【作者】 高彩荣；

【导师】 王英元；

【作者基本信息】 山西医科大学 ， 法医学， 2008， 博士

【摘要】 流行病学调查资料表明,随着工业化的发展,过敏性疾病的发病率明显增高,并正在快速增长,已经成为21世纪严重影响人类健康的全球性疾病。因过敏反应而致的过敏性休克或猝死在临床医学及法医学鉴定中也较为常见,据文献报道,国际上平均每年1000,000住院病人中发生致命性的过敏性休克有154例,在美国过敏性反应的死亡率高达3%。而且在法医学鉴定中因其他原因猝死者也常常被怀疑为过敏性休克死亡。因此过敏性休克猝死在法医学鉴定中占有很重要的地位,据本教研室资料统计近年来涉及过敏性猝死的法医学鉴定占医疗纠纷法医学鉴定的10%左右。虽然过敏性疾病一直是临床医学研究的热点,国内外学者就其发病机制、临床诊断和临床治疗做了大量研究。但在法医学实践中,过敏性猝死尸检时仅见猝死的一般征象而无特异性的病理形态学改变,法医学鉴定时要诊断过敏性休克死亡必须在排除自然性疾病、中毒及其它暴力性死亡原因的基础上,结合接触过敏原史、临床突发休克等症状才可诊断,因此给法医学鉴定带来很大困难。寻找一种客观、准确的过敏反应的诊断指标和方法便成为临床医学和法医学鉴定领域亟待解决的问题。因此,本研究利用豚鼠和小鼠制备过敏性猝死的动物模型,采用酶联免疫吸附试验、放射免疫法和免疫组化ABC法分别测定死后不同时间过敏性猝死豚鼠血清IgE、P物质含量及其在肺组织内的免疫表达;采用原位杂交的方法检测小鼠过敏性休克后不同时间咽喉、肺、胃组织类胰蛋白酶mRNA、ECPmRNA及P物质的表达。同时对115例免疫正常人血清和15例因过敏性休克死亡者采用荧光酶联免疫法(FEIA)和免疫组织化学染色方法,分别测定血清类胰蛋白酶的含量和观察类胰蛋白酶、SP在相关组织中的免疫表达,以探讨SP与过敏性休克的关系及类胰蛋白酶、IgE、SP在过敏性休克死亡法医学诊断中的应用价值。第一部分过敏性休克豚鼠、小鼠体内IgE、类胰蛋白酶和SP的变化目的测定豚鼠过敏性休克后血清IgE的含量和在肺组织中的免疫表达,并观察在死后不同时间血清IgE含量的变化规律;观察小鼠致敏前和过敏性休克后咽喉、肺和胃肠组织类胰蛋白酶mRNA和ECPmRNA的免疫表达;测定过敏性休克豚鼠血清P物质的含量并观察其在咽喉、肺和胃肠组织的免疫表达。方法分别采用多人混合血清和卵白蛋白(OVA)制备豚鼠和小鼠过敏性休克动物模型;采用酶联免疫吸附试验(ELISA)测定过敏性休克猝死的豚鼠血清IgE的含量及在死后0h、12h、24h、48h的变化规律,采用免疫组织化学方法观察IgE在肺组织中的表达:采用原位杂交的方法检测小鼠过敏性休克30min、1h、6h、24h咽喉、肺和胃组织类胰蛋白酶mRNA、ECPmRNA的表达;采用放射免疫法和免疫组化ABC法分别测定和观察过敏性休克死亡豚鼠血清P物质的含量和在咽喉、肺及胃肠道组织中的免疫组化染色。结果(1)对照组豚鼠血清IgE＜1U/ml,而且在死后12、24、48 h血清IgE含量变化没有明显差异。过敏性猝死豚鼠血清IgE含量在死亡0、12、24、48h分别为13.25±4.32U/ml、15.87±4.66U/ml、12.31±3.13U/ml、14.61±5.46U/ml,与对照组相比其差异具有显著性意义(P＜0.001),过敏性猝死豚鼠死后0、12、24、48h血清IgE含量相比无明显变化(各组之间比较P＞0.05)。实验组豚鼠肺小血管壁、血管周围和肺泡壁可见IgE棕黄色染色,对照组豚鼠相应组织染色阴性。采用BI-2000病理图像分析系统对肺组织IgE免疫组化染色进行分析,与对照组相比,过敏性休克猝死各组豚鼠肺组织IgE平均密度显著增高(P＜0.001),实验各组豚鼠肺组织IgE平均分布密度无显著性差异(P＞0.05)。(2)过敏性休克猝死豚鼠血浆P物质浓度为131.01±18.93pg/μl,对照组血浆P物质浓度为87.70±7.60pg/μl,与对照组相比,猝死组血浆P物质浓度显著升高(P＜0.01)。过敏性猝死豚鼠咽喉、肺、胃、结肠粘膜P物质免疫组化染色均呈阳性或强阳性,而对照组豚鼠相应组织中的免疫组化染色呈阴性或弱阳性(P＜0.01)。(3)致敏后小鼠咽喉、肺、胃组织类胰蛋白酶mRNA、ECPmRNA平均光密度均高于正常对照组。各组织类胰蛋白酶mRNA在致敏后30′达到高峰,随后逐渐下降,24h后仍高于正常水平。咽喉ECPmRNA于致敏后30′开始升高,并保持较高水平;肺组织ECPmRNA在致敏后6h达到高峰;胃组织则在致敏后24h后达高峰。结论4℃下冷藏,在死后48h内血清IgE含量无明显变化,血清IgE含量升高可以作为过敏性猝死法医学鉴定的客观依据。SP作为一种能够被神经系统和免疫系统共同识别的信号物质,参与了过敏性休克的发生和发展。类胰蛋白酶mRNA在过敏性休克小鼠体内的表达高峰早于血清的类胰蛋白酶高峰,能更早地说明过敏性休克的发生,ECPmRNA的达峰时间较类胰蛋白酶晚,是过敏反应迟发相的重要标志。第二部分人(尸)体过敏性猝死法医病理学诊断研究目的测定免疫正常人群血清类胰蛋白酶的含量,建立正常值范围;观察已诊断为过敏性猝死的尸体血清类胰蛋白酶、IgE的含量及类胰蛋白酶和P物质在咽喉、肺和胃肠道组织的表达与非过敏性死亡者之间的差异,探讨过敏性猝死的死后诊断方法。方法应用荧光酶联免疫法(FEIA)测定115例正常人和6例过敏性休克猝死者的血清类胰蛋白酶;采用酶联免疫吸附试验(ELISA)测定7例过敏性猝死者血清IgE含量;采用免疫组织化学的方法观察15例过敏性休克猝死者的咽喉、肺、胃肠组织的类胰蛋白酶和P物质的免疫组织化学染色并采用BL-2000病理图像分析系统进行定量分析。结果(1) 115例免疫正常人血清中肥大细胞类胰蛋白酶最低值为0.80μg/L,最高值为8.86μg/L,算术均数为3.06μg/L,几何均数为2.69μg/L,95%分位数为6.00μg/L。男女性别之间无明显差异(P＞0.05)。20岁以下组、21—40岁组和41—60岁组之间血清中类胰蛋白酶含量无明显差别(P＜0.05),而60岁以上组血清中类胰蛋白酶含量高于其他年龄组(P＜0.05);(2) 6例过敏性猝死者血清类胰蛋白酶含量分别为25.01μg/L、8.91μg/L、19.70μg/L、6.01μg/L、10.15μg/L、19.52μg/L,均高于免疫正常人血清中类胰蛋白酶正常值;(3)测定7例过敏性猝死者血清IgE,有6例血清IgE水平高于IgE正常值(大于333U/ml),1例血清IgE水平在正常值范围内(260.82U/ml);(4)过敏性猝死者咽喉、肺和胃肠道组织类胰蛋白酶含量升高,与对照组之间的差异具有显著性(P＜0.01)。(5)过敏性猝死者咽喉、肺和胃肠组织的P物质PI值分别为129.839、99.2880、113.6031,而对照组分别为44.8630、39.5439、68.7888(P＜0.001)。结论115例免疫正常人血清中类胰蛋白酶含量的正常参考值＜6.00μg/L,含量无男女性别之间差异,60岁以下无年龄差异。过敏性猝死者血清类胰蛋白酶含量及咽喉、肺和小肠组织类胰蛋白酶含量升高,过敏性猝死者咽喉、肺和胃组织的SP表达明显升高。因此说明,死后测定尸体血清IgE含量并结合血清类胰蛋白酶的测定可以确诊过敏性猝死,肺组织的类胰蛋白酶免疫组化染色在一定程度上反映了机体类胰蛋白酶的活性和含量,对那些失去条件检测血清中IgE和类胰蛋白酶、而又高度怀疑过敏性猝死案例的死后法医学鉴定具有一定的参考价值。更多还原

