Abstract:目的建立稳定分泌抗H5N1亚型禽流感病毒血凝素单克隆抗体的杂交瘤细胞系,为进一步研究禽流感诊断技术奠定基础。方法以纯化的H5亚型禽流感病毒按常规方法免疫BALBc小鼠,最后一次免疫后第3天取其脾细胞与SP20细胞在聚乙二醇作用下融合,用选择性培养、有限稀释法克隆和血凝抑制试验进行筛选,对获得阳性克隆株用ELISA方法进行亚型鉴定,并用37株H5、H7、H9亚型AIV测定其特异性、覆盖性。结果最后获得了3株分泌特异性抗体的杂交瘤细胞,命名为1E5、4A4、4B1,经长期体外培养和冻存后复苏能稳定地分泌抗体。经鉴定,其亚型均为IgG1、kappa链。腹水HI效价1∶210～1∶216,细胞培养上清HI效价1∶26～1∶28。3株杂交瘤所分泌的单克隆抗体均能与本中心保存的全部20株H5亚型禽流感病毒分离株发生反应,而与15株H9亚型禽流感病毒分离株、2株H7亚型禽流感病毒分离株以及H1H4、H6H15亚型禽流感病毒标准毒株均不反应,与鸡新城疫病毒、鹅新城疫病毒、鹅腺病毒和鸡产蛋下降综合征病毒等均无交叉反应。结论所获3株单克隆抗体可用于禽流感病毒特异性诊断试剂的研制。

Abstract:Objective To prepare the monoclonal antibodies specific to subtype of H5N1 avian influenza virus and offer tool for correlative researches.Methods Monoclonal antibodies specific to subtype of H5N1 avian influenza virus were developed by fusing SP2/0 myeloma cell with spleen cell of BALB/c mice immunized with AIV_H5N1, the characterization of three McAbs can specifically react with AIV_H5N1 by hemagglutination inhibit test(HI), enzyme linked immunosorbent assay(ELISA) and immunofluorescence. The inmmunoglobulin subclass of the McAbs was then identified. Results Three colone hybridoma secreting McAbs against AIV_H5N1 were obstained as F8,F9,G11 respectively, the inmmunoglobulin subclass of the McAbs was identified as IgG1,IgG2a and IgG2b. Conclusion The highly effective and specific McAbs G11 can play an important role in diagnosis, treatment and prevention of the infection of AIV_H5N1

Abstract:目的建立一种单克隆抗体介导的、经生物素—亲和素系统放大的H5亚型禽流感病毒捕获ELISA检测方法,为进一步研究检测试剂盒提供基础。方法用亲和层析法纯化抗禽流感病毒H5亚型血凝素单克隆抗体,包被微量反应板,用于捕获病毒抗原,再用生物素标记的单抗和酶标亲和素来检测病毒血凝素抗原,经方阵试验优化ELISA反应体系。用该方法检测H1—H15亚型AIV标准毒株和H5、H7、H9亚型AIV分离株,并与血凝和血凝抑制试验比较,评价其敏感性和特异性。结果纯化后的单抗具有良好的反应活性,生物素标记单抗工作浓度为1∶5000;ELISA对H5亚型AIV的检出限为025个血凝单位。该ELISA反应体系能检出H5N3标准株和所有20株国内H5亚型AIV分离株,而与其他14个血凝素亚型的AIV标准株、15个H9亚型AIV分离株和2个H7亚型AIV分离株均无交叉反应。结论初步建立了检测H5亚型禽流感病毒的单抗—生物素捕获ELISA方法,为研制试剂盒和进一步应用试验提供了基础。

Abstract:目的表达H5N1亚型禽流感病毒(AIV)NS1蛋白,用于AIV感染与注射灭活疫苗鸡的鉴别诊断和NS1蛋白功能研究。方法采用RT_PCR方法对H5N1亚型AIVNS1基因进行扩增,将PCR产物克隆于pGEM_T_easy载体,将该基因插入pGEX_4T_1中构建NS1基因原核表达载体,转化BL21大肠杆菌后,在IPTG诱导下表达NS1蛋白,Westernblot鉴定表达NS1蛋白。结果成功克隆H5N1亚型AIV的NS1基因,其核苷酸序列长度为690bp,编码230个氨基酸残基。构建NS1基因原核表达载体在大肠杆菌内表达出约51×103的NS1融合蛋白。Westernblot鉴定表明表达NS1蛋白与H7N2AIV感染鸡血清有反应性。结论在大肠杆菌中成功表达了H5N1亚型AIVNS1基因蛋白,具有与感染H7N2亚型AIV阳性血清反应原性。

