forensic practice.
This study included two parts:
1. Expression of heart-type fatty acid-binding protein in myocardial ischaemic and study on its sensitivity. Methods: The present study used an animal model of myocardial ischemia by ligating the left anterior descending branch of coronary artery of rats. we detected the expression of immunohistochemical markers H-FABP in myocardium at different interval . Briefly, 45 SD rats weighting 250-350g of either sex, were randomly distributed into8 study groups and 1 control group. Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation.
The study group ratswere killed at 15min, 30min, l h, 2h, 3h,4h, 6h,8h after ligation and the control rats killed after operation. Then all hearts were dissected and fixed in 10% formalin over 24h. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. Then treated with HE staining and H-FABP immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the intensity of positive reaction and the depletion area of H-FABP in each sample of the two groups.
Results:
1. HE staining: The control group and myocardial ischemia group within 3h were stained clearly, with distinct cross striation and oval, dense nuclei. Mild cellular swelling or/and interstitial edema, increased eosinophilia were found in ischaemic zones after 4h’s period of ischaemia. No typical necrotic myocardial cells and inflammatory cells were found in all groups. In 8h myocardial ischemia that myocardial eosinophilia has noticeably increased, and some of myocardial cells ischemic necrosis, inflammatory cells increased.
2. IHC staining: In control group, cytoplasm of all cardiomyocytes were stained homogeneous brown (positive for H-FABP), after counterstaining with hematoxylin, the nuclei stained light blue. No positive expression of H-FABP was found in interstitial tissue or other cell component. In study groups, the depletion of H-FABP from cardiomyocytes in infarcted areas at varying post-infarction intervals exhibited dentical pattern. Briefly, partial depletion occurred in a few disseminated cells in the subendo cardial layer and papillary muscles in 15min group. In 30min group, diminished or absent immuno staining cells appeared, but still located in subendo cardial layer and papillary muscles. Patchy depletion happened in lh group, extended to the middle myocardium in 2h group and extended to the outer myocardium in 3h group. By 4h after ligation completely depletion involving almost all myocardium which was distinct from normal cell started and then the depletion area increased with the prolongation of infarcted time. The comparison and statistical analysis between the depletion areas of H-FABP in various ischemic time group show: the depletion area of H-FABP in every study group is significant different from that in their relative control group; there is no significant difference between the depletion area of H-FABP within 3hours; but the depletion area of H-FABP in 3h group is significant different from that in 4h group. The sensitivity of H-FABP manifested mainly in the 3h prior myocardial ischemia.
2.Study on the postmortem stability of heart fatty-acid-binding protein for the diagnosis of the early myocardial infraction
Methods:
The present study used an 3h animal model of myocardial ischemia by ligating the left anterior descending branch of coronary artery of rabbits. We detected the expression of immunohistochemical markers H-FABP in myocardium at different postmortem interval. Briefly, 20 healthy adult rabbits weighting 2-2.5kg of either sex, were randomly distributed into study groups and control group. Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation. The study group rabbits were killed at 3h after ligation and the control rabbits killed were sacrificed directly without ligation. The myocardium was fixed after 1h, 2h、4h、8h、16h、1d、2d、3d、4d、5d、6d、7d、10d and 14d of postmortem stored at 4℃. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. Then treated with HE staining and H-FABP immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the intensity of positive reaction and the depletion area in each sample of the two groups stored at 4℃for different time intervals after death.
Results:
1. HE staining: There some pathological changes of early myocardial ischemia such as cellular cloudy swelling, hydropic degeneration, blur or disappearance of transverse striation, hypereosinophilia, nucleus contract with deeper staining, waviness of muscle fiber in ischemic myocardium, no definite indicators of myocardial infraction. During 3-4 days, the cellular cloudy swelling blur or disapearanse of transverse striation in ischemic myocardium are similar to control group’s changes. While samples stored at 4℃for 7-14d after death, the two groups experienced similar changes in morphous of tissue and in ischemic myocardial cell were similar to control group’s.
2. IHC staining: Myocardial tissue in the control group 4℃for 3 d, H-FABP in myocardial cells of a strong expression, and there was no obvious loss of District 4℃for 4 d endometrial myocardial under some small pieces missing. It showed irregularity punctiform, focus, patch depletion regions with ununiform distribution in ischemic myocardial cytoplasm, and the positive reaction was weakened in partial regions. Compared with the serial paraffin section stained with HE, the endochylema of cadiocytes showed fortified eosnophilia, nucleus contract with deep staining. There is a clear boundary between the normal positive expression of H-FABP in normal myocardium and the weakened positive expression of H-FABP at the junction of infracted zone and non-infracted myocardium. The comparison and statistical analysis between the depletion areas of H-FABP in various postmortem interval group show: While samples stored at 4℃for 3 days after death, the intensity of positive expression without significant weakened and the depletion areas without increased of H-FABP in two groups. The results of image analysis indicate that the intensity of positive reaction trends weaker as well as bigger in the depletion area along with prolongation of postmortem interval. Stored at 4℃for 4 days after death, there were significant differences in the depletion areas of H-FABP between the ischemic groups and the control groups(p<0.05). After 5 day , the depletion area of H-FABP in every study group is not significant different from that in their relative control group(p>0.05).
