用黄曲霉毒素B1单抗介导免疫组化法检测雏鸭感染黄曲霉毒素的分布规律研究  

Research on the Invading Process and Distribution of Aflatoxin B1 in Artificially Infected Ducklings by Development and Application of Monoclonal-Antibody Mediated Immunohistochemistry

作　　者：程安春[1] 刘艳丽[1] 朱德康[1] 汪铭书[1] 胡骑[1] 陈孝跃[1] 

机构地区：[1]四川农业大学动物医学院禽病防治研究中心

出　　处：《中国农业科学》2008年第8期2460-2466,共7页Scientia Agricultura Sinica

基　　金：国家农业科技成果转化资金项目(2006GB2F000249);国家“十一五”科技支撑计划(2007Z06-017);教育部“新世纪优秀人才支持计划”项目(NCET-04-0906/NCET-06-0818);四川省重点建设学科项目(SZD0418)

摘　　要：【目的】(1)建立单抗介导的、能够对黄曲霉毒素AFB1进行亚细胞定位的免疫组化方法;(2)探明AFB1侵染和分布于雏鸭靶器官(细胞)规律,为阐明AFB1对雏鸭的致病机理提供基础实验数据,为AFB1感染人类提供研究模型。【方法】(1)利用AFB1单抗、免疫组化理论和方法,结合石蜡切片技术,建立检测感染雏鸭组织器官和细胞中AFB1方法;(2)7日龄樱桃谷鸭饲喂黄曲霉毒素(AFB1)含量为150μg·kg-1的全价饲料,分别于6、12、24、48、72、96、120、144、168和192h各剖杀2只,取心、肝、脾、肺、肾、脑、十二指肠、法氏囊、胸腺和胰腺等组织器官多聚甲醛固定、石蜡包埋,应用建立的单抗介导免疫组化对AFB1在感染雏鸭体内侵染的组织器官和细胞进行定位检测。【结果】(1)建立的单抗介导的免疫组化能够特异性检测到感染雏鸭组织中的AFB1,而对鸭病毒性肝炎病毒、鸭疫里默氏杆菌、鸭多杀性巴氏杆菌、鸭沙门氏菌和鸭大肠杆菌感染鸭组织呈现阴性反应;(2)雏鸭采食含AFB1饲料后,24h可在肝脏和肾脏中检测到AFB1,随后在脾脏(48h)、胰腺(96h)、十二指肠(120h)、心肌(144h)及法氏囊(168h)检测到AFB1,其中肝脏和肾脏的阳性结果最强;AFB1分布于肝脏肝窦及汇管区周围炎性反应带的肝细胞,肾脏肾小管、集合管上皮细胞以及肾小球毛细血管,脾脏白髓及炎性细胞,胰腺腺上皮细胞,十二指肠脱落的黏膜上皮细胞,心脏血管周围和心脏发生空泡变性的心肌纤维中;AFB1主要集中分布于细胞核内,而细胞膜和细胞浆内也有少量的分布。【结论】(1)单抗介导免疫组化能够特异检测AFB1感染雏鸭组织石蜡切片中的AFB1和亚细胞定位,还可用于AFB1感染雏鸭的实验室诊断和甲醛固定组织的回顾性诊断;(2)AFB1可广泛侵害感染雏鸭各个组织器官,以肝脏和肾脏最为严重。AFB1主要蓄积于被感染的细胞核。[Objective ] （1） To develop a monoclonal-antibody mediated immunohistochemistry to detect AFB1 in sub-localization of infected cell. （2） To study the regularity invasion and distribution of aflatoxin B1 in artificially infected ducklings, to provide experimental data for explaination of pathogenesis of aflatoxin B1 in duckling, to provide research model for human. [Method] （1） Based on the monoclonal-antibody, theory and method of immunohistochemistry, and paraffin slice technology, a immunohistochemistry which can detect and localize aflatoxin B 1 in tissues and cells of infected duckling was developed. （2） The 7-day-old Cherry Valley Ducklings which were fed with complete feed containing 150μg·kg^-1 aflatoxin B1 （AFB1） and two ducklings were killed on 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h and 192 h after treatment respectively, and the specimens of heart, liver, spleen, lung, kidney, brain, duodenum, bursa of Fabricius, thymus and pancreas were collected. These specimens were fixed with paraformaldehyde, embedded by paraffin, and used to establish the monoclonal-antibody mediated immunohistochemistry to detect and localize AFB l in infected ducklings. [ Result ] （1） The method can detect AFB 1 in sections from paraffin embedded specimens and showed accuracy in localization of AFB1 in tissues and organs of infected ducklings specifically, but negative results were noticed in died ducks liver specimens infected with duck viral hepatitis, duck Riemerella anatipestifer, duck pasteurella multocida, duck Escherichia coli and duck Salmonella. （2） 24h post treatment, AFB 1 was firstly detected in the organs of liver and kidney, then in spleen （48 h）, pancreas （96 h）, duodenum （120 h）, cardiac muscle and bursa of Fabricius （168 h）, and the most obvious signals were detected in liver and kidney. The distribution and localization of AFB 1 were as follows： the hepatocytes in the inflammatory band around hepatocyte sinus and protral area of hepatic lobules, re

关 键 词：免疫组化 黄曲霉毒素 雏鸭 侵染 

分 类 号：S858.32[农业科学—临床兽医学]