XPG基因表达用于法医学年龄推断

目的探讨着色性干皮病G组(xeroderma pigmentosum group G,XPG)基因在不同年龄段健康汉族人群中的表达情况,分析XPG mRNA和蛋白表达量与年龄之间的相关性,以期为法医学年龄推断提供新的分子生物学指标。方法收集150名不同年龄段健康汉族人的外周血样,采用TRIzol法提取外周血单个核细胞(peripheral blood mononuclear cell,PBMC)总RNA,通过实时荧光定量PCR检测XPG mRNA在PBMC的相对表达量,酶联免疫吸附试验检测XPG蛋白在血浆中的表达量。结果 XPG mRNA及其蛋白表达量在≤18岁组与19~45岁组之间、≤18岁组与≥46岁组之间的差异均有统计学意义(P0.05),但19~45岁组与≥46岁组之间的差异无统计学意义(P0.05)。XPG mRNA和蛋白表达量均无性别差异(P0.05)。结论 XPG mRNA在PBMC的相对表达量在低龄段内随年龄增加而下降,其血浆中蛋白随年龄增加而升高;XPG基因有望成为法医学年龄推断的新型指标之一。     

大鼠骨骼肌挫伤后Fzd2表达与损伤时间的关系

目的检测大鼠骨骼肌挫伤后卷曲受体蛋白2(frizzled-2,Fzd2)mRNA及其蛋白表达量与损伤时间的关系,探讨其是否可以作为损伤时间推断的指标。方法利用RT-qPCR和Western印迹法检测正常对照组及损伤后4~48 h每4 h大鼠骨骼肌中Fzd2 mRNA和蛋白水平的表达量。结果 Fzd2 mRNA在损伤后24 h、36 h、40 h相对表达量增高,24 h组表达量达正常对照组的两倍(P0.05);而Fzd2蛋白在损伤后相对表达量变化不明显(P0.05)。结论大鼠骨骼肌损伤后Fzd2 mRNA在一定时间内的表达变化情况可以作为多指标联合推断损伤时间的依据。     

9-CpG年龄推断在不同海拔地域和性别人群中的验证及优化

目的本研究旨在对早先建立的9-CpG年龄推断模型在不同海拔地域和不同性别人群中年龄推断的适用性进行验证及优化,提升年龄推断的准确性,并研究检测体系的灵敏度。方法采集367份不同海拔地域、不同性别个体的血液样本,使用EpiTYPER技术平台检测9个CpG位点的甲基化值,利用年龄推断模型预测样本提供者的年龄,综合评估年龄预测的准确性。结果所有测试样本均获得相对准确的年龄预测值(N=367, MAD=4.0岁,R~2=0.83)。平原女性样本(N=40),MAD=3.63岁,R~2=0.88。平原男性样本(N=123)MAD=3.42岁,R~2=0.83;其中随机选取平原男性样本(N=40)MAD=3.39岁,R~2=0.83;高原男性样本(N=204)MAD=4.42岁,R~2=0.86。加入平原与高原变量,新建立8-CpG年龄推断模型,MAD=2.90岁,R~2=0.86(验证集MAD=3.31岁,R~2=0.83)。灵敏度分析中转化前最低DNA用量为250ng(转化后最低DNA用量约为8ng)可获得9个位点完整甲基化信息。结论本研究证明了9-CpG年龄推断模型在不同性别间无显著差异。同一模型应该考虑高原和平原人群血液样本DNA甲基化差异。加入平原与高原变量,新建立8-CpG年龄推断模型可提高高原地区人群年龄预测准确性。     

