【作者】 雷红宇；

【导师】 袁慧；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2009， 博士

【摘要】 圆弧青霉菌毒素-青霉酸(Penicillic Acid,PA)是由圆弧青霉菌产生的主要有毒代谢产物之一。许多饲料原料如高粱、大麦、燕麦、小麦、玉米和大米上都适合青霉酸的生产,且产量较高,是霉变饲料中的主要真菌毒素。青霉酸对多种动物有不同程度的毒性作用,且与其他霉菌毒素有累加、协同甚至增强作用,给动物和人们的身体健康带来很大的危害,因此迫切需要开展免疫学快速检测技术和毒理学的研究。本文通过化学方法合成了青霉酸完全抗原,并通过动物免疫和细胞融合技术制备青霉酸的单克隆抗体,研究了青霉酸在体内和体外对小鼠免疫器官和细胞的毒性效应和机理。主要研究内容和结果如下:1.通过碳二亚胺法将青霉酸(PA)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)联结,得到青霉酸人工完全抗原PA-BSA和PA-OVA。采用紫外扫描光谱法、SDS-PAGE和动物免疫试验对合成的抗原进行鉴定。结果显示偶联后的人工抗原特征性吸收峰出现偏移,人工抗原分子量比其相应载体的分子质量略大,PA与BSA的偶联比为23.2:1,PA与OVA的偶联比为10.4:1。通过动物免疫,以PA-BSA为免疫抗原免疫小鼠,制备了多抗血清。以PA-OVA为包被抗原,采用间接ELISA检测抗血清,其效价达到1:12800。表明合成的青霉酸人工完全抗原具有良好的免疫原性。2.以合成的完全抗原PA-BSA免疫小鼠,采用杂交瘤技术,经细胞融合、克隆筛选、扩大培养获得一株针对青霉酸的杂交瘤细胞4C2-F8-D2。通过动物体内诱生腹水法获得了单克隆抗体。纯化后的抗体效价为1:2×10~5,抗体类型为IgG1型。单克隆抗体与黄曲霉毒素B1、玉米赤霉烯酮、T-2毒素和伏马毒素B1均无交叉反应率,单抗的亲和常数为1.54x10~8 L/mol,为进一步建立青霉酸的免疫学快速检测方法提供了基础。3.通过体外染毒法,观察了1、3、5和10μg/ml剂量的青霉酸对小鼠脾脏淋巴细胞抗氧化酶含量、细胞增殖和DNA损伤的影响。结果表明,染毒后细胞培养液中SOD活力降低,MDA含量增加,NO浓度增加,并且有染毒剂量依赖性,说明青霉酸能降低抗氧化酶活性而影响机体的抗氧化系统。通过MTT法观察青霉酸对小鼠脾脏淋巴细胞增殖的影响,结果表明不同浓度的青霉酸对淋巴细胞的增殖均有抑制作用。通过单细胞凝胶电泳观察青霉酸对小鼠脾脏淋巴细胞DNA损伤的情况,表明高剂量的青霉酸可以引起淋巴细胞DNA损伤,说明青霉酸县一种潜在的致癌物质。4.用0.3125mg/ml(低)、1.25mg/ml(中)、5mg/ml(高)三个剂量的青霉酸人工染毒小鼠,对染毒后小鼠的免疫器官脾脏和胸腺进行了组织病理学和超微结构的观察,并通过TUNEL法进行了细胞凋亡的检测。结果显示,青霉酸对胸腺的损伤较轻微,仅在高剂量组出现胸腺的萎缩和淋巴细胞的减少;青霉酸对脾脏的损伤较严重,随着染毒剂量的增加,脾脏的损伤加重,在高剂量组出现明显的淋巴细胞死亡。超微结构观察到中、高剂量组脾脏淋巴细胞的异常凋亡和细胞器发生病变,TUNEL法检测出中、高剂量组脾脏韵细胞凋亡数量增加。说明青霉酸能引起脾脏的细胞凋亡。5.通过RT-PCR半定量检测方法对凋亡基因Bcl-2、Bax、p53、Fas和FasL基因在染毒后小鼠脾脏和胸腺组织中mRNA表达进行了研究。结果表明,中高剂量青霉酸染毒后,脾脏组织的Bcl-2的表达降低,Bax、Fas和FasL的表达升高,说明Bcl-2和Bax、Fas/FasL基因可能在青霉酸致使脾脏细胞凋亡过程中起重要作用。在胸腺组织中,凋亡因子的表达与脾脏不同,染毒后所有染毒组Bcl-2和p53表达增加、Fas表达降低,Bax表达在高剂量组增加,其他组降低,FasL在低剂量组增加,其他组降低,说明青霉酸可能会抑制胸腺的细胞凋亡。更多还原

