外伤性癫痫灶中泛素与泛素激活酶的定量分析

目的 定量测定人体外伤性癫痫灶中泛素(ubiquitin,Ub)和泛素激活酶(ubiquitin-activating enzyme,UbE1)的表达,研究泛素蛋白酶体系在外伤性癫痫(post-traumatic epilepsy,PTE)发病机制中所起的作用,探索其在PTE鉴定中的应用价值. 方法 分别收集颅脑外伤致癫痫患者脑癫痫灶组织(外伤性癫痫组)、非颅脑外伤性癫痫灶组织(非外伤性癫痫组)和交通事故死亡者正常脑组织(非癫痫组)各15例,应用RT-PCR、Western印迹技术检测3组中Ub和UbE1的含量,并对所得数据进行统计学分析. 结果 RTPCR、Western印迹技术测定显示Ub和UbE1的mRNA和蛋白质在3组中含量为外伤性癫痫组>非外伤性癫痫组>非癫痫组,两两比较,组间差异具有统计学意义(P<0.05).结论 Ub和UbE1在癫痫灶组织中表达增加,且在外伤性癫痫灶组织中增加最为明显.推断泛素蛋白酶体系的活化是PTE神经元病理改变的重要机制之一.     

外伤后大脑癫痫灶中SLIT-2的表达变化

目的通过测定人体大脑外伤后癫痫灶中轴突再生调控因子SLIT-2的表达情况,探讨其在外伤后癫痫鉴定中的应用意义。方法 75例人大脑皮质样本分为3组:①实验组:为脑外伤后癫痫患者(30例),②对照组1:为非外伤性癫痫患者(30例),③对照组2:为车祸致颅脑损伤死亡者(15例)。实验组和对照组1样本来源于癫痫手术所切除的脑皮质(患者或其近亲属知情,自愿提供);对照组2样本来源于尸体解剖的大脑皮质。通过HE染色、免疫组化染色、westernblot和图像分析技术,观察、检测上述样本SLIT-2的表达情况,所得数据经统计学处理分析。结果 SLIT-2在实验组与对照组1癫痫灶皮质的神经细胞膜上呈阳性表达,在对照组2中仅有微量表达。实验组SLIT-2的表达高于对照组1,两组间的差异具有显著性意义(P0.05);显著高于对照组2,两组间的差异具有显著性意义(P0.05)。结论 SLIT-2有望作为区别外伤后癫痫与非外伤性癫痫的病理指标之一,通过进一步研究,逐步运用到法医学的鉴定实践之中,为外伤后癫痫的法医病理学鉴定提供客观依据。     

外伤性癫痫患者癫痫灶中GABABR1和GluR1的表达

目的探讨外伤性癫痫患者颞叶癫痫灶组织γ-氨基丁酸B受体亚单位（gamma-aminobutyric acid B receptorsubunit,GABABR1）和谷氨酸受体亚单位（glutamic acid receptor subunit,GluR1）的表达变化,以期为阐释外伤性癫痫发生机制和法医学鉴定提供依据。方法收集15例外伤性癫痫患者脑皮质（外伤性癫痫组）1、5例非外性癫痫患者脑皮质（非外伤性癫痫组）、15例交通事故死亡者脑皮质（交通事故死亡对照组）,运用荧光免疫组化技术和RT-PCR技术观察GABABR1和GluR1的表达水平。结果外伤性癫痫组GABAB R1表达水平明显低于对照组,但高于非外伤性癫痫组;外伤性癫痫组GluR1表达水平明显高于对照组,但低于非外伤性癫痫组。结论 GABABR1和GluR1的表达水平的变化在外伤性癫痫病的发病过程中起着一定的作用,可以作为区分外伤性癫痫与非外伤性癫痫的病变化指标之一。     

外伤性癫痫病灶中caspase-10的表达

【摘要】目的观察凋亡基因caspase-10存外伤性癫痫病灶中的表达变化,探讨其作用机制。方法收集外伤性、非外性癫痫患者手术切除的恼皮质及交通事故死亡者脑皮质各15例,运用RT-PCR技术观察比较各实验组凋亡基因caspase-10表达水平,采用荧光免疫组织化学染色技术观察各实验组凋亡基因对应蛋白的表达情况。结果外伤性癫痫组（：aspase-10基冈和蛋白表达水平比非外伤性癫痫组和交通事故死亡组高,差异有统计学意义（P〈0．05）;非外伤性癫痫组caspase-10基因和蛋白表达水平亦显著高于交通事故死亡组（P〈0．05）。结论凋亡基因caspase－10能促进神经元凋亡,对外伤性癫痫的发病过程有促进作用。     