血清蛋白芯片表达谱在DVT疾病法医鉴定中的应用研究

目的利用蛋白芯片技术获取人体和大鼠深静脉血栓(deep venous thrombosis, DVT)组与对照组血清蛋白表达谱的蛋白峰差异,探讨蛋白表达谱与深静脉血栓形成的相关性,为DVT临床和法医学诊断提供依据。方法采用蛋白芯片技术检测DVT患者和正常人(n=10)的血清样本,获得血清蛋白芯片表达谱。建立标准化SD大鼠下腔静脉血栓模型,大鼠被随机分为DVT组和对照组(n=10),蛋白芯片技术检测获得各组血清蛋白表达谱;采用SIMCA-P 14.1软件的正交偏最小二乘判别(orthogonal partial least square-discriminant analysis,OPLS-DA)模型和Python软件的随机森林(Random forest, RF)算法模型对人体和大鼠数据进行判别分析。结果 DVT患者和正常人血清蛋白表达峰存在差异,OPLS-DA模型对DVT组和对照组准确判别,R~2X=0.631,R~2Y=0.928,Q~2=0.796;动物模型DVT组与对照组的血清蛋白表达峰亦存在差异,OPLS-DA模型可以将DVT组和对照组准确判别,R~2X=0.735,R~2Y=0.953,Q~2=0.900;人体和动物模型血清中共有8个分子量相似的血清蛋白峰,RF算法模型准确率为100%,人体预测准确率为65%。结论蛋白芯片技术可以快速获取DVT人体和大鼠血清蛋白表达谱,建立OPLS-DA模型和RF模型能对DVT组和对照组进行分类预测分析,为DVT疾病诊断和法医学鉴定提供新的辅助方法。     

AChE、BChE、PON-1和FOS mRNA在甲拌磷急性中毒致死大鼠脏器中的表达

目的通过对甲拌磷急性中毒致死大鼠脏器中乙酰胆碱(ACh E)、丁酰胆碱(BCh E)、对氧磷酶1(PON-1)和原癌基因蛋白(FOS)m RNA的表达研究,筛选出表达变化有统计学意义的脏器和中毒相关基因,探讨其在死亡原因推断中的价值。方法健康成年雌性SD大鼠,随机分为空白对照组和急性中毒死亡组,分别取心、肝、肺、脑和肠,提取总RNA,加入目的基因ACh E、BCh E、PON-1、FOS和内标基因β-肌动蛋白(β-actin)引物逆转录成c DNA后,利用实时荧光定量PCR技术(RT-PCR)检测各组c DNA的相对表达量的变化,并对各组结果进行统计学分析。结果肝脏中ACh E、BCh E和FOS m RNA的表达均上调(P0.05),ACh E m RNA在肺和脑中的表达均下调(P0.05)。结论肝脏和ACh E分别可以作为甲拌磷急性中毒致死的靶器官和基因表达标志,能为今后急性甲拌磷中毒的临床诊断与死亡推断提供依据。     

人外周血FoxO3a mRNA表达随龄变化规律的初步研究

目的评估人外周血中FoxO3a mRNA表达的随龄变化规律,为法医学个体年龄推断寻找新的分子指标。方法采集80个健康无关个体血样,按年龄分为8组:≤9岁、10～19岁、20～29岁、30～39岁、40～49岁、50～59岁、60～69岁、≥70岁组,每组10人。提取总RNA后,用实时定量RT-PCR技术检测FoxO3a mRNA的表达水平。结果人外周血FoxO3a mRNA表达随年龄的增长而逐渐增加,具有良好的相关性(r=0.841,P0.01),以年龄分组为自变量,FoxO3a mRNA表达水平为因变量,可得拟合曲线Y=0.001e0.418x。结论人外周血中FoxO3a mRNA表达具有较强的年龄特征,有望成为年龄推断的有用指标之一。     

PTEN在冠心病心肌组织中的表达

目的观察冠状动脉粥样硬化性心脏病(冠心病)患者心肌组织中人第10号染色体缺失的磷酸酶与张力蛋白同源基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)的表达,探讨PTEN的表达与冠心病发生发展的相关性。方法选取经病理学确诊为冠心病死亡案例16例作为冠心病组,非心肌病变死亡案例19例作为对照组,采用免疫组织化学以及实时荧光定量PCR法检测心肌组织中PTEN蛋白及其m RNA的表达。结果冠心病组PTEN蛋白的表达量低于对照组(P0.05);与对照组相比,冠心病组PTEN m RNA表达的差异无统计学意义(P0.05)。结论 PTEN可能与冠心病的发生发展有关。     