Conclusion:
1. There can be loss of H-FABP in myocardial ischemia cells in rats after 15 minutes, and with the ischemia intervals prolonged, the deletion areas increased gradually of myocardial cells. The deletion areas of H-FABP reached a peak at four hours of myocardial ischemia. H-FABP is a sensitive detection of myocardial ischemia, and can be detected the deletion area in the ischemic or infracted myocardial cells during 3 hours.
2. The stability of H-FABP was relatively small influenced of autolysis. While stored at 4℃within four days, the depletion areas of H-FABP in ischemic myocardial of rabbit is significant different from that in the normal myocardial. So it can be conclude that the immunohistochemical detection of H-FABP can be used in corpse stored at 4℃for 4d or less than 4d after death, and is helpful for postmortem diagnosis of AMI.
引文
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[1]Glatz JF, van der Vusse GJ. Cellular fatty acid-binding proteins: their function and physiological significance. [J] Prog Lipid Res 1996;35:243–82.
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[3]Hassan M.E. Azzazy, Maurice M.A.L. Pelsers, Robert H. Christenson Unbound Free Fatty Acids and Heart-Type Fatty Acid–Binding Protein: Diagnostic Assays and Clinical Applications [J] Clinical Chemistry 2006 52:119-29
[4]Tan N-S, Shaw NS, Vinckenbosch N, Liu P, et al. Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription. [J] Moll Cell Biol 2002 22: 5114–5127.
[5]冯爱娟,陈东风 脂肪酸结合蛋白研究进展 [J] 世界华人消化杂志 2003; 11(9):1457-1459
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[3]Hassan M.E. Azzazy, Maurice M.A.L. Pelsers, Robert H. Christenson Unbound Free Fatty Acids and Heart-Type Fatty Acid–Binding Protein: Diagnostic Assays and Clinical Applications [J] Clinical Chemistry 2006 52:119-29
[4]Tan N-S, Shaw NS, Vinckenbosch N, Liu P, et al. Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription. [J] Moll Cell Biol 2002 22: 5114–5127.
[5]冯爱娟,陈东风 脂肪酸结合蛋白研究进展 [J] 世界华人消化杂志 2003; 11(9):1457-1459
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[9]Monique JMG,Wodzig KWH,Maarten LS,et a1.Measurement of myocardial infarct size from plasma fatty acid binding protein or myoglobin, using individually estimated clearance rates.[J] Cardiovascular Research,1999,44: 247—248.
[10]Seino Y,Tomita Y,Takano T,et a1. Office cardiologists cooperative study on whole blood rapid panel tests in patients with suspicious acute myocardial infarction: comparison between heart—type fatty acid—binding protein and troponin T tests.[J] Circ J,2004,68:144— 148.
[11]Tomoaki Nakataa Akiyoshi Hashimotoa , et a1 Human Heart-Type Fatty Acid-Binding Protein as an Early Diagnostic and Prognostic Marker in Acute Coronary Syndrome [J] Cardiology 2003;99:96–104
[12]杨文东,魏树华大面积严重烧伤患者早期检测血清脂肪酸结合蛋白价值的探讨[J]中国烧伤创疡杂志,2002,14(3):141-143
[13]杨燕,张维国等FABP在放疗致心肌损伤中的诊断价值[J]山东医药,2005,45(13):38
[14]徐云兴, 单岩小儿先天性心脏病手术中心肌特异脂肪酸结合蛋白监测[J]郑州大学学报(医学版) 2005, 40,11(6):1135-1137
[15]刘宏,董国华心型脂肪酸结合蛋白在瓣膜置换术后心肌损伤早期评价中的应用[J]实用医学杂志2005 2l(l0):1019-1-22
[16]宋一璇.猝死[A].祝家镇.法医病理学[M].北京:人民卫生出版社,2001.346—347.
[17].Kazuo WA,Hironao WA,Veerkamp JH,et al. Immunohistochem- ical distribution of heart-type fatty acid-binding protein immuno-reaetivity in normal human tissue and in acute myocardial infarct[J].J Path,1993,170(1):59—65.
[18] 明媚,孟祥志.大鼠心脏型脂肪酸结合蛋白在早期心肌缺血时的含量变化 [J].中国法医学杂志,2004,19(2): 68—71.
[19] 郎博娟, 孟祥志, 王琼大鼠死亡后外周血和尿液中心脏型脂肪酸结合蛋白的研究 [J] 医学研究生学报 2005 18(7):599-608