外伤性癫痫灶组织的基因表达谱分析

目的比较外伤性和非外伤性癫痫灶组织的基因表达谱,探索外伤性癫痫的发病机制,为相关鉴定提供依据。方法收集外伤性和非外伤性癫痫灶组织样本各15例。分别提取总RNA,经逆转录标记不同的探针与人类cDNA表达谱芯片杂交,获得扫描图像,分析信号的强度和比值,对实验数据按照RNA微阵列分析法标准化,对差异表达基因进行聚类分析。结果两组样本中共有超过2 990条基因明显表达（表达超过背景2～5倍以上）。以30%样本中的表达调节水平大于1.5倍作为差异基因筛选标准,与非外伤性癫痫组比较,外伤性癫痫组样本中存在显著性表达差异的基因有192个,其中表达上调的有115个,表达下调的有77个。结论采用基因表达谱芯片技术可以有效地识别外伤性癫痫和非外伤性癫痫的差异表达基因,并可用于外伤性癫痫发病机制和分子分型的研究。     

Bcl-2蛋白在外伤性癫痫病灶中的表达

目的观察Bcl-2蛋白在外伤性癫痫病灶中的表达变化,探讨其作用机制。方法收集外伤性癫痫、非外性癫痫患者及交通事故死亡者脑皮质各15例,采用免疫组织化学和荧光免疫组织化学技术,观察各实验组Bcl-2蛋白表达水平,测定阳性表达的灰度值,数据采用SPSS 12.0软件进行统计分析。结果外伤性及非外伤性癫痫组Bcl-2蛋白表达水平均高于交通事故死亡组（P〈0.05）;而非外伤性癫痫组表达明显高于外伤性癫痫组,差异亦具有显著统计学意义（P〈0.05）。结论 Bcl-2蛋白能抑制神经元凋亡,对外伤性癫痫的发病过程具有抑制作用。     

大鼠脑外伤后大脑皮质脑红蛋白的表达

目的探讨脑外伤后大脑脑红蛋白（Ngb）的时空变化规律及其法医学意义。方法实验大鼠分为打击后15min、30min、1h、3h、6h、12h、1d、2d 8组,并设正常对照组。采用硬膜外局部打击方法复制SD大鼠外伤性局灶性脑挫伤模型,在各组预设时间点断颈处死,并按损伤区、打击周边缘区和非损伤区皮质分别取材,采用半定量反转录聚合酶链式反应（半定量RT-PCR）方法和图像分析技术观测脑外伤后Ngb mRNA表达的变化规律,并对所测数据进行统计学分析。结果大鼠脑外伤后脑损伤区Ngb mRNA表达在伤后15min开始下降,24h降至最低水平;打击周边损伤区NgbmRNA表达于伤后15min即可见表达增高,12h增高达峰值;非损伤区皮质Ngb mRNA表达强于正常皮质。结论 Ngb在脑损伤的急性阶段对神经细胞有保护作用。     

大鼠外伤性癫痫模型皮层SPP1蛋白的表达

目的检测分泌型焦磷酸蛋白1 (Secreted phosphoprotein 1,SPP1)在外伤性癫痫(post-traumaticepilepsy,PTE)大鼠皮层脑组织中的表达变化,探讨其与外伤性癫痫的关联。方法随机将18只大鼠分为正常对照组、生理盐水组和实验组。建立外伤性癫痫模型。采用免疫组化法检测GFAP蛋白,采用Western blot法检测SPP1蛋白。结果大鼠行为学平均评分为4分;EEG显示明显痫性放电;免疫组化结果显示,实验组大鼠脑组织GFAP阳性细胞与正常对照组和生理盐水组比较,显著增高(P <0.05);生理盐水组GFAP阳性细胞与正常对照组比较,差异不具有统计学意义(P> 0.05),以上结果综合表明成功建立了大鼠外伤性癫痫模型。Western blot结果显示,实验组SPP1蛋白表达量较正常对照组和生理盐水组显著增高(P <0.01);生理盐水组SPP1蛋白表达量较正常对照组不显著,差异无统计学意义(P> 0.05)。结论 SPP1在外伤性癫痫大鼠皮层脑组织中表达显著上调,外伤性癫痫的发生机制可能与SPP1的表达异常增高有关。     