Abstract:Objective To compare the pathogenicity of avian influenza virus to different mammals. Methods\ Four different species of mammal were selected, receiving the AIV through nasal cavity; then the changes of body temperature,body weight,clinical signs and pathology were observed, the virus from the experimental animals isolated and the antibody detected. Results Among mouse,rat,cavia cobaya and gerbil, mouse is the most sensitive animal model to AIV. Conclusion ICR and NIH mouse are suitable animal models for Avian influenza infection.

Abstract:Objective To explore techniques of isolating embryonic stem-like cells (ESLCs) from mouse blastocyst inner cell mass and the utilization of ESLCs to generate chimerical mice. Methods\ ESLCs from mouse blastocyst inner cell masses of 3\^5 days were isolated as donor' cells to inject receptors'blastocysts to generate chimerical mice by micromanipulating techniques, and all those blastocysts were transfered to the uterine of pseudopregnant female mice to gernerate chimerical mice. Results All the ESLCs isolated from 36 inner cell masses were injected into 256 blastocysts of Kunming mice ,and those blastocysts were transferred to uterine of 32 pseudopregnant female mice, 2 chimerical mice from 12 gernerations were obtained. Conclusion \ Technique of isolating ESLCs was a new approach in generating chimerical animals, especially in some animals in which Esc lines were not constructed.

Abstract:目的建立大鼠体外循环实验模型。方法雄性成年SD大鼠10只(450～550g),麻醉后给予气管切开,呼吸机辅助通气,股动脉置管接监护仪实时监测动脉血压并按时采集动脉血标本,股静脉置管用于持续补液和静脉血样采集。经右颈静脉置多孔引流管至右心房行中心静脉引流,血液经特制微型膜肺氧合后,由蠕动泵经右颈动脉实施灌注。本模型采用林格氏液和贺斯进行无血预充,总量为16ml,晶胶比为1∶1,灌注流量100～150ml(kg·min),转流时间60min。结果实验中膜肺氧合满意,血流动力学稳定,所有动物都成功脱机。结论利用大鼠可以建立简单、廉价的体外循环动物模型。本模型适用于研究与体外循环相关的全身炎症反应和多脏器功能损伤的病理生理机制及其防治策略。

Abstract:Objective To set up the model of multiple channels gastric electrical stimulation and gastric emptying. Methods\ Twelve British Beagle dogs were involved in this study .Four pairs of bipolar electrodes were implanted on the stomach serosa along the greater curvature . A jejunal cannula was implanted 20cm beyond the pylorus for the assessment of gastric emptying. Results\ 1. Chyme was easily collected from the jejunal cannula, which represented the effect of various gastric electrical stimulation and various stimulation parameters on gastric motility. 2. Gastric slow waves were recorded clearly and steadily via gastric serosal multiple electrodes. It could record a variety of gastric slow wave in various experiments and various periods. 3. Single_channel and multi_channel electrical stimulation of long pulse via stomach serosal electrodes was able to entrain gastric slow wave. Conclusion\ Multiple channels gastric electrical stimulation is an available method for investigating gastric electrophysiology, gastric pacing and effect of gastric pacing on gastric emptying. British Beagle dogs are ideal materials for this model.

Abstract:Objective To discuss the method of establishment of cardiac ischemic model in rats. Methods The left anterior descending coronary artery (LAD) was ligated to form an ischemic area which located at the anterolateral left ventricular free wall, accounting for 20%-50% of it. Results\ 85 animals' LAD were stitched, and 74 survived. Among these survived animals, 60 with heart dysfunction were confirmed as qualified ischemic model by operative observation, electrocardiogram and histopathology. Conclusion\ This method can easily create the ischemic heart model with acceptable death rate, but the model's stability is varying, which requires more candidate samples.

