大鼠皮肤切创后E-选择素表达及稳定性研究

目的探讨大鼠皮肤切创后E-选择素表达规律及法医学意义。方法健康SD大鼠90只,随机分成4组：正常对照组、活体切创组（30min～7d12个时间点）、死后切创组（30min～3h3个时间点）、死后稳定性组（-20％6h-7d9个时间点,25％6h～3d5个时间点）。在大鼠头部建立皮肤切创模型,按设定的时间点取皮肤检材,运用免疫组化和图像分析技术,检测血管内皮E-选择素的表达规律。结果在活体切创组中,伤后1hE-选择素在血管内皮细胞内即呈阳性表达,持续至伤后7d,且随时间变化呈规律性表达。在正常对照及死后切创组未见阳性表达。死后-20％稳定性组各时间点E-选择素表达与死后即刻比较无显著性差异（P〉0．05）。25％各时间点与死后即刻比较有显著性差异（P〈0．01）。结论E-选择素在创伤后血管内皮细胞内特异性的表达具有时序性规律,且低温条件下稳定性较好。     

Preliminary study of the effect of acute antemortem haemorrhage on postmortem abdominal impedance in rats

Studies over many years have revealed a consistent, inverse relationship between extracellular impedance of the rat abdomen and postmortem interval (PMI). Recent studies have shown that this relationship can be improved by correcting measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C, thus rendering them independent of the tissues' temperature at the time of impedance measurement. The present study, undertaken as a part of an ongoing effort to identify variables which might influence postmortem rate of change in abdominal impedance, was aimed at studying the possible effect of acute antemortem haemorrhage on abdominal impedance. Sudden loss of approximately 30% of the total blood volume, immediately prior to the death was without statistically significant effect on the pattern of postmortem change in abdominal impedance. Thus, in the control and experimental rats, respectively, impedance decreased progressively from 58.7 +/- 1.8 and 68.0 +/- 11.3 omega, 1 h postmortem, to 31.5 +/- 2.2 and 33.7 +/- 4.7 omega at a postmortem interval of 120 h (n = 6 in each group). In both groups, the relationship between impedance and postmortem interval was either linear or curvilinear. These findings are believed to mitigate in favour of continued effort to examine the potential usefulness of abdominal impedance, as an index of postmortem interval, under conditions encountered in routine forensic investigation.     

大鼠骨骼肌损伤修复中FZD4时序性变化及其应用

目的以卷曲受体4(FZD4)为例,探索细胞膜上受体蛋白的表达变化能否作为损伤时间推断的指标。方法 78只大鼠随机分为对照组和损伤后4h、8h、12h、16h、20h、24h、28h、32h、36h、40h、44h和48 h组(n=6)。麻醉后,采用重力锤自由落体方式砸伤大鼠右后肢,取股四头肌处挫伤的组织。应用免疫荧光、real-time PCR和western blotting方法检测肌肉中FZD4蛋白及m RNA的表达量。结果免疫荧光结果显示FZD4表达于骨骼肌细胞膜上,胞浆和细胞核内均未见表达;Real-time PCR结果表明FZD4 m RNA在损伤后8h、12h、36h和40h显著上调(P0.05),均超过对照组的两倍;Western blotting结果示FZD4蛋白的表达在8h、36h、40h、44h和48h组明显增加(P0.05),但均未超过对照组的两倍。结论 FZD4 m RNA和蛋白在肌肉挫伤后呈时序性变化规律可作为损伤时间推断的指标,在一定程度上FZD4 m RNA比FZD4更适用于损伤时间推断。     

Interleukin-6 and C-reactive protein serum levels in sepsis-related fatalities during the early postmortem period

