MALDI-TOF-MS成像技术及其在法医损伤标志物筛选中的应用

传统的免疫学和分子生物学技术在寻找损伤分子标志物方面存在成果孤立、效价模糊以及相互间作用机制不明等缺点.质谱成像技术集筛选、分析和图像化于一体,更适合基于形态学的损伤分子标志物研究.本文综述了基于基质辅助激光解吸/电离-飞行时间质谱（matrix-assisted laser desorption/ionizationtime-of flight mass spectrometry,MALDI-TOF-MS）成像技术筛选并识别特征性蛋白质的方法和最新进展,阐述了其在法医学损伤研究中的应用价值.     

MALDI-TOF-IMS在生物医学中的研究进展及法医学应用展望

基质辅助激光解吸/电离-飞行时间质谱成像(matrix-assisted laser desorption/ionization time-offlight imaging mass spectrometry,MALDI-TOF-IMS)技术通过对生物组织切片的扫描,分析切片中的未知组分,获取组织的分子成像图,已成为研究生物标志物、脂类分布、药物代谢等方面的有力手段。本文对该技术在蛋白质鉴定、临床应用、药物研发、脂类研究及脑损伤方面的应用进行了综述。     

MALDI-TOF-IMS在蛋白质组学研究中的新进展

基质辅助激光解吸/电离-飞行时间质谱成像(matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry,MALDI-TOF-IMS)技术已成为目前蛋白质组学研究中的经典技术,是一种研究生物组织切片中蛋白质和小分子物质分布的工具。MALDI-TOF-IMS通过单次扫描就能够同时分析生物组织切片中的各种未知组分,在保持组织中细胞和分子完整性的同时获取组织的分子成像图。近年来,质谱成像技术在生物医学各领域中有了较快发展,本文综合文献资料对MALDI-TOF-IMS技术目前的发展水平、质谱成像原理、方法学以及在法医病理学中的应用前景进行了综述。     

MALDI-TOF-IMS分析DAI大鼠脑组织内差异蛋白的分布

目的基于基质辅助激光解吸/电离-飞行时间质谱成像(matrix assisted laser desorption/ionizationtime of flight imaging mass spectrometry,MALDI-TOF-IMS)技术建立弥漫性轴索损伤(diffuse axonal injury,DAI)大鼠脑组织内蛋白差异化表达的质谱成像方法。方法应用AutoflexⅢ MALDI-TOF质谱仪在质荷比1 000~20 000范围内对DAI组和对照组脑组织进行质谱扫描,利用Clin Pro Tool 2.2软件对两组数据进行统计学分析,挑选出存在表达差异的蛋白质分子进行质谱成像,观察不同质荷比的蛋白质分子在脑组织中的分布情况。结果质荷比为4 963、5 634、6 253、6 714及7 532的5个蛋白质分子在DAI大鼠脑组织内表达量存在差异。结论 MALDI-TOF-IMS可用于大鼠DAI后脑组织内差异蛋白的研究,初步建立了质谱成像技术检测DAI大鼠脑组织中差异蛋白分布的分析方法。     

MALDI-TOF-MS对DAI大鼠脑干蛋白质组学的分析

目的应用基质辅助激光解吸/电离-飞行时间质谱(matrix-assisted laser desorption/ionization timeof-flight mass spectrometry,MALDI-TOF-MS)技术建立弥漫性轴索损伤(diffuse axonal injury,DAI)诊断模型,筛选DAI相关蛋白或多肽,为寻找DAI的分子标志物提供理论基础。方法将15只雄性SD大鼠随机分为DAI组(n=10)和对照组(n=5),采用MALDI-TOF-MS技术对两组大鼠脑干组织的蛋白或多肽表达谱进行检测,运用Clin Pro Tools 2.2分析软件筛选出具有差异的分子峰,应用遗传算法建立诊断模型。结果筛选出有差异的分子峰61个(P0.05),其中DAI组有9个分子峰下调,52个分子峰上调。筛选出5个分子峰建立诊断模型,交叉验证的特异性为96.14%、敏感性为95.98%,盲法验证结果的特异性为73.33%、敏感性为70.00%。结论应用MALDI-TOF-MS技术不仅能够建立特异性和敏感性较高的DAI诊断模型,还可以筛选差异蛋白或多肽,为DAI的法医学鉴定奠定理论基础。     

