无菌豚鼠的培育及无菌豚鼠EV71比较医学研究
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摘要
豚鼠是一种应用广泛的实验动物。可以用于免疫学、传染病、药物学、儿科学等方面的研究。无菌豚鼠在生物医学实验中因为排除了病原体和环境因素的影响,受抗原刺激时,免疫反应强烈且单一,因此在生命科学研究中有着广阔的应用前景。1932年Reyniers获得无菌豚鼠。我所自1980年开始研究无菌豚鼠并培育成功,为我国无菌豚鼠的发展和应用奠定了基础。但限于当时的生物医学发展水平,国内对无菌豚鼠的需求较少,而没能进行规范化培育与推广、应用。随着生命科学的发展和生物医学的进步,重大疾病特别是重大传染病研究的深入,无菌豚鼠将逐渐显示出其重要价值。
EV71是一种含有正向RNA单链无包膜病毒,属于肠道病毒属,小核糖核酸病毒科。EV71和CA10、CA16等共属于A性肠道病毒,容易引起手足口病。但是EV71的感染有时会产生严重的神经系统疾病,如无菌性脑炎、脑干脑炎、和脊椎灰质炎相似的麻痹。自1974年首次报道以来，EV71已在世界范围内引起十多次爆发与流行。例如澳大利亚、欧洲、亚洲和美国。近年来在亚太地区有上升的趋势。虽然HFMD的流行性很强,但是对其致病机理以及感染后比较医学方面的研究很少。其主要原因之一是EV71很难感染动物,制作成稳定的动物模型。并且国内外建立成功的动物模型多为出生七日以内的乳鼠,难以采集血液、器官材料进行比较医学方面的研究。
基于以上理由,本论文开展了无菌豚鼠制作技术的规范化和无菌豚鼠EV71比较医学的研究。
无菌豚鼠制作技术的规范化实验采取子宫摘除术的方法对普通级豚鼠进行剖腹净化,在无菌隔离器内剥离子宫取仔,用人工哺乳的方法将仔豚鼠培育成无菌豚鼠,并每隔一段时期进行无菌检测。以保证无菌豚鼠的质量。同时对孕豚鼠、豚鼠子宫摘除手术日期、豚鼠人工乳、人工饲料等进行对比选择。40日左右时,分别对动物心脏取血,自动生化分析仪测定无菌豚鼠、普通级豚鼠血液学参数。比较三组动物血常规、血生化有无差异。为无菌豚鼠的应用和推广奠下基础。
无菌豚鼠EV71比较医学研究实验利用第一部分得到的无菌豚鼠作为模型动物,利用多种接种方法接种病毒。分别进行临床观察、病原学检查,用来验证动物感染动物模型成立。同时对不同组感染后动物模型的一些免疫指标如：EV71特异性抗体,动物基础性IgG、IgA、IL-2、IL-6水平及CD4/CD8变化进行比较分析。感染了EV71的无菌豚鼠出现疱疹、腹泻等临床症状,普通豚鼠无明显的临床症状。病原学检测无菌豚鼠100%感染病毒,在多个组织中可检测到病毒；普通豚鼠病毒检出率低并无明显的规律性。免疫学检测中,首次发现无菌豚鼠在EV71感染后血清中无特异性抗体产生,但是基础抗体值增加；普通豚鼠血清中检测出特异性抗体,且基础抗体值增高程度较无菌豚鼠高,具体原因不明。CD4/CD8比值升高,且CD4、CD8细胞数均有不同程度的降低。疱疹出现处的组织有明显的水肿现象,无炎性因子浸润。结果表明无菌豚鼠更易感染EV71病毒。无菌豚鼠和普通豚鼠在感染病毒后免疫指标检测结果差异大,需要进一步的探讨其原因。本文首次尝试使用无菌豚
Guinea pigs are widely used in immunology、infectious diseases、pharmacology and pediatrics. Because of pathogen free, the immunologic system Germ-free guinea pig is in quiescenxe, so that the immune reaction of germ free animal is intensive. The breeding of germ free guinea pigs was successfully initiated in 1932 by Reyniers. Although we have successfully cultivated germ free guinea in 1980s., because of the limited devolping of biomedicine, the needs of germ free animals was small.Now as the develoging of life science and biomedicine, especially the intensive studies in infection disease,the requirement of high quality laboratory animals is increasing. Its great economic value and application potential are displaying gradually.
