【作者】 陈继梁；

【导师】 刘敏；

【作者基本信息】 四川大学 ， 法医学， 2007， 硕士

【摘要】 研究背景及目的支气管哮喘是一种以呼吸道炎症、呼吸道高反应和呼吸道重塑为特征的常见多发疾病。近年来，其发病率和死亡率呈逐年上升趋势；法医学实践中因哮喘引起的猝死案例也逐年增加，是目前法医学死因鉴定中的难点之一。以往，法医学对哮喘猝死的研究报道多认为与哮喘并发症和细支气管粘液栓引发窒息或呼吸衰竭等因素有关。近年有研究表明，哮喘猝死案例的心肌组织有一定程度的损害，但至今哮喘猝死的心肌损害的病理机制尚不清楚。另外，哮喘猝死的法医学诊断还存在一些争议，如，生前发病史不清楚的死者是否确因哮喘发作引起猝死、哮喘疾病诊断仅从肺部的病理改变来确定是否具有局限性、哮喘疾病是不是引起死者死亡的根本原因等。因此，研究支气管哮喘心肌损害机制不仅有利于临床对哮喘疾病的治疗，还有利于法医学实践中支气管哮喘猝死的诊断。本研究在成功建立SD大鼠支气管哮喘动物模型的基础上，采用常规HE染色、心肌组织HSP70免疫组织化学染色、TUNEL染色及流式细胞术相结合的方法，研究各种实验条件下心肌组织的损害规律，以期探讨支气管哮喘时心肌细胞损害的病理机制，为哮喘猝死的法医学死因鉴别诊断提供形态学辅助依据。方法健康雄性SD大鼠120只(四川大学华西实验动物中心提供)，一月龄左右，体重100±15克。在成功建立SD大鼠支气管哮喘模型的基础上，随机分6组：支气管哮喘发作肾上腺素组、支气管哮喘发作氨茶碱组、支气管哮喘发作肾上腺素加氨茶碱组、支气管哮喘不给予药物组、实验对照组及正常对照组。每组又依据断颈处死时间的不同，分为2周、4周、6周、8周四个小组，每小组5只大鼠。取心室段心肌组织，其中一份用10％甲醛溶液固定，常规石蜡包埋、切片，行常规HE染色、心肌热休克蛋白70(HSP70)免疫组织化学染色、末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记技术(TUNEL染色技术)检测，在光学显微镜下观察并作图像分析及相应的统计学处理。另外一份心肌组织不固定，行流式细胞术检测。结果1．心肌组织常规HE染色各实验组与对照组之间无明显区别。心肌细胞染色清晰，横纹清楚，细胞核染色质分布均匀，部分区域可见心肌纤维呈波浪状改变及心肌纤维断裂，心肌间质未见炎性细胞浸润及心肌细胞坏死等改变。2．心肌组织HSP70的免疫组织化学染色实验对照组与正常对照组SD大鼠心肌细胞在各时间段内HSP70阳性信号表：达基本呈阴性，实验组在实验第2周始，心肌细胞浆内即可见HSP70染色阳性信号表达，随哮喘发作时间的延长，心肌细胞浆HSP70染色表达强度(PI％—HSP70值)逐渐增强，至第8周时达最高。另外，依据实验各组干预条件的不同，在哮喘发作的相同时间段内，心肌细胞浆HSP70染色阳性信号表达强度(PI％—HSP70值)有显著差异，依次为：支气管哮喘发作肾上腺素组＞支气管哮喘发作肾上腺素加氨茶碱组＞支气管哮喘不给予药物组＞支气管哮喘发作氨茶碱组。实验对照组和正常对照组之间，心肌细胞浆HSP70染色表达强度没有明显差异。3．末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记技术实验对照组与正常对照组SD大鼠心肌细胞核在各时间段内TUNEL染色阳性信号表达基本呈阴性。实验组在实验第2周始，心肌细胞核即有TUNEL染色阳性信号表达，随哮喘发作时间的延长，心肌细胞核TUNEL染色表达强度(PI％—TUNEL值)逐渐增强，至第8周时达最高峰。据实验各组实验干预条件的不同，在哮喘发作的相同时间段内的心肌细胞核TUNEL染色阳性信号表达强度(PI％—TUNEL值)有显著差异，依次为：支气管哮喘发作肾上腺素组＞支气管哮喘发作肾上腺素加氨茶碱组＞支气管哮喘不给予药物组＞支气管哮喘发作氨茶碱组。实验对照组和正常对照组之间，心肌细胞核TUNEL染色阳性信号表达强度无明显差异。4．流式细胞仪检测实验组心肌细胞凋亡率明显高于对照组。在实验组中，哮喘心肌细胞在第2周始即出现明显的细胞凋亡峰，且随着哮喘发作时间的延续，心肌细胞凋亡率呈逐渐上升趋势，至第8周时最高。各实验组中，依据实验各组实验干预条件的不同，在哮喘发作的相同时间段内心肌细胞凋亡率有显著差异，依次为：支气管哮喘发作肾上腺素组＞支气管哮喘发作肾上腺素加氨茶碱组＞支气管哮喘不给予药物组＞支气管哮喘发作氨茶碱组。实验对照组和正常对照组之间没有明显差别。结论1．支气管哮喘的SD大鼠，其心肌细胞有一定程度的损害，心肌细胞的损害程度随着哮喘发作时间的延长而加重。2．哮喘促进心肌细胞凋亡机制的激活引起心肌细胞凋亡是哮喘心肌损害的病理机制中一个重要因素。心肌细胞凋亡数量和表达强度随哮喘发作时间的延长而增加。3某些哮喘治疗药物的使用可能促进心肌细胞凋亡的发生如肾上腺素。4法医学哮喘猝死的死因鉴别诊断中，哮喘诱发心肌细胞的凋亡而使心功能受损是一个值得重视的因素。在哮喘猝死的法医病理检查中，除了对心肌组织常规的HE检查外，可以考虑采用心肌组织免疫组织化学染色、TUNEL染色和FCM的方法来作为哮喘心肌组织损害的形态学辅助诊断依据。更多还原

【Abstract】 Background and purpose:The bronchus asthma is a familiar disease, its paroxysm characteristic is an inflammation to respiratory airway with airway high reaction and airway remodeling.In recent years, the reports of the outbreak rate and death rate about the bronchus asthma were on the increase year by year.In the forensic medicine, the reports of disputing case examples about sudden death caused by asthma also were increased gradually, there were still much difficulties on the analysis of cause of death now.Although there were many reports of damage for expressing, asthma sufferer in addition to lung a pathological changes as well having myocardium, these researching discovers about sudden death had been caused by asthma all did not had the particularity on the analysis of cause of death in forensic medicine, and the molcule pathology mechanism of myocardial damage had been caused by bronchus asthma was still not pure, the related cultural heritage reports were too more seldom seen.On the basis of successfully establishing the SD rat bronchus asthma animal