生物质谱技术在法医遗传学中的应用展望

随着基质辅助激光解吸电离质谱 (MALDI MS)和电喷雾离子化质谱 (ESI MS)两种技术的迅速发展 ,生物质谱技术已经成为生命科学研究中的重要工具。由于直接检测分子的质量 ,质谱技术同其他方法相比更加准确。生物质谱技术在核酸序列分析中具有快速性、微量化、和高通量的特点。利用MALDI TOF MS技术可以分析降解检材的SNP遗传标记 ,这在法医学中将有着重要的意义。本文对生物质谱技术的原理、进展、面临的困难以及在法医遗传学中的应用前景作了评述     

DESI-HRMS-HDI在添改字迹鉴定中的应用研究

目的 通过电喷雾解吸电离(DESI)—高分辨飞行时间质谱(HRMS-HDI)成像技术建立了 一种新型添改字迹鉴别技术.方法 对10支黑色签字笔书写的"11"被添改成"47"的90个样本用分子成像技术测试.结果 该技术可将84个被添改的原始字迹"11"以二维成像方式显现.结论 DESI-HRMS-HDI技术无需检材预处理...     

LC-MS~n分析新乌头碱在兔尿液中的代谢产物

乌头属(aconitum)中药临床应用广泛,治疗风湿痹痛等症疗效显著。但该属中药所含乌头类生物碱既是有效成分又是剧毒成分,其治疗量与中毒、致死量接近[1],因此服用过量、误服或投毒等都可引起乌头类生物碱中毒[2,3]。乌头碱中毒无特异性临床表现,且在体内迅速分解,给检测带来困难。在乌头属中药中新乌头碱(mesaconitine)多与乌头碱(aconi-tine)共存,且新乌头碱含量高于乌头碱。本文应用LC-MSn分析家兔灌服新乌头碱中毒死亡前尿液中的代谢产物,为检测新乌头碱的代谢产物提供新方法。1材料与方法1.1仪器与材料Finnigan公司LCQ液相色谱-质谱…     

基质辅助激光解吸飞行时间质谱分析生物样品中甲基苯丙胺

目的建立尿样和头发中甲基苯丙胺的基质辅助激光解吸飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS）分析方法。方法尿样采用液液提取,头发经0.1mol/L盐酸水解后采用液液提取,以碳纳米管为基质应用MALDI-TOF-MS法检测。结果尿样中甲基苯丙胺的最低检测限（LOD）为0.5μg/mL,线线范围为线性范围为0.5～100μg/mL（R2=0.9970）;毛发中甲基苯丙胺的最低检测限（LOD）为0.4ng/mg,线性范围为0.4～60ng/mg（R2=0.9976）,对送检案例中尿样和头发检材进行检测,效果良好。结论本方法适用于尿样和头发中甲基苯丙胺的分析,与传统气相色谱质谱联用和液相色谱-质谱联用相比,分析速度更快,适合大批量样品同时分析。     

苯二氮卓类新精神活性物质去氯依替唑仑的定性检验方法研究

目的建立苯二氮卓类新精神活性物质去氯依替唑仑的气相色谱-质谱(GC-MS)和超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF MS)定性检验方法。方法未知样品用甲醇和水提取,取上清液,采用GC-MS和UPLC-Q-TOF MS进行分析。结果经GC-MS检测,保留时间为17.73 min的未知组分的质谱碎片主要特征离子峰有m/z 279,308,239,252,225,77,126。经UPLC-Q-TOF MS检测,保留时间为4.781 min的未知组分的准分子离子峰为309.1173,碰撞诱导解离(CID)模式下二级质谱主要离子有m/z 280.0776,255.0952,240.0719,225.0604,206.0748。经缴获毒品分析科学工作组(Scientific Working Group for the Analysis of Seized Drugs,SWGDRUG)分析谱库检索和文献查询获得的信息资料进行比对,鉴定为去氯依替唑仑。结论该方法具有分析简便、快速的特点,可以用于实际案件的检测。     