Postmortem interleukin-6 (IL-6) and C-reactive protein (CRP) serum levels were investigated prospectively in sepsis-related fatalities and non-septic fatalities by using a linear regression model. At least three blood samples were collected between 0.3 and 139 h postmortem from sepsis-related fatalities (n=8) and non-septic fatalities (n=16). In addition, one antemortem blood sample was collected shortly before death from the septic patients. Antemortem and postmortem IL-6 and CRP levels were highly elevated in all individuals included in the sepsis group. An excessive postmortem increase of IL-6 serum levels associated with progressive time after death was observed in five out of the eight septic patients. Both, IL-6 and CRP serum concentrations seem to be suitable biochemical postmortem markers of sepsis. The determination of IL-6 serum levels above 1500 pg/ml in peripheral venous blood obtained in the early postmortem interval can be considered as a diagnostic hint towards an underlying septic condition. A more precise postmortem discrimination between sepsis and non-septic underlying causes of death is provided by the postmortem measurement of serum CRP in peripheral venous blood: on condition that at least two postmortem CRP values have been determined at different time points postmortem, the CRP level of a deceased at the time of death can be calculated by using linear regression analysis. When assessing postmortem IL-6 and CRP concentrations as biochemical postmortem markers of sepsis, various clinical conditions, such as a preceding trauma or burn injury going along with elevated IL-6 and/or CRP levels prior to death as a result of the systemic inflammatory response syndrome (SIRS) should be taken into consideration, thus adding relevant information for the practical interpretation of the results.     

Quantitative analysis of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) in human skin wounds

Proinflammatory cytokines play an important role in the mediation of inflammation and trauma. They could be useful for the determination of vitality and wound age. In the present study, 144 human skin wounds due to sharp force were investigated. The material was collected during operations (N=96) and postmortem examinations (N=48). The wound age varied from several seconds or minutes to 9 days. Control skin was available in each individual. The tissue specimens were homogenized and extracted in a solution of PBS and protease inhibitors. Interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) were measured by quantitative ELISA analysis. Statistical evaluation was performed by the t-test using the quotients of levels (wound sample/control skin). In surgical specimens the cytokine levels revealed a clear tendency to increase with wound age. IL-1beta in early skin wounds (24 h, P<0.05). The quantitative analysis of proinflammatory cytokines in wound extracts can contribute to the determination of vitality and wound age, in particular in the very early post-traumatic interval (classic stab wounds).     

A preliminary study on the changes of expression of PDGF-beta,PDGFR-beta,TGF-beta 1, TGFR,bFGF and its relationship with the wound age in wound healing

OBJECTIVE: To explore the relationship between the expression change of cytokines and the wound age during the healing process of rats skin wound. METHODS: Immunohistochemical and image-analysis methods were performed on vital skin wounds(after incision 0.5-168 h am) and postmortem damage(after incision 0.5-6 h pm). RESULTS: The expression of the cytokines PDGF-beta, PDGFR-beta, TGF-beta 1, and bFGF in the epithelial cells was already enhanced since 0.5 h am after damage and their strongest expression reaction was seen at 24-96 h am. In addition, the expression of PDGF-beta, PDGFR-beta, TGF-beta 1 and bFGF was also found in the macrophages and the fibroblasts of the granulation tissue, and the expression changes in the postmortem damage group showed that the skin tissue within 0.5-3 h after incision showed immunohistochemical changes but weakly expression and 3 h thereafter no any change was found. CONCLUSION: The expression characteristics of the above mentioned cytokines in wound repair should be related to the wound age and it reminds therefore that they may be used as immunohistochemical criteria for accurate determining the wound age.     

Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1beta,IL-6, TNF-alpha) in human skin wounds

The proinflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) hold important functions in the early and late courses of inflammation, trauma and wound healing. In the present study, human skin wounds due to sharp force (n = 105) were collected during surgery and autopsy. The wound age mainly varied from several minutes to 5 h, some specimens aged up to 6 weeks. Control specimens from uninjured skin were available in each case. After preparation of cryostat sections, immunohistochemistry was performed according to the APAAP technique, using monoclonal and polyclonal antibodies. The results were evaluated semiquantitatively. All markers were weakly expressed in normal human skin constitutively. However, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands. IL-1beta and IL-6 showed enhanced expression after 15 and 20 min at the earliest (increase of epidermal reactivity). After 30-60 and 60-90 min, respectively, marked expression was observed with these markers. Similar alterations were detectable with TNF-alpha after 15 and 60-90 min. The reactivity of all three markers persisted over several hours, then decreased to basal levels again and sometimes reappeared after days and in granulation tissue. Leukocytes reacting with IL-1beta and IL-6 appeared after approximately 2 h. CONCLUSION: proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early post-traumatic interval prior to leukocyte reaction. Autolysis did not play a role in the samples investigated (postmortem interval up to 8 days). Problems could sometimes rise from constitutive expression. Therefore, it is recommended to examine control samples from the same individual and to compare the reactivity with wound specimens.     