胶原纤维、Ⅳ型胶原及CD34诊断外伤性迟发性脾破裂

外伤性迟发性脾破裂的鉴定难点在于准确判断脾破裂与腹部受到外力事件之间是否存在直接延续的因果关系。经典的组织病理检查的结果虽然具有十分重要的作用,但存在假阴性的可能,因此需要寻找其他辅助诊断指标。本文通过一例明确的外伤性迟发性脾破裂病例,在损伤及正常脾组织中,利用HE染色、Masson染色、免疫组化染色,针对胶原纤维、Ⅳ型胶原、CD34三个指标进行组织形态学对比,并量化免疫组化结果。HE染色发现出血灶周围已经形成了肉芽组织;Masson染色发现出血区周围的胶原纤维密度明显高于远端正常组织,且形成包膜状纤维结构;免疫组化结果发现,出血区域的Ⅳ型胶原及CD34相比正常对照组织均呈强阳性,平均光密度具有统计学差异。在判断脾损伤后经过时间方面,HE染色判断为7~14d,Masson染色判断其大于6d,免疫组化判断为7~14d,三者结果基本一致。说明HE染色、Masson染色以及Ⅳ型胶原和CD34免疫组化染色均可获得较为准确的结果。与HE染色相比,胶原纤维、Ⅳ型胶原、CD34更为直观并易于识别判断,这为今后此类案件的鉴定提供了新的辅助指标。     

GFAP及NSE阳性表达与早期PMI的相关性

目的通过对神经元及胶质细胞进行HE染色、免疫组化染色,观察GFAP/NSE的阳性表达,探讨推断早期死亡时间(postmortem interval,PMI)的可行性。方法健康白兔36只,分为6组,处死后对兔脑组织取材并进行HE染色及相应免疫组化染色,观察10℃条件下镜下0~24h的变化,分别测定0h、1h、4h、8h、16h、24h阳性细胞的数目和OD值。结果在10℃条件下,HE染色在0~24h内无明显变化;脑组织AST中GFAP的阳性表达在24h内无统计学意义;神经元NSE的表达逐渐降低,并具有统计学意义。结论在10℃条件下NSE的阳性表达与PMI具有相关性,可以用于早期PMI推断;GFAP的阳性表达与早期PMI无相关性。     

肺动脉血栓栓塞猝死23例法医病理学分析

目的分析肺动脉血栓栓塞(pulmonary thromboembolism,PTE)猝死的法医病理学特点,探讨血栓的演变过程及外伤等邻近事件与PTE猝死的因果关系判定方法。方法对四川华西法医学鉴定中心1998—2008年23例PTE猝死案件进行回顾分析。结果PTE猝死合并外伤、手术、制动等危险因素,其中外伤12例,手术21例;院内发生22例,以伤后住院时间1～2周及术后1周多见。PTE中17例为反复性栓子栓塞,余为一次性栓塞。栓子来源于下肢深静脉系统16例,左侧多见。结论明确栓子的来源、外伤及外科手术等邻近事件对于确定PTE猝死是至关重要的。     

Urinary excretion profiles of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol: a Delta9-THC-COOH to creatinine ratio study #2