Enterovirus 71 (EV71) is a small nonenveloped virus with a genome of single-strand positive RNA of about 7,500 nucleotides(nt); it belongs to the genus Enterovirus of the family Picornaviridae. EV71 is classified as Human enterovirus species A, along with some coxsackie A viruses, such as CA10 and CA16, which cause hand, foot, and mouth disease and herpangina. However, EV71 infection is sometimes associated with severe neurological diseases, such as brain stem encephalitis and poliomyelitis-like paralysis EV71 were first emphasized during an outbreak in Bulgaria in 1975, after that it cause several larger scale outbreak.Although EV71 has strong epidemicity, the research of pathogenic mechanism and post-infection is rarely disscused. The most impotant reason is its diffcuty in infecting animals. Although infection models of EV71 have been established with young mice (1 to 7 days old),the lab staff have trouble in collection blood and other esperimental materials.
For these reseasons, we carried out the study on normalization for cultivating germ-free guinea pigs and comparative study on EV71 infected germ-free guinea pigs.
The Germ-free guinea pigs were cleansed by cesarean section, and the offspring were fed with artificial milk by hand in germfree isolator. Detect the gem-free situation at every fixed time by hematology analyzer. Hematological Parameters were compared between Germ-free and conventional guinea pig by SPSS 12.0.
EV71 was combined inoculated on the germ-free guinea pigs, which gained from the first part of the research. After that we detected the virus replication of germ free guinea pigs. Then the change of total IgG、IgA、IL-2、IL-6 in blood serum and CD4/CD8 were detected before and after EV71 infection. The result indicated that germ free guinea pigs are more susceptive to EV71. There are significant difference between germ free and conventional guinea pigs on immune indices after EV71 infection.Because the reason is not clear, more research is needed to be done in this area.It is the first time we use germ free guinea pigs as EV71 infection animal model,which can be helpful references in the usage of germ free guinea pigs as infection animal model.
EV71是一种含有正向RNA单链无包膜病毒,属于肠道病毒属,小核糖核酸病毒科。EV71和CA10、CA16等共属于A性肠道病毒,容易引起手足口病。但是EV71的感染有时会产生严重的神经系统疾病,如无菌性脑炎、脑干脑炎、和脊椎灰质炎相似的麻痹。自1974年首次报道以来，EV71已在世界范围内引起十多次爆发与流行。例如澳大利亚、欧洲、亚洲和美国。近年来在亚太地区有上升的趋势。虽然HFMD的流行性很强,但是对其致病机理以及感染后比较医学方面的研究很少。其主要原因之一是EV71很难感染动物,制作成稳定的动物模型。并且国内外建立成功的动物模型多为出生七日以内的乳鼠,难以采集血液、器官材料进行比较医学方面的研究。
基于以上理由,本论文开展了无菌豚鼠制作技术的规范化和无菌豚鼠EV71比较医学的研究。
无菌豚鼠制作技术的规范化实验采取子宫摘除术的方法对普通级豚鼠进行剖腹净化,在无菌隔离器内剥离子宫取仔,用人工哺乳的方法将仔豚鼠培育成无菌豚鼠,并每隔一段时期进行无菌检测。以保证无菌豚鼠的质量。同时对孕豚鼠、豚鼠子宫摘除手术日期、豚鼠人工乳、人工饲料等进行对比选择。40日左右时,分别对动物心脏取血,自动生化分析仪测定无菌豚鼠、普通级豚鼠血液学参数。比较三组动物血常规、血生化有无差异。为无菌豚鼠的应用和推广奠下基础。
无菌豚鼠EV71比较医学研究实验利用第一部分得到的无菌豚鼠作为模型动物,利用多种接种方法接种病毒。分别进行临床观察、病原学检查,用来验证动物感染动物模型成立。同时对不同组感染后动物模型的一些免疫指标如：EV71特异性抗体,动物基础性IgG、IgA、IL-2、IL-6水平及CD4/CD8变化进行比较分析。感染了EV71的无菌豚鼠出现疱疹、腹泻等临床症状,普通豚鼠无明显的临床症状。病原学检测无菌豚鼠100%感染病毒,在多个组织中可检测到病毒；普通豚鼠病毒检出率低并无明显的规律性。免疫学检测中,首次发现无菌豚鼠在EV71感染后血清中无特异性抗体产生,但是基础抗体值增加；普通豚鼠血清中检测出特异性抗体,且基础抗体值增高程度较无菌豚鼠高,具体原因不明。CD4/CD8比值升高,且CD4、CD8细胞数均有不同程度的降低。疱疹出现处的组织有明显的水肿现象,无炎性因子浸润。结果表明无菌豚鼠更易感染EV71病毒。无菌豚鼠和普通豚鼠在感染病毒后免疫指标检测结果差异大,需要进一步的探讨其原因。本文首次尝试使用无菌豚
Guinea pigs are widely used in immunology、infectious diseases、pharmacology and pediatrics. Because of pathogen free, the immunologic system Germ-free guinea pig is in quiescenxe, so that the immune reaction of germ free animal is intensive. The breeding of germ free guinea pigs was successfully initiated in 1932 by Reyniers. Although we have successfully cultivated germ free guinea in 1980s., because of the limited devolping of biomedicine, the needs of germ free animals was small.Now as the develoging of life science and biomedicine, especially the intensive studies in infection disease,the requirement of high quality laboratory animals is increasing. Its great economic value and application potential are displaying gradually.