死后肌肉切创创壁纤维蛋白的扫描电镜观察

用扫描电镜观察大鼠肌肉组织切创创壁纤维蛋白网的形成和形态,发现生前和死后8小时内的切创壁,均有纤维蛋白网形成,两者形态无显著差异。死后16小时,切创创壁无纤维蛋白网形成。     

兔急性肺损伤后肺内VEGF时序性表达的研究

目的观察大潮气量机械通气致兔急性肺损伤(acutelung injury,ALI)后,肺组织中血管内皮生长因子(VEGF)的动态变化,探讨其在损伤时间推断中应用的价值。方法57只雄性新西兰大白兔随机分为正常组(3只)、对照组(27只)和实验组(27只)。机械通气(V_T 60mL/kg)致AL后于0,1,3,6,12,18,24,36,48h各处死动物3只。取肺组织,测量右上肺组织湿干重比(W/D),肺组织进行HE及免疫组织化学染色并进行图像定量分析,观察VEGF变化规律。结果正常组VEGF有低水平表达;大潮气量机械通气1min后至肺损伤后12h,实验组VEGF表达水平与正常组无明显差异(P>0.05);12～48h随损伤时间的延长VEGF表达水平持续上升,48h仍处于较高水平,其间各时间点VEGF表达水平与正常组比较均有显著性差异(P<0.05)。结论大潮气量机械通气致兔ALI 2d内,VEGF在肺组织炎症发生、发展和愈合修复过程中存在时间相关性,对法医病理推断呼吸机所致的ALI时间具有参考价值。     

Estimation of postmortem interval using an electric impedance spectroscopy technique: A preliminary study

The objective of this study was to develop a rapid method for the estimation of postmortem interval (PMI) using electric impedance spectroscopy. Postmortem rat spleens were studied at 10 °C, 20 °C, and 30 °C; The results obtained demonstrated that postmortem interval negatively correlated with the absolute value of Im Z^// (capacitive reactance component) in electrical impedance. This suggests that electric impedance spectroscopy may be a sensitive tool to determine the postmortem interval.     

Immunohistochemical detection of fibronectin and tenascin in incised human skin injuries

Immunohistochemical detection of molecules involved in inflammatory reaction can be useful for the diagnosis of vitality in skin wounds. We studied the expression of fibronectin (FN) and tenascin (TN) in 58 human skin wounds (48 vital and 10 postmortem). The age of vital injuries ranged from 3 min to 8 h and postmortem specimens were collected after a postinfliction interval of 15-180 min. One hundred thirty-seven formalin-fixed paraffin-embedded sections (mean: 2.3 sections per case) were stained with each of two monoclonal antibodies against FN and TN using the streptABC technique. A reticular staining for FN in wound edge and dermis was observed in 50% of vital specimens versus 0% in postmortem cases. Immunoreactivity was reduced in 10 autolysed cases. FN positivity exclusively at the injury margin was observed in 39.4% of vital wounds and 10% of postmortem cases. TN was negative in all specimens. Vital and postmortem hemorrhage areas showed positivity for FN and TN. Due to its low sensitivity, immunohistochemical analysis of FN is useful for determining vitality only in a minority of cases. Different factors in everyday practice, including autolysis and technical problems often produce false negative reactions with the result that FN cannot be regarded as a reliable parameter of vitality. Positive reactions (network staining) are more valuable than negativity but are not pathognomonic. Both vital and postmortem hemorrhages show an enhanced positivity for FN and TN, thus impeding the diagnosis.     

