【作者】 郑娜；

【导师】 刘良；

【作者基本信息】 华中科技大学 ， 法医学， 2010， 博士

【摘要】 背景：心肌梗死(myocardial infarction, MI)是心肌严重缺血所导致的心肌细胞坏死。MI是心肌缺血的严重并发症之一,占所有死亡的10-15%,占猝死的60%。因此,MI作为心血管系统的重要病变之一,成为世界范围内的健康隐患。依据病变时间差异,MI可分为急性心肌梗死(acute myocardial infarction, AMI)及陈旧性心肌梗死(old myocardial infarction, OMI).AMI往往表现为心肌细胞的溶解性坏死,心肌细胞肿胀,肌原纤维和细胞器广泛溶解、消失,甚至只有核和胞膜的存留,整个心肌细胞犹如一个空鞘,同时细胞周围出现纤维增生、变粗,形成网架结构。最终核消失,网架塌陷崩解；心肌细胞的再生能力极差,坏死区域最常见的修复形式为瘢痕形成，即坏死组织完全为胶原纤维所取代,纤维排列不规则。陈旧的瘢痕组织较致密,其胶原纤维可发生玻璃样变性,或钙盐沉着,这就是OMI的主要病理学改变。从组织病理学角度,MI导致急性炎症,并且坏死组织逐渐被胶原瘢痕所取代,该过程大概需要6-8周时间。疾病的进程均为正常组织-微小病变-典型病变,早期AMI从组织病理学水平难以诊断,而且AMI向OMI发展过程中表现出来的组织病理学改变愈加复杂,难以进行有效的区分。在实际工作中,无论大体标本的宏观观察,还是组织切片的微观观察,所见到的病理改变往往比较复杂,单纯依靠主观的组织病理学检查,往往难以对微小病变或轻度病变加以准确的区分和辨认。因此亟需寻找一种便捷、敏感、可重复性的方法,以适用于法医病理学鉴定的需要。傅立叶变换红外光谱(Fourier transform infrared spectroscopy, FTIR)技术兴起于20世纪80年代,是确定分子组成和结构的有效技术工具,具有分析过程简便、噪声低、光通量高、测量速度快、波数精度高、频率测量范围宽、操作费用低、无损检测等优点,广泛应用于化工、冶金、地矿、石油、医药、珠宝鉴定、法庭科学等诸多领域。对于固体、液体或气体样本,单一组分的纯净物及多种组分的混合物都可以应用FTIR技术进行测定。FTIR既可以用于样品的定性分析,也可用于定量分析。除此之外,FTIR检测过程制样简单,生物样本无需额外的染色技术,极大地缩短了测定时间。基于法医病理学特定的保留样本及复审需要,FTIR的无损性、可重复性尤为适用。当有机分子吸收红外线后,会产生分子振动能级的跃迁,这种跃迁因不同有机分子的振动形式、振动方向及振幅大小而异,其振动能级跃迁有所差异,因此不同结构的化合物在红外线的照射下呈现出不同波长的吸收带,即红外光谱。FTIR即是将干涉图应用数学上的傅立叶转换所形成的红外光谱。对于多原子的有机物来讲,有些振动来自于整个分子,有些振动来自于分子局部的化学键或官能团,同一类型的化学键的振动具有相近的振动频率。因此,FTIR依据分子共价键的不同运动方式所产生的红外吸收的特征峰,来确定该化合物的化学结构或分子中的官能团特征。细胞由细胞膜、细胞质、细胞核构成,各部分主要化学组成为蛋白质及脂质、糖原、核酸,这些组分的含量、构型及构象在不同组织细胞表现不同。在许多疾病过程中,这些组分会发生变化,而这些变化可以通过红外光谱的差异表现出来。目前FTIR应用于肿瘤、动脉粥样硬化斑块、骨钙化异常等疾病的分子特征进行研究,并发现了一些特征性的变化。国外学者曾利用MRI技术对活体内陈旧性心肌梗死及急性心肌梗死进行区别,但利用FTIR及面成像技术进行心肌梗死的分子特征测定并初步进行区分,目前国内外尚未见报道。本研究突破传统分子特征研究思路,在国际上首创性的联用FTIR、红外显微镜及面成像技术对心肌梗死的分子特征进行研究；将尸体样本引入分子水平研究,在同一张切片上设定实验组及对照组,消除了死后变化的影响；快速、敏感的对细胞各主要大分子成分的含量及空间构型进行同时分析,消除了单次分析一个指标的操作差异,并最大程度节省了检测时间；在前期开创性工作的基础上,对蛋白质、糖原、核酸及脂质等成分的变化关系进行探讨,进一步用于探测陈旧性及急性心肌梗死的分子特征,并为最终在分子水平区分陈旧性及急性心肌梗死奠定基础。面成像技术的应用使得化学图像能直观的反映各大分子的分布及变化情况,并建立了与组织病理学的良好对应关系,延伸了“红外病理学”这门交叉学科的涵盖范围。本研究属于化学技术与法医学研究的交叉性创新研究。目的：本研究针对现今法医学心肌梗死的鉴定、陈旧性及急性心肌梗死的时间区分问题,通过将光谱学FTIR、红外显微镜及面成像技术的联合应用,对陈旧性及急性心肌梗死的分子变化进行测定,主要探讨红外光谱反映出来的蛋白质、脂质、糖原及核酸的变化,以及相互之间的变化关系。并通过红外化学图像的特点,研究化学图像与组织病理学的对应关系,为最终应用FTIR技术在分子水平上对陈旧性心肌梗死及急性心肌梗死加以区分奠定基础。方法：实验材料及仪器1999年-2008年本系检案案例陈旧性心肌梗死及急性心肌梗死心脏样本。傅立叶变换红外光谱仪(Nicolet 6700, Thermo Fisher Scientific,美国)；傅立叶变换红外显微镜附件(Nicolet continuum microscope, Thermo Fisher Scientific,美国)；傅立叶变换红外光谱仪(Nicolet iN10, Thermo Fisher Scientific,美国)；红外光谱工作站(OMNIC 8.0, Thermo Fisher Scientific,美国)；虚拟显微成像系统(Dotslide 2.1, Olympus,日本)；脱水机(AP-280X, Microm,德国)包埋机(STP-120, Microm,德国)；切片机(HM-340E, Microm,德国)；展片机(ZPJ-1,天津天利航空机电有限公司)；烤片机(KPJ-1,天津天利航空机电有限公司)；苏木素、伊红、天然树胶(上海试剂一厂)；二甲苯、乙醇、氨水、盐酸(分析纯)。实验方法FTIR及红外显微镜联用检测根据鉴定结果确定陈旧性心肌梗死部位,各心脏样本在心肌梗死边缘取材2块,包括心肌梗死区及邻近正常组织区。样本常规制作蜡块,行连续切片,厚6μm。将其中一张切片平铺于普通载玻片上,行常规HE染色。将HE染色切片经Dotslide虚拟显微成像系统在40分辨率下进行扫描,并依据病理学改变设定心肌梗死病变区域为实验组,同一张切片无明显病变区域为对照组。另一张连续切片平铺于表面镀金的特殊载玻片上,此切片仅行常规脱蜡、脱水处理,不经任何染色方法处理,晾干行FTIR检测。应用Nicolet 6700 FTIR光谱仪、Nicolet continuum红外显微镜(物镜15×,目镜10×)及其匹配工作站OMNIC 8.0检测。反射模式,经液氮冷却,在室温24℃下进行,测定范围为4000 cm-1-650 cm-1。参数设置为单张光谱覆盖面积为25μm×25μm,分辨率为4cm-1,扫描次数为32次。每个样品在测试前先进行背景扫描,并储存该光谱作为固定背景。