短柄乌头急性中毒——乌头碱在大鼠体内分布的研究

本文用高效液相色谱法检测了大白鼠短柄乌头急性中毒后(LD_(50)剂量灌胃),乌头碱在心、肝、肾、血、脑内的含量分布。结果表明;体内乌头碱含量甚微或检不出。30例大鼠LD_(50)剂量灌胃后,16例2h内中毒死亡。其肝、肾内的乌头碱检出率明显高于心、血、脑,且中毒表现明显。另14例于2h整处死,其肝、肾、血中检出较高。16例2h内死亡组肝中乌头碱含量分析提示乌头碱在体内代谢很快。此结果为法医工作中乌头碱中毒案件调查、检材提取、毒物分析结果的评价提供了一定的依据。     

Age estimation methods using anthropological parameters on human teeth-(A0736)

The research was conducted on the 160 intact extracted human teeth with one and two roots of the known age and sex. The teeth were disinfected, dried and X-rayed. After that the section of the longitudinal cut through the teeth was performed in order to facilitate monitoring of all tissues and morphological characteristics of the teeth. The age was determined in three ways: Method 1 [G.Bang, E. Ramm, Determination of age in humans from root dentin transparency, Acta Odontol. Scand. 28 (1970) 3-35]--analysis of the translucency of the root dentine, Method 2 [S. Kvaal, T. Solheim, A non-destructive dental method for age estimation, J. Forensic Odonto-stomatol. 12 (1994) 6-11]--analysis of the root and the root canal from the X-ray, Method 3 [G. Johanson, Age determination from human teeth, Odontol. Revy. 22 (1971) 1-126]--analysis of six parameters on each teeth. All data were subject to the correlation and regression analysis which showed the following: all of the three applied methods were in the significant correlation with the real age, and the best of them proved to be Method 3 where the coefficient of correlation was 0.85, p<0.001. The teeth of the maxilla are more convenient for the age determination than the teeth of mandible. They are in the significant strong correlation with the known real age, and in Method 3, the coefficient of correlation is 0.78, p<0.001. Age determination of the teeth with two roots is in significant correlation with the known real age p<0.001 in relation to the determined age on the teeth with one root. The results show that sex too, is in significant correlation with the real age, p<0.001. In practice, the methods used and the results achieved in this research have been enabling the dental age estimation of human remains from mass graves after the 1991 war in Croatia.     

用引物Y_3,Y_4和DNA扩增技术作血液(痕)和毛根性别鉴定的研究

作者用引物Y_3、Y_4和DNA聚合酶链式反应(PCR)作微量人类血液(痕)和毛根的性别鉴定。扩增的靶序列位于Y染色体DNA特异3.4kb重复序列中,扩增产物为460bp。检材用量为:新鲜血液0.5μl、血痕纱纤维1mm、毛根单个。20例保存4个月的血痕与2例保存6年半的血痕性别判定结果均正确,无性别记载的保存9～11年的3例血痕显现了清晰的460bpY特异DNA扩增带。15例保存20天的自然脱落毛根性别判定结果均正确。本法省略了检材处理中的酚-氯仿抽提DNA等纯化步骤,既简化了实验操作,又减少了检验过程中外源DNA的污染机会和样品DNA的损耗,使这一性别鉴定方法更符合法医学实践的需要。     

Possibilities of DNA sex determination in hair roots

Species- and sex-determination on hair roots were simultaneously performed using extracted DNA and a human Y chromosomespecific probe. (pH Y 2.1) After Southern hybridization, Hae III-digested DNA fragments were detected by non-radioactive digoxigenin detection system. DNA was extracted from one to five freshly plucked hair roots. The specific 2.12 kb fragment was successfully detected in male DNA samples from a single hair root. A positive identification of female DNA was more difficult. The hair root DNA was revealed to be stable at room temperature for at least 2 weeks (examination time) and produced the same specific band pattern as the DNA of fresh hair roots. In the blind tests with DNA samples from randomly plucked one to four hair roots, the rate of successful sex-determination was 95.8% on male samples (23 out of 24 samples) and 25% on female samples (4 out of 16 samples).     