死亡人体组织中rRNA亚基的稳定性及影响因素评价

目的探讨人尸体组织中rRNA亚基表达水平的稳定性及影响因素。方法选取3例成人、3例婴幼儿大脑皮质和脾组织,在尸检即刻、48、72、96、120、144、168、192、216、240h,采用实时荧光定量RT-PCR法检测5s、5.8s、18s、28s rRNA的表达水平。结果两个年龄组两种组织中4种rRNA亚基表达量相比较均显示有差异(P0.05)。同种组织比较:脑组织中两个年龄组最后一个时间点与第一时间点4种rRNA亚基的表达量均有差异(P0.05),其他各时间点与第一时间点无显著性差异(P0.05);脾组织中婴幼儿5s rRNA在最后一个时间点,其余3种rRNA亚基最后两个时间点与第一时间点比较有差异(P0.05);成人5s rRNA最后一个时间点,5.8s rRNA亚基最后两个时间与第一时间点比较差异(P0.05),其余两种亚基各时间点与第一时间点相比较均无差异(P0.05)。结论尸体组织中rRNA亚基表达稳定性好,适于作为晚期死亡时间推断的内参基因使用,但需注意指标、组织和年龄段差异性。     

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。     

miR-17及其靶基因HIF-1α、STAT3在心源性猝死心肌组织中的表达及其意义

目的观察微小RNA17 (miR-17)与HIF-1α、STAT3在心脏性猝死(SCD)心肌组织中的表达水平,并探讨其意义。方法选择因心血管系统疾病猝死的20例心脏标本作为SCD组;选取20例死因为非心脏疾患的心脏标本作为对照组。应用免疫组织化学染色方法分别检测两组心肌组织中HIF-1α和STAT3表达,采用荧光定量PCR方法检测两组心肌组中miR-17、HIF-1α和STAT3的表达。结果免疫组化染色结果显示,两组心肌组织中HIF-1α、STAT3的表达比较,差异均有统计学意义(Z=-3.636、-3.552,P 0.01);两组心肌组织中HIF-1α与STAT3的表达水平呈负相关(r=-0.996,P 0.05)。荧光定量PCR方法检测结果显示,SCD组心肌组织中miR-17与HIF-1α相对表达量呈正相关(r=0.706, P 0.05),miR-17与STAT3相对表达量呈负相关(r=-0.730, P 0.05)。结论 SCD心肌组织中miR-17、HIF-1α高表达,STAT3低表达,miR-17、HIF-1α和STAT3联合应用有望作为诊断SCD较为客观的指标。     

A validation study of mRNA markers for skin cell identification

The objectives of this study were to develop and optimize a multiplex of three skin specific gene markers; loricrin (LOR), corneodesmosin (CDSN) and keratin 9 (KRT9) and 1 house-keeping marker, β-actin (ACTB) using an endpoint PCR assay to analyze expression data from a range of relevant samples. Marker specificity and suitability were evaluated for their inclusion in future forensic casework. The presence of the three skin mRNA markers was successfully confirmed from swabs of human skin obtained from 20 individuals at each of 6 different body sites (forehead, neck, arm, palm, leg and sole). Significant variation was observed in the relative expression of the three genes across the body sites, with some individuals consistently failing to express one or more of the targets. Inter-individual variation was also evident. Accordingly, these markers must be used with caution in the identification of skin in forensic samples.     

Age estimation using cytochrome c oxidase activity analysis

Recently, in the field of forensic medicine the number of unidentified cadavers has increased due to mass disasters and international terrorism. In addition to the conventional anthropological methods, a simple and precise method to estimate the age of these unidentified cadavers to assist in the personal identification is necessary. On the other hand, many researchers have reported that mitochondrial respiratory activity decreases with aging because of the production of reactive oxidative species in the process of ATP generation. Therefore, it may be possible for us to estimate human age by analyzing mitochondrial activity. In this report we attempted to analyze cytochrome c oxidase (CCO) activity, and the amount of protein and mRNA expression in various aged rats. The age of human subjects was estimated through the analysis of human CCO activity from 28 actual forensic cases. The CCO activity, the amount of protein and the mRNA expression increased in the 3rd week and decreased afterwards in rats. Furthermore, human CCO activity was decreased gradually with aging. Therefore, CCO activity analysis may be useful for age estimation in forensic cases.     