【Abstract】 Investigation of epidemiological indicated that incidence of the hypersensitivity which is 21 per 100,000 person is increasing with the development of industrialization and the hypersensitivity disease have become the global disease that threated people health acutely.It is common that anaphylactic shock or sudden death result from allergy in clinical medicine and forensic medicine.Every year there are approximate 154 fatal anaphylacticus episodes during 1,000,000 hospitalzed subjects internationally and death rate of anaphylactic shock in the United States is 1%to 3%according to references.The sudden deaths from other causes are possiblebly suspended as the sudden death from hypersensitivity in medicolegal expertise. Therefore identification of anaphylactic shock and sudden death from hypersensitivity is very significant in medicolegal expertise.The medicolegal expertise involving in sudden death from hypersensitivity is about 10%in the medicolegal expertise of medical tangle at resent years according to our laboratory data.Hypersensitivity disease is the study hot spot.Although scholars have researched much on pathogenesy,clinical diagnosis and clinical therapy of hypersensitivity disease,we still can not find the specialpathological changes on autopsy and histological examination in the dead body sudden death from hypersensitivity.It is difficult to diagnosis the sudden death from hypersensitivity in the medicolegal expertise.Diagnosis of anaphylaxis is based on the occurrence of characteristic symptoms,especially when they develop upon exposure to a potential trigger.If we diagnose sudden death from hypersensitivity,we must do exclude the death from natural disease,poisoning and other violence,and combine the history of touching anaphylactogens.It is urgent to search a subject and accurate method of diagnosis in clinic medicine and forensic medicine.Therefore,the present study makes the animal mold of anaphylactic shock and sudden death from hypersensitivity with rats and guinea pigs,detects and analyses the content of IgE and P substance in the serum and to make staining in P substance in the lung utilizing enzyme linked immunosorbent assay and radioimmunity and immunohistchemistry respectively;to detect content of tryptase mRNA and of ECPmRNA and make staining in P substance in throat,lung,stomach of rats dead from anaphylactic shock at different time with hybridization in situ.In addition,we detect the serum level of the mast cell tryptase in 115 healthy adults and 15 cases dead from hypersensitivity with fluor euzymelinked immunosorbent assay and survey immunostaining of the mast cell tryptase and P substance in tissue with immunohistochemistry in order to discuss P substance relating to anaphylactic shock and the application value of the level of tryptase,IgE and P substance by measurements in medicolegal expertise.Part 1.The change of IgE,Tryptase and P substance in rats and guinea-pigs of anaphylactic shockObjective To detect the content and change rules of IgE in the serum and IgE immunostaining in the lung of the guinea-pigs in the anaphylactic shock expriments and observe change rules of IgE in the serum at different times after guinea-pig died.To survey tryptase mRNA and ECPmRNA immunostaining in throat,lung,stomach of rats in pre- and post-anaphylaxis.To measure the content of P substance in the serum of the guinea-pigs of anaphylactic shock and observe the immune-state of P substance in throat,lung,stomach of the guinea-pigs of anaphylactic shock.Methods Make the anaphylactic shock model of the guinea-pigs and the rat with respectively the mixed serum of different people and Ovalbumin(OVA).To detect the content of IgE in the serum of the guinea-pigs death from anaphylactic shock utilizing enzyme