过敏性猝死豚鼠体内肥大细胞超微结构及P物质变化的法医病理学研究
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摘要
目的 建立豚鼠过敏性休克动物模型，观察其死后病理变化，并检测血浆中P物质（Substance P, SP）的浓度，探讨P物质在过敏性休克中的作用，试图为过敏性休克的法医学诊断提供客观的诊断依据。
方法 18只豚鼠，随机分为实验组和对照组（每组各9只）。实验组采用多人混合血清致敏豚鼠，建立过敏性休克死亡的动物模型。取豚鼠血浆及脏器组织（咽喉、肺、胃、结肠）。（1）放射免疫学方法测定血浆P物质含量；（2）石蜡切片，常规HE染色；（3）应用免疫组织化学SABC法进行P物质染色，并应用BI-2000图像分析系统进行图像分析，计算阳性指数（PI）；（4）透射电镜观察肥大细胞形态。
结果（1）实验组血浆中P物质的含量明显高于对照组（实验组131.01±18.93pg/μl，对照组87.70±7.60pg/μl，P＜0.01）；（2）实验组HE染色在光镜下形态表现为急死的非特异性改变，对照组未见明显病理变化；（3）免疫组化染色，P物质在实验组的咽喉、肺、胃、结肠等组织中表达明显增加（咽喉组织实验组PI＝63.59±14.51，对照组PI=33.32±8.04 ，P＜0.01；肺组织实验组PI=55.98±14.8，对照组PI＝20.51±6.76，P＜0.01；胃组织实验组PI＝18.97±6.69，对照组PI=13.32±2.48，＜0.01；结肠组织实验组PI＝89.22±13.87，对照组PI=53.20±15.32，P＜0.01）；（4）过敏性休克豚鼠肥大细胞超微结构呈脱颗粒状，对照组豚鼠肥大细胞的胞浆内颗粒丰富，未见脱颗粒现象。
结论 过敏性休克时，豚鼠血浆中P物质含量增高，咽喉、肺、胃、结肠等组织中P物质表达增加，肥大细胞呈脱颗粒的表现。因此，应用放射免疫法测定血浆中P物质的含量和免疫组化法对咽喉、肺、胃、结肠中P物质进行染色，并结合肥大细胞脱颗粒的超微结构，可为过敏性休克的法医学诊断提供客观的血清学和形态学依据。
Objective Guinea pig anaphylactic shock modes were established using heterologous serum. The morphologic changes were observed, and the concentrations of Substance P (SP) in plasma were tested. We are trying to seek objective and sensitive methods to diagnose anaphylactic shock for forensic experts.
Method 18 guinea pigs were divided into two groups randomly, the experimental group and the control group, each of which contains 9 guinea pigs. Guinea pigs in the experimental group were made into anaphylactic shock modes with human sera. Plasma, throat, lung, stomach and colon were kept. We did the follow investigations, (1) Levels of SP in plasma were tested using radioimmunologic method; (2) Routine HE staining; (3) SP was stained using immunohistochemical method, and the images of SP were analyzed by BI-2000 image analyzer, and the positive indexes (PI) were calculated; (4) The form of mast cells were observed under transmission electron microscope.
Results (1) The concentrations of SP of the experimental group are higher than that of the control group; (2) The specimens of the anaphylactic shock cavies show non-specificity sudden death changes, while the ones of the control group have no obvious changes; (3) Expressions of SP in throat, lung, stomach and colon of the experimental group’s cavies are stronger than that in the control group’s ones (P＜0.01); (4) Mast cells have released their granules.
Conclusion Staining SP and measuring the plasma level of SP, combined with the ultrastructure of mast cell, may be a helpful method to diagnose anaphylactic shock for forensic experts.
方法 18只豚鼠，随机分为实验组和对照组（每组各9只）。实验组采用多人混合血清致敏豚鼠，建立过敏性休克死亡的动物模型。取豚鼠血浆及脏器组织（咽喉、肺、胃、结肠）。（1）放射免疫学方法测定血浆P物质含量；（2）石蜡切片，常规HE染色；（3）应用免疫组织化学SABC法进行P物质染色，并应用BI-2000图像分析系统进行图像分析，计算阳性指数（PI）；（4）透射电镜观察肥大细胞形态。
结果（1）实验组血浆中P物质的含量明显高于对照组（实验组131.01±18.93pg/μl，对照组87.70±7.60pg/μl，P＜0.01）；（2）实验组HE染色在光镜下形态表现为急死的非特异性改变，对照组未见明显病理变化；（3）免疫组化染色，P物质在实验组的咽喉、肺、胃、结肠等组织中表达明显增加（咽喉组织实验组PI＝63.59±14.51，对照组PI=33.32±8.04 ，P＜0.01；肺组织实验组PI=55.98±14.8，对照组PI＝20.51±6.76，P＜0.01；胃组织实验组PI＝18.97±6.69，对照组PI=13.32±2.48，＜0.01；结肠组织实验组PI＝89.22±13.87，对照组PI=53.20±15.32，P＜0.01）；（4）过敏性休克豚鼠肥大细胞超微结构呈脱颗粒状，对照组豚鼠肥大细胞的胞浆内颗粒丰富，未见脱颗粒现象。
结论 过敏性休克时，豚鼠血浆中P物质含量增高，咽喉、肺、胃、结肠等组织中P物质表达增加，肥大细胞呈脱颗粒的表现。因此，应用放射免疫法测定血浆中P物质的含量和免疫组化法对咽喉、肺、胃、结肠中P物质进行染色，并结合肥大细胞脱颗粒的超微结构，可为过敏性休克的法医学诊断提供客观的血清学和形态学依据。
Objective Guinea pig anaphylactic shock modes were established using heterologous serum. The morphologic changes were observed, and the concentrations of Substance P (SP) in plasma were tested. We are trying to seek objective and sensitive methods to diagnose anaphylactic shock for forensic experts.
