To isolate and identify avian influenza virus in crested ibis(Nipponian nippon),specimens were collected from dead crested ibis.The suspension of brain tissue of crested ibis was used to inoculate SPF chicken embryo,then the AIV was isolated.Hemagglutination test(HA),hemagglutination inhibition(HI),the death time of chicken embryos(MDT),median lethal dosage(LD50) and the intracerebral pathogenicity index(ICPI) tests were carried out respectively to identify the serotype of AIV.The strain could agglutinate the erythrocytes of chicken,duck,sheep,goat,swine,human and rabbit.The hemagglutination of viruses could be inhibited by H9N2 antiserum.The MDT of isolated strain was 48 h,index of LD50 was 50%,and ICPI was 1.544.The results showed that the serotype of this strain was identified as H9,which belonged to LPAIV.This strain of AIV derived from crested ibis was LPAIV,and its virulence was very weak.

Objective:To investigate the role of phosphatidylinositol 3-kinase(PI3K)/AKT signaling pathway in bone homeostasis in vivo via conditionally disrupting the Pten gene in osteoblasts using Cre-LoxP system and to create a mouse model of bone metabolism-associated disease.Methods: Soft X ray,bone mineral density analysis and histological analysis were used to compare the bone mass of wild-type mice with that of Pten gene knockout mice.Besides,real-time RT-PCR was also employed to explore the expression level of osteoblast differentiation markers.Results: Pten mutant mice were of normal size.However,soft X-ray,bone mineral density and histological analysis demonstrated a dramatically and progressively increased bone mineral density throughout the life of mutant mice,which were similar to osteopetrosis in humans.Accordingly,real-time RT-PCR revealed a significant increase in the expression levels of osteoblast differentiation markers such as osteocalcin,collagen Ⅰ and alkaline phosphatase.Conclusion:A mouse model of osteopetrosis was established,which will facilitate studies on the molecular mechanisms underlying this disorder.

According to GenBank of the rabies virus glycoprotein gene sequence,two pairs of specific primers were designed using Oliga software.Amplified the full-length cDNA fragment of GP gene of Flury strain by reverse transcription-polymerase chain reaction(RT-PCR)and cloned into pMD-18T vector for sequencing.Sequence and homology analysis showed that the cDNA of RGP encoding 524 amino acids was consisted of 1 574 nucleic acids.The homologies of nucleic acid and amino acid sequences to those reported were 82.3%~96.3% and 88.4%~94.3%,respectively.

Objective To evaluate the usefulness of electron microscopy in detection of virus in animal samples. Methods Negative staining and ultrathin sections were used for electron microscopic examination. Results Plentiful mouse poxvirus particles were observed under the electron microscope by both staining methods. Conclusion Electron microscopy is a reliable and useful technique and can be successfully used in animal virus detection.

狂犬病(rabies)是一种自然疫源性疫病,可感染所有温血动物,从上百年的流行规律分析和实验研究已证实犬科动物是本病的主要宿主,人与犬的接触(玩赏伴侣关系居多)已是狂犬病散发的主要原因,人则主要通过病犬咬伤而感染.其主要临床表现有恐水怕惊、咽肌痉挛、持续或阵发性抽搐等症状.狂犬病是迄今为止人类疾病病死率最高的急性传染病,一旦发病,病死率高达100%,全世界每年约有3.5万～5万人死于狂犬病,该病主要分布在亚洲、非洲和拉丁美洲等发展中国家.中国在2000多年前就有狂犬病的记载,也是受狂犬病危害最为严重的国家之一.狂犬病在我国曾一度得到有效控制,但近年来狂犬病疫情又有抬头和快速回升的趋势.

狂犬病是一种急性传染性疾病,狂犬病病毒属于弹状病毒科,可感染所有温血动物,其主要临床特征表现为恐水怕惊、咽肌痉挛、持续性或阵发性抽搐等症状。世界各地均有狂犬病的发生,其主要传染源是犬科和猫科动物。中国近十年来狂犬病发病率有所上升,主要原因是养犬数量剧增、免疫接种比率下降。目前,狂犬病的发病机制还不是很清楚,期望在阐明发病机理的基础上研制出特效药物。目前惟一能够控制狂犬病的方法是进行狂犬病疫苗的有效预防接种,文章就有关狂犬病疫苗的研制进展进行了分析与述评。

应用本中心选育的兔出血症病毒(RHDV)配制成油佐剂抗原,按特定基础免疫与强化免疫程序接种陕西关中驴,适时采集血浆,选用改进的提纯工艺和质检方法,研制用于预防和治疗驴抗兔瘟免疫球蛋白(IgG)生物制剂获得理想结果.经在全国6个省、市、自治区试用后疗效显著,治愈率85%,紧急预防有效率为100%.