linked immunosorbent assay,and observe the ch-ange rules of in the serum of the guinea-pigs death from anaphylactic shock in 0h、12h、24h、48h after the guinea-pigs died.To observe expression of IgE in lung of the guinea-pigs death from anaphylactic shock utilizing immunohistochemistry. To survey expression of tryptase mRNA and ECPmRNA in throat,lung,stomach of rats of anaphylactic shock at different times with hybridization in situ.To detect the content of P substance in the serum of the guinea-pigs of anaphylactic shock with radio-immunity.To survey the expression of P substance in throat,lung,stomach of the guinea-pigs of anaphylactic shock with immunohistochemistry.Results(1) The content of IgE in control group＜1 Iu/ml,and the content of IgE have not obvious difference at 12h、24h、48h after the guiea-pigs of control groups died.The content of IgE in the guiea-pigs dead from anaphylactic shock is 13.25±4.32 Iu/ml、15.87±4.66 Iu/ml、12.31±3.13 Iu/ml、14.61±5.46 Iu/ml respectively at 0h、12h、24h、48h after the guiea-pigs died. There is the significant difference between the control group and the experimental group(P＜0.001).The content of IgE is not obvious difference at 12h、24h、48h after the guiea-pigs died from anaphylactic shock(each groups compare P＞0.05).The result of immunohistochemistry dyeing indicates that there are some pale yellow granulas in the lung small vessle wall、circum-vessel and alveolar wall of the experimental groups.It is not change accordingly in the experimental group.To compare with the control group,the average density of IgE in the experimental groups increases significant by analysis with BI-2000 patho-image analysis system(P＜0.001),there is not significant difference in experimental groups(P＞0.05).(2) The content of P substance in the serum of the guinea-pigs of anaphylactic shock is 131.01±18.93pg/μl,the content of P substance in the control groups is 87.70±7.60pg/μl.To compare to the control group,the content of P substance in the serum of the guinea-pigs of anaphylactic shock increases significantly(P＜0.01).Immunohistochemistry expression of P substance in throat,lung,stomach of the guinea-pigs of anaphylactic shock present masculine or hardro-masculine,but immunohistochemistry expression of P substance in throat,lung,stomach of the control group present negative or weakly positive(P＜0.01).(3) The average density of tryptase mRNA and of ECPmRNA in throat,lung,stomach of rats of anaphylactic shock is higher than the control groups.The content of tryptase mRNA in each tissues achieve peak in 30 minutes after allergize,sequentia the content of tryptase mRNA fall-off,still is higher than normal level in 24 hours after allergize.The content of ECPmRNA in throat begin to increase,and maintain a higher level in 30 minutes after allergize,The content of ECPmRNA in lung achieve peak in 6 hours after allergize,The content of ECPmRNA in stomach achieve peak in 24 hours after allergize.Conclusion The content of IgE in the serum have no marked change in 4℃,measuring the serum level of IgE can provide an objective diadynamic criteria to diagnose anaphylactic shock for forensic experts.P substance as the signal materil which can be recognized by nervous system and immune system participate in anaphylactic shock’ happening and development.The content of tryptase mRNA in rats of anaphylactic shock can achieve peak earlier than the content of ECPmRNA,tryptase can earlier illustrate the happening of anaphylactic shock.ECPmRNA