根据HE染色切片划分对照组及实验组,在红外显微镜指导下将相同区域划分为对照组(A组)及实验组(B组),各组随机选取10个检测区域,所采集光谱经OMNIC 8.0工作站存储并处理分析。应用OMNIC软件对光谱进行自动基线校正、自动平滑处理后,分析蛋白质酰胺、脂质、糖原、核酸等谱带的峰位、峰强度,及部分特征性谱带的峰高比,以诠释陈旧性心肌梗死及急性心肌梗死的分子特征,并建立与组织病理学的对应关系。FTIR、红外显微镜及面成像(mapping)技术联用检测切片样本制样过程同上。联用Nicolet iN10 FTIR光谱仪、Nicolet continuum红外显微镜(物镜15×,目镜10×)及其匹配工作站OMNIC 8.0检测。反射模式,经液氮冷却,在室温24℃下进行,测定范围为4000 cm-1-650 cm-1。参数设置为单张光谱覆盖面积为100μm×100μm,分辨率为16cm-1,扫描次数为1次。背景采集及分组同上。对整张切片组织进行扫描采集化学图像。应用OMNIC Picta对化学图像进行自动基线校正、自动平滑处理,并研究其与形态学的对应关系。统计学分析及图像处理所得数据经SPSS 13.0软件进行统计学一维方差分析,以均数±标准差表示,P<0.05具有统计学差异。结果：FTIR及红外显微镜检测在陈旧性心肌梗死病变区,蛋白质酰胺A、B、Ⅰ、Ⅱ各谱带变化趋势一致,均为峰位蓝移、峰强度增加。脂质CH3反对称及对称伸缩振动、CH2反对称及对称伸缩振动谱带变化趋势一致,均为峰位无移动、峰强度增加。核酸PO2反对称伸缩振动谱带的峰位出现蓝移,但对称伸缩振动谱带的峰位无移动,峰强度均降低。糖原C-O伸缩振动谱带的峰位无移动、峰强度降低。I1652/I1634、I1650/11550比值均增高；I1550／I3300、I1160／I1080比值均降低；I2920/I2960比值无变化。在急性心肌梗死病变区,蛋白质酰胺A、B、Ⅰ、Ⅱ各谱带变化趋势一致,均为峰位无移动、峰强度降低。脂质仅CH3反对称伸缩振动谱带的峰位出现红移、峰强度降低,其余CH3对称、CH2反对称及对称伸缩振动各谱带变化趋势一致,均为峰位无移动、峰强度降低。核酸PO2反对称伸缩振动谱带的峰位无移动,而PO2对称伸缩振动谱带的峰位红移,两谱带的峰强度均降低。糖原C-O伸缩振动谱带的峰位无移动、峰强度降低。I1652/I1643比值降低；I1650/I1550比值增高；I1550／I3300、I2920/I2850、I1160／I1080均无变化。FTIR及mapping检测面扫描成像技术采集的化学图像基本可以反映MI各大分子分布特征,并且与陈旧性心肌梗死的组织病理学分布特征基本一致,与急性心肌梗死的组织病理学分布特征较一致。应用数学模型一阶导数、二阶导数对化学图像进行处理,有利于改善化学图像与形态学变化的对应性。结论：应用FTIR技术可以揭示陈旧性心肌梗死及急性心肌梗死的分子特征,并依据红外病理学改变有效地区分陈旧性及急性心肌梗死病理变化。化学图像直观、敏感的建立了与组织病理学的对应关系,结合红外光谱可以初步区分陈旧性及急性心肌梗死病理变化。更多还原

【Abstract】 Introduction:Myocardial infarction (myocardial infarction, MI) is necrosis of the myocardium as a result of severe ischaemia. MI is extremely common, accounting for 10-15% of all deaths and about 60% of sudden unexpected deaths. Therefore, as an important disease of the cardiovascular system, MI is a worldwide health risks and burdens.According to the interval, MI can be divided into acute myocardial infarction (acute myocardial infarction, AMI) and old myocardial infarction (old myocardial infarction, OMI). AMI is often manifested in the lytic necrosis of cadiocytes. Cadiocytes displays swelling, and myofibrils are widely dissolved, disappeared, or only the nuclear and the membrane left, the cadiocyte looks like an empty sheath. Finally, the nucleus is dismissed and the frame is disrupted. Due to the poor regeneration capacity, the most common repair is scar formation, which necrotic tissue completely replaced by collagen fibers, fibers arranged irregularly. Old scar is dense, and the collagen fibers can occur hyaline degeneration, or calcium calm, which are the main pathological changes of OMI. At the hispathological level, MI leads to acute inflammation, and necrotic tissue is gradually replaced by scar, the process continued for about 6-8 weeks.The processes of diseases are from normal tissues to small lesions, and then typical lesions. Not only the diagnosis of early stage of AMI by pathological change is difficult, but also the complex pathological changes of OMI and AMI increase the difficulty to distinguish effectively. In practice, both in macro-observation of macro-samples and microscopic observation of sections, due to the complicated pathologic changes, it is difficult to