【作者】 刘杨；

【导师】 王英元；

【作者基本信息】 山西医科大学 ， 法医学， 2009， 博士

【摘要】 研究背景:肌动蛋白是构成细胞骨架的主要蛋白质之一,几乎可表达于所有的真核细胞。现已证实心肌和肝脏内肌动蛋白的死后降解规律具有时间相关性,可用于死亡时间推断。但是机体死后各脏器之间肌动蛋白的降解规律是否具有差异性?环境因素(如温度、湿度)、人为因素(如致死方式)是否会对肌动蛋白的死后降解造成的影响?肌动蛋白的死后形态学改变是否也与死亡时间具有相关关系?以及在人体组织内,肌动蛋白的死后降解规律是否也具有时间相关性?目前有关这些问题还未见有研究报道。因此,本次实验将对上述这些问题进行探讨。实验目的:(1)观察大鼠死后不同时间心、肝、脾、肺、肾、脑和骨骼肌等组织内肌动蛋白的含量变化,为晚期死亡时间推断寻找参考指标。(2)探讨大鼠骨骼肌肌动蛋白纤维的形态学变化与死亡时间的相关性。(3)研究不同温度条件下大鼠骨骼肌肌动蛋白死后降解规律的差异性。(4)探讨环境湿度对大鼠骨骼肌肌动蛋白死后降解规律的影响。(5)比较不同死亡原因对大鼠骨骼肌肌动蛋白死后降解规律的影响。(6)探讨大鼠骨骼肌肌动蛋白的降解规律与离体时间的关系。(7)探讨人体胸大肌肌动蛋白含量变化与离体时间的关系。实验方法:(1)大鼠经机械性窒息法致死后随机分为7组,分别于死后0、24、48、72、96、120和168h剖取心、肝、脾、肺、肾、脑和骨骼肌等组织,采用免疫印迹及图像分析技术检测上述组织内肌动蛋白的含量,应用SPSS11.5软件对所得数据进行统计学分析。(2)分别采用光学显微镜、激光扫描共聚焦显微镜和透射电镜对大鼠死后不同时间骨骼肌(右后肢的内收大肌)肌动蛋白纤维的形态学变化进行观察。(3)应用免疫印迹及计算机图像分析技术,测量并分析在不同温度条件下(10℃、20℃和30℃)大鼠死后168h内骨骼肌肌动蛋白的含量变化差异。(4)对不同湿度条件下(30%、50%和70%)大鼠死后0、24、48、72、96、120和168 h骨骼肌肌动蛋白的含量进行检测。(5)将大鼠分别采用机械性窒息、失血性休克和电击法致死,采用免疫印迹及计算机图像分析技术检测并分析不同致死方式组大鼠死后168h内骨骼肌肌动蛋白的含量变化差异。(6)采用免疫印迹及计算机图像分析技术检测大鼠骨骼肌离体0、24、48、72、96、120和168h时肌动蛋白的含量,观察其含量变化与离体时间的关系。(7)收集外科手术中切除的人体胸大肌组织,置于人工气候箱内保存(恒温20℃,湿度恒定50%),采用免疫印迹及计算机图像分析技术检测其离体0、24、48、72、96、120和168h后肌动蛋白的含量。实验结果:(1)大鼠死后,其心肌、肝、脾、肺、肾、脑和骨骼肌等组织内肌动蛋白含量均随死亡时间的延长而逐渐下降,与死亡时间具有相关性。但肌动蛋白在上述各器官内的死后降解速度有差异(P<0.05),在脑组织的降解速度最快,其余依次为肺、脾、肝、肾、心肌和骨骼肌。(2)随着死亡时间的延长,大鼠骨骼肌纤维的横纹逐渐模糊、消失,细胞核固缩、溶解(光学显微镜观察);由肌动蛋白构成的细肌丝逐渐崩解、紊乱,直至肌小节和细肌丝结构消失(透射电镜观察);骨骼肌肌动蛋白纤维在死后24h即可发生不同程度的抗肌动蛋白抗体的缺染,缺染面积的变化趋势与死亡时间相关,至死后168h,几乎观察不到抗肌动蛋白抗体的阳性产物(激光扫描共聚焦显微镜观察)。(3)不同温度组大鼠骨骼肌肌动蛋白的降解速度进行组间比较时,组间差异具有统计学意义(P<0.05);环境温度与死亡时间之间具有交互作用(F=8.894,P=0.001)。肌动蛋白相对含量与环境温度、PMI的多元回归方程为:Y=136689.8-465.5X1-1377.7X2,R2 =0.823(Y为肌动蛋白的IOD值,X1为PMI,下同;X2为环境温度)。(4)不同湿度组大鼠骨骼肌肌动蛋白的降解速度进行组间比较时,组间差异具有统计学意义(P<0.05),环境湿度的升高,肌动蛋白的降解速度加快。肌动蛋白相对含量与环境湿度、PMI的多元回归方程为:Y=136689.8-465.5X1-1377.7X2,R2 =0.823(X2为环境湿度)。(5)不同死亡原因组大鼠骨骼肌肌动蛋白降解速度进行组间比较时,失血性休克组大鼠骨骼肌肌动蛋白的死后降解速度慢于其它两组(P<0.05)。肌动蛋白相对含量与死亡原因、PMI的多元回归方程为:Y=118112.8-438.6X1-4972.5X2,R2 =0.793(X2为死亡原因:1=机械性窒息、2=失血性休克、3=电击致死)。(6)大鼠骨骼肌肌动蛋白含量变化与离体时间呈负相关关系,肌动蛋白含量变化趋势与在体组比较,差异具有统计学意义(P<0.05)。肌动蛋白相对含量与标本保存方式、PMI(或离体时间)的多元回归方程为:Y=82422.6-420.5X1+15884.3X2,R2 =0.865(X2为标本保存方式:1=在体、2=离体保存)。(7)人体胸大肌肌动蛋白的含量变化与离体时间呈负相关关系,其降解规律与大鼠骨骼肌肌动蛋白死后降解规律有差异(P<0.05),但在大多数离体时间点,人体胸大肌与大鼠骨骼肌肌动蛋白的含量差值不具有统计学意义(P=0.334)。人体胸大肌肌动蛋白相对含量与PMI的回归方程及R2值为:Y=108288.8-342.28X,R2=0.89( X为离体时间)。结论:(1)大鼠死后心、肝、脾、肺、肾、脑和骨骼肌肌动蛋白的降解规律具有组织差异性,有助于较晚期死亡时间的推断。但是肌动蛋白在上述组织中的含量变化均与死亡时间具有相关性。(2)大鼠骨骼肌肌动蛋白纤维的形态学改变与死亡时间具有相关性,有助于早期死亡时间推断。(3)骨骼肌肌动蛋白的死后降解速度会受环境温度影响,在一定范围内有随温度升高而加快的趋势。(4)环境湿度会影响骨骼肌肌动蛋白的死后降解速度,二者在一定范围内呈正相关关系。(5)死亡原因会影响大鼠骨骼肌肌动蛋白的死后降解速度,骨骼肌肌动蛋白的死后降解规律可能会有助于某些死亡原因的判断。(6)大鼠离体骨骼肌肌动蛋白的含量变化与离体时间呈负相关,在离体保存状态下,肌动蛋白的降解速度被延缓。(7)人体胸大肌离体后肌动蛋白的降解趋势与离体时间同样具有相关关系,证实骨骼肌肌动蛋白死后降解规律可以作为法医学死亡时间推断的生物学指标。更多还原

【Abstract】 BackgroudActin is structural subunit of microfilaments and is the most abundant intracellular protein in a eukaryotic cell, which often constitutes as much as 50% of total cellular protein. Experiments had shown that actin in the liver tissue of rats could be detected at 8 days postmortem, but could not be examined after 10 days postmortem and within a certain postmortem interval ( <54h postmortem), the extent of anti-actin monoclonal antibody staining depletion in cardiac and skeletal muscle cells increases with the extension of postmortem interval. These observations indicate that the degradation pattern of the actin may be used as a parameter for PMI estimation. However, whether there are variability of actin postmortem degradation in different tissues? Whether the environmental factors( temperature and humidity), anthropic factors(modes of death) could affact the postmortem degradation of actin ? Whether there are correlationship between the morphologic changes of actin and the time since death? Whether the actin content in human tissues also decreases with the extension of the post-isolation time ? At present, we can’t find reports in these filds. So, in this study, we try to find the answer for the above problems.Objective(1)To observe the postmortem degradation of actin in cardiac muscle, liver, spleen, lung, kidney, brain and skeletal muscle of rats,for the purpose of finding a new method of estimating the time since death.