achieve peak late,it is an important signal of anaphylactic response late onset phasing.Part 2.Study of forensic pathologic diagnosis in human(dead) body sudden death from hypersensitivityObjective To detect the content in the serunm of health adult,eatablish range of normal values.To observe the tryptase’and IgE’ content in the serum of dead body who be diagnosed sudden death from hypersensitivity,and to observe the difference of P substance’expression in throat,lung,stomach between health adults and dead bodies died from hypersensitivity in order to approach the diagnosis method of sudden death from hypersensitivity.Methods To detect the serum level of the mast cell tryptase in 115 healthy adults and 15 cases dead from hypersensitivity with fluor euzymelinked immunosorbent assay and to detect the content of IgE in the serum of 7 cases dead from hypersensitivity with enzyme linked immunosorbent assay,and to survey immunostaining of the mast cell tryptase and P substance in throat,lung,stomach of 15 cases dead from hypersensitivity with immunohistochemistry, and to make quantitative analysis with BI-2000 patho- image analysis system. Results(I) The content of Mast cell tryptase in the serum of 115 healthy adults,the nadir is 0.80μg/L,the maximum is 8.86μg/L,the arithmetic mean is 3.06μg/L,the geometric mean is 2.69μg/L,the 95 percentage quantile is 6.00μg/L.There is not obvisous difference between man and woman(P＞0.05).There is not obvisous difference among healthy individuals＜21 years, healthy individuals 21 to 40 years,healthy individuals 41 to 60 years,healthy individuals＞60 years(P＜0.05).(2) The content of tryptase in the serum of 6 cases sudden dead from hypersensitivity is respectively 25.01μg/L,8.91μg/L,19.70μg/L,6.01μg/L,10.15μg/L, 19.52μg/L,the content of tryptase in the serum of 6 cases is higher than the content of tryptase in the serum of healthy individuals.(3) The content of IgE in the serum of 7 cases dead from hypersensitivity there are 6 cases which higher than normal level(＞333U/ml),1 case is within normal(260.82 U/ml).(4) The content of tryptase in throat,lung,stomach of the dead bodies died from hypersensitivity is higher than healthy individual,there is significant difference compare with control group(P＜0.01).PI of P substance in the throat,lung, stomach respectively is 129.839、99.2880、113.6031、PI of the control group respectively is 44.8630、39.5439、68.7888(P＜0.001).Conclusion The normal range of tryptase in the serum of 115 healthy individuals is 0μg/L～6μg/L,there is not obvisous difference between man and woman.There is not obvisous difference among the people whose age are below 60 years,the content in the serum of the people whose ages are above 60 years.The content of tryptase in the serum of the individuals dead from hypersensitivity and the content of tryptase in throat,lung,stomach of the dead bodies died from hypersensitivity is higher than normal level.The expression of P substance in the throat,lung,stomach of the dead bodies died from hypersensitivity enhance.Accordingly, to detect the content of IgE and tryptase in the serum of the dead body can diagnosis sudden death from hypersensitivity,immunohistochemistry dyeing of tryptase in the lung can reflect tryptase’activity and content.In these cases which have no condition to detect IgE and tryptase in the serum but is highly suspected sudden death from hypersensitive,we can observe immunohistochemistry dyeing of tryptase in the lung.更多还原