accurately identify and distinguish the histopathological minimal changes only relying on subjective experiences. Therefore it is useful to find a convenient, sensitive and reproducible method to fit for the needs of forensic pathology.Fourier transform infrared spectroscopy (FTIR) technology emerged in 80’s of 20th century. It is an effective technology to determine the moleculer composition and structure. The advantages include simple analysis process, low noise, high light quantity, high precision of wave number, wide measuring range, low cost and nondestructive test. It is widely used in chemical, metallurgy, mining, petroleum, pharmaceuticals, forensic science and other areas. For the solid, liquid or gas samples, the purity of a single component and multi-component mixture of materials can be measured using by FTIR techniques. FTIR can be used for qualitative analysis and quantitative analysis of samples. In addition, the advantage of simple sample-preparation and no staining significantly reduces measurement time. Based on samples reservation and re-identify of the samples, non-destructive and repeatable characteristics made the FTIR particularly important.After absorbsorption of infrared light, the organic molecules will produce molecular vibration for transition. Due to the diversity of vibrational forms, vibrational directions and vibrational amplitudes of organic molecules, the transition displays variously. The different structures of the compounds showed absorption bands with different wavelength, which is IR. FTIR is the IR applied mathematic Fourier’s transform. For polyatomic organical, the same type of chemical bond vibrations have similar frequencies, some vibration derive from the whold molecule, and some from the vibration of partial chemical bonds or functional groups. Based on characteristic absorption peak of infrared absorption generated by different modes of motion, FTIR determines the characteristics of chemical structure or molecular functional group.Cell is composed of cell membrane, cytoplasm and nucleus. Its main chemical compositions are protein, lipid, glycogen and nucleic acid. The contents, configurations and conformations of these components are different in different cells. In the course of many diseases, these components change, which could be shown by difference infrared spectra. The diagnosis of cancer, COPD, Alzheimer’s disease and other diseases are studied by FTIR. Foreign scholars have differentiated the old myocardial infarction and acute myocardial infarction used MRI’in vivo, but no reports was seen on study of molecular characteristics of myocardial infarction by FTIR and mapping technology in national and international region.Our study breaked through the traditional thinking on molecular characteristics, detected the technology molecular characteristics of myocardial infarction applied by FTIR pioneeringly, eliminated the impact of the changes after death, and detected the relationship of protein, glycogen, nucleic acids and lipid