当前医疗损害鉴定制度存在问题与对策

随着《侵权责任法》的实施,医疗纠纷司法鉴定将在医疗损害鉴定中起着越来越重要的作用。我国的医疗损害鉴定制度经历了一系列的制度变迁过程。但是,基于医疗的专业性、复杂性和技术性,医疗损害的鉴定不是一般法医能胜任的鉴定工作。医疗事故技术鉴定制度仍是一项可利用的制度资源。当前医疗损害鉴定制度的构建,应警惕形式意义上的鉴定公正带来实质意义上的鉴定不公正;警惕法律对患者的保护带来医疗对患者的伤害。建议对医疗事故技术鉴定制度进行改造,使之成为类似于美国的审前审查机制,或者采取医疗损害司法鉴定特别许可制度,以完善我国的医疗损害鉴定制度。     

Forensic aspects of maxillofacial radiology

Radiology has been used extensively in conventional dental identification, anatomically based identification and identification using maxillofacial skeletal landmarks such as the frontal sinus. Examples of these are well documented in the literature. The purpose of this paper was to revisit the methods where radiographic methods may be used to determine identity using the teeth, the root structures and the frontal sinuses. Additionally suggestions are offered for management of radiography in mass disasters and cases where age determination is required. Computer assisted tomography can be used in the assessment of the degree of fit of a weapon to a wound in cases of blunt force skull injury and plane films can assist in depicting the pattern of post mortem skull fractures. Micro-computed tomography has been used in matching weapons to wounds in sharp-force injury cases. The radiologist's role in cases of civil litigation and fraud is discussed and case examples are given. There are gaps in the science where radiological methods are used. The author offers several suggestions for possible research projects to close some of these gaps.     

Analysis of rosin and modified rosin esters in adhesives by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The optimum method for analyzing glyceryl rosinate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. The performances of dithranol, anthracene, and 2,5-dihydroxybenzoic acid, pyrene, trans,trans-1,4-diphenyl-1,3-butadiene, 9-nitroanthracene, indole-3-acrylic acid and retinoic acid as a matrix agent, and those of sodium trifluoroacetate (NaTFA) and silver trifluoroacetate (AgTFA) as a cationization agent were measured. Dithranol showed higher signal to noise ratio and lower base-line, and AgTFA showed better performance than NaTFA. In this study, however, NaTFA was chosen since the ester peaks with high signal to noise ratios were observed, and the spectra of sodium-cationized molecules are simpler than those of silver-cationized molecules. Rosin esters and modified rosin esters in 22 rubber-based pressure sensitive adhesives and a paper-cement were analyzed. Glyceryl rosinate, glyceryl disproportionated rosinate, pentaerythrityl rosinate and pentaerythrityl hydrogenated rosinate were easily detected despite the fact that rubber-based adhesives are complex mixtures of elastomers, tackifiers, antioxidants and other additives. The detection limit of this method was studied and 2% of glyceryl rosinate in a rubber-based pressure sensitive adhesive was detected. It has been proved that MALDI-TOF-MS is a useful analytical method to analyze rosin and modified rosin esters in adhesives.     

Postmortem microscopic changes observed at the human head hair proximal end

Only two types of human hair roots (proximal ends) derived from decomposing scalps are reported in the literature. The most common representation of the putrid root includes a postmortem dark root band in published photomicrographs. In this study, 22 cases were reviewed in which there was reliable time of death documentation from medical investigator reports. A review of these cases finds that the most common putrid hair proximal end change does not contain the postmortem root band. Four primary types of hair proximal end postmortem change were identified. This study finds no correlation of time of death with scalp hair proximal end decomposition. In addition two examples are presented that suggest that hair roots do not decompose after fresh removal from the scalp and exposure to the outside elements.     

DNA identification in mass fatality incidents

DNA identification has become an important aspect of mass fatality management as well as in other instances of difficult identification of human remains. Most large mass fatality incidents will require DNA identification. Medical examiners should prepare for such potential eventuality. Whether DNA is tested, in mass fatality incidents, DNA specimens should be obtained from remains as well as from next-of-kin for potential testing. DNA identification is neither as slow nor as expensive relative to the overall fatality management as is commonly assumed. This article sought to provide medical examiners with a framework for DNA identification in mass fatality incidents.