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.     

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。     

大鼠皮肤切创愈合过程中Cygb及HIF-1α mRNA的表达

目的探讨皮肤切创愈合过程中Cygb及HIF-1αmRNA表达的相关性及时间变化规律。方法 60只健康雄性SD大鼠随机分为9个实验组和1个正常对照组,建立大鼠皮肤全层切创模型,实验组分别于术后0、1、3、6、12、24、48、72、96h脱颈处死大鼠,提取创周皮肤总RNA,采用实时荧光定量PCR技术检测Cygb及HIF-1αmRNA的表达,实验组与对照组及各组间进行比较分析。结果 Cygb与HIF-1αmRNA创伤后表达的总体变化趋势基本一致,均于伤后12h首次达峰,分别为对照组的1.6倍和5.4倍(P<0.05);随后下降,并分别于伤后48h、72h再次升高,至96h达对照组的2.8倍和5.6倍(P<0.05)。结论大鼠皮肤切创愈合过程中Cygb与HIF-1 mRNA的表达呈现时序性变化且有一定的相关性,可作为皮肤损伤时间推断的指标。     

纤维联接蛋白的析出与大鼠皮肤早期损伤的时间关系

采用免疫组化(PAP)方法,检测大鼠皮肤损伤区的FN,发现在生前创伤后15min,创壁FN即呈明确阳性;随着伤后经历时间的延长,创壁FN逐渐增多,并滑创壁呈条带状沉积;而在死后5min的创伤,创壁FN则呈阴性。本文为区别生前与死后皮肤创伤提出了一种新的方法。     

不同死亡方式大鼠心肌组织中ATP、ADP和AMP含量比较

目的比较不同死亡方式对大鼠心肌组织能量物质变化的影响,为判断不同死亡方式及死亡时间提供依据。方法180只大鼠随机分为三组,以不同方式(失血、窒息、断颈)处死,高效液相色谱法检测死后0、0.5、1、1.5、2、3、4、6、8、12、18和24h大鼠心肌组织中的ATP、ADP、AMP含量。结果三组不同死亡方式大鼠心肌组织的ATP、ADP、AMP的含量在大部分时间点做组间比较有明显差异(P<0.05);每组不同死亡方式的大鼠心肌组织中ATP、AMP的含量随时间的变化有明显的差异(P<0.01),而各组中的ADP在死亡一定时间后无明显差异。结论不同死亡方式的大鼠心肌组织中能量物质死后变化可以用作对死亡方式及某种方式不同死亡时间做出判断的依据。     

大鼠皮肤切创愈合过程中ICAM-1及P选择素的表达

目的　探讨细胞间粘附分子 1(ICAM 1)及P选择素的表达变化与损伤时间的关系。方法　应用免疫组织化学技术 ,对实验大鼠不同损伤时间的生前伤 ( 5min～ 7d)及死后伤 ( 5～ 3 0min)皮肤组织中ICAM 1及P选择素的表达变化进行观察。结果　在生前损伤组中 ,ICAM 1最早在伤后 1h ,最迟至伤后 3d在表皮层中呈阳性表达 ;P选择素最早在伤后 10min ,最迟至伤后 5h即在血管内皮细胞中呈阳性表达。此外 ,ICAM 1还在炎症细胞及纤维母细胞中表达 ,且随损伤时间变化而呈规律性变化。在时间段分组 ,ICAM 1在第Ⅰ组 ( 5min～ 1h)中阳性细胞率极低( 0 41± 0 73 % ) ,第Ⅱ组 ( 3h～ 7h)及第Ⅲ组 ( 9h～ 12h)均呈显著性增高 ( 9 79± 3 74% ,2 3 3 3± 1 10 % ) ,至第Ⅳ组 ( 1d)达高峰 ( 3 0 5 8± 2 65 % ) ,其后逐渐减少。ICAM 1及P选择素在死后伤中均呈阴性表达。结论　ICAM 1及P选择素可作为法医学损伤时间判定的有效指标。     