changes of old myocardial infarction and acute myocardial infarction. The mapping technology allowed chemical images directly reflecting the distribution of major elements and changes, and established a-good correspondence with histopathologic changes, and extended the areas of "infrared pathology". The present study is the innovation with cross-application of chemical technology and forensic science.Objective:The present study focused on forensic identification of myocardial infarction, and the distinction of acute myocardial infarction and old myocardial infarction. The present study detected the molecular characteristics of old myocardial infarction and acute myocardial infarction by FTIR and mapping technology. The aim was to find the changes of protein, lipid, glycogen and nucleic acid changes and the relationships reflected by infrared spectra, and explore its relationship with the corresponding of histopathology changes and the chemical images.Material and Method:Meterial and machines:The heart samples of old myocardial infarction and acute myocardial infarction of autospy cases in our departmet from 1999 to 2008.Nicolet 6700 FTIR, Thermo Fisher Scientific, USA;Nicolet continuum microscope, Thermo Fisher Scientific, USA;Nicolet iN10 FTIR, Thermo Fisher Scientific, USA;OMNIC workstation 8.0, Thermo Fisher Scientific, USA;Dotslide virtural micro-imaging systerm 2.1, Olympus, Japan;Spin-drier, AP-280X, Microm, Germany;Embedding machine, STP-120, Microm, Germany;Microtome, HM-340E, Microm, Germany;Expanding machine, ZPJ-1, tianli aviation machinery electrical company, China;Toasting machine, KPJ-1, tianli aviation machinery electrical company, China.Methods:Detection by FTIR and micro-spectroscopy methodSamples were conventionally made wax block, and cross-section of 6μm. One section was mounted on a glass slide, conventionally stained with H&E. The section was scanned by Dotslide the virtual imaging system at resolutions of 40, and identified as the control group and experimental group according to pathological changes of myocardial infarction lesion. Another section was mounted on a special infrared reflective slide, only routinely done with deparaffinageing and dehydration, without any staining processing, and dried for FTIR.The research was applied by combined Nicolet 6700 FTIR, Nicolet continuum infrared microscope (objective×15, eyepiece×10) and OMNIC workstation 8.0. the present research was in reflection mode, and cooled by liquid nitrogen, at room temperature 24℃. The infrared range was 4000 cm-1 to 650 cm-1. The parameters were setted that covered area was 25μm×25μm, with the resolution of 4cm-1, scanning time of 32 times. The background of each sample was scanned first and the spectra were saved. According to the virtual imaging of HE stained section, the control group and experimental group were setted. Randomly chosen ten test points from each group, the collected infrared spectra were saved and processed by OMNIC 8.0 workstations. Automatic baseline correction