本文利用in viuo治疗模型对关于5-Fu和IFN-a抗肿瘤协同作用机理进行了研究。人肝细胞癌细胞株PLC／PLE／5，接种到裸鼠背部皮下，复制裸鼠模型，分为对照组、5-Fu治疗组、IFN-a治疗组以及5-Fu／IFN-a联合用药组并检测5-Fu体内代谢相关酶的活性。5-Fu以0．5mg／只，腹腔注射，IFN-a以10万IU／只，皮下注射，3次／w。用药后1h、48h、7d分别取各组裸鼠肿瘤组织进行5-Fu体内代谢相关酶—thymidylate synthase(TS)，dihydropyrimidine dehydrogenase(DPD)，thymidinphos phorylase(TP)、orotate phosphoribosyl transferase(OPRT)、uridine phosphorylase(UP)等酶的活性测定。结果5-Fu治疗组、IFN-a治疗组以及联合用药组之间的5-Fu代谢相关酶活性未发现显著性差异，由此推测肝细胞癌治疗中5-Fu+IFN-a联合治疗中，IFN-a对5-Fu代谢相关酶的生物调节作用并不显著。

目的:探讨经脾内同种异体移植培养的原代胎肝前体细胞与成体肝细胞悬液对大鼠药物性肝衰竭的疗效,并观察脾内移植肝细胞的生物学特性.方法:采用D-氨基半乳糖(D-gal)建立大鼠急性肝衰竭模型,24 h后随机分为三组进行治疗.A组.经脾内移植体外培养7d的肝细胞2×107;B组:经脾内移植肝细胞悬液2×107;C组:经脾内注射生理盐水1 ml.观察受体大鼠的存活率、肝脏功能和病理变化及移植肝细胞的生物学特性.结果:A组、B组大鼠存活率(77%、59%)与C组大鼠存活率(22%)相比具有显著性差异,肝功能各项指标均有明显改善,A、B组与C组大鼠的肝功能改变方面有统计学差异.经HE和PAS染色证实,移植的肝细胞在受体脾内结构和功能保持较好.结论:经脾内移植的培养的原代胎肝前体细胞和肝细胞悬液均能提高大鼠药物性肝衰竭的存活率、改善肝功能及肝脏组织病理变化,但培养原代胎肝前体细胞优于成体肝细胞悬液.

OBJECTIVE:To study whether the porcine alpha1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte negatively expresses GT mRNA and resists to the cytotoxicity of nature antibody in human serum.METHODS:The porcine alpha1, 3 galactosyltransferase gene siRNA targeted vector (pPNTloxPGTsiRNA) were construct with pPNTloxPGT and pMXSV/U6 vector. Positive-negative selection was used to produce a heterozygous pPNTloxPGTsiRNA knockout (+/-) clone. The GT mRNA expressions were detected with northern blot. Complement-mediated NAb cytotoxicity after incubation of hepatocytes with NAbs and complement was determined using 3- (4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS, tetrazolium salt) colorimetric assay.RESULTS:The pPNTloxPGTsiRNA targeted porcine hepatocyte (+/-) negative express GT mRNA. Only 14% to 18% cytotoxicity can be detected at the highest serum concentration. The pPNTloxPGT targeted porcine hepatocyte (+/-) express GT mRNA just as the wild type porcine cells and the cytotoxicity are 77% to 83%.CONCLUSION:The porcine a1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte (+/-) negative express GT and resisted to nature antibody in human serum.

To prepare anti-porcine reproductive and respiratory syndrome virus IgG injection, we vaccinated GuanZhong donkey with inactivated porcine reproductive and respiratory syndrome virus antigen which is accompanied with adjuvant, according to basic immune and booster immune procedure.After 9～11 days,we extracted the blood plasma from the donkeys ,then isolating and,purificating as well as identification anti-PRRSV IgG.Finally,we carried out the serum neutralization test on the IgG ,the antibody value in the animal serum is reached over 1∶320,indicating that this method can successfully produce the anti-porcine reproductive and respiratory syndrome virus IgG injection.

采用重复手术方法,对经过激素刺激超数排卵的家兔成熟卵母细胞进行收集和筛选评价,探索不同组合激素处理对家兔进行超数排卵,获得大量的高质量卵母细胞简单有效的方法,为进行兔转基因、核移植等相关方面的研究奠定基础.采用FSH+HCG和FSH混合PVP(25%)两种激素处理组合,并用正常排卵雌兔作对照,经过三次手术法取卵(每次间隔至少50 d以上).经输卵管插管,由输卵管伞端收集卵母细胞并进行筛选.结果,采用激素处理的两组获取总卵母细胞数和可用卵数比对照组均高出3倍以上(P＜0.05),两种超排方法之间超排效果无显著差异(P＞0.1);三次连续手术均成活的家兔中,对照组和FSH+HCG组三次取卵效果无显著差异(P＞0.05),FSH混合PVP(25%)组首次手术与第三次手术出现显著差异(P＜0.01).重复采用FSH+HCG和FSH混合PVP(25%)两种激素处理组合超数排卵,手术法收集卵母细胞,简化超排程序,增加超排效果,提高实验动物利用率.