Estimation of postmortem interval. Temperature-correction of extracellular abdominal impedance during the first 21 days of death

Extracellular impedance of the intact abdomen of rats increased from 79.0+/-7.4Omega, 1h postmortem (i.e. 0.04 day), to 130.5+/-14.4Omega at postmortem interval (PMI)=1 day. Impedance then decreased with time, reaching 64.2+/-9.9Omega at PMI=21 days. The time taken for mean abdominal impedance to decrease below the value at PMI=0.04 day averaged 16 days. It is therefore impossible, using extracellular abdominal impedance alone, to distinguish (in terms of interpolating PMI) between numerically equal impedances on the rising and falling phases of curves depicting impedance as a function of PMI.Correction of measured impedances to their theoretically-predicted values at an arbitrarily chosen temperature of 40 degrees C appreciably diminished the magnitude of the increase in impedance following death. Thus, temperature-corrected abdominal impedance increased from 56.2+/-4.8Omega at PMI=0.04 day to 59.5+/-6.2Omega at PMI=1 day. Impedance then decreased, reaching 29.2+/-4.1Omega at PMI=21 days. The time taken for mean, temperature-corrected abdominal impedance to decrease below the value at PMI=0.04 day averaged 3 days (as opposed to 16 days (see above) in the absence of temperature-correction).These findings are believed to improve the usefulness of extracellular abdominal impedance as a potential tool for estimation of postmortem interval.     

小鼠皮肤切创IL-10和IL-4的表达及其与损伤时间关系

目的　探讨IL 10、IL 4在小鼠皮肤切创愈合过程中的表达变化规律及其与损伤时间的关系。方法　应用免疫组织化学及图像分析技术 ,观察实验小鼠不同时程的生前伤 ( 0 5～ 168h)及死后伤 ( 1～ 6h)皮肤创伤局部IL 10、IL 4的表达情况。结果　生前伤 ,IL 10在伤后 1～ 3h阳性反应逐渐增强 ,3h达峰值 ,2 4h降至较低水平 ,伤后 48h再次升高 ,72h又达新峰值 ,其阳性表达部位主要在表皮细胞及单核 /巨噬细胞 ;IL 4于伤后 2 4h出现明显阳性反应 ,96h表达水平达峰值 ,168h仍维持在较高水平 ,其阳性细胞主要为创伤局部的成纤维细胞。死后伤 ,IL 10仅于死后 1～ 3h呈阳性染色 ,IL 4未见阳性染色。结论　IL 10、IL 4在小鼠皮肤切创愈合过程中的表达呈现一定的时序性变化。     

兔皮肤钝器伤IL-1β、COX-2和MCP-1 mRNA表达与法医学应用

目的应用荧光定量PCR技术检测兔皮肤钝器伤后IL-1β、COX-2和MCP-1mRNA的时序性表达规律,分析其与损伤时间的关系。方法分别在兔耳皮肤钝器伤后<0.5h,0.5h,1h,2h,3h,4h,5h,6h,8h,12h,24h,1d,3d,7d和死后10min,0.5h,1h取材,一步法提取组织总RNA,逆转录合成cDNA第一条链,荧光定量PCR检测IL-1β、COX-2和MCP-1mRNA逆转录合成第一条cDNA链的拷贝数,作统计学分析。结果IL-1βmRNA在钝器伤后<0.5h表达显著增强(P<0.001),0.5h达到峰值,至第2d降至基本水平;COX-2mRNA于创伤后0.5h表达显著增强(P<0.05)并持续强表达至24h开始下降,第2d降至正常;MCP-1 mRNA在钝器伤后于1h表达显著增强(P<0.05),于3h出现表达峰,至第3d降至正常;3个指标在死后各时间组与正常对照无明显表达差异。结论检测皮肤IL-1β、COX-2和MCP-1mRNA可对早期损伤时间的推断提供帮助,并且这三个指标都可以鉴别生前伤和死后伤。