Subjects with a history of chronic marijuana use were screened for cannabinoids in urine specimens with the EMIT((R)) II Plus cannabinoids assay with a cut-off value of 50 ng/ml. All presumptively positive specimens were submitted for confirmatory analysis for the major urinary cannabinoid metabolite (Delta(9)-THC-COOH) by GC-MS with a cut-off value of 15 ng/ml. Creatinine was analyzed in each specimen as an index of dilution. Huestis and Cone [J. Anal. Toxicol. 22 (1998) 445] reported that serial monitoring of Delta(9)-THC-COOH to creatinine ratios in paired urine specimens collected at least 24h apart could differentiate new drug use from residual Delta(9)-THC-COOH excretion. The best accuracy (85.4%) for predicting new marijuana use was a Delta(9)-THC-COOH/creatinine ratio > or =0.5 (dividing the Delta(9)-THC-COOH to creatinine ratio of specimen 2 by the specimen 1 ratio). In a previous study in this laboratory [J. Anal. Toxicol. 23 (1999) 531], urine specimens were collected from chronic marijuana users at least 24h apart and dilute urine specimens (creatinine values <2.2 micromol/l) were excluded from the data analysis. The objective of the present study was to determine whether creatinine corrected urine specimens positive for cannabinoids could differentiate new marijuana use from the excretion of residual Delta(9)-THC-COOH in chronic users of marijuana based on the Huestis 0.5 ratio. Urine specimens (N=946) were collected from 37 individuals with at least 48h between collections. All urine specimens were included in the data review irrespective of creatinine concentration. The mean urinary Delta(9)-THC-COOH concentration was 302.4 ng/ml, mean Delta(9)-THC-COOH/creatinine ratio (ng/ml Delta(9)-THC-COOH/(mmol/l) creatinine) was 29.3 and the Huestis ratio calculation indicated new drug use in 83% of all sequentially paired urine specimens. The data were sub-divided into three groups (A-C) based on the mean Delta(9)-THC-COOH/creatinine values. Interindividual Delta(9)-THC-COOH/creatinine mean values ranged from 2.2 to 13.8 in group A (264 specimens, N=15 subjects) where 80.7% of paired specimens indicated new drug use. In group B, mean Delta(9)-THC-COOH/creatinine values ranged from 15.3 to 37.8 in 444 specimens (N=14 subjects) and 83.3% of paired specimens indicated new drug use. In group C, individual mean Delta(9)-THC-COOH/creatinine values were >40.1 (41.3-132.5) in 238 urine specimens (N=8 subjects) and 85.3% of paired urine specimens indicated new marijuana use. Correcting Delta(9)-THC-COOH excretion for urinary dilution and comparing Delta(9)-THC-COOH/creatinine concentration ratios of sequentially paired specimens (collected at least 48h apart) provided an objective indicator of new marijuana use in this population.     

The association between body mass index and pulmonary thromboembolism in an autopsy population

Abstract: To evaluate the association between obesity and pulmonary thromboembolism (PTE) in a forensic context, 160 autopsy cases of fatal PTE were compared with age‐ and gender‐matched controls. The mean age of cases was 66 years (range 26–98 years; M/F 74:86). The mean body mass index (BMI) of cases with PTE was 30.88 (range 14.95–79.51), which was significantly higher than in the controls (mean BMI = 25.33; range 12.49–61.84) (p < 0.0001). Comparing the group with PTE with controls showed that five (3.1%) compared to 20 (12.5%) were underweight, 39 (24.4%) compared to 67 (41.88%) were of normal weight, 49 (30.63%) compared to 43 (26.88%) were overweight, 43 (26.88%) compared to 24 (15%) were obese, and 24 (15.0%) compared to six (3.75%) were morbidly obese. In each category of above‐normal BMIs, there were significantly greater numbers in the groups with PTE: overweight (p < 0.01), obese (p < 0.001), and morbidly obese (p < 0.0001).     

Is There a Relationship Between Bladder Outlet Obstruction due to Benign Prostatic Hyperplasia and Pulmonary Thromboembolism?

Benign prostatic hyperplasia with chronic bladder outlet obstruction has been associated with deep venous thrombosis (DVT) and fatal pulmonary thromboembolism (PTE). To evaluate this further, 60 autopsy cases of men with PTE were compared with 60 age-matched controls. The criteria for outlet obstruction were macroscopic prostatic enlargement with bladder trabeculation and benign prostatic hyperplasia on microscopy. Ten of the 60 men (16.7%) with fatal PTE had evidence of bladder outlet obstruction (age 57-78 years; mean 71.4 years). Of the 60 controls, 12 had evidence of bladder outlet obstruction (20%) (age 67-86 years; mean 75.5 years). No significant relationship could be demonstrated between bladder outlet obstruction and fatal PTE cases (p = 0.8). Given reports of this association, however, it is possible that bladder distension with venous compression may act as a risk modifier in certain individuals in association with other significant comorbidities, but this risk appears low.     