【Abstract】 Penicillic Acid(PA) is one of the main metabolites generated from Penicillium cyclopium.Many feedstuff raw materials,such as sorghum,barley,oats,wheat,corn and rice were suitable for the production of PA with a high output as the main mycotoxin in moldy feedstuff.To some extent,PA has toxic effect on many animals and the interaction of PA and other mycotoxin on animals will contribute to cumulative toxicity and enhance its toxicity.Hence,rapid and effective approaches of testing should be discovered and research on its toxicology to cope with the serious threat of PA posed to animals and humans.In this article,complete antigens of perficillic acid were synthesized by chemical methods and monoclonal antibodies were prepared through animal immunization and cell fusion technology.Toxicity of penicillic acid to immune organs and cells of mice was researched in vivo and vitro. The main research content and results are as follows:1.PA was conjugated to bovine serum album(BSA) and ovalbumin(OVA) by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride(EDC).The artificial antigens PA-BSA and PA-OVA were identified by Ultra-violet spectrometric scanning, SDS-PAGE and immunization.Results showed that the absorption peak of conjugation were different from that of the carrier protein alone and of the PA.The conjugated ratio of PA and BSA was 23.2:1 and that of PA and OVA was 10.4:1. Balb/c mice were immunized by the artificial antigen of PA-BSA,with PA-OVA as coating antigen.It was experimentally found that the average titer of antiserums was more than 12800 by indirect ELISA.It indicated that the complete antigens were obtained.2.Hybridoma cell strains were selected after fusion of mouse SP2/0 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with Penicillic Acid(PA) conjugated to bovine serum albumin(BSA) by ELISA.A monoclonal antibody specific for PA was produced in vivo by the hybridoma cell. Analysis revealed that the monoclonal antibody was of the IgG1 type,with the titer was 1:2×10~5.The relative cross-reactivity of PA with AFB-1,ZON,T-2,FB were less than 0.01%and with an affinity constant for PA of about 1.54×10~8 liters per mol.It offered a foundation for developing immunoassay method for PA. 3.Spleen lymphocytes were separate and exposed to PA at the dosage of 1,3,5 and 10μg/ml for identify the toxicity of PA in vitro.Anti-oxidase content,the rate of the cell proliferation and DNA damage were examined.Results showed that the activity of is lower than the control,but the concentration of MDA and NO increased which was dose-dependent on PA.Penicillic acid can reduce the activity of antioxidant enzymes.MTT was adopted to observe the spleen lymphocyte proliferation,which showed the proliferation of lymphocytes decreased when exposed to PA.PA with higher dose can cause lymphocyte DNA damage by single cell gel electrophoresis,which indicated PA is a potential carcinogen.4.Three groups of mice were exposed to different dose of PA,and then the effect of PA on mice in vivo,apoptosis,histological and ultra-structural changes of the immune organs of the mice were observed.Observation of transmission electron microscope was carried out to study the ultra-structural changes,and Tunnel analysis was used to study the apoptosis of mice spleen.Results showed that no obvious changes were found in the thymus except partial atrophy and lymphocyte reduced in the highest dose group.More serious injures were found in spleen by seen the lymphocyte death and aggravated with the dose of PA increased.Typical apoptotic lymphocytes were found both in middle and high dose group,and also a significant increase of apoptosis rate compared with the control.5.The expression of apoptosis gene Bcl-2,Bax,p53,Fas and FasL mRNA were measured by RT-PCR in spleen and thymus of mice exposed to PA at different dose. Results showed that the expression of Bcl-2 decreased and Bax,Fas,FasL increased in spleen at middle and high dose group,which induced Bcl-2,Bax,Fas/FasL may play an important role in the process of apoptosis in spleen.But it was different in thymus from the spleen.The expression of Bcl-2 and p53 increased,Fas decreased in thymus.We supposed that PA may inhibit apoptosis in thymus.更多还原