and auto-smoothing were performed by OMNIC software. Wave number and peak intensity of the protein, carbohydrate and lipid and nucleic acid bands’and the peak height ratio of some characteristic bands were analysed. The molecular characteristics were used to interpret the instinct of old myocardial infarction and acute myocardial infarction, and to establish the corresponding relations of histopathology and infrared spectra.Detection by FTIR and mapping techologyThe sections were treated the same as the research above-mentioned. The research was performed by combined Nicolet iN10 FTIR, Nicolet continuum micro-spectroscopy and OMNIC workstation 8.0. The parameters were setted that covered area was 100μm×100μm, with the resolution of 16cm-1, scanning time of 1 time. Other parameters were the same as the research mentioned above. The chemical image was collected for the whole section. The chemical image was treated with auto-baseline correction and auto-smothing to establish the corresponding relations of histopathology and the chemical image.Stastatical analysis and image processingThe data were statistically by SPSS 13.0 for one-way AVON analysis, with the mean±standard deviation, the P<0.05 was considered as statistical significance.Result:In old myocardial infarction region, all the amide bands displayed the same trend, what were the wave number diaplayed blue shift and the intensity increasing. The antisymmetry and symmetry of CH3 and CH2 displayed intensity increasing. The antisymmetry of PO2 displayed blue shift and the intensity decreasing, but the symmetry of the bond had no wave number shift. The C-O bond of glycogen displayed intensity decreasing. The ratio of I1652/I1634、I1650/I1550 showed heighten, the ratio of I1550/I3300、I1160/I1080 showed the opposite, and the ratio of I2920/I2960 showed no difference.In acute myocardial infarction region, all the amide bands displayed the same trend, what were the wave number diaplayed intensity decreasing. Except the antisymmetry CH3 bond displayed red shift, all the other stretching vibration of CH3 and CH2 displayed no shift and intensity decreasing. The symmetry of PO2 displayed red shift and the intensity decreasing, but the antisymmetry of the bond had no wave number shift. The C-O bond of glycogen displayed intensity decreasing. The ratio of I1652/I1634showed lower, the ratio of I1650/I1550 showed the opposite, and the ratio of I1550/I3300、I1160/I1080、I2920/I2960 showed no difference.Conclusion:The molecular characteristics of old myocardial infarction and acute myocardial infarction were revealed by FTIR and mapping technology. The old myocardial infarction and acute myocardial infarction were distinguished according to infrared pathology. It intuitively and sensitively established the corresponding relationship of histopathology and Chemical images. Combined with infrared spectroscopy, the chemical image could initially distinguish the pathological changes of old myocardial infarction and acute myocardial infarction.更多还原