眼球突出度测量方法的比较及其法医学意义

目的比较Hertel突眼度计及CT测量两种眼球突出度测量方法的差异,探讨其法医学应用价值。方法选取56例眼部无损伤及疾病的正常人作为正常组,在确定规范的CT影像工作站上测量眼球突出度绝对值,并比较双眼眼球突出度有无差异。选取47例单侧眼眶骨折的伤者,根据两眼有无眼眶骨折,将其分为伤眼组及健眼组,分析Hertel突眼度计及CT测量两种方法测得同一眼眼球突出度绝对值的差异以及同一受检者双眼眼球突出度相对差值的差异。结果 CT法测量正常人双眼间眼球突出度差异无统计学意义。健眼组CT法测得眼球突出度绝对值为(16.66±5.41)mm,Hertel突眼度计测得眼球突出度绝对值为(16.16±4.45)mm,两组测量结果之间差异无统计学意义(P0.05)。伤眼组应用两种方法测量的眼球突出度绝对值之间差异具有统计学意义(P0.05)。两种测量方法测得的眼球突出度相对差值在伤眼和健眼组之间差异无统计学意义(P0.05)。结论 CT法与突眼度计法具有较好的一致性,可以运用于法医学鉴定实践。     

Evaluation of vaginal mRNA markers in women from different age groups: A GeFI collaborative study

Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Although it is known that vagina undergoes characteristic age-related morphological and physiological changes over a lifetime, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups.In this collaborative study involving ten GeFI (Italian working group of ISFG) laboratories, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of vaginal swabs obtained from female volunteer donors in their reproductive years (n = 84) and postmenopausa (n = 55).Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa “observed” in the tested sample according to scoring protocol) b) average peak height ratios between vaginal-specific markers within mRNA profiling replicates.Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated.     

Cutaneous (tPA) and Skeletal (TnI) mRNA as Markers of Aging in Contused Wound

Wound age estimation is one of the most important forensic aspects. Troponin I (TnI) and many cytokines, for example, tissue plasminogen activator (tPA), are involved in wound inflammation and healing. Skeletal (TnI) and cutaneous (tPA) mRNA was detected using real‐time PCR in 25 female albino rats. They were divided into 5 groups: control and 4 injured groups. Injured groups were sacrificed 1, 6, 24, and 30 h after inflicting contused wound. The expression levels of cutaneous (tPA) were decreased significantly at 1, 6, and 30 h after contusion (71.7%, 30.7 and 16.9%), while the expression levels of skeletal (TnI) were increased significantly at 1 and 6 h post‐traumatic, then they gradually decreased until reaching normal levels at 24 h and assumed significantly lower levels at 30 h postcontusion. These results suggested that the determination of cutaneous (tPA) and skeletal (TnI) mRNA levels was useful for wound age estimation.     

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.     

缺血修饰白蛋白在急性缺血性心脏病中的法医学应用

目的探讨心包液中缺血修饰白蛋白(ischemia modified albumin,IMA)水平在心脏性猝死诊断中的应用价值及法医学意义。方法应用白蛋白-钴离子结合法检测急性缺血性心脏病组(36例)、急性心肌梗死组(6例)、心肌病组(4例)、对照组(15例)死者的心包液中IMA水平,比较各组之间IMA水平的差异。通过ROC曲线获得最佳IMA水平的截断值以及区分急性缺血性心脏病和对照组的敏感度和特异度。结果急性缺血性心脏病组心包液中IMA水平高于对照组(P0.05);而与急性心肌梗死组、心肌病组心包液中IMA水平比较,差异均无统计学意义(P0.05)。应用ROC曲线分析得出识别急性心肌缺血的IMA的截断值为40.65 U/m L,其诊断急性心肌缺血的敏感度为60.0%,特异度为80.5%。结论心包液中IMA有望作为诊断急性心肌缺血的参考指标,为心脏性猝死的法医学诊断提供客观依据。     

Time‐dependent Expression of MMP‐2 and TIMP‐2 after Rats Skeletal Muscle Contusion and Their Application to Determine Wound Age

The ability to determine vitality and estimate the survival period after a wound is critical in routine forensic practice. The mRNA levels of MMP‐2 and TIMP‐2 were examined using quantitative real‐time RT‐PCR to determine the age of a wound. Furthermore, the colocalization of them with Macrophage Marker, respectively, was detected by double immunofluorescence, and a standardized rat model of skeletal muscle contusion was established. In the antemortem contused groups, a large number of macrophages showed positive staining for MMP‐2 and TIMP‐2, and the expression of MMP‐2 and TIMP‐2 mRNA increased sharply at 3 days postinjury, with relative quantities of 5.75 and 2.98. No samples in the other groups showed relative quantities of >5.75 and 2.98; therefore, relative quantities exceeding 5.75 and 2.98 were strongly indicated 3 days after contusion. In addition, there was a significant decrease in the relative quantity in the postmortem contused groups, indicating that they were useful for diagnosing vitality.