model, this study has associatively adopted routine HE, myocardium organizes HSP70 immunohistochemistry,TUNEL and flow cytometry methods,and to study the myocardial damage that every kind of medicine interfere the expression regulation of the term bottom,and to inquiry into the pathologic mechanism in molcule that bronchus asthma myocardial cell has been injuried with period,to provide adequate morphologic grounds for the differential diagnosis of cause of sudden death caused by asthma in the forensic medicine.Methods:There were one hundred and twenty male Sparague-Dawely rates,they were one-month-year-old and 100±15 gram weight or so. On the basis of successfully establishing the SD rat bronchus asthma animal model, they were randomly divided into six groups: a group of adrenaline to deal with asthmatic syndrome, a group of amonophylline to deal with asthmatic syndrome, a group of adrenaline and amoniphylline to deal with asthmatic syndrome, a group of no drugs to deal with asthmatic syndrome, experimental control, normal control. Every group again was divided as four subgroups(2W,4W,6W,8W) according to death time by breaking off the cervical spine .Every subgroup had five rats .Each rat had been drawed its myocardium of cardiac ventricle after death.A slice of myocardium has been put into 10% Formalin solution to fixed, or conventional paraffin embedding and dicing,and has been taken routine HE with myocardium organizes HSP70 immunohistochemistry and TUNEL methods to examine their damage regulation.Another myocardium has been not taken fixing to keep fresh phase,and has been taken flow cytometry (FCM) to examine.All related experiment results has been studied by using microscope with IMAGE analytical system and homologous statisticses.Results:1 routine haematoxylin and eosin dyeingThe comparison groups and other experimental groups are not obviously distinctive.Myocardium cell dyeing and cell transverse striation are clear, and the cell nucleuses are compact, then have not seen any myocardium cell necrosis.Between the myocardium cell the nature has not seen the inflammation germ cell to infiltrate, the partial regions obviously myocardium fiber assumed the wave shape changing and myocardium fiber breaking off.2 myocardium organizes HSP70 immunohistochemistryIn the experimental control and normal control the positive test signal expressing of myocardium cytoplasm HSP70 of SD rat mostly were negative in each period of time.In the other experimental groups the positive test signal expressing of myocardium cytoplasm HSP70 of SD rat had begun to appear at experimental second week,and along with time lengthening,the myocardium cytoplasm HSP70 expression (PI-HSP70 value) had been strengthened gradually, to 8th week it was highest.According to different medicine intervention on the experimental groups, SD rat myocardium cytoplasm HSP70 positive signal expression intensity (PI - HSP70 value) had the remarkable difference in same period of time,were in turn: a group of adrenaline to deal with asthmatic syndrome > a group of adrenaline and amoniphylline to deal with asthmatic syndrome > a group of no drugs to deal with asthmatic syndrome >a group of amonophylline to deal with asthmatic syndrome.The experiment compares