Cannabinoid concentrations in hair from documented cannabis users

Fifty-three head hair specimens were collected from 38 males with a history of cannabis use documented by questionnaire, urinalysis and controlled, double blind administration of delta9-tetrahydrocannabinol (THC) in an institutional review board approved protocol. The subjects completed a questionnaire indicating daily cannabis use (N=18) or non-daily use, i.e. one to five cannabis cigarettes per week (N=20). Drug use was also documented by a positive cannabinoid urinalysis, a hair specimen was collected from each subject and they were admitted to a closed research unit. Additional hair specimens were collected following smoking of two 2.7% THC cigarettes (N=13) or multiple oral doses totaling 116 mg THC (N=2). Cannabinoid concentrations in all hair specimens were determined by ELISA and GCMSMS. Pre- and post-dose detection rates did not differ statistically, therefore, all 53 specimens were considered as one group for further comparisons. Nineteen specimens (36%) had no detectable THC or 11-nor-9-carboxy-THC (THCCOOH) at the GCMSMS limits of quantification (LOQ) of 1.0 and 0.1 pg/mg hair, respectively. Two specimens (3.8%) had measurable THC only, 14 (26%) THCCOOH only, and 18 (34%) both cannabinoids. Detection rates were significantly different (p<0.05, Fishers' exact test) between daily cannabis users (85%) and non-daily users (52%). There was no difference in detection rates between African-American and Caucasian subjects (p>0.3, Fisher's exact test). For specimens with detectable cannabinoids, concentrations ranged from 3.4 to >100 pg THC/mg and 0.10 to 7.3 pg THCCOOH/mg hair. THC and THCCOOH concentrations were positively correlated (r=0.38, p<0.01, Pearson's product moment correlation). Using an immunoassay cutoff concentration of 5 pg THC equiv./mg hair, 83% of specimens that screened positive were confirmed by GCMSMS at a cutoff concentration of 0.1 pg THCCOOH/mg hair.     

Urinary excretion profiles of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol. Study III. A Delta9-THC-COOH to creatinine ratio study

Huestis and Cone reported in [J. Anal. Toxicol. 22 (1998) 445] that serial monitoring of Delta9-THC-COOH/creatinine ratios in paired urine specimens collected at least 24h apart could differentiate new drug use from residual Delta(9)-THC-COOH excretion following acute marijuana use in a controlled setting. The best accuracy (85.4%) for predicting new marijuana use was for a Delta(9)-THC-COOH/creatinine ratio > or = 0.5 (dividing the Delta9-THC-COOH/creatinine ratio of specimen no. 2 by the specimen no. 1 ratio). In previous studies in this laboratory [J. Anal. Toxicol. 23 (1999) 531 and Forensic Sci. Int. 133 (2003) 26], urine specimens were collected from chronic marijuana users > or = 24 h or > = 48 h apart in an uncontrolled setting. Subjects with a history of chronic marijuana use were screened for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/ml) followed by confirmation for Delta9-THC-COOH by GC-MS (cut-off 15 ng/ml). Creatinine was analyzed as an index of dilution. The objective of the present study was to evaluate whether creatinine corrected specimens could differentiate new marijuana or hashish use from the excretion of residual Delta(9)-THC-COOH in chronic marijuana users based on the Huestis 0.5 ratio. Urine specimens (N=376) were collected from 29 individuals > or = 96 h between urine collections. The mean urinary Delta9-THC-COOH concentration was 464.4 ng/ml, mean Delta9-THC-COOH/creatinine ratio (ng/(ml Delta9-THC-COOH mmoll creatinine)) was 36.8 and the overall mean Delta9-THC-COOH/creatinine ratio of specimen 2/mean Delta9-THC-COOH/creatinine ratio of specimen 1 was 1.37. The Huestis ratio calculation indicated new drug use in 83% of all sequentially paired urine specimens. The data were sub-divided into three groups (Groups A-C) based on mean Delta9-THC-COOH/creatinine values. Interindividual mean Delta9-THC-COOH/creatinine values ranged from 4.7 to 13.4 in Group A where 80% of paired specimens indicated new drug use (N=10) and 20.4-39.6 in Group B where 83.6% of paired specimens indicated new drug use (N=7). Individual mean Delta9-THC-COOH/creatinine values ranged from 44.2 to 120.2 in Group C where 84.5% of paired urine specimens indicated new marijuana use (N=12). Correcting Delta9-THC-COOH excretion for urinary dilution and comparing Delta9-THC-COOH/creatinine concentration ratios of sequentially paired specimens (collected > or = 96 h apart) may provide an objective indicator of ongoing marijuana or hashish use in this population.