Method 18 guinea pigs were divided into two groups randomly, the experimental group and the control group, each of which contains 9 guinea pigs. Guinea pigs in the experimental group were made into anaphylactic shock modes with human sera. Plasma, throat, lung, stomach and colon were kept. We did the follow investigations, (1) Levels of SP in plasma were tested using radioimmunologic method; (2) Routine HE staining; (3) SP was stained using immunohistochemical method, and the images of SP were analyzed by BI-2000 image analyzer, and the positive indexes (PI) were calculated; (4) The form of mast cells were observed under transmission electron microscope.
Results (1) The concentrations of SP of the experimental group are higher than that of the control group; (2) The specimens of the anaphylactic shock cavies show non-specificity sudden death changes, while the ones of the control group have no obvious changes; (3) Expressions of SP in throat, lung, stomach and colon of the experimental group’s cavies are stronger than that in the control group’s ones (P＜0.01); (4) Mast cells have released their granules.
Conclusion Staining SP and measuring the plasma level of SP, combined with the ultrastructure of mast cell, may be a helpful method to diagnose anaphylactic shock for forensic experts.
引文
郭景元. 实用法医学[M]. 第一版. 上海：上海科学技术出版社，1982.356－361.
Van Hagen PM, Hofland LJ, ten Bokum AM. Neuropeptides and their receptors in the immune system[J]. Ann Med, 1999, 31(Suppl 2):15－22.
Sun W, Tadmori I, Yang l, et al. Vasoactive intestinal peptides (VIP) inhibits TGF-beta Ⅰproduction in murine macrophages[J]. J Neuroi- mmunol,2000, 107:88－89.
郭薇,陈玉川,刘水平等. 肥大细胞体外脱颗粒的检测方法[J]. 中国病理生理杂志，2002,18(8):1023-1024.
林文棠，朱平. 实用临床免疫学[M]. 第一版.西安：第四军医大学出版社，2003.1-18.
Bauer O, Razin E. Mast cell-nerve interactions[J]. News Physiol Sci 2000, 15: 213–218.
Church MK, Clough GF. Human skin mast cells: in vitro and in vivo studies[J]. Ann-Allergy-Asthma-Immunol. 1999, 83(5): 471-475.
路长林. 神经肽基础与临床[M]. 上海：第二军医大学出版社，2000.131-146.
方秀斌. 神经肽与神经营养因子[M].第一版.北京：人民卫生出版社，2002. 50－57.
Cocchiara R, Lampiasi N, Albeggiani G,et al. Mast cell production of TNF-alpha induced by substance P evidence for a modulatory role of substance P-antagonists [J]. J Neuroimmunol. 1999 ,101(2):128-136.
Joos -G-F， Germonpre -P-R， Pauwels -R-A. Neural mechanisms in asthma[J]. Clin-Exp-Allergy. 2000，30 (Suppl 1): 60-65.
Joos -G-F，Germonpre -P-R， Pauwels -R-A. Role of tachykinins in asthma[J]. Allergy. 2000，55(4): 321-337.
Canning -B-J, Fischer –A. Neural regulation of airway smooth muscle tone
[J]. Respir-Physiol. 2001, 125(1-2): 113-127.
Piedimonte G.. Neuroimmune interactions in respiratory syncytial virus-infected airways[J]. Pediatr Infect Dis J. 2002，21: 462–467.
Santoni G，Perfumi MC，Spreghini E，et al. Neurokinin type-1 receptor antagonist inhibits enhancement of T cell functions by substance P in normal and neuromanipulated capsaicin treated rats[J]. J Neuroimmunol, 1999, 93(1-2):15－25.
Goode T，O’Connell J，Ho WZ，et al. Differential expression of neurokinin-1 receptor by human mucosal and peripheral lymphoid cells[J]. Clin Diagn Lab Immunol，2000，7(3)：371－376.
Piedimonte, Giobanni MD. Contribution of neuroimmune mechanisms to airway inflammation and remodeling during and after respiratory syncytial virus infection[J]. Pediatr Infect Dis J. 2003, 22(Suppl 2): S66-S74.