Abstract:Objective To describe the pathology of liver tumors in rabbits induced by diethylnitrosamine and the expression of c myc, p53 genes and AFP in the tumors and pericarcinomatous tissues. Method 25 nodules in 5 rabbit livers and 5 pieces of pericarcinomatous tissues each from one liver were observed by light microscopy and electron microscopy, and the expressions of c myc gene, p53 gene and AFP were also observed in these tissues with immunohistochemical staining.Results and Conclusion There were 10 nodules of adenomatous hyperplasia, 11 of well differentiated hepatocellular carcinoma(HCC)and 4 of moderate differentiated HCC among the 25 nodules. The tumorigenesis of HCC in rabbits induced by diethylnitrosamine was related to the over expressions of c myc gene, AFP and the mutation of p53 gene. C myc over expression appeared first and p53 mutation the latest during the tumorigenesis of HCC. AFP over expression reached the top positive reaction ratio(93 3%) among the 3 detected parameters.

Abstract:目的探索建立与人类乳腺增生病相类似动物模型的最佳方法。方法采用手术、己烯雌酚、黄体酮处理动物。检测血液中雌二醇(E2)、黄体酮(P)、促卵泡生成激素(FSH)、促黄体生成激素(LH)水平;乳腺组织作组织形态学观察与评价。结果与对照组比较,各实验组动物的乳腺组织切片均有典型的乳腺增生;血清中各激素的水平也有不同的变化。结论采用“己烯雌酚 黄体酮”方法,以家兔为实验对象,建立乳腺增生病的动物模型是目前最好的方法。

Abstract:Objective To determine susceptibility of adult strain Kunming mouse to rotavirus (RV) infection. Methods Adult strain Kunming mice were experimentally perorally infected by group A rotavirus, human Wa and rhesus SA11. Clinical response was observed and RV antigen in animal stools was detected. Results Apparent diarrhea was observed in mice 2 days post infection and peaked at 4 days; RV antigen could be detected in stool samples collected from infected mice at least from the second to sixth day post infection. Conclusion\ Kunming mouse is susceptible to RV infection and could be used as an adult animal model for evaluation of efficacy of medicinal treatment and vaccine protection to RV infection.

Abstract:目的观察室温下21～25℃DMEM、血浆和血液 DMEM有氧灌注对离体兔肾的形态学影响,探讨离体肾在体外灌注情况下的细胞脱落规律。方法50只大耳白兔随机分为正常对照(5只)、DMEM组(15只)、血浆组(15只)、血液 DMEM组(15只),实验组观察时相点为灌注后1、3、6h。各组离体肾分别用相应的灌注液进行有氧灌注,通过大体观察、含水率、病理切片、脱落细胞记数、原位标记法(TerminaldeoxynucleotidyltransferaseTdTmediatedbiotindUTPnickendlabeling,TUNEL)凋亡染色进行灌注肾脏的细胞脱落规律分析。结果各组灌注后肾脏外观明显水肿,正常肾脏含水率为786%,灌注后肾脏含水率显著升高,DMEM组肾脏灌注6h后含水率为848%。光镜下见肾脏间质水肿、肾小球肾小管上皮细胞脱落、细胞核崩解、细胞密度减小。TUNEL染色正常对照凋亡细胞率为23%,DMEM组灌注1h细胞凋亡率即达159%。灌注液中脱落细胞数随灌注时间进行性增加。结论室温下离体肾脏有氧灌注后有较严重的肾脏上皮细胞脱落现象,凋亡和坏死均参与了其发生机制。

Abstract:This paper discusses current study status and existing problems of animal models of hepatic fibrosis. Rat is taken as a classical example to elaborate several available methods, pathogenesis, pathway, effect of each method with details and compare the advantages and disadvantages among them and different usage. In the end we advance the development trend in the future.

Abstract:目前非人灵长类结核病的诊断方法单一,给饲养和管理带来影响。通过总结和讨论人的结核病诊断技术,结合非人灵长类的实际情况,指出在结核病检疫中,虽然结核菌素是经典的早期诊断的方法,但血清抗体的检测是结核菌素试验的有效辅助手段,尤其对无反应猴群的结核病检测具有独特的优势。然而,IFNγ的检测有望替代结核菌素试验,实现体外检测结核病。传统的结核菌培养技术时间长,假阴性多,污染率高,给猴的结核病的确诊带来困难。原位PCR技术,能加快诊断速度,提高检出率,特别有利于对小病灶区的病源诊断。虽然目前该技术还不能替代细菌学诊断技术,但两种方法的有效结合将有利于提高非人灵长类的结核病的病源诊断。