Enterovirus 71 (EV71) is a small nonenveloped virus with a genome of single-strand positive RNA of about 7,500 nucleotides(nt); it belongs to the genus Enterovirus of the family Picornaviridae. EV71 is classified as Human enterovirus species A, along with some coxsackie A viruses, such as CA10 and CA16, which cause hand, foot, and mouth disease and herpangina. However, EV71 infection is sometimes associated with severe neurological diseases, such as brain stem encephalitis and poliomyelitis-like paralysis EV71 were first emphasized during an outbreak in Bulgaria in 1975, after that it cause several larger scale outbreak.Although EV71 has strong epidemicity, the research of pathogenic mechanism and post-infection is rarely disscused. The most impotant reason is its diffcuty in infecting animals. Although infection models of EV71 have been established with young mice (1 to 7 days old),the lab staff have trouble in collection blood and other esperimental materials.
For these reseasons, we carried out the study on normalization for cultivating germ-free guinea pigs and comparative study on EV71 infected germ-free guinea pigs.
The Germ-free guinea pigs were cleansed by cesarean section, and the offspring were fed with artificial milk by hand in germfree isolator. Detect the gem-free situation at every fixed time by hematology analyzer. Hematological Parameters were compared between Germ-free and conventional guinea pig by SPSS 12.0.
EV71 was combined inoculated on the germ-free guinea pigs, which gained from the first part of the research. After that we detected the virus replication of germ free guinea pigs. Then the change of total IgG、IgA、IL-2、IL-6 in blood serum and CD4/CD8 were detected before and after EV71 infection. The result indicated that germ free guinea pigs are more susceptive to EV71. There are significant difference between germ free and conventional guinea pigs on immune indices after EV71 infection.Because the reason is not clear, more research is needed to be done in this area.It is the first time we use germ free guinea pigs as EV71 infection animal model,which can be helpful references in the usage of germ free guinea pigs as infection animal model.
引文
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[2]Meir Eir Lev, Jack R. BA.Impaired Delayed Hypersensitivity in Germ-Free Guinea Pigs Immunology,1970,19 (47)
[3]Backhed F, Ley RE, et;Host-bacterial mutualism in the human intestine [J]. Science. 2005; 307:1915-1920
[4]Hooper LV, Bry L, Falk PG,et.Host-microbial symbiosis in the mammalian intestine exploring an internal ecosystem[J]. Bioessays.1998;20:336-343
[5]Hooper LV,Gordon JI.Comnnensalhost-bacterial relationship in the gut[J]. Science. 2001; 292:1115-1118
[6]MIYAKAWA, M., KISHIMOTO, H., ITAYA, J., YEI, Y.and KASHIO, T. (1958). 'Homo-transplantationexperiment in germ-free animals.'Acta path. Jap.,8,177.
[7]ROZMIAREK, H., CHORPENNING, F. W., GISLER, D. B.and FREDERICK, G. I. (1969).'Presence of a normally occurring antibody in germ-free guinea pigs.'Abstracts of 8th Annual Meeting of the Assoc. of Gnotobiotics,p.8.
[8]张旭帆卢金星李革等,悉生豚鼠体内复方双歧杆菌制剂对福氏志贺氏菌的作用[J]中国预防医学杂志,1990，1(8)：18-23
[9]Helen R. Jervis, DSc,et.Animal Model:Experimental Entamoeba histolytica Infection in the Germfree Guinea Pig
[10]R. Higgins, J.P. Descoteaux and R. Degre.Pathogenesis of Experimental Leptospira interrogans Serotype icterohaemorrhagiae Infection in the Guinea Pig:Possible Role of Endotoxin of Intestinal Bacteria in the Development of Lesions[J]Can. J. comp. Med.44:304-308
[11]STEPHEN P. COBURN.*J, et.Role of Intestinal Microflora in the Metabolism of Vitamin B-6 and 4'-Deoxypyridoxine Examined Using Germfree Guinea Pigs and Rats.[J]Nutritio,1989,88:181-188
[12]无菌豚鼠的饲育方法及其特征和应用的研究
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