张万会，朱运龙. P物质的免疫调节效应[J].生理科学进展. 1992,23(2):116－121.
Farthing MJ, Casburn-Jones A, Banks MR. Getting control of intestinal secretion: thoughts for 2003[J]. Dig Liver Dis. 2003, 35(6):378-85.
rowicki ZK， Hornby PJ. Substance P in the dorsal motor nucleus of the vagus evokes gastric motor inhibition via neurokinin 1 receptor in rat[J]. J Pharmacol Exp Ther 293, 214－221.
McRitchie DA，T?rk I. Distribution of substance P-like immunoreactive neurons and terminals throughout the nucleus of the solitary tract in the human brainstem[J]. J Comp Neurol. 343, 83 – 101.
杨清玉，彭绍华．实用猝死病理学[M]. 第一版. 北京：群众出版社，1992. 335-337.
黄飞骏，刘世沧. 过敏性休克猝死的法医学鉴定7例[J]. 法医学杂志. 1996（12）:97.
Van Hagen PM, Hofland LJ, ten Bokum AM. Neuropeptides and their receptors in the immune system[J]. Ann Med, 1999, 31(Suppl 2):15－22.
Sun W, Tadmori I, Yang l, et al. Vasoactive intestinal peptides (VIP) inhibits TGF-beta Ⅰproduction in murine macrophages[J]. J Neuroi- mmunol,2000, 107:88－89.
郭薇,陈玉川,刘水平等. 肥大细胞体外脱颗粒的检测方法[J]. 中国病理生理杂志，2002,18(8):1023-1024.
林文棠，朱平. 实用临床免疫学[M]. 第一版.西安：第四军医大学出版社，2003.1-18.
Bauer O, Razin E. Mast cell-nerve interactions[J]. News Physiol Sci 2000, 15: 213–218.
Church MK, Clough GF. Human skin mast cells: in vitro and in vivo studies[J]. Ann-Allergy-Asthma-Immunol. 1999, 83(5): 471-475.
路长林. 神经肽基础与临床[M]. 上海：第二军医大学出版社，2000.131-146.
方秀斌. 神经肽与神经营养因子[M].第一版.北京：人民卫生出版社，2002. 50－57.
Cocchiara R, Lampiasi N, Albeggiani G,et al. Mast cell production of TNF-alpha induced by substance P evidence for a modulatory role of substance P-antagonists [J]. J Neuroimmunol. 1999 ,101(2):128-136.
Joos -G-F， Germonpre -P-R， Pauwels -R-A. Neural mechanisms in asthma[J]. Clin-Exp-Allergy. 2000，30 (Suppl 1): 60-65.
Joos -G-F，Germonpre -P-R， Pauwels -R-A. Role of tachykinins in asthma[J]. Allergy. 2000，55(4): 321-337.
Canning -B-J, Fischer –A. Neural regulation of airway smooth muscle tone
[J]. Respir-Physiol. 2001, 125(1-2): 113-127.
Piedimonte G.. Neuroimmune interactions in respiratory syncytial virus-infected airways[J]. Pediatr Infect Dis J. 2002，21: 462–467.
Santoni G，Perfumi MC，Spreghini E，et al. Neurokinin type-1 receptor antagonist inhibits enhancement of T cell functions by substance P in normal and neuromanipulated capsaicin treated rats[J]. J Neuroimmunol, 1999, 93(1-2):15－25.
Goode T，O’Connell J，Ho WZ，et al. Differential expression of neurokinin-1 receptor by human mucosal and peripheral lymphoid cells[J]. Clin Diagn Lab Immunol，2000，7(3)：371－376.
Piedimonte, Giobanni MD. Contribution of neuroimmune mechanisms to airway inflammation and remodeling during and after respiratory syncytial virus infection[J]. Pediatr Infect Dis J. 2003, 22(Suppl 2): S66-S74.
张万会，朱运龙. P物质的免疫调节效应[J].生理科学进展. 1992,23(2):116－121.
Farthing MJ, Casburn-Jones A, Banks MR. Getting control of intestinal secretion: thoughts for 2003[J]. Dig Liver Dis. 2003, 35(6):378-85.
rowicki ZK， Hornby PJ. Substance P in the dorsal motor nucleus of the vagus evokes gastric motor inhibition via neurokinin 1 receptor in rat[J]. J Pharmacol Exp Ther 293, 214－221.
McRitchie DA，T?rk I. Distribution of substance P-like immunoreactive neurons and terminals throughout the nucleus of the solitary tract in the human brainstem[J]. J Comp Neurol. 343, 83 – 101.
杨清玉，彭绍华．实用猝死病理学[M]. 第一版. 北京：群众出版社，1992. 335-337.
黄飞骏，刘世沧. 过敏性休克猝死的法医学鉴定7例[J]. 法医学杂志. 1996（12）:97.