(2)To investigate the correlation between morphologic changes of actin in skeletal muscle of rats and the time since death.(3)To study the effects of ambient temperature on the actin postmortem degradation of rats.(4)To investigate the variability of actin postmortem degradation in skeletal muscle of rats at different ambient humidity.(5)The content changes of actin in the skeletal muscle of rats killed by different manners were investigated to elucidate evidence that can be used to determine the modes of death.(6)To study the relationship between the actin content in ex vivo skeletal muscle of rats and the time since isolation.(7)To observe the degradation of actin in ex vivo human pectoralis major within 168h after being excised. Methods(1) Twenty eight clear Sprague Dawley rats were killed by suffocating and put into an artificial climate incubator(set at 20℃)for 0, 24, 48, 72, 96, 120 and 168h, respectively. The actin contents in the above tissues were quantitated by western-blot assay and collected by Image Pro Plus 5.0 image analysis system, and the datas were then statistically analyzed with the SPSS 11.5 software.(2) The morphologic changes of actin filament in the skeletal muscle (adductor magnus of right hind leg) of twenty eight rats were observed by optical microscope, laser scanning confocal microscope (LSCM) and transmission electron microscope (TEM) at different postmortem intervals (0, 24, 48, 72, 96, 120 and 168h).(3) The differences of the content of actin in the adductor magnus of rats at different ambient temperature (10℃, 20℃and 30℃) and different postmortem intervals (0, 24, 48, 72, 96, 120 and 168h) were measured and analyzed with western-blot assay and image analysis technology.(4) The content changes of actin in the adductor magnus of rats at different ambient humidity (30%, 50% and 70%) were determined by using western-blot assay and image analysis technology, in the course of 168h after death.(5)Twelve SD rats were randomly allocated into 3 groups and killed by bleeding,suffocating,and electrocution,respectively.The contents of actin in the adductor magnus of rats killed by different manners at different postmortem intervals (0, 24, 48, 72, 96, 120 and 168h) were measured by western-blot.(6)The adductor magnus of four SD rats killed by suffocating were dissected and put into the artificial climate incubator set at 20℃. The actin content in the ex vitro specimens were quantitated with western blot assay and image analysis techniques at differernt time after being excised (0, 24, 48, 72, 96, 120 and 168h).(7) The actin content in the ex vitro human pectoralis major (n = 4) were quantitated with western-blot assay, in the course of 168h after being excised.Results(1)Actin content in all these tissues decreased gradually with the prolonged postmortem intervals(PMI). The degradation differences between the groups was statistically significant(P<0.05), with the degradation in the brain fastest,then the lung, the spleen,the liver, the kidney, the cardiac muscle and the skeletal muscle in order. There was a strong correlation between actin degradation and PMI, and the coefficient of determination (R2) exceeded 0.75 in all these tissues.(2)After death, the disappearance of the transverse striation and karyopycnosis, hypochromatosis of the skeletal muscle of rats can be observed by the optical microscope, and all that became more and more notable with the extension of PMI; the results of the TEM revealed that actin filament began to disintegrate with the lapse of PMI, and at last, the structure of sarcomere and actin filament disappeared; the results of LSCM showed that skeletal muscles revealed depletion of anti-actin antibody staining and the extent of staining depletion increased with extension of PMI, and nearly no actin positive product can be detected until 168h after death.