本实验用配有佐剂的猪瘟病毒抗原,按特定基础免疫与强化免疫程序接种关中毛驴,用两次盐析法沉淀提取抗猪瘟病毒IgG,其中对提纯制备工艺所涉及的pH值、温度、硫酸铵浓度等工艺进行探索比较,继之对单位体积内的蛋白浓度、酶标抗体效价和稀释度进行了标化,同时对质量监控进行了平行性分析.本实验以陕西关中毛驴作为免疫反应供浆动物;研制用于预防和治疗猪瘟的抗病毒免疫球蛋白(IgG)生物制剂获得理想结果.经6省、市、区试用后,深受用户和兽医防疫部门的一致好评和欢迎.

目的:了解粒细胞集落刺激因子(G-CSF)对肝干细胞的动员作用影响.方法:用CCl4法复制大鼠肝硬化模型,分为2组,行2/3肝切除3h后,实验组皮下注射G-CSF,对照组注射等量生理盐水,分别于24h、48h、7d、10d、2周后处死,取肝脏切片,免疫组化方法观测动物模型肝脏中CD34、Thy-1表达情况.结果:皮下注射G-CSF 5d后,G-CSF实验组和对照组肝脏内均未见Thy-1+细胞,CD34+细胞仅存在于门脉区血管内皮,实验组和对照组无差异.结论:G-CSF短期内应用对于肝干细胞的动员无明显作用.

用驴制备抗猪瘟病毒(HCV)免疫球蛋白,预防和治疗猪HCV感染.制备HCV抗原复合物,通过免疫驴,用纯化方法从驴血清中提取特异性抗体(IgG),经过理化学检定、血清学、动物效力实验.免疫驴均可产生特异IgG,Dot*.ELISA效价大于1∶640(IgG浓缩浓度为60 mg/mL);可保护猪免受HCV攻击;室温保存半年,4℃两年效价不变.已成功地应用HCV免疫驴制备出高效价特异性抗HCV*.IgG.

AIM: To investigate the function parameters of tissue engineered bladders with marrow stromal cells(MSCs). METHODS: The MSCs and the bladder-shaped accellular organ specific matrix (BAMG) were obtained from the New Zealand White rabbits. The exterior surface of BAMG was seeded with the resuspended MSCs. A total of 20 rabbits underwent a trigone-sparing cystectomy. The animals were randomly assigned to three groups. Group A ( n =6) underwent a closure of the trigone without a reconstructive procedure. Group B ( n =7) underwent a reconstruction using a BAMG only and Group C ( n =7) underwent a reconstruction using a BAMG plus autologous MSCs. Preoperative and postoperative cystometry and radiography studies were performed serially. Animals were euthanized at 12 weeks. Gross and histological analyses were also performed. RESULTS: The average time elapsed between the initial marrow biopsy and the final implantation of the tissue-engineered neo bladders was (21±3) days ( x±s ). Post operation, the average capacities in Group A, B and C were 22%, 85% and 95%, respectively and showed a marked difference in bladder compliance (11%, 104%, and 107% respectively). Histologically, group C showed a normal cellular organization consisting of a trilayer of urothelium, submucosa and muscle. The Group A, B presented a pattern of normal urothelial cells with a thickened fiberotic submucosa and a thin layer of muscle fibers. CONCLUSION: Our study demonstrates that the MSCs can be successfully used in rabbits bladder augmentations and replacement by tissue engineering techniques.

以关中驴为试验动物,用猪瘟病毒(HCV)、犬细小病毒(CPV)、兔瘟病毒(RHDV)、鸡新城疫病毒(NDV)、伪狂犬病毒(PRV)等病毒进行免疫,检测所产生的抗体效价,并与猪、鸡、兔、犬、及猫等动物进行比较.试验结果表明,关中驴与对照动物产生的抗体效价一致,证明了关中驴可替代其它动物用于生物制品研究.

陕西省周至县楼观台野生动物抢救中心人工饲养的8只幼龄朱鹮突然发病,迅速死亡,死前无特征性症状.根据剖检变化、实验室检验,确诊此次朱鹮发病为新城疫病毒感染所致.