between the experimental control and normal control, there were not obvious difference on the PI -HSP70 value.3 TdT-mediated dUTP-biotin nick end labeling-TUNELIn each period of time, the majority of SD rat myocardium cell nucleus TUNEL positive signal expression intensity were negative in experimental control and normal control.In the other experimental groups the positive test signal expressing of myocardium cell nucleus TUNEL of SD rat had begun to appear at experimental second week,and along with time lengthening,the myocardium cell nucleus TUNEL expression (PI-TUNEL value) had been strengthened gradually, to 8th week it was highest.According to different medicine intervention on the experimental groups, SD rat myocardium cell nucleus TUNEL positive signal expression intensity (PI - TUNEL value) had the remarkable difference in same period of time,were in turn: a group of adrenaline to deal with asthmatic syndrome > a group of adrenaline and amoniphylline to deal with asthmatic syndrome > a group of no drugs to deal with asthmatic syndrome >a group of amonophylline to deal with asthmatic syndrome.The experiment compares between the experimental control and normal control, there were not obvious difference on the PI -TUNEL value.4 flow cytometer-FCMMyocardium cell apoptosis rates of all experimental groups obviously were higher than the controls.It had begun to increase gradually at experimental second week,and along with time lengthening, to 8th week it was highest.According to different medicine intervention on the experimental groups, SD rat myocardium cell apoptosis rate had the remarkable difference in same period of time,were in turn: a group of adrenaline to deal with asthmatic syndrome > a group of adrenaline and amoniphylline to deal with asthmatic syndrome > a group of no drugs to deal with asthmatic syndrome >a group of amonophylline to deal with asthmatic syndrome.The experiment compares between the experimental control and normal control, there were not obvious difference on the myocardium cell apoptosis rate. Conclusion:1 Myocardium cell of bronchia asthma SD rat has the certain degrees of damage,and it goes into action the extension of the time along with asthma but aggravation.2 The myocardium cell apoptosis caused by myocardium cell apoptosis mechanism activation promoted by the asthma that is an important of factor in the molecular pathology mechanism of asthma myocardium cell damage.The quantity and the expression of myocardium cell apoptosis goes into action the extension of the time along with asthma but aggravation.3 Some medicines about asthma may promote the myocardium cell apoptosis,such asβ2-acceptors excited medicinal preparations - adrenalin.4 In the forensic medicine about the antidiastole of cause of sudden death caused by asthma, the myocardium cell apoptosis caused by asthma has taken the heart function to suffer injury was a factor which was worth taking.In the forensic pathologic examination about the sudden death caused by asthma, as an assistant diagnosis basis of the myocardium cell damaging.,besides taking the routine HE examination method to examine the myocardium organization, and may consider taking the immunohistochemical method as well as TUNEL and the FCM method.更多还原