(3)Within 168h postmortem, actin content in the adductor magnus of rats in different ambinet temperature groups all reduced gradually with the extension of the PMI, and significant differences can be observed between the different temperature groups at all the intervals (P<0.05). The interaction effect on the postmortem dagradation of actin between the ambinet temperature and PMI can be found via the statistical analysis. The multiple regression equation between actin content and ambient temperature, PMI were: Y=136689.8-465.5X1-1377.7X2,R2 =0.823(Y was the IOD value of the actin content, X1 was the PMI,which was the same as the below in the whole thesis; X2 was the ambient temperature).(4)The postmortem degradation rate of actin in the different humidity groups were differernt, and there were significant differences in actin content can be observed between the rats in different humidity groups at most of intervals. The multiple regression equation between actin content and ambient humidity, PMI and the R2 were: Y=136689.8-465.5X1-1377.7X2,R2 =0.823 (X2 was the ambient humidity).(5)There were significant differences in contents of actin in the adductor magnus between the hemorrhage shock group and suffocation group,electrocution group at most of the intervals (P<0.05). The multiple regression equation between actin content and modes of death, PMI were: Y=118112.8-438.6X1-4972.5X2,R2 =0.793 (X2 was the modes of death: 1- by bleeding,2-by suffocating,3-by electrocution).(6)Actin content in the ex vivo adductor magnus of rats reduced gradually with the extension of the PMI, and there was a proximate linear relationship between actin content and post-isolation time, and significant differences can be observed between ex vivo and in vitro groups(P<0.05). The multiple regression equation were:Y=82422.6-420.5X1+15884.3X2,R2 =0.865(X2 :the modes of preservation of the specimen: 1- in vitro, 2- ex vivo).(7)There were significant differences in actin content in human pectoralis major (P<0.05) after being excised, but no differences can be found in the reminers of the actin content in human pectoralis major and adductor magnus of rats at most of the intervals(F=1.22,P=0.334). The multiple regression equation between actin content and PMI and the R2 were: Y=108288.8-342.28X,R2=0.89(X was the time after isolation). Conclusion(1) Although with tissue disparity in degradation, the actin contents in cardiac muscle, liver, spleen, lung, kindey, brain and skeletal muscle of rats decreased gradually with prolonged PMI, which may potentially be a useful parameter for the late PMI estimation.(2)To observe the morphologic changes of the actin filament of skeletal muscle may be expected to be a useful method for the early PMI estimation.(3)The influence of temperature on the postmortem degradation of actin in skeletal muscle was evident, and the degradation rate tended to to be raised by higher temperature.(4)The postmortem degradation rate of actin in skeletal muscle were different between different humidity groups, and the rise of the ambient humidity tended to speed up the degradation rate.(5)The manners of death could influence the postmortem degradation rate of actin in the skeletal muscle of rats, the content changes of actin may provide a basis for determination of the manner of death.(6)There was a negative correlativity between actin content in the skeletal muscle of rats and the post-isolation time, and the postmortem degradation rate of actin was higher in vitro group than in ex vivo group.(7)The actin content in human pectoralis major decreased with the extension of the post-isolation time, which seems to be a useful biological indicator for the late PMI estimation.更多还原